Translocation of dopamine and binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane (WIN 35,428) measured under identical conditions in rat striatal synaptosomal preparations. Inhibition by various blockers.
Translocation of dopamine and binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane (WIN 35,428) measured under identical conditions in rat striatal synaptosomal preparations. Inhibition by various blockers.
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在相同条件下，在大鼠纹状体突触体制备物中测量多巴胺的易位和 2β-甲氧甲氧基-3β-(4-氟苯基)托烷 (WIN 35,428) 的结合。
DOI：
10.1016/0006-2952(94)00485-5
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发表时间：
1995
影响因子：
5.8
通讯作者：
Reith,ME
中科院分区：
文献类型：
--
作者：
Xu,C;Coffey,LL;Reith,ME
在相同的实验缓冲液（磷酸盐- krebs）和温度（25°）条件下，测定了大鼠纹状体粗突触体制剂中[3H]多巴胺的易位和2 β-碳甲氧基-3 β-（4-氟苯基）[3H]-tropane （[3H]WIN 35,428）的结合。[3H]多巴胺摄取作为时间的函数至少在8分钟内接近线性，而[3H]WIN 35,428结合在1分钟内达到平衡，并在其平台值保持至少20分钟。以下抑制剂在摄取和结合试验中进行了测试，与相同的突触体制备平行进行：可卡因，win35,428，苯托品，诺米芬辛，马辛多，哌醋甲酯，和灰[1-（2-苯并[b]-噻吩基）环己基]哌啶（BTCP）， Lu 19-005（茚丙林），1-（2-（4-氟苯基）-甲氧基）-乙基)-4-（3-苯基-2-丙基）哌嗪（GBR 12909）， 1-（2-（二苯甲氧基）-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[1,2-a]喹诺啉（CGS 12066B）。当与[3H]多巴胺或[3H]WIN 35,428一起作用8 min时，观察到的结合ic50值普遍高于摄取ic50值（平均1.4倍），在结合摄取ic50的线性回归分析中有显著的y轴截距。对于缓慢平衡抑制剂，摄取ic50值估计过高，仅在抑制剂存在8分钟的最后一分钟监测[3H] 1分钟多巴胺摄取，获得相对较低的值；在这些条件下，结合与摄取的ic50比平均为2.3。考虑到放射配体和抑制剂平衡动力学的动力学计算表明，结合和摄取测量之间的后一种比较最相关，并表明复杂性超出了多巴胺转运的简单竞争性抑制，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，根据用于测量这些比率的实验条件，应该谨慎地解释结合超过摄取的ic50比率。
Translocation of [3H]dopamine and binding of 2 β-carbomethoxy-3 β-(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 °). [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, ndash[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 ic50values were generally higher (average 1.4-fold) than the uptake ic50values, with a significant y-axis intercept in linear regression analysis of binding on uptake ic50. For slowly equilibrating inhibitors, estimates of uptake ic50values 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 ic50ratios 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 ic50ratios should be interpreted with caution, depending on the experimental conditions used to measure these ratios.