奈比洛尔舒张血管及保护自发性高血压大鼠血管内皮的作用
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摘要
研究背景：奈比洛尔是一个高度选择性β1受体阻断剂,具有独特的舒血管作用。前期研究表明,奈比洛尔的舒血管作用具有一氧化氮(NO)依赖性,但其具体信号转导途径并不清楚。PI3K/Akt/eNOS是细胞内重要的信号转导通路。Akt可磷酸化内皮型一氧化氮合酶(eNOS),促进NO释放。同时,血管内皮NO的释放还受不同亚型K+通道的调节。PI3K/Akt/eNOS信号传导通路或K+通道是否参与奈比洛尔的舒血管作用还不得而知。另外,实验显示,奈比洛尔促进内皮NO释放可能有血管差异。
目的：本研究拟通过离体血管张力实验,观察奈比洛尔对Wistar大鼠不同血管(主动脉、颈动脉、肾动脉、肠系膜动脉和股动脉)舒张作用的差异；观察NOS抑制剂,不同亚型K+通道阻断剂和PI3K/Akt阻断剂对奈比洛尔舒张作用的影响,进一步探讨奈比洛尔舒血管的机制。
方法：成年雄性Wistar大鼠,220-250g,击头致昏,立即取主动脉、颈动脉、肾、肠系膜和股动脉,置于O2饱和的4℃生理盐溶液(physiological salt solution, PSS)中。主动脉和颈动脉剔除脂肪及周围结缔组织后,剪成3-4mm的血管环。血管环用两根不锈钢微型挂钩贯穿血管管腔,水平横向悬挂在浴管内,下方固定,上方以一细钢丝连于张力换能器(PowerLab),经PowerLab计算机生物信号采集分析系统记录血管的舒缩变化。浴管内含有通以100%O2、37℃的PSS 10ml。为了使标本处于长度-张力曲线的最适点开始收缩,主动脉和颈动脉环悬挂在浴管后,立即分别给予2.0g和1.0g的前负荷,温育2h。在温育期间不断调整张力,使之维持稳定,每20min更换一次新鲜的PSS。所有动脉环用60 mmol/L KCl多次刺激,当标本对刺激稳定时,即相邻连续两次同样的刺激所引起的收缩幅度差别<10%；且在此基础上10-5mol／L乙酰胆碱(acetylcholine, Ach)所引起的舒张幅度≥30%时,认为血管环组织结构完整,功能正常,开始正式实验,观察药物干预的作用。
用大头针固定肠系膜动脉,肾动脉和股动脉主干和周围组织,去除动脉周围的脂肪组织,选取动脉的三级分支血管,剪取2mm左右的血管环。将两根直径为40μm的钨丝穿入管腔,固定血管环在Multi Myograph System-610M浴槽内传感器上。浴槽内含有5ml PSS,持续通以100%O2,温度控制在37℃。为了使血管环处于最佳反应状态,调整肠系膜动脉、肾动脉和股动脉的跨壁压,使其分别保持在相当于100mmHg、80mmHg和100mmHg的基础压力状态,平衡60min,每20min更换新鲜的37℃PSS液。血管环的张力变化通过DMT换能系统采集,并用Chart 5.3生物信号分析处理软件记录。为了检测血管环在离体状态下对血管活性物质的反应性,在开始正式实验之前,用60 mmol/L KC1预收缩血管。当收缩达稳定的平台时,用Ach(10-5mol/L)舒张,连续二次。当相邻两次刺激的收缩幅度差别不大于10%时；在此基础上当Ach所引起的舒张幅度≥20%,认为血管环组织结构完整,功能正常,开始正式实验。
用60 mmol/L KCl或苯肾上腺素(phenylephrine, Phe)(主动脉和颈动脉用10-6mol／L,肾动脉、肠系膜动脉和股动脉用10-5mol／L)预收缩不同动脉。收缩达平台后,累计加入奈比洛尔(10-7-10-5 mol／L),观察其舒张作用,建立奈比洛尔的累积对数浓度—反应曲线(Logarithm Cumulative Concentration Response Curve, LCCR)。PSS液冲洗3次,使血管张力达原来基线水平。稳定60min后,再次用60 mmol/L KC1或Phe收缩动脉。收缩达平台后,分别在浴管中加入不同亚型的K+通道阻断剂iberiotoxin (100nmol/L)、格列本脲(glibenclamide,0.1 mmol/L)、4-氨基吡啶(4-aminopyridine,1 mmol/L)和氯化钡(BaCl2, lmmol/L); PI3K阻断剂Wortmannin (5×10-7 mol/L); Akt阻断剂(lL-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate,10-5 mol/L);NOS阻断剂左旋硝基精氨酸甲酯(L-NAME,100μmol/L)。当血管活动再次达到平台后,在浴管中累计加入奈比洛尔(10-7-10-5mol／L)。以60 mmol/L KC1或Phe引起的张力升高为100%,评价NOS阻断剂,K+通道阻断剂和PI3K/Akt阻断剂对奈比洛尔的舒血管效应的影响。
结果：奈比洛尔可浓度依赖性地舒张Phe或KCl收缩的大鼠不同动脉,其最大舒张效应(Emax)因收缩剂不同而有差异。Phe收缩血管时,奈比洛尔Emax顺序为：肠系膜动脉(96.7±12.8%)≈主动脉(94.2±5.4%)>股动脉(84.84±12.1%)≈肾动脉(80.3±3.4%)>颈动脉(74.9±10.1%)。KCl收缩血管时,奈比洛尔对肠系膜动脉的舒张作用最大(90.5±15.2%),其余动脉Emax无显著性差异：股动脉(75.7±17.7%),主动脉(73.1±11.3%),肾动脉(71.6±6.2%),颈动脉(70.9±7.1%)。奈比洛尔对不同动脉的舒张作用均可以被eNOS抑制剂L-NAME (100μmol/L)抑制,但不受P13K抑制剂wortmannin (5×10-7 mol/L)和选择性Akt抑制剂(lL-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate,10-5 mol/L)的影响。同样,K+通道阻断剂：iberiotoxin (100 nmol/L),格列本脲(0.1 mmol/L),4-氨基吡啶(1mmol/L), BaCl2 (1 mmol/L)对奈比洛尔的舒血管作用也无显著性影响。
结论：奈比洛尔可浓度依赖性地舒张Phe或KCl收缩的大鼠不同动脉。无论是K+通道还是PI3K/Akt/eNOS信号通路均不参与奈比洛尔对大鼠主动脉、颈动脉、肾动脉、肠系膜动脉和股动脉的舒张作用。
研究背景：高血压是严重威胁人类健康和生命的疾病。实验证实,高血压患者和高血压动物模型存在血管内皮功能减退,后者又加速了高血压靶器官的损害。血管内皮功能是“内皮-高血压-心血管事件”链的始动因子和载体。血管内皮功能不全可表现为血管舒缩失衡,血管内皮细胞分泌的活性物质改变,内皮结构损伤,血管重构等方面。逆转高血压内皮功能不全已经成为降压之外的另一治疗靶点。β受体阻断剂在高血压治疗中有广泛的应用。奈比洛尔是高度选择性β1受体阻滞剂,既有β1受体阻滞作用,又能增加一氧化氮(NO)生物利用度,扩张血管,没有一般β受体阻断剂代谢方面的缺点,是当前较佳β受体阻断剂。奈比洛尔可改善自发性高血压大鼠(SHR)主动脉的血管反应性,增加其血浆中NO的含量。但是,有关奈比洛尔对SHR血管重构、内皮损伤、血管中活性物质水平、小动脉血管反应性等影响的研究比较少。
目的：本研究拟通过观察奈比洛尔对SHR主动脉和肾小叶间动脉重构、血管内皮损伤、血管活性物质水平及不同动脉血管反应性的影响,进一步探讨奈比洛尔对SHR血管内皮的保护作用。
方法：SHR和同源正常血压大鼠Wister-Kyoto(WKY),随机分成4组：(1)SHR+Nebivolol组(n=6)：奈比洛尔(8mg/kg/day, i.g.); (2) SHR+Atenolol组(n=6)：阿替洛尔(80mg/kg/day, i.g.); (3) SHR空白对照组(n=6)；(4)WKY空白对照组(n=6)。给药过程中,每周在同一时间测量各组大鼠的体重及尾动脉收缩压。给药8周后,用3%戊巴比妥钠(30 mg/kg, i.p.)麻醉大鼠。腹主动脉采血后,迅速取主动脉、颈动脉、肾、肠系膜动脉和股动脉。肾脏及一部分主动脉用4%多聚甲醛固定,用于HE染色,进行形态学观察和血管内径(luminal radius, L),中膜厚度(media thicknes, M),中膜／内径(M／L)的测量；一部分主动脉切片经免疫组织化学染色分析,观察主动脉内皮细胞八因子相关抗原(FⅧ)表达。一部分主动脉制成10%组织匀浆,与血浆离心后,测定主动脉和血浆NO(硝酸还原酶法),血管紧张素Ⅱ(AngⅡ)和内皮素(ET-1)(放射免疫法)和血浆vWF(ELISA)水平。另一部分主动脉和其他动脉(颈动脉、肾动脉、肠系膜动脉、股动脉)用于离体血管张力实验,观察SHR不同动脉对Phe、KCl、ET-1、AngⅡ的收缩反应及对Ach和硝普钠(SNP)的舒张反应性。
结果：
(1)实验期间,SHR体重及收缩压均明显高于同龄WKY。奈比洛尔和阿替洛尔对SHR大鼠体重无影响。与阿替洛尔比较,奈比洛尔可明显降低SHR收缩压,此降压作用在给药后4周最显著。
(2)主动脉形态学观察结果：各组大鼠主动脉L无显著性差异,但SHR主动脉M,M／L较WKY显著增高。奈比洛尔可降低SHR主动脉M和M／L,但阿替洛尔对此无影响。
(3)肾小叶间动脉形态学观察结果：与WKY比较,SHR肾小叶间动脉L减小,M与M／L增高。奈比洛尔治疗后,SHR肾小叶间动脉L增加,M和M／L显著降低,但阿替洛尔对此无影响。
(4)主动脉内皮FⅧ免疫组织化学：SHR主动脉FⅧ阳性表达较WKY弱,棕色内皮层不完整。奈比洛尔给药后,SHR主动脉内皮细胞FⅧ阳性表达增强,棕色层显色较清晰,内皮完整。阿替洛尔给药后对SHR主动脉FⅧ表达无影响。
(5)血浆vWF含量：与WKY组比较,SHRⅧ浆vWF水平显著升高。奈比洛尔可降低SHR血浆vWF,而阿替洛尔对SHR血浆vWF水平无显著性影响。
