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.

Undernutrition Affects Cell Survival, Oxidative Stress, Ca2+ Handling and Signaling Pathways in Vas Deferens, Crippling Reproductive Capacity

“…In this regard, the huge increase in PKA-mediated phosphorylation of PMCA in the CM group compared to the lesser increase in PKC-mediated phosphorylation (Figure 6B and 6D) could account for this imbalance. We demonstrated that PKA is a key activator of PMCA in the innervated faces of electrocytes from Electrophorus electricus L. [31] and in renal cells [28,29], whereas PKC acts as an inhibitor [41], and the undernutrition-induced imbalance between these two kinases could account for an altered Ca 2+ handling. To date, it has not been demonstrated that SERCA is a substrate for PKA.…”

“…Kinase-mediated phosphorylation of Ca 2+ -ATPase in homogenized vas deferens tissue was determined as previously described [28,29]. The membranes (1 mg/ml) were preincubated for 10 min at 37 o C in a reaction medium containing 50 mM Hepes-Tris (pH 7.4), 0.5 mM ouabain, 5 mM MgCl 2 , 10 mM NaN 3 , 10 mM NaF, 0.3 mM EGTA, 0.34 mM CaCl 2 (10 µM free Ca 2+ ) and 1.1 M hydroxylamine in the absence of protein kinase inhibitors, or in the presence of either 10 nM PKAi 5-24 (to block PKA activity) or 10 nM calphostin C (to ensure inhibition of the diacylglycerol-dependent PKC isoforms).…”

Signalling in response to sub‐picomolar concentrations of active compounds: Pushing the boundaries of GPCR sensitivity

“…The authors suggest that both receptors are required for this ultra‐sensitive response, which fits with identification of functional heterodimers between AT 1 and AT 2 receptors (Axelband et al, ). The ultra‐sensitive Ang II inhibition of Ca 2+ ‐ATPase activity was also abolished by ultra‐low concentrations of an Ang II metabolite, Ang‐(3–4) (Axelband et al, ). In fact, 10 fM Ang‐(3–4) completely restored normal Ca 2+ ‐ATPase activity via a Gα s –cAMP–PKA pathway (Axelband et al, ).…”

“…Thus, opposing phosphorylation of the Ca 2+ ‐ATPase (by Ang II–PKC or Ang‐(3–4)–PKA) in response to ultra‐low concentrations of angiotensin ligands controls its activity. Interestingly, the ultra‐sensitive Ang‐(3–4) response appeared to be dependent on the AT 2 receptor only, as it was blocked in the presence of the AT 2 receptor antagonist, PD123319, but unaffected by the AT 1 receptor antagonist, losartan (Axelband et al, ). The AT 2 receptor was found to co‐immunoprecipitate with Gα s proteins under basal conditions (Axelband et al, ).…”

“…Interestingly, the ultra‐sensitive Ang‐(3–4) response appeared to be dependent on the AT 2 receptor only, as it was blocked in the presence of the AT 2 receptor antagonist, PD123319, but unaffected by the AT 1 receptor antagonist, losartan (Axelband et al, ). The AT 2 receptor was found to co‐immunoprecipitate with Gα s proteins under basal conditions (Axelband et al, ). This is particularly interesting, as we have shown that responses to femtomolar concentrations of relaxin, isoprenaline, and carbachol are all dependent on the pre‐assembly of a multi‐component protein complex (that includes G proteins) at the plasma membrane (Civciristov et al, ; Halls & Cooper, ).…”