Preliminary Characterization of the 125I‐Angiotensin 1‐7 Binding Site in Rodent Liver

Abstract

Angiotensin 1–7 (Ang 1‐7) is a product of the renin‐angiotensin system that acts centrally and peripherally to reduce blood pressure and improve baroreflex sensitivity. For more than a decade Mas has been viewed as the receptor for Ang 1‐7, however, Ang 1‐7 binding has not been pharmacologically characterized in tissue membrane preparations. In this study, we show that 125I‐Ang 1‐7 binds to a site for which Ang 1‐7 has low affinity. To assess the possibility that the low affinity of Ang 1‐7 to compete for 125/127I‐Ang 1‐7 binding was due to its susceptibility to metabolism by peptidases in the tissue membrane homogenate, the integrity of Ang 1‐7 as well as 127I‐Ang 1‐7 (iodoAng 1‐7) was evaluated by HPLC analysis. Both Ang 1‐7 and iodoAng 1‐7 were substantially metabolized in liver membrane homogenates at room temperature (RT). However, when incubated on ice (~0 C), 125I‐Ang 1‐7 binds with moderately higher affinity (~3 nM versus 10–20 nM at RT) to a binding site in rat liver membranes that is displaceable by iodoAng 1‐7 at nanomolar concentrations, but Ang 1‐7 still shows a low affinity to compete (1.4μM for Ang 1‐7and 69.4nM for iodoAng 1‐7). At RT 125I‐Ang 1‐7 binding is partially displaceable by metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) inhibitors, e.g., marimastat, batimastat, and GM‐6001 at nanomolar IC50 values, similar to their IC50 values for MMP activity, suggesting that 125I‐Ang 1‐7 binds to MMPs and/or ADAMs as well as other tissue elements at RT. However, when incubated at ~0 C, the MMP and ADAM inhibitors had micromolar IC50 values, suggesting that 125I‐Ang 1‐7 binding to these peptidases is temperature dependent. Our results continue to suggest 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. Continuing studies are underway to identify the peptidase(s) or other binding moieties to which 125I‐Ang 1‐7 binds to determine their potential therapeutic importance as druggable targets.