scholarly journals Haem and nitric oxide: synergism in the modulation of the endothelial haem oxygenase-1 pathway

2003 ◽  
Vol 372 (2) ◽  
pp. 381-390 ◽  
Author(s):  
Roberta FORESTI ◽  
Martha HOQUE ◽  
Sandip BAINS ◽  
Colin J. GREEN ◽  
Roberto MOTTERLINI
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Sodium Nitroprusside ◽  
Nitrosative Stress ◽  
No Production ◽  
Oxidative And Nitrosative Stress ◽  
No Donors ◽  
Aortic Endothelial Cells ◽  
Oxygenase Activity ◽  
Haem Oxygenase

NO potently up-regulates vascular haem oxygenase-1 (HO-1), an inducible defensive protein that degrades haem to CO, iron and the antioxidant bilirubin. Since several pathological states are characterized by increased NO production and liberation of haem from haem-containing proteins, we examined how NO influences HO-1 induction mediated by haemin. Aortic endothelial cells treated with S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP) or diethylenetriamine-NONOate (DETA/NO) and haemin exhibited higher levels of haem oxygenase activity compared with cells exposed to NO donors or haemin alone. This was accompanied by a marked increase in bilirubin production and, notably, by a strong magnification of cellular haem uptake. A role for haem metabolites in modulating HO-1 expression by NO was assessed by exposing cells to SNAP, SNP or DETA/NO in medium derived from cells treated with haemin, which contained increased bilirubin levels. This treatment considerably potentiated HO-1 expression and haem oxygenase activity mediated by NO and the use of a haem oxygenase inhibitor abolished this effect. Both iron liberated during haem breakdown and the formation of nitroxyl anion from NO appeared to partially contribute to the amplifying phenomenon; in addition, medium from haemin-treated cells significantly augmented the release of NO by NO donors. Thus we have identified novel mechanisms related to the induction of HO-1 by NO indicating that the signalling actions of NO vary significantly in the presence of haem and haem metabolites, ultimately increasing the defensive abilities of the endothelium to counteract oxidative and nitrosative stress.

scholarly journals Thiol-metabolizing proteins and endothelial redox state: differential modulation of eNOS and biopterin pathways

2010 ◽  
Vol 298 (1) ◽  
pp. H194-H201 ◽  
Author(s):  
Toru Sugiyama ◽  
Thomas Michel
Keyword(s):  
Endothelial Cells ◽  
Redox State ◽  
Nitrosative Stress ◽  
Redox Balance ◽  
Vascular Wall ◽  
Differential Regulation ◽  
No Production ◽  
Oxidative And Nitrosative Stress ◽  
Aortic Endothelial Cells ◽  
Intracellular Redox

The intracellular redox state is stringently maintained by thiol-based antioxidants to establish a balance for the physiological and pathophysiological roles of reactive oxygen species. The relative contributions of the thioredoxin (Trx) and glutathione/glutaredoxin systems to intracellular redox balance are incompletely understood, as are the consequences of altered thiol metabolism on endothelial nitric oxide (NO) synthase (eNOS) and NO-dependent pathways in the endothelium. We designed duplex small interfering RNA (siRNA) constructs to specifically “knock down” the expression of three key thiol-metabolizing enzymes in cultured aortic endothelial cells. Transfection of siRNA constructs targeting glutathione reductase (GR), cytosolic Trx reductase (TrxR1), or mitochondrial Trx reductase (TrxR2) significantly decreased the intracellular reduced glutathione-to-oxidized glutathione ratio. siRNA-mediated knockdown of either GR, TrxR1, or TrxR2 markedly suppressed VEGF-induced NO production (measured by an electrochemical NO sensor) and also blocked eNOS enzyme activity (using the [3H]arginine/[3H]citrulline assay). Pretreatment of endothelial cells with N, N′-bis(2-chloroethyl)- N-nitrosourea, an inhibitor of GR and TrxR, significantly decreased VEGF-induced NO production. siRNA-mediated TrxR2 knockdown led to a marked increase in hydrogen peroxide (H2O2) production in endothelial cells. In contrast, knockdown of GR or TrxR1 only slightly increased H2O2 production. Supplementation of endothelial cells with tetrahydrobiopterin prevented the increase in H2O2 generation seen with siRNA-mediated knockdown of GR. These studies show that the differential regulation of thiol-metabolizing proteins leads to critical changes in oxidative and nitrosative stress pathways. Greater understanding of the differential regulation of thiol-metabolizing proteins may lead to the development of new pharmacological targets for diseases associated with oxidative stress in the vascular wall.

