Interactions of peroxynitrite with uric acid in the presence of ascorbate and thiols: implications for uncoupling endothelial nitric oxide synthase

It has been suggested that uric acid acts as a peroxynitrite scavenger although it may also stimulate lipid peroxidation. To gain insight into how uric acid may act as an antioxidant, we used electron spin resonance to study the reaction of uric acid and plasma antioxidants with ONOO-. Peroxynitrite reacted with typical plasma concentrations of urate 16-fold faster than with ascorbate and 3-fold faster than cysteine. Xanthine but not other purine-analogs also reacted with peroxynitrite. The reaction between ONOO- and urate produced a carbon-centered free radical, which was inhibited by either ascorbate or cysteine. Moreover, scavenging of ONOO- by urate was significantly increased in the presence of ascorbate and cysteine. An important effect of ONOO- is oxidation of tetrahydrobiopterin, leading to uncoupling of nitric oxide synthase. The protection of eNOS function by urate, ascorbate and thiols in ONOO(-)-treated bovine aortic endothelial cells (BAECs) was, therefore, investigated by measuring superoxide and NO using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) and the NO-spin trap Fe[DETC]2. Peroxynitrite increased superoxide and decreased NO production by eNOS indicating eNOS uncoupling. Urate partially prevented this effect of ONOO- while treatment of BAECs with the combination of either urate with ascorbate or urate with cysteine completely prevented eNOS uncoupling caused by ONOO-. We conclude that the reducing and acidic properties of urate are important in effective scavenging of peroxynitrite and that cysteine and ascorbate markedly augment urate's antioxidant effect by reducing urate-derived radicals.     

TNFα reduces eNOS activity in endothelial cells through serine 116 phosphorylation and Pin1 binding: Confirmation of a direct,inhibitory interaction of Pin1 with eNOS

Production of NO by the endothelial nitric oxide synthase (eNOS) has a major role in blood pressure control and suppression of atherosclerosis. In a previous study, we presented evidence implicating the Pin1 prolyl isomerase in negative modulation of eNOS activity in bovine aortic endothelial cells (BAECs). Pin1 recognizes phosphoserine/phosphothreonine–proline motifs in target proteins and catalyzes prolyl isomerization at the peptide bond. In the present study, we show, first, with purified proteins, that Pin1 binds to eNOS directly via the Pin1 WW domain. Binding is enhanced by mimicking phosphorylation of eNOS at S116. Interaction of Pin1 with eNOS markedly reduces eNOS enzymatic activity. Second, in BAECs, we show that TNFα induces ERK 1/2-mediated S116 phosphorylation of eNOS, accompanied by Pin1 binding. TNFα treatment of BAECs results in a reduction in NO release from the cells in a manner that depends on the activities of both Pin1 and ERK 1/2. Evidence is also presented that this mechanism of eNOS regulation cannot occur in rat and mouse cells because there is no proline residue in the mouse and rat amino acid sequences adjacent to the putative phosphorylation site. Moreover, we find that phosphorylation of this site is not detectable in mouse eNOS.     

内外源性EETs对血管内皮一氧化氮合酶的表达及其在Thr-495位磷酸化的作用

目的　研究细胞色素P4 5 0表氧化酶基因转染和直接加入EETs对内皮细胞eNOS表达及其在Thr 4 95位磷酸化的影响。方法　在原代培养的牛主动脉内皮细胞中 ,分别加入生理浓度的 8,9 EET(1 0 0nmol·L-1 )、1 1 ,1 2 EET(1 0 0nmol·L-1 )、1 4 ,1 5 EET(1 0 0nmol·L-1 )孵育 4h ,或直接用重组腺相关病毒介导的花生四烯酸表氧化酶转染牛主动脉内皮细胞2wk ,以产生内源性EETs ,用Westernblot法检测总eNOS蛋白的表达及eNOSThr 4 95磷酸化的水平 ;此外 ,从大鼠尾静脉注射真核表达质粒pCB6、pCB6 2C1 1OR、pCB6 2J2和pCB6 F87V ,2wk后检测大鼠主动脉总eNOS表达及eNOSThr 4 95磷酸化的水平。结果　与空白和溶媒组比较 ,外源性EETs明显促进内皮细胞总eNOS表达 ,增加eNOSThr 4 95的磷酸化 ,而CYP4 5 0抑制剂 (1 7 ODYA)则可明显降低eNOS表达和eNOSThr 4 95的磷酸化水平 ;与相应的对照组比较 ,体内和体外转染不同的表氧化酶基因均能明显上调内皮细胞eNOS的表达 ,增加eNOSThr 4 95的磷酸化水平。结论　EETs和CYP表氧化酶不仅能明显促进血管内皮细胞总eNOS蛋白的表达 ,而且还通过其翻译后修饰来增加其Thr 4 95位蛋白磷酸化水平     