(6)血浆及主动脉NO、ET-1、NO/ET-1:与WKY比较,SHR血浆和主动脉NO含量降低；主动脉ET-1含量增加,但是血浆ET-1水平在两组间无明显改变。而且,SHR主动脉和血浆NO/ET-1较WKY显著降低。奈比洛尔可增加SHR血浆及主动脉NO含量和NO／ET-1,但对血浆和主动脉ET-1含量无影响。阿替洛尔对SHR血浆及主动脉NO、ET-1、NO/ET-1无显著影响。
(7)血浆及主动脉AngⅡ、NO/AngⅡ:SHR血浆和主动脉AngⅡ水平较WKY显著增加,NO/AngⅡ降低。奈比洛尔对血浆AngⅡ含量无影响,但可降低主动脉AngⅡ含量,使血浆和主动脉NO/AngⅡ增加。阿替洛尔对血浆及主动脉AngⅡ、NO/AngⅡ无显著影响。
(8)不同动脉血管反应性：
SHR主动脉、颈动脉、肾动脉、肠系膜动脉和股动脉对Phe(10-8-10-5mol／L)、KCl(20-120m mol/L);颈动脉对ET-1 (10-11-10-8mol/L)、AngⅡ(10-9-10-5mol/L)及主动脉对AngⅡ的收缩反应显著高于WKY；不同动脉对Ach (10-10-10-5 mol/L)的舒张效应则弱于WKY,而对SNP (10-10-10-4 mol/L)的舒张反应在两组无显著性差异。
奈比洛尔可以降低SHR不同动脉对四种收缩剂的反应,其程度因血管不同而异：对KCl收缩的抑制作用在颈动脉和股动脉尤为明显；对Phe的抑制在主动脉、肾动脉、股动脉显著；对ET-1和AngⅡ的抑制在主动脉显著。同时,奈比洛尔可增强SHR不同动脉对Ach的舒张反应,尤其在肾动脉；但是奈比洛尔对不同动脉SNP的舒张无影响。
阿替洛尔对SHR不同动脉对四种收缩剂收缩反应的抑制作用有血管差异。阿替洛尔可抑制除肾动脉和肠系膜动脉外其余动脉对KCl的收缩；抑制除肠系膜动脉外其余动脉对Phe的收缩；减少主动脉和颈动脉对ET-1, AngⅡ的收缩。阿替洛尔可增强SHR肾动脉对Ach的舒张,对SNP的舒张无影响。
结论：奈比洛尔在显著降压的同时,可改善SHR血管重构,减少主动脉内皮损伤,增加SHR血浆和主动脉舒血管物质水平,降低缩血管物质含量,改善不同动脉的血管反应性,重建血管舒缩平衡。
研究背景：血管内皮功能不全在心血管疾病的发生发展中起重要作用,有效预防内皮功能受损可延缓乃至逆转相关病变进程。一氧化氮(nitric oxide, NO)在内皮功能调节中发挥重要作用,被认为是维持血管结构和功能的主要因子。内源性一氧化氮合酶(NOS)的抑制剂非对称性二甲基精氨酸(asymmetric dimethylarginine, ADMA)竞争性抑制NOS活性,减少NO生成。大量资料显示,ADMA水平的增高与多种病理状况下血管内皮功能不全有显著的相关性,可作为一个新的内皮功能不全预测因子。通过降低ADMA从而改善血管内皮功能为心血管疾病防治提供了新途径。我们第二部分的研究显示,奈比洛尔可以保护SHR血管内皮功能。但有关奈比洛尔血管内皮保护作用与ADMA系统关系的研究较少。
研究目的：本实验拟从组织和细胞水平探讨奈比洛尔对内皮细胞的保护作用与ADMA系统的关系。
研究方法：
1、奈比洛尔对SHR ADMA系统的影响。SHR大鼠及WKY大鼠随机分成4组：(1)SHR+Nebivolol组(n=6)：给予奈比洛尔8 mg/kg/day; (2) SHR+Atenolol组(n=6)：给予阿替洛尔80mg/kg/day; (3) SHR空白对照组(n=6)；(4)WKY空白对照组(n=6)。奈比洛尔,阿替洛尔溶于蒸馏水中灌胃,对照组给予等体积蒸馏水灌胃。给药8周后,3%戊巴比妥钠(30mg/kg, i.p.)麻醉大鼠,腹主动脉取血后,迅速分离主动脉,肠系膜动脉,将其置于液氮中冷冻后-70℃保存。离心分离血浆,测ADMA (ELISA法)和NO(硝酸还原酶法)含量,NOS活性(化学法)。提取主动脉和肠系膜动脉总RNA,用RT-PCR分析eNOS,DDAH-2和PRMT-1 mRNA表达。提取主动脉和肠系膜动脉总蛋白,用Western bloting分析DDAH-2和PRMT-1蛋白表达。采用冰冻切片荧光染色行主动脉原位活性氧(ROS)检测。
2、奈比洛尔对AngⅡ诱导损伤的人脐静脉内皮细胞(HUVECs)的影响。用AngⅡ1μmol/L培养HUVECs 24h,不加或加入阿替洛尔20μmol／L、奈比洛尔(5、10、20μmol/L)预处理1h。0.25%胰酶消化细胞,获取细胞悬液,离心收集细胞上清液,测ADMA (ELISA)和NO水平(硝酸还原酶法)水平,NOS活性(化学法)。提取HUVECs,总RNA,用RT-PCR分析eNOS, DDAH-2和PRMT-1 mRNA表达。提取HUVECs,总蛋白,用Western bloting分析DDAH-2和PRMT-1蛋白表达,同时检测细胞内DDAH活性。
3、奈比洛尔对ADMA损伤HUVECs的拮抗作用。用ADMA 16μmol/L培养HUVECs 24h,不加或加入阿替洛尔20μmol／L、奈比洛尔(5、10、20μmol/L)预处理1h。0.25%胰酶消化细胞,获取细胞悬液,离心收集细胞上清液,测NO水平(硝酸还原酶法)水平、NOS活性(化学法)。提取HUVECs,总RNA,用RT-PCR分析eNOS mRNA表达,同时测细胞ROS水平。
4、ADMA对离体大鼠主动脉血管反应性的影响及奈比洛尔对此的影响。成年雄性Wistar大鼠,击头致昏,快速分离主动脉。主动脉剔除脂肪及周围结缔组织后,剪成3-4mm的血管环,悬挂在浴管内,经PowerLab计算机生物信号采集分析系统记录血管的舒缩变化。观察ADMA (10-7-10-4 mol/L)对大鼠主动脉静息张力、Phe(10-6mol／L)预收缩主动脉张力、Ach(10-10-10-5mol／L)舒血管作用的影响及奈比洛尔(10-7,10-6和10-5mol／L)对此的影响。
结果：
1、与WKY比较,SHR血浆ADMA水平显著升高,NO水平和NOS活性明显下降。SHR主动脉和肠系膜动脉eNOS及DDAH-2 mRNA表达下降,PRMT-1 mRNA表达增加；DDAH-2蛋白表达下降,PRMT-1蛋白表达增加；主动脉原位ROS荧光强度增强。奈比洛尔可降低SHR血浆ADMA水平,增加NO含量和NOS活性,上调SHR主动脉和肠系膜动脉eNOS mRNA及DDAH-2 mRNA/蛋白表达,下调PRMT-1 mRNA/蛋白表达,降低SHR主动脉原位ROS荧光强度。阿替洛尔对SHR上述改变无影响。
2、AngⅡ1μmol/L培养HUVECs 24h可明显增加上清液ADMA水平,降低NO水平和NOS、DDAH活性,下调eNOS mRNA及DDAH-2 mRNA/蛋白表达,上调RMT-1 mRNA/蛋白表达。奈比洛尔(5、10、20μmol／L)可剂量依赖性抑制AngⅡ1μmol/L所致的上述改变。预先给予阿替洛尔20μmol／L则没有起到类似的作用。
3、ADMA 16μmol/L培养HUVECs 24h后,明显增加上清液ADMA水平,降低NO含量和NOS活性,升高细胞内ROS水平,下调eNOS mRNA表达。奈比洛尔(5、10、20、μmol/L)可剂量依赖性抑制ADMA 16μmol/L所致的上述效应。预先给予阿替洛尔20μmol/L则没有起到类似的作用。
4、离体血管张力实验表明,ADMA (10-7-10-4 mol/L)对离体大鼠主动脉静息张力无影响,但可剂量依赖性升高Phe(10-6 mol/L)预收缩大鼠主动脉的张力,抑制Ach(10-10-10-5mol／L)的舒张反应。奈比洛尔(10-7,10-6,10-5mol／L)可剂量依赖性抑制ADMA对Phe预收缩大鼠主动脉张力的升高,减少ADMA 10-5 mol/L对大鼠主动脉Ach舒张反应的抑制。
结论：奈比洛尔可通过下调PRMT-1表达,增加DDAH活性和表达,降低ADMA水平,保护内皮细胞。同时,奈比洛尔可拮抗ADMA导致的细胞损伤和血管收缩。
Background:Nebivolol is a highly selectiveβ1 adrenoceptor blocker with additional vasodilating properties. It has been shown that the nebivolol-induced vasorelaxation is nitric oxide (NO) dependent. However, the nature of the transduction pathway that mediates nebivolol's activation remains elusive. PI3K/Akt/eNOS is an important cell signal transduction pathway. The serine/tyrosine protein kinase Akt phosphorylates endothelial cell nitric oxide synthase (eNOS) and enhances the ability of eNOS to generate NO. Previous studies have shown that the release of NO from the endothelium may be ascribed to the modulation of different types of K+ channels. It has also been show that there was a difference in the NO release induced by nebivolol among different arteries.
