RESUMO
Previous data showed that prostaglandin E2 (PGE2) mediates the inhibitory effect of bradykinin (BK) on proximal tubule (PT) Na+-ATPase activity. The aim of this work was to investigate the molecular mechanisms involved in the effect of PGE2 on PT Na+-ATPase. We used isolated basolateral membrane (BLM) from pig PT, which expresses several components of different signaling pathways. The inhibitory effect of PGE2 on PT Na+-ATPase activity involves G-protein and the activation of protein kinase A (PKA) because: (1) PGE2 increased [³5S]GTPγS binding; (2) GDPßS abolished the inhibitory effect of PGE2; (3) PGE2 increased PKA activity; (4) the inhibitory effect of PGE2 was abolished by PKA inhibitor peptide. We observed that the PKA-mediated inhibitory effect of PGE2 on PT Na+-ATPase activity requires previous activation of protein kinase C. In addition, we observed that PGE2 stimulates Ca²+-independent phospholipase A2 activity representing an important positive feedback to maintain the inhibition of the enzyme. These results open new perspectives to understanding the mechanism involved in the effect of PGE2 on proximal tubule sodium reabsorption.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/enzimologia , Proteína Quinase C/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Bradicinina/farmacologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Dinoprostona/metabolismo , Inibidores Enzimáticos/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Técnicas In Vitro , Túbulos Renais Proximais/metabolismo , Fosfolipases A2 Independentes de Cálcio/metabolismo , Multimerização Proteica/efeitos dos fármacosRESUMO
In a previous paper we showed that bradykinin (BK), interacting with its B2 receptor, inhibits proximal tubule Na+ -ATPase activity but does not change (Na+ +K+)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B2-mediated modulation of proximal tubule Na+ -ATPase by BK. To abolish B1 receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg9-[Leu8]-BK (DALBK), a specific antagonist of B1 receptor. A dual effect on the Na+ -ATPase activity through the B2 receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C beta (PI-PLCbeta)/protein kinase C (PKC); its inhibitory action is mediated by Ca2+ -independent phospholipase A2 (iPLA2). Prior activation of the PI-PLCbeta/PKC pathway is required to activate the iPLA2-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B2 receptor and activation of membrane-associated iPLA2.