RESUMEN
BACKGROUND: Apoptotic death of endothelial cells (EC) plays a crucial role for the development of ischemic injury. In the present study we investigated the impact of extracellular Adenosine-5'-triphosphate (ATP), either released from cells or exogenously added, on ischemia-induced apoptosis of human EC. METHODS AND RESULTS: To simulate ischemic conditions, cultured human umbilical vein endothelial cells (HUVEC) were exposed to 2 h of hypoxia (Po(2)<4mm Hg) in serum-free medium. Ischemia led to a 1.7-fold (+/-0.4; P<0.05) increase in EC apoptosis compared to normoxic controls as assessed by immunoblotting and immunocytochemistry of cleaved caspase-3. Ischemia-induced apoptosis was accompanied by a 2.3-fold (+/-0.5; P<0.05) increase of extracellular ATP detected by using a luciferin/luciferase assay. Addition of the soluble ecto-ATPase apyrase, enhancing ATP degradation, increased ischemia-induced caspase-3 cleavage. Correspondingly, inhibition of ATP breakdown by addition of the selective ecto-ATPase inhibitor ARL67156 significantly reduced ischemia-induced apoptosis. Extracellular ATP acts on membrane-bound P2Y- and P2X-receptors to induce intracellular signaling. Both, ATP and the P2Y-receptor agonist UTP significantly reduced ischemia-induced apoptosis in an equipotent manner, whereas the P2X-receptor agonist αß-me-ATP did not alter caspase-3 cleavage. The anti-apoptotic effects of ARL67156 and UTP were abrogated when P2-receptors were blocked by Suramin or PPADS. Furthermore, extracellular ATP led to an activation of MEK/ERK- and PI3K/Akt-signaling pathways. Accordingly, inhibition of MEK/ERK-signaling by UO126 or inhibition of PI3K/Akt-signaling by LY294002 abolished the anti-apoptotic effects of ATP. CONCLUSION: The data of the present study indicate that extracellular ATP counteracts ischemia-induced apoptosis of human EC by activating a P2Y-receptor-mediated signaling reducing caspase-3 cleavage.
Asunto(s)
Adenosina Trifosfato/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Citoprotección , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Isquemia/enzimología , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y/metabolismo , Butadienos/farmacología , Células Cultivadas , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Isquemia/patología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Morfolinas/farmacología , Nitrilos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
AIMS: Since adenine nucleotide translocase 1 (ANT1) overexpression improved cardiac function in rats with activated renin-angiotensin system (RAS) and angiotensin II is known to enhance transforming growth factor ß (TGFß) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGFß/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGFß(1)-induced apoptosis, whether mitochondrial permeability transition pore (MPTP) regulation is involved, and SMAD signaling pathway is affected. METHODS AND RESULTS: Ventricular cardiomyocytes isolated from wild-type (WT) and ANT1 transgenic rats were treated with the apoptosis-inducing agent TGFß(1) (1 ng/ml). TGFß(1) treatment of WT cells enhanced the number of apoptotic cells by 31.8 ± 11.7% (p<0.01 vs. WT) measured by chromatin condensation. Apoptosis was blocked by 1µM cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 overexpression on TGFß(1)-induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFß(1) decreased mitochondrial membrane potential as measured by JC-1 staining by 18.0 ± 3.7% in WT cardiomyocytes, but only by 7.2 ± 2.8% (p<0.05 vs. WT) in ANT1 cardiomyocytes. Cyclosporine A also attenuated the decline in mitochondrial membrane potential under TGFß(1) in WT cardiomyocytes. Determination of MPTP opening by Calcein assay in isolated cardiomyocytes and calcium retention assay in isolated mitochondria revealed a reduced open probability of MPTP after ANT1 overexpression. In addition to the effects of ANT1 on MPTP opening we investigated if ANT1 may interfere with the classical TGFß signaling pathway. Interestingly, ANT1-transgenic cardiomyocytes expressed less TGFß receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGFß(1) stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFß(1) treatment, SMAD signaling was still responsive to TGFß(1) indicated by an upregulation of SMAD7, a TGFß(1) target protein. CONCLUSION: Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFß(1) by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFß signaling.
