Richfield E K
Unit of Functional Neuroanatomy, National Institute of Mental Health, Bethesda, MD 20892.
Brain Res. 1991 Feb 1;540(1-2):1-13. doi: 10.1016/0006-8993(91)90486-f.
The dopamine uptake complex was examined in the rat central nervous system using [3H]GBR 12935 and in vitro quantitative autoradiography to determine all binding data. [3H]GBR 12935 labels two unique binding sites, the dopamine uptake complex and a piperazine acceptor site. These two sites differ in their pharmacologic properties, anatomical distributions, densities, and response to lesions. Using appropriate binding conditions, [3H]GBR 12935 can be used to specifically label the dopamine uptake complex. [3H]GBR 12935 labeled a single binding site with characteristics of the dopamine uptake complex when mazindol (25 microM) was used as a blank. The specific binding and autoradiographic appearance of [3H]GBR 12935 to the dopamine uptake complex was improved by including trans-flupentixol (0.75 microM) to displace binding to a previously described piperazine acceptor site, recently determined to be a site on cytochrome P450IID1. Binding was saturable and reversible to the dopamine uptake complex. The equilibrium dissociation constant (1.4 +/- 0.7 nM), maximal number of binding sites (6.0 +/- 1.3 pmol/mg protein), and Hill coefficient (1.1 +/- 0.1) of [3H]GBR 12935 in rat striatum using mazindol to define non-specific binding was not significantly altered by the inclusion of trans-flupentixol (0.75 microM). Using GBR 12909 as a blank produced a greater maximal number of binding sites (8.4 +/- 2.3 pmol/mg protein), but no significant difference in the equilibrium dissociation constant (1.6 +/- 0.3 nM) or Hill coefficient (1.1 +/- 0.1). A series of drugs that bind to the dopamine uptake complex displaced [3H]GBR 12935 in a rank order consistent with other binding and behavioral studies of this complex. The rank order of these drugs was GBR 12909 greater than mazindol greater than nomifensine greater than benztropine greater than desipramine greater than amphetamine greater than dopamine; all these drugs displayed a Hill coefficient near one and were best modeled as a single site. Cocaine and WIN 35,428 (a cocaine congener) were unique in their competition for [3H]GBR 12935 binding, displaying biphasic curves, low Hill coefficients, and were best modeled as two site fits. Lesioning of the dopaminergic median forebrain bundle resulted in a dramatic loss of the dopamine uptake complex in the striatum, nucleus accumbens, olfactory tubercle, and substantia nigra. Other dopaminergic projection areas were decreased to a lesser extent. Striatal ibotenate lesions did not decrease the density of the dopamine uptake complex, despite a large decrease in the dopamine D1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
使用[3H]GBR 12935和体外定量放射自显影技术在大鼠中枢神经系统中检测多巴胺摄取复合物，以确定所有结合数据。[3H]GBR 12935标记两个独特的结合位点，即多巴胺摄取复合物和哌嗪受体位点。这两个位点在药理学特性、解剖分布、密度以及对损伤的反应方面存在差异。在适当的结合条件下，[3H]GBR 12935可用于特异性标记多巴胺摄取复合物。当使用吗茚酮（25 microM）作为空白时，[3H]GBR 12935标记了一个具有多巴胺摄取复合物特征的单一结合位点。通过加入反式氟奋乃静（0.75 microM）以取代与先前描述的哌嗪受体位点（最近确定为细胞色素P450IID1上的一个位点）的结合，[3H]GBR 12935与多巴胺摄取复合物的特异性结合和放射自显影外观得到改善。与多巴胺摄取复合物的结合是可饱和且可逆的。使用吗茚酮定义非特异性结合时，[3H]GBR 12935在大鼠纹状体中的平衡解离常数（1.4±0.7 nM）、最大结合位点数（6.0±1.3 pmol/mg蛋白质）和希尔系数（1.1±0.1），在加入反式氟奋乃静（0.75 microM）后没有显著改变。使用GBR 12909作为空白产生了更大的最大结合位点数（8.4±2.3 pmol/mg蛋白质），但平衡解离常数（1.6±0.3 nM）或希尔系数（1.1±0.1）没有显著差异。一系列与多巴胺摄取复合物结合的药物以与该复合物的其他结合和行为研究一致的顺序取代[3H]GBR 12935。这些药物的顺序为GBR 12909＞吗茚酮＞诺米芬辛＞苯海索＞地昔帕明＞苯丙胺＞多巴胺；所有这些药物的希尔系数接近1，并且最好被模拟为单一位点。可卡因和WIN 35,428（一种可卡因同系物）在竞争[3H]GBR 12935结合方面是独特的，显示出双相曲线、低希尔系数，并且最好被模拟为双位点拟合。多巴胺能中脑前束的损伤导致纹状体、伏隔核、嗅结节和黑质中多巴胺摄取复合物的显著丧失。其他多巴胺能投射区域的减少程度较小。纹状体注射鹅膏蕈氨酸损伤并没有降低多巴胺摄取复合物的密度，尽管多巴胺D1受体有大量减少。（摘要截断于400字）
