Relative contribution of different l-arginine sources to the substrate supply of endothelial nitric oxide synthase

In certain cases of endothelial dysfunction l-arginine becomes rate-limiting for NO synthesis in spite of sufficiently high plasma concentrations of the amino acid. To better understand this phenomenon, we investigated routes of substrate supply to endothelial nitric oxide synthase (eNOS). Our previous data with human umbilical vein (HUVEC) and EA.hy.926 endothelial cells demonstrated that eNOS can obtain its substrate from the conversion of l-citrulline to l-arginine and from protein breakdown. In the present study, we determined the quantitative contribution of proteasomal and lysosomal protein degradation and investigated to what extent extracellular peptides and l-citrulline can provide substrate to eNOS. The RFL-6 reporter cell assay was used to measure eNOS activity in human EA.hy926 endothelial cells. Individual proteasome and lysosome inhibition reduced eNOS activity in EA.hy926 cells only slightly. However, the combined inhibition had a pronounced reducing effect. eNOS activity was fully restored by supplementing either l-citrulline or l-arginine-containing dipeptides. Histidine prevented the restoration of eNOS activity by the dipeptide, suggesting that a transporter accepting both, peptides and histidine, mediates the uptake of the extracellular peptide. In fact, the peptide and histidine transporter PHT1 was expressed in EA.hy926 cells and HUVECs (qRT/PCR). Our study thus demonstrates that l-citrulline and l-arginine-containing peptides derived from either intracellular protein breakdown or from the extracellular space seem to be good substrate sources for eNOS.     

Protective effects of isorhamnetin on apoptosis and inflammation in TNF-α-induced HUVECs injury

Little is known about the role of isorhamnetin on endothelial cell apoptosis and inflammation when insulted by TNF-α injury. In our study, HUVECs were treated with TNF-α for 6 hours. HUVECs apoptosis were detected using flow cytometry. The expressions of ICAM-1, VCAM-1, E-selectin, NF-κB, AP-1 and eNOS were determined with western blotting or flow cytometry. The results showed TNF-α increased of apoptosis and the expression of ICAM-1, VCAM-1 and E-selectin in HUVECs, accompanied by significant augmentation of NF-κB and AP-1 expression. Pretreatment with isorhamnetin significantly reduced apoptosis in TNF-α-treated HUVECs. Moreover, isorhamnetin significantly attenuated TNF-α-induced upregulation of ICAM-1, VCAM-1, AP-1, E-selectin and NF-κB expression. Meanwhile, isorhamnetin also increased the expression of eNOS. So, isorhamnetin could suppress TNF-α-induced apoptosis and inflammation by blocking NF-κB and AP-1 signaling in HUVECs, which might be one of the underlying mechanisms for treatment of coronary heart disease.     

葛根素对心肌梗死大鼠心肌iNOS，eNOS蛋白表达及AKT磷酸化水平的影响

目的：探讨葛根素诱导血管生成的分子机制。方法：通过大鼠心肌梗死动物模型，运用Western-blot的方法检测心肌iNOS、eNOS，AKT，p-AKT的蛋白表达。结果：葛根素120mg／kg腹腔注射四周能不同程度增加心肌梗死大鼠缺血区域和非缺血区域心肌eNOS蛋白表达以及AKT磷酸化水平，而对iNOS蛋白表达没有明显的影响。结论：葛根素诱导血管生成作用的分子机制与其促进AKT磷酸化水平，从而激活eNOS，使NO生成增加有关。     

缺氧复氧损伤对血管内皮细胞一氧化氮合成酶基因表达的影响及辛伐他汀的干预作用

目的探讨缺氧、复氧损伤对血管内皮细胞一氧化氮合成酶基因表达的影响及辛伐他汀的干预作用,并探讨其可能的机制。方法体外培养人脐静脉内皮细胞株ECV304,使用自制的缺氧小室对ECV304进行缺氧、复氧处理。第1组:仅进行缺氧、复氧处理。第2组:不同浓度的辛伐他汀(0.1、1.0、10.0μmol/L)及10.0μmol/L辛伐他汀 0.2mmol/L甲羟戊酸预处理ECV304,再行缺氧2h、复氧2h处理。RT-PCR分别检测内皮细胞eNOS、iNOS基因的相对表达量。结果缺氧2h、复氧2hECV304eNOS相对表达量明显减少(均P≤0.01);缺氧2hiNOS相对表达量明显减少(P≤0.01),复氧2、4、8hiNOS相对表达量明显增加(均P<0.05);辛伐他汀(0.1、1.0、10.0μmol/L)预处理后再给予缺氧、复氧刺激则均使eNOS相对表达量增加(均P<0.05),并呈浓度依赖增加,同时iN-OS相对表达量减少;同时用辛伐他汀和甲羟戊酸预处理后则以上辛伐他汀的作用消失。结论缺氧、复氧应激下血管内皮细胞eNOS表达减少,iNOS表达增加;辛伐他汀可以阻止因缺氧、复氧刺激导致的eNOS低表达和iNOS高表达,该作用可被甲羟戊酸逆转。     

