Article

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.

Download full-text

Full-text

Available from: Marianne Verhaar, Jul 20, 2015
0 Followers
 · 
108 Views
  • Source
    • "Guanosine triphosphate cyclohydrolase I (GTPCH I) is the rate-limiting enzyme of the biosynthesis of tetrahydrobiopterin (BH 4 ), which is an essential cofactor of NOS. In the presence of subsaturating levels or deficiency of BH 4 , electron transfer in NOS becomes uncoupled from L-arginine oxidation and NO formation, with subsequently less NO and more ROS generation, the so-called NOS uncoupling [65]. Furthermore, ROS serve as an amplifying mechanism to further exacerbate NOS uncoupling. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Reactive oxygen species (ROS) are generated by several different cellular sources, and their accumulation within the myocardium is widely considered to cause harmful oxidative stress. On the other hand, their role as second messengers has gradually emerged. The equilibrium of the nitroso/redox balance between reactive nitrogen species and ROS is crucial for the health of cardiomyocytes. This review provides a comprehensive overview of sources of oxidative stress in cardiac myocytes and describes the role of the nitroso/redox balance in cardiac pathophysiology. Although the exact mechanism of ROS production by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox's) is not completely understood, Nox2 and Nox4 have particularly important roles within the myocardium. Increasing evidence suggests that Nox2 produces superoxide and Nox4 generates only hydrogen peroxide. We also discuss the key role of nitric oxide synthases (NOSs) in the maintenance of the nitroso/redox balance: uncoupled endothelial NOS has been suggested to shift from nitric oxide to ROS production, contributing to increased oxidative stress within the myocardium. Furthermore, we highlight the importance of sequentially targeting and/or regulating the specific sources of oxidative and nitrosative stress to prevent and/or reverse myocardial dysfunction. Inhibition of NADPH oxidase-dependent ROS is considered to be a potential strategy for treatment of cardiomyopathy. Neither in vivo nor clinical data are available for NADPH oxidase inhibitors. Specifically targeting the mitochondria with the antioxidant MitoQ would be a very promising translation approach, because it could prevent mitochondrial permeability transition pore opening when ROS are produced during heart reperfusion. Enhancing NO signaling could also be a promising therapeutic approach against myocardial dysfunction.
    Free Radical Biology and Medicine 07/2012; 53(8):1531-40. DOI:10.1016/j.freeradbiomed.2012.07.010 · 5.71 Impact Factor
  • Source
    • "Therefore, tight coupling of eNOS is likely to be important to maintain normal cardiovascular function and prevent the development of cardiovascular disease. A number of potential mechanisms are responsible for uncoupling eNOS, although the most consistent evidence suggests that is caused by a deficiency in tetrahydrobiopterin (BH4) (Bendall et al., 2005; Bevers et al., 2006). eNOS uncoupling also occurs in the following situations: (i) a shortage of substrate, L-Arg (Vergnani et al., 2000); (ii) an increase in asymmetric dimethyl-L-arginine (ADMA) concentration (Antoniades et al., 2009); (iii) dysregulation of protein–protein interactions (Pritchard et al., 2001); (iv) dephosphorylation of eNOS on threonine residue 495 (Lin et al., 2003); and (v) eNOS redistribution to the cytosolic fraction of the cell. "
    [Show abstract] [Hide abstract]
    ABSTRACT: One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O(2) (•-) ) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism. HUVECs were incubated with ox-LDL with/without pyridoxine, N(G) -nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O(2) (•-) was measured using lucigenin chemiluminescence, and O(2) (•-) -sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot. Ox-LDL significantly increased O(2) (•-) production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O(2) (•-) generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments. Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.
    British Journal of Pharmacology 07/2011; 165(3):754-64. DOI:10.1111/j.1476-5381.2011.01607.x · 4.99 Impact Factor
  • Source
    • "inactivation likely plays an important role in increasing vascular tone , contributing to increased arterial resistance . The uncoupling of eNOS from its cofactors through relative L - arginine deficiency as well as by reduced tetrahydropterin , causes eNOS to produce reactive oxygen species ( ROS ) rather than NO , thereby reducing NO synthesis ( Bevers , Braam , Post , et al . , 2006 ) and increasing oxidative stress . Nitric oxide inactivation owing to excess generation of reactive oxygen species , increased production of endogenous vasoconstrictors such as angiotensin - II and endothelin , decreased bioavailability of L - arginine , as well as defects in intracellular transduction pathways are several proposed mec"
    [Show abstract] [Hide abstract]
    ABSTRACT: Salt sensitivity is associated with a rise in blood pressure (BP) occurring during sodium loading and/or a fall in BP during sodium restriction that exceeds random fluctuations in BP. Salt sensitivity is more common in African American than Caucasian hypertensives and is also present, in normotensive African Americans. The mechanism or mechanisms resulting in salt-sensitive hypertension are multiple and include both activation of the renin angiotensin system via increases in angiotensin II and reductions in the endogenous vasodilator, nitric oxide (NO). An important means of NO downregulation is through asymmetric dimethylarginine (ADMA), an endogenous NO inhibitor, which is largely metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). The activity of DDAH is impaired by oxidative stress, thereby permitting ADMA to accumulate thus resulting in further inhibition of NO. Increases in oxidative stress, reduction in DDAH activity, and augmented action of ADMA on depressing NO production represents a plausible mechanism in human salt sensitivity. The study investigates and characterizes the above mechanism through which salt-induced depression of NO synthesis occurs in normotensive African Americans. The study population was a cohort of mostly young (mean age 45 years, SD=6.0), female (87%), African Americans who were normotensive, and overweight. The DDAH level and NO metabolites came down after sodium exposure (6.17% and 11.53%, respectively), while the BP rose (SBP: +2.8 mm Hg; DBP +0.8 mm Hg) and the augmentation index (a measure of arterial stiffness) increased by almost 12% after sodium exposure, though not statistically significant. The difference in sodium:creatinine ratio was directly proportional to the change in BP (SBP: p=0.01; DBP p=0.13), which likely mediated the BP effect. Additionally, NO metabolites and DDAH levels were positively correlated to each other (r=0.90; p=
Show more