内皮源性超极化因子对内皮一氧化氮合酶基因表达的调节

目的　以内皮细胞产生NO的关键酶———eNOS(内皮一氧化氮合酶 )为研究目标 ,探讨外源性内皮源性超极化因子EDHF(EETs)对内皮细胞合成NO的影响。方法　在原代培养 3～ 4代以内的牛主动脉内皮细胞中 ,分别加入不同浓度 (5 0～ 2 0 0nmol·L-1)的 8,9 EET、11,12 EET、14 ,15 EET ,作用 1h后用不同的方法收获细胞。用WesternBlot以及NorthernBlot方法检测EETs对eNOS基因表达的影响 ;同时通过检测L [3 H] 精氨酸转化为L [3 H] 瓜氨酸的量研究EETs对NOS活性的影响。结果　显示 8,9 EET、11,12 EET、14 ,15 EET均呈浓度依赖性地增加eNOS蛋白质的表达 ,并提高eNOSmRNA表达水平以及NOS酶活性。结论　外源性EDHF对eNOS基因表达是一种正反馈调节作用 ,从而能够促进内皮细胞NO的产生 ,通过药物调节内皮表氧化酶进而促进eNOS基因表达可作为防治心血管疾病的新策略     

Serine 1179 phosphorylation of endothelial nitric oxide synthase caused by 2,4,6-trinitrotoluene through PI3K/Akt signaling in endothelial cells

Although 2,4,6-trinitrotoluene (TNT) has been found to uncouple nitric oxide synthase (NOS), thereby leading to reactive oxygen species (ROS), cellular response against TNT still remains unclear. Exposure of bovine aortic endothelial cells (BAECs) to TNT (100 microM) resulted in serine 1179 phosphorylation of endothelial NOS (eNOS). With specific inhibitors (wortmannin and LY294002), we found that PI3K/Akt signaling participated in the eNOS phosphorylation caused by TNT, whereas the ERK pathway did not. ROS were generated following exposure of BAECs to TNT. However, TNT-mediated phosphorylation of either eNOS or Akt was drastically blocked by NAC and PEG-CAT. Interestingly, pretreatment with apocynin, a specific inhibitor for NADPH oxidase, diminished the phosphorylation of eNOS and Akt. These results suggest that TNT affects NADPH oxidase, thereby generating hydrogen peroxide, which is capable of activating PI3K/Akt signaling associated with eNOS Ser 1179 phosphorylation.     

Chronic nicotine exposure attenuates proangiogenic activity on human umbilical vein endothelial cells

The pathogenic mechanism of nicotine, a major product of smoking, on vascular endothelial cells is not well defined yet. The purpose of this study was to determine whether chronic exposure to nicotine alters angiogenic activity in human umbilical vein endothelial cells and to identify a potential role for endothelial nitric oxide synthase (eNOS) expression. Our study demonstrated that acute nicotine treatment enhanced nitric oxide release, eNOS activation, and proangiogenic activity. However, chronic nicotine exposure impaired proangiogenic function (decreased cell migration and tubular structure formation) in human umbilical vein endothelial cells compared with acute exposure, but sustained the antiapoptotic effect. These findings seem to be related to eNOS gene expression and nitric oxide production, which may be involved in the pathophysiology of chronic nicotine addicts.     

N-acetylcysteine attenuates TNF-alpha-induced human vascular endothelial cell apoptosis and restores eNOS expression

The circulatory inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in pathological conditions, such as diabetes, which initiate or exacerbate vascular endothelial injury. Both nitric oxide (NO) and reactive oxygen species may play a dual role (i.e., inhibiting or promoting) in TNF-alpha-induced endothelial cell apoptosis. We investigated the effects of the antioxidant N-acetylcysteine on TNF-alpha-induced apoptosis in human vascular endothelial cell (cell line ECV304) apoptosis, NO production and lipid peroxidation. Cultured vascular endothelial cell (ECV304) were either not treated (control), or treated with TNF-alpha (40 ng/ml) alone or TNF-alpha in the presence of N-acetylcysteine at 30 mmol/l or 1 mmol/l, respectively, for 24 h. Cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was assessed by flow cytometry. TNF-alpha-induced endothelial cell apoptosis was associated with increased inducible NO synthase but reduced endothelial NO synthase (eNOS) protein expression. NO production and the levels of the lipid peroxidation product malondialdehyde were concomitantly increased. Treatment with NAC at 30 mmol/l restored eNOS expression and further increased NO production as compared to TNF-alpha alone, resulting in improved cell viability and reduced apoptosis. This was accompanied by increased superoxide dismutase activity, increased glutathione peroxidase production and reduced malondialdehyde levels. N-acetylcysteine at 1 mmol/l, however, did not have significant effects on TNF-alpha-induced endothelial cell apoptosis and cell viability despite it slightly enhanced glutathione peroxidase production. N-acetylcysteine attenuation of TNF-alpha-induced human vascular endothelial cell apoptosis is associated with the restoration of eNOS expression.     

