Critical Role for Tetrahydrobiopterin Recycling by Dihydrofolate Reductase in Regulation of Endothelial Nitric-oxide Synthase Coupling RELATIVE IMPORTANCE OF THE DE NOVO BIOPTERIN SYNTHESIS VERSUS SALVAGE PATHWAYS*
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.
Pharmacology, Toxicology and Pharmaceutical Science
5
5%
Immunology and Microbiology
2
2%
Other
10
9%
Unknown
29
27%
Attention Score in Context
Attention Score in Context
This research output has an Altmetric Attention Score of 15. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 04 December 2025.
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#3,209,289
of 34,115,020 outputs
Outputs from Journal of Biological Chemistry
#2,226
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Outputs of similar age
#12,578
of 172,496 outputs
Outputs of similar age from Journal of Biological Chemistry
#26
of 485 outputs
Altmetric has tracked 34,115,020 research outputs across all sources so far. Compared to these this one has done particularly well and is in the 90th percentile: it's in the top 10% of all research outputs ever tracked by Altmetric.
So far Altmetric has tracked 29,812 research outputs from this source. They typically receive a little more attention than average, with a mean Attention Score of 6.4. This one has done particularly well, scoring higher than 92% of its peers.
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We're also able to compare this research output to 485 others from the same source and published within six weeks on either side of this one. This one has done particularly well, scoring higher than 94% of its contemporaries.
