Pharmacological characterization of a novel non‐AT1, non‐AT2 angiotensin binding site

Abstract

The recent discovery of a novel non‐AT1, non‐AT2 binding site for angiotensins in the rat brain and testis has catalyzed efforts to purify and characterize this protein. To assess the specificity of this binding site for angiotensins, various neuropeptides were evaluated with competition binding assays in brain and testis using 125I‐Sar1, Ile8 angiotensin II (Ang II) as the radioligand in the presence of saturating concentrations of AT1 and AT2 receptor antagonists and 100 μM parachloromercuribenzoate. Primary screening of 25 neuropeptides at 10 uM concentration revealed seven peptides that inhibited > 50%. Orphanin and two of its congeners (tyr1, and tyr14) exhibited IC50 values ~ 1–5 μM in the brain and testis. Sar1,thr8 Ang II an Ang II antagonist showed high affinity for the binding site in brain and testis, consistent with previous observations of high affinity for Sar1,Ile8 Ang II. Ang I Pro11,D‐Ala12 moderately inhibited 125I‐Sar1, Ile8 Ang II binding in brain and testis at 10 μM confirming the preference of this binding site for Ang II receptor‐active peptides. The selective neurolysin inhibitor Pro‐Ile, was inactive at 10 μM. These results further establish the specificity of this novel non‐AT1, non‐AT2 binding site for angiotensins II and III and suggest that this protein is heretofore uncharacterized. Supported by NHLBI HL‐096357.