RESUMEN
RATIONALE: During the transition from compensated hypertrophy to heart failure, the signaling between L-type Ca(2+) channels in the cell membrane/T-tubules and ryanodine receptors in the sarcoplasmic reticulum becomes defective, partially because of the decreased expression of a T-tubule-sarcoplasmic reticulum anchoring protein, junctophilin-2. MicroRNA (miR)-24, a junctophilin-2 suppressing miR, is upregulated in hypertrophied and failing cardiomyocytes. OBJECTIVE: To test whether miR-24 suppression can protect the structural and functional integrity of L-type Ca(2+) channel-ryanodine receptor signaling in hypertrophied cardiomyocytes. METHODS AND RESULTS: In vivo silencing of miR-24 by a specific antagomir in an aorta-constricted mouse model effectively prevented the degradation of heart contraction, but not ventricular hypertrophy. Electrophysiology and confocal imaging studies showed that antagomir treatment prevented the decreases in L-type Ca(2+) channel-ryanodine receptor signaling fidelity/efficiency and whole-cell Ca(2+) transients. Further studies showed that antagomir treatment stabilized junctophilin-2 expression and protected the ultrastructure of T-tubule-sarcoplasmic reticulum junctions from disruption. CONCLUSIONS: MiR-24 suppression prevented the transition from compensated hypertrophy to decompensated hypertrophy, providing a potential strategy for early treatment against heart failure.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Acoplamiento Excitación-Contracción/efectos de los fármacos , Insuficiencia Cardíaca/prevención & control , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , MicroARNs/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Oligonucleótidos Antisentido/uso terapéutico , Animales , Estenosis Aórtica Subvalvular/complicaciones , Canales de Calcio Tipo L/fisiología , Señalización del Calcio/fisiología , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Hipertrofia Ventricular Izquierda/complicaciones , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/fisiología , Modelos Cardiovasculares , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Oligonucleótidos Antisentido/farmacología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/fisiología , Retículo Sarcoplasmático/ultraestructuraRESUMEN
RATIONALE: Failing cardiomyocytes exhibit decreased efficiency of excitation-contraction (E-C) coupling. The downregulation of junctophilin-2 (JP2), a protein anchoring the sarcoplasmic reticulum to T-tubules, has been identified as a major mechanism underlying the defective E-C coupling. However, the regulatory mechanism of JP2 remains unknown. OBJECTIVE: To determine whether microRNAs regulate JP2 expression. METHODS AND RESULTS: Bioinformatic analysis predicted 2 potential binding sites of miR-24 in the 3'-untranslated regions of JP2 mRNA. Luciferase assays confirmed that miR-24 suppressed JP2 expression by binding to either of these sites. In the aortic stenosis model, miR-24 was upregulated in failing cardiomyocytes. Adenovirus-directed overexpression of miR-24 in cardiomyocytes decreased JP2 expression and reduced Ca(2+) transient amplitude and E-C coupling gain. CONCLUSIONS: MiR-24-mediated suppression of JP2 expression provides a novel molecular mechanism for E-C coupling regulation in heart cells and suggests a new target against heart failure.
