Angiotensin-(3–4) counteracts the Angiotensin II inhibitory action on renal Ca2+-ATPase through a cAMP/PKA pathway

We recently demonstrated that Angiotensin-(3−4) [Ang-(3−4)], an Ang II-derived dipeptide, overcomes inhibition of plasma membrane Ca2+-ATPase promoted by nanomolar concentrations of Ang II in basolateral membranes of renal proximal tubule cells, with involvement of a so far unknown AT2R-dependent and NO-independent mechanism. The present study investigates the signaling pathway triggered by Ang-(3−4) that is responsible for counteracting the inhibitory effect of Ang II, and attempts to elucidate the functional interaction of the dipeptide with Ang II at the level of AT2R. Stimulation by cholera toxin of Gsα protein structurally linked to AT2R − as revealed by their co-immunoprecipitation − mimicked the effect of Ang-(3−4) on Ca2+-ATPase activity. Furthermore, addition of dibutyril-cAMP (db-cAMP) mimicked Ang-(3−4), whereas the specific PKA inhibitor, PKAi(5–24) peptide, suppressed the counter-regulatory effect of Ang-(3−4) and the AT2R agonist, CGP42112A. Membrane-associated PKA activity was stimulated by Ang-(3−4) or CGP42112A to comparable levels as db-cAMP, and the Ang-(3–4) effect was abrogated by the AT2R antagonist PD123319, whereas the AT1R antagonist Losartan had no effect. Ang-(3−4) stimulated PKA-mediated phosphorylation of Ca2+-ATPase and activated PKA to comparable levels. Binding assays demonstrated that Ang-(3−4) could not displace 3H-Ang II from HEK 293T cells expressing AT2R, but 10−10mol/L Ang-(3−4) resulted in the appearance of a probable higher-affinity site (picomolar range) for Ang II. The results presented herein demonstrate that Ang-(3−4), acting as an allosteric enhancer, suppresses Ang II-mediated inhibition of Ca2+-ATPase through an AT2R/cAMP/PKA pathway, after inducing conformational changes in AT2R that results in generation of higher-affinity sites for Ang II. ► Signaling pathway triggered by Ang-(3−4) in renal cells. ► Partnership among Ang-(3−4), type 2 angiotensin II receptors and protein kinase A. ► Ang-(3−4) and renal calcium handling.