转人M受体亚型基因CHO细胞系在新药筛选中的应用

综述了转入M受体亚型基因CHO细胞系在M受体亚型选择性新筛选中的应用以及应用过程中应注意的问题。,并已筛选出的M受体亚型选择性激动剂和拮抗剂作简要介绍。     

M受体及相关选择性药物研究进展

毒草碱受体（M受体）是体内重要的G蛋白偶联受体之一,有M1～M5五种药理学亚型,各亚型在体内的分布和功能不同,受体蛋白结构和信号转导机制也有差异。对M受体、相关选择性药物及受体一配体作用位点的研究,将为设计以M受体各亚型为靶标的选择性药物提供帮助,对临床治疗多种M受体功能紊乱的疾病,如阿尔茨海默病等具有重要意义。     

抗胆碱类药物在帕金森病治疗中的研究进展

帕金森氏病(PD)是一类困扰人类健康的重大疾病,属中老年神经系统退行性病变疾病,患者脑内多巴胺能系统功能低下,胆碱能系统功能亢进,影响患者的运动功能。PD是一种慢性进展性疾病,尚无根治方法,中枢抗胆碱药是早于左旋多巴用于帕金森氏病一类药物,目前因其临床用药无M受体亚型选择性,毒性作用多见,致使临床该类药物的研究滞后。大量研究发现M4受体亚型为中脑黑质纹状体内主要分布的M受体亚型,对中脑纹状体多巴胺(DA)的合成和释放具有重要的调节作用,对DA参与的运动功能调控发挥着重要的作用,很有可能成为帕金森病治疗抗胆碱类药物研发的一个重要靶标。本课题组研究发现具有M4受体亚型选择性的中枢抗胆碱药物盐酸去甲基苯环壬酯在小鼠震颤模型、6-OHDA黑质纹状体定向损毁大鼠模型及MPTP特异损伤C57BL/6小鼠模型均表现出良好的缓解帕金森病运动功能障碍作用,与临床常用的中枢抗胆碱药盐酸苯海索比较,其选择性增加纹状体内DA递质释放作用更显著,抗帕金森病药效更好,无认知损伤、胃肠道反应轻等副作用更低的特点。提示该药效学差异与二药间受体亚型选择性差异有关,本研究为临床合理开发具有M4受体亚型选择性的新型抗胆碱药物提供重要的理论依据。     

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。     

M受体的研究进展

M受体是机体中最重要的受体之一，根据药理学分类可分为M1，M2，M3和M4 4种药理学亚,发子克隆技术可将其分为m1,m2,m3,m4和m5，其中M1，M2和M3分别对应于m1,m2和m3。各种亚型基本结构相似，主要差别在于胞浆内3环（i3环）的不同，这决定了它们功能的不同。m1，m3和m5结构功能相似，可激活PI系统和cAMP，m2和m4则可抑制腺苷酸环化酶系统。不同部位各种亚型的分布和功能也有所不同。M受体亚型的分布和功能及其活性药物对临床具有重要意义。     

M5受体参与中脑奖赏的研究进展

在五种毒蕈碱受体亚型中,M5毒蕈碱受体是最后被发现的,M5受体在大脑和内脏组织中含量最少[1]。至今未找到M5受体的高亲和性的选择性配体和毒素,也没有发现哪个大脑区域强烈表达M5受体mRNA[2]。因此,尽管对M1～M4受体的药理学作用有详尽的了解,但对M5受体的药理学特性却知之甚少。全脑免疫组化研究可以粗略地了解大脑M5受体的含量,但不能反映其在解剖学上的详尽分布[1]。另外,有研究者用树眼睛蛇毒素和AQ-RA741封闭M1～M4受体,了解各个脑区和一些内脏组织的剩余M5受体的分布状况[2]。即便是这样,残存的约15%的M3受体仍可能干扰对M5受…     

M胆碱能受体和GABAB受体在脊髓背角Ⅱ板层神经元上的表达

目的观察M胆碱能受体亚型M2和B型γ-氨基丁酸（GABAB）受体在脊髓Ⅱ板层神经元的表达情况。方法SD雄性大鼠,体重160～180g,麻醉后快速取下L4-6脊髓,进行冰冻切片,M2受体和GABAB受体进行免疫荧光组化双重染色。结果在激光共聚焦显微镜下观察,在脊髓Ⅱ板层神经元上,表达有丰富的M2受体和GABAB受体,其中56．57％的神经元可同时表达2种受体。结论脊髓背角Ⅱ板层神经元可接受M胆碱能受体与GABAB受体的共调节。     

