RESUMEN
Peripheral neuropathy is the most frequent dose-limiting adverse effect of oxaliplatin. Acute pain symptoms that are induced or exacerbated by cold occur in almost all patients immediately following the first infusions. Evidence has shown that oxaliplatin causes ion channel expression modulations in dorsal root ganglia neurons, which are thought to contribute to peripheral hypersensitivity. Most dysregulated genes encode ion channels involved in cold and mechanical perception, noteworthy members of a sub-group of potassium channels of the K2P family, TREK and TRAAK. Downregulation of these K2P channels has been identified as an important tuner of acute oxaliplatin-induced hypersensitivity. We investigated the molecular mechanisms underlying this peripheral dysregulation in a murine model of neuropathic pain triggered by a single oxaliplatin administration. We found that oxaliplatin-mediated TREK-TRAAK downregulation, as well as downregulation of other K+ channels of the K2P and Kv families, involves a transcription factor known as the neuron-restrictive silencer factor (NRSF) and its epigenetic co-repressors histone deacetylases (HDACs). NRSF knockdown was able to prevent most of these K+ channel mRNA downregulation in mice dorsal root ganglion neurons as well as oxaliplatin-induced acute cold and mechanical hypersensitivity. Interestingly, pharmacological inhibition of class I HDAC reproduces the antinociceptive effects of NRSF knockdown and leads to an increased K+ channel expression in oxaliplatin-treated mice.
Asunto(s)
Regulación hacia Abajo/fisiología , Epigénesis Genética/fisiología , Hiperalgesia/metabolismo , Oxaliplatino/toxicidad , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Transcripción Genética/fisiología , Animales , Antineoplásicos/toxicidad , Regulación hacia Abajo/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Hiperalgesia/inducido químicamente , Hiperalgesia/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Canales de Potasio/biosíntesis , Canales de Potasio/genética , Canales de Potasio de Dominio Poro en Tándem/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Pulmonary hypertension (PH) is a progressive and potentially serious lung disease, defined by an abnormal elevation of pulmonary arterial pressure. PH occurs for many reasons, and hypoxia is considered as an important stimulus for the disease. Proliferation and migration of pulmonary artery smooth muscular cells (PASMCs) in the small peripheral pulmonary arteries are common characteristic features in hypoxia-induced PH (HPH). However, the mechanisms involved in the hypoxia-induced cell proliferation and migration are not clear. The aim of the present study was to investigate the role of lncRNA Gas5 in the hypoxia-stimulated proliferation and migration of human PASMCs (hPASMCs). We found that the expression of Gas5 was down-regulated in a rat model with hypoxia and in cultured hypoxic hPASMCs, and silence of Gas5 significantly promoted hPASMCs proliferation and migration in both normal and hypoxia condition. Subsequent studies revealed that miR-23b-3p interacted with Gas5 by directly targeting the miRNA-binding site in the Gas5 sequence, and qRT-PCR results showed miR-23b-3p and Gas5 could affect each other's expression, respectively. Further study demonstrated that Gas5 acted as a competing endogenous RNA (ceRNA) for miR-23b-3p to modulate the KCNK3 expression, and these interactions led to promotion of hPASMCs proliferation and migration. This study identified that Gas5/miR-23b-3p/KCNK3 axis may be a mechanism that hypoxia-induced PASMCs proliferation and migration, providing a strategy for clinical treatment of HPH in the future.
Asunto(s)
Regulación hacia Abajo/fisiología , Miocitos del Músculo Liso/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Arteria Pulmonar/metabolismo , ARN Largo no Codificante/biosíntesis , Animales , Hipoxia de la Célula/fisiología , Células Cultivadas , Expresión Génica , Masculino , Proteínas del Tejido Nervioso/genética , Canales de Potasio de Dominio Poro en Tándem/genética , Arteria Pulmonar/citología , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , Ratas , Ratas Sprague-DawleyRESUMEN
TWIK-related acid-sensitive K+ (TASK) homomeric channels, TASK1 and TASK3, are present in PC12 cells. The channels do not heteromerize due plausibly to a lack of p11 protein. Single-channel recording reveals that most of the rat carotid body (CB) glomus cells express heteromeric TASK1-TASK3 channels, but the presence of p11 in glomus cells has not yet been verified. TASK1, but not TASK3, binds to p11, which has a retention signal for the endoplasmic reticulum. We hypothesized that p11 could facilitate heteromeric TASK1-TASK3 formation in glomus cells. We investigated this hypothesis in isolated immunocytochemically identified rat CB glomus cells. The findings were that glomus cells expressed p11-like immunoreactive (IR) material, and TASK1- and TASK3-like IR material mainly coincided in the cytoplasm. The proximity ligation assay showed that TASK1 and TASK3 heteromerized. In separate experiments, supporting evidence for the major role of p11 for channel heteromerization was provided in PC12 cells stimulated by nerve growth factor. p11 production took place there via multiple signaling pathways comprising mitogen-activated protein kinase and phospholipase C, and heteromeric TASK1-TASK3 channels were formed. We conclude that p11 is expressed and TASK1 and TASK3 heteromerize in rat CB glomus cells.
