Tetrahydrobiopterin, but not L-arginine, decreases NO synthase uncoupling in cells expressing high levels of endothelial NO synthase.

Department of Nephrology and Hypertension, Institute and Graduate School of Biomembranes, University Medical Centre, Utrecht, The Netherlands.
Hypertension (Impact Factor: 7.63). 01/2006; 47(1):87-94. DOI: 10.1161/01.HYP.0000196735.85398.0e
Source: PubMed

ABSTRACT Endothelial NO synthase (eNOS) produces superoxide when depleted of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-arginine by uncoupling the electron flow from NO production. High expression of eNOS has been reported to have beneficial effects in atherosclerotic arteries after relatively short periods of time. However, sustained high expression of eNOS may have disadvantageous vascular effects because of uncoupling. We investigated NO and reactive oxygen species (ROS) production in a microvascular endothelial cell line (bEnd.3) with sustained high eNOS expression and absent inducible NOS and neuronal NOS expression using 4,5-diaminofluorescein diacetate and diacetyldichlorofluorescein as probes, respectively. Unstimulated cells produced both NO and ROS. After stimulation with vascular endothelial growth factor (VEGF), NO and ROS production increased. VEGF-induced ROS production was even further increased by the addition of extra L-arginine. Nomega-nitro-L-arginine methyl ester decreased ROS production. These findings strongly suggest that eNOS is a source of ROS in these cells. Although BH4 levels were increased as compared with another endothelial cell line, eNOS levels were >2 orders of magnitude higher. The addition of BH4 resulted in increased NO production and decreased generation of ROS, indicating that bEnd.3 cells produce ROS through eNOS uncoupling because of relative BH4 deficiency. Nevertheless, eNOS-dependent ROS production was not completely abolished by the addition of BH4, suggesting intrinsic superoxide production by eNOS. This study indicates that potentially beneficial sustained increases in eNOS expression and activity could lead to eNOS uncoupling and superoxide production as a consequence. Therefore, sustained increases of eNOS or VEGF activity should be accompanied by concomitant supplementation of BH4.

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    ABSTRACT: Tetrahydrobiopterin (BH4) is an essential cofactor for the production of nitric oxide (NO) and supplementation with BH4 improves NO-dependent vasodilation. NO also reduces sympathetic vasoconstrictor responsiveness in resting and contracting skeletal muscle. Thus, we hypothesized that supplementation with BH4 would blunt sympathetic vasoconstrictor responsiveness in resting and contracting skeletal muscle. Sprague-Dawley rats (n = 15, 399 ± 57 g) were anesthetized and instrumented with an indwelling brachial artery catheter, femoral artery flow probe, and a stimulating electrode on the lumbar sympathetic chain. Triceps surae muscles were stimulated to contract rhythmically at 30% and 60% of maximal contractile force (MCF). The percentage change of femoral vascular conductance (%FVC) in response to sympathetic stimulations delivered at 2 and 5 Hz was determined at rest and during muscle contraction in control and acute BH4 supplementation (20 mg·kg−1 + 10 mg·kg−1·h−1, IA) conditions. BH4 reduced (P < 0.05) the vasoconstrictor response to sympathetic stimulation (i.e., decrease in FVC) at rest (Control: 2 Hz: −28 ± 5%FVC; 5 Hz: −45 ± 5%; BH4: 2 Hz: −17 ± 4%FVC; 5 Hz: −34 ± 7%FVC) and during muscular contraction at 30% MCF (Control: 2 Hz: −14 ± 6%FVC; 5 Hz: −28 ± 11%; BH4: 2 Hz: −6 ± 6%FVC; 5 Hz: −16 ± 10%) and 60% MCF (Control: 2 Hz: −7 ± 3%FVC; 5 Hz: −16 ± 6%FVC; BH4: 2 Hz: −2 ± 3%FVC; 5 Hz: −11 ± 6%FVC). These data are consistent with our hypothesis that acute BH4 supplementation decreases sympathetic vasoconstrictor responsiveness in resting and contracting skeletal muscle.
    Physiological Reports. 10/2014; 2(10).
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    ABSTRACT: Endothelial dysfunction plays a key role in stroke in chronic kidney disease patients. To explore the underlying mechanisms, we evaluated the effects of two uremic toxins on cerebral endothelium function. bEnd.3 cells were exposed to indoxyl sulfate (IS) and inorganic phosphate (Pi). Nitric oxide (NO), reactive oxygen species (ROS) and O2•- were measured using specific fluorophores. Peroxynitrite and eNOS uncoupling were evaluated using ebselen, a peroxide scavenger, and tetrahydrobiopterin (BH4), respectively. Cell viability decreased after IS or Pi treatment (p < 0.01). Both toxins reduced NO production (IS, p < 0.05; Pi, p < 0.001) and induced ROS production (p < 0.001). IS and 2 mM Pi reduced O2•- production (p < 0.001). Antioxidant pretreatment reduced ROS levels in both IS- and Pi-treated cells, but a more marked reduction of O2•- production was observed in Pi-treated cells (p < 0.001). Ebselen reduced the ROS production induced by the two toxins (p < 0.001); suggesting a role of peroxynitrite in this process. BH4 addition significantly reduced O2•- and increased NO production in Pi-treated cells (p < 0.001), suggesting eNOS uncoupling, but had no effect in IS-treated cells. This study shows, for the first time, that IS and Pi induce cerebral endothelial dysfunction by decreasing NO levels due to enhanced oxidative stress. However, Pi appears to be more deleterious, as it also induces eNOS uncoupling.
    Toxins 06/2014; 6(6):1742-1760. · 2.48 Impact Factor

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