内皮细胞乙酰胆碱靶标不同于M受体的药理学特征

目的 研究内皮细胞乙酰胆碱靶标与M受体药理学特征的差异。方法 采用离体血管环实验方法,观察抗M_3受体的单克隆抗体和M受体激动剂毛果芸香碱对乙酰胆碱引起的内皮依赖性舒张血管作用的影响。结果 毛果芸香碱能够剂量依赖性拮抗乙酰胆碱诱导的内皮依赖性血管舒张作用,其拮抗性质为非竞争性拮抗;抗M_3受体的单克隆抗体不影响乙酰胆碱诱发的内皮依赖性舒张血管作用,但能拮抗乙酰胆碱诱发的离休肠肌收缩作用。结论 内皮细胞乙酰胆碱靶标的药理学特征不同于经典M受体。     

牛主动脉内皮细胞乙酰胆碱激活蛋白介导的胞内钙离子变化

目的　研究主动脉内皮细胞乙酰胆碱激活蛋白(EPA)介导胞内钙离子变化的特征 ,并探讨培养牛主动脉内皮细胞EPA功能的变化。方法　以Fluo 3,Frua 2为荧光探针 ,采用共聚焦和荧光光度法测定内皮细胞 [Ca2 + ]i。结果　Ach能够引起体外培养 6代以内内皮细胞 [Ca2 + ]i 的增加 ;Ach激发的内皮细胞 [Ca2 + ]i 信号表现为瞬时性单次或连续 2次的钙振荡 ,具有异时性和快速耐受的特点。未观察到烟碱和其它M受体激动剂引起的 [Ca2 + ]i 变化。Ach对胞内 [Ca2 + ]i 浓度的作用以原代细胞增加最为明显 ,但随培养时间的延长 ,此效应逐渐减弱 ,至 13代后无效应。结论　血管内皮细胞乙酰胆碱激活蛋白激活能介导胞内钙离子的增高 ,经培养后EPA功能丧失。     

牛主动脉内皮细胞乙酰胆碱激活蛋白介导的胸内钙离子变化

目的：研究主动脉内皮细胞乙酰胆碱激活蛋白（EPA）介导胞内钙离子变化的特征,并探讨培养牛主动脉内皮细胞EPA功能的变化。方法：以Fluo-3,Frua-2为荧光探针,采用共聚焦和荧光光度法测定内皮细胞[Ca^2 ]。结果：Ach能够引起体外培养6代以内内皮细胞[Ca^2 ]i的增加;Ach能够引起体外培养6代以内内皮细胞[Ca^2 ]i信号表现为瞬时性单次或连续2次的钙振荡,具有异时性和快速耐受的特点。未观察到烟碱和其它M受体激动剂引起的[Ca^2 ]i变化。Ach对胞内[Ca^2 ]i浓度的作用以原代细胞增加最为明显,但随培养时间的延长,此效应逐渐减弱,至13代后无效应。结论：血管内皮细胞乙酰胆碱激活蛋白激活能介导胞内钙离子的增高,经培养后EPA功能丧失。     

卡维地洛对高血压心脏病患者的M2-乙酰胆碱能受体自身抗体的影响

目的 探讨卡维地洛对高血压心脏病患者的M2-乙酰胆碱能受体自身抗体的影响.方法 2011年1月至2012年1月期间,我院诊治的74例高血压心脏病患者,随机将其分为对照组(常规治疗)和观察组(对照组基础上,加用卡维地洛),平均随访半年,对两组患者的心功能,以及M2-乙酰胆碱能受体自身抗体的变化情况,进行观察和比较.结果 与治疗前相比,治疗后两组LVEDD、LVESD、LVEF,以及心功能均有所改善.与治疗后对照组相比,观察组心功能分级、LVEDD、LVESD明显降低,而LVEF升高,血清中M2-乙酰胆碱受体自身抗体滴度明显降低,P<0.05,有统计学意义.结论 M2-乙酰胆碱受体与高血压心脏病的发病过程有着密切的联系,卡维地洛通过阻断M2-乙酰胆碱受体自身抗体对心脏的不利影响,发挥心脏保护作用.     