Asunto(s)
Estenosis de la Válvula Aórtica/metabolismo , Insuficiencia Cardíaca/metabolismo , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Miocitos Cardíacos/metabolismo , Regulación hacia Arriba , Animales , Estenosis de la Válvula Aórtica/patología , Calcio/metabolismo , Células Cultivadas , Biología Computacional , Acoplamiento Excitación-Contracción/fisiología , Insuficiencia Cardíaca/patología , Proteínas de la Membrana/genética , MicroARNs/genética , Modelos Animales , Miocitos Cardíacos/patología , ARN Mensajero/metabolismo , Ratas , Retículo Sarcoplasmático/fisiologíaRESUMEN
AIM: To study and establish a proteome reference map and regulation network of neonatal rat cardiomyocyte. METHODS: Cultured cardiomyocytes of neonatal rats were used. All proteins expressed in the cardiomyocytes were separated and identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Biological networks and pathways of the neonatal rat cardiomyocytes were analyzed using the Ingenuity Pathway Analysis (IPA) program (www.ingenuity.com). A 2-DE database was made accessible on-line by Make2ddb package on a web server. RESULTS: More than 1000 proteins were separated on 2D gels, and 148 proteins were identified. The identified proteins were used for the construction of an extensible markup language-based database. Biological networks and pathways were constructed to analyze the functions associate with cardiomyocyte proteins in the database. The 2-DE database of rat cardiomyocyte proteins can be accessed at http://2d.bjmu.edu.cn. CONCLUSION: A proteome reference map and regulation network of the neonatal rat cardiomyocytes have been established, which may serve as an international platform for storage, analysis and visualization of cardiomyocyte proteomic data.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Miocitos Cardíacos/química , Proteoma/análisis , Proteómica/métodos , Animales , Células Cultivadas , Masculino , Redes y Vías Metabólicas , Proteoma/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
To test the hypothesis that AMP-activated protein kinase (AMPK) is possibly the downstream signaling molecule of certain subtypes of adrenergic receptor (AR) in the heart, we evaluated AMPK activation mediated by ARs in H9C2 cells, a rat cardiac source cell line, and rat hearts. The AMPK-alpha subunit and the phosphorylation level of Thr(172)-AMPK-alpha subunit were subjected to Western blot analysis. Osmotic minipumps filled with norepinephrine (NE), phenylephrine (PE) or vehicle [0.01% (W/V) vitamin C solution] were implanted into male Sprague-Dawley rats subcutaneously. The pumps delivered NE or PE continuously at the rate of 0.2 mg/kg per hour. After 7-day infusion, the activity of AMPK was examined following immunoprecipitation with anti-AMPK-alpha antibody. At the cellular level, we found that NE elevated AMPK phosphorylation level in a dose- and time-dependent manner, with the maximal effect at 10 micromol/L NE after 10-minute treatment. This effect was insensitive to propranolol, a specific beta-AR antagonist, but abolished by prazosin, an alpha(1)-AR antagonist, suggesting that alpha(1)-AR but not beta-AR mediated the phosphorylation of AMPK. Moreover, the results from rat models of 7-day-infusion of AR agonists demonstrated that the activity of AMPK was significantly higher in NE (7.4-fold) and PE (6.0-fold) infusion groups than that in the vehicle group (P<0.05, n=6). On the other hand, no obvious cardiac hypertrophy and tissue fibrosis changes were observed in PE-infused rats. Taken together, our results demonstrate that alpha(1)-AR stimulation enhances the activity of AMPK, indicating an important role of alpha(1)-AR stimulation in the regulation of AMPK in the heart. Understanding the activation of AMPK mediated by alpha(1)-AR might have clinical implications in the therapy of heart failure.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos alfa/fisiología , Animales , Línea Celular , Ventrículos Cardíacos , Masculino , Miocardio/citología , Norepinefrina/farmacología , Fenilefrina/farmacología , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
To investigate the subcellular distribution of three alpha(1)-adrenergic receptor subtypes and their internalization and trafficking upon agonist stimulation in human embryonic kidney (HEK) 293A cell line, saturation radioligand binding assay, laser confocal imaging, and Western blot were applied to examine the distribution and changes in localization of three alpha(1)-AR subtypes in transfected HEK 293A cells prior to and after treatment with phenylephrine. The results are as follows: (1) The transfection efficiency was over 90%and was equal among three alpha(1)-AR subtypes. alpha(1B ) -AR expression in cell membrane was the highest, and alpha(1D ) -AR was the lowest, as determined by (125)I-BE2254 binding assay, however, K(d)s were not significantly different among the three receptor subtypes. (2) Without agonist stimulation, alpha(1A ) -AR was detected not only on the cell surface but also in the cytosol, alpha(1B ) -AR was predominantly located on the cell surface, whereas alpha(1D ) -AR was mostly detected in the cytosol. (3) After 1 h of stimulation with phenylephrine, as observed using confocal microscope, less alpha(1A)- and alpha(1B ) -AR were detected on the cell surface but more in the cytosol. The change was more remarkable in alpha(1B)-AR than that in alpha(1A)-AR, whereas no change of distribution was detected in alpha(1D)-AR in response to phenylephrine. However, when examined by Western blot, no change in distribution was detected in alpha(1A)- and alpha(1D)-AR, only alpha(1B)-AR showed the same change as that shown in confocal imaging. It is suggested that the characteristics of localization and changes of distribution are different among three alpha(1)-AR subtypes in HEK293A cells upon phenylephrine stimulation.
