RESUMEN
Background: Variants in the MYBPC3 gene are a frequent cause of hypertrophic cardiomyopathy (HCM) but display a large phenotypic heterogeneity. Founder mutations are often believed to be more benign as they prevailed despite potential negative selection pressure. We detected a pathogenic variant in MYBPC3 (del exon 23-26) in several probands. We aimed to assess the presence of a common haplotype and to describe the cardiac characteristics, disease severity and long-term outcome of mutation carriers. Methods: Probands with HCM caused by a pathogenic deletion of exon 23-26 of MYBPC3 were identified through genetic screening using a gene panel encompassing 59 genes associated with cardiomyopathies in a single genetic center in Belgium. Cascade screening of first-degree relatives was performed, and genotype positive relatives were further phenotyped. Clinical characteristics were collected from probands and relatives. Cardiac outcomes included death, heart transplantation, life-threatening arrhythmia, heart failure hospitalization or septal reduction therapy. Haplotype analysis, using microsatellite markers surrounding MYBPC3, was performed in all index patients to identify a common haplotype. The age of the founder variant was estimated based on the size of the shared haplotype using a linkage-disequilibrium based approach. Results: We identified 24 probands with HCM harbouring the MYBPC3 exon 23-26 deletion. Probands were on average 51 ± 16 years old at time of clinical HCM diagnosis and 62 ± 10 years old at time of genetic diagnosis. A common haplotype of 1.19 Mb was identified in all 24 probands, with 19 of the probands sharing a 13.8 Mb haplotype. The founder event was estimated to have happened five generations, or 175-200 years ago, around the year 1830 in central Flanders. Through cascade screening, 59 first-degree relatives were genetically tested, of whom 37 (62.7%) were genotype positive (G+) and 22 (37.3%) genotype negative (G-). They were on average 38 ± 19 years old at time of genetic testing. Subsequent clinical assessment revealed a HCM phenotype in 19 (51.4%) G+ relatives. Probands were older (63 ± 10 vs. 42 ± 21 years; p < 0.001) and had more severe phenotypes than G+ family members, presenting with more symptoms (50% vs. 13.5%; p = 0.002), arrhythmia (41.7% vs. 12.9%, p = 0.014), more overt hypertrophy and left ventricular outflow tract obstruction (43.5% vs. 3.0%; p < 0.001). Male G+ relatives more often had a HCM phenotype (78.6% vs. 34.8%; p = 0.010) and were more severely affected than females. At the age of 50, a penetrance of 78.6% was observed, defined as the presence of HCM in 11 of 14 G+ relatives with age ≥50 years. Overall, 20.3% of all variant carriers developed one of the predefined cardiac outcomes after a median follow-up of 5.5 years with an average age of 50 (±21) years. Conclusion: A Belgian founder variant, an exon 23-26 deletion in MYBPC3, was identified in 24 probands and 37 family members. The variant is characterized by a high penetrance of 78.6% at the age of 50 years but has variable phenotypic expression. Adverse outcomes were observed in 20.3% of patients during follow-up.
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INTRODUCTION: Impending paradoxical embolism (IPDE) is a rare condition where a thrombus straddles the foramen ovale with a high risk of arterial embolism. CASES REPORT: We report two cases of impending paradoxical embolism, an uncommon condition with a high mortality rate. The first in a young woman with acute right heart failure operated emergently, the second, in an old and frail lady presenting an ischemia of the left arm, treated by anticoagulants. 3 D echocardiography imaging is presented and treatment modality is discussed. CONCLUSION: Emergent treatment is mandatory for IPDE, a serious disease with a high early mortality. This paper is a reminder of how to deal with such a rare condition.
