Increasing dihydrobiopterin causes dysfunction of endothelial nitric oxide synthase in rats in vivo

Abstract

An elevation of oxidized forms of tetrahydrobiopterin (BH(4)), especially dihydrobiopterin (BH(2)), has been reported in the setting of oxidative stress, such as arteriosclerotic/atherosclerotic disorders, where endothelial nitric oxide synthase (eNOS) is dysfunctional, but the role of BH(2) in the regulation of eNOS activity in vivo remains to be evaluated. This study was designed to clarify whether increasing BH(2) concentration causes endothelial dysfunction in rats. To increase vascular BH(2) levels, the BH(2) precursor sepiapterin (SEP) was intravenously given after the administration of the specific dihydrofolate reductase inhibitor methotrexate (MTX) to block intracellular conversion of BH(2) to BH(4). MTX/SEP treatment did not significantly affect aortic BH(4) levels compared with control treatment. However, MTX/SEP treatment markedly augmented aortic BH(2) levels (291.1 ± 29.2 vs. 33.4 ± 6.4 pmol/g, P < 0.01) in association with moderate hypertension. Treatment with MTX alone did not significantly alter blood pressure or BH(4) levels but decreased the BH(4)-to-BH(2) ratio. Treatment with MTX/SEP, but not with MTX alone, impaired ACh-induced vasodilator and depressor responses compared with the control treatment (both P < 0.05) and also aggravated ACh-induced endothelium-dependent relaxations (P < 0.05) of isolated aortas without affecting sodium nitroprusside-induced endothelium-independent relaxations. Importantly, MTX/SEP treatment significantly enhanced aortic superoxide production, which was diminished by NOS inhibitor treatment, and the impaired ACh-induced relaxations were reversed with SOD (P < 0.05), suggesting the involvement of eNOS uncoupling. These results indicate, for the first time, that increasing BH(2) causes eNOS dysfunction in vivo even in the absence of BH(4) deficiency, demonstrating a novel insight into the regulation of endothelial function.

Guinea pig ascorbate status predicts tetrahydrobiopterin plasma concentration and oxidation ratio in vivo

“…Thus, our data provide in vivo support for a relationship between ascorbate status and biopterin bioavailability and may therefore indirectly explain clinical observations showing a negative impact of vitamin C deficiency on cardiovascular health since a high BH 2 -to-BH 4 ratio has been linked to endothelial dysfunction in several studies [5,6,[8][9][10][11]. However, the human relevance of the ascorbate-dependent maintenance of a reduced BH 4 pool clearly needs to be confirmed in controlled clinical studies.…”

“…Superoxide can oxidize BH 4 directly or through the formation of peroxynitrite [7] leading to decreased production of NO and further increased production of superoxide. It has been suggested that the ratio of BH 2 to BH 4 , rather than the absolute levels of BH 4 and BH 2 , is the best predictor of endothelial function [5,6,[8][9][10][11]. Vitamin C deficiency has been associated with increased risk of cardiovascular disease in many epidemiological studies [12] and this has prompted investigations into the potential mechanisms by which ascorbate may influence cardiovascular function.…”

Biopterin Metabolism and eNOS Expression during Hypoxic Pulmonary Hypertension in Mice

“…In the absence of BH 4 deficiency, increased BH 2 levels (thus resulting in a decrease of BH 4 /BH 2 ratio) is a determinant of eNOS uncoupling in intact cells [7-9]. We show here that the lung content of BH 2 remained unchanged at the different time of hypoxia exposure, with no changes of DHFR protein expression (the enzyme which recycles BH 2 into BH 4 ).…”

“…BH 4 bioavailability depends on a balance between de novo synthesis by GTP cyclohydrolase-1 (GTPCH-1, the first and limiting step for BH 4 biosynthesis from GTP), loss of BH 4 (due to oxidation of BH 4 into the dihydrobiopterin BH 2 ) and recycling of BH 2 into BH 4 by dihydrofolate reductase (DHFR) [7]. Even in the absence of BH 4 deficiency, elevated levels of BH 2 can compete with BH 4 for binding to eNOS, resulting in eNOS uncoupling [8,9]. Rather than the absolute BH 4 concentration, both eNOS / BH 4 stoicheiometry and biopterin redox status appear as key determinants of eNOS uncoupling [7,10].…”

Kinetics of acid-induced degradation of tetra- and dihydrobiopterin in relation to their relevance as biomarkers of endothelial function

“…While BH 2 may bind to NOS just like BH 4 (Vásquez-Vivar et al, 2002;Crabtree et al, 2008), it does not support the formation of nitric oxide (Presta et al, 1998) and instead superoxide is formed (Vásquez-Vivar et al, 2002). Studies have suggested that the ratio of BH 4 to BH 2 correlates better with endothelial function than the absolute concentration of BH 4 (Noguchi et al, 2011;Crabtree et al, 2008;Takeda et al, 2009;Kar & Kavdia, 2011). Consequently, appropriate stabilization and analysis of both BH 4 and BH 2 is a prerequisite.…”