有机磷类杀虫剂与毒蕈碱乙酰胆碱M2受体的磷酸化

目的探讨有机磷类杀虫剂对大鼠脑G蛋白偶联受体激酶2介导的毒蕈碱乙酰胆碱M2受体磷酸化的影响。方法采用亲和层析法从大鼠脑组织纯化毒蕈碱乙酰胆碱M2受体;将纯化的毒蕈碱乙酰胆碱M2受体或β-肾上腺素受体、G蛋白偶联受体激酶-2,[γ-p~（32）]ATP与对氧磷（PO）、氧毒死蜱（CPO）共同保温,聚丙烯酰胺凝胶电泳分离蛋白,放射性自显影检测M2受体磷酸化结果。结果氧毒死蜱完全抑制大鼠脑M2受体的磷酸化,其半数抑制浓度（IC_（50））为70μmol/L;对氧磷不抑制M2受体的磷酸化;氧毒死蜱和对氧磷都不抑制β-肾上腺素受体的磷酸化。结论氧毒死蜱可选择性地抑制大鼠脑M2受体的磷酸化;某些有机磷杀虫剂存在其他靶分子或作用途径。     

血管内皮细胞非神经性M受体药理学与疾病治疗的研究进展

血管内皮细胞上存在介导内皮依赖性血管舒张反应的血管内皮细胞非神经性毒蕈碱样受体(M受体)。对血管内皮细胞非神经性M受体亚型的定性一直存在争议。多数药理实验结果显示血管舒张反应是M3受体介导的;另外M1,M2受体也在不同组织类型的血管上介导了血管舒张反应。基因敲除小鼠的结果显示,主动脉、冠状动脉上介导舒张反应的受体以M3亚型为主;脑动脉上以M5为主。药理实验发现,介导血管舒张效应的内皮细胞非神经性M受体具有相对的组织、器官选择性和动物种属差异,与神经性M受体药理学特性有差别。激活血管内皮细胞非神经性M受体可促使内皮细胞分泌多种血管活性因子,介导血管的舒张、维持血管的正常生理状态;同时还可促进组织型纤溶酶原激活物的生成,降低多种粘附分子的分泌,产生抗动脉粥样硬化和抗血栓形成的功能。与传统药物作用机制不同。     

1,2,5,6-4H-1-烷基-3-取代吡啶类新衍生物对血管内皮细胞功能的调节作用及其构效关系

目的：以槟榔碱为结构母核，设计合成1，2，5，6-4H-1-烷基-3-取代吡啶类新衍生物，分析其对血管内皮细胞功能的影响。方法：采用离体大鼠主动脉环和离体豚鼠回肠收缩实验，分别观察新结构化合物对血管和回肠平滑肌张力的影响；并进一步观察一氧化氮合酶抑制剂L-NAME、环氧酶抑制剂吲哚美辛、M受体亚型非选择性激动剂毛果芸香碱和M受体亚型非选择性拮抗剂阿托品对新结构化合物诱发的血管舒张反应的影响。结果：从100个新化合物中发现4个有舒血管反应，但不激活M受体的化合物，分别是HH91、HH95、HH98、HH103。新化合物HH103诱发的内皮依赖性舒血管反应可被L-NAME所拮抗，但不被吲哚美辛、阿托品和毛果芸香碱拮抗。结论：新化合物可诱发内皮依赖性舒张反应并具有特定构效关系；HH103通过促进内皮细胞释放NO发挥其效应；但其作用特征又与乙酰胆碱不同。     