Purpose:The present study was designed to determine the relaxation of nebivolol on different arteries (aorta, carotid artery, renal artery, mesenteric artery and femoral artery); to observe the effects of NOS inhibitor, K+ channels inhibitors or PI3K/Akt inhibitors on the vasorelaxation induced by nebivolol in different rat arteries.
Methods:Male Wistar rats (200-250g) were killed by cervical dislocation. Then thoracic aortas, carotid arteries, renal arteries, mesenteric arteries and femoral arteries were removed and placed in chilled (4℃) physiological salt solution (PSS) which was gassed with 100% O2. Aortas, carotid arteries were cleaned of connective tissue and fat, and cut into 3-4mm long rings. For the recording of tension the vessel rings were mounted, between 2 L-shaped stainless steel hooks, in 10ml organ baths filled with oxygenated PSS solution (bubbled with 100%O2 and maintained at 37℃) (pH 7.4). Each preparation was fixed, via a silk thread, to an isometric force transducer (A.D. Instruments, PowerLab, Australia) and force was recorded via PowerLab computer system (A.D. Instruments). Each vessel ring was subjected to an initial resting tension (aorta,2g; carotid artery, 1g). A 2h equilibration period was allowed before any experimental intervention, and during equilibration, the bath was flushed every 20 minutes with the fresh PSS. After equilibration, the rings were activated 2 times with 60mmol/L KCl. The integrity of endothelium was presumed by observation that the relaxation induced by 10-5mol/L acetylcholine (Ach) on 60 mmol/L KCl induced contraction was greater than 30%. When the contraction induced by 60 mmol/L KCl and the relaxation induced by 10-5 mol/L Ach were repeatable (the change was less than 10% between successive contractions or relaxation), the effects of the drugs to be tested were observed.
The femoral artery, the third-order branches of the superior mesenteric artery and renal artery were isolated and cut into 2mm-long rings. The rings were mounted in separate 5ml tissue baths filled with PSS solution (bubbled with 100% O2 and maintained at 37℃) (pH 7.4) using 40μm steel wire in a small-vessel myograph (Multi Myograph System-610M, Danish Myo Technology A/S, Denmark) for tension recordings. The rings were normalized according to standard procedures and stretched to a state equal to 100mmHg,80mmHg, 100mmHg, respectively. The rings were equilibrated for at least for 1h, and during equilibration, the bath was flushed every 20 minutes with the fresh PSS. After equilibration, the rings were activated 2 times with 60mmol/L KC1. The integrity of endothelium was presumed by observation that the relaxation induced by 10"5 mol/L Ach on contraction induced by 60 mmol/L KCl was greater than 20%. When the contraction induced by 60 mmol/L KCl and the relaxation induced by 10-5 mol/L Ach were repeatable (the change was less than 10% between successive contractions or relaxation), the effects of the drugs to be tested were observed.
Vessels were then washed extensively, and subsequently contracted with phenylephrine (Phe) (aorta and carotid artery were pre-contracted with 10"6 mol/L, mesenteric artery, femoral artery and renal artery were pre-contracted with 10"5 mol/L) or with 60 mmol/L KCl. Following attainment of plateau constriction in response to Phe or KCl, the logarithm cumulative concentration response curve (LCCRC) to nebivolol (10-7-10-5 mol/L) was performed. Then the vessels were contracted with Phe or 60mmol/L KCl again. Following attainment of plateau constriction, specific inhibitors of each type of K+channels:iberiotoxin (100 nmol/L), glibenclamide (0.1 mmol/L),4-aminopyridine (4-AP,1 mmol/L), BaCl2 (1 mmol/L), wortmannin (5×10"7 mol/L, a selective inhibitor of phosphatidylinositol 3-kinase (PI3K)), Akt inhibitor (lL-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate,10-5 mol/L, a selective inhibitor of Akt) and NG-nitro-L-arginine methyl ester (L-NAME,100μmol/L), an inhibitor of NO synthase were added to the bathing medium respectively. When the plateau constriction was attained, nebivolol (10-7-10'5 mol/L) was added cumulatively. So the effects of NOS inhibitor, K+channels inhibitors or PI3K/Akt inhibitors on the vasorelaxation induced by nebivolol in different rat arteries were studied.
Results:Nebivolol (10-7-10-5 mol/L) concentration-dependently relaxed pre-contractions induced by KC1 and PE in different rat arteries. The Emax of the relaxation of nebivolol was different according to the contractors. The rank order of vasodilator efficacy as measured in relation to the maximal vasodilation induced by nebivolol (10"7-10-5 mol/L) was:mesenteric artery (96.7±12.8%)≈aorta (94.2±5.4%)>femoral artery (84.8±12.1%)≈renal artery (80.3±3.4%)>carotid artery (74.9±10.1%) pre-contracted with Phe. In arteries contracted with 60mmol/L KCl, the maximal vasodilation was similar, expect in mesenteric artery (90.57±15.2%):femoral artery (75.7±17.7%), aorta (73.1±11.3%), renal artry (71.6±6.2%), carotid artery (70.9±7.1%). The vasodilator effect of nebivolol, in concentrations of 3 and 10μmol/L, was significantly blocked by 100μmol/L of the NOS inhibitor L-NAME in all arteries The exposure of the vessel rings to a selective inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin (5×10-7 mol/L) or a selective inhibitor of Akt (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate,10-5 mol/L) did not influence nebivolol-induced vasorelaxation. Similarly, K+channels blockers:iberiotoxin (100 nmol/L), glibenclamide (0.1 mmol/L),4-aminopyridine (1 mmol/L), or BaCl2(1 mmol/L) had no influence on the relaxation of nebivolol in arteries precontracted by Phe or KCl.
Conclusion:Nebivolol produced a concentration-dependent vasodilation in different rat arteries pre-contracted by Phe or KCl. In the isolated rat aorta, carotid artery, femoral artery, mesenteric artery and renal artery, neither K+channels nor PI3K/Akt/eNOS pathway was involved in the relaxation induced by nebivolol.
Background:Hypertension is a major risk factor for cardiovascular disease. Experiments show that there is endothelial dysfunction in animal and human hypertension, which in turn accelerates the damage of target organs. Endothelia dysfunction, which may manifest as vasomotor imbalance and changes in vaso-active substances and so on, is the initiator factors and carrier of "endothelial-high blood pressure-cardiovascular events" chain. Vascular endothelial damage enables the vascular remodeling. The remodeling can be an early manifestation of vascular structural changes and the basis for a number of cardiovascular events. It has been became a new target for hypertension treatment to improve the endothelia function. Nebivolol is a highly selectiveβ1-adrenergic receptor antagonist that can blockβ1-adrenergic receptor and promote endothelium-dependent vasodilation by increasing bioavailability of nitric oxide (NO). Compared with other traditionalβ-adrenergic receptor antagonists, nebivolol, without having the metabolic defects, is a bestβ-adrenergic receptor antagonist at present. Studies show that nebivolol can ameliorate the vascular reaction of aorta and improve the plasma NO concentration of SHR. But the data is few about the effect of nebivolol on the reaction of arteriole, the vaso-activesubstance in artery and the reconstruction of vascular.
Purpose:The aim of the study was to assess the protective effect of nebivolol on endothelial dysfunction in spontaneously hypertensive rats (SHR) by observing the reconstruction of arteries, vascular endothelial damage, levels of vaso-activesubstance and the vascular responses of different arteries.
Methods:SHR and age-matched Wister-Kyoto (WKY) rats were randomly divided into 4 groups:(1) SHR+Nebivolol group(n=6):SHR treated with nebivolol(8 mg/kg/day, i.g.); (2) SHR+Atenolol group (n=6):SHR treated with Atenolol (80 mg/kg/day, i.g.); (3) SHR control group (n=6):SHR treated with vehicle once daily, i.g.; (4) WKY control group (n=6):WKY treated with vehicle once daily, i.g. Therapy continued for 8 weeks, body weight and tail systolic blood pressure were measured weekly. At the end of this study, animals were anesthetized with 3% sodium pentobarbital (30 mg/kg, i.p.). Blood was draw by abdominal aortic puncture, then the aorta, carotid artery, renal, mesentery and femoral artery were moved rapidly. Renal and a part of aorta were fixed with 4% paraformaldehyde to observe the vascular morphological changes, including vessel lumen diameter (L), media thickness (M) and M/L. Expression of FVM in aorta was performed by immunohistochemistry analysis. Levels of NO (nitric acid reducdase assay), endothelin-1 (ET-1, radioimmunoassay), angiotensin II (AngⅡ, radioimmunoassay) in plasma, aorta and von Willebrand factor (vWF, ELISA) in plasma were measured. The contraction of different arteries (aorta, carotid artery, renal artery, femoral artery and mesentery artery) to Phe, KCl, ET-1, AngⅡand relaxation to acetylcholine (Ach) and sodium nitroprusside (SNP) were also studied in vitro.