Asunto(s)
Ecocardiografía Tridimensional , Embolia Paradójica , Foramen Oval Permeable , Embolia Pulmonar , Ecocardiografía , Ecocardiografía Transesofágica , Embolia Paradójica/diagnóstico por imagen , Embolia Paradójica/etiología , Femenino , Foramen Oval Permeable/diagnóstico , Foramen Oval Permeable/diagnóstico por imagen , HumanosAsunto(s)
Válvula Aórtica/cirugía , Cateterismo Cardíaco/métodos , Fístula , Insuficiencia Cardíaca , Imagen Multimodal/métodos , Dispositivo Oclusor Septal , Cirugía Asistida por Computador/métodos , Anciano , Disección Aórtica/cirugía , Ecocardiografía , Endocarditis/diagnóstico , Endocarditis/etiología , Endocarditis/fisiopatología , Femenino , Fístula/diagnóstico por imagen , Fístula/etiología , Fístula/cirugía , Fluoroscopía/métodos , Atrios Cardíacos/diagnóstico por imagen , Atrios Cardíacos/patología , Atrios Cardíacos/cirugía , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/cirugía , Implantación de Prótesis de Válvulas Cardíacas/efectos adversos , Implantación de Prótesis de Válvulas Cardíacas/métodos , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/cirugía , Humanos , Reoperación/efectos adversos , Reoperación/métodos , Resultado del Tratamiento , Injerto Vascular/métodosRESUMEN
Connexin-43 (Cx43), a predominant cardiac connexin, forms gap junctions (GJs) that facilitate electrical cell-cell coupling and unapposed/nonjunctional hemichannels that provide a pathway for the exchange of ions and metabolites between cytoplasm and extracellular milieu. Uncontrolled opening of hemichannels in the plasma membrane may be deleterious for the myocardium and blocking hemichannels may confer cardioprotection by preventing ionic imbalance, cell swelling and loss of critical metabolites. Currently, all known hemichannel inhibitors also block GJ channels, thereby disturbing electrical cell-cell communication. Here we aimed to characterize a nonapeptide, called Gap19, derived from the cytoplasmic loop (CL) of Cx43 as a hemichannel blocker and examined its effect on hemichannel currents in cardiomyocytes and its influence in cardiac outcome after ischemia/reperfusion. We report that Gap 19 inhibits Cx43 hemichannels without blocking GJ channels or Cx40/pannexin-1 hemichannels. Hemichannel inhibition is due to the binding of Gap19 to the C-terminus (CT) thereby preventing intramolecular CT-CL interactions. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 prevented metabolic inhibition-enhanced hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and modestly decreased the infarct size after myocardial ischemia/reperfusion in mice in vivo. We conclude that preventing Cx43 hemichannel opening with Gap19 confers limited protective effects against myocardial ischemia/reperfusion injury.
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Conexina 43/antagonistas & inhibidores , Canales Iónicos/efectos de los fármacos , Daño por Reperfusión Miocárdica/prevención & control , Fragmentos de Péptidos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Uniones Comunicantes/efectos de los fármacos , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , PorcinosRESUMEN
We present a case of acute anterior myocardial infarction in a breastfeeding woman, 10 days after delivery. The presumed cause was proximal left anterior artery vasospasm, induced by a combination of smoking a first cigarette in the early morning and salbutamol inhalation, in the particular context of peripartum. We discuss briefly the epidemiology, pathophysiology, risk factors, diagnosis, treatment and prognosis of myocardial infarction related to pregnancy and the postpartum period.
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Infarto de la Pared Anterior del Miocardio/etiología , Complicaciones Cardiovasculares del Embarazo/etiología , Trastornos Puerperales/etiología , Agonistas de Receptores Adrenérgicos beta 2/efectos adversos , Adulto , Albuterol/efectos adversos , Infarto de la Pared Anterior del Miocardio/fisiopatología , Lactancia Materna , Vasoespasmo Coronario/complicaciones , Vasoespasmo Coronario/fisiopatología , Femenino , Humanos , Embarazo , Complicaciones Cardiovasculares del Embarazo/fisiopatología , Trastornos Puerperales/fisiopatología , Fumar/efectos adversosRESUMEN
The cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) is an important factor determining the functional state of blood-brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca(2+)](i) changes on BBB function. We applied different agonists that trigger [Ca(2+)](i) oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca(2+)](i) oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca(2+)](i) with BAPTA, indicating that [Ca(2+)](i) oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca(2+)](i) oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca(2+)](i) oscillations provoked by exposure to adenosine 5' triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations.
