Las determinaciones de glutamato utilizando Amplex Red Glutamic Acid Assay están afectadas por el agonista P2X BzATP

Amplex Red (AR) reagent is the most stable and sensitive fluorogenic substrate known for horseradish peroxidase (HRP). Thus, in the presence of hydrogen peroxide (H2O2), AR (a nonfluorescent compound) is converted to resorufin (a strongly fluorescing product) by the action of HRP. In the last years, multiple assays have been developed using this reagent to quantify a diverse assortment of analyses and also to detect the activity of many different enzymes. We recently showed that BzATP, agonist of several ionotropic nucleotide receptors (P2X), interfered with Amplex Red oxidation catalyzed by HRP. In the present work we reported that glutamate determinations using Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit are also strongly interfered by BzATP. These data are really interesting because demonstrate that resorufin fluorescence, in the presence of BzATP, is not proportional to glutamate concentration and, therefore, it must not be used as method to measure glutamate concentration.ÿEl compuesto Amplex Red (AR) es el sustrato fluorogénico para la peroxidasa de rábano (HRP) más estable y sensible. Así, en presencia de peróxido de hidrógeno (H2O2), el AR (un compuesto no fluorescente) es convertido en resorufina (un producto fuertemente fluorescente) por la acción de la HRP. En los últimos años se han realizado muchos ensayos utilizando este compuesto para cuantificar un variado surtido de análisis, así como para detectar la actividad de muchas y variadas enzimas. Recientemente hemos demostrado que el BzATP, un agonista de varios receptores ionotrópicos de nucleótidos (P2X), interfiere con la oxidación del Amplex Red catalizada por la HRP. En el presente trabajo mostramos que las determinaciones de glutamato utilizando el kit Amplex Red Glutamic Acid/Glutamate Oxidase Assay están fuertemente interferidas por el BzATP. Estos datos son realmente interesantes porque demuestran que la fluorescencia de la resorufina, en presencia del BzATP, no es proporcional a la concentración de glutamato y, por lo tanto, no debe ser utilizada como un método para medir la concentración de glutamato

Cultural competence as a dimension of professional identity: A qualitative study with teachers and healthcare professionals

Las competencias culturales (CC) son habilidades profesionales esenciales en comunidades diversas, especialmente en el ámbito educativo y sanitario. Diversos programas de formación en CC han mostrado resultados variados. En este artículo se defiende que las CC deben integrarse en la identidad profesional para que sean eficaces. Analizamos las distintas formas de integración de las CC en seis profesionales de un barrio multicultural mediante una técnica cualitativa de análisis de posicionamientos sociales. Se entrevistó tres profesores de primaria y tres profesionales de la salud de un centro de atención primaria del mismo barrio. Los resultados mostraron tres formatos de integración de las CC en las identidades profesionales como: Competencia cultural crítica, como igualitarismo cultural y asistencialismo, y como conocimiento cultural relacionado con la práctica profesional.Cultural Competences (CC) are key professional skills in heterogeneous communities, particularly in the educational and healthcare fields. A diversity of educational programs in CC have resulted in various degrees of success. In this article we pose that CCs should become part of a professional identity in order to be efficacious. We analyze the various ways in which CCs may be integrated into six professionals in a multicultural neighborhood using the qualitative analysis technique of social positioning. We interviewed three primary school teachers and three healthcare professionals that work in a primary healthcare unit in the same neighborhood. Our results reveal the existence of three CC integration formats in professional identities: as a key cultural competence; as cultural egalitarianism and care-giving, and as cultural awareness as it pertains to the professional practice.Ministerio de Ciencia e Innovación del Gobierno de España PSI2011 -2555

Caracterización farmacológica y análisis funcional de los receptores nicotínicos α9α10 en células cromafines aisladas de la médula adrenal de rata

The identification of acetylcholine nicotinic receptors (nAChRs) formed by α9 and α10 subunits in the sensory cells of the vestibular and auditory systems, prompted us to investigate their presence in adrenomedullary chromaffin cells, in which they could mediate membrane hyperpolarization through the activation of small-conductance Ca2+-dependent K+ channels (SK channels). The aim of the current study has been to pharmacologically identify α9α10 nAChRs and initiate their functional characterization in isolated chromaffin cells from the rat adrenal medulla. We have employed the patch clamp technique to record either the ionic currents generated by the activation of nAChRs or the associated changes in membrane potential. We took advantage of the specificity of α-conotoxin RgIA for the nAChRs formed by α9 and α10 subunits. Our pharmacological results suggest that the rat chromaffin cells express functional α9α10 nAChRs that would influence the electrical behaviour of these cells.El descubrimiento de receptores nicotínicos (nAChRs) formados por las subunidades α9 y α10 en las células de los epitelios sensitivos del sistema vestibular y auditivo ha motivado su búsqueda en estructuras del sistema nervioso autónomo, como las células cromafines de la médula adrenal, en las que podrían inducir la hiperpolarización de la membrana celular mediante la activación de canales de K+ dependientes de Ca2+ de baja conductancia iónica (canales SK). El objetivo fundamental de nuestro trabajo ha sido determinar la presencia y, en su caso, caracterizar funcionalmente el nAChR α9α10 en las células cromafines de la médula adrenal de la rata. Con ese propósito, hemos empleado cultivos primarios de células cromafines obtenidas de la glándula adrenal de rata y recurrido a la técnica electrofisiológica de patch-clamp para registrar las corrientes iónicas generadas por la activación de los nAChRs del conjunto de la membrana celular. Asimismo, hemos empleado la α-conotoxina RgIA, un péptido capaz de bloquear de forma selectiva los nAChRs formados por las subunidades α9 y α10. Los resultados obtenidos aportan evidencias farmacológicas que permiten concluir que las células cromafines de la rata expresan nAChRs α9α10 funcionales que, además, desempeñarían un papel modulador comportamiento eléctrico de dichas células

Immunolocalisation of P2Y receptors in the rat eye

Nucleotides present an important role in ocular physiology which has been demonstrated by recent works that indicate their involvement in many ocular processes. P2Y are important among P2 receptors since they can control tear production, corneal wound healing, aqueous humour dynamics and retinal physiology. Commercial antibodies have allowed us to investigate the distribution of P2Y receptors in the cornea, anterior and posterior chamber of the eye and retina. The P2Y1 receptor was present mainly in cornea, ciliary processes, and trabecular meshwork. The P2Y2 receptors were present in cornea, ciliary processes and retinal pigmented epithelium. P2Y4 was present in cornea, ciliary processes, photoreceptors, outer plexiform layer and ganglion cell layer. The P2Y6 presented almost an identical distribution as the P2Y4 receptor. The P2Y11 was also detectable in the retinal pigmented epithelium. The detailed distribution of the receptors clearly supports the recent findings indicating the relevant role of nucleotides in the ocular function

Papel fisiológico de los nucleótidos extracelulares en el sistema nervioso central: señalización vía receptores P2X y P2Y

In the last few years nucleotide receptors, the ionotropic P2X1-7 subunits and the metabotropic P2Y1, 2, 4, 6, 11, 12, 13, 14, have acquired an excepcional importance due to their strategic location in organs and tissues, their great variety along with the complexity of the associated signalling pathways and the first evidence of the serious alterations entailed in their dysfunctions. Our group has been pioneer in the characterization of these receptors in the nervous system, where we defined their location and functionality. The abundant presence, at a presynaptic level, of P2X3 and P2X7 should be emphasized, where by means of calcium intake they induce neurotransmitter exocytosis, such as glutamate, GABA, catecholamines and acetylcholine among others, as described in previous works by our group. In addition, they induce an extensive remodeling of the terminal’s cytoskeleton and exocytotic mechanisms through CaMKII and they can interact widely with other ionotropic and metabotropic receptors co-existing in nearby areas. Neural cells also exhibit the presence of most P2Y receptors signalling through a large variety of intracellular cascades. Recently we have demostrated that P2Y metabotropic receptors of the sub-family activated by ADP, especially P2Y13, are connected with the signalling towards GSK3 and â-catenin, opening new ways of understading the nucleotide function in survival and maintenance of neural cells. In addition both P2X and P2Y receptors play a role in early developmental stages and neural maturation where their function has to be fully understanded. Nucleotide receptors are also very abundant in glial cells, and our group has shown that most P2Y receptors are present and fully functional in cultured astrocytes, where, depending on the subtype receptor they activate a large variety of signalling cascades.En los ultimos anos los receptores de nucleotidos, receptores ionotropicos P2X1-7 y metabotropicos P2Y1, 2, 4, 6, 11, 12, 13, 14, han adquirido una importancia excepcional debido a su localizacion estrategica en organos y tejidos, a su gran variedad junto con la complejidad de vias de senalizacion a las que estan asociados y a las primeras evidencias de importantes alteraciones debidas a su mal funcionamiento. Nuestro grupo ha sido pionero en la caracterizacion estos receptores en el sistema nervioso, donde definimos su localizacion y su funcionalidad. La abundante presencia, a nivel presinaptico, de las subunidades P2X3 y P2X7 debe ser resaltada, donde gracias a la entrada de calcio inducen la exocitosis de varios neurotransmisores, como glutamato, GABA, catecolaminas y acetilcolina entre otros, como ha sido descrito por nuestro grupo en trabajos previos. Ademas, estos receptores inducen una profunda remodelacion del citoesqueleto de las terminales nerviosas y de los mecanismos exocitoticos a traves de la CaMKII y pueden interactuar con otros receptores ionotropicos y metabotropicos co-existentes en sus cercanias. La mayoria de los receptores P2Y tambien estan presentes en las celulas nerviosas, activando vias de senalizacion a traves de una gran variedad de cascadas intracelulares. Recientemente hemos demostrado que los receptores metabotropicos P2Y pertenecientes a la sub-familia de receptores activados por ADP, especialmente el P2Y13, estan conectados con la senalizacion hacia GSK3 y ƒÀ-catenina, lo que abre nuevas vias para la comprension de la funcion de los nucleotidos en la supervivencia y el mantenimiento de las celulas nerviosas. Ademas, tanto los receptores P2X como los P2Y juegan un papel en los estadios iniciales del desarrollo y en la maduracion neuronal donde su funcion aun ha de ser plenamente comprendida. Los receptores de nucleotidos son tambien muy abundantes en las celulas gliales, y nuestro grupo ha demostrado que la mayoría de los receptores P2Y están presentes y son plenamente funcionales en astrocitos en cultivo, donde, dependiendo del subtipo de receptor, activan una gran variedad de cascadas de señalización

Farmapas: mapas conceptuales en Farmacología

El uso de mapas conceptuales tiene como objetivo conseguir, con el apoyo de los profesores-tutores, que los estudiantes de Veterinaria lleguen a alcanzar conocimientos significativos y perdurables de los contenidos de una asignatura de notoria complejidad como es la Farmacología, Farmacia y Terapéutica. Los alumnos inscritos voluntariamente (n= 11) en el Seminario desarrollado durante el curso 2011/2012, dentro de las actividades propuestas por el Aula Virtual de Farmacología en el Campus Virtual de la UCM, participaron activamente en el desarrollo de las siguientes actividades. En primer lugar, un seminario o sesión tutorial de adiestramiento en las técnicas de elaboración de mapas conceptuales. A continuación, se formaron grupos de estudiantes que, con el uso de esta herramienta de estudio, desarrollaron mapas temáticos sobre las distintas secciones que conforman el programa de la asignatura. Estos mapas fueron expuestos y discutidos en reuniones de todos los miembros para, posteriormente, incorporarlos a otro mapa conceptual integrador de toda la Farmacología General. Por último, se ha efectuado un análisis crítico del interés, utilización y ayuda, que este tipo de metodología ha supuesto para los estudiantes y su repercusión en el rendimiento académico. De los resultados obtenidos, se deduce que la instauración de este tipo de técnicas de enseñanza-aprendizaje mejora la capacidad de razonamiento de los estudiantes, promueve en éstos la adquisición de aprendizaje significativo y colaborativo y facilita la construcción de una estructura de conocimientos perdurable en el tiempo, que les ayudará en el desarrollo de su futuro profesional

Brain-derived neurotrophic factor and the course of experimental cerebral malaria

The role of neurotrophic factors on the integrity of the central nervous system (CNS) during cerebral malaria (CM) infection remains obscure, but the long-standing neurocognitive sequelae often observed in rescued children can be attributed in part to the modulation of neuronal survival and synaptic plasticity. To discriminate the contribution of key responses in the time-sequence of the pathogenic events that trigger the development of neurocognitive malaria syndrome we defined four stages (I–IV) of the neurological progression of CM in C57BL/6 mice infected with Plasmodium berghei ANKA. Upregulation of ICAM-1, VCAM-1, e-selectin and p-selectin expression was detected in all cerebral regions before parasitized red blood cells (pRBC) accumulation. As the severity of symptoms increased, BDNF mRNA progressively diminished in several brain regions, earliest in the thalamus–hypothalamus, cerebellum, brainstem and cortex, and correlated with a four-stage disease sequence. Immunohistochemical confocal microscopy revealed changes in the BDNF distribution pattern, suggesting altered axonal transport. During CM progression, molecular markers of neurological infection and inflammation in the parasite and the host, respectively, were accompanied by a switch in the brain constitutive proteasome to the immunoproteasome, which could impede normal protein turnover. In parallel with BDNF downregulation, NCAM expression also diminished with increased CM severity. Together, these data suggest that changes in BDNF availability could be involved in the pathogenesis of CM.Depto. de Bioquímica y Biología MolecularFac. de VeterinariaFALSEpu

Altered Nucleotide Receptor Expression in a Murine Model of Cerebral Malaria

In cerebral malaria, the most severe complication of malaria, both neurotransmission mechanisms and energy metabolism are affected. To understand how metabolic changes modify neurotransmission, we examined P2 receptor expression in a murine model of cerebral malaria. Quantitative polymerase chain reaction experiments revealed that parasite deposition was greatest in the cerebellum, compared with other areas of the brain, suggesting a correlation between brain parasitemia and loss of control of movement. Infected mice showed modified patterns of expression of P2 receptor subtype messenger RNA (mRNA), depending on both the specific purinergic receptor and the cerebral region analyzed. Immunohistochemical studies indicated altered levels of protein expression by these receptors in infected brains and, in some cases, a pattern of expression different from that noted in control mice. These differences in both the amount of mRNA and the protein distribution of P2 receptors observed in the different brain sites in infected mice suggest an important role for P2 receptors in either provoking cerebral damage or conferring neuroprotection.Depto. de Bioquímica y Biología MolecularFac. de VeterinariaFALSEpu

P2X7 Receptors Trigger ATP Exocytosis and Modify Secretory Vesicle Dynamics in Neuroblastoma Cells*

Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca2+-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca2+ concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca2+ and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation