Tetrahydrobiopterin, but Not L-Arginine, Decreases NO Synthase Uncoupling in Cells Expressing High Levels of Endothelial NO Synthase

Leiden University, Leyden, South Holland, Netherlands
Hypertension (Impact Factor: 6.48). 01/2006; 47(1):87-94. DOI: 10.1161/01.HYP.0000196735.85398.0e
Source: PubMed


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.

Download full-text


Available from: Marianne Verhaar
  • Source
    • "Interestingly, in certain conditions such as reduced levels of BH4, eNOS itself can produce superoxide, a process referred to as “eNOS uncoupling,” in which oxygen becomes terminal electron acceptor instead of L-arginine [107]. NO does not influence the function of BBB during normoxia, but seems to confer protection during ischemia [108]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: As fundamental research advances, it is becoming increasingly clear that a clinically expressed disease implies a mixture of intertwining molecular disturbances. Oxidative stress is one of such pathogenic pathways involved in virtually all central nervous system pathologies, infectious, inflammatory, or degenerative in nature. Since brain homeostasis largely depends on integrity of blood-brain barrier (BBB), many studies focused lately on BBB alteration in a wide spectrum of brain diseases. The proper two-way molecular transfer through BBB depends on several factors, including the functional status of its tight junction (TJ) complexes of proteins sealing neighbour endothelial cells. Although there is abundant experimental work showing that oxidative stress associates BBB permeability alteration, less is known about its implications, at molecular level, in TJ protein expression or TJ-related cell signalling. In this paper, oxidative stress is presented as a common pathway for different brain pathogenic mechanisms which lead to BBB dysregulation. We revise here oxidative-induced molecular mechanisms of BBB disruption and TJ protein expression alteration, in relation to ageing and neurodegeneration.
    Full-text · Article · Mar 2013 · Oxidative Medicine and Cellular Longevity
    • "Vitamin C has been shown to preserve the iNOS cofactor tetrahydrobiopterin (BH 4 ), preventing its oxidation. This activity also prevents the formation of superoxide anion derived from molecular oxygen reduction in the absence of BH 4 (Bevers et al., 2006; Kuzkaya, Weissmann, Harrison, & Dikalov, 2003). Synergistic effects have been described between vitamin C and E in the defense against oxidative stress (Packer & Obermüller-Jevic, 2002). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The authors studied the effects of antioxidant diet supplementation with an almond-based beverage on neutrophil antioxidants, nitrite, and protein oxidative alterations after exercise. Fourteen trained male amateur runners were randomly assigned in a double-blind fashion to receive antioxidant supplementation (152 mg/d vitamin C and 50 mg/d vitamin E) or placebo using an almond-based beverage for 1 mo and participated in a half-marathon race. Blood samples were taken before and after the half-marathon and after 3 hr recovery. Supplementation significantly increased basal neutrophil vitamin C compared with placebo (p < .05). Exercise increased neutrophil vitamin E levels in the supplemented group and decreased vitamin C in both groups after recovery (p < .05). Neutrophil catalase and glutathione peroxidase gene expression and nitrite levels were significantly increased as result of exercise (p < .05). Nitrotyrosine and protein carbonyl derivates increased only in the placebo group after exercise (p < .05), and these values remained high at recovery. No significant differences were evidenced in caspase-3 activity and DNA damage. Antioxidant supplementation with vitamins C and E reduced the exercise-induced oxidation of proteins in neutrophils, without altering the antioxidant adaptive response, as evidenced by the increased catalase and glutathione peroxidase gene expression.
    No preview · Article · Feb 2013 · International Journal of Sport Nutrition and Exercise Metabolism
  • Source
    • "The activity of NOS is regulated by substrate, cofactor availability, and electron transfer rate. The regulating factors such as arginine (Gornik and Creager, 2004) and BH4 (Bevers et al., 2006) can be affected by ROS that can lead to dysfunctional eNOS. As summarized in the Figure 1, in pathological states involving oxidative stress such as hypertension NOS could be uncoupled (Schulz et al., 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Endothelial dysfunction is the hallmark of hypertension, which is a multifactorial disorder. In the cardiovascular system reactive oxygen species play a pivotal role in controlling the endothelial function and vascular tone. Physiologically, the endothelium-derived relaxing factors (EDRFs) and endothelium-derived contractile factors (EDCFs) that have functions on the vascular smooth muscle cells. The relaxation induced by the EDRFs nitric oxide (NO), prostacyclin, and the endothelium-derived hyperpolarization factor (EDHF) could be impaired in hypertension. The impaired ability of endothelial cells to release NO along with enhanced EDCFs production has been described to contribute to the endothelium dysfunction, which appears to lead to several cardiovascular diseases. The present review discusses the role of oxidative stress, vascular endothelium, and vascular tone control by EDRFs, mainly NO, and EDCFs in different models of experimental hypertension.
    Full-text · Article · Dec 2012 · Frontiers in Physiology
Show more