Regulation of Ca(2+)-dependent nitric oxide synthase in bovine aortic endothelial cells

1995 ◽  
Vol 269 (3) ◽  
pp. C757-C765 ◽  
Author(s):  
B. J. Buckley ◽  
Z. Mirza ◽  
A. R. Whorton
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
No Synthase ◽  
No Production ◽  
Intact Cells ◽  
Aortic Endothelial Cells ◽  
A 23187 ◽  
Transient Rise ◽  
Synthase Inhibitor ◽  
Sustained Increase

Vascular endothelium responds to Ca(2+)-mobilizing agonists by producing nitric oxide (NO), a potent vasodilator and inhibitor of platelet aggregation. Regulation of constitutively expressed endothelial NO synthase (eNOS) in intact cells is not well understood. We investigated the kinetics of NO formation in response to Ca(2+)-mobilizing agonists, the requirement for extracellular L-arginine, and the role of NO in regulating eNOS activity. When endothelial cells were stimulated with bradykinin and ATP in the presence of 100 microM L-arginine, we observed a rapid and transient rise in intracellular Ca2+ concentration ([Ca2+]i) from 50 +/- 8 nM to 698 +/- 74 and 637 +/- 53 nM, respectively, and a rapid and transient rise in NO production from a basal level of 37 pmol.min-1.mg protein-1 to 256 and 275 pmol.min-1.mg protein-1, respectively. When cells were stimulated with A-23187 or thapsigargin in the presence of 100 microM L-arginine, we observed a sustained increase in [Ca2+]i and a sustained increase in NO production. The rate of NO synthesis was linear over 30 min, rising above control levels of 7 pmol/min to 53 pmol/min for A-23187 and 62 pmol/min for thapsigargin. Thapsigargin stimulated NO production and [Ca2+]i with 50% effective concentration values of 0.01 and 0.05 microM, respectively. Ca(2+)-stimulated NO production was attenuated by the NO synthase inhibitor NG-monomethyl-L-arginine, the removal of extracellular L-arginine, and the Ca(2+)-chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. When we exposed cells to NO gas (3.1 mM for 15 min) and S-nitrosoglutathione (10 mM for 1 h) thapsigargin-stimulated NO production was decreased by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The Nitric Oxide (NO) Donor Sodium Nitroprusside (SNP) and Its Potential for the Schizophrenia Therapy: Lights and Shadows

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3196
Author(s):  
Elli Zoupa ◽  
Nikolaos Pitsikas
Keyword(s):  
Nitric Oxide ◽  
Sodium Nitroprusside ◽  
Cognitive Deficits ◽  
Negative Symptoms ◽  
Mental Disease ◽  
No Production ◽  
No Donor ◽  
No Donors ◽  
Worldwide Population ◽  
The Brain

Schizophrenia is a severe psychiatric disorder affecting up to 1% of the worldwide population. Available therapy presents different limits comprising lack of efficiency in attenuating negative symptoms and cognitive deficits, typical features of schizophrenia and severe side effects. There is pressing requirement, therefore, to develop novel neuroleptics with higher efficacy and safety. Nitric oxide (NO), an intra- and inter-cellular messenger in the brain, appears to be implicated in the pathogenesis of schizophrenia. In particular, underproduction of this gaseous molecule is associated to this mental disease. The latter suggests that increment of nitrergic activity might be of utility for the medication of schizophrenia. Based on the above, molecules able to enhance NO production, as are NO donors, might represent a class of compounds candidates. Sodium nitroprusside (SNP) is a NO donor and is proposed as a promising novel compound for the treatment of schizophrenia. In the present review, we intended to critically assess advances in research of SNP for the therapy of schizophrenia and discuss its potential superiority over currently used neuroleptics.

