Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling

Jennifer Whitsett, Artur Rangel Filho, Savitha Sethumadhavan, Joanna Celinska, Michael Widlansky, Jeannette Vasquez-Vivar

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 BH 4, 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 BH 4 therapies, which may be further aggravated by folate supplements.

Original languageEnglish (US)
Pages (from-to)143-150
Number of pages8
JournalFree Radical Biology and Medicine
Volume63
DOIs
StatePublished - 2013
Externally publishedYes

Fingerprint

Tetrahydrofolate Dehydrogenase
Endothelial cells
Recycling
Blood Vessels
Endothelial Cells
Folic Acid
Oxidation
Nitric Oxide Synthase
Superoxides
Assays
Fluorescence
High Pressure Liquid Chromatography
Clinical Trials
Kinetics
sapropterin
Enzymes
Therapeutics

Keywords

  • Dihydrobiopterin
  • Dihydrofolate
  • Endothelial drug metabolism
  • Free radicals
  • Methotrexate
  • Nitric oxide synthase

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

Cite this

Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling. / Whitsett, Jennifer; Filho, Artur Rangel; Sethumadhavan, Savitha; Celinska, Joanna; Widlansky, Michael; Vasquez-Vivar, Jeannette.

In: Free Radical Biology and Medicine, Vol. 63, 2013, p. 143-150.

Research output: Contribution to journalArticle

Whitsett, Jennifer ; Filho, Artur Rangel ; Sethumadhavan, Savitha ; Celinska, Joanna ; Widlansky, Michael ; Vasquez-Vivar, Jeannette. / Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling. In: Free Radical Biology and Medicine. 2013 ; Vol. 63. pp. 143-150.
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AU - Widlansky, Michael

AU - Vasquez-Vivar, Jeannette

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N2 - 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 BH 4, 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 BH 4 therapies, which may be further aggravated by folate supplements.

AB - 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 BH 4, 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 BH 4 therapies, which may be further aggravated by folate supplements.

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KW - Methotrexate

KW - Nitric oxide synthase

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