选择性作用于肾上腺素受体的药物

简述肾上腺素受体的分型、功能及两种新的肾上腺素受体亚型。阐述选择性作用于肾上腺素受体药物的开发及其临床应用情况，详细介绍了几种新型的选择性肾上腺素受体药物。     

大鼠心室肌M3受体与间隙连接蛋白43作为抗心律失常药物靶点的综合研究

目的在蛋白质分子水平研究心律失常相关蛋白质M₃受体与间隙连接蛋白43之间的结构相互作用，并为其作为筛选药物靶点提供依据。方法通过免疫组化结合激光共聚焦显微镜，及免疫沉淀与免疫印迹技术，研究M受体与间隙连接蛋白43的结构性共定位关系。结果证实了大鼠心室肌细胞膜蛋白中M₁～M₅等5个亚型的存在；观察到大鼠单个心肌细胞膜上M₃受体与间隙连接蛋白43的结构性共定位；发现M受体各亚型与间隙连接蛋白43均存在结构整合，且一定浓度离子型去垢剂可破坏M₃受体与间隙连接蛋白43的结构整合关系，并进一步发现参与M₃受体结构整合的是间隙连接蛋白43的磷酸化形式。结论大鼠心室肌M受体亚型与间隙连接蛋白43的磷酸化形式存在结构性共定位关系，且可被一定浓度离子型去垢剂破坏。     

选择性毒蕈碱受体激动剂的研究

随着毒蕈碱受体（M受体）亚型及能甄别这些受体亚型的化合物的不断发现,为寻找新型、高效、选择性高且副作用小的用于治疗阿尔茨海默病的M受体激动剂提供了理论依据。综述选择性M受体激动剂槟榔碱及其衍生物的结构修饰、构效关系以及临床研究新进展,其中结构改造主要包括四氢吡啶环的单或双氮杂环取代及以肟醚基、醚基或五元杂环作为生物电子等排体取代酯基。     

Muscarinic interactions of bisindolylmaleimide analogues

We have used radioligand binding studies to determine the affinities of seven bisindolylmaleimide analogues, six of which are selective inhibitors of protein kinase C, at human muscarinic M₁–M₄ receptors. The compounds were most potent at M₁ receptors, and Ro-31-8220 was the most potent analogue, with a K_d of 0.6 μM at M₁ receptors. The weakest compounds, bisindolylmaleimide IV and bisindolylmaleimide V, had K_d values of 100 μM. If it is necessary to use protein kinase C inhibitors at concentrations of 10 μM or more in studies involving muscarinic receptors then bisindolylmaleimide IV may be the most appropriate inhibitor to use.     

Prejunctional M1 and postjunctional M3 muscarinic receptors in the circular muscle of the guinea-pig ileum

The effects of subtype-selective muscarinic receptor antagonists on electrically evoked release of acetylcholine and muscle contraction were compared in circular muscle preparations of the guinea-pig ileum. Incubation of the preparation with [³H]choline resulted in the formation of [³H]acetylcholine. Electrical stimulation caused the release of [³H]acetylcholine which was abolished by tetrodotoxin and omission of calcium from the medium. 5-Hydroxytryptamine (10 M) and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (300 M) did not change acetylcholine release. The muscarinic antagonists pirenzepine (M1 selective), AF-DX 116 (M2 selective) and hexahydrosiladifenidol (M3 selective) caused concentration-dependent increases in the evoked release of acetylcholine, and inhibitions of the circular muscle contraction. The postjunctional affinity constants (pA₂ values) obtained for hexahydrosiladifenidol (8.06), pirenzepine (6.95) and AF-DX 116 (6.60) identified the muscular receptor as an M3 subtype. Pirenzepine was more potent in facilitating the evoked release than hexahydrosiladifenidol and AF-DX 116. These findings suggest that the release of acetylcholine in the circular muscle is inhibited by M1 muscarinic autoreceptors whereas muscle contraction is mediated by M3 receptors.     