Results:
(1) During the study, the body weight and systolic blood pressure were higher in SHR than in age-matched WKY. Nebivolol and atenolol had no effect on the body weight of SHR. Compared with atenolol, treatment with nebivolol induced an acute and significant reduction in systolic blood pressure; this reached a maximum in the fourth week.
(2) Morphometry study of aorta:There was no difference in L among different groups, but M and M/L increased in SHR than in WKY. Treatment with nebivolol reduced M and M/L in SHR while Atenolol had no effect.
(3) Morphometry study of interlobular artery in renal:The L decreased, but M and M/L of interlobular artery increased in SHR. Treatment with nebivolol raised L and lessened M and M/L in SHR, but atenolol had no effect.
(4) Thoracic aorta immunohistochemistry assay to identify FⅧin endothelia:The expression of FⅧwas weakly positive in SHR than in WKY, the brown endothelium was not intact and continuous. Treated with nebivolol, the expression of FⅧincreased and the brown endothelium was clear and intact, however atenolol had no effect.
(5) Level of vWF in plasma:The level of vWF increased in SHR than in WKY. Nebivolol reduced the level of vWF in SHR, but atenolol had no influence.
(6) Levels of NO, ET-1 and NO/ET-1 in plasma and aorta:The levels of NO, NO/ET-1 in plasma and aorta were lowered and ET-1 in aorta was increased in SHR than in WKY; but no difference was found in plasma ET-1 between SHR and WKY. Nebivolol increased the level of NO and NO/ET-1 in plasma and aorta, but had no influence on level of ET-1 in aorta and plasma. Atenolol had no effect on theses changes in SHR.
(7) Levels of Ang II and NO/AngⅡin plasma and aorta:The level of Ang II was increased and NO/Ang II was reduced in SHR plasma and aorta than in WKY. Nebivolol had no effect on Ang II in plasma, but reduced it in aorta. So the NO/Ang II in plasma and aorta were increased after nebivolol treatment. Atenolol had no effect on the changes in SHR.
(8) Vascular reaction of different arteries:
The contractions to Phe, KCl in different arteries; to ET-1 in carotid artery; to AngⅡin aorta and carotid artery were increased in SHR than in WKY. The relaxation to Ach decreased in SHR than in WKY, but there was no difference of relaxation to SNP among groups.
Nebivolol reduced the contraction of different arteries to Phe, KCl, AngⅡand ET-1 in SHR, but there was difference in the reduction based on the kind of arteries. It was marked to KCl in carotid artery and femoral artery, to Phe in aorta, renal artery, femoral artery, and to ET-1 and Ang II in aorta. Nebivolol increased the relaxation of different arteries to Ach, especially in renal artery, but had no effect on the relaxation of different arteries to SNP.
Atenolol just decreased the contraction of different arteries to KCl apart from renal artery and mesenteric artery; to Phe besides mesenteric artery; to ET-1, AngⅡin aorta and carotid artery. Atenolol increased the relaxation of renal artery to Ach in SHR, but had no effect on the relaxation to SNP.
Conclusion:The antihypertensive effect of nebivolol in SHR was accompany by protect on endothelial function:retarding vascular remodeling; reducing the injury of aorta endothelia cell; modulate the unbalance of vasoactive substances in aorta and plasma; improving the vascular reaction to different vasomotors, resuming the balance of relaxation and contraction.
Objectives:Endothelial dysfunction plays an important role in the development of cardiovascular diseases. Nitric oxideis is recognized as a major mediator in regulating vascular construction and function. Aasymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor that compatetively inhibits nitric oxide synthase (NOS) activity and reduces NO level, has been considered a new predictor of endothelial dysfunction. In a variety of pathophysiological conditions, plasm ADMA has been found rising significantly, and then may induce endothelial dysfunction. With the understanding of endothelial dysfunction and pathological roles of ADMA in cardiovascular disease, it may become a new therapeutic avuenues to improve endothelial function by reducing ADMA level. Nebivolol can protect vascular endothlia function. But data is few about the relationship of protective effect of nebivolol on vascular and ADMA.
Aim:To investigate the relationship of protective effect of nebivolol on vascular and ADMA.
Methods:
1. Effect of nebivolol on ADMA system of SHR. SHR and age-matched Wister-Kyoto (WKY) rats were randomly divided into 4 groups:(1) SHR+Nebivolol group (n=6):SHR treated with nebivolol(8 mg/kg/day, i.g.); (2) SHR+Atenolol group (n=6):SHR treated with Atenolol(80 mg/kg/day, i.g.); (3) SHR control group (n=6):SHR treated with vehicle once daily, i.g.; (4) WKY control group (n=6):WKY treated with vehicle once daily, i.g. After treatment for 8 weeks, rats were anesthetized with 3% sodium pentobarbital (30 mg/kg, i.p.). Blood was draw by abdominal aortic puncture, then the aorta, mesentery artery were moved rapidly and kept in-70℃. The levels of ADMA (ELISA), NO (nitric acid reducdase assay) and NOS activity (chemical method) in plasma were measured. The mRNA expression of eNOS, DDAH-2, PRMT-1 in aorta and mesenteric artery were measured by RT-PCR. The protein expression of DDAH-2 and PRMT-1 in aorta and mesenteric artery were measured by Western blot. ROS level in aorta were also measured.
2. Effect of nebivolol on HUVECs cultured with AngⅡ. HUVECs were cultured with Ang II 1μmol/L for 24h in the absence or presence of nebivolol (5,10 or 20μmol/L) or atenolol (20μmol/L) for 1 h. The supernaent in the conditioned medium was collected by centrifugation for determination of ADMA (ELISA), NO (nitric acid reducdase assay) and NOS activity (chemical method). The expression of eNOS, DDAH-2, PRMT-1 mRNA (RT-PCR), the protein expression of DDAH-2 and PRMT-1 (Western blot), activity of DDAH in HUVECs were also measured.
3. Effect of nebivolol on HUVECs cultured with ADMA. HUVECs were cultured with ADMA 16μmol/L for 24h in the absence or presence of nebivolol (5,10 or 20μmol/L) or atenolol (20μmol/L) for 1 h. The supernaent in the conditioned medium was collected by centrifugation for determination of NO and NOS activity. The expression of eNOS mRNA (RT-PCR), ROS in HUVECs were also measured.
4. Effect of nebivolol on influence of ADMA on vascular reponse of rat aorta. Male Wistar rats were killed by cervical dislocation. Then thoracic aortas were removed quickly. Aortas was cleaned of connective tissue and fat, and cut into 3-4mm long rings. Each preparation was fixed, via a silk thread, to an isometric force transducer and force was recorded via PowerLab computer-7 -4 system (A.D. Instruments). The effects of ADMA (10-10 mol/L) on aorta resting tone, aorta pre-contracted by Phe (10-6mol/L), relaxation induced by Ach (10-10-10-5 mol/L) and the effect of nebivolol (10-5,10-6,10-5mol/L) on the function of ADMA on rat aorta were observed in virto.
Rusults:
1. Compared with WKY, the level of plasma ADMA was elevated significantly, plsma NO, activity of NOS were decreased in SHR. In aorta and mesenteric artery of SHR, the mRNA expression of eNOS, mRNA/protein expression of DDAH-2 reduced, but mRNA/protein expression of PRMT-1 increased. ROS level in aorta also increased in SHR than in WKY. Nebivolol treatment increased plasma NO and activity of NOS, decreased ADMA leve. Nebivlolol up regulated mRNA expression of eNOS and mRNA/protein expression of DDAH-2; down regulated mRNA/protein expression of PRMT-1 in aorta and mesenteric artery. Nebivolol reduced ROS level of SHR aorta. Atenolol had no effect on the changes in SHR.
2. AngⅡ(1μmol/L) for 24h significantly decreased NO level and NOS activity, increased the mRNA/protein expression of PRMT-1, decreased DDAH activity, mRNA expression of eNOS and mRNA/expression of DDAH-2, resulting in elevation of ADMA level. Pretreatment with nebivolol (5,10 or 20μmol/L) concentration-dependently attenuated the above effects by AngⅡ. Atenolol (20μmol/L) had no influence on the effects induced by AngⅡ.
3. ADMA (16μmol/L) for 24h significantly markedly decreased NO level, NOS activity and eNOS mRNA; and increased intracellular ROS generation. Pretreatment with nebivolol (5,10 or 20μmol/L) concentration-dependently attenuated the above effects by ADMA. Atenolol (20μmol/L) had no influence on the effects induced by ADMA.
4. ADMA (10-7-10-4 mol/L) had no effect on the restiong tone of rat aorta, but ADMA concentration-dependently increased the tone of aorta pre-contracted by Phe (10 mol/L) and inhibited Ach (10-10-10-5 mol/L) induced relaxation in vitro. Nebivolol (10-5,10-6,10-5 mol/L) dose-dependently decreased the elevation of ADMA on pre-contracted rat aorta and the inhibition of ADMA 10-5 mol/L on relaxation induced by Ach.