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Barrera Hematoencefálica/metabolismo , Calcio/metabolismo , Conexinas/metabolismo , Células Endoteliales/metabolismo , Adenosina Trifosfato/farmacología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Bradiquinina/farmacología , Señalización del Calcio/efectos de los fármacos , Carbenoxolona/farmacología , Bovinos , Línea Celular , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Células Endoteliales/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Humanos , Permeabilidad/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Vasodilatadores/farmacologíaRESUMEN
Monolayer cultures of primary hepatocytes, isolated from freshly removed livers, represent widely used in vitro tools in the area of liver physiology and pathology, pharmacology and toxicology. However, a major shortcoming of these systems is that they cope with dedifferentiation, which is accompanied by spontaneous cell death. The goal of the present study was to elucidate the mechanisms that drive the process of self-generated cell demise in primary hepatocyte cultures. For this purpose, isolated rat hepatocytes were cultivated under conventional conditions, and the occurrence of apoptosis and necrosis was monitored during 4 days by performing a set of acknowledged cell death assays. These included examination of cell morphology by light microscopy, quantification of apoptotic and necrotic cell populations by Hoechst 33342 and propidium iodide in situ staining, assessment of apoptotic and necrotic activities by measuring caspase 3-like activity and extracellular leakage of lactate dehydrogenase, and studying the expression of apoptosis regulators through immunoblot analysis. In essence, two cell death peaks were observed, namely shortly after cell seeding and in the final stages of the cultivation period, both involving apoptotic and necrotic actions. The outcome of this study not only sheds new light onto the molecular processes that underlie spontaneous cell death in primary hepatocyte cultures, but also opens perspectives for the establishment of strategies to increase cell survival in these popular in vitro systems.
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Apoptosis/fisiología , Desdiferenciación Celular/fisiología , Hepatocitos/metabolismo , Cultivo Primario de Células , Animales , Muerte Celular/fisiología , Células Cultivadas , Masculino , Necrosis/patología , Ratas , Ratas Sprague-DawleyRESUMEN
The excitatory amino acid L-ß-N-oxalyl-α,ß-diaminopropionic acid (L-ß-ODAP) in Lathyrus sativus L. is proposed as the causative agent of the neurodegenerative disease neurolathyrism. We investigated the effect of L-ß-ODAP on [Ca2+]i handling, redox homeostasis, and cell death in rat spinal motor neurons. L-ß-ODAP and L-glutamate triggered [Ca2+]i transients, which were inhibited by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor blockers; 2,3-dioxo-6-nitro-1,2,3, 4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide and 1-naphthyl acetylspermine, the latter specifically blocking Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. In addition, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, and to a lesser extent 1-naphthyl acetylspermine, protected the neurons against cell death induced by L-ß-ODAP or L-glutamate. Methionine and cysteine were also protective against neuronal cell death. We conclude that deregulation of [Ca2+]i homeostasis and oxidative stress contribute to motor neuron cell death in neurolathyrism.
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Latirismo/inducido químicamente , Enfermedad de la Neurona Motora/inducido químicamente , Neuronas Motoras/efectos de los fármacos , beta-Alanina/análogos & derivados , Animales , Células Cultivadas , Latirismo/metabolismo , Latirismo/patología , Enfermedad de la Neurona Motora/metabolismo , Enfermedad de la Neurona Motora/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , beta-Alanina/toxicidadRESUMEN
Coordinated electrical activity in the heart is supported by gap junction channels located at the intercalated discs of cardiomyocytes. Impaired gap junctional communication between neighbouring cardiomyocytes contributes to the development of re-entry arrhythmias after myocardial ischaemia. Current antiarrhythmic therapy is hampered by a lack of efficiency and side effects, creating the need for a new generation of drugs. In this review, we focus on compounds that increase gap junctional communication, thereby increasing the conduction velocity and decreasing the risk of arrhythmias. Some of these compounds also inhibit connexin 43 (Cx43) hemichannels, thereby limiting adenosine triphosphate loss and volume overload following ischaemia/reperfusion, thus potentially increasing the survival of cardiomyocytes. The compounds discussed in this review are: (i) antiarrythmic peptide (AAP), AAP10, ZP123; (ii) GAP-134; (iii) RXP-E; and (vi) the Cx mimetic peptides Gap 26 and Gap 27. None of these compounds have effects on Na(+) , Ca(2+) and K(+) channels, and therefore have no proarrhythmic activity associated with currently available antiarrhythmic drugs. GAP-134, RXP-E, Gap 26 and Gap 27 are pharmalogical agents with a favorable clinical safety profile, as already confirmed in phase I clinical trials for GAP-134. These agents show an excellent promise for treatment of arrhythmias in patients with ischaemic cardiomyopathy.