RESUMEN
In the heart, stimulation of beta-adrenergic receptors (betaAR) serves as the most powerful means to increase cardiac contractility and relaxation in response to stress or a "fight-or-flight" situation. However, sustained beta-adrenergic stimulation promotes pathological cardiac remodeling such as myocyte hypertrophy, apoptosis and necrosis, thus contributing to the pathogenesis of chronic heart failure. Over the past decade, compelling evidence has demonstrated that coexisting cardiac betaAR subtypes, mainly beta(1)AR and beta (2)AR, activate markedly different signaling cascades. As a result, acute beta(1)AR stimulation activates the G(s) -adenylyl cyclase-cAMP-PKA signaling that can broadcast throughout the cell, whereas beta(2)AR-evoked cAMP signaling is spatially and functionally compartmentalized, due to concurrent G(i) activation. Chronic stimulation of beta(1)AR and beta(2)AR elicits opposing effects on the fate of cardiomyocytes: beta(1)AR induces hypertrophy and apoptosis; but beta(2)AR promotes cell survival. The cardiac protective effect of beta(2)AR is mediated by a signaling pathway sequentially involving G(i), G(betagamma), PI3K and Akt. Unexpectedly, beta(1)AR-induced myocyte hypertrophy and apoptosis are independent of the classic cAMP/PKA pathway, but require activation of Ca(2+)/calmodulin-dependent kinase II (CaMK II). The outcomes of cardiac-specific transgenic overexpression of either beta AR subtype in mice have reinforced the fundamentally different functional roles of these betaAR subtypes in governing cardiac remodeling and performance. These new insights regarding betaAR subtype stimulation not only provide clues as to cellular and molecular mechanisms underlying the beneficial effects of beta AR blockers in patients with chronic heart failure, but also delineate rationale for combining selective beta(1)AR blockade with moderate beta(2)AR activation as a potential novel therapy for the treatment of chronic heart failure.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos beta/fisiología , Adenilil Ciclasas/metabolismo , Animales , Proteínas de Unión al GTP/metabolismo , Corazón/fisiología , Insuficiencia Cardíaca/fisiopatología , Humanos , Receptores Adrenérgicos beta/clasificación , Receptores Adrenérgicos beta 1/fisiología , Receptores Adrenérgicos beta 2/fisiología , Transducción de SeñalRESUMEN
The expression of beta-adrenergic receptor subtypes and its effect on neonatal rat cardiac fibroblast proliferation were investigated by radioligand binding assay and [(3)H]-thymidine incorporation analysis, respectively. The results indicated that there was no significant difference in the beta-adrenergic receptor density (B(max)) and affinity (K(D)) between cardiomyocytes and cardiac fibroblasts. The [(125)I]-pindolol competitive inhibition curves (ICI 118551 and CGP 20712A) were significantly better fit in a one-site model in membrane preparation of cardiac fibroblasts. In cultured cardiac fibroblasts, 0.1 micromol/L isoproterenol-induced [(3)H]-thymidine incorporation was completely inhibited by a selective beta (2)-AR antagonist ICI 118551, or a non-selective beta-AR antagonist propranolol, but not by CGP 20712A, a selective beta(1)-AR antagonist. These results suggest that isoproterenol-induced cardiac fibroblast proliferation is mediated by beta(2)-AR, the preponderant beta-AR subtype in cardiac fibroblasts.