动态观察实验性高脂血症对血管内皮活性物质含量及其基因表达的影响以及DDPH的内皮细胞保护作用

目的　观察在高脂血症动物模型形成过程中不同程度的高血脂以及DDPH对动脉血中NO、ET 1含量以及动脉壁eNOS、内皮素 1基因表达的影响。方法　在实验性家兔高脂血症模型基础上 ,采用半定量逆转录多聚酶链式反应(RT PCR)等技术 ,观察了不同时期的高脂血症对内皮素及一氧化氮代谢的影响以及DDPH的干预作用。结果　不同时期的高脂饮食可造成不同程度的高脂血症 ,相应的血管内皮细胞的内皮素 1和一氧化氮的代谢紊乱也有所不同 :轻、中度高脂血症可引起eNOS及内皮素 1mRNA表达水平升高和外周血ET 1含量增高 ;而重度高脂血症可引起eNOS及内皮素mRNA表达水平下降和外周血NOP、ET 1浓度降低 ;DDPH(ig)可明显降低实验性家兔的高血脂 ,并同时促进一氧化氮代谢产物 (NOP)、ET 1含量的升高和eNOS、ET 1mRNA的表达。结论　不同程度的高脂血症对血管内皮活性物质及其基因表达的影响是不同的。DDPH可保护血管内皮细胞 ,恢复血管活性物质合成和释放 ,从而恢复血管的反应性     

Icariin enhances endothelial nitric-oxide synthase expression on human endothelial cells in vitro

Icariin, a flavonoid isolated from Epimedii herba, is considered to be the major therapeutical constituent of E. herba. The aim of this study was to investigate the possible protective effects and to clarify the mechanism of icariin on endothelial cells in vitro. Incubation of human umbilical vein endothelial cells (HUVEC) derived EA. hy926 cells with icariin(0.1, 1, 10 micromol l(-1)) from 6 h to 72 h, then the production of NO was measured to evaluate the protective effects of icariin. RT-PCR was employed to confirm the mRNA expression of endothelial nitric oxide synthase (eNOS). Western blotting was used to evaluate the protein expression of eNOS. NO production was enhanced in a time- and concentration-dependent manner (P<0.05), which was well matched with the expression of eNOS mRNA (up to 2.4-fold) and protein (up to 2.5-fold) after long-term incubation with icariin in endothelial cells (P<0.05). Moreover, activated NF-kappaB was increased in EA. hy926 cells incubated with icariin for 24 h, in association with an increase in the expression of eNOS gene. In addition to its long-term effects on eNOS expression, icariin also enhanced the production of bioactive NO in the short-term (after a 5 min incubation, P<0.05). In concert with other effects, the protective effects of icariin on endothelial cells may contribute to the cardiovascular protective effects.     

Role of heat shock protein 90 in bradykinin-stimulated endothelial nitric oxide release.

Previously we described ENAP-1, a 90-kDa protein that is tyrosine-phosphorylated in endothelial cells in response to bradykinin (BK) stimulation and is associated with endothelial nitric oxide synthase (eNOS). Subsequently, other investigators demonstrated that eNOS interacts with heat shock protein 90 (Hsp90) following stimulation of endothelial cells with vascular endothelial growth factor (VEGF), histamine, or fluid shear stress. Therefore, we tested the hypotheses that ENAP-1 and Hsp90 are the same protein and that BK activation of eNOS is dependent on Hsp90. Immunoblotting of immunoprecipitated Hsp90 with anti-phosphotyrosine antibody shows that Hsp90 is tyrosine-phosphorylated in response to BK stimulation of bovine aortic endothelial cells (BAECs). Coimmunoprecipitation of Hsp90 with anti-eNOS antibody reveals a Hsp90-eNOS complex in endothelial cells under basal conditions that is increased following BK stimulation. Taken together with the tyrosine phosphorylation data, these data suggest that ENAP-1 is Hsp90. BK-stimulated nitric oxide (NO) release is completely blocked by pretreatment with geldanamycin, a specific inhibitor of Hsp90, illustrating the importance of the Hsp90-eNOS interaction. In vitro binding assays with Hsp90-glutathione-S-transferase fusion proteins show direct binding of eNOS with the middle domain (residues 259-615) of Hsp90.     

