Development of an Automated, High‐Throughput Assay to Detect Angiotensin AT2‐Receptor Agonistic Activity by Nitric Oxide Measurement In Vitro

Abstract

BackgroundThe angiotensin AT2‐receptor (AT2R) is a key component within the protective arm of the renin‐angiotensin system inducing vasodilation by nitric oxide (NO) production. Currently, no high‐throughput assay is available to identify AT2R agonists in vitro, which could explain the low number of AT2R selective ligands in drug development programs.ObjectiveTo design and validate a high‐throughput method for detection of AT2R activation in vitro based on NO release.MethodsHuman aortic endothelial cells (HAEC) were seeded into 96‐well plates at a density of 5000 cells per well. 48 hours after seeding, cells were loaded for 30 minutes with the NO probe, DAF‐FM diacetate (5µM). Cells were stimulated for 15 minutes with the AT2R‐agonist C21 (1µM) or with classical NO‐releasing agents as positive controls: bradykinin (BK, 0.1µM), acetylcholine (ACh, 1µM) or Ang‐(1‐7) (MasR agonist, 0.1µM). Cells were fixed and fluorescence signals captured by fluorescence microscopy with automated, built‐in image analysis (ImageXpress Pico, Molecular Devices, USA). AT2R‐transfected (AT2R‐CHO) or non‐transfected (NT‐CHO) Chinese hamster ovary cells were cultured and loaded with DAF‐FM diacetate according to the same protocol as HAEC. Concentration‐response curves were obtained for C21, BK, Ang II and Ang‐(1‐5) (0.1nM to 10µM each) using the automated protocol for ImageXpress Pico described above on AT2R‐CHO. To evaluate whether effects of the compounds in AT2R‐CHO were AT2R specific, the response to these compounds at the highest concentration (10µM) was also tested in NT‐CHO.ResultsUsing primary HAEC we could show that the automated, high‐throughput workflow was able to detect NO release by AT2R agonists as efficiently as by classical NO‐releasing agents [C21: 16.16 ± 6.84%; Ang‐(1‐7): 25.45 ± 3.37%, BK: 19.30 ± 2.92%; ACh: 24.01 ± 4.24%; one‐way ANOVA followed by Dunnett’s multiple comparison test, p≤0.05 vs control, ≥3 independent experiments for all compounds). In AT2R‐CHO, we detected that both C21 and Ang II (AT2R agonists) increased NO release in a concentration‐dependent manner, while BK had no effect (AT2R‐CHO lacks BK receptors) at any tested concentration. Surprisingly Ang‐(1‐5), a degradation product of Ang II thought to be inactive, induced NO release in a concentration‐dependent manner in AT2R‐CHO. In NT‐CHO, none of the compounds stimulated NO release, indicating that the effects by C21, Ang II and Ang‐(1‐5) in AT2R‐CHO were AT2R specific.ConclusionMeasurement of NO release from AT2R‐CHO or HAEC by DAF‐FM diacetate in an automated way is suitable as high‐throughput assay for the identification of AT2R‐agonistic compounds in vitro. Application of the assay revealed that Ang‐(1‐5), which has been regarded as an inactive metabolite of Ang II, has AT2R agonistic properties.