Asunto(s)
Fibroblastos/citología , Miocitos Cardíacos/citología , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/fisiología , Animales , Animales Recién Nacidos , Proliferación Celular , Células Cultivadas , Fibroblastos/metabolismo , Miocitos Cardíacos/metabolismo , Ratas , Ratas Wistar , Receptores Adrenérgicos beta 2/genéticaRESUMEN
Using matchmaker yeast two-hybrid system, it has been demonstrated that there exists an interaction between the cellular C terminal of alpha(1A)-adrenergic receptor (alpha(1A)-AR) and a segment of bone morphogenetic protein-1 (BMP-1). In the present study binding between the two proteins was further determined in human embryonic cell 293 (HEK293), a mammalian expression system. Mammalian expression vector PCP3HA was constructed by PCR and consisted of segments of BMP-1 cDNA, and vector PDT-alpha(1A) consisted of the full-length cDNA of human alpha(1A)-AR. They were transfected to HEK293 cells and examined by Western blot. alpha(1A)-AR and the segment of BMP-1 could be detected in the lysis of transfected cells. Then binding between alpha(1A)-AR and the segment of BMP-1 in HEK293 cell was determined by enzyme-linked immunosorbent assays (ELISA) and co-immunoprecipitation. In ELISA experiment, the ELISA microwell plate was first coated with anti-FLAG M2 antibody, which recognizes the FLAG-tagged alpha(1A)-AR, then the cell lysis, anti-HA rabbit polyclonal antibody and HRP conjugated anti-rabbit antibody were added in turn. The OD(490) values among the control group, PDT-alpha(1A) transfection group and PCP3HA transfection group, exhibited no significant difference (0.034+/-0.027, 0.042+/-0.019, 0.030+/-0.0096), but the OD(490) values of PDT-alpha(1A) and PCP3HA co-transfection group (0.57+/-0.12) were significantly higher than those of the other groups (P<0.001, respectively). In co-immunoprecipitation experiments, HEK293 cells expressing alpha(1A)-AR or/and segment of BMP-1 were lysed and incubated with anti-FLAG M2 antibody, then the immunoprecipitation pellet was immunoblotted with either the HRP conjugated anti-FLAG antibody or the anti-HA antibody, which recognizes the HA-tagged segment of BMP-1. Segment of BMP-1 was present in the pellet immunoprecipitation of PDT-alpha(1A) and PCP3HA co-transfected group. In conclusion, the results indicate that alpha(1A)-AR and the segment of BMP-1 are present in the same complex in HEK293 cells.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Riñón/metabolismo , Metaloendopeptidasas/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Proteína Morfogenética Ósea 1 , Proteínas Morfogenéticas Óseas/genética , Células Cultivadas , Embrión de Mamíferos , Ensayo de Inmunoadsorción Enzimática , Humanos , Riñón/citología , Metaloendopeptidasas/genética , Unión Proteica , Receptores Adrenérgicos alfa 1/genética , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The aim of the present study was to investigate the effects of beta-adrenergic receptor (beta-AR) activation on metabolism in cultured neonatal rat cardiomyocytes. The protein synthesis and total protein content of cardiomyocytes were determined by [(3)H]-leucine incorporation and BCA protein content assay. Cardiomyocyte glucose uptake was measured by [(3)H]-2-deoxy-D-glucose uptake analysis. Adenosine monophosphate activated protein kinase (AMPK) phosphorylation was detected by Western blot. The results showed that sustained stimulation with isoproterenol (ISO), a beta-adrenoceptor agonist, had no effect on [(3)H]-leucine incorporation and total protein content in cardiomyocytes. With beta-AR activation by ISO or NE (pretreated with a selective blocker of the alpha(1)-adrenoceptor prazosin) for 48 h, both the glucose uptake and AMPK phosphorylation increased significantly compared with unstimulated cardiomyocytes. These results suggest that although sustained beta-AR activation has no effect on cardiomyocyte protein metabolism, glucose uptake and AMPK activity are increased significantly. The role of these beta-AR activation-induced changes in cardiac hypertrophy remains to be further investigated.
Asunto(s)
Glucosa/metabolismo , Proteínas Musculares/biosíntesis , Miocitos Cardíacos/metabolismo , Proteínas Quinasas Activadas por AMP , Animales , Animales Recién Nacidos , Células Cultivadas , Complejos Multienzimáticos/metabolismo , Miocitos Cardíacos/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos betaRESUMEN
This study was aimed to determine the in vivo signal transduction pathway responsible for isoproterenol (ISO)-induced cardiac hypertrophy or remodeling. Mice were treated with ISO (15 mg/kg body weight) or vehicle by intraperitoneal injection (i.p.). Activation of mitogen-activted protein kinase (MAPK), NF-kappaB and JAK/STAT pathway in the left ventricular myocardium was measured by Western blot analysis. ISO significantly activated MAPK (ERK1/2 and p38) at early phase (5 min); biphasic activation of NF-kappaB was observed in our in vivo study; and ISO caused a delayed STAT3 activation (at 60 to 240 min) in mouse myocardium. Taken together, these results indicate that ISO activates these signal transduction pathways in different time course.
