Development of a Novel Radioligand for Characterization of the Mas Receptor for Angiotensin 1-7

Nova Southeastern University, Davie, Florida, USA

Objective: To use radioligand binding to characterize Mas receptor binding in tissue. Background: Mas is the receptor for angiotensin 1-7 (Ang 1-7), however, no studies have characterized Mas receptor radioligand binding in tissue membranes. While the presence of a single iodine-125 molecule on the tyrosine of AngII does not impair its binding to the AT1 and AT2 receptors, it is not known whether iodine-125 on the tyrosine of Ang 1-7 is equally innocuous. Methods: To address this question, we prepared a novel analog Tyr0-Ang 1-7, to provide an alternate site for radioiodination of Ang 1-7. Tyr0-Ang 1-7 was radioiodinated using chloramine T (Hunter and Greenwood, 1962) with a 7-times excess of peptide to iodine to minimize di-iodination of the peptide. Two radioiodinated peaks, presumed to be mono 125I-Y0-Ang 1-7 and mono 125I-Y4-Ang 1-7, were resolved from uniodinated peptide by HPLC (C18 reverse phase column, 14.5% acetonitrile:triethylamine phosphate, pH 3.0 mobile phase). Saturation radioligand binding assays were run for both peaks using rat liver membranes ± 10 μM Ang 1-7. Results: Both 125I-Y-Ang 1-7 peaks displayed high affinity, saturable binding to rat liver membranes: early peak: KD=7.7±2.3 nM, Bmax=3.3±0.72 fmol/mg wet weight; and late peak KD=11±2, Bmax=3.6±0.4 fmol/mg wet weight. Conclusion: These results suggest that addition of a tyrosine at the amino terminus of Ang 1-7 increases its binding affinity for Mas and that the presence of an iodine-125 on either Y0 or Y4 of Ang 1-7 does not preclude high affinity, saturable binding to Mas.