在Sf9昆虫细胞-杆状病毒系统中表达毒蕈碱型M_2及M_5受体重组突变体

目的为乙酰胆碱毒蕈碱(M)受体亚型特异性的变构调节剂及基因工程的研究提供实验平台。方法用PCR及搭桥PCR法对乙酰胆碱M2及M5受体作以下突变:①将N-糖基化位点Asp突变为Asn;②删除对蛋白酶敏感的M受体的第三个细胞内环;③在C端添加凝血酶识别位点(CMV)和6-His标记。将PCR扩增出重组嵌合蛋白基因亚克隆到杆状病毒转移载体,制备重组杆状病毒并感染昆虫细胞表达M2/M5受体蛋白。Western印迹及放射性配体受体结合实验验证受体的正确表达及功能。结果通过搭桥PCR,成功扩增出1018 bp的重组M2受体和1041 bp重组M5受体核酸序列;使用pUC/M13的扩增引物成功构建M2/M5重组转移载体。将重组载体质粒与线性化病毒DNA共转染昆虫细胞Sf9,制备重组杆状病毒并感染昆虫细胞,见细胞空泡样病变。Western印迹分析确定重组杆状病毒感染昆虫细胞M2/M5蛋白表达,放射性配体受体饱和实验结果表明,表达的重组受体蛋白与[3H]N-甲基-东莨菪碱具有特异性结合能力。结论 Sf9昆虫细胞能够表达M2及M5重组受体蛋白,M2及M5重组受体蛋白的病毒样颗粒可用于M受体的新药研究。     

类叶升麻苷对东莨菪碱所致记忆获得性障碍的改善作用

目的　观察类叶升麻苷对小鼠学习记忆能力的影响。方法　用东莨菪碱致小鼠记忆获得性障碍模型 ,以行为学实验 (跳台法、水迷路法 )、大脑皮层和纹状体乙酰胆碱酯酶活性和M受体的最大结合力为指标观察类叶升麻苷的作用。结果　在东莨菪碱致小鼠记忆障碍模型中 ,类叶升麻苷可延长跳台实验的平台停留期 ,并显著减少错误次数 ;在水迷路实验中可提高正确反应百分率 ;并能拮抗东莨菪碱所引起的大脑皮层乙酰胆碱酯酶的增高及皮层、纹状体M受体最大结合力的降低。结论　类叶升麻苷对东莨菪碱所引起的小鼠学习记忆能力障碍有改善作用 ,其作用机制可能与抑制乙酰胆碱酯酶活性和激动M受体的作用有关     

类叶升麻苷对东莨菪碱所致记忆获得性障碍的改善作用

目的观察类叶升麻苷对小鼠学习记忆能力的影响.方法用东莨菪碱致小鼠记忆获得性障碍模型,以行为学实验(跳台法、水迷路法)、大脑皮层和纹状体乙酰胆碱酯酶活性和M受体的最大结合力为指标观察类叶升麻苷的作用.结果在东莨菪碱致小鼠记忆障碍模型中,类叶升麻苷可延长跳台实验的平台停留期,并显著减少错误次数;在水迷路实验中可提高正确反应百分率;并能拮抗东莨菪碱所引起的大脑皮层乙酰胆碱酯酶的增高及皮层、纹状体M受体最大结合力的降低.结论类叶升麻苷对东莨菪碱所引起的小鼠学习记忆能力障碍有改善作用,其作用机制可能与抑制乙酰胆碱酯酶活性和激动M受体的作用有关.     

Vascular adenosine monophosphate‐activated protein kinase: Enhancer,brake or both?

Adenosine monophosphate‐activated protein kinase (AMPK), expressed/present ubiquitously in the body, contributes to metabolic regulation. In the vasculature, activation of AMPK is associated with several beneficial biological effects including enhancement of vasodilatation, reduction of oxidative stress and inhibition of inflammatory reactions. The vascular protective effects of certain anti‐diabetic (metformin and sitagliptin) or lipid‐lowering (simvastatin and fenofibrate) therapeutic agents, of active components of Chinese medicinal herbs (resveratrol and berberine) and of pharmacological agents (AICAR, A769662 and PT1) have been attributed to the activation of AMPK (in endothelial cells, vascular smooth muscle cells and/or perivascular adipocytes), independently of changes in the metabolic profile (eg glucose tolerance and/or plasma lipoprotein levels), leading to improved endothelium‐derived nitric oxide‐mediated vasodilatation and attenuated endothelium‐derived cyclooxygenase‐dependent vasoconstriction. By contrast, endothelial AMPK activation with pharmacological agents or by genetic modification is associated with reduced endothelium‐dependent relaxations in small blood vessels and elevated systolic blood pressure. Indeed, AMPK activators inhibit endothelium‐dependent hyperpolarization (EDH)‐type relaxations in superior mesenteric arteries, partly by inhibiting endothelial calcium‐activated potassium channel signalling. Therefore, AMPK activation is not necessarily beneficial in terms of endothelial function. The contribution of endothelial AMPK in the regulation of vascular tone, in particular in the microvasculature where EDH plays a more important role, remains to be characterized.     

