Xu C, Coffey L L, Reith M E
Department of Basic Sciences, University of Illinois College of Medicine, Peoria 61656.
Biochem Pharmacol. 1995 Jan 31;49(3):339-50. doi: 10.1016/0006-2952(94)00485-5.
Translocation of [3H]dopamine and binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]-tropane ([3H]WIN 35,428) were measured in crude synaptosomal preparations from rat striatum under identical conditions of assay buffer (phosphate-Krebs) and temperature (25 degrees). [3H]Dopamine uptake as a function of time was close to linear for at least 8 min, whereas [3H]WIN 35,428 binding had reached equilibrium within 1 min and remained at its plateau value for at least 20 min. The following inhibitors were tested in uptake and binding assays run in parallel with the same synaptosomal preparation: cocaine, WIN 35,428, benztropine, nomifensine, mazindol, methylphenidate, N-[1-(2-benzo[b]-thiophenyl)cyclohexyl]piperidine (BTCP), Lu 19-005 (Indatraline), 1-(2-(di(4-fluorophenyl)-methoxy)-ethyl)-4-(3-phenyl-2-propyl)piperazine (GBR 12909), 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenyl-2-propyl)piperazine (GBR 12935) and 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo [1,2-a]quinoxaline (CGS 12066B). When present together with [3H]dopamine or [3H]WIN 35,428 for 8 min, the observed binding IC50 values were generally higher (average 1.4-fold) than the uptake IC50 values, with a significant y-axis intercept in linear regression analysis of binding on uptake IC50. For slowly equilibrating inhibitors, estimates of uptake IC50 values were overestimates, and relatively lower values were obtained by monitoring [3H]dopamine uptake for 1 min only during the last minute of the 8-min presence of inhibitor; under these conditions, binding over uptake IC50 ratios were on the average 2.3. Kinetic calculations, taking into account both radioligand and inhibitor equilibration kinetics, indicated that the latter comparison between binding and uptake measurements was most relevant, and suggested the involvement of complexities beyond simple competitive inhibition of dopamine transport, such as different binding domains for substrate and blocker recognition, or spare receptors for blockers. The present data indicate that binding over uptake IC50 ratios should be interpreted with caution, depending on the experimental conditions used to measure these ratios.
在相同的测定缓冲液（磷酸盐 - 克雷布斯缓冲液）和温度（25摄氏度）条件下，测量大鼠纹状体粗制突触体制剂中[3H]多巴胺的转运以及2β - 甲氧羰基 - 3β - （4 - 氟苯基）[3H] - 托烷（[3H]WIN 35,428）的结合。[3H]多巴胺摄取随时间的变化在至少8分钟内接近线性，而[3H]WIN 35,428结合在1分钟内达到平衡，并在至少20分钟内保持在平台值。在与相同突触体制剂平行进行的摄取和结合测定中测试了以下抑制剂：可卡因、WIN 35,428、苯海索、诺米芬辛、马吲哚、哌甲酯、N - [1 - （2 - 苯并[b] - 噻吩基）环己基]哌啶（BTCP）、Lu 19 - 005（茚达曲林）、1 - （2 - （二（4 - 氟苯基） - 甲氧基） - 乙基） - 4 - （3 - 苯基 - 2 - 丙基）哌嗪（GBR 12909）、1 - （2 - （二苯基甲氧基） - 乙基） - 4 - （3 - 苯基 - 2 - 丙基）哌嗪（GBR 12935）和7 - 三氟甲基 - 4 - （4 - 甲基 - 1 - 哌嗪基） - 吡咯并[1,2 - a]喹喔啉（CGS 12066B）。当与[3H]多巴胺或[3H]WIN 35,428一起存在8分钟时，观察到的结合IC50值通常高于摄取IC50值（平均1.4倍），在结合对摄取IC50的线性回归分析中有显著的y轴截距。对于缓慢平衡的抑制剂，摄取IC50值的估计值被高估，通过仅在抑制剂存在时间的8分钟的最后一分钟监测[3H]多巴胺摄取1分钟获得相对较低的值；在这些条件下，结合与摄取IC50的比率平均为2.3。考虑到放射性配体和抑制剂平衡动力学的动力学计算表明，结合与摄取测量之间的后一种比较最相关，并表明涉及超出多巴胺转运简单竞争性抑制的复杂性因素，例如底物和阻滞剂识别的不同结合域，或阻滞剂的备用受体。目前的数据表明，结合与摄取IC50比率的解释应谨慎，这取决于用于测量这些比率的实验条件。