Asunto(s)
Apoptosis/efectos de los fármacos , Translocasas Mitocondriales de ADP y ATP/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Animales , Apoptosis/genética , Células Cultivadas , Expresión Génica , Masculino , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Ratas , Ratas Sprague-Dawley , Transducción de Señal , TransgenesRESUMEN
BACKGROUND AND PURPOSE: Intermedin is a member of the calcitonin gene-related-peptide (CGRP) family expressed in endothelial cells and acts via calcitonin receptor-like receptors (CLRs). Here we have analysed the receptors for intermedin and its effect on the endothelial barrier in monolayers of human umbilical vein endothelial cells (HUVECs). EXPERIMENTAL APPROACH: We analysed the effect of intermedin on albumin permeability, contractile machinery, actin cytoskeleton and VE-cadherin in cultured HUVECs. KEY RESULTS: Intermedin concentration-dependently reduced basal endothelial permeability to albumin and antagonized thrombin-induced hyperpermeability. Intermedin was less potent (EC(50) 1.29 ± 0.12 nM) than adrenomedullin (EC(50) 0.24 ± 0.07 nM) in reducing endothelial permeability. These intermedin effects were inhibited by AM(22-52) and higher concentrations of αCGRP(8-37), with pA(2) values of αCGRP(8-37) of 6.4 for both intermedin and adrenomedullin. PCR data showed that HUVEC expressed only the CLR/RAMP2 receptor complex. Intermedin activated cAMP/PKA and cAMP/Epac signalling pathways. Intermedin's effect on permeability was blocked by inhibition of PKA but not of eNOS. Intermedin antagonized thrombin-induced contractile activation, RhoA activation and stress fibre formation. It also induced Rac1 activation, enhanced cell-cell adhesion and antagonized thrombin-induced loss of cell-cell adhesion. Treatment with a specific inhibitor of Rac1 prevented intermedin-mediated barrier stabilization. CONCLUSION AND IMPLICATIONS: Intermedin stabilized endothelial barriers in HUVEC monolayers via CLR/RAMP2 receptors. These effects were mediated via cAMP-mediated inactivation of contractility and strengthening of cell-cell adhesion. These findings identify intermedin as a barrier stabilizing agent and suggest intermedin as a potential treatment for vascular leakage in inflammatory conditions.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Endotelio Vascular/fisiología , Hormonas Peptídicas/farmacología , Trombina/farmacología , Uniones Adherentes/efectos de los fármacos , Uniones Adherentes/fisiología , Péptido Relacionado con Gen de Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/metabolismo , AMP Cíclico/genética , AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , ARN/genética , ARN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Adrenomedulina/genética , Receptores de Adrenomedulina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Growth differentiation factor 15 (GDF15) is induced during heart failure development, and may influence different processes in cardiac remodeling. While its anti-apoptotic action under conditions of ischemia-reperfusion have been shown, it remained unclear if this is a broadly protective effect applicable to other apoptotic stimuli. Furthermore, effects on cardiac hypertrophy remained obscure. Therefore, we investigated the effects of GDF15 on induction of hypertrophy and apoptosis in ventricular cardiomyocytes. GDF15 (3 ng/ml) enhanced hypertrophic growth of cardiomyocytes as determined by an increase in cell size by 27 +/- 5% and rate of protein synthesis by 47 +/- 15%. In addition, a time and dose-dependent increase in SMAD-binding affinity was found, as well as enhanced phosphorylation of R-SMAD1. Inhibition of SMADs by transformation of cardiomyocytes with SMAD-decoy oligonucleotides abolished the hypertrophic growth effect. Specific inhibitors of PI3K (10 microM LY290042 or 10 nM wortmannin) or ERK (10 microM PD98059) also blocked GDF15-induced hypertrophy and SMAD activation. Apoptosis induction by three different agents, 100 nM angiotensin II, 1 ng/ml TGFbeta(1), or the NO-donor SNAP (100 microM) was blocked by addition of GDF15 (3 ng/ml). Scavenging of SMADs by transformation of cardiomyocytes with SMAD-decoy oligonucleotides abolished the anti-apoptotic effect of GDF15. In conclusion, GDF15 protects ventricular cardiomyocytes against different apoptotic stimuli and enhances hypertrophic growth. Hypertrophic signaling is thereby mediated via the kinases PI3K and ERK and the transcription factor R-SMAD1. Thus, GDF15 may influence cardiac remodeling via two different mechanisms, apoptosis protection and induction of hypertrophy.
Asunto(s)
Apoptosis , Cardiomegalia/metabolismo , Cardiomegalia/patología , Factor 15 de Diferenciación de Crecimiento/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Transducción de Señal , Factores de Edad , Angiotensina II/metabolismo , Animales , Apoptosis/efectos de los fármacos , Cardiomegalia/prevención & control , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Miocitos Cardíacos/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Oligonucleótidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Proteína Smad1/genética , Proteína Smad1/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta1/metabolismo , Remodelación VentricularRESUMEN
Ischemia-induced apoptosis of endothelial cells may contribute to tissue injury, organ failure, and transplantation rejection. However, little is known about survival mechanisms capable to counteract endothelial apoptosis. This study investigated the potential role of an endogenous anti-apoptotic response elicited by transient hypoxia, capable to avert ongoing apoptosis in endothelial cells. Experiments were carried out in three different types of cultured endothelial cells (human umbilical vein, pig aorta, and from rat coronary microvasculature). As a pro-apoptotic challenge endothelial cells were cultured in serum-free medium and subjected to hypoxia for 2 h. We found that transient hypoxia reduced caspase 3 activation within 1 h of hypoxia. Accordingly, the number of apoptotic cells was reduced after 24 h of reoxygenation. This was true for all three cell types analyzed. Analysis of Akt and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways revealed that hypoxia induced a transient activation of ERK 2 but not of Akt. ERK 2 phosphorylation preceded the phosphorylation of pro-apoptotic molecule Bad at Ser112, an inhibitory phosphorylation site specific for ERK. The protective effects of hypoxia regarding Bad phosphorylation, caspase 3 activation, and apoptosis were abolished by MEK 1/2 inhibitors, PD98059 or UO126, as well as by antisense oligonucleotides directed against ERK 1/2. Furthermore, inhibition of this pathway inhibited hypoxia-induced increase in mitochondrial membrane potential. The present study demonstrates that transient hypoxia induces a novel survival mechanism that protects endothelial cells against apoptosis. This endogenous process involves MEK/ERK-mediated inhibition of the pro-apoptotic molecule Bad and caspase 3.
Asunto(s)
Apoptosis , Células Endoteliales/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Hipoxia de la Célula , Supervivencia Celular , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/genética , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Potencial de la Membrana Mitocondrial , Oligonucleótidos Antisentido/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Transducción de Señal , Porcinos , Factores de Tiempo , Proteína Letal Asociada a bcl/metabolismoRESUMEN
Ischemic preconditioning has a powerful protective potential against a reperfusion-induced injury of the post-ischemic myocardium. Cardiomyocyte hypercontracture, i.e. excessive cell shortening, is an essential mechanism of the reperfusion-induced injury. Rigor contracture, i.e. Ca(2+)-independent contracture, has been shown to be an import component of the reperfusion-induced hypercontracture. Since rigor contracture is dependent on the rapidity of the metabolic recovery during reoxygenation, we hypothesized that preconditioning of the cardiomyocyte mitochondria may improve mitochondrial function to restore the energy balance during the initial phase of reoxygenation and may thus prevent rigor contracture. For this purpose adult rat cardiomyocytes were exposed to anoxia with subsequent reoxygenation. For preconditioning, cells were pre-treated with the mitochondrial ATP-sensitive K(+) channel opener diazoxide. Pre-treatment with 100 micromol/l diazoxide significantly reduced the reoxygenation-induced hypercontracture of cardiomyocytes due to an attenuation of the Ca(2+)-independent rigor-type contracture, which was accompanied by an acceleration of the phosphocreatine resynthesis during the initial phase of reoxygenation. Treatment with the mitochondrial ATP-sensitive K(+) channel antagonist 5-hydroxydecanoate (500 micromol/l) during preconditioning phase abolished these protective effects. Similarly, partial suppression of the mitochondrial function with 100 micromol/l NaCN during the reoxygenation phase abolished the diazoxide effects. Finally, in isolated rat hearts, preconditioning with diazoxide prior to global ischemia significantly improved left ventricular function and attenuated hypercontracture during reperfusion. This effect could be abolished by the treatment with 100 micromol/l NaCN during reperfusion. Taken together, pharmacological preconditioning of cardiomyocytes with diazoxide protects against the reoxygenation-induced rigor hypercontracture due to an improvement of the energy recovery at the onset of reoxygenation.
Asunto(s)
Diazóxido/farmacología , Precondicionamiento Isquémico Miocárdico , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Animales , Ácidos Decanoicos/farmacología , Hidroxiácidos/farmacología , Hipoxia/fisiopatología , Canales KATP/antagonistas & inhibidores , Canales KATP/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Fosfocreatina/metabolismo , Ratas , Ratas Wistar , Cianuro de Sodio/farmacología , Vasodilatadores/farmacologíaRESUMEN
alpha-Adrenergic stimulation results in a positive adaptation of cardiomyocytes to increased cardiac work load by induction of hypertrophy and enhanced contraction. However, sustained adrenergic stimulation causes progression to heart failure. Under simultaneous activation of alpha- and beta-adrenoceptors by the naturally occurring catecholamine noradrenaline, beta1-stimulation inhibits alpha-adrenergic-stimulated hypertrophy. If beta-adrenergic stimulation may also influence cardiomyocyte contraction is not known yet. We now demonstrate that exposure of cardiomyocytes to noradrenaline or isoprenaline for 24 h results in a reduced cell shortening at low beating frequencies (0.5 Hz). At high beating frequencies (2 Hz), cell shortening was normal. beta-adrenergic stimulation enhances SERCA2A expression at the messenger RNA and protein level. This induction of the Ca(2+) pump SERCA2A by the transcription factor NFAT is responsible for maintenance of normal cell contraction at high beating frequencies since inhibition of NFAT by decoy-oligonucleotides impairs SERCA2A expression and cell shortening after beta-adrenergic stimulation. In conclusion, although reduced cell shortening is found under low beating frequencies, we demonstrate preservation of cardiomyocyte contraction at 2 Hz after exposure to beta-adrenergic stimuli, which indicate that adrenergic stimulation a priori does not cause impaired heart function. The increase of SERCA expression indicates an even improved Ca(2+) handling of the cells.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Agonistas Adrenérgicos beta/farmacología , Cardiotónicos/farmacología , Isoproterenol/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Norepinefrina/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Animales , Calcio/metabolismo , Estimulación Cardíaca Artificial , Células Cultivadas , Inducción Enzimática , Frecuencia Cardíaca , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/enzimología , Masculino , Miocitos Cardíacos/enzimología , Factores de Transcripción NFATC/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Factores de TiempoAsunto(s)
