Tetrahydrobiopterin-dependent formation of endothelium-derived relaxing factor (nitric oxide) in aortic endothelial cells

Schmidt, Kurt; Werner, Ernst R.; Mayer, Bernd; Wächter, Helmut; Kukovetz, W. R.

doi:10.1042/bj2810297

Cited by 149 publications

(73 citation statements)

References 30 publications

(30 reference statements)

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“…21 Thus, a deficiency of BH 4 produces two additive physiologic consequences: (a) uncoupling of the L-arginine NO pathway resulting in increased formation of oxygen-free radicals by eNOS 22 and (b) loss of NO production. 23 In a recent study, we found that deoxycorticosterone acetate salt hypertension caused BH 4 oxidation in aortas of mice, leading to a marked increase in superoxide production and a significant reduction in NO production. Treatment of these mice with oral BH 4 reduced vascular O 2 KÀ production, increased NO production and blunted the increase in BP due to deoxycorticosterone acetate salt hypertension.…”

Section: Potential Mechanismsmentioning

confidence: 92%

Tetrahydrobiopterin: a novel antihypertensive therapy

et al. 2008

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Tetrahydrobiopterin (BH 4 ) is a cofactor for the nitric oxide (NO) synthase enzymes, such that its insufficiency results in uncoupling of the enzyme, leading to release of superoxide rather than NO in disease states, including hypertension. We hypothesized that oral BH 4 will reduce arterial blood pressure (BP) and improve endothelial function in hypertensive subjects. Oral BH 4 was given to subjects with poorly controlled hypertension (BP 4135/85 mm Hg) and weekly measurements of BP and endothelial function made. In Study 1, 5 or 10 mg kg À1 day À1 of BH 4 (n ¼ 8) was administered orally for 8 weeks, and in Study 2, 200 and 400 mg of BH 4 (n ¼ 16) was given in divided doses for 4 weeks. Study 1: significant reductions in systolic (P ¼ 0.005) and mean BP (P ¼ 0.01) were observed with both doses of BH 4 . Systolic BP was 15 ± 15 mm Hg (P ¼ 0.04) lower after 5 weeks and persisted for the 8-week study period. Study 2: subjects given 400 mg BH 4 had decreased systolic (P ¼ 0.03) and mean BP (P ¼ 0.04), with a peak decline of 16±19 mm Hg (P ¼ 0.04) at 3 weeks. BP returned to baseline 4 weeks after discontinuation. Significant improvement in endothelial function was observed in Study 1 subjects and those receiving 400 mg BH 4 . There was no significant change in subjects given the 200 mg dose. This pilot investigation indicates that oral BH 4 at a daily dose of 400 mg or higher has a significant and sustained antihypertensive effect in subjects with poorly controlled hypertension, an effect that is associated with improved endothelial NO bioavailability.

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Section: Potential Mechanismsmentioning

confidence: 92%

Tetrahydrobiopterin: a novel antihypertensive therapy

et al. 2008

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“…Fig. 8 shows that the 35 S-labeled band running at 135 kDa was specific for ecNOS and was not different between control and ascorbate-pretreated cells suggesting that ecNOS synthesis was not altered by ascorbic acid.…”

Section: Effect Of Ascorbic Acid On Ecnos Protein Content In Endothelmentioning

confidence: 94%

“…Medium 199 (M199), human serum, fetal calf serum, collagenase, and human serum albumin (HSA) were from Boehringer Ingelheim Bioproducts (Heidelberg, Germany). 35 S-label and methionine-free RPMI medium were from ICN Pharmaceuticals (Costa Mesa, CA); nitrocellulose was from Millipore (Eschborn, Germany); specific monoclonal antibodies against human ecNOS (clone 3) or against murine macrophage-inducible NOS (iNOS, clone 6) were from Transduction Laboratories (Lexington, KY); the fluorescein isothiocyanate-conjugated secondary antibody (rabbit anti-mouse IgG (H ϩ L)) with minimal cross-reaction to human serum proteins was from Dianova (Hamburg, Germany); NADPH, tetrahydrobiopterin, and L-nitroarginine methyl ester (L-NAME) were from Alexis Corp. (Lä ufelfingen, Switzerland). Endothelial cell growth supplement, peroxidase-labeled anti-mouse IgG (Fabspecific), anti-mouse agarose, FAD, FMN, calmodulin, ionomycin, thrombin, EDTA, EGTA, trypsin/EDTA solution (0.05/0.02%, v/v), leupeptin, phenylmethylsulfonyl fluoride (PMSF), L-ascorbic acid, dehydro-L-ascorbic acid, ascorbate 2-phosphate, ascorbate 2-sulfate, L-gulonolactone, and other reagents were purchased from Sigma (Deisenhofen, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Metabolic Labeling and Immunoprecipitation of ecNOS-HUVEC were incubated with 100 M ascorbic acid for 6 h. Subsequently, 50 Ci of Tran 35 S-label reagent in methionine-free RPMI medium containing 100 M ascorbate was added for 3 h. Untreated control cells were processed in parallel. The labeled cells were lysed by the addition of Hepes buffer (pH 7.4) containing 0.5% Triton X-100, 0.05% SDS, and protease inhibitors (1 mM PMSF, 0.6 mM leupeptin, 0.025 mM pepstatin).…”