Conclusion:Nebivolol reduced ADMA by increasing DDAH activity/expression and reducing PRMT-1 expression. Nebivolol also can inhibit cell injury and contraction induced by ADMA.
目的：本研究拟通过离体血管张力实验,观察奈比洛尔对Wistar大鼠不同血管(主动脉、颈动脉、肾动脉、肠系膜动脉和股动脉)舒张作用的差异；观察NOS抑制剂,不同亚型K+通道阻断剂和PI3K/Akt阻断剂对奈比洛尔舒张作用的影响,进一步探讨奈比洛尔舒血管的机制。
方法：成年雄性Wistar大鼠,220-250g,击头致昏,立即取主动脉、颈动脉、肾、肠系膜和股动脉,置于O2饱和的4℃生理盐溶液(physiological salt solution, PSS)中。主动脉和颈动脉剔除脂肪及周围结缔组织后,剪成3-4mm的血管环。血管环用两根不锈钢微型挂钩贯穿血管管腔,水平横向悬挂在浴管内,下方固定,上方以一细钢丝连于张力换能器(PowerLab),经PowerLab计算机生物信号采集分析系统记录血管的舒缩变化。浴管内含有通以100%O2、37℃的PSS 10ml。为了使标本处于长度-张力曲线的最适点开始收缩,主动脉和颈动脉环悬挂在浴管后,立即分别给予2.0g和1.0g的前负荷,温育2h。在温育期间不断调整张力,使之维持稳定,每20min更换一次新鲜的PSS。所有动脉环用60 mmol/L KCl多次刺激,当标本对刺激稳定时,即相邻连续两次同样的刺激所引起的收缩幅度差别<10%；且在此基础上10-5mol／L乙酰胆碱(acetylcholine, Ach)所引起的舒张幅度≥30%时,认为血管环组织结构完整,功能正常,开始正式实验,观察药物干预的作用。
用大头针固定肠系膜动脉,肾动脉和股动脉主干和周围组织,去除动脉周围的脂肪组织,选取动脉的三级分支血管,剪取2mm左右的血管环。将两根直径为40μm的钨丝穿入管腔,固定血管环在Multi Myograph System-610M浴槽内传感器上。浴槽内含有5ml PSS,持续通以100%O2,温度控制在37℃。为了使血管环处于最佳反应状态,调整肠系膜动脉、肾动脉和股动脉的跨壁压,使其分别保持在相当于100mmHg、80mmHg和100mmHg的基础压力状态,平衡60min,每20min更换新鲜的37℃PSS液。血管环的张力变化通过DMT换能系统采集,并用Chart 5.3生物信号分析处理软件记录。为了检测血管环在离体状态下对血管活性物质的反应性,在开始正式实验之前,用60 mmol/L KC1预收缩血管。当收缩达稳定的平台时,用Ach(10-5mol/L)舒张,连续二次。当相邻两次刺激的收缩幅度差别不大于10%时；在此基础上当Ach所引起的舒张幅度≥20%,认为血管环组织结构完整,功能正常,开始正式实验。
用60 mmol/L KCl或苯肾上腺素(phenylephrine, Phe)(主动脉和颈动脉用10-6mol／L,肾动脉、肠系膜动脉和股动脉用10-5mol／L)预收缩不同动脉。收缩达平台后,累计加入奈比洛尔(10-7-10-5 mol／L),观察其舒张作用,建立奈比洛尔的累积对数浓度—反应曲线(Logarithm Cumulative Concentration Response Curve, LCCR)。PSS液冲洗3次,使血管张力达原来基线水平。稳定60min后,再次用60 mmol/L KC1或Phe收缩动脉。收缩达平台后,分别在浴管中加入不同亚型的K+通道阻断剂iberiotoxin (100nmol/L)、格列本脲(glibenclamide,0.1 mmol/L)、4-氨基吡啶(4-aminopyridine,1 mmol/L)和氯化钡(BaCl2, lmmol/L); PI3K阻断剂Wortmannin (5×10-7 mol/L); Akt阻断剂(lL-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate,10-5 mol/L);NOS阻断剂左旋硝基精氨酸甲酯(L-NAME,100μmol/L)。当血管活动再次达到平台后,在浴管中累计加入奈比洛尔(10-7-10-5mol／L)。以60 mmol/L KC1或Phe引起的张力升高为100%,评价NOS阻断剂,K+通道阻断剂和PI3K/Akt阻断剂对奈比洛尔的舒血管效应的影响。
结果：奈比洛尔可浓度依赖性地舒张Phe或KCl收缩的大鼠不同动脉,其最大舒张效应(Emax)因收缩剂不同而有差异。Phe收缩血管时,奈比洛尔Emax顺序为：肠系膜动脉(96.7±12.8%)≈主动脉(94.2±5.4%)>股动脉(84.84±12.1%)≈肾动脉(80.3±3.4%)>颈动脉(74.9±10.1%)。KCl收缩血管时,奈比洛尔对肠系膜动脉的舒张作用最大(90.5±15.2%),其余动脉Emax无显著性差异：股动脉(75.7±17.7%),主动脉(73.1±11.3%),肾动脉(71.6±6.2%),颈动脉(70.9±7.1%)。奈比洛尔对不同动脉的舒张作用均可以被eNOS抑制剂L-NAME (100μmol/L)抑制,但不受P13K抑制剂wortmannin (5×10-7 mol/L)和选择性Akt抑制剂(lL-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate,10-5 mol/L)的影响。同样,K+通道阻断剂：iberiotoxin (100 nmol/L),格列本脲(0.1 mmol/L),4-氨基吡啶(1mmol/L), BaCl2 (1 mmol/L)对奈比洛尔的舒血管作用也无显著性影响。
结论：奈比洛尔可浓度依赖性地舒张Phe或KCl收缩的大鼠不同动脉。无论是K+通道还是PI3K/Akt/eNOS信号通路均不参与奈比洛尔对大鼠主动脉、颈动脉、肾动脉、肠系膜动脉和股动脉的舒张作用。
研究背景：高血压是严重威胁人类健康和生命的疾病。实验证实,高血压患者和高血压动物模型存在血管内皮功能减退,后者又加速了高血压靶器官的损害。血管内皮功能是“内皮-高血压-心血管事件”链的始动因子和载体。血管内皮功能不全可表现为血管舒缩失衡,血管内皮细胞分泌的活性物质改变,内皮结构损伤,血管重构等方面。逆转高血压内皮功能不全已经成为降压之外的另一治疗靶点。β受体阻断剂在高血压治疗中有广泛的应用。奈比洛尔是高度选择性β1受体阻滞剂,既有β1受体阻滞作用,又能增加一氧化氮(NO)生物利用度,扩张血管,没有一般β受体阻断剂代谢方面的缺点,是当前较佳β受体阻断剂。奈比洛尔可改善自发性高血压大鼠(SHR)主动脉的血管反应性,增加其血浆中NO的含量。但是,有关奈比洛尔对SHR血管重构、内皮损伤、血管中活性物质水平、小动脉血管反应性等影响的研究比较少。
目的：本研究拟通过观察奈比洛尔对SHR主动脉和肾小叶间动脉重构、血管内皮损伤、血管活性物质水平及不同动脉血管反应性的影响,进一步探讨奈比洛尔对SHR血管内皮的保护作用。
方法：SHR和同源正常血压大鼠Wister-Kyoto(WKY),随机分成4组：(1)SHR+Nebivolol组(n=6)：奈比洛尔(8mg/kg/day, i.g.); (2) SHR+Atenolol组(n=6)：阿替洛尔(80mg/kg/day, i.g.); (3) SHR空白对照组(n=6)；(4)WKY空白对照组(n=6)。给药过程中,每周在同一时间测量各组大鼠的体重及尾动脉收缩压。给药8周后,用3%戊巴比妥钠(30 mg/kg, i.p.)麻醉大鼠。腹主动脉采血后,迅速取主动脉、颈动脉、肾、肠系膜动脉和股动脉。肾脏及一部分主动脉用4%多聚甲醛固定,用于HE染色,进行形态学观察和血管内径(luminal radius, L),中膜厚度(media thicknes, M),中膜／内径(M／L)的测量；一部分主动脉切片经免疫组织化学染色分析,观察主动脉内皮细胞八因子相关抗原(FⅧ)表达。一部分主动脉制成10%组织匀浆,与血浆离心后,测定主动脉和血浆NO(硝酸还原酶法),血管紧张素Ⅱ(AngⅡ)和内皮素(ET-1)(放射免疫法)和血浆vWF(ELISA)水平。另一部分主动脉和其他动脉(颈动脉、肾动脉、肠系膜动脉、股动脉)用于离体血管张力实验,观察SHR不同动脉对Phe、KCl、ET-1、AngⅡ的收缩反应及对Ach和硝普钠(SNP)的舒张反应性。
结果：
(1)实验期间,SHR体重及收缩压均明显高于同龄WKY。奈比洛尔和阿替洛尔对SHR大鼠体重无影响。与阿替洛尔比较,奈比洛尔可明显降低SHR收缩压,此降压作用在给药后4周最显著。
(2)主动脉形态学观察结果：各组大鼠主动脉L无显著性差异,但SHR主动脉M,M／L较WKY显著增高。奈比洛尔可降低SHR主动脉M和M／L,但阿替洛尔对此无影响。
(3)肾小叶间动脉形态学观察结果：与WKY比较,SHR肾小叶间动脉L减小,M与M／L增高。奈比洛尔治疗后,SHR肾小叶间动脉L增加,M和M／L显著降低,但阿替洛尔对此无影响。
(4)主动脉内皮FⅧ免疫组织化学：SHR主动脉FⅧ阳性表达较WKY弱,棕色内皮层不完整。奈比洛尔给药后,SHR主动脉内皮细胞FⅧ阳性表达增强,棕色层显色较清晰,内皮完整。阿替洛尔给药后对SHR主动脉FⅧ表达无影响。
(5)血浆vWF含量：与WKY组比较,SHRⅧ浆vWF水平显著升高。奈比洛尔可降低SHR血浆vWF,而阿替洛尔对SHR血浆vWF水平无显著性影响。
