Chen N H, Ding J H, Wang Y L, Reith M E
Department of Pharmacology, School of Basic Medical Sciences, Nanjing Medical University, People's Republic of China.
J Neurosci Methods. 1997 Aug 22;75(2):179-86. doi: 10.1016/s0165-0270(97)00070-8.
Binding of the cocaine analog 3 beta-(4-[125I]iodophenyl)tropane-2 beta-carboxylic acid isopropyl ester ([125I]RTI-121) to filters was studied in order to assess its contribution to labeling dopamine transporters on rat striatal synaptosomal membranes in filtration assays. Filter binding (FB) decreased with increasing Na+. Cocaine (30 and 100 microM) substantially reduced the FB at low Na+ with much less of an effect at higher Na+. Similar results were observed with K+. At 10 mM Na+, RTI-121 (1 microM) displaced the FB to the same degree as cocaine (100 microM); mazindol (10 microM), BTCP (1 microM), and dopamine (1 mM) did so to a lesser degree; and GBR12935 (1 microM) did not. If the specific binding was calculated without deducting the FB displaced with cocaine (DFB), the DFB accounted for 15-19% of the 'specific binding' at 10 mM Na+ in the assay. This additional binding population resulted in an upward curvilinear Scatchard plot and incorrect estimation of equilibrium binding parameters and ion potencies. At 10 mM Na+, without deduction of DFB, the high-affinity component had a Kd of 3.4 nM and Bmax of 2.4 pmol/mg protein, and the respective values for the low-affinity component were 84 nM and 16 pmol/mg protein; when DFB was deducted, one component was observed with a Kd of 4.4 nM and Bmax of 3.3 pmol/mg protein. The presence of higher Na+ in the assay diminished these artifacts. Thus, at 150 mM Na+, without deduction of DFB, there was one binding component with a Kd of 3.9 nM and Bmax of 4.6 pmol/mg protein; these values became 3.3 nM and 3.8 pmol/mg protein when DFB was deducted.
为了评估可卡因类似物3β-(4-[¹²⁵I]碘苯基)托烷-2β-羧酸异丙酯([¹²⁵I]RTI-121)在过滤试验中对大鼠纹状体突触体膜上多巴胺转运体标记的贡献，研究了其与滤膜的结合情况。滤膜结合(FB)随Na⁺浓度升高而降低。可卡因(30和100μM)在低Na⁺浓度时显著降低FB，在高Na⁺浓度时作用较小。K⁺也观察到类似结果。在10 mM Na⁺时，RTI-121(1μM)与可卡因(100μM)使FB位移的程度相同；吗茚酮(10μM)、BTCP(1μM)和多巴胺(1 mM)使FB位移的程度较小；而GBR12935(1μM)则没有。如果在不扣除可卡因置换的FB(DFB)的情况下计算特异性结合，在试验中10 mM Na⁺时，DFB占“特异性结合”的15 - 19%。这种额外的结合群体导致Scatchard图呈向上的曲线，并对平衡结合参数和离子效力进行错误估计。在10 mM Na⁺时，不扣除DFB，高亲和力成分的Kd为3.4 nM，Bmax为2.4 pmol/mg蛋白质，低亲和力成分的相应值为84 nM和16 pmol/mg蛋白质；扣除DFB后，观察到一个成分，Kd为4.4 nM，Bmax为3.3 pmol/mg蛋白质。试验中较高Na⁺的存在减少了这些假象。因此，在150 mM Na⁺时，不扣除DFB，有一个结合成分，Kd为3.9 nM，Bmax为4.6 pmol/mg蛋白质；扣除DFB后，这些值变为3.3 nM和3.8 pmol/mg蛋白质。
