Neurocentre Magendie


14 publication(s) depuis Janvier 2006:

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03/2017 | Glia   IF 6
Astrocytic IP3 Rs: Contribution to Ca2+ signalling and hippocampal LTP.
Sherwood MW, Arizono M, Hisatsune C, Bannai H, Ebisui E, Sherwood JL, Panatier A, Oliet SH, Mikoshiba K

Astrocytes regulate hippocampal synaptic plasticity by the Ca2+ dependent release of the N-methyl d-aspartate receptor (NMDAR) co-agonist d-serine. Previous evidence indicated that d-serine release would be regulated by the intracellular Ca2+ release channel IP3 receptor (IP3 R), however, genetic deletion of IP3 R2, the putative astrocytic IP3 R subtype, had no impact on synaptic plasticity or transmission. Although IP3 R2 is widely believed to be the only functional IP3 R in astrocytes, three IP3 R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP3 R and the contribution of the three IP3 R subtypes to Ca2+ signalling. As a proxy for gliotransmission, we found that long-term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP3 R blocker heparin, and rescued by exogenous d-serine, indicating that astrocytic IP3 Rs regulate d-serine release. To explore which IP3 R subtypes are functional in astrocytes, we used pharmacology and two-photon Ca2+ imaging of hippocampal slices from transgenic mice (IP3 R2-/- and IP3 R2-/- ;3-/- ). This approach revealed that underneath IP3 R2-mediated global Ca2+ events are an overlooked class of IP3 R-mediated local events, occurring in astroglial processes. Notably, multiple IP3 Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca2+ release by astrocytic IP3 Rs. GLIA 2017;65:502-513.

16/12/2015 | Cell Rep   IF 7.9
Bidirectional Control of Synaptic GABAR Clustering by Glutamate and Calcium.
Bannai H, Niwa F, Sherwood MW, Shrivastava AN, Arizono M, Miyamoto A, Sugiura K, Levi S, Triller A, Mikoshiba K

GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABAA receptor (GABAAR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3 receptor-dependent calcium release and protein kinase C to promote GABAAR clustering and GABAergic transmission. This GABAAR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses.

10/06/2015 | Cell Calcium   IF 2.9
Both RyRs and TPCs are required for NAADP-induced intracellular Ca release.
Gerasimenko JV, Charlesworth RM, Sherwood MW, Ferdek PE, Mikoshiba K, Parrington J, Petersen OH, Gerasimenko OV

Intracellular Ca2+ release is mostly mediated by inositol trisphosphate, but intracellular cyclic-ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are important messengers in many systems. Whereas cADPR generally activates type 2 ryanodine receptors (RyR2s), the NAADP-activated Ca2+ release mechanism is less clear. Using knockouts and antibodies against RyRs and Two-Pore Channels (TPCs), we have compared their relative importance for NAADP-induced Ca2+ release from two-photon permeabilized pancreatic acinar cells. In these cells, cholecystokinin-elicited Ca2+ release is mediated by NAADP. TPC2-KO reduced NAADP-induced Ca2+ release by 64%, but the combination of TPC2-KO and an antibody against TPC1, significantly reduced Ca2+ release by 86% (64% vs. 86%, p<0.0002). In RyR3-KO, NAADP-evoked Ca2+ release reduced by approximately 50% but, when combined with antibodies against RyR1, responses were 90% inhibited. Antibodies against RyR2 had practically no effect on NAADP-evoked Ca2+ release, but reduced release in response to cADPR by 55%. Antibodies to RyR1 inhibited NAADP-induced Ca2+ liberation by 81%, but only reduced cADPR responses by 30%. We conclude that full NAADP-mediated Ca2+ release requires both TPCs and RyRs. The sequence of relative importance for NAADP-elicited Ca2+ release from the all stores is RyR1>TPC2>RyR3>TPC1>>RyR2. However, when assessing NAADP-induced Ca2+ release solely from the acidic stores (granules/endosomes/lysosomes), antibodies against TPC2 and TPC1 virtually abolished the Ca2+ liberation as did antibodies against RyR1 and RyR3. Our results indicate that the primary, but very small, NAADP-elicited Ca2+ release via TPCs from endosomes/lysosomes triggers the detectable Ca2+-induced Ca2+ release via RyR1 and RyR3 occurring from the granules and the ER.

03/04/2012 | sci signal   IF 7.4
Receptor-selective diffusion barrier enhances sensitivity of astrocytic processes to metabotropic glutamate receptor stimulation.
Arizono M, Bannai H, Nakamura K, Niwa F, Enomoto M, Matsu-Ura T, Miyamoto A, Sherwood MW, Nakamura T, Mikoshiba K

Metabotropic glutamate receptor (mGluR)-dependent calcium ion (Ca(2)+) signaling in astrocytic processes regulates synaptic transmission and local blood flow essential for brain function. However, because of difficulties in imaging astrocytic processes, the subcellular spatial organization of mGluR-dependent Ca(2)+ signaling is not well characterized and its regulatory mechanism remains unclear. Using genetically encoded Ca(2)+ indicators, we showed that despite global stimulation by an mGluR agonist, astrocyte processes intrinsically exhibited a marked enrichment of Ca(2)+ responses. Immunocytochemistry indicated that these polarized Ca(2)+ responses could be attributed to increased density of surface mGluR5 on processes relative to the soma. Single-particle tracking of surface mGluR5 dynamics revealed a membrane barrier that blocked the movement of mGluR5 between the processes and the soma. Overexpression of mGluR or expression of its carboxyl terminus enabled diffusion of mGluR5 between the soma and the processes, disrupting the polarization of mGluR5 and of mGluR-dependent Ca(2)+ signaling. Together, our results demonstrate an mGluR5-selective diffusion barrier between processes and soma that compartmentalized mGluR Ca(2)+ signaling in astrocytes and may allow control of synaptic and vascular activity in specific subcellular domains.

01/06/2011 | Biochem J   IF 3.6
InsP(3)receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences.
Lur G, Sherwood MW, Ebisui E, Haynes L, Feske S, Sutton R, Burgoyne RD, Mikoshiba K, Petersen OH, Tepikin AV

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca(2)(+) entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP(3)Rs (InsP(3) receptors). The co-localization between Orai1 and IP(3)Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca(2)(+) store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca(2)(+) store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP(3)Rs and a basolateral pool that interacts with STIM1 following the Ca(2)(+) store depletion. Experiments on IP(3)R knockout animals demonstrated that the apical Orai1 localization does not require IP(3)Rs and that IP(3)Rs are not necessary for the activation of SOCE. However, the InsP(3)-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP(3)Rs could have an inhibitory effect on this Ca(2)(+) entry mechanism.

05/04/2011 | Proc Natl Acad Sci U S A   IF 9.6
Calmodulin protects against alcohol-induced pancreatic trypsinogen activation elicited via Ca2+ release through IP3 receptors.
Gerasimenko JV, Lur G, Ferdek P, Sherwood MW, Ebisui E, Tepikin AV, Mikoshiba K, Petersen OH, Gerasimenko OV

Alcohol abuse is a major global health problem, but there is still much uncertainty about the mechanisms of action. So far, the effects of ethanol on ion channels in the plasma membrane have received the most attention. We have now investigated actions on intracellular calcium channels in pancreatic acinar cells. Our aim was to discover the mechanism by which alcohol influences calcium homeostasis and thereby understand how alcohol can trigger premature intracellular trypsinogen activation, which is the initiating step for alcohol-induced pancreatitis. We used intact or two-photon permeabilized acinar cells isolated from wild-type mice or mice in which inositol trisphosphate receptors of type 2 or types 2 and 3 were knocked out. In permeabilized pancreatic acinar cells even a relatively low ethanol concentration elicited calcium release from intracellular stores and intracellular trypsinogen activation. The calcium sensor calmodulin (at a normal intracellular concentration) markedly reduced ethanol-induced calcium release and trypsinogen activation in permeabilized cells, effects prevented by the calmodulin inhibitor peptide. A calmodulin activator virtually abolished the modest ethanol effects in intact cells. Both ethanol-elicited calcium liberation and trypsinogen activation were significantly reduced in cells from type 2 inositol trisphosphate receptor knockout mice. More profound reductions were seen in cells from double inositol trisphosphate receptor (types 2 and 3) knockout mice. The inositol trisphosphate receptors, required for normal pancreatic stimulus-secretion coupling, are also responsible for the toxic ethanol action. Calmodulin protects by reducing calcium release sensitivity.

30/06/2009 | Proc Natl Acad Sci U S A   IF 9.6
Pancreatic protease activation by alcohol metabolite depends on Ca2+ release via acid store IP3 receptors.
Gerasimenko JV, Lur G, Sherwood MW, Ebisui E, Tepikin AV, Mikoshiba K, Gerasimenko OV, Petersen OH

Toxic alcohol effects on pancreatic acinar cells, causing the often fatal human disease acute pancreatitis, are principally mediated by fatty acid ethyl esters (non-oxidative products of alcohol and fatty acids), emptying internal stores of Ca(2+). This excessive Ca(2+) liberation induces Ca(2+)-dependent necrosis due to intracellular trypsin activation. Our aim was to identify the specific source of the Ca(2+) release linked to the fatal intracellular protease activation. In 2-photon permeabilized mouse pancreatic acinar cells, we monitored changes in the Ca(2+) concentration in the thapsigargin-sensitive endoplasmic reticulum (ER) as well as in a bafilomycin-sensitive acid compartment, localized exclusively in the apical granular pole. We also assessed trypsin activity in the apical granular region. Palmitoleic acid ethyl ester (POAEE) elicited Ca(2+) release from both the ER as well as the acid pool, but trypsin activation depended predominantly on Ca(2+) release from the acid pool, that was mainly mediated by functional inositol 1,4,5- trisphosphate receptors (IP(3)Rs) of types 2 and 3. POAEE evoked very little Ca(2+) release and trypsin activation when IP(3)Rs of both types 2 and 3 were knocked out. Antibodies against IP(3)Rs of types 2 and 3, but not type 1, markedly inhibited POAEE-elicited Ca(2+) release and trypsin activation. We conclude that Ca(2+) release through IP(3)Rs of types 2 and 3 in the acid granular Ca(2+) store induces intracellular protease activation, and propose that this is a critical process in the initiation of alcohol-related acute pancreatitis.