scholarly journals Conjugated Metabolites of Hydroxytyrosol and Tyrosol Contribute to the Maintenance of Nitric Oxide Balance in Human Aortic Endothelial Cells at Physiologically Relevant Concentrations

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7480
Author(s):  
Gabriele Serreli ◽  
Melanie Le Sayec ◽  
Camilla Diotallevi ◽  
Alice Teissier ◽  
Monica Deiana ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Leukocyte Adhesion ◽  
Virgin Olive Oil ◽  
Guanosine Monophosphate ◽  
No Production ◽  
Aortic Endothelial Cells ◽  
Human Aortic Endothelial Cells ◽  
Conjugated Metabolites ◽  
Aortic Endothelial

Nitric oxide (NO) is an important signaling molecule involved in many pathophysiological processes. NO mediates vasodilation and blood flow in the arteries, and its action contributes to maintaining vascular homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. Dietary antioxidants and their metabolites have been found to be directly and/or indirectly involved in the modulation of the intracellular signals that lead to the production of NO. The purpose of this study was to investigate the contribution of conjugated metabolites of hydroxytyrosol (HT) and tyrosol (TYR) to the release of NO at the vascular level, and the related mechanism of action, in comparison to their parental forms. Experiments were performed in human aortic endothelial cells (HAEC) to evaluate the superoxide production, the release of NO and production of cyclic guanosine monophosphate (cGMP), the activation of serine/threonine-protein kinase 1 (Akt1), and the activation state of endothelial nitric oxide synthase (eNOS). It was observed that the tested phenolic compounds enhanced NO and cGMP concentration, inhibiting its depletion caused by superoxide overproduction. Moreover, some of them enhanced the activation of Akt (TYR, HT metabolites) and eNOS (HT, HVA, TYR-S, HT-3S). Overall, the obtained data showed that these compounds promote NO production and availability, suggesting that HT and TYR conjugated metabolites may contribute to the effects of parental extra virgin olive oil (EVOO) phenolics in the prevention of cardiovascular diseases.

scholarly journals Phytoestrogen Genistein Up-Regulates Endothelial Nitric Oxide Synthase Expression Via Activation of cAMP Response Element-Binding Protein in Human Aortic Endothelial Cells

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3190-3198 ◽  
Author(s):  
Hongwei Si ◽  
Jie Yu ◽  
Hongling Jiang ◽  
Hazel Lum ◽  
Dongmin Liu
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
No Production ◽  
Enos Expression ◽  
Camp Response Element ◽  
Aortic Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Human Aortic Endothelial Cells

We previously reported that genistein, a phytoestrogen, up-regulates endothelial nitric oxide synthase (eNOS) and prevents hypertension in rats that are independent of estrogen signaling machinery. However, how genistein regulates eNOS expression is unknown. In the present study, we show that genistein enhanced eNOS expression and NO synthesis in primary human aortic endothelial cells. Inhibition of extracellular signal regulated kinase, phosphoinositol-3 kinase, or protein kinase C did not affect genistein-enhanced eNOS expression and NO synthesis. However, chemical inhibition of protein kinase A (PKA) or adenoviral transfer of the specific endogenous PKA inhibitor gene completely abolished PKA activity and genistein-stimulated eNOS expression and NO production. Accordingly, genistein induced PKA activity and subsequent phosphorylation of cAMP response element (CRE)-binding protein (CREB) at Ser133. Suppression of CREB by small interfering RNA transfection abolished genistein-enhanced eNOS expression and NO production. Consistently, deletion of the CRE site within human eNOS promoter eliminated genistein-stimulated eNOS promoter activity. These findings provide the first evidence to our knowledge that genistein may play a beneficial role in vascular function through targeting the PKA/CREB/eNOS/NO signaling pathway.