阿司匹林对人主动脉血管内皮细胞炎性损伤的保护作用

目的：研究阿司匹林（aspirin,Asp）对脂多糖（lipopolysaccharide,LPS）诱导人主动脉内皮细胞（human aortic endothelial cells,HAECs）损伤的保护作用,并进一步阐明其对一氧化氮合酶（NOS）及血管内皮生长因子（VEGF）及其相关受体信号的调控。方法：LPS建立HAECs损伤模型。苏木精-伊红（HE）染色观察细胞形态;MTT法、划痕实验分析HAECs损伤修复能力;ELISA测定一氧化氮（NO）含量;Western blot检测内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）、VEGF和血管内皮生长因子受体-2（VEGFR-2）蛋白表达。结果：给药12 h后Asp明显改善LPS（5 mg·L^-1）导致的细胞损伤、提高修复能力（P<0.05）,并上调NO分泌量及VEGF、VEGFR-2的蛋白表达（P<0.01）;升高eNOS蛋白的表达（P<0.01）。而给药24 h后阿司匹林显著下调LPS导致的NO分泌量及iNOS、VEGF、VEGFR-2的蛋白表达升高,同时升高eNOS蛋白的表达（P<0.01）。结论：阿司匹林对LPS诱导的血管内皮细胞炎性损伤的保护作用与调节NOS/NO和VEGF及其受体的动态平衡密切相关。     

Chronic activation of AMP-activated protein kinase prevents 20-hydroxyeicosatetraenoic acid-induced endothelial dysfunction

1. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor involved in vascular dysfunction and blood pressure regulation. Studies have revealed strong associations between 20-HETE and endothelial dysfunction; however, the signalling mechanisms are largely unknown. Therefore, the aim of the present study was to investigate the effect of 20-HETE on the association between endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (Hsp90). 2. In mouse aortic rings, 20-HETE significantly enhanced the constriction to phenylephrine and inhibited the relaxation to acetylcholine (P=0.05 vs control rings). In mice with chronic AMP-activated protein kinase (AMPK) activation, this protected against the negative effects of 20-HETE (P<0.05). Immunoprecipitation of eNOS in human umbilical vein endothelial cells treated with 20-HETE revealed a decrease in basal and vascular endothelial growth factor-stimulated Hsp90 association with eNOS (P<0.05). Pretreatment of cells with 5'-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR; a chronic activator of AMPK) prevented the loss of Hsp90 association with eNOS following 20-HETE treatment. Treatment with 20-HETE for 24 h induced an increase in eNOS phosphorylation that was not seen following acute treatment (30 min). The increased eNOS phosphorylation was accompanied by transient changes in Akt phosphorylation. 3. In conclusion, 20-HETE impairs eNOS-Hsp90 association, which can be reversed by chronic activation of AMPK. This provides a mechanism for reduced nitric oxide bioactivity and endothelial dysfunction in diseases with elevated 20-HETE levels, such as hypertension.     

Endothelial nitric oxide synthase activation through obacunone-dependent arginase inhibition restored impaired endothelial function in ApoE-null mice

Endothelial arginase constrains the activity of endothelial nitric oxide synthase (eNOS) by substrate depletion and reduces nitric oxide bioavailability. During the screening course of arginase inhibitor, we found obacunone as an arginase inhibitor. We tested the hypothesis that obacunone regulates vascular endothelial NO production. Obacunone incubation inhibited arginase I and II activities in liver and kidney lysates, respectively, in dose-dependent manner. Obacunone reciprocally increased nitrite/nitrate (NOx) production in HUVECs. In isolated aortic rings, obacunone increased intracellular l-arginine concentration and enhanced eNOS coupling, leading to increased NO and decreased superoxide production, with no changes in protein expression. Vasoconstriction response to U46619 was attenuated in obacunone-treated aortic vessels compared to that in untreated vessels. Endothelium-dependent vasorelaxant response to acetylcholine was significantly increased in obacunone-treated vessels and was modulated by the NO-dependent signaling cascade. The dose-dependent vasorelaxant response to Ach was reduced in the aortic vessels of ApoE^?/? mice fed a high-cholesterol diet. Obacunone incubation increased vasorelaxation to the level of a WT mouse, although the endothelium-independent response to sodium nitroprusside was identical among the groups. Therefore, obacunone may help treat cardiovascular diseases derived from endothelial dysfunction and may be useful for designing pharmaceutical compounds.     

