Estudio del efecto de dos modos de activación del receptor de melanocortinas tipo 4 sobre los canales de calcio operados por voltaje y su impacto sobre la actividad neuronal

Los canales de calcio operados por voltaje (CaV) se localizan a lo largo de toda la estructura de una neurona regulando la liberación de neurotransmisores, la expresión génica y el potencial de membrana, entre otras funciones. La actividad de los CaV es modulada por el voltaje, por la concentración del ion Ca+2 y por distintas proteínas, entre las que se destacan los receptores acoplados a proteína G (GPCR). En este trabajo de tesis estudiamos cómo un GPCR, el receptor de melanocortinas tipo 4 (MC4R), modula la actividad de distintos subtipos de CaV, y los posibles efectos en el control de la actividad neuronal. El MC4R se expresa en distintos núcleos del cerebro y cuando se activa por agonistas disminuye la ingesta de alimento, entre otras funciones, aunque los mecanismos moleculares que median su acción anorexigénica son aún desconocidos. El MC4R, presenta una característica peculiar conocida para algunos GPCRs: se encuentra activo en forma constitutiva aun en ausencia de sus agonistas. Pero una propiedad distintiva, conocida hasta el momento sólo para el sistema de melanocortinas, es que presenta un agonista inverso endógeno, la proteína relacionada a agouti (AgRP), que controla su actividad constitutiva. Además, AgRP funciona como antagonista del MC4R. Al estudiar el efecto de la actividad del MC4R sobre distintos subtipos de CaV, nosotros encontramos que la activación del MC4R evocada por agonista inhibe la ICa de los CaV2.2 en un sistema de expresión heteróloga y en neuronas de amígdala en cultivo. Esta inhibición señaliza a través de Gs y es independiente del voltaje. Encontramos que, a consecuencia de la inhibición de los CaV2.2 por la activación del MC4R, disminuye la liberación de GABA en neuronas de la amígdala en cultivo y podría ser relevante en circuitos GABAérgicos de la amígdala controlando el tono inhibitorio en la amígdala central. En contraste, observamos que la actividad constitutiva del MC4R inhibe las ICa de los CaV1.2, CaV1.3 y CaV2.1 en un sistema de expresión heteróloga y en neuronas de amígdala en cultivo. Esta inhibición es ocluida por AgRP a largo plazo, depende de la cantidad de MC4R expresado, señaliza por la proteína Gi/o y depende de la fosforilación de la proteína ERK1/2. Estudios preliminares indican que la inhibición de los CaV1.2/CaV1.3 podría disminuir la activación de la expresión génica de neuronas en cultivo, mientras que la inhibición de los CaV2.1 disminuiría la cantidad de este CaV en las sinapsis de neuronas en cultivos primarios de amígdala. La inhibición diferencial de los distintos subtipos de CaV por los dos modos de activación del MC4R contribuye a la regulación precisa de la actividad neuronal.Facultad de Ciencias Exacta

Impact of A118G polymorphism on the Mu opioid receptor function in pain

Mu Opioid Receptor (MOR) activation by exogenous or endogenous agonists causes reduction of pain threshold after a noxious stimulus, relieving pain sensation.MOR is encoded by OPRM1 gene and its messenger RNA suffers extensible modifications by alternative splicing and single nucleotide polymorphisms (SNPs). A118G (N40D) is the most frequent encoding MOR SNP in humans. In this review we discuss the impact of this polymorphism at molecular, cellular and clinical levels. Since some SNPs are unequally distributed among human populations, we also discuss the utility of A118G as an ethnicity marker among worldwide human populations. As an example, we evaluate A118G frequency in an Argentinean humanpopulation and compare it with worldwide frequencies extracted from HapMap database.Instituto Multidisciplinario de Biología Celula

Impact of A118G polymorphism on the Mu opioid receptor function in pain

Mu Opioid Receptor (MOR) activation by exogenous or endogenous agonists causes reduction of pain threshold after a noxious stimulus, relieving pain sensation.MOR is encoded by OPRM1 gene and its messenger RNA suffers extensible modifications by alternative splicing and single nucleotide polymorphisms (SNPs). A118G (N40D) is the most frequent encoding MOR SNP in humans. In this review we discuss the impact of this polymorphism at molecular, cellular and clinical levels. Since some SNPs are unequally distributed among human populations, we also discuss the utility of A118G as an ethnicity marker among worldwide human populations. As an example, we evaluate A118G frequency in an Argentinean humanpopulation and compare it with worldwide frequencies extracted from HapMap database.Instituto Multidisciplinario de Biología Celula