Tumor necrosis factor-α reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells

2007 ◽  
Vol 293 (2) ◽  
pp. H1115-H1121 ◽  
Author(s):  
Bonnie L. Goodwin ◽  
Laura C. Pendleton ◽  
Monique M. Levy ◽  
Larry P. Solomonson ◽  
Duane C. Eichler
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Elevated Serum ◽  
Proximal Promoter ◽  
No Production ◽  
Aortic Endothelial Cells ◽  
Tnf Α ◽  
Argininosuccinate Synthase ◽  
Endothelial Nitric Oxide ◽  
Aortic Endothelial

Endothelial dysfunction associated with elevated serum levels of TNF-α observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-α (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-α with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-α suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-α may be mediated, in part, via the NF-κB signaling pathway. These findings demonstrate that TNF-α coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-α is an added insult to endothelial function because of its important role in NO production and in endothelial viability.

Regulation of nitric oxide synthesis by oxygen in vascular endothelial cells

1997 ◽  
Vol 272 (6) ◽  
pp. L1161-L1166 ◽  
Author(s):  
A. R. Whorton ◽  
D. B. Simonds ◽  
C. A. Piantadosi
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Oxidation Products ◽  
No Oxidation ◽  
No Production ◽  
Vascular Endothelial ◽  
Intact Cells ◽  
Aortic Endothelial Cells ◽  
Basal Rate

Vascular endothelial cells synthesize nitric oxide (NO) in response to agonists that elevate cytosolic free Ca2+ concentrations. Once activated, NO synthase (NOS) requires arginine, NADPH, and O2 as cosubstrates. In this study, we investigated the role of O2 in regulating endothelial NOS activity in intact bovine aortic endothelial cells by measuring the rate of nitrite (NO2-) and nitrate (NO3-) production after conversion of NO2- to S-nitrosoglutathione before analysis or after reduction of NO2- and NO3- to NO using acidic vanadium chloride. The basal rate of NO2- production was 6.5 +/- 0.8 pmol.min-1.mg protein-1. Thapsigargin (TG, 1 microM) elevated free cytosolic Ca2+ concentration and increased the rate of NO2- synthesis. At maximal concentrations of TG, the rate of stimulated NO2- production was linear for at least 20 min and was eightfold higher than the basal rate (53.5 +/- 1.8 pmol.min-1.mg protein-1). Incubation of cells in gas mixtures chosen to produce PO2 values in the physiological range led to a progressive fall in the rate of TG-stimulated NO2- production, as O2 concentrations were reduced from that of room air. The half-maximal effective concentration for NO2- production by intact cells was found to occur at 38 Torr. PO2 values higher than that of room air did not lead to a change in the rate of TG-stimulated NO2- production. To confirm that measurement of NO2- accurately reflects total NO production, both NO2- plus NO3- were measured in buffer samples from cells incubated in either room air or N2. The sum of these NO oxidation products was inhibited similarly by hypoxia. These findings suggest that O2 is an important determinant of NOS activity in hypoxic tissues or in vascular beds such as the pulmonary arterial or fetal circulation where PO2 values in the range of 40 Torr are encountered normally.

Heparan sulfate proteoglycan is essential to thrombin-induced calcium transients and nitric oxide production in aortic endothelial cells

2008 ◽  
Vol 100 (09) ◽  
pp. 483-488 ◽  
Author(s):  
Chiwaka Kimura ◽  
Masahiro Oike
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
No Production ◽  
Sulfate Proteoglycan ◽  
Protease Activated Receptors ◽  
Aortic Endothelial Cells ◽  
Polysaccharide Chain ◽  
Intracellular Signals ◽  
Aortic Endothelial

SummaryThrombin induces Ca2+ transients and subsequent nitric oxide (NO) production in vascular endothelial cells. Thrombin cleaves protease-activated receptors, resulting in activation of intracellular signals, but it is not clarified how the extracellular thrombin stays around the cells to exert its enzyme activities. This study aimed to investigate the possible involvement of heparin sulfate proteoglycan (HSPG) in the effects of thrombin on vascular endothelium. Heparinase III completely removed the polysaccharide chain of HSPG in bovine aortic endothelial cells (BAECs).Thrombin induced Ca2+ transients in control BAECs, but not in heparinase III-treated BAECs. In contrast, ATP induced Ca2+ transients both in control and heparinase III-treated BAECs. Thrombin that was pre-incubated with heparin also failed to induced Ca2+ transients in BAECs. Furthermore, thrombin-induced NO production, as assessed with DAF-2 flu-orescence, was suppressed in heparinase III-treated BAECs and by the pre-incubation of thrombin with heparin. ATP-induced NO production was, however, not affected in heparinase III-treated BAECs. These results indicate that it is essential for thrombin to bind to the polysaccharide chain of HSPG for inducing Ca2+ transients and NO production in BAECs.