Antihypertensive methyldopa,labetalol, hydralazine,and clonidine reversed tumour necrosis factor‐α inhibited endothelial nitric oxide synthase expression in endothelial‐trophoblast cellular networks

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor‐α (TNF‐α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre‐incubated with (or without) low dose TNF‐α (0.5 ng/mL) or TNF‐α plus soluble fms‐like tyrosine kinase‐1 (sFlt‐1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR‐8/SVneo cells were co‐cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF‐α on eNOS mRNA expression. After pre‐incubating endothelial cells with TNF‐α and sFlt‐1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF‐α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF‐α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF‐α. The anti‐angiogenic molecule sFlt‐1 may antagonise the potential benefit of these medications by interfering with the NOS pathway.     

Exercise-induced modulation of endothelial nitric oxide production

In the arterial wall nitric oxide (NO) is the key transmitter for endothelium-dependent regulation of vascular tone. It is produced in intact endothelial cells by endothelial NO synthase (eNOS) as the key enzyme from L-arginine. Endothelial NO generation is highly regulated by mechanical, humoral, and metabolic factors. The regulation of NO synthesis occurs at different levels: ENOS gene polymorphisms are related to eNOS expression and activity and may potentially increase coronary event rate, mRNA expression is influenced by estrogen status and shear stress, mRNA stability is enhanced by vascular endothelial growth factor (VEGF), and final enzyme activity is regulated by the phosphorylation status at serine/threonine residues. Released from endothelial cells NO is rapidly transported to the neighboring vascular smooth muscle cells (VSMCs), where it induces the production of cGMP as a second messenger. CGMP in turn increases Ca2+ uptake into intracellular calcium stores thereby lowering [Ca2+]i and inducing VSMC relaxation and vasodilation. On its way to the VSMCs NO may be prematurely degraded by reactive oxygen species. On the other hand, chronic endurance exercise with regular bouts of increased laminar flow along the endothelium has the potential to increase eNOS mRNA expression and phosphorylation via AKT (protein kinase B) and to reduce oxidative stress by improving antioxidative protection. The growing knowledge about the complex regulation of NO synthesis and degradation in cardiovascular diseases and its response to exercise has led to a new understanding of the protective effects of long-term habitual physical activity against atherosclerotic heart disease and vascular aging.     

Lack of involvement of extracellular signal-regulated kinase (ERK) in the agonist-induced endothelial nitric oxide synthesis

In a recent paper, it was shown that stimulation of endothelial cells with bradykinin (BK) leads to phosphorylation of endothelial nitric oxide synthase (eNOS) mediated by extracellular signal-regulated kinase (ERK) (J. Biol. Chem. 275 (2000) 30707). Since in vitro phosphorylation by ERK reduced the catalytic activity of eNOS, it was suggested that this mechanism may be an important determinant of nitric oxide signalling in endothelial cells. To explore the physiological role of ERK as regulator of nitric oxide synthesis in intact cells, we measured the effects of the kinase inhibitor PD 98059 on BK- and ATP-induced nitric oxide formation in cultured endothelial cells and isolated vascular smooth muscle strips. PD 98059 completely inhibited ERK activation by BK and ATP in porcine aortic endothelial cells without affecting eNOS activation. Moreover, PD 98059 did not potentiate relaxation of isolated porcine pulmonary arteries to BK or ATP, indicating that ERK-catalysed eNOS phosphorylation does not contribute to the regulation of nitric oxide formation in intact cells or tissues.     

普伐他汀对人内皮祖细胞一氧化氮合成的影响

目的观察普伐他汀对人内皮祖细胞（EPCs）一氧化氮（NO）合成的影响。方法密度梯度离心法获取外周血单个核细胞,培养7d后,收集贴壁细胞并分别加入普伐他汀,10μmol/L及100μmol/L干预48h,免疫组化、荧光显微镜和流式细胞仪鉴定EPC,用RT-PCR方法测定对细胞内皮型一氧化氮合酶（eNOS）mRNA表达的影响,并用硝酸还原酶法测定培养液中一氧化氮（NO）的水平。结果普伐他汀组的人内皮祖细胞eNOS mRNA的表达、NO的合成明显增加。结论普伐他汀可增加人内皮祖细胞eNOS mRNA的表达和NO的合成