| Autor: |
Silva, Igor Maciel Souza, Kjærgaard, Kenneth, Mortensen, Christina, Santos, Robson A. S., Verano-Braga, Thiago, Stage, Tore Bjerregaard, Steckelings, Ulrike Muscha |
| Jazyk: |
angličtina |
| Rok vydání: |
2021 |
| Zdroj: |
Silva, I M S, Kjærgaard, K, Mortensen, C, Santos, R A S, Verano-Braga, T, Stage, T B & Steckelings, U M 2021, ' A High-throughput Nitric Oxide Measurement Assay Reveals That Angiotensin-(1-5) Is An AT2 Receptor Agonist ', Hypertension, vol. 78, no. Suppl. 1, P250 . https://doi.org/10.1161/hyp.78.suppl_1.p250 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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