Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT2 receptor ligands reveals that EMA401 and PD123319 have agonistic properties
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Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT2 receptor ligands reveals that EMA401 and PD123319 have agonistic properties. / Peluso, A. Augusto; Souza-Silva, Igor M.; Villela, Daniel C.; Hansen, Pernille B.L.; Hallberg, Anders; Bader, Michael; Santos, Robson; Sumners, Colin; Steckelings, U. Muscha.
I: Biochemical Pharmacology, Bind 216, 115793, 2023.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT2 receptor ligands reveals that EMA401 and PD123319 have agonistic properties
AU - Peluso, A. Augusto
AU - Souza-Silva, Igor M.
AU - Villela, Daniel C.
AU - Hansen, Pernille B.L.
AU - Hallberg, Anders
AU - Bader, Michael
AU - Santos, Robson
AU - Sumners, Colin
AU - Steckelings, U. Muscha
N1 - Publisher Copyright: © 2023
PY - 2023
Y1 - 2023
N2 - With the discovery of the protective arm of the renin-angiotensin system (RAS), interest has grown in protective RAS-related receptors such as the angiotensin AT2-receptor [AT2R] as potential new drug targets. While it is known that AT2R couple to Gi, it is also apparent that they do not signal via inhibition of adenylyl cyclase/decrease in cAMP, as do many Gi-coupled receptors. Thus, standard commercially-available assays cannot be applied to test for agonistic or antagonistic properties of AT2R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT2R. Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT2R ligands, primarily for distinguishing between AT2R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT2R-transfected CHO cells is a reliable assay for the characterization of AT2R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT2R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT2R-transfected HEK-293 cells are unresponsive to AT2R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT2R antagonists, are partial agonists at the AT2R. Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT2R-CHO cells that is suitable for the characterisation of novel and established AT2R ligands.
AB - With the discovery of the protective arm of the renin-angiotensin system (RAS), interest has grown in protective RAS-related receptors such as the angiotensin AT2-receptor [AT2R] as potential new drug targets. While it is known that AT2R couple to Gi, it is also apparent that they do not signal via inhibition of adenylyl cyclase/decrease in cAMP, as do many Gi-coupled receptors. Thus, standard commercially-available assays cannot be applied to test for agonistic or antagonistic properties of AT2R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT2R. Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT2R ligands, primarily for distinguishing between AT2R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT2R-transfected CHO cells is a reliable assay for the characterization of AT2R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT2R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT2R-transfected HEK-293 cells are unresponsive to AT2R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT2R antagonists, are partial agonists at the AT2R. Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT2R-CHO cells that is suitable for the characterisation of novel and established AT2R ligands.
KW - Angiotensin AT-receptor
KW - EMA401
KW - Nitric oxide
KW - PD123319
U2 - 10.1016/j.bcp.2023.115793
DO - 10.1016/j.bcp.2023.115793
M3 - Journal article
C2 - 37689272
AN - SCOPUS:85171473735
VL - 216
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
SN - 0006-2952
M1 - 115793
ER -
ID: 368251237