Abstract P240: Decarboxylation of Ang-(1-7) to Ala 1 -ang-(1-7) Leads to Major Changes in Pharmacodynamics

Abstract

Background: Within the renin-angiotensin system, angiotensin (Ang)-(1-7) is cardiovascular protective, stimulates regeneration, and opposes the often detrimental effects of Ang II. We identified two receptors for the heptapeptide; the G protein-coupled receptors Mas and MrgD. Recently, a decarboxylated form of Ang-(1-7), Ala 1 -Ang-(1-7) (Alamandine), has been described as having similar vasorelaxing effects as Ang-(1-7) but distinctively stimulating the MrgD receptor. Methods and Results: The aim of this study was to elucidate the consequences of the lack in the carboxyl group in amino acid one on intracellular signalling, to discover the receptor fingerprint for Ala 1 -Ang-(1-7), and to characterize the consequences for pharmacodynamics. Therfore cAMP was used as the main readout in receptor-transfected HEK293 cells as well as primary cells (mesangial cells and HUVEC). Ala 1 -Ang-(1-7) elevated cAMP concentrations in primary endothelial and mesangial cells. However, the dose-response curves clearly discriminated from the curves generated with Ang-(1-7) (EC 50 = 1.1 x 10 -8 M), with much lower EC 50 (EC 50 = 3.6 x 10 -11 M) and bell-shape for Ala 1 -Ang-(1-7). We provided pharmacological proof that both, Mas (EC 50 = 6.31 x 10 -12 M) and MrgD (EC 50 = 3.98 x 10 -13 M), are functional receptors for Ala 1 -Ang-(1-7). Consequently, the heptapeptide failed to increase cAMP concentration in primary mesangial cells with genetic deficiency in both receptors. As for Ang-(1-7), the AT2 blocker PD123319 also blocked the Ala 1 -Ang-(1-7) effects on Mas and MrgD receptors and in primary cells. The very distinct dose-response curves for both heptapeptides could be explained by in silico modelling, energy calculations, and an involvement of G alpha i for higher concentrations of Ala 1 -Ang-(1-7). Conclusions: Our results identify Ala 1 -Ang-(1-7) as a peptide with specific pharmacodynamic properties and build the basis for the design of more potent and efficient Ang-(1-7) analogues for therapeutic interventions in a rapidly growing number of diseases.