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: 8822771
• 负责人: Ann M Decker
• 金额: $ 10.9万
• 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 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822771
• 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; 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; public health relevance; 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）：拟议项目的目标是开发基于细胞的检测方法来分析人类多巴胺，去甲肾上腺素和血清素生物胺转运体（BATs）的神经递质释放。两种类型的BAT配体（再摄取抑制剂和底物型释放剂）都能提高细胞外神经递质浓度，但再摄取抑制剂与转运体结合，而底物型释放剂在神经元内运输并诱导神经递质释放。由于许多精神兴奋剂和“浴盐”等设计药物与bat相互作用，因此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活性的典型试验涉及使用新鲜大鼠脑突触体或转染细胞，这两种方法都有尚未解决的问题。虽然大鼠脑突触体具有天然的转运机制，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，但该试验不测量人类转运体的活性。使用大鼠突触体也降低了效率，增加了成本，阻碍了转运蛋白活性的高通量筛选，并且由于使用动物引起了伦理问题。目前基于细胞的检测使用缺乏天然转运机制的通用细胞系，并且含有转染的人类转运蛋白；这两个特征导致无法区分BAT释放剂和再摄取抑制剂。因此，有必要改进现有的方法，以产生更好的人类转运神经递质释放数据，同时降低成本，消除伦理问题，提高整体分析效率。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。继评价神经递质释放和摄取活性的细胞系，配体