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Cited by 210Nitric Oxide and Endothelin EffectsFolate and B Vitamins ResearchBiochemical and Molecular Research

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Critical Role for Tetrahydrobiopterin Recycling by Dihydrofolate Reductase in Regulation of Endothelial Nitric-oxide Synthase Coupling

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Crabtree, Mark J., Tatham, Amy L., Hale, Ashley B., Alp, Nicholas J., Channon, Keith M.·2009·Journal of Biological Chemistry
DOI 10.1074/jbc.m109.041483PMC PMC2788863
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Abstract

Tetrahyrobiopterin (BH4) is a required cofactor for the synthesis of nitric oxide by endothelial nitric-oxide synthase (eNOS), and BH4 bioavailability within the endothelium is a critical factor in regulating the balance between NO and superoxide production by eNOS (eNOS coupling). BH4 levels are determined by the activity of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in de novo BH4 biosynthesis. However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but the functional importance of DHFR in maintaining eNOS coupling remains unclear. We investigated the role of DHFR in regulating BH4 versus BH2 levels in endothelial cells and in cell lines expressing eNOS combined with tet-regulated GTPCH expression in order to compare the effects of low or high levels of de novo BH4 biosynthesis. Pharmacological inhibition of DHFR activity by methotrexate or genetic knockdown of DHFR protein by RNA interference reduced intracellular BH4 and increased BH2 levels resulting in enzymatic uncoupling of eNOS, as indicated by increased eNOS-dependent superoxide but reduced NO production. In contrast to the decreased BH4:BH2 ratio induced by DHFR knockdown, GTPCH knockdown greatly reduced total biopterin levels but with no change in BH4:BH2 ratio. In cells expressing eNOS with low biopterin levels, DHFR inhibition or knockdown further diminished the BH4:BH2 ratio and exacerbated eNOS uncoupling. Taken together, these data reveal a key role for DHFR in eNOS coupling by maintaining the BH4:BH2 ratio, particularly in conditions of low total biopterin availability.

