RESUMEN
The voltage-dependent potassium channel Kv1.3 participates in peripheral insulin sensitivity. Genetic ablation of Kv1.3 triggers resistance to diet-induced weight gain, thereby pointing to this protein as a pharmacological target for obesity and associated type II diabetes. However, this role is under intense debate because Kv1.3 expression in adipose tissue raises controversy. We demonstrated that Kv1.3 is expressed in white adipose tissue from humans and rodents. Moreover, other channels, such as Kv1.1, Kv1.2, Kv1.4 and especially Kv1.5, from the same Shaker family are also present. Although elevated insulin levels and adipogenesis remodel the Kv phenotype, which could lead to multiple heteromeric complexes, Kv1.3 markedly participates in the insulin-dependent regulation of glucose uptake in mature adipocytes. Adipocyte differentiation increased the expression of Kv1.3, which is targeted to caveolae by molecular interactions with caveolin 1. Using a caveolin 1-deficient 3T3-L1 adipocyte cell line, we demonstrated that the localization of Kv1.3 in caveolar raft structures is important for proper insulin signaling. Insulin-dependent phosphorylation of the channel occurs at the onset of insulin-mediated signaling. However, when Kv1.3 was spatially outside of these lipid microdomains, impaired phosphorylation was exhibited. Our data shed light on the putative role of Kv1.3 in weight gain and insulin-dependent responses contributing to knowledge about adipocyte physiology.
Asunto(s)
Adipocitos/metabolismo , Insulina/genética , Canal de Potasio Kv1.3/genética , Obesidad/genética , Células 3T3-L1 , Adipogénesis/genética , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Caveolas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Resistencia a la Insulina/genética , Canal de Potasio Kv1.3/metabolismo , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Obesidad/metabolismo , Obesidad/patologíaRESUMEN
Voltage-dependent K(+) channels (Kv) are involved in a number of physiological processes, including immunomodulation, cell volume regulation, apoptosis as well as differentiation. Some Kv channels participate in the proliferation and migration of normal and tumor cells, contributing to metastasis. Altered expression of Kv1.3 and Kv1.5 channels has been found in several types of tumors and cancer cells. In general, while the expression of Kv1.3 apparently exhibits no clear pattern, Kv1.5 is induced in many of the analyzed metastatic tissues. Interestingly, evidence indicates that Kv1.5 channel shows inversed correlation with malignancy in some gliomas and non-Hodgkin's lymphomas. However, Kv1.3 and Kv1.5 are similarly remodeled in some cancers. For instance, expression of Kv1.3 and Kv1.5 correlates with a certain grade of tumorigenicity in muscle sarcomas. Differential remodeling of Kv1.3 and Kv1.5 expression in human cancers may indicate their role in tumor growth and their importance as potential tumor markers. However, despite of this increasing body of information, which considers Kv1.3 and Kv1.5 as emerging tumoral markers, further research must be performed to reach any conclusion. In this review, we summarize what it has been lately documented about Kv1.3 and Kv1.5 channels in human cancer.
RESUMEN
Kv, which play a role in the immune system, are remodeled during carcinogenesis. Leukocytes present a limited Kv repertoire, with Kv1.3 and Kv1.5 as isoforms that are involved in neoplastic processes, such as proliferation and migration. In this study, we identified Kv1.5 in B-lymphocytes, characterized its role in proliferation and migration, and analyzed Kv1.3 and Kv1.5 expression in human non-Hodgkin lymphomas. DLBCL, F, MCL, ALCL, and T, along with control N specimens, were analyzed. Kv1.3 and Kv1.5 were found to be remodeled differentially; whereas Kv1.3 expression did not correlate with the state of dedifferentiation or the nature of lymphomatous cells, Kv1.5 abundance correlated inversely with clinical aggressiveness. Whereas indolent F expressed noticeable levels of Kv1.5, aggressive DLBCL showed low Kv1.5 levels. In addition, control LNs expressed heterogeneous high levels of Kv1.3, which could indicate some reactivity, whereas Kv1.5 abundance was low and quite homogeneous. Our data show that Kv1.5 is a determinant of human B cell proliferation and migration, thereby identifying this channel as a new target for immunomodulation. Our work also provides new insights into the use of Kv1.3 and Kv1.5 as potential targets during tumorigenesis.
