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Axelband F.,Federal University of Rio de Janeiro | Axelband F.,Institute Nacional Of Ciencia E Tecnologia Em Biologia Estrutural E Bioimagem | Dias J.,Federal University of Rio de Janeiro | Dias J.,Institute Nacional Of Ciencia E Tecnologia Em Biologia Estrutural E Bioimagem | And 10 more authors.
Regulatory Peptides

We recently demonstrated that Angiotensin-(3-4) [Ang-(3-4)], an Ang II-derived dipeptide, overcomes inhibition of plasma membrane Ca 2+-ATPase promoted by nanomolar concentrations of Ang II in basolateral membranes of renal proximal tubule cells, with involvement of a so far unknown AT 2R-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 AT 2R. Stimulation by cholera toxin of G sα protein structurally linked to AT 2R - as revealed by their co-immunoprecipitation - mimicked the effect of Ang-(3-4) on Ca 2+-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 AT 2R 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 AT 2R antagonist PD123319, whereas the AT 1R antagonist Losartan had no effect. Ang-(3-4) stimulated PKA-mediated phosphorylation of Ca 2+-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 AT 2R, 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 Ca 2+-ATPase through an AT 2R/cAMP/PKA pathway, after inducing conformational changes in AT 2R that results in generation of higher-affinity sites for Ang II. © 2012 Elsevier B.V. Source

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