Cardiología/historia , Isquemia Miocárdica/terapia , Cardiología/economía , Alemania , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Isquemia Miocárdica/historia , Reperfusión Miocárdica/historia , Reperfusión Miocárdica/métodos , Sociedades Médicas/economía , Sociedades Médicas/historiaRESUMEN
Rapid interventional restoration of coronary blood flow is the most effective therapy to limit infarct size in today}s cardiology. The early phase of reperfusion represents, however, a window of therapeutic opportunities largely unused in the clinic. Experimentally it has been clearly shown that the modalities of reperfusion have a substantial impact on infarct size, since reperfusion itself can damage the myocardium (reperfusion injury). The major cause for acute injury of the cardiomyocytes in reperfusion is their hypercontracture. Cytosolic Ca (2+) overload and malfunction of cell organelles, i. e. sarcoplasmic reticulum and mitochondria, determine the pathophysiology of reperfusion injury. The underlying mechanisms can be influenced in the first minutes of reperfusion by activation of protective signalling pathways (reperfusion therapy). First clinical studies confirm the efficacy of acute reperfusion therapy.
Asunto(s)
Daño por Reperfusión Miocárdica/fisiopatología , Reperfusión Miocárdica/métodos , Calcio/metabolismo , Circulación Coronaria , Humanos , Mitocondrias Cardíacas/metabolismo , Transducción de Señal , Resultado del TratamientoRESUMEN
AIM: Angiotensin-converting enzyme (ACE) inhibitors like ramiprilat bind to ACE expressed on the cell surface of endothelial cells and induce cell-specific signalling including the activation of activator protein (AP)-1. The present study addressed the question whether ramiprilat exerts a similar effect on adult ventricular cardiomyocytes, i.e. activates the AP-1 or modifies contractile performance. It was further aimed to decide whether such effects depend on bradykinin receptors or whether they are directly mediated via ACE. METHODS: Adult rat ventricular cardiomyocytes were isolated and cultured. mRNA expression of ACE was investigated by RT-PCR, AP-1 activation by gel mobility shift assays, and cardiac contractile performance by electrical pacing of isolated cells and analysis of cell shortening via a line-camera. RESULTS: Cardiomyocytes stably express ACE. Ramiprilat increased maximal contraction velocity and shortened the time-to-peak of contraction. In contrast to effects evoked by bradykinin, such effects caused by ramiprilat were not attenuated by HOE 140, a bradykinin-receptor antagonist. These effects were also not attenuated in the presence of l-nitro-arginine, used to mimic bradykinin-dependent signalling. In cardiomyocytes, bradykinin but not ramiprilat activated AP-1. Ramiprilat activates AP-1 in endothelial cells that are known to respond to ramiprilat in this way. CONCLUSION: Ramiprilat exerts direct, bradykinin-receptor independent effects on cardiomyocytes that improve cellular function without a corresponding effect on AP-1 activation or induction of AP-1 dependent effects. This newly described effect of ramiprilat may contribute to the protective effects seen by application of ACE inhibitors.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Contracción Miocárdica/fisiología , Miocitos Cardíacos/fisiología , Peptidil-Dipeptidasa A/metabolismo , Ramipril/análogos & derivados , Factor de Transcripción AP-1/metabolismo , Función Ventricular , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ventrículos Cardíacos/citología , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Ramipril/administración & dosificación , Ratas , Ratas WistarRESUMEN
A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.
Asunto(s)
Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Vuelo Espacial , Animales , Biopelículas/crecimiento & desarrollo , Femenino , Expresión Génica , Genes Bacterianos , Proteína de Factor 1 del Huésped/fisiología , Hierro/metabolismo , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteómica , Regulón , Salmonelosis Animal/etiología , Salmonella typhimurium/fisiología , Virulencia , Simulación de IngravidezRESUMEN
OBJECTIVES: Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile machinery of endothelial cells. This inactivation relies on dephosphorylation of the regulatory myosin light chain (MLC) due to an activation of the MLC phosphatase (MLCP). To date, activation and function of MLCP in endothelial cells are only partially understood. METHODS: Here, the mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human endothelial cells from umbilical veins. Cells were transfected with the endogenous protein phosphatase 1 (PP1)-specific inhibitor-2 (I-2). RESULTS: Overexpression of I-2 led to inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing the MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP recruits the PP1delta catalytic subunit and the myosin phosphatase targeting subunit (MYPT1) to form a complex. ATP stimulated dephosphorylation of MYPT1 at the inhibitory phosphorylation sites threonine 850 and 696. However, extracellular ATP failed to stimulate MYPT1 dephosphorylation in I-2-overexpressing cells. CONCLUSIONS: The present study shows for the first time that, in endothelial cells, extracellular ATP causes activation of MLCP through recruitment of PP1delta and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1.
