Effect of calcium on nicotine-induced current expressed by an atypical alpha-bungarotoxin-insensitive nAChR2

Two distinct native alpha-bungarotoxin (alpha-Bgt)-insensitive nicotinic acetylcholine receptors (nAChRs), named nAChR1 and nAChR2, were identified in the cockroach Periplaneta americana dorsal unpaired median (DUM) neurons. They differed in their electrophysiological, pharmacological properties and intracellular regulation pathways. nAChR2 being an atypical nicotinic receptor closed upon agonist application and its current-voltage relationship resulted from a reduction in potassium conductance. In this study, using whole-cell patch-clamp technique, we demonstrated that calcium modulated nAChR2-mediated nicotine response. Under 0.5 microM alpha-Bgt and 20 mM d-tubocurarine, the nicotine-induced inward current amplitude was strongly reduced in the presence of intracellularly applied BAPTA or bath application of calcium-free solution. In addition, using cadmium chloride, we showed that nicotine response was modulated by extracellular calcium through plasma membrane calcium channels. Moreover, extracellular application of caffeine and thapsigargin reduced nAChR2-mediated response. Together these experiments revealed a complex calcium-dependent regulation of nAChR2

Ca2+ tunnelling through the ER lumen as a mechanism for delivering Ca2+ entering via store-operated Ca2+ channels to specific target sites

Ca2+ signaling is perhaps the most universal and versatile mechanism regulating a wide range of cellular processes. Because of the many different calcium-binding proteins distributed throughout cells, signaling precision requires localized rises in the cytosolic Ca2+ concentration. In electrically non-excitable cells, for example epithelial cells, this is achieved by primary release of Ca2+ from the endoplasmic reticulum via Ca2+ release channels placed close to the physiological target. Because any rise in the cytosolic Ca2+ concentration activates Ca2+ extrusion, and in order for cells not to run out of Ca2+, there is a need for compensatory Ca2+ uptake from the extracellular fluid. This Ca2+ uptake occurs through a process known as store-operated Ca2+ entry. Ideally Ca2+ entering the cell should not diffuse to the target site through the cytosol, as this would potentially activate undesirable processes. Ca2+ tunneling through the lumen of the endoplasmic reticulum is a mechanism for delivering Ca2+ entering via store-operated Ca2+ channels to specific target sites, and this process has been described in considerable detail in pancreatic acinar cells and oocytes. Here we review the most important evidence and present a generalized concept

Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons

Caractérisation des mécanismes de régulation intracellulaires des récepteurs de l'acétylcholine de type nicotinique résistants à l'a-bungarotoxine exprimés sur les dorsal unpaired median (DUM) neurones de la blatte Periplaneta americana

