Effects of homocysteine on endothelial nitric oxide production

2000 ◽  
Vol 279 (4) ◽  
pp. F671-F678 ◽  
Author(s):  
Xiaohui Zhang ◽  
Hong Li ◽  
Haoli Jin ◽  
Zachary Ebin ◽  
Sergey Brodsky ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Redox State ◽  
Calcium Ionophore ◽  
No Production ◽  
Cellular Redox ◽  
A Cell ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Peroxynitrite Scavengers

Hyperhomocysteinemia (HHCy) is an independent and graded cardiovascular risk factor. HHCy is prevalent in patients with chronic renal failure, contributing to the increased mortality rate. Controversy exists as to the effects of HHCy on nitric oxide (NO) production: it has been shown that HHCy both increases and suppresses it. We addressed this problem by using amperometric electrochemical NO detection with a porphyrinic microelectrode to study responses of endothelial cells incubated with homocysteine (Hcy) to the stimulation with bradykinin, calcium ionophore, or l-arginine. Twenty-four-hour preincubation with Hcy (10, 20, and 50 μM) resulted in a gradual decline in responsiveness of endothelial cells to the above stimuli. Hcy did not affect the expression of endothelial nitric oxide synthase (eNOS), but it stimulated formation of superoxide anions, as judged by fluorescence of dichlorofluorescein, and peroxynitrite, as detected by using immunoprecipitation and immunoblotting of proteins modified by tyrosine nitration. Hcy did not directly affect the ability of recombinant eNOS to generate NO, but oxidation of sulfhydryl groups in eNOS reduced its NO-generating activity. Addition of 5-methyltetrahydrofolate restored NO responses to all agonists tested but affected neither the expression of the enzyme nor formation of nitrotyrosine-modified proteins. In addition, a scavenger of peroxynitrite or a cell-permeant superoxide dismutase mimetic reversed the Hcy-induced suppression of NO production by endothelial cells. In conclusion, electrochemical detection of NO release from cultured endothelial cells demonstrated that concentrations of Hcy >20 μM produce a significant indirect suppression of eNOS activity without any discernible effects on its expression. Folates, superoxide ions, and peroxynitrite scavengers restore the NO-generating activity to eNOS, collectively suggesting that cellular redox state plays an important role in HCy-suppressed NO-generating function of this enzyme.

scholarly journals Cilostazol Induces eNOS and TM Expression via Activation with Sirtuin 1/Krüppel-like Factor 2 Pathway in Endothelial Cells

2021 ◽  
Vol 22 (19) ◽  
pp. 10287
Author(s):  
Chih-Hsien Wu ◽  
Yi-Lin Chiu ◽  
Chung-Yueh Hsieh ◽  
Guo-Shiang Tsung ◽  
Lian-Shan Wu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
Umbilical Vein ◽  
Sirtuin 1 ◽  
No Production ◽  
Beneficial Effects ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Cilostazol was suggested to be beneficial to retard in-stent atherosclerosis and prevent stent thrombosis. However, the mechanisms responsible for the beneficial effects of cilostazol are not fully understood. In this study, we attempted to verify the mechanism of the antithrombotic effect of cilostazol. Human umbilical vein endothelial cells (HUVECs) were cultured with various concentrations of cilostazol to verify its impact on endothelial cells. KLF2, silent information regulator transcript-1 (SIRT1), endothelial nitric oxide synthase (eNOS), and endothelial thrombomodulin (TM) expression levels were examined. We found cilostazol significantly activated KLF2 expression and KLF2-related endothelial function, including eNOS activation, Nitric oxide (NO) production, and TM secretion. The activation was regulated by SIRT1, which was also stimulated by cilostazol. These findings suggest that cilostazol may be capable of an antithrombotic and vasculoprotective effect in endothelial cells.

scholarly journals Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase

2008 ◽  
Vol 86 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Syamantak Majumder ◽  
Ajit Muley ◽  
Gopi Krishna Kolluru ◽  
Samir Saurabh ◽  
K. P. Tamilarasan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Function ◽  
Egg Yolk ◽  
Cellular Migration ◽  
No Production ◽  
Enos Phosphorylation ◽  
Low Levels ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.

scholarly journals Endoplasmic Reticulum Ca2+ Release Modulates Endothelial Nitric-oxide Synthase via Extracellular Signal-regulated Kinase (ERK) 1/2-mediated Serine 635 Phosphorylation

