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.

We previously demonstrated that Ang II inhibits the renal plasma membrane Ca(2+)-ATPase. In the present work we have studied the effect of Ang II, at concentrations similar to those found in the renal interstitium, on the Ca(2+)-ATPase from proximal tubule cells. High Ang II concentration (5 x 10(-7) mol/L) led to the recovery of Ca(2+)-ATPase activity previously inhibited by 50% at low Ang II concentration (10(-10) mol/L). Reactivation occurred in parallel with: (i) formation of only two dead-end metabolites [Ang-(3-4) and Tyr] after incubation of isolated membranes with micromolar Ang II; and (ii) dissociation of constitutive AT(1)R/AT(2)R heterodimers, which are preserved with 10(-10) mol/L Ang II. When the membranes were incubated with 10(-14) mol/L Ang-(3-4), inhibition by 10(-10) mol/L Ang II was no longer observed. The counteracting effect of Ang-(3-4) was abolished by PD123319, an antagonist of AT(2)R, and mimicked by CGP42112A, an agonist of AT(2)R. Ang-(1-7) is an intermediate in the formation of Ang-(3-4) via a pathway involving angiotensin-converting enzyme (ACE), and complete dipeptide breakdown to Tyr and Val is impaired by low Ang II. We conclude that Ang-(3-4) may be a physiological regulator of active Ca(2+) fluxes in renal proximal cells by acting within the renin-angiotensin axis.

In a previous paper we demonstrated that Ang-(3-4) counteracts inhibition of the Ca(2+)-ATPase by Ang II in the basolateral membranes of kidney proximal tubules cells (BLM). We have now investigated the enzymatic routs by which Ang II is converted to Ang-(3-4). Membrane-bound angiotensin converting enzyme, aminopeptidases and neprilysin were identified using fluorescent substrates. HPLC showed that Plummer's inhibitor but not Z-pro-prolinal blocks Ang II metabolism, suggesting that carboxypeptidase N catalyzes the conversion Ang II--> Ang-(1-7). Different combinations of bestatin, thiorphan, Plummer's inhibitor, Ang II and Ang-(1-5), and use of short proteolysis times, indicate that Ang-(1-7)--> Ang-(1-5)--> Ang-(1-4)--> Ang-(3-4) is a major route. When Ang III was combined with the same inhibitors, the following pathway was demonstrated: Ang III--> Ang IV--> Ang-(3-4). Ca(2+)-ATPase assays with different Ang II concentrations and different peptidase inhibitors confirm the existence of these pathways in BLM and show that a prolyl-carboxypeptidase may be an alternative catalyst for converting Ang II to Ang-(1-7). Overall, we demonstrated that BLM have all the peptidase machinery required to produce Ang-(3-4) in the vicinity of the Ca(2+)-ATPase, enabling a local RAS axis to effect rapid modulation of active Ca(2+) fluxes.

Objetivo: estimar a prevalência do risco de lesão por pressão, identificar os fatores associados e estabelecer modelo de predição para seu desenvolvimento em pacientes atendidos por um programa de atenção domiciliar. Método: trata-se de um estudo transversal, realizado entre junho de 2017 e janeiro de 2018, com 131 pacientes atendidos pelo Programa Melhor em Casa de uma cidade do norte de Minas Gerais. Com auxí­lio de um instrumento foram coletados dados socioeconômicos, demográficos e clí­nicos. Realizou-se análise descritiva, bivariada e regressão de Poisson robusta e ajustada. Resultados: dos 131 pacientes, 76,3% apresentaram risco de lesão por pressão. As variáveis independentes que impactaram, de forma significativa e conjunta, no risco de lesão por pressão foram: "limitação", "relato de memória prejudicada", "incontinência", "lesão sacral" e "proeminência óssea". Conclusão: o risco de lesão por pressão é um achado comum em pacientes da atenção domiciliar e cuidados para sua prevenção devem ser estabelecidos.

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