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

摘要

In order to compare the potential selectivity of R-(-)-DM-phencynonate hydrochloride with its raeemate (±)-DM-pbencynonate hydrochloride on acetylcholine muscarinie receptor subtypes, the five human acetylcholine muscarinic receptor subtypes (M1-M5) (CHO-hml-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-hml-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-hml-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 (nil) 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.%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-(-)去甲基盐酸苯环千酯及其消旋体对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,Ms:1179±87.0 pmol/mg·pro,[3H]-NMS的Kd值M1-M5分别足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犁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-(-)去甲基盐酸苯环千酯对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.