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.
Highlights
In vascular disease states such as atherosclerosis and diabetes, endothelial nitric oxide (NO) bioactivity is reduced, and oxidative stress is increased, resulting in endothelial dysfunction
We investigated the role of dihydrofolate reductase (DHFR) in regulating BH4 versus BH2 levels in endothelial cells and in cell lines expressing endothelial nitric-oxide synthase (eNOS) combined with tet-regulated GTP cyclohydrolase I (GTPCH) expression in order to compare the effects of low or high levels of de novo BH4 biosynthesis
In this study we used both pharmacologic inhibition and genetic manipulation to determine the role of DHFR in the regulation of BH4 levels in sEnd.1 endothelial cells and in cell lines which express tetracycline-regulatable human GTCPH and an eNOS-GFP fusion construct, described previously [7]
Summary
In addition to key roles in folate metabolism, dihydrofolate reductase (DHFR; EC 1.5.1.3) can reduce BH2, regenerating BH4 [9, 10]. It is, likely that net BH4 bioavailability within the endothelium reflects the balance between de novo BH4 synthesis, loss of BH4 by oxidation to BH2, and the regeneration of BH4 by DHFR. The requirement for DHFR in regulating intracellular BH4 homeostasis and the quantitative relationships that relate BH4 de novo synthesis versus BH4 recycling to eNOS coupling remain uncertain. We report that GTPCH is the key regulator of the total amount of intracellular biopterins, DHFR is critical to eNOS function by determining BH4:BH2 ratio and, in maintaining eNOS coupling. DHFR is important in preventing “self-propagated” eNOS uncoupling in conditions of low total biopterin levels, when eNOS-dependent oxidation of BH4 that would further exacerbate eNOS uncoupling can be rescued by DHFR
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