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

Abstract

It is well established that Mas (NP_002368) is the receptor for angiotensin 1–7 (Ang 1–7), however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has repeatedly failed to reliably demonstrate high affinity, saturable binding of 125I‐Ang 1–7 in liver, kidney, brain and testes membrane preparations from both rats and mice. While the presence of a single iodine‐125 molecule on the tyrosine of Ang II does not impair its binding to the AT1 and AT2 receptors, it is not known whether the presence of an iodine‐125 molecule on the tyrosine of Ang 1–7 is equally innocuous. To begin to address this question, we prepared a novel analog of Ang 1–7: Tyr0‐Ang 1–7, to provide an alternate site for radioiodination of Ang 1–7. Tyr0‐Ang 1–7 was radioiodinated using the chloramine T procedure of 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 mono125I‐Y0‐Ang 1–7 and mono125I‐Y4‐Ang 1–7, were resolved from the uniodinated peptide peak by HPLC (C18 reverse phase column, 14.5% acetonitrile: triethylamine phosphate, pH 3.0 isocratic mobile phase). Saturation and competition radioligand binding assays were run for each peak using rat liver membrane preparations in the presence and absence of 10 μM Ang 1–7. While the identity of the early and late 125I‐Y0‐ Ang 1–7 peaks has not yet been established, preliminary studies indicate that both 125I‐Y0‐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. Competition binding assays run using the late peak 125I‐Y0‐Ang 1–7 revealed IC50's of 32±15 nM for Ang 1–7 and 15 ± 9.6 nM for Y0‐Ang 1–7. Comparison of the ability of a variety of Ang peptides to compete for late peak 125I‐Y0‐Ang 1–7 binding at a 10 μM concentration gave a rank order of Ang 1–7>Ang 3–7>Ang IV>Ang III>Ang 2–7>Ang II>Ang I. These results suggest that addition of a tyrosine at the amino terminus of Ang 1–7 may increase 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.Support or Funding InformationThis work was supported by the Cardiovascular Neuroscience Fund, Nova Southeastern University and the Peptide Radioiodination Shared Resource, Georgetown University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.