β-Pro7Ang III is a novel highly selective angiotensin II type 2 receptor (AT2R) agonist, which acts as a vasodepressor agent via the AT2R in conscious spontaneously hypertensive rats

Abstract

We have previously shown that individual β-amino acid substitution in angiotensin (Ang) II reduced Ang II type 1 receptor (AT1R) but not Ang II type 2 receptor (AT2R)-binding and that the heptapeptide Ang III exhibited greater AT2R:AT1R selectivity than Ang II. Therefore, we hypothesized that β-amino-acid-substituted Ang III peptide analogues would yield highly selective AT2R ligands, which we have tested in binding and functional vascular assays. In competition binding experiments using either AT1R- or AT2R-transfected human embryonic kidney (HEK)-293 cells, novel β-substituted Ang III analogues lacked appreciable AT1R affinity, whereas most compounds could fully displace (125)I-Sar(1)Ile(8) Ang II from AT2R. The rank order of affinity at AT2R was CGP42112 > Ang III > β-Pro(7) Ang III=Ang II > β-Tyr(4) Ang III ≥ PD123319 >> β-Phe(8) Ang III >> β Arg(2) Ang III=β-Val(3) Ang III >> β-Ile(5) Ang III. The novel analogue β-Pro(7) Ang III was the most selective AT2R ligand tested, which was >20,000-fold more selective for AT2R than AT1R. IC50 values at AT2R from binding studies correlated with maximum vasorelaxation in mouse aortic rings. Given that β-Pro(7) Ang III was an AT2R agonist, we compared β-Pro(7) Ang III and native Ang III for their ability to reduce blood pressure in separate groups of conscious spontaneously hypertensive rats. Whereas Ang III alone increased mean arterial pressure (MAP), β-Pro(7) Ang III had no effect. During low-level AT1R blockade, both Ang III and β-Pro(7) Ang III, but not Ang II, lowered MAP (by ∼30 mmHg) at equimolar infusions (150 pmol/kg/min for 4 h) and these depressor effects were abolished by the co-administration of the AT2R antagonist PD123319. Thus, β-Pro(7) Ang III has remarkable AT2R selectivity determined in binding and functional studies and will be a valuable research tool for insight into AT2R function and for future drug development.

Update on angiotensin AT2 receptors

“…Interestingly, previous studies showed that the heptapeptide Ang II metabolite, Ang III, elicited a biphasic BP response with an initial pressor response followed by a depressor response that was blocked by AT 2 R antagonist PD (32). Building on these findings, Del Borgo and colleagues (**33) recently synthesized a new AT 2 R ligand, β-Pro-Ang III, with >20,000-fold AT 2 R to AT 1 R selectivity. This AT 2 R agonist evoked vasorelaxation in vitro and lowered BP acutely by ~ 35 mmHg during low-level AT 1 R blockade in conscious SHR.…”

“…This AT 2 R agonist evoked vasorelaxation in vitro and lowered BP acutely by ~ 35 mmHg during low-level AT 1 R blockade in conscious SHR. These vascular actions of β-Pro-Ang III were abolished by AT 2 R blockade with PD and also by BK B 2 R antagonist icatibant or NOS inhibitor L-NAME, indicating that they were caused by AT 2 R activation via the well-recognized BK-NO AT 2 R signaling pathway in the vasculature (**33). Of primary importance, the vasodepressor actions of Ang III and β-Pro-Ang III were much more impressive than those of Ang II under the same experimental conditions (in the presence of low-level AT 1 R blockade) (**33).…”

“…These vascular actions of β-Pro-Ang III were abolished by AT 2 R blockade with PD and also by BK B 2 R antagonist icatibant or NOS inhibitor L-NAME, indicating that they were caused by AT 2 R activation via the well-recognized BK-NO AT 2 R signaling pathway in the vasculature (**33). Of primary importance, the vasodepressor actions of Ang III and β-Pro-Ang III were much more impressive than those of Ang II under the same experimental conditions (in the presence of low-level AT 1 R blockade) (**33). Similar to reports on natriuresis, these results demonstrate the differential effects of Ang III over Ang II on AT 2 R activation in the vasculature (8–10,15, **33).…”

“…Of primary importance, the vasodepressor actions of Ang III and β-Pro-Ang III were much more impressive than those of Ang II under the same experimental conditions (in the presence of low-level AT 1 R blockade) (**33). Similar to reports on natriuresis, these results demonstrate the differential effects of Ang III over Ang II on AT 2 R activation in the vasculature (8–10,15, **33). …”

β3-tripeptides act as sticky ends to self-assemble into a bioscaffold

“…36 Protease activity was quenched in spiked samples by the addition of acetonitrile (water/acetonitrile, 1:1) at selected intervals and stored at À20 C for analysis. Samples were centrifuged at 10,000g for 5 min, and the supernatant was collected for liquid chromatography-mass spectrometry (LC-MS) analysis.…”