Asunto(s)
Adenosina Trifosfato/farmacología , Células Endoteliales/enzimología , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Adenosina Trifosfato/análogos & derivados , Amidas/farmacología , Western Blotting , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Toxinas Marinas , Fosfatasa de Miosina de Cadena Ligera/antagonistas & inhibidores , Nucleotidasas/antagonistas & inhibidores , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 1 , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas/genética , Proteínas/metabolismo , Antagonistas de Receptores Purinérgicos P1 , Piridinas/farmacología , Teofilina/análogos & derivados , Teofilina/farmacología , Trombina/farmacología , Transfección/métodos , Quinasas Asociadas a rhoRESUMEN
Nitric oxide (NO) is increased under several pathophysiological, mainly inflammatory processes in the heart and has been characterized as an inducer of apoptosis in cardiomyocytes. The transcription factor activating protein-1 (AP-1) has been identified as a mediator of NO-induced apoptosis. Genes that are regulated by AP-1 under apoptotic conditions have not been identified yet. Therefore, we performed a microarray analysis with subsequent real-time polymerase chain reaction (PCR) to identify genes regulated by AP-1 in NO-induced ventricular cardiomyocytes of rats and tested the functional role of these genes in apoptosis. Cardiomyocytes were transformed with AP-1 decoy oligonucleotides for inhibition of AP-1 activity. These, as well as non-transformed control cells, were stimulated with the NO donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 100 microM) for 2 h. Some of the genes with differential gene expression on microarrays were further analysed by real-time PCR. Genes that are induced by SNAP were not identified. However, four genes, pyridoxal kinase, heat shock protein 10 (Hsp10), antigen identified by monoclonal antibodies 4F2 (4F2) and myosin light chain 2, were downregulated by SNAP in presence of AP-1. Pyridoxal kinase, Hsp10 and 4F2 have anti-apoptotic effects in unstimulated cells because downregulation of their expression by antisense oligos induced apoptosis in cardiomyocytes. An involvement of these genes in NO-induced apoptosis could only be proven for pyridoxal kinase. In conclusion, using microarray technology, we identified three anti-apoptotic genes (Hsp10, 4F2 and pyridoxal kinase) in ventricular cardiomyocytes, which may help the cells to resist some apoptotic stimuli. The downregulation of these genes results in cardiomyocyte apoptosis. Prevention of their downregulation may protect cardiomyocytes against apoptotic stimuli, and this may be of therapeutic benefit.
Asunto(s)
Apoptosis/fisiología , Chaperonina 10/genética , Proteína-1 Reguladora de Fusión/genética , Regulación de la Expresión Génica/fisiología , Miocitos Cardíacos/fisiología , Piridoxal Quinasa/genética , Factor de Transcripción AP-1/fisiología , Animales , Apoptosis/genética , Sistemas de Computación , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Ventrículos Cardíacos , Masculino , Análisis por Micromatrices , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Donantes de Óxido Nítrico/farmacología , Oligonucleótidos Antisentido/farmacología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , S-Nitroso-N-Acetilpenicilamina/farmacología , Factor de Transcripción AP-1/antagonistas & inhibidoresRESUMEN
Elevations in angiotensin II (AngII) and transforming growth factor (TGF-beta1) levels are often found under conditions leading to progression of heart failure. From several studies, it is evident that AngII enhances TGF-beta1 expression via activator protein 1 (AP-1) activation, and that this pathway is involved in hypertrophic growth of the heart muscle and in the development of cardiac fibrosis. We now continued characterization of the signaling pathway stimulated by AngII in ventricular cardiomyocytes of rat and analyzed if the enhancement of TGF-beta1 expression by AngII may also contribute to apoptosis induction, which is another predictor of heart failure progression. Stimulation of cardiomyocytes with 100 nM AngII for 2 h activated the transcription factors AP-1 and GATA by 68.6+/-23.9 or 70.7+/-9.8%. Induction of both factors was mediated by p38 mitogen-activated protein kinase (MAPK) because it was totally blocked using a specific inhibitor of the kinase (SB202190). When GATA was inhibited by transformation of cardiomyocytes with decoy oligonucleotides, AngII could not enhance TGF-beta1 expression. This inhibition was observed on the mRNA level in real-time polymerase chain reaction and on the protein level in Western blots. As a consequence, upon AngII stimulation for 24 h, release of TGF-beta1 from cardiomyocytes was also reduced from 240.5+/-50.4 to 130.5+/-22.1% (p<0.05). In contrast to the early induction of GATA and AP-1, the transcription factor similar to mothers against decapentaplegic homolog (SMAD) was induced by AngII after 24 h. This stimulation was dependent on TGF-beta1 because it was blocked by antibodies specific for TGF-beta1. Twenty-four hours after AngII addition, the number of apoptotic cardiomyocytes increased by 6.5+/-1.2%, and this apoptosis induction was blocked when SMAD activity was inhibited by transformation of cardiomyocytes with SMAD decoy oligonucleotides. In conclusion, the transcription factors AP-1 and GATA are activated by p38 MAPK upon AngII stimulation, and both are needed to enhance TGF-beta1 expression in ventricular cardiomyocytes. TGF-beta1 acts in an autocrine loop on the cells to induce apoptosis via SMAD signaling. Thus, the often-found correlation between AngII, TGF-beta1, AP-1, and SMAD in pathogenesis of heart disease reflects the proapoptotic signaling pathway induced by AngII in cardiomyocytes.