(6)血浆及主动脉NO、ET-1、NO/ET-1:与WKY比较,SHR血浆和主动脉NO含量降低；主动脉ET-1含量增加,但是血浆ET-1水平在两组间无明显改变。而且,SHR主动脉和血浆NO/ET-1较WKY显著降低。奈比洛尔可增加SHR血浆及主动脉NO含量和NO／ET-1,但对血浆和主动脉ET-1含量无影响。阿替洛尔对SHR血浆及主动脉NO、ET-1、NO/ET-1无显著影响。
(7)血浆及主动脉AngⅡ、NO/AngⅡ:SHR血浆和主动脉AngⅡ水平较WKY显著增加,NO/AngⅡ降低。奈比洛尔对血浆AngⅡ含量无影响,但可降低主动脉AngⅡ含量,使血浆和主动脉NO/AngⅡ增加。阿替洛尔对血浆及主动脉AngⅡ、NO/AngⅡ无显著影响。
(8)不同动脉血管反应性：
SHR主动脉、颈动脉、肾动脉、肠系膜动脉和股动脉对Phe(10-8-10-5mol／L)、KCl(20-120m mol/L);颈动脉对ET-1 (10-11-10-8mol/L)、AngⅡ(10-9-10-5mol/L)及主动脉对AngⅡ的收缩反应显著高于WKY；不同动脉对Ach (10-10-10-5 mol/L)的舒张效应则弱于WKY,而对SNP (10-10-10-4 mol/L)的舒张反应在两组无显著性差异。
奈比洛尔可以降低SHR不同动脉对四种收缩剂的反应,其程度因血管不同而异：对KCl收缩的抑制作用在颈动脉和股动脉尤为明显；对Phe的抑制在主动脉、肾动脉、股动脉显著；对ET-1和AngⅡ的抑制在主动脉显著。同时,奈比洛尔可增强SHR不同动脉对Ach的舒张反应,尤其在肾动脉；但是奈比洛尔对不同动脉SNP的舒张无影响。
阿替洛尔对SHR不同动脉对四种收缩剂收缩反应的抑制作用有血管差异。阿替洛尔可抑制除肾动脉和肠系膜动脉外其余动脉对KCl的收缩；抑制除肠系膜动脉外其余动脉对Phe的收缩；减少主动脉和颈动脉对ET-1, AngⅡ的收缩。阿替洛尔可增强SHR肾动脉对Ach的舒张,对SNP的舒张无影响。
结论：奈比洛尔在显著降压的同时,可改善SHR血管重构,减少主动脉内皮损伤,增加SHR血浆和主动脉舒血管物质水平,降低缩血管物质含量,改善不同动脉的血管反应性,重建血管舒缩平衡。
研究背景：血管内皮功能不全在心血管疾病的发生发展中起重要作用,有效预防内皮功能受损可延缓乃至逆转相关病变进程。一氧化氮(nitric oxide, NO)在内皮功能调节中发挥重要作用,被认为是维持血管结构和功能的主要因子。内源性一氧化氮合酶(NOS)的抑制剂非对称性二甲基精氨酸(asymmetric dimethylarginine, ADMA)竞争性抑制NOS活性,减少NO生成。大量资料显示,ADMA水平的增高与多种病理状况下血管内皮功能不全有显著的相关性,可作为一个新的内皮功能不全预测因子。通过降低ADMA从而改善血管内皮功能为心血管疾病防治提供了新途径。我们第二部分的研究显示,奈比洛尔可以保护SHR血管内皮功能。但有关奈比洛尔血管内皮保护作用与ADMA系统关系的研究较少。
研究目的：本实验拟从组织和细胞水平探讨奈比洛尔对内皮细胞的保护作用与ADMA系统的关系。
研究方法：
1、奈比洛尔对SHR ADMA系统的影响。SHR大鼠及WKY大鼠随机分成4组：(1)SHR+Nebivolol组(n=6)：给予奈比洛尔8 mg/kg/day; (2) SHR+Atenolol组(n=6)：给予阿替洛尔80mg/kg/day; (3) SHR空白对照组(n=6)；(4)WKY空白对照组(n=6)。奈比洛尔,阿替洛尔溶于蒸馏水中灌胃,对照组给予等体积蒸馏水灌胃。给药8周后,3%戊巴比妥钠(30mg/kg, i.p.)麻醉大鼠,腹主动脉取血后,迅速分离主动脉,肠系膜动脉,将其置于液氮中冷冻后-70℃保存。离心分离血浆,测ADMA (ELISA法)和NO(硝酸还原酶法)含量,NOS活性(化学法)。提取主动脉和肠系膜动脉总RNA,用RT-PCR分析eNOS,DDAH-2和PRMT-1 mRNA表达。提取主动脉和肠系膜动脉总蛋白,用Western bloting分析DDAH-2和PRMT-1蛋白表达。采用冰冻切片荧光染色行主动脉原位活性氧(ROS)检测。
2、奈比洛尔对AngⅡ诱导损伤的人脐静脉内皮细胞(HUVECs)的影响。用AngⅡ1μmol/L培养HUVECs 24h,不加或加入阿替洛尔20μmol／L、奈比洛尔(5、10、20μmol/L)预处理1h。0.25%胰酶消化细胞,获取细胞悬液,离心收集细胞上清液,测ADMA (ELISA)和NO水平(硝酸还原酶法)水平,NOS活性(化学法)。提取HUVECs,总RNA,用RT-PCR分析eNOS, DDAH-2和PRMT-1 mRNA表达。提取HUVECs,总蛋白,用Western bloting分析DDAH-2和PRMT-1蛋白表达,同时检测细胞内DDAH活性。
3、奈比洛尔对ADMA损伤HUVECs的拮抗作用。用ADMA 16μmol/L培养HUVECs 24h,不加或加入阿替洛尔20μmol／L、奈比洛尔(5、10、20μmol/L)预处理1h。0.25%胰酶消化细胞,获取细胞悬液,离心收集细胞上清液,测NO水平(硝酸还原酶法)水平、NOS活性(化学法)。提取HUVECs,总RNA,用RT-PCR分析eNOS mRNA表达,同时测细胞ROS水平。
4、ADMA对离体大鼠主动脉血管反应性的影响及奈比洛尔对此的影响。成年雄性Wistar大鼠,击头致昏,快速分离主动脉。主动脉剔除脂肪及周围结缔组织后,剪成3-4mm的血管环,悬挂在浴管内,经PowerLab计算机生物信号采集分析系统记录血管的舒缩变化。观察ADMA (10-7-10-4 mol/L)对大鼠主动脉静息张力、Phe(10-6mol／L)预收缩主动脉张力、Ach(10-10-10-5mol／L)舒血管作用的影响及奈比洛尔(10-7,10-6和10-5mol／L)对此的影响。
结果：
1、与WKY比较,SHR血浆ADMA水平显著升高,NO水平和NOS活性明显下降。SHR主动脉和肠系膜动脉eNOS及DDAH-2 mRNA表达下降,PRMT-1 mRNA表达增加；DDAH-2蛋白表达下降,PRMT-1蛋白表达增加；主动脉原位ROS荧光强度增强。奈比洛尔可降低SHR血浆ADMA水平,增加NO含量和NOS活性,上调SHR主动脉和肠系膜动脉eNOS mRNA及DDAH-2 mRNA/蛋白表达,下调PRMT-1 mRNA/蛋白表达,降低SHR主动脉原位ROS荧光强度。阿替洛尔对SHR上述改变无影响。
2、AngⅡ1μmol/L培养HUVECs 24h可明显增加上清液ADMA水平,降低NO水平和NOS、DDAH活性,下调eNOS mRNA及DDAH-2 mRNA/蛋白表达,上调RMT-1 mRNA/蛋白表达。奈比洛尔(5、10、20μmol／L)可剂量依赖性抑制AngⅡ1μmol/L所致的上述改变。预先给予阿替洛尔20μmol／L则没有起到类似的作用。
3、ADMA 16μmol/L培养HUVECs 24h后,明显增加上清液ADMA水平,降低NO含量和NOS活性,升高细胞内ROS水平,下调eNOS mRNA表达。奈比洛尔(5、10、20、μmol/L)可剂量依赖性抑制ADMA 16μmol/L所致的上述效应。预先给予阿替洛尔20μmol/L则没有起到类似的作用。
4、离体血管张力实验表明,ADMA (10-7-10-4 mol/L)对离体大鼠主动脉静息张力无影响,但可剂量依赖性升高Phe(10-6 mol/L)预收缩大鼠主动脉的张力,抑制Ach(10-10-10-5mol／L)的舒张反应。奈比洛尔(10-7,10-6,10-5mol／L)可剂量依赖性抑制ADMA对Phe预收缩大鼠主动脉张力的升高,减少ADMA 10-5 mol/L对大鼠主动脉Ach舒张反应的抑制。
结论：奈比洛尔可通过下调PRMT-1表达,增加DDAH活性和表达,降低ADMA水平,保护内皮细胞。同时,奈比洛尔可拮抗ADMA导致的细胞损伤和血管收缩。
Background:Nebivolol is a highly selectiveβ1 adrenoceptor blocker with additional vasodilating properties. It has been shown that the nebivolol-induced vasorelaxation is nitric oxide (NO) dependent. However, the nature of the transduction pathway that mediates nebivolol's activation remains elusive. PI3K/Akt/eNOS is an important cell signal transduction pathway. The serine/tyrosine protein kinase Akt phosphorylates endothelial cell nitric oxide synthase (eNOS) and enhances the ability of eNOS to generate NO. Previous studies have shown that the release of NO from the endothelium may be ascribed to the modulation of different types of K+ channels. It has also been show that there was a difference in the NO release induced by nebivolol among different arteries.