Asunto(s)
Linfocitos B/fisiología , Canal de Potasio Kv1.5/metabolismo , Linfoma/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Inmunohistoquímica , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.5/genética , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Linfoma/genética , Linfoma/patología , Ratones , Persona de Mediana Edad , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adulto JovenRESUMEN
Glioma-initiating cells (GICs) represent a potential important therapeutic target because they are likely to account for the frequent recurrence of malignant gliomas; however, their identity remains unsolved. Here, we characterized the cellular lineage fingerprint of GICs through a combination of electrophysiology, lineage marker expression, and differentiation assays of 5 human patient-derived primary GIC lines. Most GICs coexpressed nestin, NG2 proteoglycan, platelet-derived growth factor receptor-α, and glial fibrillary acidic protein. Glioma-initiating cells could be partially differentiated into astrocytic but not oligodendroglial or neural lineages. We also demonstrate that GICs have a characteristic electrophysiologic profile distinct from that of well-characterized tumor bulk cells. Together, our results suggest that GICs represent a unique type of cells reminiscent of an immature phenotype that closely resembles but is not identical to NG2 glia with respect to marker expression and functional membrane properties.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Glioma/genética , Glioma/patología , Inmunofenotipificación , Neuroglía/fisiología , Animales , Neoplasias Encefálicas/química , Línea Celular Tumoral , Células Cultivadas , Glioma/química , Humanos , Potenciales de la Membrana/genética , Ratones , Neuroglía/patología , Células Tumorales CultivadasRESUMEN
Voltage-dependent K+ channels (Kv) are involved in the proliferation and differentiation of mammalian cells, since Kv antagonists impair cell cycle progression. Although myofibers are terminally differentiated, some myoblasts may re-enter the cell cycle and proliferate. Since Kv1.3 and Kv1.5 expression is remodeled during tumorigenesis and is involved in smooth muscle proliferation, the purpose of this study was to analyze the expression of Kv1.3 and Kv1.5 in smooth muscle neoplasms. In the present study, we examined human samples of smooth muscle tumors together with healthy specimens. Thus, leiomyoma (LM) and leiomyosarcoma (LMS) tumors were analyzed. Results showed that Kv1.3 was poorly expressed in the healthy muscle and indolent LM specimens, whereas aggressive LMS showed high levels of Kv1.3 expression. Kv1.5 staining was correlated with malignancy. The findings show a remodeling of Kv1.3 and Kv1.5 in human smooth muscle sarcoma. A correlation of Kv1.3 and Kv1.5 expression with tumor aggressiveness was observed. Thus, our results indicate Kv1.5 and Kv1.3 as potential tumorigenic targets for aggressive human LMS.
RESUMEN
Because Kv1.3 and Kv1.5 K(+) channels are remodeled during tumorigenesis and participate in skeletal muscle proliferation, we analyzed their expression in human skeletal muscle sarcomas. Aggressive alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) were studied. Kv1.5 expression was moderate in adult muscle and low in ERMS, whereas it was notable in ARMS and embryonic samples. Kv1.3 expression showed no major differences between RMS and healthy samples. We found a correlation of Kv1.3 and Kv1.5 expression with the tumor malignancy.
Asunto(s)
Canal de Potasio Kv1.3/análisis , Canal de Potasio Kv1.5/análisis , Músculo Esquelético/patología , Rabdomiosarcoma/metabolismo , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Fase G1 , Humanos , Inmunohistoquímica , Canal de Potasio Kv1.3/fisiología , Canal de Potasio Kv1.5/fisiología , Masculino , Persona de Mediana Edad , Rabdomiosarcoma/patologíaRESUMEN
Voltage-dependent K(+) channels (Kv) control repolarization and membrane potential in electrically excitable cells. In addition, Kv channels are involved in the maintenance of vascular smooth muscle tone, insulin release, epithelial K(+) transport, cell proliferation and leukocyte activation. Kv1.3 and Kv1.5 are widely distributed throughout the body and are involved in a variety of physiological processes taking place in the immune system, brain and muscle. Since the developmental pattern of Kv channels has an essential role in the maturing human, we aimed to study Kv1.3 and Kv1.5 channels in 8-10 week human fetal tissues. We chose that gestational age because all organs are in place and the nervous system, although not fully developed. However, the human embryo is undergoing major changes, which will lead to a defined adult pattern. Our results indicated that numerous tissues expressed Kv1.3 and Kv1.5. While Kv1.3 overlapped with the central and peripheral nervous systems, Kv1.5 was mostly localized in the central nervous system. In addition, both channels were abundantly expressed in the hematopoietic fetal liver. Finally, Kv1.5 heavily stained skeletal muscle and heart, whereas Kv1.3 was slightly present. This is the first study to analyze Kv1.3 and Kv1.5 in human during the beginning of fetal development.