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Antiarrítmicos/farmacología , Conexinas/fisiología , Uniones Comunicantes/fisiología , Cardiopatías/tratamiento farmacológico , Péptidos/farmacología , Animales , Antiarrítmicos/efectos adversos , Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Ensayos Clínicos como Asunto , Cardiopatías/metabolismo , Cardiopatías/patología , Humanos , Terapia Molecular Dirigida , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , Péptidos/efectos adversos , Péptidos/uso terapéuticoRESUMEN
The pannexin (Panx) family of proteins, which is co-expressed with connexins (Cxs) in vertebrates, was found to be a new GJ-forming protein family related to invertebrate innexins. During the past ten years, different studies showed that Panxs mainly form hemichannels in the plasma membrane and mediate paracrine signalling by providing a flux pathway for ions such as Ca²(+), for ATP and perhaps for other compounds, in response to physiological and pathological stimuli. Although the physiological role of Panxs as a hemichannel was questioned, there is increasing evidence that Panx play a role in vasodilatation, initiation of inflammatory responses, ischemic death of neurons, epilepsy and in tumor suppression. Moreover, it is intriguing that Panxs may also function at the endoplasmic reticulum (ER) as intracellular Ca²(+)-leak channel and may be involved in ER-related functions. Although the physiological significance and meaning of such Panx-regulated intracellular Ca²(+) leak requires further exploration, this functional property places Panx at the centre of many physiological and pathophysiological processes, given the fundamental role of intracellular Ca²(+) homeostasis and dynamics in a plethora of physiological processes. In this review, we therefore want to focus on Panx as channels at the plasma membrane and at the ER membranes with a particular emphasis on the potential implications of the latter in intracellular Ca²(+) signalling.
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Adenosina Trifosfato/metabolismo , Conexinas/fisiología , Retículo Endoplásmico/metabolismo , Animales , Señalización del Calcio/fisiología , Membrana Celular/metabolismo , Humanos , RatonesRESUMEN
It is nowadays well established that gap junctions are critical gatekeepers of cell proliferation, by controlling the intercellular exchange of essential growth regulators. In recent years, however, it has become clear that the picture is not as simple as originally anticipated, as structural precursors of gap junctions can affect cell cycling by performing actions not related to gap junctional intercellular communication. Indeed, connexin hemichannels also foresee a pathway for cell growth communication, albeit between the intracellular compartment and the extracellular environment, while connexin proteins as such can directly or indirectly influence the production of cell cycle regulators independently of their channel activities. Furthermore, a novel set of connexin-like proteins, the pannexins, have lately joined in as regulators of the cell proliferation process, which they can affect as either single units or as channel entities. In the current paper, these multifaceted aspects of connexin-related signalling in cell cycling are reviewed.
