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. 2003 Apr;111(8):1201-9.
doi: 10.1172/JCI14172.

Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension

Ulf Landmesser  1 , Sergey Dikalov, S Russ Price, Louise McCann, Tohru Fukai, Steven M Holland, William E Mitch, David G Harrison
Affiliations

Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension

Ulf Landmesser et al. J Clin Invest. 2003 Apr.

Abstract

Tetrahydrobiopterin is a critical cofactor for the NO synthases, and in its absence these enzymes become "uncoupled," producing reactive oxygen species (ROSs) rather than NO. In aortas of mice with deoxycorticosterone acetate-salt (DOCA-salt) hypertension, ROS production from NO synthase is markedly increased, and tetrahydrobiopterin oxidation is evident. Using mice deficient in the NADPH oxidase subunit p47(phox) and mice lacking either the endothelial or neuronal NO synthase, we obtained evidence that hypertension produces a cascade involving production of ROSs from the NADPH oxidase leading to oxidation of tetrahydrobiopterin and uncoupling of endothelial NO synthase (eNOS). This decreases NO production and increases ROS production from eNOS. Treatment of mice with oral tetrahydrobiopterin reduces vascular ROS production, increases NO production as determined by electron spin resonance measurements of nitrosyl hemoglobin, and blunts the increase in blood pressure due to DOCA-salt hypertension. Endothelium-dependent vasodilation is only minimally altered in vessels of mice with DOCA-salt hypertension but seems to be mediated by hydrogen peroxide released from uncoupled eNOS, since it is inhibited by catalase. Tetrahydrobiopterin oxidation may represent an important abnormality in hypertension. Treatment strategies that increase tetrahydrobiopterin or prevent its oxidation may prove useful in preventing vascular complications of this common disease.

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Figures

Figure 1
Figure 1
Role of the endothelial NO synthase as a source of ROSs in hypertensive vessels. (a) Effect of endothelial removal and NO synthase inhibition (L-NAME, 1 mM) on vascular superoxide (O2•–) production estimated by lucigenin chemiluminescence in sham-operated and DOCA-salt–hypertensive rats (n = 6–10 per group). (b) Vascular O2•– production estimated by lucigenin chemiluminescence in sham-operated and DOCA-salt–hypertensive wild-type (C57BL/6), eNOS–/–, and nNOS–/– knockout mice (n = 6–10 per group). (c) Vascular O2•– production measured by the SOD-inhibitable cytochrome c reduction assay in sham-operated and DOCA-salt–hypertensive wild-type, eNOS–/–, and nNOS–/– knockout mice (n = 5–13 per group).
Figure 2
Figure 2
Tetrahydrobiopterin and oxidized biopterin content in aortas from sham-operated and DOCA-salt–hypertensive mice: effect of oral treatment with tetrahydrobiopterin, p47phox, or eNOS deficiency. Tetrahydrobiopterin (H4B) and oxidized biopterin (7,8-H2B plus biopterin) were determined using HPLC analysis after differential oxidation (n = 3–4 per group). Three to six aortas were pooled for each measurement. *P < 0.05 versus sham, **P < 0.05 versus DOCA (C57BL/6).
Figure 3
Figure 3
Effect of NADPH oxidase deficiency (p47phox–/–) and treatment with tetrahydrobiopterin (H4B) on vascular O2•– production in DOCA-salt hypertension. (a) Vascular O2•– production estimated by lucigenin chemiluminescence in sham-operated and DOCA-salt–hypertensive mice (n = 6–10 per group). (b) Vascular O2•– production measured by the SOD-inhibitable cytochrome c reduction assay (n = 5–13 per group).
Figure 4
Figure 4
Effect of NADPH oxidase deficiency (p47phox–/–) and tetrahydrobiopterin (H4B) treatment on NO production. NO production was analyzed by ESR measurements of nitrosyl hemoglobin levels (n = 5).
Figure 5
Figure 5
Endothelium-dependent vascular relaxation in DOCA-salt hypertension: role of hydrogen peroxide. Effect of catalase (1200 U/ml) on endothelium-dependent vasorelaxations to calcium ionophore A23187 (a) and endothelium-independent vasorelaxations to nitroglycerin (b) in aortas from sham-operated, DOCA-salt–hypertensive mice and hypertensive mice treated with H4B (n = 6–10, *P < 0.05). (c) H2O2 production in aortas from sham-operated, DOCA-salt–hypertensive mice (with and without oral H4B treatment) and DOCA-salt–treated eNOS–/– and p47phox–/– mice. H2O2 production was determined by the fluorometric Amplex red assay in the presence of A23187 (10–6 M). The effect of NO synthase inhibition (1 mM L-NAME) and PEG catalase (1000 U) was studied (n = 3–6).
Figure 6
Figure 6
Proposed concept of eNOS uncoupling in hypertension. Hypertension stimulates O2•– formation from the vascular NADPH oxidase. This results in oxidation of the eNOS cofactor tetrahydrobiopterin (H4B). Our data suggest that peroxynitrite (ONOO–) or oxidants derived from peroxynitrite contribute to tetrahydrobiopterin oxidation. Cofactor-deficient eNOS or eNOS with oxidized forms of H4B produces large amounts of O2•– upon stimulation of the enzyme.
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