Asunto(s)
Angiotensina II/metabolismo , Apoptosis , Comunicación Autocrina , Sistema de Señalización de MAP Quinasas , Miocitos Cardíacos/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Angiotensina II/farmacología , Animales , Apoptosis/efectos de los fármacos , Comunicación Autocrina/efectos de los fármacos , Western Blotting , Cardiomiopatías/metabolismo , Células Cultivadas , Factores de Transcripción GATA/biosíntesis , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Miocitos Cardíacos/efectos de los fármacos , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Smad/metabolismo , Factores de Tiempo , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Parathyroid hormone-related peptide (PTHrP) was found to improve contractile function of stunned myocardium in pigs. The peptide is released from coronary endothelial cells during ischemia and significantly improves post-ischemic recovery. The present study was aimed to decide whether such an induction of contractile responsiveness of the heart requires co-activation of adjacent cells or is a genuine phenomenon of cardiomyocytes. A second aim of this study was to decide whether such an improvement is linked to depressed cell function in general or oxidative inhibition. Isolated adult ventricular cardiomyocytes from rats were constantly paced at 0.5 Hz for 10 min. Cells were exposed to a brief oxidative inhibition by addition of 0.5 mmol/l potassium cyanide (KCN) in the presence of glucose. Under these conditions, cells stopped beating after 280 s on average. 30 s before they stopped to beat, cells had already developed a reduction in cell shortening, maximal relaxation and contraction velocity. In the co-presence of PTHrP (1-34) (100 nmol/l) cells continued to beat regular and did not develop reduced cell shortening, irrespectively of oxidative inhibition. In a second attempt, cells were exposed to the NO donor SNAP (100 micromol/l) or 8-bromocGMP (1 mmol/l). As expected both agents reduced cell shortening significantly. This reduction in cell shortening was attenuated in co-presence of PTHrP, too. Finally, we investigated the effect of PTHrP on cell shortening at different extracellular concentrations of calcium. Although, PTHrP increased intracellular calcium at 2 and 5 mmol/l extracellular calcium, respectively, it improved cell shortening only at 5 mmol/l extracellular calcium. Thus, the beneficial effect of PTHrP on cell shortening was independent from intracellular calcium but dependent on the steepness of the calcium gradient between intra- and extracellular calcium. In conclusion, our study shows that PTHrP is able to improve cell shortening rapidly and directly irrespectively of the reason for the reduced cell function. Improved electromechanical coupling rather than intracellular calcium handling seems to be the most important mechanism.