Section: Methodsmentioning

confidence: 99%

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l-Ascorbic Acid Potentiates Nitric Oxide Synthesis in Endothelial Cells

Heller

Münscher-Paulig

Gräbner

et al. 1999

Journal of Biological Chemistry

210

106

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Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 M ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 M) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 M and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid. Endothelium-derived nitric oxide (NO)1 exerts several vasoprotective activities including smooth muscle relaxation, inhibition of platelet activation, and regulation of endothelial cell permeability and adhesivity (1-4). NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of an NADPH-dependent NO synthase (NOS) that requires tetrahydrobiopterin, FAD, and FMN as cofactors (5). The endothelial NOS isoform (ecNOS) is constitutively expressed and is activated upon an increase of intracellular calcium following cell stimulation with receptor-dependent stimuli such as thrombin and bradykinin or with receptor-independent stimuli like calcium ionophore (6).Since NO interferes with key processes in atherogenesis (7), a lack of NO might promote the development of atherosclerosis. Indeed, clinical studies have confirmed an impairment of vascular NO synthesis in patients with atherosclerosis or with increased atherogenic risk factors (8). The dysfunction of the endothelial NO pathway may involve impaired signal transduction mechanisms, decreased ecNOS activity, reduced intracellular a...

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“…Furthermore, low levels of tetrahydrobiopterin (BH 4 ) were detected during the onset and progression of PD (2,16,17). BH 4 is a critical cofactor for NOS activity (18,19). Evidence indicates that BH 4 , by acting as a "redox switch," plays a critical role in increasing not only the rate of ⅐ NO generation by NOS, but also in controlling the formation of superoxide and hydrogen peroxide (20 -22).…”

mentioning

confidence: 99%

1-Methyl-4-phenylpyridinium-induced Apoptosis in Cerebellar Granule Neurons Is Mediated by Transferrin Receptor Iron-dependent Depletion of Tetrahydrobiopterin and Neuronal Nitric-oxide Synthase-derived Superoxide

Shang

Kotamraju

Kalivendi

et al. 2004

Journal of Biological Chemistry

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In this study, we investigated the molecular mechanisms of toxicity of 1-methyl-4-phenylpyridinium (MPP ؉ Parkinson's disease (PD) 1 is characterized by mitochondrial complex I defects, elevated iron levels in brain tissue, tetrahydrobiopterin (BH 4 ) and dopamine deficiencies, and ␣-synuclein accumulation in Lewy body aggregates (1-4). The exact molecular mechanisms leading to the pathophysiology of PD are not well understood. 1-Methyl-4-phenylpyridinium ion (MPP ϩ ), a mitochondrial complex I inhibitor, produces Parkinson-like symptoms in humans and laboratory animals, and has been used to investigate the mechanism of pathogenesis of PD (5, 6). MPP ϩ is the ultimate metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant found in illicit narcotics (7,8). MPP ϩ is taken up into dopaminergic neurons via the dopamine transporter and accumulates into mitochondria, where it inhibits complex I activity. Although the mechanism of MPP ϩ -induced neurotoxicity is not fully understood, there is increasing evidence supporting the involvement of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (1, 9).)Previous studies using the neuronal nitric-oxide synthase (nNOS) knockout mice implicate nitric oxide ( ⅐ NO) and peroxynitrite (ONOO Ϫ ) in MPP ϩ -induced neurodegeneration (10). However, therapeutic intervention studies with nitric-oxide synthase (NOS) inhibitors in MPTP-treated mice demonstrated the opposite result, i.e. the attenuation of ⅐ NO was more deleterious than protective (11)(12)(13)(14). Decreased formation of ⅐ NOderived metabolites (nitrite and nitrate) in cerebrospinal fluids was observed in PD (15). Furthermore, low levels of tetrahydrobiopterin (BH 4 ) were detected during the onset and progression of PD (2,16,17). BH 4 is a critical cofactor for NOS activity (18,19). Evidence indicates that BH 4 , by acting as a "redox switch," plays a critical role in increasing not only the rate of ⅐ NO generation by NOS, but also in controlling the formation of superoxide and hydrogen peroxide (20 -22). Pharmacological manipulation of BH 4 has been suggested as a therapeutic strategy to modulate ⅐ NO and superoxide generation in neuronal cells and endothelial cells (23)(24)(25)(26)(27). De novo biosynthesis of BH 4 is catalyzed by guanosine 5Ј-triphosphate cyclohydrolase I (GT-PCH I) (28,29). Mammalian cells can also generate BH 4 by

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Tetrahydrobiopterin-dependent formation of endothelium-derived relaxing factor (nitric oxide) in aortic endothelial cells

Cited by 149 publications

References 30 publications

Tetrahydrobiopterin: a novel antihypertensive therapy

Tetrahydrobiopterin: a novel antihypertensive therapy

l-Ascorbic Acid Potentiates Nitric Oxide Synthesis in Endothelial Cells

1-Methyl-4-phenylpyridinium-induced Apoptosis in Cerebellar Granule Neurons Is Mediated by Transferrin Receptor Iron-dependent Depletion of Tetrahydrobiopterin and Neuronal Nitric-oxide Synthase-derived Superoxide

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