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Assessing Vesicular Monoamine Transport and Toxicity Using Fluorescent False Neurotransmitters
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2020-12-30 , DOI: 10.1021/acs.chemrestox.0c00380 Carlie A Black 1 , Meghan L Bucher 2 , Joshua M Bradner 2 , Lauren Jonas 1 , Kenny Igarza 1 , Gary W Miller 2
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2020-12-30 , DOI: 10.1021/acs.chemrestox.0c00380 Carlie A Black 1 , Meghan L Bucher 2 , Joshua M Bradner 2 , Lauren Jonas 1 , Kenny Igarza 1 , Gary W Miller 2
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Impairments in the vesicular packaging of dopamine result in an accumulation of dopamine in the cytosol. Cytosolic dopamine is vulnerable to two metabolic processes—enzymatic catabolism and enzymatic- or auto-oxidation—that form toxic metabolites and generate reactive oxygen species. Alterations in the expression or activity of the vesicular monoamine transporter 2 (VMAT2), which transports monoamines such as dopamine from the cytosol into the synaptic vesicle, result in dysregulated dopamine packaging. Here, we developed a series of assays using the fluorescent false neurotransmitter 206 (FFN206) to visualize VMAT2-mediated vesicular packaging at baseline and following pharmacological and toxicological manipulations. As a proof of principle, we observed a significant reduction in vesicular FFN206 packaging after treatment with the VMAT2 inhibitors reserpine (IC50: 73.1 nM), tetrabenazine (IC50: 30.4 nM), methamphetamine (IC50: 2.4 μM), and methylphenidate (IC50: 94.3 μM). We then applied the assay to investigate the consequences on vesicular packaging by environmental toxicants including the pesticides paraquat, rotenone, and chlorpyrifos, as well as the halogenated compounds unichlor, perfluorooctanesulfonic acid, Paroil, Aroclor 1260, and hexabromocyclododecane. Several of the environmental toxicants showed minor impairment of the vesicular FFN206 loading, suggesting that the toxicants are weak VMAT2 inhibitors at the concentrations tested. The assay presented here can be applied to investigate the effect of additional pharmacological compounds and environmental toxicants on vesicular function, which will provide insight into how exposures to such factors are involved in the pathogenesis of monoaminergic diseases such as Parkinson’s disease, and the assay can be used to identify pharmacological agents that influence VMAT2 activity.
中文翻译：
使用荧光假神经递质评估囊泡单胺转运和毒性
多巴胺囊泡包装的损害导致多巴胺在细胞质中的积累。细胞溶质多巴胺易受两种代谢过程的影响——酶促分解代谢和酶促或自氧化——形成有毒代谢物并产生活性氧。囊泡单胺转运蛋白 2 (VMAT2) 的表达或活性的改变，将多巴胺等单胺从胞质溶胶转运到突触小泡，导致多巴胺包装失调。在这里，我们使用荧光假神经递质 206 (FFN206) 开发了一系列检测方法，以在基线和药理学和毒理学操作后可视化 VMAT2 介导的囊泡包装。作为原则证明，50：73.1 nM）、丁苯那嗪（IC 50：30.4 nM）、甲基苯丙胺（IC 50：2.4 μM）和哌醋甲酯（IC 50: 94.3 微米)。然后，我们应用该检测方法研究环境毒物对囊泡包装的影响，包括杀虫剂百草枯、鱼藤酮和毒死蜱，以及卤代化合物单氯、全氟辛烷磺酸、Paroil、Aroclor 1260 和六溴环十二烷。几种环境毒物显示出对囊泡 FFN206 负载的轻微损害，表明这些毒物在测试浓度下是弱 VMAT2 抑制剂。此处介绍的测定可用于研究其他药理化合物和环境毒物对囊泡功能的影响，这将有助于深入了解这些因素的暴露如何参与帕金森病等单胺能疾病的发病机制，
更新日期：2020-12-30
中文翻译：
使用荧光假神经递质评估囊泡单胺转运和毒性
多巴胺囊泡包装的损害导致多巴胺在细胞质中的积累。细胞溶质多巴胺易受两种代谢过程的影响——酶促分解代谢和酶促或自氧化——形成有毒代谢物并产生活性氧。囊泡单胺转运蛋白 2 (VMAT2) 的表达或活性的改变，将多巴胺等单胺从胞质溶胶转运到突触小泡，导致多巴胺包装失调。在这里，我们使用荧光假神经递质 206 (FFN206) 开发了一系列检测方法，以在基线和药理学和毒理学操作后可视化 VMAT2 介导的囊泡包装。作为原则证明，50：73.1 nM）、丁苯那嗪（IC 50：30.4 nM）、甲基苯丙胺（IC 50：2.4 μM）和哌醋甲酯（IC 50: 94.3 微米)。然后，我们应用该检测方法研究环境毒物对囊泡包装的影响，包括杀虫剂百草枯、鱼藤酮和毒死蜱，以及卤代化合物单氯、全氟辛烷磺酸、Paroil、Aroclor 1260 和六溴环十二烷。几种环境毒物显示出对囊泡 FFN206 负载的轻微损害，表明这些毒物在测试浓度下是弱 VMAT2 抑制剂。此处介绍的测定可用于研究其他药理化合物和环境毒物对囊泡功能的影响，这将有助于深入了解这些因素的暴露如何参与帕金森病等单胺能疾病的发病机制，
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