Asunto(s)
Anexina A2/biosíntesis , Cuerpo Carotídeo/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Proteínas S100/biosíntesis , Animales , Cuerpo Carotídeo/citología , Inmunohistoquímica , Masculino , Células PC12 , Ratas , Ratas WistarRESUMEN
BACKGROUND: Despite an increasing understanding of atrial fibrillation (AF) pathophysiology, translation into mechanism-based treatment options is lacking. In atrial cardiomyocytes of patients with chronic AF, expression, and function of tandem of P domains in a weak inward rectifying TASK-1 (K+ channel-related acid-sensitive K+ channel-1) (K2P3.1) atrial-specific 2-pore domain potassium channels is enhanced, resulting in action potential duration shortening. TASK-1 channel inhibition prevents action potential duration shortening to maintain values observed among sinus rhythm subjects. The present preclinical study used a porcine AF model to evaluate the antiarrhythmic efficacy of TASK-1 inhibition by adeno-associated viral anti-TASK-1-siRNA (small interfering RNA) gene transfer. METHODS: AF was induced in domestic pigs by atrial burst stimulation via implanted pacemakers. Adeno-associated viral vectors carrying anti-TASK-1-siRNA were injected into both atria to suppress TASK-1 channel expression. After the 14-day follow-up period, porcine cardiomyocytes were isolated from right and left atrium, followed by electrophysiological and molecular characterization. RESULTS: AF was associated with increased TASK-1 transcript, protein and ion current levels leading to shortened action potential duration in atrial cardiomyocytes compared to sinus rhythm controls, similar to previous findings in humans. Anti-TASK-1 adeno-associated viral application significantly reduced AF burden in comparison to untreated AF pigs. Antiarrhythmic effects of anti-TASK-1-siRNA were associated with reduction of TASK-1 currents and prolongation of action potential durations in atrial cardiomyocytes to sinus rhythm values. Conclusions Adeno-associated viral-based anti-TASK-1 gene therapy suppressed AF and corrected cellular electrophysiological remodeling in a porcine model of AF. Suppression of AF through selective reduction of TASK-1 currents represents a new option for antiarrhythmic therapy.
Asunto(s)
Antiarrítmicos/uso terapéutico , Fibrilación Atrial/genética , Remodelación Atrial/fisiología , Regulación de la Expresión Génica , Terapia Genética/métodos , Atrios Cardíacos/fisiopatología , Proteínas del Tejido Nervioso/genética , Canales de Potasio de Dominio Poro en Tándem/genética , Potenciales de Acción/fisiología , Animales , Fibrilación Atrial/metabolismo , Fibrilación Atrial/terapia , Modelos Animales de Enfermedad , Electrocardiografía , Miocitos Cardíacos/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , ARN/genética , Ratas , PorcinosRESUMEN
TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA)-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK) inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment.
