Abstract P2027: Preliminary Characterization Of The 125 I-angiotensin 1-7 Binding Site In Rat Liver

Abstract

Angiotensin 1-7 (Ang 1-7) is a product of the renin-angiotensin system (RAS) that can reduce blood pressure and fibrosis. For more than a decade Mas has been viewed as the receptor for Ang 1-7, however, there are lingering doubts regarding the nature of the interaction between the heptapeptide and Mas. To date, no one has pharmacologically characterized Ang 1-7 binding in tissue membrane preparations. In this study we show that 125 I-Ang 1-7 binds to a site for which Ang 1-7 has low (μmolar) affinity, suggesting that the addition of an iodine molecule to the tyrosine in position 4 of Ang 1-7 drastically changes the characteristics of this peptide making it unsuitable for characterization of Ang 1-7 receptors. 125 I-Ang 1-7 binds with moderately high affinity (10-20 nM) and saturability to a binding site in rat liver membranes that is displaceable by 127 I-Ang 1-7 (iodoAng 1-7) at nanomolar concentrations. This binding is also displaceable by inhibitors of metalloproteases. To test the hypothesis that 125 I-Ang 1-7 binds to a peptidase, a series of peptidase inhibitors were evaluated for their ability to inhibit 125 I-Ang 1-7 binding. The non-selective matrix metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) inhibitors marimastat, batimastat, and GM-6001, which inhibit MMP1,2,3,7,8,13,14 and ADAM 9,17, partially inhibited 125 I-Ang 1-7 binding with nanomolar affinities, similar to their IC 50 values for MMPs, suggesting that 125 I-Ang 1-7 binds to MMPs and/or ADAMs as well as other tissue elements. Further testing with the specific MMP-2 (ARP100), MMP-9 and ADAM-17 inhibitors indicate that 125 I-Ang 1-7 is not binding to these peptidases. Continuing studies are underway to establish conditions under which the 125 I-Ang 1-7 ligand is not metabolized by peptidases, while retaining the ability to bind to peptidases is underway to enable the identification of the specific peptidase(s) or other binding moieties to which 125 I-Ang 1-7 binds to determine their potential therapeutic importance as a druggable targets.