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)：该项目的目标是开发基于细胞的分析方法，以分析人类多巴胺、去甲肾上腺素和5-羟色胺生物胺转运体(BAT)的神经递质释放。两种类型的BAT配体(重摄取抑制剂和底物类型释放器)都能提高细胞外神经递质浓度，但重摄取抑制剂与转运体结合，而底物类型释放器在神经元内转运并诱导神经递质释放。由于许多心理刺激剂和特制药物(如“浴盐”)与蝙蝠相互作用，了解这种相互作用的机制对于确定如何更好地治疗成瘾障碍非常有用。目前，评估一般化合物BAT活性的典型方法包括使用新鲜的大鼠脑突触体法或转基因细胞法，这两种方法都存在尚未解决的问题。虽然大鼠脑突触体具有天然的运输机制，并根据人类使用情况在预期的效力范围内区分释放和再摄取抑制，但该实验并不测量人类转运蛋白的活性。大鼠突触体的使用也降低了效率，增加了成本，阻止了对转运蛋白活性的高通量筛选，并因使用动物而引起了伦理问题。目前基于细胞的分析使用的是缺乏天然运输机制的通用细胞系，含有转基因的人类转运蛋白；这两个特征导致无法区分BAT释放剂和再摄取抑制剂。因此，有必要改进目前的方法，以产生更好的人类转运蛋白神经递质释放数据，同时降低成本，消除伦理顾虑，并提高整体检测效率。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本地运输机制的细胞系。在评估神经递质释放和摄取细胞系、配体的活性之后 在老鼠蝙蝠已知活动的情况下，将对其进行测试。来自人类细胞的结果将与大鼠脑突触体数据进行比较，以确定模型之间的配基效力如何比较，以及人类细胞对释放和再摄取抑制的区分程度。还将在HEK293细胞中分析这些配体，以比较内源性转运蛋白活性和转基因转运蛋白活性。