eNOS genotype is without effect on circulating nitrite/nitrate level in healthy male population

INTRODUCTION: Nitric oxide (NO) plays an important role in the regulation of the cardiovascular system. It is produced by endothelial nitric oxide synthase (eNOS), which exhibits genetic polymorphisms. Although the clinically relevant polymorphism T(+IhI-786)C reduces eNOS-promoter activity, it is not clear whether circulating nitrite/nitrate (NOx) are affected by this polymorphism. MATERIALS AND METHODS: We addressed this issue by studying a homogeneous group of 200 healthy subjects (males, Caucasians, nonsmokers, 18+IBM-56 years of age, and not taking any medication). Genotypes were determined by restriction fragment length polymorphism and circulating NOx were determined by chemiluminescence. RESULTS: We found nonsignificant effects of the T(+IhI-786)C polymorphism on circulating NOx (mean+ALE-S.D.=52.2+ALE-21.4, 49.0+ALE-17.8, and 45.9+ALE-16.8 +A7w-mol/L for genotypes +IBw-TT,+IB0 +IBw-TC,+IB0 and +IBw-CC,+IB0 respectively) and on total plasma cholesterol concentrations (both P>.05). No correlation was found between circulating NOx and total plasma cholesterol concentrations (P>.05). CONCLUSIONS: Our study provides strong evidence that the T(+IhI-786)C polymorphism does not affect plasma NOx concentrations, which are believed to reflect endogenous production of NO. Therefore, our results suggest that this polymorphism does not affect endogenous NO production.     

Endothelium-dependent and -independent relaxation and VASP serines 157/239 phosphorylation by cyclic nucleotide-elevating vasodilators in rat aorta

Endothelium-dependent vasodilation is thought to be mediated primarily by the NO/cGMP signaling pathway whereas cAMP-elevating vasodilators are considered to act independent of the endothelial cell layer. However, recent functional data suggest that cAMP-elevating vasodilators such as beta-receptor agonists, adenosine or forskolin may also be endothelium-dependent. Here we used functional and biochemical assays to analyze endothelium-dependent, cGMP- and cAMP-mediated signaling in rat aorta. Acetylcholine and sodium nitroprusside (SNP) induced a concentration-dependent relaxation of phenylephrine-precontracted aorta. This response was reflected by the phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), a validated substrate of cGMP- and cAMP-dependent protein kinases (cGK, cAK), on Ser(157) and Ser(239). As expected, the effects of acetylcholine were endothelium-dependent. However, relaxation induced by the beta-receptor agonist isoproterenol was also almost completely impaired after endothelial denudation. At the biochemical level, acetylcholine- and isoproterenol-evoked cGK and cAK activation, respectively, as measured by VASP Ser(239) and Ser(157) phosphorylation, was strongly diminished. Furthermore, the effects of isoproterenol were repressed by eNOS inhibition when endothelium was present. We also observed that the relaxing and biochemical effects of forskolin were at least partially endothelium-dependent. We conclude that cAMP-elevating vasodilators, i.e. isoproterenol and to a lesser extent also forskolin, induce vasodilation and concomitant cyclic nucleotide protein kinase activation in the vessel wall in an endothelium-dependent way.     