Asunto(s)
Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Animales , Calcio/metabolismo , GMP Cíclico/fisiología , Masculino , Miocitos Cardíacos/fisiología , Óxido Nítrico/fisiología , Oxidación-Reducción , Cianuro de Potasio/farmacología , Ratas , Ratas WistarRESUMEN
OBJECTIVE: Reperfusion injury of the myocardium is characterised by development of cardiomyocyte hypercontracture. Previous studies have shown that cGMP-mediated stimuli protect against reperfusion injury, but the cellular mechanism is still unknown. METHODS: To simulate ischemia/reperfusion, adult rat cardiomyocytes were incubated anoxically (pH(o) 6.4) and then reoxygenated (pH(o) 7.4). Cytosolic calcium [Ca(2+)](i) (fura-2 ratio), pH(i) (BCECF ratio), cell length, and phospholamban phosphorylation were analysed. Under simulated ischemia cardiomyocytes develop [Ca(2+)](i) overload. When reoxygenated they rapidly undergo hypercontracture, triggered by oscillations of [Ca(2+)](i). We investigated whether cGMP-mediated stimuli can modulate [Ca(2+)](i) or pH(i) recovery and whether this contributes to their protective effect. Membrane-permeable cGMP analogues, 8-bromo-cGMP (1 mmol/L) or 8-pCPT-cGMP (10 micrommol/L), or a receptor-mediated activator of particulate guanylyl cyclase, urodilatin (1 micromol/L), were applied. RESULTS: The investigated stimuli protect against reoxygenation-induced hypercontracture (cell length as percent of end-ischemic length; control: 68+/-1.6; 8-bromo-cGMP: 88+/-1.5*; 8-pCPT-cGMP: 84+/-2.9*; urodilatin: 87+/-1.1*; n=24; *p<0.05). Recovery from [Ca(2+)](i) overload after 2 min reoxygenation [fura-2 ratio (a.u.); control: 1.43+/-0.15; 8-bromo-cGMP: 1.86+/-0.15*; 8-pCPT-cGMP: 1.92+/-0.19*; urodilatin: 1.93+/-0.24*; n=25; *p<0.05] was accelerated, and the frequency of [Ca(2+)](i) oscillations (min(-1)) was significantly reduced (control: 49+/-5.0 min(-1); 8-bromo-cGMP: 18+/-3.5* min(-1); 8-pCPT-cGMP: 18+/-4.5* min(-1); urodilatin: 16+/-4.1* min(-1); n=24; *p<0.05). cGMP-mediated stimuli increased sarcoplasmic Ca(2+) sequestration (caffeine-releasable Ca(2+) pool: 2-3 fold increase vs. control). Inhibition of sarcoplasmic Ca(2+)-ATPase (SERCA) by thapsigargin (150 nmol/L) or of protein kinase G with KT-5823 (1 micromol/L) abolished the effect of these stimuli on [Ca(2+)](i) recovery. The investigated stimuli significantly enhanced phospholamban phosphorylation. CONCLUSIONS: We conclude that cGMP-dependent signals activate SERCA via a protein kinase G-dependent phosphorylation of phospholamban. The increase in SERCA activity seems to reduce peak [Ca(2+)](i) and [Ca(2+)](i) oscillation during reoxygenation and to attenuate the excessive activation of the contractile machinery that otherwise leads to the development of hypercontracture.
Asunto(s)
Calcio/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Carbazoles/farmacología , Tamaño de la Célula/efectos de los fármacos , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Citosol/metabolismo , Concentración de Iones de Hidrógeno , Indoles/farmacología , Masculino , Microscopía Fluorescente , Miocitos Cardíacos/efectos de los fármacos , Ratas , Ratas Wistar , Retículo Sarcoplasmático/metabolismo , Tapsigargina/farmacologíaRESUMEN
AIMS: Endothelin-1 (ET-1) promotes endothelial cell growth. Endothelial cell proliferation involves the activation of Ca2+-activated K+ channels. In this study, we investigated whether Ca2+-activated K+ channels with big conductance (BK(Ca)) contribute to endothelial cell proliferation induced by ET-1. METHODS: The patch-clamp technique was used to analyse BK(Ca) activity in endothelial cells derived from human umbilical cord veins (HUVEC). Endothelial proliferation was examined using cell counts and measuring [3H]-thymidine incorporation. Changes of intracellular Ca2+ levels were examined using fura-2 fluorescence imaging. RESULTS: Characteristic BK(Ca) were identified in cultured HUVEC. Continuous perfusion of HUVEC with 10 nmol L(-1) ET-1 caused a significant increase of BK(Ca) open-state probability (n = 14; P < 0.05; cell-attached patches). The ET(B)-receptor antagonist (BQ-788, 1 micromol L(-1)) blocked this effect. Stimulation with Et-1 (10 nmol L(-1)) significantly increased cell growth by 69% (n = 12; P < 0.05). In contrast, the combination of ET-1 (10 nmol L(-1)) and the highly specific BK(Ca) blocker iberiotoxin (IBX; 100 nmol L(-1)) did not cause a significant increase in endothelial cell growth. Ca2+ dependency of ET-1-induced proliferation was tested using the intracellular Ca2+-chelator BAPTA (10 micromol L(-1)). BAPTA abolished ET-1 induced proliferation (n = 12; P < 0.01). In addition, ET-1-induced HUVEC growth was significantly reduced, if cells were kept in a Ca2+-reduced solution (0.3 mmol L(-1)), or by the application of 2 aminoethoxdiphenyl borate (100 micromol L(-1)) which blocks hyperpolarization-induced Ca2+ entry (n = 12; P < 0.05). CONCLUSION: Activation of BK(Ca) by ET-1 requires ET(B)-receptor activation and induces a capacitative Ca2+ influx which plays an important role in ET-1-mediated endothelial cell proliferation.