Asunto(s)
Feto/metabolismo , Canal de Potasio Kv1.3/análisis , Canal de Potasio Kv1.5/análisis , Embrión de Mamíferos/metabolismo , Humanos , Inmunohistoquímica , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.5/metabolismoRESUMEN
The KCNQ1 (Kv7.1) channel plays an important role in cardiovascular physiology. Cardiomyocytes co-express KCNQ1 with KCNE1-5 proteins. KCNQ1 may co-associate with multiple KCNE regulatory subunits to generate different biophysically and pharmacologically distinct channels. Increasing evidence indicates that the location and targeting of channels are important determinants of their function. In this context, the presence of K(+) channels in sphingolipid-cholesterol-enriched membrane microdomains (lipid rafts) is under investigation. Lipid rafts are important for cardiovascular functioning. We aimed to determine whether KCNE subunits modify the localization and targeting of KCNQ1 channels in lipid rafts microdomains. HEK-293 cells were transiently transfected with KCNQ1 and KCNE1-5, and their traffic and presence in lipid rafts were analyzed. Only KCNQ1 and KCNE3, when expressed alone, co-localized in raft fractions. In addition, while KCNE2 and KCNE5 notably stained the cell surface, KCNQ1 and the rest of the KCNEs showed strong intracellular retention. KCNQ1 targets multiple membrane surface microdomains upon association with KCNE peptides. Thus, while KCNQ1/KCNE1 and KCNQ1/KCNE2 channels target lipid rafts, KCNQ1 associated with KCNE3-5 did not. Channel membrane dynamics, analyzed by fluorescence recovery after photobleaching (FRAP) experiments, further supported these results. In conclusion, the trafficking and targeting pattern of KCNQ1 can be influenced by its association with KCNEs. Since KCNQ1 is crucial for cardiovascular physiology, the temporal and spatial regulations that different KCNE subunits may confer to the channels could have a dramatic impact on membrane electrical activity and putative endocrine regulation.
Asunto(s)
Canal de Potasio KCNQ1/metabolismo , Microdominios de Membrana/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Línea Celular , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Canal de Potasio KCNQ1/genética , Cinética , Microscopía Confocal , Canales de Potasio con Entrada de Voltaje/genética , Subunidades de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Kv1.3 plays a crucial role in the activation and proliferation of T-lymphocytes and macrophages. While Kv1.3 is responsible for the voltage-dependent potassium current in T-cells, in macrophages this K(+) current is generated by the association of Kv1.3 and Kv1.5. Patients with autoimmune diseases show a high number of effector memory T cells that are characterized by a high expression of Kv1.3 and Kv1.3 antagonists ameliorate autoimmune disorders in vivo. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) used in patients who suffer from painful autoimmune diseases such as rheumatoid arthritis. In this study, we show that diclofenac impairs immune response via a mechanism that involves Kv1.3. While diclofenac inhibited Kv1.3 expression in activated macrophages and T-lymphocytes, Kv1.5 remained unaffected. Diclofenac also decreased iNOS levels in Raw 264.7 cells, impairing their activation in response to lipopolysaccharide (LPS). LPS-induced macrophage migration and IL-2 production in stimulated Jurkat T-cells were also blocked by pharmacological doses of diclofenac. These effects were mimicked by Margatoxin, a specific Kv1.3 inhibitor, and Charybdotoxin, which blocks both Kv1.3 and Ca(2+)-activated K(+) channels (K(Ca)3.1). Because Kv1.3 is a very good target for autoimmune therapies, the effects of diclofenac on Kv1.3 are of high pharmacological relevance.