Asunto(s)
Neoplasias de la Mama/metabolismo , Senescencia Celular , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Canales de Potasio de Dominio Poro en Tándem/genéticaRESUMEN
BACKGROUND Changes in expression and activity of ion channels are important pathophysiological mechanisms underlying detrusor overactivity (DO) in partial bladder outlet obstruction (PBOO). The objective of this study was to examine the expression of TREK-1 channel in the bladder and central nervous system of DO rats. MATERIAL AND METHODS Thirty Sprague-Dawley rats were subjected to PBOO operations and those displaying non-voiding contractions (NVCs) in cystometry were classified as DO. Sham-operated rats without NVCs in cystometry served as controls. The expression and distribution of TREK-1 in the bladder, spinal cord, and dorsal root ganglion (DRG) were detected by real time-PCR, western blot, and immunohistochemistry. RESULTS TREK-1 channel expression in the DRG was significantly increased at the mRNA level (11.20±3.762 vs. 3.209±1.505, P<0.01) and protein level (2.195±0.058 vs. 1.713±0.066, P<0.01) in DO rats as compared to control rats. However, the expression of TREK-1 mRNA in the bladder (1.380±0.810 vs. 4.206±3.827, P>0.05) and spinal cord (0.764±0.357 vs. 0.696±0.188, P>0.05) was comparable between the 2 groups. Immunohistochemistry showed enhanced immunoreactive signals of TREK-1 channel in the DRG, but not in the spinal cord and bladder. CONCLUSIONS TREK-1 channel was upregulated in the DRG of DO rats after chronic PBOO, which might suppress neuronal excitability and play a protective role in bladder overactivity in PBOO.
Asunto(s)
Ganglios Espinales/metabolismo , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Obstrucción del Cuello de la Vejiga Urinaria/metabolismo , Vejiga Urinaria Hiperactiva/metabolismo , Animales , Femenino , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Regulación hacia Arriba , Vejiga Urinaria/metabolismo , Obstrucción del Cuello de la Vejiga Urinaria/genética , Vejiga Urinaria Hiperactiva/genéticaAsunto(s)
Regulación de la Expresión Génica , Mucosa Nasal/metabolismo , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Rinitis/metabolismo , Sinusitis/metabolismo , Enfermedad Crónica , Humanos , Mucosa Nasal/inmunología , Rinitis/inmunología , Rinitis/patología , Sinusitis/inmunología , Sinusitis/patologíaRESUMEN
BACKGROUND: Mutations in the KCNK3 gene have been identified in some patients suffering from heritable pulmonary arterial hypertension (PAH). KCNK3 encodes an outward rectifier K(+) channel, and each identified mutation leads to a loss of function. However, the pathophysiological role of potassium channel subfamily K member 3 (KCNK3) in PAH is unclear. We hypothesized that loss of function of KCNK3 is a hallmark of idiopathic and heritable PAH and contributes to dysfunction of pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, leading to pulmonary artery remodeling: consequently, restoring KCNK3 function could alleviate experimental pulmonary hypertension (PH). METHODS AND RESULTS: We demonstrated that KCNK3 expression and function were reduced in human PAH and in monocrotaline-induced PH in rats. Using a patch-clamp technique in freshly isolated (not cultured) pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, we found that KCNK3 current decreased progressively during the development of monocrotaline-induced PH and correlated with plasma-membrane depolarization. We demonstrated that KCNK3 modulated pulmonary arterial tone. Long-term inhibition of KCNK3 in rats induced distal neomuscularization and early hemodynamic signs of PH, which were related to exaggerated proliferation of pulmonary artery endothelial cells, pulmonary artery smooth muscle cell, adventitial fibroblasts, and pulmonary and systemic inflammation. Lastly, in vivo pharmacological activation of KCNK3 significantly reversed monocrotaline-induced PH in rats. CONCLUSIONS: In PAH and experimental PH, KCNK3 expression and activity are strongly reduced in pulmonary artery smooth muscle cells and endothelial cells. KCNK3 inhibition promoted increased proliferation, vasoconstriction, and inflammation. In vivo pharmacological activation of KCNK3 alleviated monocrotaline-induced PH, thus demonstrating that loss of KCNK3 is a key event in PAH pathogenesis and thus could be therapeutically targeted.
Asunto(s)
Hipertensión Pulmonar/fisiopatología , Proteínas del Tejido Nervioso/fisiología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Adventicia/patología , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , División Celular , Endotelio Vascular/patología , Fibroblastos/patología , Predisposición Genética a la Enfermedad , Hemodinámica , Humanos , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/complicaciones , Hipertensión Pulmonar/genética , Hipertrofia Ventricular Derecha/etiología , Inflamación , Masculino , Potenciales de la Membrana , Monocrotalina/toxicidad , Mutación , Miocitos del Músculo Liso/patología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/genética , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Sulfonamidas/farmacología , Resistencia Vascular , ortoaminobenzoatos/farmacologíaRESUMEN
Müller cells are principal glial cells in rat retina and have attracted much attention in glaucoma studies. However, it is not clear whether adenosine and adenosine receptor (AR) antagonists play any roles in the regulation of potassium channels in Müller cells and subsequently in the promotion of glutamine synthetase (GS) and L-Glutamate/L-Aspartate Transporter (GLAST) functions. We found that chronic ocular hypertension (COH) in rat down-regulated Müller cells Kir2.1, Kir4.1, TASK-1, GS and GLAST expressions and attenuated the peak of inward potassium current. Retinal ganglion cells (RGC) count was lower in the COH rats than that in the sham operation animals. Intravitreal injection of selective A2A AR antagonist SCH442416 up-regulated Müller cell Kir4.1, TASK-1, GS and GLAST expressions and enhanced inward potassium currents compared with those in the COH rats with vehicle control. Meanwhile, the RGC count was higher following intravitreal injection of SCH442416 in the COH rats than that after vehicle injection. The fact that PKA inhibitor H-89 blocked these SCH442416 effects suggested that the PKA signaling pathway was involved in the observed ocular responses following the intravitreal SCH442416 injection.
Asunto(s)
Adenosina/farmacología , Células Ependimogliales/metabolismo , Glaucoma/patología , Canales de Potasio/efectos de los fármacos , Antagonistas de Receptores Purinérgicos P1/farmacología , Sistema de Transporte de Aminoácidos X-AG/biosíntesis , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Glutamato-Amoníaco Ligasa/biosíntesis , Isoquinolinas/farmacología , Masculino , Proteínas del Tejido Nervioso , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Pirazoles/farmacología , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Sulfonamidas/farmacologíaRESUMEN
BACKGROUND: Antiarrhythmic management of atrial fibrillation (AF) remains a major clinical challenge. Mechanism-based approaches to AF therapy are sought to increase effectiveness and to provide individualized patient care. K(2P)3.1 (TASK-1 [tandem of P domains in a weak inward-rectifying K+ channel-related acid-sensitive K+ channel-1]) 2-pore-domain K+ (K(2P)) channels have been implicated in action potential regulation in animal models. However, their role in the pathophysiology and treatment of paroxysmal and chronic patients with AF is unknown. METHODS AND RESULTS: Right and left atrial tissue was obtained from patients with paroxysmal or chronic AF and from control subjects in sinus rhythm. Ion channel expression was analyzed by quantitative real-time polymerase chain reaction and Western blot. Membrane currents and action potentials were recorded using voltage- and current-clamp techniques. K(2P)3.1 subunits exhibited predominantly atrial expression, and atrial K(2P)3.1 transcript levels were highest among functional K(2P) channels. K(2P)3.1 mRNA and protein levels were increased in chronic AF. Enhancement of corresponding currents in the right atrium resulted in shortened action potential duration at 90% of repolarization (APD90) compared with patients in sinus rhythm. In contrast, K(2P)3.1 expression was not significantly affected in subjects with paroxysmal AF. Pharmacological K(2P)3.1 inhibition prolonged APD90 in atrial myocytes from patients with chronic AF to values observed among control subjects in sinus rhythm. CONCLUSIONS: Enhancement of atrium-selective K(2P)3.1 currents contributes to APD shortening in patients with chronic AF, and K(2P)3.1 channel inhibition reverses AF-related APD shortening. These results highlight the potential of K(2P)3.1 as a novel drug target for mechanism-based AF therapy.