2011 ◽  
Vol 286 (22) ◽  
pp. 20100-20108 ◽  
Author(s):  
Zhihong Xiao ◽  
Tingting Wang ◽  
Honghua Qin ◽  
Chao Huang ◽  
Youmei Feng ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Nitric Oxide Synthase ◽  
Protein Phosphorylation ◽  
Endothelial Nitric Oxide Synthase ◽  
No Production ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca2+ and protein phosphorylation, but the interrelationship between intracellular Ca2+ mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca2+ release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca2+ release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca2+ release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca2+ and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca2+ release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca2+/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca2+ release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca2+ release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca2+ is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca2+-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.

scholarly journals Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase

2006 ◽  
Vol 174 (3) ◽  
pp. 369-377 ◽  
Author(s):  
Carlos Fernández-Hernando ◽  
Masaki Fukata ◽  
Pascal N. Bernatchez ◽  
Yuko Fukata ◽  
Michelle I. Lin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Cdna Clones ◽  
No Production ◽  
Human Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [3H]-palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.

scholarly journals Src Kinase Activates Endothelial Nitric-oxide Synthase by Phosphorylating Tyr-83

2005 ◽  
Vol 280 (43) ◽  
pp. 35943-35952 ◽  
Author(s):  
David Fulton ◽  
Jarrod E. Church ◽  
Ling Ruan ◽  
Chunying Li ◽  
Sarika G. Sood ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Tyrosine Phosphorylation ◽  
Endothelial Nitric Oxide Synthase ◽  
Phosphorylation Site ◽  
Src Kinase ◽  
No Production ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

The endothelial nitric-oxide synthase (eNOS) is regulated in part by serine/threonine phosphorylation, but eNOS tyrosine phosphorylation is less well understood. In the present study we have examined the tyrosine phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) exposed to oxidant stress. Hydrogen peroxide and pervanadate (PV) treatment stimulates eNOS tyrosine phosphorylation in BAECs. Phosphorylation is blocked by the Src kinase family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, eNOS and c-Src can be coimmunoprecipitated from BAEC lysates by antibodies directed against either protein. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and then treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site. Tyr-83 phosphorylation is associated with a 3-fold increase in basal NO release from cotransfected cells. Furthermore, the Y83F eNOS mutation attenuated thapsigargin-stimulated NO production. Taken together, these data indicate that Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 modulates eNOS activity in endothelial cells.

Abstract P198: Differential Regulation of Endothelial Nitric Oxide Synthase Phosphorylation by Protease-Activated Receptors in Adult Human Endothelial Cells

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Lakeisha C Tillery ◽  
Evangeline D Motley-Johnson
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
Umbilical Vein ◽  
Differential Regulation ◽  
No Production ◽  
Protease Activated Receptors ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Protease-activated receptors (PARs) have been shown to regulate endothelial nitric oxide synthase (eNOS) through the activation of specific sites on the enzyme. It has been established that phosphorylation of eNOS-Ser-1177 leads to the production of the potent vasodilator nitric oxide (NO), and is associated with PAR-2 activation; while phosphorylation of eNOS-Thr-495 decreases NO production, and is coupled to PAR-1 activation. In this study, we demonstrate a differential regulation of the eNOS/NO pathway by the PARs using primary adult human coronary artery endothelial cells (HCAEC). Thrombin and the PAR-1 activating peptide, TFLLR, which are known to phosphorylate eNOS-Thr-495 in bovine and human umbilical vein endothelial cells, phosphorylated eNOS-Ser-1177 in HCAECs, and increased NO production. The PAR-1 responses were blocked using SCH-79797, a PAR-1 inhibitor, and L-NAME was used to inhibit NO production. A PAR-2 specific ligand, SLIGRL, which has been shown to phosphorylate eNOS-Ser-1177 in bovine and human umbilical vein endothelial cells, primarily regulated eNOS-Thr-495 phosphorylation and suppressed NO production in the HCAECs. PAR-3, known for its non-signaling potential, was activated by TFRGAP, a PAR-3 mimicking peptide, and only induced phosphorylation of eNOS-Thr-495 with no effect on NO production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was calcium-dependent using the calcium chelator, BAPTA, and eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632. These data suggest a vascular bed specific differential coupling of PARs to the signaling pathways that regulate eNOS and NO production that may be responsible for the modulation of endothelial function associated with cardiovascular disease.

