RESUMEN
We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting ß-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by ß-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on ß-arrestin 2 mediated internalization.
Asunto(s)
Endocitosis/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Arrestinas/antagonistas & inhibidores , Arrestinas/genética , Arrestinas/metabolismo , Western Blotting , Células Cultivadas , Concanavalina A/farmacología , Endocitosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas para Inmunoenzimas , Riñón/citología , Riñón/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Adrenérgicos alfa 1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Arrestina beta 2 , beta-ArrestinasRESUMEN
INTRODUCTION: Prolonged catecholamine overstimulation of the myocardium in chronic heart failure causes a reduction in the number and functionality of beta1-adrenoceptors (beta1-AR) of the heart. Desensitization of beta1-AR is mediated by their phosphorylation by a group of cytosolic kinases (G-protein-coupled receptor kinases GRK). In advanced heart failure, an increase in GRK levels associated with the severity of the disease has been observed. OBJECTIVE: The objective of this study was to analyze messenger RNA (mRNA) levels of beta1-AR in the myocardium of patients who underwent transplantation for advanced heart failure and their correlation with expression of the major cardiac isoenzymes of GRK. MATERIALS AND METHODS: Myocardial tissue samples were obtained from the left ventricles of 14 explanted hearts of patients who underwent transplantation for dilated (n = 7) and ischemic (n = 7) cardiomyopathy. RT-PCR techniques were used to analyze mRNA levels of beta1-AR and the isoenzymes GRK2, GRK3, and GRK5. RESULTS: We observed a significant correlation between beta1-AR and the 3 subtypes of GRK (R(2) = 0.668, 0.71, and 0.318, respectively). CONCLUSIONS: In patients with advanced heart failure pretransplantation, we observed a significant correlation between beta1-AR and GRK2 and GRK3 levels. GRK5, the subtype predominantly expressed in the myocardium, showed a lesser correlation with beta1-AR levels.