Effects of phorbol ester on carbachol-induced contraction in bovine ciliary muscle: possible involvement of protein kinase C

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M₃ subtype. Acute exposure to phorbol esters (1 μM phorbol 12,13-dibutyrate, PDB, or 0.1 μM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 μM) and H₇ [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 μM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [³H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Muscarinic receptor antagonists for the treatment of chronic obstructive pulmonary disease

Introduction: Long-acting muscarinic receptor antagonists (LAMAs) are central to the treatment of chronic obstructive pulmonary disease (COPD) because of the important role of the cholinergic system in the pathophysiology of this disorder.

Areas covered: LAMAs show clinically meaningful effects in lung function and other important supportive outcomes, such as exacerbations, health-related quality of life, dyspnea, rescue medication use and nighttime/early morning symptoms, and are safe. Muscarinic receptor antagonists could exert other useful actions such as the anti-inflammatory, anti-remodeling, mucus-modifying, and anti-cough effects. Concerns have been raised about possible associations of muscarinic receptor antagonists with cardiovascular morbidity and mortality. Muscarinic receptor antagonists can be combined with long-acting β₂-agonists (LABAs), inhaled corticosteroids (ICSs) and LABA + ICS. There are number of LAMAs that are being identified but only few have reached the clinical development. Fixed-dose combination formulations of both novel and established LAMAs with LABAs are being developed.

Expert opinion: There are important questions concerning the use of LAMAs in the treatment of patients suffering from stable COPD that need conclusive answers.     

MUSCARINIC RECEPTOR DIFFERENTIATION

1. Several selective antagonists are available to differentiate between muscarinic receptors. 2. Further subdivision of M1 and M2 muscarinic receptors appears possible and is supported by studies with cloned receptors. 3. Reasons for differences between affinity constants determined in functional and binding studies and whether receptor subtypes couple exclusively with a particular cellular mechanism are still to be determined.     

Effect of subchronic galantamine treatment on neuronal nicotinic and muscarinic receptor subtypes in transgenic mice overexpressing human acetylcholinesterase

Overexpression of acetylcholinesterase (AChE) in mice causes cholinergic deficits with memory impairment. In this study, AChE overexpressing (hAChE-Tg) and control (FVB/N) mice were treated with the AChE inhibitor (AChEI) galantamine (4 mg/kg/day) for 10 days. The concentration of galantamine in plasma was 75-80 ng/ml. The inhibition of AChE was 20% in red blood cells (RBC) and 30% in brain cortical tissue. A significant increase in [(3)H]cytisine (alpha4 nicotinic receptor) binding was measured in the CA1 and CA3 area of the hippocampus of FVB/N mice following galantamine treatment. Similarly, a significant increase in [(125)I]alphabungarotoxin (alpha7 nicotinic receptor) binding was found in the frontal cortex, retrosplenial gr. cortex, motor cortex and thalamus in galantamine treated FVB/N compared to saline treated mice. No significant changes in nicotinic receptor binding sites were observed in galantamine treated hAChE-Tg mice. Significant decreases in the muscarinic receptors measured by [(3)H]AF-DX-384 (M2 muscarinic receptor) and [(3)H]pirenzepine (M1 muscarinic receptor) were observed in several brain regions of galantamine treated FVB/N and hAChE-Tg mice. This study shows regional and receptor subtype specific changes in the nicotinic receptor subtypes compared to the muscarinic receptors following galantamine treatment in FVB/N and hAChE-Tg mice.     

海马M_2受体调节杏仁核内谷氨酸的释放与学习记忆的研究

目的研究海马内M1和M2受体对学习记忆功能的影响及其可能机制。方法SD大鼠分为3组,分别海马内注射等体积的M1-ASODN(M1-AS组)、M2-ASODN(M2-AS组)和生理盐水(对照组),以被动逃避反应的步入潜伏期作为衡量学习记忆功能的指标,以CZE-LIFD法检测杏仁核内游离谷氨酸的浓度。结果M1-ASODN缩短步入潜伏期,为对照组的25%(P<0.01),M2-ASODN使步入潜伏期较对照组增加了75%(P<0.05)。双侧海马内注射ASODN后与对照组相比,M2-AS组杏仁核内谷氨酸的含量增加164%(P<0.01),而M1-AS组无变化(P>0.05)。结论海马内M受体参与恐惧性记忆的调节,M2受体抑制学习记忆,M1受体促进学习记忆。海马内M2受体可能通过调节杏仁核内谷氨酸含量的变化介导被动逃避反应的记忆过程。     

