Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y₂ receptor ligands

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• 作者：Ralf Ziemek ; Albert Brennauer ; Erich Schneider ; Chiara Cabrele ; Annette G. Beck-Sickinger ; Gü ; nther Bernhardt and Armin Buschauer
• 关键词：Fluorescent probe ; Neuropeptide Y ; Y₂ receptor ; Flow cytometry ; Chimeric G protein ; Aequorin ; BIIE0246
• 刊名：European Journal of Pharmacology
• 出版年：2006
• 期刊代码：24_00142999
• 类别：neu
• 出版时间：3 December 2006
• 卷：551
• 期：1-3
• 页码：10-18
• 文件大小：612 K

摘要

With respect to the discovery and characterization of neuropeptide Y₂ receptor ligands as pharmacological tools or potential drugs, fluorescence- and luminescence-based assays were developed to determine both the affinity and the activity of receptor agonists and antagonists. A flow cytometric binding assay is described for the hY₂ receptor stably expressed in CHO cells using cy5-labeled porcine neuropeptide Y and compared with a radioligand binding assay. Binding of the fluorescent ligand was visualized by confocal microscopy. Stable co-transfection with the chimeric G protein Gq_i5 enabled the establishment of a spectrofluorimetric fura-2 and a flow cytometric fluo-4 calcium assay. Further stable expression of apoaequorin targeted to the mitochondria allowed the establishment of an aequorin assay which could be performed in the 96-well format. The shape of the concentration–response curves of porcine neuropeptide Y in the presence of the Y₂-selective receptor antagonist BIIE0246, characteristic of either competitive or insurmountable antagonism, depended on the period of incubation with the cells. Functional data of Y₂ receptor agonists and antagonists determined in the fluorescence- and luminescence-based assays were in good agreement.