Jørgensen Susanne, Nielsen Elsebet Østergaard, Peters Dan, Dyhring Tino
NeuroSearch A/S, Drug Discovery, 93 Pederstrupvej, Ballerup DK-2750, Denmark.
J Neurosci Methods. 2008 Mar 30;169(1):168-76. doi: 10.1016/j.jneumeth.2007.12.004. Epub 2007 Dec 17.
Pre-synaptic dopamine, norepinephrine and serotonin transporters (DAT, NET and SERT) terminate synaptic catecholamine transmission through reuptake of released neurotransmitter. Common approaches for studying these transporters involve radiolabeled substrates or inhibitors which, however, have several limitations. In this study we have used a novel neurotransmitter transporter uptake assay kit. The assay employs a fluorescent substrate that mimics the biogenic amine neurotransmitters and is taken up by the cell through the specific transporters, resulting in increased fluorescence intensity. In order to validate the assay, a variety of reference and proprietary neurotransmitter transporter ligands from a number of chemical and pharmacological classes were tested. The ability of these compounds to inhibit the selective transporter-mediated uptake demonstrated a similar rank order of potency and IC(50) values close to those obtained in radiolabeled neurotransmitter uptake assays. The described assay enables monitoring of dynamic transport activity of DAT, NET and SERT and is amenable for high-throughput screening and compound characterization.
突触前多巴胺、去甲肾上腺素和5-羟色胺转运体（DAT、NET和SERT）通过再摄取释放的神经递质来终止突触儿茶酚胺传递。研究这些转运体的常用方法涉及放射性标记底物或抑制剂，然而，这些方法存在若干局限性。在本研究中，我们使用了一种新型神经递质转运体摄取检测试剂盒。该检测采用一种荧光底物，它模拟生物胺神经递质，并通过特定转运体被细胞摄取，导致荧光强度增加。为了验证该检测方法，测试了来自多个化学和药理学类别的多种参考和专利神经递质转运体配体。这些化合物抑制选择性转运体介导摄取的能力显示出类似的效价顺序，且IC(50)值接近在放射性标记神经递质摄取检测中获得的值。所描述的检测方法能够监测DAT、NET和SERT的动态转运活性，适用于高通量筛选和化合物表征。
