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 125 I‐Sar 1 , Ile 8 angiotensin II (Ang II) as the radioligand in the presence of saturating concentrations of AT 1 and AT 2 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 (tyr 1 , and tyr 14 ) exhibited IC50 values ~ 1–5 μM in the brain and testis. Sar 1 ,thr 8 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 Sar 1 ,Ile 8 Ang II. Ang I Pro 11 ,D‐Ala 12 moderately inhibited 125 I‐Sar 1 , Ile 8 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‐AT 1 , non‐AT 2 binding site for angiotensins II and III and suggest that this protein is heretofore uncharacterized. Supported by NHLBI HL‐096357.
These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.
We showed ACE (angiotensin-converting enzyme) 2 is higher in the kidney of male compared with female mice. To further investigate this sex difference, we examined the role of ACE2 in Ang-[1-8] (angiotensin [1-8])-induced hypertension and regulation of the renin-angiotensin system in the kidney of WT (wild type) and Ace2 KO (knockout) mice. Mean arterial pressure rose faster in WT male than WT female mice after Ang-[1-8] infusion. This sex difference was attenuated in ACE2 KO mice. Ang-[1-8] infusion reduced glomerular AT1R (angiotensin type 1 receptor) binding in WT female mice by 30%, and deletion of Ace2 abolished this effect. In contrast, Ang-[1-8] infusion increased glomerular AT1R binding in WT male mice by 1.2-fold, and this effect of Ang-[1-8] persisted in Ace2 KO male mice (1.3-fold). ACE2 also had an effect on renal protein expression of the neutral endopeptidase NEP (neprilysin), the enzyme that catabolizes Ang-[1-10] (angiotensin [1-10]), the precursor of Ang-[1-8]. Ang-[1-8] infusion downregulated NEP protein expression by 20% in WT male, whereas there was a slight increase in NEP expression in WT female mice. Deletion of Ace2 resulted in lowered NEP expression after Ang-[1-8] infusion in both sexes. These findings suggest sex-specific ACE2 regulation of the renin-angiotensin system contributes to female protection from Ang-[1-8]-induced hypertension. These findings have ramifications for the current coronavirus disease 2019 (COVID-19) pandemic, especially in hypertension since ACE2 is the SARS-CoV-2 receptor and hypertension is a major risk factor for poor outcomes.