Asunto(s)
Potenciales de Acción/fisiología , Fibrilación Atrial/metabolismo , Fibrilación Atrial/fisiopatología , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Regulación hacia Arriba/fisiología , Anciano , Anciano de 80 o más Años , Fibrilación Atrial/diagnóstico , Enfermedad Crónica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas del Tejido NerviosoRESUMEN
BACKGROUND: Effective treatment of atrial fibrillation (AF) remains an unmet need. Human K2P3.1 (TASK-1) K(+) channels display atrial-specific expression and may serve as novel antiarrhythmic targets. In rodents, inhibition of K2P3.1 causes prolongation of action potentials and QT intervals. We used a porcine model to further elucidate the significance of K2P3.1 in large mammals. OBJECTIVE: The purpose of this study was to study porcine (p)K2P3.1 channel function and cardiac expression and to analyze pK2P3.1 remodeling in AF and heart failure (HF). METHODS: The porcine K2P3.1 ortholog was amplified and characterized using voltage-clamp electrophysiology. K2P3.1 mRNA expression and remodeling were studied in domestic pigs during AF and HF induced by atrial burst pacing. RESULTS: Porcine K2P3.1 cDNA encodes a channel protein with 97% identity to human K2P3.1. K(+) currents recorded from Xenopus oocytes expressing pK2P3.1 were functionally and pharmacologically similar to their human counterparts. In the pig, K2P3.1 mRNA was predominantly expressed in atrial tissue. AF and HF were associated with reduction of K2P3.1 mRNA levels by 85.1% (right atrium) and 77.0% (left atrium) at 21-day follow-up. In contrast, ventricular K2P3.1 expression was low and not significantly affected by AF/HF. CONCLUSION: Porcine K2P3.1 channels exhibit atrial expression and functional properties similar to their human orthologs, supporting a general role as antiarrhythmic drug targets. K2P3.1 down-regulation in AF with HF may indicate functional relevance of the channel that remains to be validated in prospective interventional studies.
Asunto(s)
Fibrilación Atrial/genética , Regulación de la Expresión Génica , Insuficiencia Cardíaca/genética , Proteínas del Tejido Nervioso/genética , Canales de Potasio de Dominio Poro en Tándem/genética , ARN Mensajero/genética , Animales , Fibrilación Atrial/metabolismo , Fibrilación Atrial/fisiopatología , Modelos Animales de Enfermedad , Femenino , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Proteínas del Tejido Nervioso/biosíntesis , Técnicas de Placa-Clamp , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , PorcinosRESUMEN
TWIK-related acid-sensitive potassium channels (TASK3) are pharmacological targets of CNS inflammation induced by acidification. They function as molecular switches between survival and death of neurons. In this report, TASK3 cloned from human brain cDNA was tagged with enhanced green fluorescent protein (eGFP), and the fusion gene was transiently expressed in human neuroblastoma SH-SY5Y cells. A cell line stably expressing TASK-eGFP fusion proteins was generated from transient expression cells by using fluorescence-activated cell sorting followed by antibiotic selection. The uniform expression of TASK3 fusion proteins was further confirmed by flow cytometry. Moreover, the localization of TASK3 tagged with eGFP was checked by confocal microcopy. TASK3-eGFP fusion proteins are observed on the SH-SY5Y cell membrane. The strategies using eGFP as a fusion tag facilitate the monitoring of the TASK3 expression and enable the successful employment of FACS for screening and construction of cell lines stably expressing TASK3. The TASK3 overexpression cell line will lay a fundamental for the in vitro evaluation of TASK3 function during hypoxic/ischemic injury.
Asunto(s)
Expresión Génica , Canales de Potasio de Dominio Poro en Tándem/genética , Proteínas Recombinantes de Fusión/genética , Química Encefálica , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , ADN Complementario/genética , ADN Complementario/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Microscopía Confocal , Neuronas/citología , Neuronas/metabolismo , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
BACKGROUND: Preconditioning with volatile anaesthetic agents induces tolerance to focal cerebral ischaemia, although the underlying mechanisms have not been clearly defined. The present study analyses whether TREK-1, a two-pore domain K(+) channel and target for volatile anaesthetics, plays a role in mediating neuroprotection by sevoflurane. METHODS: Differentiated SH-SY5Y cells were preconditioning with sevoflurane and challenged by oxygen-glucose deprivation (OGD). Cell viability and expression of caspase-3 and TREK-1 were evaluated. Rats that were preconditioned with sevoflurane were subjected to middle cerebral artery occlusion (MCAO), and the expression of TREK-1 protein and mRNA was analysed. Neurological scores were evaluated and infarction volume was examined. RESULTS: Sevoflurane preconditioning reduced cell death in differentiated SH-SY5Y cells challenged by OGD. Sevoflurane preconditioning reduced infarct volume and improved neurological outcome in rats subjected to MCAO. Sevoflurane preconditioning increased levels of TREK-1 mRNA and protein. Knockdown of TREK-1 significantly attenuated sevoflurane preconditioning-induced neuroprotective effects in vitro and in vivo. CONCLUSIONS: Sevoflurane preconditioning-induced neuroprotective effects against transient cerebral ischaemic injuries involve TREK-1 channels. These results suggest a novel mechanism for sevoflurane preconditioning-induced tolerance to focal cerebral ischaemia.