Role of the endothelium in the vasodilator response of rat thoracic aorta to histamine

Despite their potent vasodilating action in vivo, acetylcholine and histamine often show a vasoconstricting action in vitro. As the endothelium has an important role in the vasodilating effect of acetylcholine, we investigated the possible role of the endothelium in the vasodilating effect of histamine in comparison to acetylcholine. Experiments were done on ring segments of rat thoracic aorta mounted for isometric tension measurements. We demonstrated that relaxation by histamine and acetylcholamine of pre-contracted rat aorta segments required the presence of endothelial cells. Acetylcholine acting on muscarinic receptors, and histamine acting on H₁-receptors seemed to initiate the production of mediator(s) from the endothelial cells, which leads to relaxation of the vascular smooth muscle cells. This production appeared to be depressed by ETYA and hydroquinone, and under hypoxic conditions.     

银杏叶片对血管内皮功能的影响

目的观察银杏叶片对血管内皮功能的保护作用。方法80例门诊体检志愿者,随机分为治疗组和对照组,分别于实验前及治疗后30、90、108d使用高频超声检测肱动脉血管内皮功能的变化。结果治疗前两组患者内皮依赖性舒张功能均无差异,而治疗30、90、108d后内皮依赖性舒张功能明显改善。结论中药银杏叶片在心脑血管疾病防治中具有重要的保护作用。     

Submaximal PPARγ activation and endothelial dysfunction: new perspectives for the management of cardiovascular disorders

PPARγ activation plays an important role in glucose metabolism by enhancing insulin sensitization. PPARγ is a primary target for thiazolidinedione-structured insulin sensitizers like pioglitazone and rosiglitazone employed for the treatment of type 2 diabetes mellitus. Additionally, PPARγ activation inhibits adhesion cascades and detrimental vascular inflammatory events. Importantly, activation of PPARγ plays a distinctive role in regulating the physiology and expression of endothelial nitric oxide synthase (eNOS) in the endothelium, resulting in enhanced generation of vascular nitric oxide. The PPARγ activation-mediated vascular anti-inflammatory and direct endothelial functional regulatory actions could, therefore, be beneficial in improving the vascular function in patients with atherosclerosis and hypertension with or without diabetes mellitus. Despite the disappointing cardiac side effect profile of rosiglitazone-like PPARγ full agonists, the therapeutic potential of novel pharmacological agents targeting PPARγ submaximally cannot be ruled out. This review discusses the potential regulatory role of PPARγ on eNOS expression and activation in improving the function of vascular endothelium. We argue that partial/submaximal activation of PPARγ could be a major target for vascular endothelial functional improvement. Interestingly, newly synthesized partial agonists of PPARγ such as balaglitazone, MBX-102, MK-0533, PAR-1622, PAM-1616, KR-62776 and SPPARγM5 are devoid of or have a reduced tendency to cause the adverse effects associated with full agonists of PPARγ. We propose that the vascular protective properties of pharmacological agents, which submaximally activate PPARγ, should be investigated. Moreover, the therapeutic opportunities of agents that submaximally activate PPARγ in preventing vascular endothelial dysfunction (VED) and VED-associated cardiovascular disorders are discussed.     

Autoradiographic localization of muscarinic acetylcholine receptors in the rat pulmonary vascular tree.