Purpose:The present study was designed to determine the relaxation of nebivolol on different arteries (aorta, carotid artery, renal artery, mesenteric artery and femoral artery); to observe the effects of NOS inhibitor, K+ channels inhibitors or PI3K/Akt inhibitors on the vasorelaxation induced by nebivolol in different rat arteries.
Methods:Male Wistar rats (200-250g) were killed by cervical dislocation. Then thoracic aortas, carotid arteries, renal arteries, mesenteric arteries and femoral arteries were removed and placed in chilled (4℃) physiological salt solution (PSS) which was gassed with 100% O2. Aortas, carotid arteries were cleaned of connective tissue and fat, and cut into 3-4mm long rings. For the recording of tension the vessel rings were mounted, between 2 L-shaped stainless steel hooks, in 10ml organ baths filled with oxygenated PSS solution (bubbled with 100%O2 and maintained at 37℃) (pH 7.4). Each preparation was fixed, via a silk thread, to an isometric force transducer (A.D. Instruments, PowerLab, Australia) and force was recorded via PowerLab computer system (A.D. Instruments). Each vessel ring was subjected to an initial resting tension (aorta,2g; carotid artery, 1g). A 2h equilibration period was allowed before any experimental intervention, and during equilibration, the bath was flushed every 20 minutes with the fresh PSS. After equilibration, the rings were activated 2 times with 60mmol/L KCl. The integrity of endothelium was presumed by observation that the relaxation induced by 10-5mol/L acetylcholine (Ach) on 60 mmol/L KCl induced contraction was greater than 30%. When the contraction induced by 60 mmol/L KCl and the relaxation induced by 10-5 mol/L Ach were repeatable (the change was less than 10% between successive contractions or relaxation), the effects of the drugs to be tested were observed.
The femoral artery, the third-order branches of the superior mesenteric artery and renal artery were isolated and cut into 2mm-long rings. The rings were mounted in separate 5ml tissue baths filled with PSS solution (bubbled with 100% O2 and maintained at 37℃) (pH 7.4) using 40μm steel wire in a small-vessel myograph (Multi Myograph System-610M, Danish Myo Technology A/S, Denmark) for tension recordings. The rings were normalized according to standard procedures and stretched to a state equal to 100mmHg,80mmHg, 100mmHg, respectively. The rings were equilibrated for at least for 1h, and during equilibration, the bath was flushed every 20 minutes with the fresh PSS. After equilibration, the rings were activated 2 times with 60mmol/L KC1. The integrity of endothelium was presumed by observation that the relaxation induced by 10"5 mol/L Ach on contraction induced by 60 mmol/L KCl was greater than 20%. When the contraction induced by 60 mmol/L KCl and the relaxation induced by 10-5 mol/L Ach were repeatable (the change was less than 10% between successive contractions or relaxation), the effects of the drugs to be tested were observed.
Vessels were then washed extensively, and subsequently contracted with phenylephrine (Phe) (aorta and carotid artery were pre-contracted with 10"6 mol/L, mesenteric artery, femoral artery and renal artery were pre-contracted with 10"5 mol/L) or with 60 mmol/L KCl. Following attainment of plateau constriction in response to Phe or KCl, the logarithm cumulative concentration response curve (LCCRC) to nebivolol (10-7-10-5 mol/L) was performed. Then the vessels were contracted with Phe or 60mmol/L KCl again. Following attainment of plateau constriction, specific inhibitors of each type of K+channels:iberiotoxin (100 nmol/L), glibenclamide (0.1 mmol/L),4-aminopyridine (4-AP,1 mmol/L), BaCl2 (1 mmol/L), wortmannin (5×10"7 mol/L, a selective inhibitor of phosphatidylinositol 3-kinase (PI3K)), Akt inhibitor (lL-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate,10-5 mol/L, a selective inhibitor of Akt) and NG-nitro-L-arginine methyl ester (L-NAME,100μmol/L), an inhibitor of NO synthase were added to the bathing medium respectively. When the plateau constriction was attained, nebivolol (10-7-10'5 mol/L) was added cumulatively. So the effects of NOS inhibitor, K+channels inhibitors or PI3K/Akt inhibitors on the vasorelaxation induced by nebivolol in different rat arteries were studied.
Results:Nebivolol (10-7-10-5 mol/L) concentration-dependently relaxed pre-contractions induced by KC1 and PE in different rat arteries. The Emax of the relaxation of nebivolol was different according to the contractors. The rank order of vasodilator efficacy as measured in relation to the maximal vasodilation induced by nebivolol (10"7-10-5 mol/L) was:mesenteric artery (96.7±12.8%)≈aorta (94.2±5.4%)>femoral artery (84.8±12.1%)≈renal artery (80.3±3.4%)>carotid artery (74.9±10.1%) pre-contracted with Phe. In arteries contracted with 60mmol/L KCl, the maximal vasodilation was similar, expect in mesenteric artery (90.57±15.2%):femoral artery (75.7±17.7%), aorta (73.1±11.3%), renal artry (71.6±6.2%), carotid artery (70.9±7.1%). The vasodilator effect of nebivolol, in concentrations of 3 and 10μmol/L, was significantly blocked by 100μmol/L of the NOS inhibitor L-NAME in all arteries The exposure of the vessel rings to a selective inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin (5×10-7 mol/L) or a selective inhibitor of Akt (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate,10-5 mol/L) did not influence nebivolol-induced vasorelaxation. Similarly, K+channels blockers:iberiotoxin (100 nmol/L), glibenclamide (0.1 mmol/L),4-aminopyridine (1 mmol/L), or BaCl2(1 mmol/L) had no influence on the relaxation of nebivolol in arteries precontracted by Phe or KCl.
Conclusion:Nebivolol produced a concentration-dependent vasodilation in different rat arteries pre-contracted by Phe or KCl. In the isolated rat aorta, carotid artery, femoral artery, mesenteric artery and renal artery, neither K+channels nor PI3K/Akt/eNOS pathway was involved in the relaxation induced by nebivolol.
Background:Hypertension is a major risk factor for cardiovascular disease. Experiments show that there is endothelial dysfunction in animal and human hypertension, which in turn accelerates the damage of target organs. Endothelia dysfunction, which may manifest as vasomotor imbalance and changes in vaso-active substances and so on, is the initiator factors and carrier of "endothelial-high blood pressure-cardiovascular events" chain. Vascular endothelial damage enables the vascular remodeling. The remodeling can be an early manifestation of vascular structural changes and the basis for a number of cardiovascular events. It has been became a new target for hypertension treatment to improve the endothelia function. Nebivolol is a highly selectiveβ1-adrenergic receptor antagonist that can blockβ1-adrenergic receptor and promote endothelium-dependent vasodilation by increasing bioavailability of nitric oxide (NO). Compared with other traditionalβ-adrenergic receptor antagonists, nebivolol, without having the metabolic defects, is a bestβ-adrenergic receptor antagonist at present. Studies show that nebivolol can ameliorate the vascular reaction of aorta and improve the plasma NO concentration of SHR. But the data is few about the effect of nebivolol on the reaction of arteriole, the vaso-activesubstance in artery and the reconstruction of vascular.
Purpose:The aim of the study was to assess the protective effect of nebivolol on endothelial dysfunction in spontaneously hypertensive rats (SHR) by observing the reconstruction of arteries, vascular endothelial damage, levels of vaso-activesubstance and the vascular responses of different arteries.
Methods:SHR and age-matched Wister-Kyoto (WKY) rats were randomly divided into 4 groups:(1) SHR+Nebivolol group(n=6):SHR treated with nebivolol(8 mg/kg/day, i.g.); (2) SHR+Atenolol group (n=6):SHR treated with Atenolol (80 mg/kg/day, i.g.); (3) SHR control group (n=6):SHR treated with vehicle once daily, i.g.; (4) WKY control group (n=6):WKY treated with vehicle once daily, i.g. Therapy continued for 8 weeks, body weight and tail systolic blood pressure were measured weekly. At the end of this study, animals were anesthetized with 3% sodium pentobarbital (30 mg/kg, i.p.). Blood was draw by abdominal aortic puncture, then the aorta, carotid artery, renal, mesentery and femoral artery were moved rapidly. Renal and a part of aorta were fixed with 4% paraformaldehyde to observe the vascular morphological changes, including vessel lumen diameter (L), media thickness (M) and M/L. Expression of FVM in aorta was performed by immunohistochemistry analysis. Levels of NO (nitric acid reducdase assay), endothelin-1 (ET-1, radioimmunoassay), angiotensin II (AngⅡ, radioimmunoassay) in plasma, aorta and von Willebrand factor (vWF, ELISA) in plasma were measured. The contraction of different arteries (aorta, carotid artery, renal artery, femoral artery and mesentery artery) to Phe, KCl, ET-1, AngⅡand relaxation to acetylcholine (Ach) and sodium nitroprusside (SNP) were also studied in vitro.
