A High-throughput Nitric Oxide Measurement Assay Reveals That Angiotensin-(1-5) Is An AT2 Receptor Agonist

<jats:p>The angiotensin AT2-receptor (AT2R) is a key component within the protective arm of the renin-angiotensin system (RAS), being involved in nitric oxide (NO) production and vasodilation. To this date, no quantitative high-throughput assay is available to identify AT2R agonists in vitro, which may be a reason for the low number of AT2R selective ligands in drug development programs. Objective: To design and validate a high-throughput method for detection of AT2R activation in vitro. Methods and Results: NO release was selected as readout for AT2R activation in AT2R transfected (CHO-AT2) and non-transfected (CHO-NT) CHO cells using DAF-FM( 5x10-6 mol/L) as NO probe. Cells were seeded on 96-well plates and stimulated for 15 minutes with C21 or Ang II (established AT2R agonists, 10-6mol/L), Ang-(1-5) (molecule with unknown biological status, 10-6 mol/L) or Ang-(1-7) (Mas-receptor agonist, 10-7 mol/L). After fixation of cells, fluorescence signals were captured by fluorescence microscopy using an automated imaging system (ImageXpress Pico, Molecular Devices, San Jose, USA) and image analysis by ImageJ. In CHO-AT2, C21 (+34.78 +/- 12.09), Ang II (+28.76 +/- 17.65) and Ang-(1-5) (+78.00 +/- 23.82) increased NO release (one-way ANOVA, at least 3 independent experiments), while Ang-(1-7) had no effect (+ 0.13 ß/- 4.74). In CHO-NT, none of the compounds stimulated release of NO (C21 -4.91 +/-10.24; Ang II -4.79 +/- 12.44; Ang (1-5) -8.88 +/- 18.16; Ang-(1-7) -11.57 +/- 19.95) indicating that the responses in CHO-AT2 were AT2R specific. Conclusions: Measurement of NO release from AT2R transfected CHO cells by DAF-FM fluorescence 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 is commonly regarded as an inactive metabolite of Ang II, has AT2R agonistic properties.</jats:p>