scholarly journals β-Cyclodextrin Inhibits Monocytic Adhesion to Endothelial Cells through Nitric Oxide-Mediated Depletion of Cell Adhesion Molecules

Molecules ◽  
2020 ◽  
Vol 25 (16) ◽  
pp. 3575
Author(s):  
Sujeong Jang ◽  
Seongsoo Lee ◽  
Heonyong Park
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Cell Adhesion ◽  
No Production ◽  
Aortic Endothelial Cells ◽  
Enos Phosphorylation ◽  
Inflammatory Processes ◽  
Cell Adhesion Molecule 1 ◽  
Endothelial Nitric Oxide ◽  
Inflammatory Effect

Cyclodextrins (CDs) are used as drug delivery agents. In this study, we examined whether CDs have an inflammatory effect on endothelial cells. First, we found that β-CD promoted cell proliferation in bovine aortic endothelial cells and elevated nitric oxide (NO) production through dephosphorylation of threonine-495 (T-495) in endothelial nitric oxide synthetase (eNOS). Dephosphorylation of T-495 is known to activate eNOS. Phosphorylation of T-495 was found to be catalyzed by protein kinase Cε (PKCε). We then found that β-CD inhibits binding of PKCε to diacylglycerol (DAG) via formation of a β-CD-DAG complex, indicating that β-CD inactivates PKCε. Furthermore, β-CD controls activation of PKCε by reducing the recruitment of PKCε into the plasma membrane. Finally, β-CD inhibits expression of intercellular and vascular cell adhesion molecule-1 by increasing NO via control of PKCε/eNOS and suppression of THP-1 cell adhesion to endothelial cells. These findings imply that β-CD plays an important role in anti-inflammatory processes.

Abstract 345: Inhibitor Kappa B Kinase Regulates Nitric Oxide Synthase Activity in Vascular Endothelial Cells

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Mohan Natarajan ◽  
Manikcam Krishnan ◽  
Ryzad Konopinski ◽  
Colleen Gaudette ◽  
Preethi Janardhanan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Binding Site ◽  
High Glucose ◽  
No Production ◽  
Aortic Endothelial Cells ◽  
Enos Activity ◽  
Hsp 90 ◽  
Oxide Synthase

The generation of nitric oxide (NO) in endothelial cells is predominantly regulated by endothelial nitric oxide synthase (eNOS) activity. The role of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity has been established. Earlier, we demonstrated an increased interaction of Hsp-90 with inhibitor kappa B kinase β (IKKβ) in aortic endothelial cells (ECs) under conditions of high glucose (HG). These studies implicated the possibility of a site-specific competition between eNOS and IKKβ to bind to Hsp-90. In this study, the binding site of IKKβ on Hsp-90β is demonstrated by generating 5’deletional mutants of Flag tagged Hsp-90 and full length enhanced yellow fluorescent protein tagged IKKβ (EYFP-IKKβ). Forceful co-expression of the Hsp-90 deletion constructs with EYFP-IKKβ plasmid followed by immunoprecipitation/immunoblotting revealed the binding site of IKKβ to be between 307-711aa on Hsp-90, the middle domain (440-600 aa) where eNOS has been shown to bind to Hsp-90. Co-immunoprecipitation experiments performed in lysates of human aortic endothelial cells incubated with 25mM high glucose exhibited enhanced IKK beta interaction with both Hsp-90 α and β isoforms. Independently blocking Hsp-90α or β expression by siRNA transfection significantly blocked VEGF-induced NO production implicating the possible mechanism that IKK beta regulates NO production by competing with eNOS for both the isoforms of Hsp-90. These results confirm the previously unknown participation of IKKβ in the downregulation of eNOS activity under the influence of HG and provide a novel therapeutic strategy of using IKKβ as an effective target to improve NO generation in diabetic arteries.

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