吗啡依赖大鼠脊髓和脑干毒蕈碱受体亚型基因的表达

目的观察吗啡依赖大鼠脊髓和脑干毒蕈碱型乙酰胆碱受体m_1～5的表达。方法以β-actin为内标,用RT-PCR方法检测m_1～5的表达。结果吗啡依赖大鼠脊髓m_1～5受体和脑干中m₁和m₂表达较正常对照组明显升高,注射纳洛酮1 h后脊髓m_1～4和脑干m₁表达较依赖组减少。东莨菪碱(0.5 mg·kg^-1,ip)或呱伦西平(10 mg·kg^-1,ip)明显减少吗啡戒断症状,呱伦西平处理后脊髓m₁,m₂,m₃和m₅表达较戒断对照组增加,东莨菪碱处理后脊髓m₂,m₃和m₄表达增加。结论脊髓M受体适应性改变是吗啡戒断症状表达的生物学基础。     

Mapping the Ligand Binding Pocket of the Human Muscarinic Cholinergic Receptor Hml: Contribution of Tyrosine-82

The ligand binding pocket of many G protein-coupled receptors is thought to be located within the core formed by their seven transmembrane domains (TMDs). Previous results suggested that muscarinic antagonists bind to a pocket located toward the extracellular region of the TMDs, primarily at TMDs 2, 3, 6, and 7. Tyrosine-82 (Y82) is located in TMD2 only one helical turn from the presumed membrane surface of Hm1, whereas a phenylalanine (F124) is found in the equivalent position of the closely related Hm3. In order to determine the contribution of Y82 to Hm1 ligand binding and selectivity versus Hm3, we constructed the point mutation Y82 F of Hm1 and measured binding affinities of various ligands, with 3H-N-methylscopolamine (3H-NMS) as the tracer. The Hm1 wild-type receptor and the Y82F mutant were transfected into human embryonic kidney U293 cells. Whereas the affinities of NMS, carbachol, and atropine were either unchanged (carbachol) or enhanced by less than twofold (atropine and NMS), the affinity of the Hm1-selective piren-zepine was reduced threefold by the Y82 F mutation. These changes parallel affinity differences of Hm1 and Hm3, indicating that the Y82 F mutation affects the binding pocket and that Y82 contributes to the binding selectivity among closely related muscarinic receptors.     

Isolated porcine bronchi provide a reliable model for development of bronchodilator anti-muscarinic agents for human use

BACKGROUND AND PURPOSE: In human airways, muscarinic acetylcholine receptors (mAChRs) exert a predominant role in the control of airways resistance and anti-muscarinic agents are currently included in the pharmacological treatment of chronic obstructive pulmonary disease (COPD). However, the development of more effective mAChR antagonists is hampered by considerable species variability in the ultrastrucural and functional control of airway smooth muscle, making extrapolation of any particular animal model questionable. This study was designed to characterize the mAChRs in a bronchial preparation from pigs, animals considered to provide close models of human biology. EXPERIMENTAL APPROACH: Smooth muscle bronchial strips were examined by electron microscopy in order to compare their neuromuscular structure with that of human bronchi and used to study the affinity of a series of selective mAChR antagonists, estimated as pKis in competition binding assays with NMS and pA2, by Schild analysis, in contractile experiments. KEY RESULTS: Pharmacodynamic binding parameters and affinity profiles of a series of antagonists were consistent with the presence of a majority of M2 mAChRs along with a minor population of M3 mAChRs. Functionally, the highly significant correlation between postjunctional pA2 affinities and corresponding affinity constants at human recombinant M1-M5 subtypes indicated that smooth muscle contraction in porcine bronchi, as in human bronchi, was dependent on the M3 subtype. CONCLUSION AND IMPLICATIONS: Based on the characterization of mAChRs, isolated porcine bronchi provide an additional experimental model for development of mAChR antagonists for the treatment of human airway dysfunctions.