Catecholamine Uptake, Micro-fluorometry/Drug Screening

描述(由申请人提供)：去甲肾上腺素(HNET)、多巴胺(HDAT)和5-羟色胺(HSERT)的人类单胺转运体(HMATs)是治疗药物(抗抑郁药)和成瘾精神兴奋剂(可卡因、苯丙胺)的靶标。有关与MATS相互作用的药物的信息主要来自放射性配体底物分析。随着第一阶段CEBRA的应用，我们将开发一种新的分析HMAT药物相互作用的方法，特别是儿茶酚胺，利用荧光底物和实时单细胞荧光显微镜和光谱分析。我们计划用一种新的儿茶酚胺底物asp[4-(4-(dimethylamino)styrl)-N-methyl-pyridinium].来分析hnet和hdat的活性。天冬氨酸在结构上与MPP相似，但天冬氨酸在可见光区有较大的斯托克斯位移(4751=6101)。我们已经证明，天冬氨酸是一种钠依赖的底物，与NET和DAT具有亲和力。在目标I中，我们将测量表达hNET和MAT的人胚胎肾细胞(HEK)中天冬氨酸的积聚，并测量针对抗抑郁药和精神兴奋剂的转运蛋白活性。ASP荧光的各向异性和猝灭可以被用来区分结合和运输，我们将测试抗抑郁药、可卡因和苯丙胺与hnet和hDAT的作用，以检查哪些过程受到影响。在目标1中，我们将首先在自然组织培养系统(颈上神经节神经元)中研究Net，并用DAT启动类似的研究。原代组织培养还提供了一种检测内源性相关蛋白影响的方法，内源性相关蛋白可能是特定的神经元治疗靶点，从而避免全身并发症。AIMS I和II的首要目标是开发ASP分析，用于高通量筛选抑制或刺激单胺转运和结合的推定药物，特别是儿茶酚胺、去甲肾上腺素和多巴胺。

DESCRIPTION (provided by applicant): The human monoamine transporters (hMATs) for norepinephrine (hNET), dopamine (hDAT), and serotonin (hSERT) are targets for therapeutic drugs (antidepressants) and addictive psycho-stimulants (cocaine, amphetamines). Information about drugs that interact with MATs comes largely from radio-ligand substrate assays. With the Phase I CEBRA application, we will develop a new assay for hMAT drug interactions, in particular the catecholamines, utilizing fluorescent substrates and real-time, single cell fluorescence microscopy and spectroscopy. We plan to analyze hNET and hDAT activity in transfected cells with a novel substrate for the catecholamines, ASP [4-(4-(dimethylamino)styrl)-N-methyl-pyridinium]. ASP is structurally similar to MPP, but ASP fluoresces in the visible range with large Stokes shift (4751 => 610 1). We have already demonstrated that ASP is a Na-dependent substrate with uM affinity for NET and DAT. In Aim I we will measure ASP accumulation in human embryonic kidney cells (HEK) expressing hNET and MAT and measure transporter activity against antidepressants and psycho-stimulants. Anisotropy and quenching of ASP fluorescence can be exploited to distinguish binding from transport and we will test antidepressants, cocaine, and amphetamines against hNET and hDAT to examine which processes is affected. In Aim 1 we will first study NET in a native tissue culture system (superior cervical ganglion neurons) and initiate similar studies with DAT. Primary tissue culture also provides a means of examining the impact of endogenous associated proteins, which may be specific neuronal therapeutic targets, avoiding systemic complications. The overarching goal of Aims I and II is to develop the ASP assay for high-throughput screening of putative drugs that inhibit or stimulate monoamine transport and binding, in particular the catecholamines, norepinephrine and dopamine.