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Ciclo Celular , Conexinas/fisiología , Animales , Proliferación Celular , Conexinas/química , Conexinas/genética , Regulación de la Expresión Génica , Humanos , Transducción de SeñalRESUMEN
Connexin-assembled gap junctions (GJs) and hemichannels coordinate intercellular signaling processes. Although the regulation of connexins in GJs has been well characterized, the molecular determinants controlling connexin-hemichannel activity are unresolved. Here we investigated the regulation of Cx43-hemichannel activity by actomyosin contractility and intracellular [Ca(2+)] ([Ca(2+)](i)) using plasma membrane-permeable TAT peptides (100 µM) designed to interfere with interactions between the cytoplasmic loop (CL) and carboxy-terminal (CT) in primary bovine corneal endothelial cells and HeLa, C6 glioma, and Xenopus oocytes ectopically expressing Cx43. Peptides corresponding to the last 10 CT aa (TAT-Cx43CT) prevented the inhibition of Cx43-hemichannel activity by contractility/high [Ca(2+)](i), whereas a reverse peptide (TAT-Cx43CTrev) did not. These effects were independent of zonula occludens-1, a cytoskeletal-associated Cx43-binding protein. In contrast, peptides corresponding to CL (TAT-L2) inhibited Cx43-hemichannel responses, whereas a mutant peptide (TAT-L2(H126K/I130N)) did not inhibit. In these assays, TAT-Cx43CT acted as a scaffold for TAT-L2 and vice versa, a finding supported by surface plasmon resonance measurements. Loop/tail interactions appeared essential for Cx43-hemichannel activity, because TAT-Cx43CT restored the activity of nonfunctional hemichannels, consisting of either Cx43 lacking the C-terminal tail (Cx43(M239)) or intact Cx43 ectopically expressed in Xenopus oocytes. We conclude that intramolecular loop/tail interactions control Cx43-hemichannel activity, laying the basis for developing hemichannel-specific blockers.
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Conexina 43/metabolismo , Canales Iónicos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Bovinos , Línea Celular , Línea Celular Tumoral , Córnea/citología , Córnea/metabolismo , Células Endoteliales/metabolismo , Productos del Gen tat/metabolismo , Células HeLa , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Espacio Intracelular/metabolismo , Canales Iónicos/efectos de los fármacos , Oocitos/metabolismo , Unión Proteica , Ratas , Trombina/metabolismo , Xenopus laevis/metabolismoRESUMEN
The neurotoxin beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (L-beta-ODAP) is an L-glutamate analogue at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors in neurons and therefore acts as an excitotoxic substance. Chronic exposure to L-beta-ODAP present in Lathyrus sativus L. (L. sativus) seeds is proposed as the cause of the neurodegenerative disease neurolathyrism, but the mechanism of its action has not been conclusively identified. A key factor in excitotoxic neuronal cell death is a disturbance of the intracellular Ca2+ homeostasis, including changes in the capacity of intracellular Ca2+ stores like the endoplasmic reticulum (ER) or mitochondria. In this study, aequorin and other Ca2+ indicators were used in N2a neuroblastoma cells to investigate alterations of cellular Ca2+ handling after 24 h exposure to L-beta-ODAP. Our data demonstrate increased mitochondrial Ca2+ loading and hyperpolarization of the mitochondrial membrane potential (Psi(m)), which was specific for L-beta-ODAP and not observed with L-glutamate. We conclude that L-beta-ODAP disturbs the ER-mitochondrial Ca2+ signaling axis and thereby renders the cells more vulnerable to its excitotoxic effects that ultimately will lead to cell death.
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Señalización del Calcio/fisiología , Calcio/metabolismo , Mitocondrias/efectos de los fármacos , Neurotoxinas/toxicidad , beta-Alanina/análogos & derivados , Aequorina , Animales , Señalización del Calcio/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Ácido Glutámico/toxicidad , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Indicadores y Reactivos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Mitocondrias/metabolismo , Factores de Tiempo , beta-Alanina/toxicidadRESUMEN
The present study was set up to investigate the fate of connexin32 and its channels in hepatocellular apoptosis. Primary hepatocyte cultures were exposed to Fas ligand and cycloheximide, and modifications in connexin32 expression and localization, and gap junction functionality were studied. We found that gap junction functionality rapidly declined upon progression of cell death, which was associated with a decay of the gap junctional connexin32 protein pool. Simultaneously, levels of newly synthesized connexin32 protein increased and gathered in a hemichannel configuration. This became particularly evident towards the end stages of the cell death process and was not reflected at the transcriptional level. We next either silenced connexin32 expression or inhibited connexin32 hemichannel activity prior to cell death induction. Both approaches resulted in a delayed termination of the cell death response. We conclude that connexin32 hemichannels facilitate the apoptotic-to-necrotic transition, which typically occurs in the final stage of hepatocellular apoptosis.