Asunto(s)
Isquemia Encefálica/prevención & control , Precondicionamiento Isquémico/métodos , Éteres Metílicos/farmacología , Fármacos Neuroprotectores/farmacología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Animales , Encéfalo/metabolismo , Isquemia Encefálica/metabolismo , Monóxido de Carbono/sangre , Diferenciación Celular/efectos de los fármacos , Hipoxia de la Célula/fisiología , Supervivencia Celular/efectos de los fármacos , Infarto Cerebral/metabolismo , Infarto Cerebral/patología , Infarto Cerebral/prevención & control , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Éteres Metílicos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Oxígeno/sangre , Presión Parcial , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control , Sevoflurano , Células Tumorales CultivadasRESUMEN
TASK3 two-pore domain potassium (K2P) channels are responsible for native leak K channels in many cell types which regulate cell resting membrane potential and excitability. In addition, TASK3 channels contribute to the regulation of cellular potassium homeostasis. Because TASK3 channels are important for cell viability, having putative roles in both neuronal apoptosis and oncogenesis, we sought to determine their behavior under inflammatory conditions by investigating the effect of TNFα on TASK3 channel current. TASK3 channels were expressed in tsA-201 cells, and the current through them was measured using whole cell voltage clamp recordings. We show that THP-1 human myeloid leukemia monocytes, co-cultured with hTASK3-transfected tsA-201 cells, can be activated by the specific Toll-like receptor 7/8 activator, R848, to release TNFα that subsequently enhances hTASK3 current. Both hTASK3 and mTASK3 channel activity is increased by incubation with recombinant TNFα (10 ng/ml for 2-15 h), but other K2P channels (hTASK1, hTASK2, hTREK1, and hTRESK) are unaffected. This enhancement by TNFα is not due to alterations in levels of channel expression at the membrane but rather to an alteration in channel gating. The enhancement by TNFα can be blocked by extracellular acidification but persists for mutated TASK3 (H98A) channels that are no longer acid-sensitive even in an acidic extracellular environment. TNFα action on TASK3 channels is mediated through the intracellular C terminus of the channel. Furthermore, it occurs through the ASK1 pathway and is JNK- and p38-dependent. In combination, TNFα activation and TASK3 channel activity can promote cellular apoptosis.
Asunto(s)
Apoptosis/fisiología , Regulación de la Expresión Génica/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo , Adenilato Quinasa/genética , Adenilato Quinasa/metabolismo , Sustitución de Aminoácidos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular , Técnicas de Cocultivo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Imidazoles/farmacología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mutación Missense , Canales de Potasio de Dominio Poro en Tándem/genética , Estructura Terciaria de Proteína , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/genética , Receptor Toll-Like 8/metabolismo , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The majority of the polytopic proteins that are synthesized at the ER (endoplasmic reticulum) are integrated co-translationally via the Sec61 translocon, which provides lateral access for their hydrophobic TMs (transmembrane regions) to the phospholipid bilayer. A prolonged association between TMs of the potassium channel subunit, TASK-1 [TWIK (tandem-pore weak inwardly rectifying potassium channel)-related acid-sensitive potassium channel 1], and the Sec61 complex suggests that the ER translocon co-ordinates the folding/assembly of the TMs present in the nascent chain. The N-terminus of both TASK-1 and Kcv (potassium channel protein of chlorella virus), another potassium channel subunit of viral origin, has access to the N-glycosylation machinery located in the ER lumen, indicating that the Sec61 complex can accommodate multiple arrangements/orientations of TMs within the nascent chain, both in vitro and in vivo. Hence the ER translocon can provide the ribosome-bound nascent chain with a dynamic environment in which it can explore a range of different conformations en route to its correct transmembrane topology and final native structure.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Canales de Potasio de Dominio Poro en Tándem/química , Señales de Clasificación de Proteína , Estructura Secundaria de Proteína , Canales de Translocación SECRESUMEN
Astrocytes play a fundamental role in the pathogenesis of ischemic neuronal death. The optimal operation of electrogenic astrocytic transporters and exchangers for some well-defined astrocyte brain homeostatic functions depends on the presence of K(+) channels in the cell membranes and the hyperpolarized membrane potential. Our previous study showed that astrocytes functionally express two-pore domain K(+) channel TREK-1, which helps to set the negative resting membrane potential. However, the roles of TREK-1 on astrocytic function under normal and ischemic conditions remain unclear. In this study, we investigated the expression of TREK-1 protein on cultured astrocytes and the effect of TREK-1 activity on astrocytic glutamate clearance capacity and release of s100ß after simulated ischemic insult. TREK-1 immunoreactivity was up-regulated after hypoxia. Suppression of TREK-1 activity inhibited the glutamate clearance capability, enhanced the inflammatory secretion of astrocytes derived s100ß and led to increased neuronal apoptosis after ischemic insult. Our results suggest that TREK-1 activity is involved in astrocytic function and neuronal survival. This would provide evidence showing astrocytic TREK-1 involvement in ischemia pathology which may serve as a potential therapeutic target in stroke.
