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基于细胞的报告基因释放分析测定钙依赖性神经毒素和神经活性药物的活性。

Cell-Based Reporter Release Assay to Determine the Activity of Calcium-Dependent Neurotoxins and Neuroactive Pharmaceuticals.

机构信息

Department of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam, 14469 Potsdam, Germany.

出版信息

Toxins (Basel). 2021 Mar 30;13(4):247. doi: 10.3390/toxins13040247.

Abstract

The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.

摘要

新开发的基于细胞的功能测定法的适用性已针对一系列通过刺激或抑制钙依赖性神经递质释放来发挥作用的神经毒素和神经活性药物的活性进行了测试。在该功能测定中,报告酶与神经递质一起从神经分泌小泡中释放出来。当前的研究表明,一种来自分化的人神经母细胞瘤报告细胞系(SIMA-hPOMC1-26-GLuc 细胞)的荧光素酶的释放可以通过毒蕈碱乙酰胆碱受体的 Gq 偶联的激动剂 carbachol 以及 Ca 通道形成蜘蛛毒素α- latrotoxin 来刺激。carbachol 刺激的荧光素酶释放完全被毒蕈碱乙酰胆碱受体拮抗剂阿托品和α- latrotoxin 介导的 Ca-螯合剂 EGTA 抑制,证明了荧光素酶释放刺激的特异性。SIMA-hPOMC1-26-GLuc 细胞在 mRNA 和蛋白水平上主要表达 L-和 N-型,并且较少表达 T-型 VGCC。与表达谱一致,高 K-缓冲液引起的去极化刺激的荧光素酶释放可被 L-型 VGCC 抑制剂有效且剂量依赖性地抑制,而 N-型和 T-型抑制剂的抑制作用较小。P/Q-和 R-型抑制剂不会影响 K-刺激的荧光素酶释放。总之,新建立的基于细胞的测定法可能代表一种通用工具,可用于分析通过调节钙依赖性神经递质释放来发挥作用的一系列神经毒素和神经活性药物的生物学效率。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ebb0/8066854/dc0cd17349f3/toxins-13-00247-g001.jpg

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