Asunto(s)
Isoproterenol/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Animales , Corazón/efectos de los fármacos , Janus Quinasa 1/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Factor de Transcripción STAT3/metabolismo , Factores de TiempoRESUMEN
Wistar rats of 8, 10 and 12-week-old were chosen for study of the relationship between cardiac growth and its gene expression profile changes during maturation. The ultrasonic parameters of rat hearts were recorded before sacrifice, then total RNA of left ventricle were extracted and gene expression profiles were analyzed by cDNA microarray. During growth from 8 weeks to 12 weeks, the body weight increased by 45.5% (287+/-13 g vs 197+/-10 g), and the increment in the first two-week period was equal to that of the second two-week period. The mass of left ventricle and the posterior wall thickness increased by 27.7% (0.60+/-0.03 g vs 0.47+/-0.02 g) and 23.6% (2.04+/-0.04 mm vs 1.65+/-0.13 mm), respectively, and their increment in the first two-week period was much more than that in the second one. Meanwhile, the gene expression profile of the left ventricle changed significantly, which involved cellular structure, metabolism, oxidative stress, signal transduction, etc. Compared with the 8-week-old rats, these genes were mostly up-regulated in 10-week-old rats, while for 12-week-old rats, the gene expression profile of the left ventricle recovered to the pattern of 8-week-old rats again on the whole. These results suggest that the relationship between the changes in cardiac function and gene expression profile can be analyzed comprehensively with the technique of microarray, and that the changes in gene expression profile of the left ventricle during rat maturation adapt to the physiological growth of heart, which is of benefit for keeping the metabolism balance between materials and energy.
Asunto(s)
Ventrículos Cardíacos/anatomía & histología , Corazón/crecimiento & desarrollo , Miocardio/metabolismo , Transcriptoma , Animales , Corazón/fisiología , Masculino , Miocardio/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas WistarRESUMEN
To get insights into the principles of gene expression changes during cardiac hypertrophy, three rat cardiac hypertrophy models were prepared, i.e., suprarenal abdominal aortic stenosis (SRS), arterial-vein fistula (AVF) and continuous jugular vein infusion of norepinephrine (NEi). The cardiac function and structure were analyzed by echocardiograph as well as histological examination. Total RNA of left ventricles was extracted and gene expression profiles were analyzed by cDNA microarray. SRS and NEi induced concentric cardiac hypertrophy and AVF induced eccentric hypertrophy in rats, among which NEi caused obvious cardiac fibrosis. The changes of gene expression profiles were compared comprehensively across different pathologic cardiac hypertrophy models. While gene expression profiles of different cardiac hypertrophy models compared with pairs, parts of the genes involved were found overlapped, and mostly the gene expression changed in the same direction between two models, but some of them changed in the opposite directions. Expression levels of 19 genes were found changed across all cardiac hypertrophy models, and genes relatively regulated in a specific model was also found when comparison of all the three models was carried out. Novel clues for further study might derive from the results mentioned above, and some genes might be the marker genes of cardiac hypertrophy or the targets of therapy.
Asunto(s)
Cardiomegalia/genética , Perfilación de la Expresión Génica , Miocitos Cardíacos/metabolismo , Animales , Aorta Abdominal/cirugía , Derivación Arteriovenosa Quirúrgica , Cardiomegalia/etiología , Constricción , Masculino , Miocitos Cardíacos/efectos de los fármacos , Norepinefrina , Análisis de Secuencia por Matrices de Oligonucleótidos , Sondas de Oligonucleótidos , Distribución Aleatoria , Ratas , Ratas Wistar , Venas Cavas/cirugíaRESUMEN
The purpose of the present study was to observe the expression of Axin protein during cardiac remodeling in rats. Cardiac remodeling animal models were prepared with the methods of jugular venous norepinephrine (NE)-infusion or arterial-vein fistula (AVF). The ultrasonic parameters of rat hearts were recorded before sacrifice. The expressions of Axin protein were determined by Western blot in rat hearts from different remodeling models as well as cultured cardiac fibroblasts from adult rats. Cardiac concentric hypertrophy and fibrosis was induced by 3-day jugular vein infusion of NE in rats. The expression of Axin in the left ventricles increased significantly compared with that of the control group. Cardiac eccentric hypertrophy without fibrosis was induced by A-V fistula for one week in rats, and no change in Axin protein expression in the left ventricles was observed. In cultured adult rat cardiac fibroblasts, NE treatment for 24 h increased significantly the Axin protein level. It is therefore concluded that Axin protein was expressed in rat heart and increased significantly in left ventricles during NE-induced rat cardiac remodeling, which may be relevant to cardiac fibrosis.
