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 language | English (US) |
|---|---|
| Pages (from-to) | 143-150 |
| Number of pages | 8 |
| Journal | Free Radical Biology and Medicine |
| Volume | 63 |
| DOIs | |
| State | Published - 2013 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling
AU - Whitsett, Jennifer
AU - Filho, Artur Rangel
AU - Sethumadhavan, Savitha
AU - Celinska, Joanna
AU - Widlansky, Michael
AU - Vasquez-Vivar, Jeannette
PY - 2013
Y1 - 2013
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.
KW - Dihydrobiopterin
KW - Dihydrofolate
KW - Endothelial drug metabolism
KW - Free radicals
KW - Methotrexate
KW - Nitric oxide synthase
UR - http://www.scopus.com/inward/record.url?scp=84884840975&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84884840975&partnerID=8YFLogxK
U2 - 10.1016/j.freeradbiomed.2013.04.035
DO - 10.1016/j.freeradbiomed.2013.04.035
M3 - Article
C2 - 23707606
AN - SCOPUS:84884840975
VL - 63
SP - 143
EP - 150
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
SN - 0891-5849
ER -