知母、黄芪活性成分对M_2受体与G蛋白偶联的影响

Effect of active compounds of Zhimu and Huangqi on the coupling of M_2 receptor to G protein

目的探讨知母活性成分 ZMS、黄芪活性成分 HQ 和两者联合应用对 M_2受体和 G 蛋白偶联的影响。方法以转染 M_2受体的 CHOm2细胞为模型,用~3H-N-甲基东莨菪碱(NMS)结合法测定 M_2受体的密度,用不被 GTP 酶水解的^(35)S-γ位硫代 GTP(GTPγS)在 M 受体激动剂氨甲酰胆碱作用下与 G 蛋白进行不可逆结合,测定结合部分的放射性,以反映 M_2受体与 Gα蛋白的偶联活性。分别测定加入 ZMS 及 HQ(10^(-5)mol/L)对偶联的影响,并以加入等体积二甲基亚砜(DMSO)为对照。以偶联活性被 M_2受体密度除,所得的商反映偶联效率。结果 ZMS 和 HQ 在浓度为10^(-5)mol/L 时都能提高 M_2受体密度和 M_2受体与 G 蛋白的偶联活性。HQ 能提高偶联效率,ZMS 无此作用,两者联合应用时偶联效率的提高更显著(偶联效率依次为1.102,1.011,1.324)。结论 ZMS 和 HQ 增强M_2受体和 G 蛋白偶联活性的机制不同,前者主要提高 M_2受体密度,从而使 G 蛋白的偶联量增加,后者则除提高 M_2受体密度外还提高偶联效率。两者联合应用时偶联效率的提高更显著。

Objective The previous studies suggested that Zhimu （ZMS） and Huangqi （HQ）, both being herb medicines, had beneficial effects on function of aged brains. The current study was designed to further explore the effect of activo compounds of ZMS and HQ on the coupling of M2 receptor to G protein. Methods CHOm2 cell line transfected with cDNA of M2 receptor was used as a model. The density of M2 receptors was determined with ^3H-N-methylscopolamine （NMS） , and the activation of G protein with ^35 S-GTPγS binding assays after carbachol stimulation. The coupling efficacy of M2 receptor to G protein was expressed as ^35S-GTPγS stimulated binding divided by M2 receptor density. The effects of ZMS and HQ were determined after adding into the cell culture medium separately or in combination at a concentration of 10^-5 moL/L （in DMSO）. The same volume of DMSO was added in the vehicle group as control. Results The density of M2 receptor as well as the amount of G protein coupled to M2 receptor was elevated in ZMS, HQ, and ZMS ＋ HQ cells as compared with that in vehicle treated cells. The coupling efficacy in HQ group was significantly higher than control （ 1. 102 vs 1. 001, t = 3. 510,P 〈 0.05） , while the coupling efficacy in ZMS group （ 1.011 ） showed no difference（ t = 0. 273, P 〉 0. 1 ）. The combined use of ZMS and HQ yielded much higher coupling efficacy （ 1. 324） than ZMS （ t = 3. 037, P 〈 0.05 ） or HQ （ t = 2. 899, P 〈 0.05 ） alone. Conclusions In the aged CHOm2 cells, both ZMS and HQ might increase the coupling of M2 reeeptor to G protein. ZMS mainly increased the M2 receptor density while HQ increased the coupling efficacy as well. Combined application of ZMS and HQ might enhance the coupling efficacy than any single treatment.