Asunto(s)
Astrocitos/fisiología , Hipoxia de la Célula/fisiología , Isquemia/metabolismo , Proteínas del Tejido Nervioso/fisiología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Animales , Apoptosis/efectos de los fármacos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Bupivacaína/farmacología , Hipoxia de la Célula/genética , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Corteza Cerebral/citología , Técnicas de Cocultivo , Medio de Cultivo Libre de Suero , Regulación de la Expresión Génica , Ácido Glutámico/metabolismo , Homeostasis , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/genética , Quinina/farmacología , Ratas , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/metabolismoRESUMEN
Astrocytes play an active and important role in the pathophysiology of cerebral ischemia. We have previously shown that mature hipppocampal astrocytes functionally express two-pore domain K(+) channel TREK-1, which significantly contributes to the passive conductance and help to set the negative resting membrane potential essential for the optimal operation of some astrocytic homeostatic functions. However, its expression under ischemic conditions remains to be determined. In this study, we examined the expression of TREK-1 in rat brain under physiological and focal ischemia conditions. The results show that TREK-1 was broadly expressed on astrocytes and neurons in the cortex, CA1 region of hippocampus. After middle cerebral artery occlusion induced focal ischemia, the TREK-1 expression was significantly increased at days 3, 7 and 30 following reperfusion, which correlated with reactive astrogliosis in the cortex and hippocampus. Cultured cortical astrocytes also express TREK-1. TREK-1 inhibitor quinine inhibited the proliferation of astrocytes exposed to hypoxia condition. These data provide evidence showing the astrocytic TREK-1 involvement in ischemia pathology.
Asunto(s)
Astrocitos/metabolismo , Región CA1 Hipocampal/metabolismo , Corteza Cerebral/metabolismo , Regulación de la Expresión Génica , Gliosis/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Canales de Potasio de Dominio Poro en Tándem/fisiología , Animales , Astrocitos/efectos de los fármacos , Región CA1 Hipocampal/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Corteza Cerebral/patología , Regulación de la Expresión Génica/efectos de los fármacos , Gliosis/genética , Gliosis/patología , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/patología , Masculino , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/biosíntesis , Canales de Potasio de Dominio Poro en Tándem/genética , Quinina/farmacología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Factores de TiempoRESUMEN
TREK1 is a widely expressed background potassium channel. Similar to mice treated with selective serotonin reuptake inhibitors (SSRIs), TREK1 knockout mice are resistant to depression-like behavior and have elevated serotonin levels leading to speculation that TREK1 inhibition may contribute to the therapeutic effects of SSRIs. This study examined how chronic fluoxetine administration and a common functional polymorphism in the serotonin-transporter-linked promoter region (5-HTTLPR) influence cortical TREK1 expression in 24 rhesus monkeys. The short rh5-HTTLPR allele as well as female gender were associated with reduced cortical TREK1 protein expression but chronic SSRI administration had no effect. These results suggest that serotonin may influence TREK1, but that chronic SSRI treatment does not result in long lasting changes in cortical TREK1 protein expression. TREK1 gender differences may be related to gender differences in serotonin and require further research.