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Apoptosis , Conexinas/metabolismo , Proteína Ligando Fas/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Activación del Canal Iónico , Animales , Apoptosis/efectos de los fármacos , Cicloheximida/farmacología , Hepatocitos/efectos de los fármacos , Humanos , Masculino , Necrosis/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína beta1 de Unión ComunicanteRESUMEN
Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca(2+) ([Ca(2+)](i)) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca(2+)](i) was in the 500nM range but the responses disappeared with larger [Ca(2+)](i) transients. Ca(2+)-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca(2+)-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca(2+)](i) triggers hemichannel opening, not by a direct action of Ca(2+) on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca(2+)](i) and acting to restrain cellular ATP loss.
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Calcio/metabolismo , Conexina 43/metabolismo , Glioma/metabolismo , Neuroglía/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Calcimicina/farmacología , Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Células Cultivadas , Conexina 43/genética , Células HeLa , Humanos , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
This study was set up to critically evaluate a commonly-used in vitro model of hepatocellular apoptotic cell death, in which freshly isolated hepatocytes, cultured in a monolayer configuration, are exposed to a combination of Fas ligand and cycloheximide for six hours. A set of well-acknowledged cell death markers was addressed: a) cell morphology was studied by light microscopy; b) apoptotic and necrotic cell populations were quantified by in situ staining with Annexin-V, Hoechst 33342 and propidium iodide (PI); c) apoptotic and necrotic activities were monitored by probing caspase 3-like activity and measuring the extracellular leakage of lactate dehydrogenase (LDH), respectively; and d) the expression of apoptosis regulators was investigated by immunoblotting. The initiation of apoptosis was evidenced by the activation of caspase 8 and caspase 9, and increased Annexin-V reactivity. Progression through the apoptotic process was confirmed by the activation of caspase 3 and Bid, the enhanced expression of Bax, and the occurrence of nuclear fragmentation. Late transition to a necrotic appearance was demonstrated by an increased number of PI-positive cells and augmented extracellular release of LDH. Thus, the in vitro model allows the study of the entire course of Fas-mediated hepatocellular apoptotic cell death, which is not possible in vivo. This experimental system can serve a broad range of in vitro pharmaco-toxicological purposes, thereby directly assisting in the reduction of animal experimentation.
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Alternativas a las Pruebas en Animales/métodos , Apoptosis/fisiología , Muerte Celular , Hepatocitos/citología , Hepatocitos/fisiología , Animales , Caspasas/metabolismo , Técnicas de Cultivo de Célula , Hepatectomía , Homeostasis , L-Lactato Deshidrogenasa/análisis , Hígado/citología , Hígado/fisiología , Necrosis , Ratas , RoedoresRESUMEN
The establishment of gap junctional intercellular communication is a prerequisite for appropriate control of tissue homeostasis. Gap junctions consist of connexin proteins, whereby a myriad of factors govern the connexin life cycle. At the transcriptional level, most attention has yet been paid to the classical cis/trans machinery (i.e. the interaction between transcription factors and regulatory elements in connexin gene promoter regions) as a gatekeeper of connexin expression. In the last few years, it has become clear that epigenetic processes are also essentially involved in connexin gene transcription. Major determinants of the epigenome include histone modifications and DNA methylation, and recently, microRNA species have also been described as key regulators of the epigenetic machinery. In the present paper, the emerging roles of epigenetic events in the control of connexin expression, and consequently of gap junctional intercellular communication, are reviewed. Besides an updated theoretical background concerning gap junctions and epigenetic phenomena, we provide an in-depth overview of their interrelationship and we demonstrate the clinical relevance of the topic.
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Comunicación Celular/genética , Fenómenos Fisiológicos Celulares/genética , Epigénesis Genética/fisiología , Uniones Comunicantes/genética , Animales , Conexinas/genética , Metilación de ADN/fisiología , Histonas/metabolismo , Homeostasis/genética , Homeostasis/fisiología , Humanos , MicroARNs/fisiología , Modelos BiológicosRESUMEN
Electroporation is generally used to transfect cells in suspension, but the technique can also be applied to load a defined zone of adherent cells with substances that normally do not permeate the plasma membrane. In this case a pulsed high-frequency oscillating electric field is applied over a small two-wire electrode positioned close to the cells. We compared unipolar with bipolar electroporation pulse protocols and found that the latter were ideally suited to efficiently load a narrow longitudinal strip of cells in monolayer cultures. We further explored this property to determine whether electroporation loading was useful to investigate the extent of dye spread between cells coupled by gap junctions, using wild-type and stably transfected C6 glioma cells expressing connexin 32 or 43. Our investigations show that the spatial spread of electroporation-loaded 6-carboxyfluorescein, as quantified by the standard deviation of Gaussian dye spread or the spatial constant of exponential dye spread, was a reliable approach to investigate the degree of cell-cell coupling. The spread of reporter dye between coupled cells was significantly larger with electroporation loading than with scrape loading, a widely used method for dye-coupling studies. We conclude that electroporation loading and dye transfer is a robust technique to investigate gap-junctional coupling that combines minimal cell damage with accurate probing of the degree of cell-cell communication.