Asunto(s)
Proteína Axina/metabolismo , Ventrículos Cardíacos/metabolismo , Remodelación Ventricular/fisiología , Animales , Células Cultivadas , Fibroblastos/citología , Masculino , Miocitos Cardíacos/citología , Norepinefrina/farmacología , Ratas , Ratas Sprague-DawleyAsunto(s)
Medicina Tradicional China , Médicos , Sociedades Médicas , China , Congresos como Asunto , Quimioterapia Combinada , HumanosAsunto(s)
Calcio/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Animales , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/fisiología , Humanos , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores Adrenérgicos alfa 1/inmunología , Receptores Adrenérgicos alfa 1/fisiología , Quinasas de Receptores Adrenérgicos betaRESUMEN
AIM: To examine the subcellular distribution of the 3 alpha1-adrenoceptor (alpha1-AR) subtypes and their internalization and trafficking upon agonist stimulation in human embryonic kidney 293A cells. METHODS: Confocal real-time imaging, enzyme linked immunosorbent assay (ELISA) and whole cell [3H]-prazosin binding assay were applied to detect the distribution and localization of the 3 alpha1-AR subtypes. RESULTS: alpha1A-AR was found both on the cell surface and in the cytoplasm; alpha1BAR, however, was predominantly detected on the cell surface, while alpha1D-AR was detected mainly in the intracellular compartments. After stimulation with phenylephrine, localization changes were detected by confocal microscopy for alpha1A- and alpha1B-AR,but the localization of alpha1D-AR were unaffected. Phenylephrine stimulation promoted a more rapid internalization of alpha1B-AR than alpha1A-AR. alpha1D-AR internalization was detected only by ELISA. Whole cell [3H]-prazosin binding assay showed that alpha1A-AR functional receptors were detected both on the cell surface and in the cytoplasm; alpha1B-AR, however, were detected predominantly on the cell surface, while alpha1D-AR were detected mainly in intracellular compartments. Phenylephrine stimulation promoted internalization of alpha1A- and alpha1B-AR. CONCLUSION: Phenylephrine stimulation induced changes in the localization of the 3 alpha1-AR.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 1 , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Fenilefrina/farmacología , Prazosina/farmacología , Receptores Adrenérgicos alfa 1/biosíntesis , Receptores Adrenérgicos alfa 1/genética , TransfecciónRESUMEN
As we previously reported, cAMP and p38 MAPK instead of protein kinase A were involved in beta-adrenergic receptor (beta-AR)-mediated interleukin-6 (IL-6) production in mouse cardiac fibroblasts. Besides kinases, phosphatases may also be involved in IL-6 gene regulation. To study the role of protein tyrosine phosphatases (PTPs) in beta-AR-mediated IL-6 production, we selected the most widely used PTP inhibitor, phenylarsine oxide (PAO). We found that PAO dose-dependently inhibited the IL-6 release in response to beta-AR agonist isoproterenol (ISO) in mouse cardiac fibroblasts. This effect was probably due to the inhibition of PTPs, resulting in increased tyrosine phosphorylation, since genistein, an inhibitor of protein tyrosine kinases further potentiated ISO-induced IL-6 production and could partially reverse the inhibitory effect of PAO. PAO also significantly inhibited the IL-6 production by forskolin, an adenylyl cyclase (AC) activator. Furthermore, PAO dose-dependently inhibited the increased cAMP accumulation by either ISO or forskolin and suppressed the phosphorylation of CREB, an important transcriptional factor for IL-6 gene expression. But PAO did not affect the activation of p38 MAPK by ISO. Although PAO was also reported to inhibit NADPH oxidase, the inhibition of NADPH oxidase by its specific inhibitor, diphenylene iodonium (DPI) could not suppress beta-AR-mediated IL-6 production, suggesting that NADPH oxidase may not contribute to the inhibitory effect of PAO on IL-6 production. To our knowledge, this is the first report that PAO can inhibit ISO-induced IL-6 expression and CREB phosphorylation, demonstrating that PTPs may negatively regulate beta-AR-mediated IL-6 production. This study may also further our understanding of beta-AR signaling and provide potential therapeutic targets for the treatment of heart diseases.
Asunto(s)
Arsenicales/administración & dosificación , Proteína de Unión a CREB/metabolismo , AMP Cíclico/metabolismo , Fibroblastos/metabolismo , Interleucina-6/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Miocardio/citologíaRESUMEN
AIM: The enhancement of intracellular Ca2+ signaling in response to alpha 1-adrenergic receptor (alpha 1-AR) stimulation is an essential signal transduction event in the regulation of cardiac functions, such as cardiac growth, cardiac contraction, and cardiac adaptation to various situations. The present study was intended to determine the role(s) of the alpha 1-AR subtype(s) in mediating this response. METHODS: We evaluated the effects of subtype-specific agonists and antagonists of the alpha 1- AR on the intracellular Ca2+ signaling of neonatal rat ventricular myocytes using a confocal microscope. RESULTS: After being cultured for 48 h, the myocytes exhibited spontaneous local Ca2+ release, sparks, and global Ca2+ transients. The activation of the alpha 1-AR with phenylephrine, a selective agonist of the alpha 1-AR, dose-dependently increased the frequency of Ca2+ transients with an EC50 value of 2.3 micromol/L. Blocking the alpha 1A-AR subtype with 5-methylurapidil (5-Mu) inhibited the stimulatory effect of phenylephrine with an IC(50) value of 6.7 nmol/L. In contrast, blockade of the alpha 1B-AR and alpha 1D-AR subtypes with chloroethylclonidine and BMY 7378, respectively, did not affect the phenylephrine effect. Similarly, the local Ca2+ spark numbers were also increased by the activation of the alpha 1-AR, and this effect could be abolished selectively by 5-Mu. More importantly, A61603, a novel selective alpha 1A-AR agonist, mimicked the effects of phenylephrine, but with more potency (EC(50) value =6.9 nmol/L) in the potentiation of Ca2+ transients, and blockade of the alpha 1A-AR by 5-Mu caused abolishment of its effects. CONCLUSION: These results indicate that alpha 1-adrenergic stimulation of intracellular Ca2+ activity is mediated selectively by the alpha 1A-AR.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1 , Antagonistas de Receptores Adrenérgicos alfa 1 , Animales , Imidazoles/metabolismo , Miocitos Cardíacos/citología , Ratas , Ratas Sprague-Dawley , Tetrahidronaftalenos/metabolismoRESUMEN
AIM: To investigate the movement of alpha(1A)-adrenergic receptors(alpha(1A)-AR) stimulated by agonist, phenylephrine (PE), and the dynamics of receptor movement in real time in single living cells with millisecond resolution. METHODS: We labeled alpha(1A)-AR using the monoclonal, anti-FLAG (a kind of tag) antibody and Cy3-conjugated goat anti-mouse IgG and recorded the trajectory of their transport process in living HEK293A cells stimulated by agonist, PE, and then analyzed their dynamic properties. RESULTS: The specific detection of alpha(1A)-AR on the surface of living HEK293A-alpha(1A) cells was achieved. alpha(1A)-AR internalize under the stimulation of PE. After the cells were stimulated with PE for 20 min, apparent colocalization was found between alpha(1A)-AR and F-actins. After 40 min stimulation of PE, trajectories of approximate linear motion in HEK293A-alpha(1A) cells were recorded, and their velocity was calculated. CONCLUSION: The specific labeling method on the living cell surface provides a convenient means of real-time detection of the behavior of surface receptors. By this method we were able to specifically detect alpha(1A)-AR and record the behavior of individual particles of receptors with 50 ms exposure time in real time in single living cells.