title: Angiotensin-(3-4) counteracts the Angiotensin II inhibitory action on renal Ca2+-ATPase through a cAMP/PKA pathway.
abstract: 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(2)R-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(2)R. Stimulation by cholera toxin of G(s)α protein structurally linked to AT(2)R--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(2)R 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(2)R antagonist PD123319, whereas the AT(1)R 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 (3)H-Ang II from HEK 293T cells expressing AT(2)R, but 10(-10) mol/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(2)R/cAMP/PKA pathway, after inducing conformational changes in AT(2)R that results in generation of higher-affinity sites for Ang II.
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Flavia
authors.initials: F
authors.lastname: Axelband
authors.forename: Juliana
authors.initials: J
authors.lastname: Dias
authors.forename: Filipe
authors.initials: F
authors.lastname: Miranda
authors.forename: Fernanda M
authors.initials: FM
authors.lastname: Ferrão
authors.forename: Rosana I
authors.initials: RI
authors.lastname: Reis
authors.forename: Claudio M
authors.initials: CM
authors.lastname: Costa-Neto
authors.forename: Lucienne S
authors.initials: LS
authors.lastname: Lara
authors.forename: Adalberto
authors.initials: A
authors.lastname: Vieyra
mesh_terms: D000494:Allosteric Regulation; D000804:Angiotensin II / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D057912:Angiotensin II Type 2 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation; D057075:Enzyme Assays; D057809:HEK293 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D047468:Immunoprecipitation; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D019808:Losartan / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D044139:Receptor, Angiotensin, Type 2 / Q000819:agonists / Q000378:metabolism*; D012756:Sheep; D015398:Signal Transduction; D014162:Transfection
chemical_list: D057912:Angiotensin II Type 2 Receptor Blockers; D007093:Imidazoles; D009842:Oligopeptides; D011725:Pyridines; D044139:Receptor, Angiotensin, Type 2; D000804:Angiotensin II; C060894:CGP 42112A; C073402:PD 123319; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000252:Calcium-Transporting ATPases; D019808:Losartan