Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT(2) receptor ligands reveals that EMA401 and PD123319 have agonistic properties

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 AT(2)-receptor [AT(2)R] as potential new drug targets. While it is known that AT(2)R 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 AT(2)R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT(2)R. Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT(2)R ligands, primarily for distinguishing between AT(2)R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT(2)R-transfected CHO cells is a reliable assay for the characterization of AT(2)R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT(2)R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT(2)R-transfected HEK-293 cells are unresponsive to AT(2)R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT(2)R antagonists, are partial agonists at the AT(2)R. Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT(2)R-CHO cells that is suitable for the characterisation of novel and established AT(2)R ligands