Asunto(s)
Colorantes/metabolismo , Electroporación/métodos , Uniones Comunicantes/metabolismo , Animales , Western Blotting , Línea Celular , Conexina 43/metabolismo , Conexinas/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Inmunohistoquímica , Peso Molecular , Ratas , Proteína beta1 de Unión ComunicanteRESUMEN
Glucose transport over the blood-brain barrier (BBB) is a nonrate-limiting step and has therefore received little attention as a possible adjustment point within the transport reaction cascade from blood glucose to brain cell glycolysis. Considerations of the normal working point of facilitated BBB glucose shuttling via the GLUT-1 protein indicate that the transport is working at about one-third of T(max) under basal conditions. Substitution of T(max) estimates indicates that the transport is then just enough to keep up with glucose consumption, maintaining the steady state. After brain activation, glucose transport has to be stimulated, and this can be accomplished by increasing the driving force or changing the T(max) and/or K(t) parameters of BBB transport. The first possibility involves a decrease of brain interstitial glucose with subsequent flow stimulation according to the law of mass action (LMA), whereas the second possibility involves signaling from activated neurons to the BBB, a regulation loop that we propose to be called "neurobarrier coupling" (NBC). Theoretical analysis of the LMA effect and comparison with data on glucose dynamics during brain activation suggest that this factor alone only covers about half of the stimulation necessary to bring glucose delivery into line with the elevated glucose consumption during activation. Adjusting glucose entry with demand thus probably involves both LMA and NBC effects, depending on the degree of brain activation. Further work is needed to demonstrate NBC effects following physiological brain activation in vivo and to identify the signals that lead to NBC in in vitro experiments.
Asunto(s)
Encéfalo/metabolismo , Glucosa/metabolismo , Animales , Barrera Hematoencefálica/fisiología , HumanosRESUMEN
Gap junction (GJ) channels are formed by two hemichannels (connexons), each contributed by the cells taking part in this direct cell-cell communication conduit. Hemichannels that do not interact with their counterparts on neighboring cells feature as a release pathway for small paracrine messengers such as nucleotides, glutamate, and prostaglandins. Connexins are phosphorylated by various kinases, and we compared the effect of various kinase-activating stimuli on GJ channels and hemichannels. Using peptides identical to a short connexin (Cx) amino acid sequence to specifically block hemichannels, we found that protein kinase C, Src, and lysophosphatidic acid (LPA) inhibited GJs and hemichannel-mediated ATP release in Cx43-expressing C6 glioma cells (C6-Cx43). Lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) inhibited GJs, but they stimulated ATP release via hemichannels in C6-Cx43. LPS and bFGF inhibited hemichannel-mediated ATP release in HeLa-Cx43 cells, but they stimulated it in HeLa-Cx43 with a truncated carboxy-terminal (CT) domain or in HeLa-Cx26, which has a very short CT. Hemichannel potentiation by LPS was inhibited by blockers of the arachidonic acid metabolism, and arachidonic acid had a potentiating effect like LPS and bFGF. We conclude that GJ channels and hemichannels display similar or oppositely directed responses to modulatory influences, depending on the balance between kinase activity and the activity of the arachidonic acid pathway. Distinctive hemichannel responses to pathological stimulation with LPS or bFGF may serve to optimize the cell response, directed at strictly controlling cellular ATP release, switching from direct GJ communication to indirect paracrine signaling, or maximizing cell-protective strategies.