Results:
(1) During the study, the body weight and systolic blood pressure were higher in SHR than in age-matched WKY. Nebivolol and atenolol had no effect on the body weight of SHR. Compared with atenolol, treatment with nebivolol induced an acute and significant reduction in systolic blood pressure; this reached a maximum in the fourth week.
(2) Morphometry study of aorta:There was no difference in L among different groups, but M and M/L increased in SHR than in WKY. Treatment with nebivolol reduced M and M/L in SHR while Atenolol had no effect.
(3) Morphometry study of interlobular artery in renal:The L decreased, but M and M/L of interlobular artery increased in SHR. Treatment with nebivolol raised L and lessened M and M/L in SHR, but atenolol had no effect.
(4) Thoracic aorta immunohistochemistry assay to identify FⅧin endothelia:The expression of FⅧwas weakly positive in SHR than in WKY, the brown endothelium was not intact and continuous. Treated with nebivolol, the expression of FⅧincreased and the brown endothelium was clear and intact, however atenolol had no effect.
(5) Level of vWF in plasma:The level of vWF increased in SHR than in WKY. Nebivolol reduced the level of vWF in SHR, but atenolol had no influence.
(6) Levels of NO, ET-1 and NO/ET-1 in plasma and aorta:The levels of NO, NO/ET-1 in plasma and aorta were lowered and ET-1 in aorta was increased in SHR than in WKY; but no difference was found in plasma ET-1 between SHR and WKY. Nebivolol increased the level of NO and NO/ET-1 in plasma and aorta, but had no influence on level of ET-1 in aorta and plasma. Atenolol had no effect on theses changes in SHR.
(7) Levels of Ang II and NO/AngⅡin plasma and aorta:The level of Ang II was increased and NO/Ang II was reduced in SHR plasma and aorta than in WKY. Nebivolol had no effect on Ang II in plasma, but reduced it in aorta. So the NO/Ang II in plasma and aorta were increased after nebivolol treatment. Atenolol had no effect on the changes in SHR.
(8) Vascular reaction of different arteries:
The contractions to Phe, KCl in different arteries; to ET-1 in carotid artery; to AngⅡin aorta and carotid artery were increased in SHR than in WKY. The relaxation to Ach decreased in SHR than in WKY, but there was no difference of relaxation to SNP among groups.
Nebivolol reduced the contraction of different arteries to Phe, KCl, AngⅡand ET-1 in SHR, but there was difference in the reduction based on the kind of arteries. It was marked to KCl in carotid artery and femoral artery, to Phe in aorta, renal artery, femoral artery, and to ET-1 and Ang II in aorta. Nebivolol increased the relaxation of different arteries to Ach, especially in renal artery, but had no effect on the relaxation of different arteries to SNP.
Atenolol just decreased the contraction of different arteries to KCl apart from renal artery and mesenteric artery; to Phe besides mesenteric artery; to ET-1, AngⅡin aorta and carotid artery. Atenolol increased the relaxation of renal artery to Ach in SHR, but had no effect on the relaxation to SNP.
Conclusion:The antihypertensive effect of nebivolol in SHR was accompany by protect on endothelial function:retarding vascular remodeling; reducing the injury of aorta endothelia cell; modulate the unbalance of vasoactive substances in aorta and plasma; improving the vascular reaction to different vasomotors, resuming the balance of relaxation and contraction.
Objectives:Endothelial dysfunction plays an important role in the development of cardiovascular diseases. Nitric oxideis is recognized as a major mediator in regulating vascular construction and function. Aasymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor that compatetively inhibits nitric oxide synthase (NOS) activity and reduces NO level, has been considered a new predictor of endothelial dysfunction. In a variety of pathophysiological conditions, plasm ADMA has been found rising significantly, and then may induce endothelial dysfunction. With the understanding of endothelial dysfunction and pathological roles of ADMA in cardiovascular disease, it may become a new therapeutic avuenues to improve endothelial function by reducing ADMA level. Nebivolol can protect vascular endothlia function. But data is few about the relationship of protective effect of nebivolol on vascular and ADMA.
Aim:To investigate the relationship of protective effect of nebivolol on vascular and ADMA.
Methods:
1. Effect of nebivolol on ADMA system of SHR. SHR and age-matched Wister-Kyoto (WKY) rats were randomly divided into 4 groups:(1) SHR+Nebivolol group (n=6):SHR treated with nebivolol(8 mg/kg/day, i.g.); (2) SHR+Atenolol group (n=6):SHR treated with Atenolol(80 mg/kg/day, i.g.); (3) SHR control group (n=6):SHR treated with vehicle once daily, i.g.; (4) WKY control group (n=6):WKY treated with vehicle once daily, i.g. After treatment for 8 weeks, rats were anesthetized with 3% sodium pentobarbital (30 mg/kg, i.p.). Blood was draw by abdominal aortic puncture, then the aorta, mesentery artery were moved rapidly and kept in-70℃. The levels of ADMA (ELISA), NO (nitric acid reducdase assay) and NOS activity (chemical method) in plasma were measured. The mRNA expression of eNOS, DDAH-2, PRMT-1 in aorta and mesenteric artery were measured by RT-PCR. The protein expression of DDAH-2 and PRMT-1 in aorta and mesenteric artery were measured by Western blot. ROS level in aorta were also measured.
2. Effect of nebivolol on HUVECs cultured with AngⅡ. HUVECs were cultured with Ang II 1μmol/L for 24h in the absence or presence of nebivolol (5,10 or 20μmol/L) or atenolol (20μmol/L) for 1 h. The supernaent in the conditioned medium was collected by centrifugation for determination of ADMA (ELISA), NO (nitric acid reducdase assay) and NOS activity (chemical method). The expression of eNOS, DDAH-2, PRMT-1 mRNA (RT-PCR), the protein expression of DDAH-2 and PRMT-1 (Western blot), activity of DDAH in HUVECs were also measured.
3. Effect of nebivolol on HUVECs cultured with ADMA. HUVECs were cultured with ADMA 16μmol/L for 24h in the absence or presence of nebivolol (5,10 or 20μmol/L) or atenolol (20μmol/L) for 1 h. The supernaent in the conditioned medium was collected by centrifugation for determination of NO and NOS activity. The expression of eNOS mRNA (RT-PCR), ROS in HUVECs were also measured.
4. Effect of nebivolol on influence of ADMA on vascular reponse of rat aorta. Male Wistar rats were killed by cervical dislocation. Then thoracic aortas were removed quickly. Aortas was cleaned of connective tissue and fat, and cut into 3-4mm long rings. Each preparation was fixed, via a silk thread, to an isometric force transducer and force was recorded via PowerLab computer-7 -4 system (A.D. Instruments). The effects of ADMA (10-10 mol/L) on aorta resting tone, aorta pre-contracted by Phe (10-6mol/L), relaxation induced by Ach (10-10-10-5 mol/L) and the effect of nebivolol (10-5,10-6,10-5mol/L) on the function of ADMA on rat aorta were observed in virto.
Rusults:
1. Compared with WKY, the level of plasma ADMA was elevated significantly, plsma NO, activity of NOS were decreased in SHR. In aorta and mesenteric artery of SHR, the mRNA expression of eNOS, mRNA/protein expression of DDAH-2 reduced, but mRNA/protein expression of PRMT-1 increased. ROS level in aorta also increased in SHR than in WKY. Nebivolol treatment increased plasma NO and activity of NOS, decreased ADMA leve. Nebivlolol up regulated mRNA expression of eNOS and mRNA/protein expression of DDAH-2; down regulated mRNA/protein expression of PRMT-1 in aorta and mesenteric artery. Nebivolol reduced ROS level of SHR aorta. Atenolol had no effect on the changes in SHR.
2. AngⅡ(1μmol/L) for 24h significantly decreased NO level and NOS activity, increased the mRNA/protein expression of PRMT-1, decreased DDAH activity, mRNA expression of eNOS and mRNA/expression of DDAH-2, resulting in elevation of ADMA level. Pretreatment with nebivolol (5,10 or 20μmol/L) concentration-dependently attenuated the above effects by AngⅡ. Atenolol (20μmol/L) had no influence on the effects induced by AngⅡ.
3. ADMA (16μmol/L) for 24h significantly markedly decreased NO level, NOS activity and eNOS mRNA; and increased intracellular ROS generation. Pretreatment with nebivolol (5,10 or 20μmol/L) concentration-dependently attenuated the above effects by ADMA. Atenolol (20μmol/L) had no influence on the effects induced by ADMA.
4. ADMA (10-7-10-4 mol/L) had no effect on the restiong tone of rat aorta, but ADMA concentration-dependently increased the tone of aorta pre-contracted by Phe (10 mol/L) and inhibited Ach (10-10-10-5 mol/L) induced relaxation in vitro. Nebivolol (10-5,10-6,10-5 mol/L) dose-dependently decreased the elevation of ADMA on pre-contracted rat aorta and the inhibition of ADMA 10-5 mol/L on relaxation induced by Ach.
Conclusion:Nebivolol reduced ADMA by increasing DDAH activity/expression and reducing PRMT-1 expression. Nebivolol also can inhibit cell injury and contraction induced by ADMA.
引文
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