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

基于细胞的人多巴胺、去甲肾上腺素和血清素转运蛋白释放测定的开发

• 批准号: 9042342
• 负责人: Ann M Decker
• 金额: $ 10.79万
• 依托单位: RESEARCH TRIANGLE INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2018-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9042342
• 关键词: Animals; Bathing; Binding; Biogenic Amines; Biological Assay; Brain; Cell Line; Cell membrane; Cells; Characteristics; Data; Designer Drugs; Development; Disease; Dopamine; Ethics; Evaluation; Generic Drugs; Glioblastoma; Goals; Health; Human; Human Cell Line; Lead; Ligands; Literature; Measures; Mediating; Methodology; Methods; Modeling; Neuroblastoma; Neurons; Neurotransmitters; Norepinephrine; Placental Choriocarcinoma; Radiolabeled; Rattus; Research Project Grants; Salts; Serotonin; Structure of thyroid parafollicular cell; Synaptosomes; Testing; Time; addiction; base; cost; dopamine transporter; extracellular; high throughput screening; immortalized cell; improved; inhibitor/antagonist; medullary thyroid carcinoma; neurotransmitter release; neurotransmitter uptake; noradrenaline transporter; prevent; psychostimulant; radiotracer; research study; reuptake; serotonin transporter; uptake

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.

描述（由申请人提供）：拟议项目的目的是开发基于细胞的测定，以分析人多巴胺，去甲肾上腺素和5-羟色胺生物源性胺转运蛋白（BAT）的神经递质释放。两种类型的蝙蝠配体（再摄取抑制剂和底物型释放器）都升高了细胞外神经递质浓度，但是再摄取抑制剂与转运蛋白结合，而底物型释放器则在神经元内运输并诱导神经递质释放。由于许多精神刺激剂和设计师药物（例如“沐浴盐”）与蝙蝠相互作用，因此了解这种相互作用的机制在确定如何更好地治疗成瘾疾病方面非常有用。当前，评估一般化合物的蝙蝠活性的典型测定法涉及使用新鲜的大鼠脑突触体或转染的细胞，这些细胞均未解决。虽然大鼠脑突触体具有天然运输机制，并根据人使用的预期效力范围区分了释放和再摄取抑制作用，但该测定不能测量人类转运蛋白活性。大鼠突触体的使用还降低了效率，提高成本，防止转运蛋白活性的高通量筛查，并由于使用动物而引起的道德问题。当前基于细胞的测定使用缺乏天然传输机械并包含转染的人类转运蛋白的通用细胞系；这两个特征都无法区分蝙蝠释放器和再摄取抑制剂。因此，需要改进当前方法，以便在降低成本，消除道德问题并提高整体测定效率的同时，生产更好的人类转运蛋白神经递质释放数据。拟议的项目将使用内源性人类转运蛋白和转运蛋白介导的释放所需的天然转运机制研究细胞系。在评估了细胞系的神经递质释放和摄取活性之后，配体 将测试在大鼠蝙蝠处的已知活性。人类细胞的结果将与大鼠脑突触体数据进行比较，以确定配体势力如何比较模型之间的效果以及人类细胞之间的区分释放和再摄取抑制作用的良好状态。配体还将在HEK293细胞中分析具有过表达的转运蛋白，以将内源性转运蛋白活性与转染的转运蛋白活性进行比较。