Abstract 3635: Inhibition of Inhibitor Kappa B Kinase Beta Augments Endothelial Nitric Oxide Synthase Activity and Nitric Oxide Production in Aortic Endothelial Cells

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Sumathy Mohan ◽  
Ryzard Konopinski ◽  
Mohan Natarajan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Vascular Endothelial Cells ◽  
No Production ◽  
Enos Activity ◽  
Hsp 90 ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hall-mark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases (inhibitor kappa B kinases α, β and γ) in non-vascular cells. In this study, we have investigated the interaction of Hsp-90-IKKβ in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKβ interacts with Hsp-90 and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (4.02 ± 0.81-folds) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.04 ± 0.06-folds). Another important and novel finding is that both IKKβ and eNOS could be co-immunoprecipitated with Hsp-90 (Figures A & B ) thus indicating the possible existence of a complex of IKKβ and eNOS interacting with single pool of Hsp-90. Inhibition of Hsp-90 with geldanamycin (2μM) or Radicicol (20μM) mitigated (0.45 ± 0.04 -fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.416-fold). Blocking of IKKβ expression by IKK inhibitor II (15μM wedelolactone) or siRNA improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.

Arecoline Increases the Production of Nitric Oxide and Post-Translational S-Nitrosoproteome in Endothelial Cells

2020 ◽  
Vol 17 (3) ◽  
pp. 172-179
Author(s):  
Chien-Yi Wu ◽  
Wun-Rong Lin ◽  
Cherng-Jye Jeng ◽  
Chien-Hsing Wu ◽  
Bin Huang
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Fluorescent Microscopy ◽  
Protein S ◽  
No Production ◽  
Absolute Quantitation ◽  
Isobaric Tag ◽  
Oxide Synthase ◽  
Biotin Switch ◽  
Endothelial Nitric Oxide

Background: Arecoline is known as a carcinogenic toxicant. The refreshment effect of arecoline is mainly due to the increase in vasodilation and blood flow. This is essential to understand whether arecoline can induce the production of Nitric Oxide (NO•) and regulate the subsequent protein S-nitrosylation in Endothelial Cells (ECs). Objective: The present study is focused on the promotion effect of arecoline in NO• production and the subsequent regulation of S-nitrosoproteome. Method: The phosphorylation of endothelial nitric oxide synthase serine 1177 residue (peNOSSer1177) was investigated by western blot. By using a specific FA-OMe fluorescent probe, the NO• molecules could be observed by fluorescent microscopy or flow cytometry. S-nitrosylated proteins were purified by biotin switch and then subjected to the Isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-labeled shotgun proteomic analysis. Results: Our study reveals that a lower concentration of arecoline can increase the phosphorylation of peNOSSer1177. Pretreatment of NG-nitro-L-arginine methyl ester (L-NAME) indicated that arecolineinduced NO• production was mediated by e-NOS. We identified 224 proteins with up-regulated S-nitrosylation and 159 proteins with down-regulated S-nitrosylation. The NO• binding sites of seven representative S-nitrosoproteins were illustrated. The effect of arecoline on the S-nitrosylation of HSP60 chaperonin and calnexin was verified. Conclusion: Our experimental results proved that a lower concentration of arecoline could modulate the production of NO• and the subsequent protein S-nitrosylation. Therefore, it is worthy for further investigation and discussion if these S-nitrosoproteomes are important in maintaining endothelium homeostasis.

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.

Ethanol Increases Endothelial Nitric Oxide Production through Modulation of Nitric Oxide Synthase Expression

1999 ◽  
Vol 81 (04) ◽  
pp. 638-642 ◽  
Author(s):  
Christo D. Venkov ◽  
Paul R. Myers ◽  
Miles A. Tanner ◽  
Ming Su ◽  
Douglas E. Vaughan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Heart Disease ◽  
Nitric Oxide Synthase ◽  
Ischemic Heart Disease ◽  
No Production ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Enos Protein ◽  
Mrna Expression Levels

SummaryModerate alcohol consumption has been shown to reduce the risk of ischemic heart disease potentially through its effect on specific endothelial-derived compounds. We tested the hypothesis that ethanol increases the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production in bovine aortic endothelial cells (BAEC). Primary cultures of BAEC grown to confluence under standard conditions were treated 3-6 h with 0.1% ethanol in the presence of indomethacin. Ethanol induced a significant increase in both basal and stimulated NO production as determined by chemiluminescence method. This effect was accompanied by a rapid increase of eNOS protein and mRNA expression levels. eNOS mRNA increased two-fold within 3 h and gradually declined, but the increased levels of mRNA persisted for >24 h. A similar increase of eNOS expression was observed in human umbilical endothelial cells exposed to ethanol. These results demonstrate that ethanol augments both basal and stimulated NO production and that this effect is associated with increased eNOS protein and mRNA expression levels. The data are consistent with the hypothesis that the reduced incidence of ischemic heart disease associated with alcohol may be related, at least in part, to the modulation of vascular endothelial cell production of NO

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