Les récepteurs de l'acétylcholine de type nicotinique (nAChRs) résistant à l'alpha-bungarotoxine exprimés sur les corps cellulaires de cellules neurosécrétrices (les DUM neurones) isolées du dernier ganglion abdominal de la blatte Periplaneta americana ont été étudiés au moyen des techniques de patch-clamp (configuration cellule entière) et de mesure de la concentration en calcium intracellulaire. Ces résultats mettent en évidence des caractéristiques entièrement nouvelles des nAChRs des insectes. Deux sous-types de récepteurs distincts (nAChR1 et nAChR2) ont été caractérisés. Ces deux nAChRs peuvent être séparés par leurs caractéristiques pharmacologiques, électrophysiologiques et par différents mécanismes de régulation intracellulaires (phosphorylation/déphosphorylation). La concentration en AMPc intracellulaire contrôle l'activité d'une protéine kinase (PKA) et d'une protéine phosphatase (PP1/2A) qui ont des effets antagonistes sur nAChR1. Le complexe calcium/calmoduline (CaM) module l'activité d'une adénylate cyclase et active une enzyme de type CaM kinase II qui potentialise la réponse de nAChR1 en partie par l'inhibition de la PP1/2A. Deux enzymes de la famille des PKC (PKC1 et PKC2) modulent nAChR1 de manière opposée et sont apparentées aux PKC " classiques " (PKC1) et " atypiques ". L'utilisation d'une sonde sensible au calcium, le fura 2, démontre que l'activation d'un récepteur de type muscarinique de sous-type M1 permet de d'augmenter la concentration en calcium intracellulaire et contrôle l'activation de ces PKC. Les effats d'un insecticide : l'imidaclopride, ont également été étudiés. Dans les conditions physiologiques, cette molécule active uniquement nAChR1 et son efficacité peut être modulée par ces processus intracellulaires de phosphorylation/déphosphorylation. L'insensibilité de nAChR2 vis-à-vis de l'imidaclopride s'explique par les caractéristiques fonctionnelles de ce récepteur. Le canal ionique de nAChR2 est perméable aux ions potassium et ouvert dans les conditions physiologiques, il se ferme en présence d'agoniste. Ces caractéristiques de nAChR2 suggèrent ainsi l'existence d'un nouveau type de récepteur ionotrope dont le canal ionique, ouvert au repos, est fermé lors de son activation.The a-bungarotoxin-resistant nicotinic acetylcholine receptors (nAChRs) expressed on the cell bodies of neurosecretory cells (DUM neurones) isolated from the terminal abdominal ganglia of the cockroach Periplaneta americana were studied using the patch-clamp technique (whole-cell recording configuration) and intracellular calcium imaging. These results demonstrate new characteristics of insect nAChRs.Two distinct nAChRs subtypes (nAChR1 et nAChR2) were characterised.These two receptors can be separated according to their pharmacological and electrophysiological properties and subtype-specific intracellular modulations (phosphorylation/dephosphorylation). First, the intracellular cAMP concentration controls both the activity of a protein kinase (PKA) and a protein phosphatase (PP1/2A) that modulate in opposite directions nAChR1. Second, the calcium/calmodulin complex (CaM) modulates adenylate cyclase and activates a CaM kinase II. This latter enzyme potentiates nAChR1 function partly through the inhibition of the PP1/2A. Third, we identified two distinct PKC that differentialy "up- and down-" regulate nAChR1 function (PKC1 and PKC2). These enzymes are related to the "classical" (PKC1) and "atypical (PKC2) PKC subtypes. Using the calcium-sensitive probe fura 2 we shown that the activation of M1 muscarinic receptor leads to an increase in intracellular calcium concentration and modulates PKC1 and PKC2 activities. The effects of the neonicotinoid insecticide imidacloprid were also studied. In physiological conditions, this compound only activates nAChR1 and its efficiency is affected by intracellular phosphorylation/dephosphorylation processes. The functional characteristics of nAChR2 can explain its insensivity to imidacloprid. The ionic channel of nAChR2 is permeable to potassium ions and open in physiological conditions. This channel closes following agonist application. nAChR2 might therefore represent a new kind of ionotropic receptor with an open channel in resting conditions that is closed when the receptor is activated.ANGERS-BU Lettres et Sciences (490072106) / SudocSudocFranceF

Les hémorragies maculaires pré-rétiniennes sous membranaires (aspect clinique, priseen charge en 2013 et étude de 5 patients traités précocement par laser Nd YAG)

Le diagnostic d'hemorragie pre-retinienne (sous membranaire) soit réalisable par un simple examen du fond de l œil, les progrès de l'imagerie retinienne et notamment l'imagerie en cohérence optique en domaine spectral, ont permis de distinguer deux localisations possibles de la collection sanguine: en position rétro-hyaloïienne, ou sous la membrane limitante interne. La simple surveillance a été pendant longtemps la seule attitude thérapeutique possible jusqu'à l'apparition des premières vitrectomies postérieures par la pars plana en 1970, basées sur les travaux de Machemer et Parel. Une alternative thérapeutique a pu être proposée dans le milieu des années 80, avec la démocratisation en ophtalmologie des lasers ND-YAG (Acronyme du nom anglais: Neodymium-doped Yttrium aluminium Garnet) et Argon. Actuellment, la membranotomie hyalloïdienne par laser trouve une place de plus en plus précoce, à condition que la collection sanguine soit de taille suffisanteAIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocSudocFranceF