Método y kit de diagnóstico para la determinación cuantitativa de la expresión del canal maxi-K

Método y kit de diagnóstico para la determinación cuantitativa de la expresión del canal maxi-K. La presente invención se refiere a un método y kit de diagnóstico para la determinación cuantitativa en leucocitos de sangre periférica de una muestra de sangre total de la expresión de los genes KCNM implicados en la hipertensión arterial humana. En concreto de la expresión, de los niveles de ARNm y/o proteínas, de las subunidades alfa y beta1 del canal iónico maxi-K, es decir, de los genes KCNMA1 y KCNMB1.Españ

Carotid body chemosensory responses in mice deficient of TASK channels

Background K+ channels of the TASK family are believed to participate in sensory transduction by chemoreceptor (glomus) cells of the carotid body (CB). However, studies on the systemic CB-mediated ventilatory response to hypoxia and hypercapnia in TASK1- and/or TASK3-deficient mice have yielded conflicting results. We have characterized the glomus cell phenotype of TASK-null mice and studied the responses of individual cells to hypoxia and other chemical stimuli. CB morphology and glomus cell size were normal in wild-type as well as in TASK1−/− or double TASK1/3−/− mice. Patch-clamped TASK1/3-null glomus cells had significantly higher membrane resistance and less hyperpolarized resting potential than their wild-type counterpart. These electrical parameters were practically normal in TASK1−/− cells. Sensitivity of background currents to changes of extracellular pH was drastically diminished in TASK1/3-null cells. In contrast with these observations, responsiveness to hypoxia or hypercapnia of either TASK1−/− or double TASK1/3−/− cells, as estimated by the amperometric measurement of catecholamine release, was apparently normal. TASK1/3 knockout cells showed an enhanced secretory rate in basal (normoxic) conditions compatible with their increased excitability. Responsiveness to hypoxia of TASK1/3-null cells was maintained after pharmacological blockade of maxi-K+ channels. These data in the TASK-null mouse model indicate that TASK3 channels contribute to the background K+ current in glomus cells and to their sensitivity to external pH. They also suggest that, although TASK1 channels might be dispensable for O2/CO2 sensing in mouse CB cells, TASK3 channels (or TASK1/3 heteromers) could mediate hypoxic depolarization of normal glomus cells. The ability of TASK1/3−/− glomus cells to maintain a powerful response to hypoxia even after blockade of maxi-K+ channels, suggests the existence of multiple sensor and/or effector mechanisms, which could confer upon the cells a high adaptability to maintain their chemosensory function

T-type Ca 2+ channels in mouse embryonic stem cells: Modulation during cell cycle and contribution to self-renewal

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and selfrenewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na + currents as well as transient Ca 2+ currents abolished by the external application of Ni 2+. Biophysical and pharmacological data indicated that the Ca 2+ current is predominantly mediated by T-type (Cav3 v2) channels. The number of cells expressing T-type channels and Cav3 v2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca 2+ currents with Ni 2+ induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Cav3 v2) Ca 2+ channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Cav3 v2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca 2+ entry mediated by Cav3 v2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal. © 2012 the American Physiological Society.Peer Reviewe

Procedimiento para detectar el precipitado de x-gal u otros precipitados opacos mediante microscopia confocal

La presente solicitud de patente se refiere a un procedimiento para la obtención de imágenes confocales de la tinción histológica Xgal, el cual se puede combinar con otros fluoróforos usados en inmunofluorescencia permitiendo por tanto el marcaje múltipl

Microscopía confocal de fluorescencia ex vivo en escala de tres colores (mcf-3cs): una nueva técnica de imagen

La microscopía confocal (MC) es una técnica de imagen que permite la visualización en tiempo real de estructuras de la piel, con una resolución comparable a la histología convencional1. Es una técnica que se ha aplicado sobre múltiples patologías dermatológicas, fundamentalmente sobre patología tumoral como el carcinoma basocelular (CB) o espinocelular. Esta tecnología puede aplicarse directamente sobre la piel (in vivo), o sobre piezas que se hayan extirpado quirúrgicamente (ex vivo). En este último caso, el uso de fluoróforos ha conseguido mejorar la calidad de las imágenes obtenidas, en lo que se conoce como MC de fluorescencia (MCF)

Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion.

Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin-deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events. Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin-deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells. These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin. Biotin is a water-soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin-deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin-deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin-deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however, they exhibit a marked decrease in the size of quantal catecholaminergic secretory events, which is not seen in AM cells. A similar differential secretory dysfunction is observed in CB cells treated with tetrabenazine, a selective inhibitor of the vesicular monoamine transporter 2 (VMAT2). VMAT2 is highly expressed in glomus cells (in comparison with VMAT1), and in biotin-deficient animals VMAT2 protein expression decreases in parallel with the decrease of biotin accumulated in CB cells. These data suggest that biotin has an essential role in the homeostasis of dopaminergic transmission modulating the transport and/or storage of transmitters within small secretory granules in glomus cells

Developmental change of T-type Ca2+ channel expression and its role in rat chromaffin cell responsiveness to acute hypoxia

Neonatal chromaffin cells of the adrenal medulla (AM) are intrinsic chemoreceptors that secrete catecholamines in response to hypoxia, thus contributing to fetal adaptation to extrauterine life. In most mammals studied, oxygen sensitivity of AM cells disappears a few days after birth, possibly due to innervation of the adrenal gland by the cholinergic fibres of the splanchnic nerve (∼postnatal day 7 in the rat). The mechanisms underlying these homeostatic changes in chromaffin cells are unknown. Low voltage-activated, T-type, Ca2+ channels regulate cell excitability and their expression is up-regulated by hypoxia. Hence, we hypothesized that these channels contribute to the developmental changes in the chemoreceptive properties of AM chromaffin cells. Using electrophysiological, immunocytochemical and molecular biology methodologies we show here that neonatal AM chromaffin cells express T-type Ca2+ channels (of α1H or Cav3.2 sub-type) and that the function of these channels is necessary for catecholamine release in response to acute hypoxia. T-type Ca2+ channel expression, as well as chromaffin cell responsiveness to hypoxia, decrease with postnatal maturation. Adult chromaffin cell sensitivity to hypoxia reappears after AM denervation in parallel with the recruitment of T-type Ca2+ channels. These observations indicate that T-type Ca2+ channels are essential for the acute response of chromaffin cells to hypoxia and help explain the disappearance of O2 sensitivity in adult AM chromaffin cells. Our results may also be relevant for understanding the pathogenesis of disorders associated with chronic hypoxia or maternal nicotine consumption