Ziemek, Ralf and Brennauer, Albert and Schneider, Erich and Cabrele, Chiara and Beck-Sickinger, Annette G. and Bernhardt, Günther and Buschauer, Armin (2006) Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y(2) receptor ligands. European Journal of Pharmacology 551 (1-3), pp. 10-8.
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With respect to the discovery and characterization of neuropeptide Y(2) 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 hY(2) 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 Y(2)-selective receptor antagonist BIIE0246, characteristic of either competitive or insurmountable antagonism, depended on the period of incubation with the cells. Functional data of Y(2) receptor agonists and antagonists determined in the fluorescence- and luminescence-based assays were in good agreement.
|Institutions:|| Chemistry and Pharmacy > Institut für Organische Chemie > Arbeitskreis Dr. Chiara Cabrele|
Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer)
|Projects:||GRK 760, Graduiertenkolleg Medizinische Chemie|
|Keywords:||Fluorescent probe; Neuropeptide Y; Y2 receptor; Flow cytometry; Chimeric G protein; Aequorin; BIIE0246|
|Subjects:||500 Science > 570 Life sciences|
600 Technology > 610 Medical sciences Medicine
|Refereed:||Yes, this version has been refereed|
|Created at the University of Regensburg:||Yes|
|Owner:||Prof. Armin Buschauer|
|Deposited On:||21 Feb 2008 08:22|
|Last Modified:||05 Aug 2009 13:42|