Rothman Richard B, Dersch Christina M, Carroll F Ivy, Ananthan Subramaniam
Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland 21224, USA.
Synapse. 2002 Mar 15;43(4):268-74. doi: 10.1002/syn.10046.
Using [125I]RTI-55 to label the dopamine transporter (DAT), our laboratory has consistently detected one binding site as well as one component of [3H]DA uptake. We report here the identification of a novel partial inhibitor of [3H]DA uptake and DAT binding (SoRI-9804). [125I]RTI-55 binding to the DAT (mouse caudate, rat caudate, HEK cells expressing the cloned DAT), the 5-HT transporter (rat brain), and [3H]DA uptake (rat caudate synaptosomes) were conducted using published procedures. 4-[(Diphenylmethyl)amino]-2-phenylquinazoline (SoRI-9804) was essentially inactive at SERT binding and resolved two DAT binding components in all three tissues, having high affinity (mean Ki of 465 nM) for about 40% of the binding sites and an essentially immeasurable Ki (> 100 microM) for the remaining 60% of the binding sites. The [3H]DA uptake experiments indicated that about 50% of uptake was SoRI-9804-sensitive. Saturation binding experiments showed that SoRI-9804 competitively inhibited [125I]RTI-55 binding to the SoRI-9804-sensitive binding component. To determine if the two binding sites discriminated by SoRI-9804 were regulated by the MAP kinase pathway, rat caudate synaptosomes were incubated in the absence or presence of 10 microM of PD98059, which inhibits activation of the MAP kinase pathway. The results indicated that inhibition of MAPK/ERK kinase decreased the total B(max) of the DAT by 90%. Treatment with PD98059 increased the proportion of the SoRI-9804-sensitive binding component from 68-80% of the total B(max). The PD98059 experiments suggest that inhibition of MAP kinase cannot explain the differential interaction of SoRI-9804 with the DAT. Viewed collectively, the present results indicate that SoRI-9804 discriminates two components of the DA transporter. Further studies will be needed to determine the underlying mechanism of this effect and if partial inhibition of DA uptake results in any unique behavioral effects.
利用[125I]RTI-55标记多巴胺转运体（DAT），我们实验室一直检测到一个结合位点以及[3H]多巴胺摄取的一个成分。我们在此报告一种新型的[3H]多巴胺摄取和DAT结合的部分抑制剂（SoRI-9804）的鉴定。使用已发表的方法进行[125I]RTI-55与DAT（小鼠尾状核、大鼠尾状核、表达克隆DAT的HEK细胞）、5-羟色胺转运体（大鼠脑）的结合以及[3H]多巴胺摄取（大鼠尾状核突触体）实验。4-[(二苯甲基)氨基]-2-苯基喹唑啉（SoRI-9804）在5-羟色胺转运体结合方面基本无活性，并且在所有三种组织中解析出两个DAT结合成分，对约40%的结合位点具有高亲和力（平均Ki为465 nM），而对其余60%的结合位点的Ki基本无法测量（>100 microM）。[3H]多巴胺摄取实验表明，约50%的摄取对SoRI-9804敏感。饱和结合实验表明，SoRI-9804竞争性抑制[125I]RTI-55与SoRI-9804敏感结合成分的结合。为了确定SoRI-9804区分的两个结合位点是否受丝裂原活化蛋白激酶（MAP激酶）途径调控，将大鼠尾状核突触体在不存在或存在10 microM PD98059（抑制MAP激酶途径的激活）的情况下孵育。结果表明，抑制MAPK/ERK激酶使DAT的总B(max)降低了90%。用PD98059处理使SoRI-9804敏感结合成分在总B(max)中的比例从68%增加到80%。PD98059实验表明，抑制MAP激酶无法解释SoRI-9804与DAT的差异相互作用。总体来看，目前的结果表明SoRI-9804区分了多巴胺转运体的两个成分。需要进一步研究以确定这种效应的潜在机制以及多巴胺摄取的部分抑制是否会导致任何独特的行为效应。