TRPV1 promotes opioid analgesia during inflammation

International audienc

Constitutive and Ghrelin-Dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest known constitutive activity of any G Protein-Coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin, and its activation increases transcriptional and electrical activity in hypothalamic neurons. Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear. The activities of the Voltage-Gated calcium channels, CaV2.1 and CaV2.2, which mediate neurotransmitter release at presynaptic terminals, are modulated by many GPCRs. Here, we show that both constitutive and Agonist-Dependent GHSR1a activity elicit a strong impairment of CaV2.1 and CaV2.2 currents in rat and mouse hypothalamic neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-Dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas Ghrelin-Dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-Dependent pathway. Thus, GHSR1a differentially inhibits CaV2 channels by Gi/o or Gq protein pathways depending on its mode of activation. Moreover, we present evidence suggesting that GHSR1a-Mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons.Instituto Multidisciplinario de Biología Celula

Constitutive and Ghrelin-Dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest known constitutive activity of any G Protein-Coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin, and its activation increases transcriptional and electrical activity in hypothalamic neurons. Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear. The activities of the Voltage-Gated calcium channels, CaV2.1 and CaV2.2, which mediate neurotransmitter release at presynaptic terminals, are modulated by many GPCRs. Here, we show that both constitutive and Agonist-Dependent GHSR1a activity elicit a strong impairment of CaV2.1 and CaV2.2 currents in rat and mouse hypothalamic neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-Dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas Ghrelin-Dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-Dependent pathway. Thus, GHSR1a differentially inhibits CaV2 channels by Gi/o or Gq protein pathways depending on its mode of activation. Moreover, we present evidence suggesting that GHSR1a-Mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons.Instituto Multidisciplinario de Biología Celula

A simple strategy for culturing morphologically-conserved rat hypothalamic tanycytes

Hypothalamic tanycytes are specialized bipolar ependymal cells that line the floor of the third ventricle. Given their strategic location, tanycytes are believed to play several key functions including being a selective barrier and controlling the amount of hypothalamic-derived factors reaching the anterior pituitary. The in vitro culture of these cells has proved to be difficult. Here, we report an improved method for the generation of primary cultures of rat hypothalamic tanycytes. Ependymal cultures were derived from tissue dissected out of the median eminence region of 10-day-old rats and cultured in a chemically defined medium containing DMEM:F12, serum albumin, insulin, transferrin and the antibiotic gentamycin. After 7 days in vitro, -30% of the cultured cells exhibited morphological features of tanycytes as observed by phase contrast or scanning electron microscopy. Tanycyte-like cells were strongly immuno-reactive for vimentin and dopamine-cAMP-regulated phospho-protein (DARPP-32) and weakly immune-reactive for glial fibrillary acidic protein. Tanycyte-like cells displayed a stable negative resting plasma membrane potential and failed to show spiking properties in response to current injections. When exposed to fluorescent beads in the culture medium, tanycyte-like cells exhibited a robust endocytosis. Thus, the present method effectively yields cultures containing tanycyte-like cells that resemble in vivo tanycytes in terms of morphologic features and molecular markers as well as electrical and endocytic activity. To our knowledge, this is the first protocol that allows the culturing of tanycyte-like cells that can be individually identified and that conserve the morphology of tanycytes in then- natural physiological environment.Instituto Multidisciplinario de Biología CelularComisión de Investigaciones Científicas de la provincia de Buenos Aire

Constitutive and Ghrelin-Dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest known constitutive activity of any G Protein-Coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin, and its activation increases transcriptional and electrical activity in hypothalamic neurons. Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear. The activities of the Voltage-Gated calcium channels, CaV2.1 and CaV2.2, which mediate neurotransmitter release at presynaptic terminals, are modulated by many GPCRs. Here, we show that both constitutive and Agonist-Dependent GHSR1a activity elicit a strong impairment of CaV2.1 and CaV2.2 currents in rat and mouse hypothalamic neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-Dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas Ghrelin-Dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-Dependent pathway. Thus, GHSR1a differentially inhibits CaV2 channels by Gi/o or Gq protein pathways depending on its mode of activation. Moreover, we present evidence suggesting that GHSR1a-Mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons.Instituto Multidisciplinario de Biología Celula

Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest constitutive activity of any G protein coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin and, its activation increases transcriptional and electrical activity in hypothalamic neurons. It is known that GHSR1a is present at some specific GABAergic presynaptic terminals; however, its impact on neurotransmitter release remains elusive. The voltage gated calcium channels, CaV2.1 and CaV2.2, control neurotransmitter release at presynaptic terminals and their activities are modulated by many GPCRs. Here we show that constitutive as well as agonist-dependent GHSR1a activation trigger a strong impairment of both CaV2.1 and CaV2.2 currents in rat and mouse neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-dependent mechanism that involves persistent reduction in channel density at plasma membrane, whereas, ghrelin-dependent GHSR1a inhibition is reversible and involves altered CaV2 current gating via a Gq-dependent pathway. Thus, we show that GHSR1a differentially inhibits CaV2 channels by Gi/o- or Gq-protein pathways depending on its activation mode. Moreover, we present evidence suggesting that GHSR1a-mediated inhibition of CaV2 impairs GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation by the disinhibition of postsynaptic neurons