The pharmacological characteristics and the anatomical localization of muscarinic receptors in the pulmonary vascular tree were investigated in lung sections of Wister-Kyoto (WKY) and spontaneously hypertensive rats (SHR). [3H]Quinuclidinyl benzylate [( 3H]QNB) was bound by sections of rat lung in a manner consistent with the labeling of muscarinic acetylcholine receptors, with a dissociation constant value (Kd) of 0.41 +/- 0.3 nM in WKY rats and of 0.37 +/- 0.2 nM in SHR. The density of muscarinic acetylcholine receptors was higher in sections of lung of WKY rats than of SHR. In the pulmonary vasculature these sites were associated with the smooth muscle of the medial layer of different size branches of the pulmonary artery and vein. No [3H]QNB binding sites were found within the endothelium in the blood vessels of either WKY rats or SHR. The density of [3H]QNB binding sites was significantly lower in the smooth muscle of pulmonary vein and its branches in SHR. There were no significant hypertension-dependent changes in the density and pattern of muscarinic receptors of pulmonary artery smooth muscle.     

尿毒症脑病和血管内皮功能障碍关系的研究进展

心脑血管系统疾病是慢性肾病最常见的并发症,也是导致患者终末期肾脏病死亡的主要原因.大量研究表明尿毒症引起的脑病和尿毒症素的蓄积、氧化应激、炎症等因素有关.尿毒症毒素引起的脑血管内皮受损,血脑屏障功能破坏是引起尿毒症脑病的病理原因之一.因此修复脑血管内皮可作为防治本病的一种治疗策略.本文就慢性肾病导致的尿毒症脑病和血管内...     

A novel in vitro endothelium-dependent vascular relaxant effect of Apocynum venetum leaf extract

1. In the present study, a novel in vitro vascular relaxant effect of Apocynum venetum leaf extract (AVLE; also called 'Luobuma'), obtained from a traditional Chinese medicinal herb with known antihypertensive effects, is reported in isometric contraction studies of rat aorta and superior mesenteric artery. At low concentrations (0.3-10 microg/mL), AVLE had no effect on the resting tension of either blood vessel and caused relaxation in agonist-precontracted vessels with functionally intact endothelium. 2. We demonstrated pharmacologically that the AVLE-induced vasorelaxation was mediated selectively by the endothelial cells in both blood vessels. Using NG-nitro-L-arginine methyl ester (L-NAME) and a low concentration of KCl (15 mmol/L), we also demonstrated that AVLE acted by releasing endothelium-derived relaxation factors; nitric oxide (NO) in the rat aorta and NO plus endothelium-derived hyperpolarizing factor in the rat mesenteric artery. 3. The vascular relaxation following brief exposure to AVLE appeared to persist even after subsequent prolonged washout; this was manifested as an attenuated contraction to subsequent application of phenylephrine (PE) compared with the PE-induced contraction after exposure to carbachol (CCh) and subsequent similar washout. The addition of L-NAME at this point in the absence of AVLE totally restored the contraction to PE, suggesting that enzymatic generation of endothelial NO persisted even after brief exposure to AVLE. 4. Unlike the endothelium-dependent NO-mediated relaxation induced by CCh, which is mediated by endothelial muscarinic receptors (and inhibited by atropine), the relaxation induced by AVLE was not inhibited by atropine and, thus, was not mediated by muscarinic receptors. However, similar to CCh-induced relaxation, AVLE-induced relaxation was associated with the activation of K+ channels. 5. These results provide a strong scientific basis for the folk use of AVLE decoction for antihypertensive therapy in traditional Chinese medicine.     

Autoradiographic localization of muscarinic receptors within the rat kidney

We used a combination of radioreceptor binding and autoradiographic techniques to study the pharmacological characteristics and anatomical localization of [3H]quinuclidinyl benzylate (QNB) in kidney sections of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). [3H]QNB was bound by sections of rat kidney in a manner consistent with the labeling of muscarinic acetylcholine receptors. Anatomically, these receptor sites were located primarily within the smooth muscle of the renal vascular tree and to a lesser extent within cortical and medullary tubules. The density of renal muscarinic acetylcholine receptors (both vascular and tubular) was decreased in hypertension but the affinity of the ligand for muscarinic receptors was unchanged. A possible role of muscarinic acetylcholine receptors in renal function is suggested.