R-(-)去甲基盐酸苯环壬酯及其消旋体对M受体亚型的选择性研究

R-(-)去甲基盐酸苯环壬酯及其消旋体对M受体亚型的选择性研究

王丽韫, 孙洪良, 牟男, 仲伯华, 刘克良, 郑建全^*

军事医学科学院毒物药物研究所, 北京 100850

收稿日期:2008-11-19 修回日期:2006-05-10 出版日期:2009-06-15 发布日期:2009-06-15
通讯作者: 郑建全*

Characterization of subtype selection properties of R-(-)-DM-phencynonate hydrochloride and its racemate on muscarinic receptors

Li-Yun Wang, Hong-Liang Sun, Nan Mou, Bo-Hua Zhong, Ke-Liang Liu, Jian-Quan Zheng^*

Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China

Received:2008-11-19 Revised:2006-05-10 Online:2009-06-15 Published:2009-06-15
Contact: Jian-Quan Zheng*

摘要/Abstract

摘要：

构建人源性5种乙酰胆碱M受体亚型(M1-M5)在中国仓鼠卵巢细胞(CHO-K1)上表达(CHO-hml-5R)稳定表达株对比分析R-(-)去甲基盐酸苯环壬酯及其消旋体对M受体亚型的选择性。在稳定培养2代CHO-hml-5R细胞上, 利用RT-PCR方法证实目的基因的表达, 检测到5种乙酰胆碱M受体亚型的mRNA, 应用放射性配体受体饱合试验检验分析各M受体亚型对氚标东莨宕碱([³H]-NMS)的亲合力及结合饱和度, 试验表明[³H]-NMS对CHO-hml-5R的最大结合力(Bmax值)分别是M1: 2110±165.1, M2: 861±90.0, M3: 1127±34.0, M4: 1055±61.5, M5: 1179±87.0 pmol/mg·pro, [³H]-NMS的Kd值M1-M5分别是0.97±0.22, 1.16±0.14, 0.99±0.06, 0.56±0.08, 1.12±0.06 nM。结果表明5种M受体亚型 (M1-M5) 稳定住株成功表达, 竞争抑制试验评价R-(-)去甲基盐酸苯环壬酯及其消旋体的亚型选择特性, 试验表明R-(-)去甲基盐酸苯环壬酯对M4受体亚型 (pD2 = 7.48) 具有较高的选择性, 高于其他M受体亚型M1(pD₂= 6.20), M₂(pD₂ = 5.99), M₃ (pD₂= 5.99), M₅ (pD₂= 6.70), 而去甲基苯环壬酯的消旋体对五种受体亚型未表现出明显的亚型选择型。R-(-)去甲基盐酸苯环壬酯及消旋体对五个受体亚型的Hill系数(nH)<1, 均表现出对M 受体的变构调节作用。该研究发现R-(-)去甲基盐酸苯环壬酯对M4亚型具有较高的选择型拮抗作用,R-(-)去甲基盐酸苯环壬酯及其消旋体与CHO-hml-5R细胞的M亚型之间的作用存在变构调节机制。

关键词: 光学异构体, 毒蕈碱乙酰胆碱受体, 受体亚型选择性, 放射性配体分析

Abstract: In order to compare the potential selectivity of R-(-)-DM-phencynonate hydrochloride with its racemate (±)-DM-phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylcholine muscarinic receptor subtypes (M1–M5) (CHO-hm1-5R) were cloned and expressed in Chinese hamster ovary (CHO-K1) cell line. The specific mRNAs of the five acetylcholine muscarinic receptor subtypes were detected by the reverse transcription-polymerase chain reaction (RT-PCR) method, demonstrating the definite expression of muscarinic receptor subtype genes (CHO-hm1-5R). The affinity and saturability of different muscarinic receptor subtypes to [³H] N-methylscopolamine ([³H]-NMS) were obtained by radioligand binding assay. Equilibrium binding assay revealed that the maximum binding capacity of [³H]-NMS (Bmax value) to CHO-hm1-5R were 40.22±3.23, 24.53±4.11, 29.65±2.65, 25.41±2.46, 32.78±4.81 pmol/mg·protein, respectively. Kd values of [³H]-NMS to muscarinic receptors M1 to M5 were 0.97±0.22, 1.16±0.14, 0.99±0.06, 0.56±0.08, 1.12±0.06 nM, respectively. R-(-)-DM-phencynonate hydrochloride was found to block the M4 receptor with a much higher potency (pD2 = 7.48) than those displayed on M1 (pD2 = 6.20), M2(pD2 = 5.99), M3(pD2 = 5.99) and M5(pD2 = 6.70) subtypes. However, for (±)-DM-phencynonate hydrochloride, no significant subtype receptor selectivity was found. Both (±)-DM- and R-(-)-DM-phencynonate hydrochloride showed allosteric effects on muscarinic receptors, the Hill coefficient (nH) of five receptor subtypes was less than 1, respectively. The results revealed that R-(-)-DM-phencynonate hydrochloride showed selectivity torwards M4 subtype, and there were allosteric effects for both R-(-)-DM-phencynonate hydrochloride and (±)-DM-phencynonate hydrochloride on muscarinic receptors.

Key words: Optical isomers, Optical isomers, Muscarinic acetylcholine receptors, Muscarinic acetylcholine receptors, Subtype receptor selectivity, Subtype receptor selectivity, Radioligand binding assay, Radioligand binding assay

中图分类号:

R962.2

Supporting:

Foundation item: National Natural Science Foundation of China (Grant No. 30672445).

^*Corresponding author. Tel.: 86-10-66931624; fax: 86-10-68211656;

e-mail: zhengjq@bmi.ac.cn

王丽韫, 孙洪良, 牟男, 仲伯华, 刘克良, 郑建全^*.

R-(-)去甲基盐酸苯环壬酯及其消旋体对M受体亚型的选择性研究

[J]. .

Li-Yun Wang, Hong-Liang Sun, Nan Mou, Bo-Hua Zhong, Ke-Liang Liu, Jian-Quan Zheng^* . Characterization of subtype selection properties of R-(-)-DM-phencynonate hydrochloride and its racemate on muscarinic receptors [J]. .

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