A non-radioactive method for angiotensin II receptor binding studies using the rat liver

Abstract

Introduction: A new non-radioactive method based on competitive ELISA has been developed for binding studies on angiotensin II (Ang II) receptors. Method: Rat liver membrane was used as the source of angiotensin receptors and FITC-angiotensin II (FITC-Ang II) was used as the labeled ligand with an affinity similar to unlabeled Ang II. The effects of different concentrations of Ang II, losartan, CGP-42112A and saralasin were studied on FITC-Ang II binding. Results: The Ki values for Ang II, losartan and CGP-42112A were calculated as 0.52 ± 0.22 nM, 6 ± 3 nM and 0.15 ± 0.07 nM, respectively. Saralasin inhibited the binding of labeled ligand biphasically, revealing two different populations of Ang receptor with different affinities for saralasin. About 74% of the binding sites were more sensitive to saralasin with a Ki value of 0.32 ± 0.04 nM while saralasin showed a Ki value of 2.7 ± 0.8 nM for the remaining binding sites. Discussion: The competitive ELISA method developed in this work yields Ki values for angiotensin antagonists similar to those obtained by others using radiolabeled ligands. The simplicity of this method makes it a suitable alternative to radioligand studies for routine analysis of interaction of drugs with angiotensin receptors.