Asunto(s)
Diclofenaco/farmacología , Sistemas de Liberación de Medicamentos/métodos , Factores Inmunológicos/farmacología , Canal de Potasio Kv1.3/metabolismo , Leucocitos/inmunología , Animales , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Inhibición de Migración Celular/efectos de los fármacos , Inhibición de Migración Celular/inmunología , Células Cultivadas , Humanos , Células Jurkat , Canal de Potasio Kv1.3/antagonistas & inhibidores , Leucocitos/efectos de los fármacos , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/patología , RatonesRESUMEN
Voltage-dependent potassium (K(v)) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of K(v) channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K(+) currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric K(v)1.3/K(v)1.5 channels, which generate the main K(v) in macrophages. An increase in K(+) current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in K(v)1.3 subunits in the K(v)1.3/K(v)1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K(v)1.3 expression. Neither of these treatments apparently altered the expression of K(v)1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K(v)1.3/K(v)1.5 hybrid channels by altering the subunit stoichiometry.
Asunto(s)
Inmunomodulación , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.5/metabolismo , Macrófagos/inmunología , Animales , Línea Celular , Dexametasona/farmacología , Humanos , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.5/antagonistas & inhibidores , Lipopolisacáridos/inmunología , Macrófagos/efectos de los fármacos , Potenciales de la Membrana , Ratones , Ratas , Venenos de Escorpión/farmacologíaRESUMEN
Modulation of the expression and activity of plasma membrane ion channels is one of the mechanisms by which immune cells can regulate their intracellular Ca(2+) signaling pathways required for proliferation and/or differentiation. Voltage-gated K+ channels, inwardly rectifying K+ channels, and Ca(2+)-activated K+ channels have been described to play a major role in controlling the membrane potential in lymphocytes and professional APCs, such as monocytes, macrophages, and dendritic cells (DCs). Our study aimed at the characterization and identification of ion channels expressed in the course of human DC differentiation from monocytes. We report in this study for the first time that immature monocyte-derived DCs express voltage-gated Na+ channels in their plasma membrane. The analysis of the biophysical and pharmacological properties of the current and PCR-based cloning revealed the presence of Nav1.7 channels in immature DCs. Transition from the immature to a mature differentiation state, however, was accompanied by the down-regulation of Nav1.7 expression concomitant with the up-regulation of voltage-gated Kv1.3 K+ channel expression. The presence of Kv1.3 channels seems to be common for immune cells; hence, selective Kv1.3 blockers may emerge as candidates for inhibiting various functions of mature DCs that involve their migratory, cytokine-secreting, and T cell-activating potential.
Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Regulación del Desarrollo de la Expresión Génica/inmunología , Canal de Potasio Kv1.3/biosíntesis , Canales de Sodio/biosíntesis , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Células Dendríticas/citología , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Humanos , Inmunofenotipificación , Activación del Canal Iónico/genética , Activación del Canal Iónico/inmunología , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/genética , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7 , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/genética , Canales de Sodio/metabolismo , Tetrodotoxina/farmacología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
PURPOSE: Tumor hypoxia has long been known to produce resistance to radiation. In this study, electron paramagnetic resonance (EPR) oxygen imaging was investigated for its power to predict the success of tumor control according to tumor oxygenation level and radiation dose. METHODS AND MATERIALS: A total of 34 EPR oxygen images were obtained from the legs of C3H mice bearing 0.5-cm(3) FSa fibrosarcomas under both normal (air breathing) and clamped tumor conditions. Under the same conditions as those during which the images were obtained, the tumors were irradiated to a variety of doses near the FSa dose at which 50% of tumors were cured. Tumor tissue was distinguished from normal tissue using co-registration of the EPR oxygen images with spin-echo magnetic resonance imaging of the tumor and/or stereotactic localization. The tumor voxel statistics in the EPR oxygen image included the mean and median partial pressure of oxygen and the fraction of tumor voxels below the specified partial pressure of oxygen values of 3, 6, and 10 mm Hg. Bivariate logistic regression analysis using the radiation dose and each of the EPR oxygen image statistics to determine which best separated treatment failure from success. RESULTS: The measurements of the dose at which 50% of tumors were cured were similar to those found in published data for this syngeneic tumor. Bivariate analysis of 34 tumors demonstrated that tumor cure correlated with dose (p = 0.004) and with a <10 mm Hg hypoxic fraction (p = 0.023). CONCLUSION: Our results have shown that, together, radiation dose and EPR image hypoxic fraction separate the population of FSa fibrosarcomas that are cured from those that fail, thus predicting curability.
