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Development of an Automated, High‐Throughput Assay to Detect Angiotensin AT2‐Receptor Agonistic Activity by Nitric Oxide Measurement In Vitro
Author(s) -
Silva Igor M. S.,
Kjærgaard Kenneth,
Mortensen Christina,
Stage Tore B.,
Steckelings Ulrike M.
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r4466
Subject(s) - chinese hamster ovary cell , angiotensin ii , nitric oxide , agonist , in vitro , chemistry , renin–angiotensin system , receptor , bradykinin , transfection , pharmacology , endocrinology , biology , biochemistry , organic chemistry , gene , blood pressure
Background The angiotensin AT 2 ‐receptor (AT 2 R) 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 AT 2 R agonists in vitro, which could explain the low number of AT 2 R selective ligands in drug development programs. Objective To design and validate a high‐throughput method for detection of AT 2 R activation in vitro based on NO release. Methods Human 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 AT 2 R‐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). AT 2 R‐transfected (AT 2 R‐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 AT 2 R‐CHO. To evaluate whether effects of the compounds in AT 2 R‐CHO were AT 2 R specific, the response to these compounds at the highest concentration (10µM) was also tested in NT‐CHO. Results Using primary HAEC we could show that the automated, high‐throughput workflow was able to detect NO release by AT 2 R 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 AT 2 R‐CHO, we detected that both C21 and Ang II (AT 2 R agonists) increased NO release in a concentration‐dependent manner, while BK had no effect (AT 2 R‐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 AT 2 R‐CHO. In NT‐CHO, none of the compounds stimulated NO release, indicating that the effects by C21, Ang II and Ang‐(1‐5) in AT 2 R‐CHO were AT 2 R specific. Conclusion Measurement of NO release from AT 2 R‐CHO or HAEC by DAF‐FM diacetate in an automated way is suitable as high‐throughput assay for the identification of AT 2 R‐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 AT 2 R agonistic properties.