Muscarinic acetylcholine receptors interactions: competition binding studies with agonists and antagonists
Muscarinic acetylcholine receptors interactions: competition binding studies with agonists and antagonists
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毒蕈碱乙酰胆碱受体相互作用：激动剂和拮抗剂的竞争结合研究
DOI：
10.1016/0006-8993(77)90936-2
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发表时间：
1977
期刊：
影响因子：
2.9
通讯作者：
M. Sokolovsky
中科院分区：
文献类型：
--
作者：
Y. Kloog;M. Sokolovsky
最近使用有效且高度特异性的拮抗剂 N-甲基-4-哌啶基苯甲酸 (4NMPB) 检测了与从小鼠大脑中制备的毒蕈碱受体的结合，该拮抗剂经过放射性标记，具有高比活性％a。正如所讨论的，这种拮抗剂的结合测定基于 Yamamura 和 Snyder 1 描述的过滤技术，使我们能够结合详细的动力学研究直接研究配体特异性。本研究旨在通过其他非放射性胆碱能配体与放射性配体 4NMPB 竞争的能力和性质，进一步探索毒蕈碱受体的一些可能的体外配体相互作用。其中包括 3-奎宁环苯甲酸 (QNB) 的两种对映体和外消旋体形式以及激动剂氧化震颤素。 N-甲基-4-哌啶苯甲酸 (4NMPB) 通常通过催化氚交换进行标记，如其他地方所述 2。比活性为 6 Ci/mmole。氧化震颤素得自 Aldrich 和 QNB，其异构体的制备方法如其他地方所述 6。化合物的化学和放射化学纯度通过分析薄层色谱法（Merck Silica 60 板，0.25 mm 厚）在两种溶剂系统（正丁醇、乙酸、水（4:1:1）；和氯仿、丙酮、二乙胺（5:4:1））中测定。所有化合物的纯度> 97~，i。雄性 ICR 小鼠（20-25 克）在两分钟内被斩首并取出大脑。使用电机驱动的聚四氟乙烯研杵(950rpm)在玻璃均质器中将每个大脑分别在冰冷的0.32M蔗糖中均质化，以产生10%匀浆(w/v)，将其以1000×g(Sorvall RC2-B)离心10分钟。丢弃沉淀，所得上清液（1 美元）用于结合测定。在 [3H] 4NMPB 的标准结合测定中，将 50/A 级分 $1 在 2 ml 改良 Krebs 溶液（25 mM Tris.HCl、118 mM NaCl、4.69 mM KCl、1.9 mM CaCl2、0.54 mM MgCl2、1.0 mM NaH2PO~、11.1 mM 葡萄糖）中于 25°C 孵育。 7.4，含有标记和未标记的配体。孵育30分钟后，加入3ml冰冷的Krebs溶液，并用玻璃过滤器（Whatman GF/C，直径25mm）通过真空快速过滤内容物。将过滤器用 3 ml 冰冷的 Krebs 溶液洗涤 3 次，并放入装有 5 ml 闪烁液的小瓶中
The binding to muscarinic receptor prepared from mouse brain was recently examined using a potent and highly specific antagonist, N-methyl-4-piperidylbenzilate (4NMPB) which was radiolabeled to a high specific activity% a. As discussed, the binding assay with this antagonist, which is based on the filtration technique described by Yamamura and Snyder 1, enabled us to investigate directly the ligand specificity in conjunction with detailed kinetic studies. The present study was undertaken to explore further some of the possible in vitro ligand interactions of the muscarinic receptors by the capacity and nature of other non-radioactive cholinergic ligands to compete with the radioactive ligand 4NMPB. These include the two enantiomers and the racemate form of 3-quinuclidinylbenzilate (QNB) and the agonist oxotremorine. N-methyl-4-piperidylbenzilate (4NMPB) was generally labeled by catalytic tritium exchange as described elsewhere 2. The specific activity was 6 Ci/mmole. Oxotremorine was obtained from Aldrich and QNB and its isomers were prepared as described elsewhere 6. The chemical and radiochemical purities of the compound were determined by analytical thin-layer chromatography (Merck Silica 60 plates, 0.25 mm thickness) in two solvent systems (n-butanol, acetic acid, water (4: 1: 1); and chloroform, acetone, diethylamine (5: 4: I)). The purity of all compounds was> 97~, i. Male ICR mice (20-25 g) were decapitated and their brains removed within two minutes. Each brain was homogenized separately in ice-cold 0.32 M sucrose using a motor driven teflon pestle (950 rpm) in a glass homogenizer to yield a l0~ homogenate (w/v), which was centrifuged for 10 min at 1000× g (Sorvall RC2-B). The pellet was discarded and the resultant supernatant fluid ($1) was used for binding assay. In the standard binding assay for [3H] 4NMPB, 50/A of fraction $1 were incubated at 25 C in 2 ml modified Krebs solution (25 mM Tris. HCl, 118 mM NaCI, 4.69 mM KCI, 1.9 mM CaC12, 0.54 mM MgCI2, 1.0 mM NaH2PO~, 11.1 mM glucose) pH 7.4, containing the labeled and unlabeled ligands. After 30 rain of incubation, 3 ml of icecold Krebs solution were added and the contents were filtered rapidly by vacuum with glass filter (Whatman GF/C, 25 mm diameter). The filters were washed three times with 3 ml ice-cold Krebs solution, and placed in vials containing 5 ml scintillation liquid