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Figures

FIGURE 1 Schematic representation of the BH4 recycling pathway and eNOScoupling. BH4 is synthesized from GTP via a series of reactions involving GTPCH, 6-pyruvoyl-tetrahydropterin synthase, sepiapterin reductase ( SR ) and DHFR. DHFR activity can be inhibited by MTX. GFRP , GTP cyclohydrolase feedback regulatory protein. PTPS , 6-pyruvoyl-tetrahydropterin synthase.
FIGURE 1 Schematic representation of the BH4 recycling pathway and eNOScoupling. BH4 is synthesized from GTP via a series of reactions involving GTPCH, 6-pyruvoyl-tetrahydropterin synthase, sepiapterin reductase ( SR ) and DHFR. DHFR activity can be inhibited by MTX. GFRP , GTP cyclohydrolase feedback regulatory protein. PTPS , 6-pyruvoyl-tetrahydropterin synthase.CC-BY · From the PDF
Image 2
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Image 3
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FIGURE 4 Inhibition of endogenous DHFR activity by MTX:nitric oxide and superoxide production. A23187-induced conversion of arginine to citrulline was used as a measure of eNOS activity. A , eNOS activity (as indicated by the inhibition of citrulline accumulation by N -methyl arginine; NMA ) was attenuated in the presence of MTX in sEnd.1 cells (*, p < 0.05). B , after exposure to MTX (1 𝛍 M) for 16 h, cells were exposed to dihydroethidium (25 𝛍 M) for 20min,andaccumulationof2-hydroxyethidiumwasmeasuredbyHPLCwith fluorescence detection. MTX exposure markedly elevated cellular superoxide production(**, p < 0.01), which was significantly inhibited after pretreatment of cells with the NOS inhibitor, L-NAME ( C ) (100 𝛍 M; *, p < 0.05). n = 6 for each experiment.
FIGURE 4 Inhibition of endogenous DHFR activity by MTX:nitric oxide and superoxide production. A23187-induced conversion of arginine to citrulline was used as a measure of eNOS activity. A , eNOS activity (as indicated by the inhibition of citrulline accumulation by N -methyl arginine; NMA ) was attenuated in the presence of MTX in sEnd.1 cells (*, p < 0.05). B , after exposure to MTX (1 𝛍 M) for 16 h, cells were exposed to dihydroethidium (25 𝛍 M) for 20min,andaccumulationof2-hydroxyethidiumwasmeasuredbyHPLCwith fluorescence detection. MTX exposure markedly elevated cellular superoxide production(**, p < 0.01), which was significantly inhibited after pretreatment of cells with the NOS inhibitor, L-NAME ( C ) (100 𝛍 M; *, p < 0.05). n = 6 for each experiment.CC-BY · From the PDF
Image 5
CC-BY · From the PDF
FIGURE 6 MTX exposure allows oxidation of BH4 in GCH cells. To test whether the effect of MTX had a differential effect at different amounts of GTPCH, a dose-response curve with doxycycline was carried out in GCH cells expressing tetracycline-regulatable GTPCH, and BH4 was quantified by HPLC. GCHcellsdemonstratedadose-dependent'switch-off'ofBH4synthesisafter exposure to increasing concentrations of doxycycline ( DOX ) for 5 days. Cells exposedtoMTX(1 𝛍 M)for16h( opensymbols , dashedline ) exhibit significantly lower amounts of BH4 compared with control cells (*, p < 0.05) ( A ) and a concomitantincreaseinBH2levels(*, p < 0.05) ( B ). n = 3for each experiment.
FIGURE 6 MTX exposure allows oxidation of BH4 in GCH cells. To test whether the effect of MTX had a differential effect at different amounts of GTPCH, a dose-response curve with doxycycline was carried out in GCH cells expressing tetracycline-regulatable GTPCH, and BH4 was quantified by HPLC. GCHcellsdemonstratedadose-dependent'switch-off'ofBH4synthesisafter exposure to increasing concentrations of doxycycline ( DOX ) for 5 days. Cells exposedtoMTX(1 𝛍 M)for16h( opensymbols , dashedline ) exhibit significantly lower amounts of BH4 compared with control cells (*, p < 0.05) ( A ) and a concomitantincreaseinBH2levels(*, p < 0.05) ( B ). n = 3for each experiment.CC-BY · From the PDF
FIGURE 7 DHFR regulates eNOS coupling in GCH/eNOS cells. GCH and GCH/eNOScells in the presence and absence of doxycycline were exposed to MTX(1 𝛍 M) for 16 h at 37 °C, and intracellular biopterin levels were quantified by HPLC as detailed under 'Experimental Procedures.' A , Western blotting shows relative abundance of eNOS, GTPCH, and DHFR in respective cell lysates. Importantly, eNOS is only observed in GCH/eNOS cells and GTPCH protein expression is abolished following exposure to doxycycline. B , BH4 levels were significantly reduced in GCH/eNOS cells after exposure to MTX (*, p < 0.05). C , inhibition of DHFR with MTX resulted in a marked elevation in BH2levelscomparedwithuntreatedcontrolcellsinbothGCHandGCH/eNOS cells (*, p < 0.05). BH2 levels were also increased in GCH/eNOS cells treated with both doxycycline and MTX compared doxycycline alone (†, p < 0.05). D , in GCH cells MTX, but not doxycycline, exposure alone was sufficient to induce a striking decrease in BH4:BH2 ratio (#, p < 0.01). In GCH/eNOS cells, doxycycline decreased the BH4:BH2 ratio (*, p < 0.05), which was further exacerbated in the presence of MTX (§, p < 0.05, n = 6).
FIGURE 7 DHFR regulates eNOS coupling in GCH/eNOS cells. GCH and GCH/eNOScells in the presence and absence of doxycycline were exposed to MTX(1 𝛍 M) for 16 h at 37 °C, and intracellular biopterin levels were quantified by HPLC as detailed under 'Experimental Procedures.' A , Western blotting shows relative abundance of eNOS, GTPCH, and DHFR in respective cell lysates. Importantly, eNOS is only observed in GCH/eNOS cells and GTPCH protein expression is abolished following exposure to doxycycline. B , BH4 levels were significantly reduced in GCH/eNOS cells after exposure to MTX (*, p < 0.05). C , inhibition of DHFR with MTX resulted in a marked elevation in BH2levelscomparedwithuntreatedcontrolcellsinbothGCHandGCH/eNOS cells (*, p < 0.05). BH2 levels were also increased in GCH/eNOS cells treated with both doxycycline and MTX compared doxycycline alone (†, p < 0.05). D , in GCH cells MTX, but not doxycycline, exposure alone was sufficient to induce a striking decrease in BH4:BH2 ratio (#, p < 0.01). In GCH/eNOS cells, doxycycline decreased the BH4:BH2 ratio (*, p < 0.05), which was further exacerbated in the presence of MTX (§, p < 0.05, n = 6).CC-BY · From the PDF
FIGURE 8 Inhibition of DHFR in GCH versus GCH/eNOS cells. After exposure to MTX (1 𝛍 M) for 16 h, cells were exposed to dihydroethidium (25 𝛍 M) for 20min,andaccumulationof2-hydroxyethidiumwasmeasuredbyHPLCwith fluorescence detection. A and C , MTX exposure markedly elevated cellular superoxide production (*, p < 0.05); however, this was not inhibited after pretreatment of cells with the NOS inhibitor, L-NAME (100 𝛍 M). B and D , MTX exposure markedly elevated cellular superoxide production (*, p < 0.05), which was significantly inhibited by L-NAME (100 𝛍 M) (†, p < 0.05, n = 6).
FIGURE 8 Inhibition of DHFR in GCH versus GCH/eNOS cells. After exposure to MTX (1 𝛍 M) for 16 h, cells were exposed to dihydroethidium (25 𝛍 M) for 20min,andaccumulationof2-hydroxyethidiumwasmeasuredbyHPLCwith fluorescence detection. A and C , MTX exposure markedly elevated cellular superoxide production (*, p < 0.05); however, this was not inhibited after pretreatment of cells with the NOS inhibitor, L-NAME (100 𝛍 M). B and D , MTX exposure markedly elevated cellular superoxide production (*, p < 0.05), which was significantly inhibited by L-NAME (100 𝛍 M) (†, p < 0.05, n = 6).CC-BY · From the PDF

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Metadata

Authors
Crabtree, M. J., Tatham, A. L., Hale, A. B., Alp, N. J., & Channon, K. M.
Affiliations
John Radcliffe Hospital (GB)
Corresponding author
Channon, Keith M.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2009-10
Volume
284
Issue
41
Pages
28128-28136
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m109.041483
PMC
PMC2788863
OpenAlex
W2058599067
Cited by
210
References
36
Citations by year
2026: 4 · 2025: 9 · 2024: 11 · 2023: 13 · 2022: 13 · 2021: 12 · 2020: 14 · 2019: 13 · 2018: 12 · 2017: 17 · 2016: 11 · 2015: 12 · 2014: 13 · 2013: 6 · 2012: 12
FWCI
11.08
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
Wellcome Trust (GR074428/Z/04/Z)
Topics
  • Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
  • Health Sciences › Nursing › Nutrition and Dietetics › Folate and B Vitamins Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Molecular Research
Keywords
tetrahydrobiopterin, dihydrofolate reductase, endothelial nitric oxide synthase, eNOS uncoupling, 7,8-dihydrobiopterin, GTP cyclohydrolase I, nitric oxide production, superoxide production, methotrexate, endothelial cells
MeSH terms
Animals, Antioxidants, Biopterins, Cell Line, Cells, Cultured, Doxycycline, Endothelium, Vascular, GTP Cyclohydrolase, Methotrexate, Oxidation-Reduction, Superoxides, Tetrahydrofolate Dehydrogenase, RNA, Small Interfering, NIH 3T3 Cells, Endothelial Cells, Mice, Nitric Oxide Synthase Type III

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