Identification of the Tyrosine Nitration Sites in Human Endothelial Nitric Oxide Synthase by Liquid Chromatography-Mass Spectrometry

Zickus, Michael A.; Fonseca, Fabiana Valéria da; Tummala, Monorama; Black, Stephen M.; Ryzhov, Victor

doi:10.1255/ejms.927

Cited by 12 publications

(11 citation statements)

References 42 publications

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“…Among the proteins known to be nitration-inhibited are MnSOD and eNOS [53-55]. Our results show that nitration of both MnSOD (Fig.…”

Section: Resultsmentioning

confidence: 56%

“…This scavenging not only compromises the vasodilatory, antiproliferative, antithrombotic, and anti-inflammatory properties of NO, but also results in the formation of highly deleterious peroxynitrite that, in turn, induces nitration of cell proteins. Many proteins are known to be susceptible to nitration and in particular the nitration of MnSOD and eNOS inhibits their activity [53-55]. MnSOD is among the most important enzymes in the first line of oxidative stress defense, and its inhibition would inevitably exacerbate the severity of further oxidative stress.…”

Section: Discussionmentioning

confidence: 99%

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Bosentan inhibits oxidative and nitrosative stress and rescues occlusive pulmonaryhypertension

Rafikova

Rafikov

Kumar

et al. 2013

Free Radical Biology and Medicine

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Pulmonary arterial hypertension (PH) is a fatal disease marked by excessive pulmonary vascular cell proliferation. Patients with idiopathic PH express endothelin-1 (ET-1) at high levels in their lungs. As the activation of both types of ET-1 receptor (ETA and ETB) leads to increased generation of superoxide and hydrogen peroxide, this may contribute to the severe oxidative stress found in PH patients. As a number of pathways may induce oxidative stress, the particular role of ET-1 remains unclear. The aim of this study was to determine whether inhibition of ET-1 signaling could reduce pulmonary oxidative stress and attenuate the progression of disease in rats with occlusive-angioproliferative PH induced by a single dose of SU5416 (200 mg/kg) and subsequent exposure to hypoxia for 21 days. Using this regimen, animals developed severe PH as evidenced by a progressive increase in right-ventricle (RV) peak systolic pressure (RVPSP), severe RV hypertrophy, and pulmonary endothelial and smooth muscle cell proliferation, resulting in plexiform vasculopathy. PH rats also had increased oxidative stress, correlating with endothelial nitric oxide synthase uncoupling and NADPH oxidase activation, leading to enhanced protein nitration and increases in markers of vascular remodeling. Treatment with the combined ET receptor antagonist bosentan (250 mg/kg/day; day 10 to 21) prevented further increase in RVPSP and RV hypertrophy, decreased ETA/ETB protein levels, reduced oxidative stress and protein nitration, and resulted in marked attenuation of pulmonary vascular cell proliferation. We conclude that inhibition of ET-1 signaling significantly attenuates the oxidative and nitrosative stress associated with PH and prevents its progression.

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“…Among the proteins known to be nitration-inhibited are MnSOD and eNOS [53-55]. Our results show that nitration of both MnSOD (Fig.…”

Section: Resultsmentioning

confidence: 56%

Section: Discussionmentioning

confidence: 99%

Bosentan inhibits oxidative and nitrosative stress and rescues occlusive pulmonaryhypertension

Rafikova

Rafikov

Kumar

et al. 2013

Free Radical Biology and Medicine

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“…Differences in the amount of peroxynitrite in the pulmonary vasculature might alter the number and location of the tyrosine residues nitrated, which could, in turn, determine whether eNOS is partially uncoupled or completely inhibited. For example, our recent study demonstrated that 19 of the 30 tyrosine residues found in eNOS could be nitrated (50). Interestingly, our data identified 4 such tyrosine residues in the region of eNOS responsible for the binding to HSP90 (13).…”

Section: Discussionmentioning

confidence: 55%

Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress

Oishi¹,

Wiseman²,

Sharma³

et al. 2008

American Journal of Physiology-Lung Cellular and Molecular Phys

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SM. Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress. Am J Physiol Lung Cell Mol Physiol 295: L756 -L766, 2008. First published August 29, 2008 doi:10.1152/ajplung.00146.2007.-Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 g/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO x), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P Ͻ 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P Ͻ 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P Ͻ 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P Ͻ 0.05). Conversely, NO x levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P Ͻ 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P Ͻ 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P Ͻ 0.05) and were greater in shunted lambs than controls at all ages (P Ͻ 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P Ͻ 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO. pulmonary circulation; oxidant stress; congenital heart disease; reactive oxygen species INFANTS AND CHILDREN with congenital cardiac defects that cause significantly increased pulmonary blood flow suffer morbidity due to early aberrations in pulmonary vascular function (17). In fact, this early pulmonary vascular dysfunction is often exacerbated in the immediate postoperative period, manifesting as increased vascular reactivity that may produce severe hypoxemia, acidosis, low cardiac output, and death if not treated immediately (7,11,31). A complete understanding of the mechanisms responsible for this pulmonary vascular dysfunction is lacking, but evidence suggests that aberrant nitric oxide (NO)-cGMP signaling and oxidative stress may participate (1,3,6,11,15,33,39,41).Basal NO production by the vascular endothelium is integral to the maintenance of the normal low resistance state of the pulmonary vasculature, and dynamic alterations in NO production modulate vascular relaxation and constriction in response to various stimuli. NO is produced in ...

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“…We have previously found both in vitro and in vivo that exogenous NO exposure causes an increase in peroxynitrite production and that increased eNOS nitration is involved in eNOS inhibition (60). Recently, using liquid chromatography/mass spectrometry, we elucidated the nitration sites on eNOS (64). In this study, we have identified four tyrosine residues that can be modified by nitration and are located in the region of eNOS responsible for the binding to heat shock protein 90 (HSP90).…”

Section: Discussionmentioning

confidence: 92%

GTP cyclohydrolase I expression is regulated by nitric oxide: role of cyclic AMP

Kumar¹,

Sun²,

Sharma³

et al. 2009

American Journal of Physiology-Lung Cellular and Molecular Phys

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Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOS-derived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH(4)) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH(4) could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH(4) levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH(4) levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH(4) levels through a cAMP/CREB-mediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH(4) levels.

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Identification of the Tyrosine Nitration Sites in Human Endothelial Nitric Oxide Synthase by Liquid Chromatography-Mass Spectrometry

Cited by 12 publications

References 42 publications

Bosentan inhibits oxidative and nitrosative stress and rescues occlusive pulmonaryhypertension

Bosentan inhibits oxidative and nitrosative stress and rescues occlusive pulmonaryhypertension

Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress

GTP cyclohydrolase I expression is regulated by nitric oxide: role of cyclic AMP

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