Asunto(s)
Hipoxia de la Célula , Fibrosarcoma/metabolismo , Fibrosarcoma/radioterapia , Oxígeno/análisis , Tolerancia a Radiación/fisiología , Animales , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Fibrosarcoma/fisiopatología , Ratones , Ratones Endogámicos C3H , Consumo de Oxígeno/fisiología , Presión Parcial , Radioterapia/métodos , Dosificación Radioterapéutica , Inducción de RemisiónRESUMEN
Tgalphaq44 mice with targeted overexpression of activated Galphaq protein in cardiomyocytes mimic many of the phenotypic characteristics of dilated cardiomyopathy in humans. However, it is not known whether the phenotype of Tgalphaq44 mice would also involve dysfunction of cardiac mitochondria. The aim of the present work was to examine changes in EPR signals of semiquinones and iron in Fe-S clusters, as compared to classical biochemical indices of mitochondrial function in hearts from Tgalphaq44 mice in relation to the progression of heart failure. Tgalphaq44 mice at the age of 14 months displayed pulmonary congestion, increased heart/body ratio and impairment of cardiac function as measured in vivo by MRI. However, in hearts from Tgalphaq44 mice already at the age of 10 months EPR signals of semiquinones, as well as cyt c oxidase activity were decreased, suggesting alterations in mitochondrial electron flow. Furthermore, in 14-months old Tgalphaq44 mice loss of iron in Fe-S clusters, impaired citrate synthase activity, and altered mitochondrial ultrastructure were observed, supporting mitochondrial dysfunction in Tgalphaq44 mice. In conclusion, the assessment of semiquinones content and Fe(III) analysis by EPR represents a rational approach to detect dysfunction of cardiac mitochondria. Decreased contents of semiquinones detected by EPR and a parallel decrease in cyt c oxidase activity occurs before hemodynamic decompensation of heart failure in Tgalphaq44 mice suggesting that alterations in function of cardiac mitochondria contribute to the development of the overt heart failure in this model.
Asunto(s)
Cardiomiopatía Dilatada/patología , Espectroscopía de Resonancia por Spin del Electrón , Mitocondrias Cardíacas/química , Mitocondrias Cardíacas/patología , Animales , Cardiomiopatía Dilatada/metabolismo , Modelos Animales de Enfermedad , Hierro/análisis , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mitocondrias Cardíacas/metabolismo , Quinonas/análisisRESUMEN
Spin trapping/electron paramagnetic resonance (EPR) spectroscopy allows specific detection of nitric oxide (NO) generation, in vivo. However, in order to detect an EPR signal in living organism, usually a stimulation of immune system with LPS is used to achieve higher than physiological NO levels. Here, we report non-invasive spin trapping of NO in tumors of non-treated, living animals. EPR spectroscopy was performed at S-band to detect NO in Cloudman S91 melanoma tumors growing in the tail of living, syngeneic hosts-DBA/2 mice. Iron (II) N-(dithiocarboxy)sarcosine Fe2+(DTCS)(2) was used as the spin trap. The results were confirmed by X-band ex vivo study. A characteristic three-line spectrum of NO-Fe(DTCS)(2) (A(N)=13 G) was observed (n=4, out of total n=6) in non-treated tumors and in tumors of animals treated with l-arginine. Substrate availability did not limit the detection of NO by spin trapping. Half-life time of the NO-Fe(DTCS)(2) in tumor tissue was about 60 min. The feasibility of non-invasive spin trapping/EPR spectroscopic detection of NO generated in tumor tissue in living animals, without additional activation of the immune system, was demonstrated for the first time.