01/2009 | acta physiol (oxf)
Downstream from calcium signalling: mitochondria, vacuoles and pancreatic acinar cell damage.
Voronina S, Sherwood M, Barrow S, Dolman N, Conant A, Tepikin A

Ca(2+) is one of the most ancient and ubiquitous second messengers. Highly polarized pancreatic acinar cells serve as an important cellular model for studies of Ca(2+) signalling and homeostasis. Downstream effects of Ca(2+) signalling have been and continue to be an important research avenue. The primary functions regulated by Ca(2+) in pancreatic acinar cells--exocytotic secretion and fluid secretion--have been defined and extensively characterized in the second part of the last century. The role of cytosolic Ca(2+) in cellular pathology and the related question of the interplay between Ca(2+) signalling and bioenergetics are important current research lines in our and other laboratories. Recent findings in these interwoven research areas are discussed in the current review.

08/2008 | Gastroenterology   IF 18.2
Direct activation of cytosolic Ca2+ signaling and enzyme secretion by cholecystokinin in human pancreatic acinar cells.
Murphy JA, Criddle DN, Sherwood M, Chvanov M, Mukherjee R, McLaughlin E, Booth D, Gerasimenko JV, Raraty MG, Ghaneh P, Neoptolemos JP, Gerasimenko OV, Tepikin AV, Green GM, Reeve JR Jr, Petersen OH, Sutton R

BACKGROUND & AIMS: Cholecystokinin (CCK) has been thought to act only indirectly on human pancreatic acinar cells via vagal nerve stimulation, rather than by direct CCK receptor activation as on rodent pancreatic acinar cells. We tested whether CCK (CCK-8 and human CCK-58) can act directly on human pancreatic acinar cells. METHODS: Human acinar cells were freshly isolated from pancreatic transection line samples, loaded with Fluo4-AM or quinacrine, and examined for Ca(2+), metabolic and secretory responses to CCK-8, human CCK-58, or acetylcholine with confocal microscopy. RESULTS: CCK-8 and human CCK-58 at physiologic concentrations (1-20 pmol/L) elicited rapid, robust, oscillatory increases of the cytosolic Ca(2+) ion concentration, showing apical to basal progression, in acinar cells from 14 patients with unobstructed pancreata. The cytosolic Ca(2+) ion concentration increases were followed by increases in mitochondrial adenosine triphosphate production and secretion. CCK-elicited Ca(2+) signals and exocytosis were not inhibited by atropine (1 mumol/L) or tetrodotoxin (100 nmol/L), showing that CCK was unlikely to have acted via neurotransmitter release. CCK-elicited Ca(2+) signals were inhibited reversibly by caffeine (5-20 mmol/L), indicating involvement of intracellular inositol trisphosphate receptor Ca(2+) release channels. Acetylcholine (50 nmol/L) elicited similar Ca(2+) signals. CONCLUSIONS: CCK at physiologic concentrations in the presence of atropine and tetrodotoxin elicits cytosolic Ca(2+) signaling, activates mitochondrial function, and stimulates enzyme secretion in isolated human pancreatic acinar cells. We conclude that CCK acts directly on acinar cells in the human pancreas.

12/2007 | am j physiol gastrointest liver physiol
Visualizing formation and dynamics of vacuoles in living cells using contrasting dextran-bound indicator: endocytic and nonendocytic vacuoles.
Voronina SG, Sherwood MW, Gerasimenko OV, Petersen OH, Tepikin AV

Here we describe a technique that allows us to visualize in real time the formation and dynamics (fusion, changes of shape, and translocation) of vacuoles in living cells. The technique involves infusion of a dextran-bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch-clamp configuration. Experiments were conducted on pancreatic acinar cells stimulated with supramaximal concentrations of cholecystokinin (CCK). The vacuoles, forming in the cytoplasm of the cell, were revealed as dark imprints on a bright fluorescence background, produced by the probe and visualized by confocal microscopy. A combination of two dextran-bound probes, one infused into the cytosol and the second added to the extracellular solution, was used to identify endocytic and nonendocytic vacuoles. The cytosolic dextran-bound probe was also used together with a Golgi indicator to illustrate the possibility of combining the probes and identifying the localization of vacuoles with respect to other cellular organelles in pancreatic acinar cells. Combinations of cytosolic dextran-bound probes with endoplasmic reticulum (ER) or mitochondrial probes were also used to simultaneously visualize vacuoles and corresponding organelles. We expect that the new technique will also be applicable and useful for studies of vacuole dynamics in other cell types.