Asunto(s)
Ácido Egtácico/análogos & derivados , Células Endoteliales/fisiología , Endotelina-1/fisiología , Canales de Potasio Calcio-Activados/fisiología , Calcio/metabolismo , Calcio/fisiología , Recuento de Células , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Quelantes/farmacología , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Conductividad Eléctrica , Células Endoteliales/efectos de los fármacos , Antagonistas de los Receptores de la Endotelina B , Humanos , Potenciales de la Membrana/fisiología , Oligopéptidos , Péptidos/farmacología , Piperidinas , Canales de Potasio Calcio-Activados/antagonistas & inhibidoresRESUMEN
cAMP-mediated signaling mechanisms may destabilize or stabilize the endothelial barrier, depending on the origin of endothelial cells. Here, microvascular coronary [coronary endothelial cells (CEC)] and macrovascular aortic endothelial cell (AEC) monolayers with opposite responses to cAMP were analyzed. Macromolecule permeability, isometric force, activation state of contractile machinery [indicated by phosphorylation of regulatory myosin light chains (MLC), activity of MLC kinase, and MLC phosphatase], and dynamic changes of adhesion complex proteins (translocation of VE-cadherin and paxillin) were determined. cAMP signaling was stimulated by the adenosine receptor agonist 5'-N-(ethylcarboxamido)-adenosine (NECA), the beta-adrenoceptor agonist isoproterenol (Iso), or by the adenylyl cyclase activator forskolin (FSK). Permeability was increased in CEC and decreased in AEC on stimulation with NECA, Iso, or FSK. The effects could be inhibited by the PKA inhibitor Rp-8-CPT-cAMPS and imitated by the PKA activator Sp-cAMPS. Under cAMP/PKA-dependent stimulation, isometric force and MLC phosphorylation were reduced in monolayers of either cell type, due to an activation of MLC phosphatase. In CEC but not in AEC, FSK induced delocalization of VE-cadherin and paxillin from cellular adhesion complexes as indicated by cell fractionation and immunofluorescence microscopy. In conclusion, decline in contractile activation and isometric force contribute to cAMP/PKA-mediated stabilization of barrier function in AEC. In CEC, this stabilizing effect is overruled by cAMP-induced disintegration of cell adhesion structures.
Asunto(s)
AMP Cíclico/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Transducción de Señal/fisiología , Animales , Aorta/citología , Aorta/fisiología , Adhesión Celular/fisiología , Células Cultivadas , Vasos Coronarios/citología , Vasos Coronarios/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico , Células Endoteliales/ultraestructura , Endotelio Vascular/ultraestructura , Activación Enzimática/fisiología , Uniones Intercelulares/ultraestructura , Masculino , Microscopía Fluorescente , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Wistar , PorcinosRESUMEN
Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of Ca(2+)-dependent MLC kinase or Ca(2+)-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP (10 microM) increased ERK2 phosphorylation from basal 17 +/- 3 to 53 +/- 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 (20 microM) or U0126 (10 microM). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic Ca(2+) rise, because it was unaltered when this was suppressed by the Ca(2+) chelator BAPTA (10 microM) or xestospongin C (3 microM), an inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido)adenosine (1 microM) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 microM). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a Ca(2+)-independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase.
Asunto(s)
Adenosina Trifosfato/metabolismo , Endotelio Vascular/enzimología , Sistema de Señalización de MAP Quinasas/fisiología , Cadenas Ligeras de Miosina/metabolismo , Animales , Aorta/citología , Calcio/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación , PorcinosRESUMEN
In some models of cardiac hypertrophy, activation of activator protein 1 (AP-1) correlates with growth. However, AP-1 is also activated by stimuli not involved in cardiac growth. This raises the following questions: does AP-1 plays a causal role for cardiomyocyte growth, and is this role model or stimulus dependent? We used a single model to address these questions, i.e., ventricular cardiomyocytes of adult rats, and two growth stimuli, i.e., alpha- and beta-adrenoceptor agonists [10 microM phenylephrine (PE) and 1 microM isoprenaline (Iso), respectively]. After 1 h of stimulation with PE, mRNA expression of c-Fos and c-Jun was upregulated to 185 +/- 32 and 132 +/- 13% of control. Fos and Jun proteins formed the AP-1 complex. PE stimulated DNA binding activity of AP-1 to 165 +/- 22% of control within 2 h and increased protein synthesis to 161 +/- 27% of control and cross-sectional area to 126 +/- 4% of control. Inhibition of AP-1 binding activity by cAMP response element (CRE) decoy oligonucleotides abolished both of these growth responses. Iso stimulated AP-1 binding activity to 203 +/- 19% of control within 2 h and stimulated protein synthesis to 145 +/- 17% of control. However, the growth effect of Iso was not abolished by CRE decoys: Iso increased protein synthesis to 158 +/- 17% of control in the presence of CRE. In conclusion, AP-1 is a causal mediator of the alpha-adrenergic, but not the beta-adrenergic, growth response of cardiomyocytes.