Akt基因治疗大鼠肝硬化门静脉高压症

目的:探讨肝硬化时肝组织内Akt和eNOS的活化是否受到抑制以及腺病毒介导的Akt基因治疗门静脉高压症的可行性.方法:以细胞内同源重组法构建复制缺陷型重组腺病毒Ad-myr-HA-Akt和Ad-EGFP.采用四氯化碳复合法制备肝硬化门静脉高压症大鼠模型.取10只正常大鼠作为对照,另取40只肝硬化大鼠随机均分为4组:未处理组、Akt治疗组、EGFP组和生理盐水组.后3组分别经尾静脉分别注射Akt重组腺病毒、Ad-EGFP和生理盐水后,3 d后,分别测定各组的门静脉压力、平均动脉压和心率.用免疫印迹法检测各组大鼠肝组织内Akt,p-Akt,eNOS,p-eNOS蛋白的表达;硝酸还原酶法检测各组肝内NO含量.EGFP组于转染后3 d处死大鼠,取肝、心、肺、肾、脑、脾和睾丸,快速冰冻切片,观察绿色荧光蛋白的表达情况.结果:Ad-myr-HA-Akt和Ad-EGFP经纯化后滴度分剐为5.5×1011vp/mL和6.0×1011 vp/mL.肝硬化大鼠肝内Akt和eNOS的活化均受到抑制,肝内NO生成减少,门静脉压力升高.Akt重组腺病毒治疗能使受到抑制的Akt和eNOS磷酸化得到恢复,同时增加肝内NO含量,降低门静脉压力.而经Ad-EGFP和生理盐水处理组,Akt和eNOS磷酸化不能恢复,肝内NO含量和门静脉压力与未经治疗大鼠相似.EGFP组在Ad-EGFP转染后3 d肝组织中可见大量绿色荧光,肺和肾组织中仅见少量荧光,而其他实质器官未见EGFP表达.结论:肝硬化时,大鼠肝内Akt和eNOS的活化均受到抑制,导致NO生成减少而肝内血管阻力增加,表现为门静脉压力升高.以腺病毒介导的Akt基因治疗门静脉高压症可行.     

褪黑素对胰岛素抵抗大鼠肾皮质内皮素、内皮型一氧化氮合酶平衡的影响

目的 研究褪黑素对胰岛素抵抗(IR)大鼠肾皮质内皮素1(ET-1)、内皮型一氧化氮合酶(eNOS)表达的影响.方法 高糖饲料喂养SD大鼠6周复制IR大鼠模型.成模后用药组(MEL组)予褪黑素10mg·kg-1·d-1灌胃,用药6周.未用药IR大鼠模型为对照(IR组).免疫组化、RT-PCR方法 检测肾皮质ET-1、eNOS蛋白和mRNA表达的改变.结果 与IR组相比,褪黑素组大鼠动脉血压(ABP)、血清TG、游离脂肪酸(FFA)、丙二醛(MDA)、胰岛素(Ins)和HOMA-IR降低(P＜0.01),血清HDL-c、超氧化物歧化酶(SOD)升高(P＜0.01).HE染色MEL组大鼠肾皮质病变明显减轻.MEL组ET-1蛋白及mRNA明显低于IR组、eNOS蛋白及mRNA明显高于IR组,差别均有显著意义(P＜0.05).结论 在IR早期应用褪黑素(10mg·kg-1·d-1)治疗可以减轻IR大鼠肾皮质ET-1与eNOS平衡的异常,对肾皮质血管内皮功能具有一定保护作用.     

阿托伐他汀与螺内酯对腹膜纤维化的作用研究

目的探讨腹膜纤维化的药物防治措施。方法50只Wister雄性大鼠随机分成5组,每组10只。A组为对照组,每日腹腔内注入0.9%生理盐水20ml。B、C、D、E组腹腔内每日注入20ml4.25%百特透析液,并于试验第7,14,21,28天分别加入乳酸盐红霉素6.25万IU;C组螺内酯100mg/（Kg·d）灌胃;D组阿托伐他汀20mg/（Kg·d）灌胃;E组两药合用。于第1、第30天测定腹膜透析液中的转化生长因子（TGF-β1）,30天后收集大鼠腹膜作常规病理（HE染色.Masson染色）,行腹膜平衡试验（PET）,免疫组化检测eNOS表达和血管生成情况。结果C、D、E组的腹膜纤维化程度比B组轻（P〈0.05）;TGF-β1）浓度明显下降（P〈0.05）;各组腹膜间皮细胞均有eNOS表达,B、C、D、E组均有新生毛细血管表达,C、D、E组较B组表达下调（P〈0.05）。结论阿托伐他汀及螺内酯能有效的防治腹膜纤维化进程。     

Recipient hypertonic saline infusion prevents cardiac allograft dysfunction

Objective

Hypertonic saline (HTS) has potent immune and vascular effects. We assessed recipient pretreatment with HTS on allograft function in a porcine model of heart transplantation and hypothesized that HTS infusion would limit endothelial and left ventricular (LV) dysfunction following transplantation.