Roberts David J, Lin Hong, Strange Philip G
School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading, RG6 6AJ, UK.
Biochem Pharmacol. 2004 May 1;67(9):1657-65. doi: 10.1016/j.bcp.2003.12.030.
This study investigated, for the D2 dopamine receptor, the relation between the ability of agonists and inverse agonists to stabilise different states of the receptor and their relative efficacies. Ki values for agonists were determined in competition versus the binding of the antagonist [3H]spiperone. Competition data were fitted best by a two-binding site model (with the exception of bromocriptine, for which a one-binding site model provided the best fit) and agonist affinities for the higher (Kh) (G protein-coupled) and lower affinity (Kl) (G protein-uncoupled) sites determined. Ki values for agonists were also determined in competition versus the binding of the agonist [3H]N-propylnorapomorphine (NPA) to provide a second estimate of Kh. Maximal agonist effects (Emax) and their potencies (EC50) were determined from concentration-response curves for agonist stimulation of guanosine-5'-O-(3-[32S]thiotriphosphate) ([35S]GTPgammaS) binding. The ability of agonists to stabilise the G protein-coupled state of the receptor (Kl/Kh determined from ligand-binding assays) did not correlate with either of two measures of relative efficacy (relative Emax, Kl/EC50) of agonists determined in [35S]GTPgammaS-binding assays, when the data for all of the compounds tested were analysed. For a subset of compounds, however, there was a relation between Kl/Kh and Emax. Competition-binding data versus [3H]spiperone and [3H]NPA for a range of inverse agonists were fitted best by a one-binding site model. Ki values for the inverse agonists tested were slightly lower in competition versus [3H]NPA compared to [3H]spiperone. These data do not provide support for the idea that inverse agonists act by binding preferentially to the ground state of the receptor.
本研究针对D2多巴胺受体，调查了激动剂和反向激动剂稳定受体不同状态的能力与其相对效能之间的关系。在与拮抗剂[3H]司哌罗宁的结合竞争中测定激动剂的Ki值。竞争数据通过双结合位点模型拟合效果最佳（溴隐亭除外，其通过单结合位点模型拟合效果最佳），并确定了激动剂对高亲和力（Kh）（G蛋白偶联）和低亲和力（Kl）（G蛋白非偶联）位点的亲和力。还在与激动剂[3H]N-丙基去甲阿朴吗啡（NPA）的结合竞争中测定激动剂的Ki值，以提供Kh的第二个估计值。通过激动剂刺激鸟苷-5'-O-(3-[32S]硫代三磷酸)（[35S]GTPγS）结合的浓度-反应曲线确定最大激动剂效应（Emax）及其效能（EC50）。当分析所有测试化合物的数据时，激动剂稳定受体G蛋白偶联状态的能力（通过配体结合试验确定的Kl/Kh）与在[35S]GTPγS结合试验中确定的激动剂相对效能的两种测量方法（相对Emax，Kl/EC50）均不相关。然而，对于一部分化合物，Kl/Kh与Emax之间存在关系。一系列反向激动剂与[3H]司哌罗宁和[3H]NPA的竞争结合数据通过单结合位点模型拟合效果最佳。与[3H]司哌罗宁相比，所测试的反向激动剂在与[3H]NPA的竞争中Ki值略低。这些数据不支持反向激动剂通过优先结合受体基态起作用的观点。
