title: A fluorimetric binding assay for angiotensin II and kinin receptors.
abstract: Angiotensin II (AngII) and kinins (bradykinin (BK) and des-Arg9-bradykinin (DBK)), are potent agents involved in the maintenance of blood pressure and several biological activities, and their better understanding is important to produce new drugs aimed to control arterial blood pressure. Previous studies on ligand-receptor binding have been based on radioactive methods, which led us to study a new method based on the fluorimetric method. A lanthanide attached to the N-terminal segment of the peptide (AngII, BK and DBK), which produces a time-resolved-fluorescent ligand, was used in a binding test with CHO cells expressing the AT1, AT2, B1 or B2 receptors in comparison with the same cell line tested with the radioactive ligand. Our findings indicated that the non-radioactive method provided a comparable result for the angiotensin receptors. On the other hand, the kinin receptors showed a slight reduction in the binding affinity, probably due to the linkage at the N-terminal segment and/or to the lower biological stability associated to the high temperature (37°C) used for the fluorimetric method, while the radioactive one was at 4°C. We can conclude that a time-resolved fluorescence assay would provide a sensitive method as an alternative tool for receptor studies.
authors.affiliation: Department of Biophysics, Federal University of São Paulo, 04023-062, Brazil. Electronic address: renan.paulo.martin@gmail.com.
authors.forename: Renan P
authors.initials: RP
authors.lastname: Martin
authors.affiliation: Department of Biophysics, Federal University of São Paulo, 04023-062, Brazil.
authors.forename: Rafael
authors.initials: R
authors.lastname: Filippelli-Silva
authors.affiliation: Department of Biophysics, Federal University of São Paulo, 04023-062, Brazil.
authors.forename: Eliete S
authors.initials: ES
authors.lastname: Rodrigues
authors.affiliation: Department of Biophysics, Federal University of São Paulo, 04023-062, Brazil.
authors.forename: Clovis R
authors.initials: CR
authors.lastname: Nakaie
authors.affiliation: Department of Biophysics, Federal University of São Paulo, 04023-062, Brazil.
authors.forename: Suma I
authors.initials: SI
authors.lastname: Shimuta
mesh_terms: D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001794:Blood Pressure / Q000502:physiology; D001920:Bradykinin / Q000031:analogs & derivatives / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D003412:Cricetulus; D005470:Fluorometry / Q000379:methods*; D011945:Receptors, Angiotensin / Q000378:metabolism
chemical_list: D011945:Receptors, Angiotensin; D000804:Angiotensin II; C024626:bradykinin, des-Arg(9)-; D001920:Bradykinin
keywords: Angiotensin; Fluorescent-labeled peptide; GPCR; Kinin; Receptor