Development of Cell-Based Human Dopamine, Norepinephrine, and Serotonin Transporter Release Assays

描述（由申请人提供）：拟定项目的目标是开发基于细胞的测定方法，以分析人类多巴胺、去甲肾上腺素和血清素生物胺转运蛋白（BAT）的神经递质释放。这两种类型的BAT配体（再摄取抑制剂和底物型释放剂）提高细胞外神经递质浓度，但再摄取抑制剂结合转运蛋白，而底物型释放剂在神经元内转运并诱导神经递质释放。由于许多精神兴奋剂和设计药物，如“浴盐”与BAT相互作用，了解这种相互作用的机制，在确定如何更好地治疗成瘾性障碍方面非常有用。目前，用于评估一般化合物的BAT活性的典型测定涉及使用新鲜的大鼠脑突触体或转染的细胞，这两者都有未解决的问题。虽然大鼠脑突触体具有天然转运机制，并在基于人类使用的预期效价范围内区分释放和再摄取抑制，但该试验未测量人类转运蛋白活性。大鼠突触体的使用也降低了效率，增加了成本，阻止了转运蛋白活性的高通量筛选，并由于使用动物而引起了伦理问题。目前基于细胞的测定使用缺乏天然转运机制并含有转染的人转运蛋白的通用细胞系;这两种特征导致无法区分BAT释放剂和再摄取抑制剂。因此，需要改进目前的方法，以产生更好的人转运蛋白神经递质释放数据，同时降低成本，消除伦理问题，并提高整体测定效率。拟议的项目将研究具有内源性人类转运蛋白和转运蛋白介导释放所需的天然转运机制的细胞系。在评价细胞系的神经递质释放和摄取活性后， 将测试在大鼠BAT中的已知活性。将人细胞的结果与大鼠脑突触体数据进行比较，以确定模型之间的配体效力如何比较以及人细胞在释放和再摄取抑制之间的区分程度。还将在过表达转运蛋白的HEK 293细胞中分析配体，以比较内源性转运蛋白活性与转染的转运蛋白活性。

DESCRIPTION (provided by applicant): The goal of the proposed project is to develop cell-based assays to analyze neurotransmitter release at the human dopamine, norepinephrine, and serotonin biogenic amine transporters (BATs). Both types of BAT ligands (reuptake inhibitors and substrate-type releasers) elevate extracellular neurotransmitter concentration, but reuptake inhibitors bind to transporters, while substrate-type releasers are transported inside the neuron and induce neurotransmitter release. Since many psychostimulants and designer drugs such as "bath salts" interact with BATs, understanding the mechanism of this interaction proves very useful in determining how to better treat addiction disorders. Currently, the typical assays for assessing the BAT activity of general compounds involve using fresh rat brain synaptosomes or transfected cells, both of which have unresolved issues. While the rat brain synaptosome has native transport machinery and distinguishes between release and reuptake inhibition in expected potency ranges based on human use, the assay does not measure human transporter activity. The use of rat synaptosomes also lowers efficiency, increases the cost, prevents high-throughput screening of transporter activity, and raises ethical concerns due to the use of animals. Current cell-based assays use generic cell lines that lack native transport machinery and contain transfected human transporters; both characteristics lead to the inability to distinguish between BAT releasers and reuptake inhibitors. Therefore, there is a need to improve upon the current methods in order to produce better human transporter neurotransmitter release data while lowering cost, removing ethical concerns, and increasing overall assay efficiency. The proposed project will investigate cell lines with endogenous human transporters and native transport machinery required for transporter-mediated release. Following evaluation of the neurotransmitter release and uptake activity of the cell lines, ligands with known activity at rat BATs will be tested. Results from the human cells will be compared with rat brain synaptosome data in order to determine how the ligand potencies compare between models and how well the human cells discriminate between release and reuptake inhibition. The ligands will also be analyzed in HEK293 cells with over-expressed transporters in order to compare endogenous transporter activity with transfected transporter activity.