Towards Identifying the Binding Site for ¹²⁵ I‐Angiotensin 1‐7

Angiotensin 1–7 (Ang 1‐7) is a product of the renin‐angiotensin system (RAS) that exhibits central actions to reduce blood pressure and improve baroreflex sensitivity. For more than a decade Mas has been considered the receptor for Ang 1‐7, however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has demonstrated high affinity (low nanomolar K D ) binding of a preparation of 125/127 I‐Ang 1‐7 in liver membranes, however, this binding is not pharmacologically specific in that the IC 50 of Ang 1‐7 is in the micromolar range and all angiotensin peptides compete for 125/127 I‐Ang 1‐7 binding equivalently. 125/127 I‐Ang 1‐7 binding to liver, kidney, brain and testes membrane preparations from both rats and mice has also failed to demonstrate a high affinity, pharmacologically specific binding site for 125/127 I‐Ang 1‐7. Considering that rapid degradation of Ang 1‐7 could be adversely affecting the binding assay, we tested different peptidase inhibitors (JMV‐390, O‐phenanthroline and a proprietary combination of peptidase inhibitors Biotools®) to prevent degradation of the radioligand. Although we were able to reduce Ang 1‐7 degradation with these inhibitors (~80% preservation), we also reduced the total and specific binding of 125 I‐Ang 1‐7. Indeed, competition binding assays for 125 I‐Ang‐(1–7) with JMV‐390, O‐phenanthroline and Biotools® showed that 125 I‐Ang‐(1–7) and all the inhibitors compete for the same binding site with JMV‐390 having a nanomolar IC 50 . It is important to note that the radioligand used for receptor binding studies contains an iodine 125 on the tyrosine in position four of Ang 1‐7, and may have different characteristics for enzymatic metabolism. To assess this possibility, we determined the metabolism of 127 I‐Ang 1‐7 (non‐radioactive iodine isotope) and indeed, the metabolism of Ang 1‐7 and 127 I‐Ang 1‐7 in liver appear to differ, with 127 I‐Ang 1‐7 being more resistant to metabolism. In addition, competition binding assays showed that 127 I‐Ang 1‐7 is ~1000‐fold more potent than Ang 1‐7 in competing for 125 I‐Ang 1‐7 binding. In conclusion, our results suggest that 125 I‐Ang‐(1–7) may be binding to a peptidase rather than a receptor and the addition of the iodine molecule to the tyrosine in position four attenuates its degradation. Studies are underway to identify this peptidase and determine its potential therapeutic importance as a druggable target. Support or Funding Information Cardiovascular Neuroscience Research Fund 337667 Speth (PI) 03/31/15‐12/31/18 ‐ Nova Southeastern Univ. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .