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Ca2+ Influx (ca2+ + influx)
Kinds of Ca2+ Influx Selected AbstractsEffects of motilin on intracellular free calcium in cultured smooth muscle cells from the antrum of neonatal ratsACTA PHYSIOLOGICA, Issue 1 2010P. Fang Abstract Aim:, The aim of this study was to determine the effects of motilin on [Ca2+]i regulation and its underlying molecular mechanism in cultured antral smooth muscle cells (ASMCs). Methods:, Antral cells were isolated and cultured from neonatal rats, and then the [Ca2+]i in these cells was evaluated by calcium fluorescent probe Fluo-3/AM on a laser scanning confocal microscope. Results:, We show that motilin dose-dependently increased [Ca2+]i concentration in cultured ASMCs. Pre-incubation of cells with either the calcium antagonist verapamil (10,5 mol L,1) or the calcium chelator Egtazic (EGTA, 0.1 mmol L,1) significantly suppressed motilin (10,6 mol L,1) induced [Ca2+]i increase as indicated by fluorescent intensity. Interestingly, after mixing with the non-selective intracellular calcium release blocker TMB-8 (10,5 mol L,1), guanosine triphosphate regulatory protein antagonist NEM (10,5 mol L,1), phospholipase C (PLC) inhibitor compound 48/80 (1.2 ,g mL,1) and ryanodine at high concentration (10,5 mol L,1), the motilin-induced [Ca2+]i increase was only partially blocked. The protein kinase C inhibitor d -sphingosine (10,6 mol L,1), however, did not show any inhibitory effect on motilin-induced [Ca2+]i elevation. Conclusions:, Our study suggests that motilin-stimulated [Ca2+]i elevation in ASMCs is probably due to sustained extracellular Ca2+ influx and Ca2+ release from Ca2+ stores via inositol tris-phosphate receptors and ryanodine receptors. Specifically, motilin-induced [Ca2+]i release is accompanied with guanosine triphosphate-binding protein-coupled receptor,PLC,inositol tris-phosphate signalling cascades. [source] Local Ca2+ influx through CRAC channels activates temporally and spatially distinct cellular responsesACTA PHYSIOLOGICA, Issue 1 2009A. B. Parekh Abstract Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels controls a disparate array of key cellular responses. In this review, recent work will be described that shows local Ca2+ influx through CRAC channels has important spatial and temporal consequences on cell function. A localized Ca2+ rise below the plasma membrane activates, within tens of seconds, catabolic enzymes resulting in the generation of the intracellular messenger arachidonic acid and the paracrine pro-inflammatory molecule LTC4. In addition, local Ca2+ entry can activate gene expression, which develops over tens of minutes. Local Ca2+ influx through CRAC channels therefore has far-reaching consequences on intra- and intercellular communication. [source] When is high-Ca2+ microdomain required for mitochondrial Ca2+ uptake?,ACTA PHYSIOLOGICA, Issue 1 2009A. Spät Abstract Ca2+ release from IP3 -sensitive stores in the endoplasmic reticulum (ER) induced by Ca2+ -mobilizing agonists generates high-Ca2+ microdomains between ER vesicles and neighbouring mitochondria. Here we present a model that describes when such microdomains are required and when submicromolar [Ca2+] is sufficient for mitochondrial Ca2+ uptake. Mitochondrial Ca2+ uptake rate in angiotensin II-stimulated H295R adrenocortical cells correlates with the proximity between ER vesicles and the mitochondrion, reflecting the uptake promoting effect of high-Ca2+ peri-mitochondrial microdomains. Silencing or inhibition of p38 mitogen-activated protein kinase (MAPK) or inhibition of the novel isoforms of protein kinase C enhances mitochondrial Ca2+ uptake and abolishes the positive correlation between Ca2+ uptake and ER-mitochondrion proximity. Inhibition of protein phosphatases attenuates mitochondrial Ca2+ uptake and also abolishes its positive correlation with ER-mitochondrion proximity. We postulate that during IP3 -induced Ca2+ release, Ca2+ uptake is confined to ER-close mitochondria, because of the simultaneous activation of the protein kinases. Attenuation of Ca2+ uptake prevents Ca2+ overload of mitochondria and thus protects the cell against apoptosis. On the other hand, all the mitochondria accumulate Ca2+ at a non-inhibited rate during physiological Ca2+ influx through the plasma membrane. Membrane potential is higher in ER-distant mitochondria, providing a bigger driving force for Ca2+ uptake. Our model explains why comparable mitochondrial Ca2+ signals are formed in response to K+ and angiotensin II (equipotent in respect to global cytosolic Ca2+ signals), although only the latter generates high-Ca2+ microdomains. [source] Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteriesACTA PHYSIOLOGICA, Issue 2 2001S. Horiguchi The contribution of the Na+/Ca2+ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (ASK) with pronounced myogenic tone and mesenteric small arteries (AMS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na+/Ca2+ exchanger, the extracellular Na+ concentration ([Na+]o) was lowered from 143 to 1.2 mM by substituting choline-Cl for NaCl. The ASK developed significant myogenic tone and constricted further in low [Na+]o. Nifedipine (1 ,M) reduced both myogenic tone and low [Na+]o-induced contraction. Because the membrane potential of ASK was not changed by low [Na+]o (,35 ± 2 mV at 143 mM [Na+]o, ,37 ± 3 mV at 1.2 mM [Na+]o), depolarization-induced Ca2+ influx was not a cause of the low [Na+]o-induced contraction. The AMS did not develop significant myogenic tone. Although low [Na+]o also constricted AMS, the magnitude of constriction was significantly weaker than that in ASK (17 ± 4 vs. 47 ± 6%, P < 0.01, at 58 mM Na+). With Bay K 8644, AMS developed myogenic tone, and low [Na+]o-induced constriction was significantly increased. In conclusion, Na+/Ca2+ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca2+ -extrusion. [source] Role of the Na+/Ca2+ exchanger in calcium homeostasis and human sperm motility regulationCYTOSKELETON, Issue 2 2006Zoltįn Krasznai Abstract A number of cell functions, such as flagellar beating, swimming velocity, acrosome reaction, etc., are triggered by a Ca2+ influx across the cell membrane. For appropriate physiological functions, the motile human sperm maintains the intracellular free calcium concentration ([Ca2+]i) at a submicromolar level. The objective of this study was to determine the role of the Na+/Ca2+ exchanger (NCX) in the maintenance of [Ca2+]i in human spermatozoa. Spermatozoa maintained in extracellular medium containing ,1 ,M Ca2+ exhibited motility similar to that of the control. In addition to several calcium transport mechanisms described earlier, we provide evidence that the NCX plays a crucial role in the maintenance of [Ca2+]i. Three chemically unrelated inhibitors of the NCX (bepridil, DCB (3,,4, -dichlorobenzamil hydrochloride), and KB-R7943) all blocked human sperm motility in a dose and incubation time dependent manner. The IC50 values for bepridil, DCB, and KB-R7943 were 16.2, 9.8, and 5.3 ,M, respectively. The treatment with the above-mentioned blockers resulted in an elevated [Ca2+]i and a decreased [Na+]i. The store-operated calcium channel (SOCC) inhibitor SKF 96365 also blocked the sperm motility (IC50 = 2.44 ,M). The presence of the NCX antigen in the human spermatozoa was proven by flow cytometry, confocal laser scanning microscopy, and immunoblotting techniques. Calcium homeostasis of human spermatozoa is maintained by several transport proteins among which the SOCC and the NCX may play a major role. Cell Motil. Cytoskeleton 2006. © 2005 Wiley-Liss, Inc. [source] Developmental characteristics of AMPA receptors in chick lumbar motoneuronsDEVELOPMENTAL NEUROBIOLOGY, Issue 11 2007Xianglian Ni Abstract Ca2+ fluxes through ionotropic glutamate receptors regulate a variety of developmental processes, including neurite outgrowth and naturally occurring cell death. In the CNS, NMDA receptors were originally thought to be the sole source of Ca2+ influx through glutamate receptors; however, AMPA receptors also allow a significant influx of Ca2+ ions. The Ca2+ permeability of AMPA receptors is regulated by the insertion of one or more edited GluR2 subunits. In this study, we tested the possibility that changes in GluR2 expression regulate the Ca2+ permeability of AMPA receptors during a critical period of neuronal development in chick lumbar motoneurons. GluR2 expression is absent between embryonic day (E) 5 and E7, but increases significantly by E8 in the chick ventral spinal cord. Increased GluR2 protein expression is correlated with parallel changes in GluR2 mRNA in the motoneuron pool. Electrophysiological recordings of kainate-evoked currents indicate a significant reduction in the Ca2+ -permeability of AMPA receptors between E6 and E11. Kainate-evoked currents were sensitive to the AMPA receptor blocker GYKI 52466. Application of AMPA or kainate generates a significant increase in the intracellular Ca2+ concentration in E6 spinal motoneurons, but generates a small response in older neurons. Changes in the Ca2+ -permeability of AMPA receptors are not mediated by age-dependent changes in the editing pattern of GluR2 subunits. These findings raise the possibility that Ca2+ influx through Ca2+ -permeable AMPA receptors plays an important role during early embryonic development in chick spinal motoneurons. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal netsDEVELOPMENTAL NEUROBIOLOGY, Issue 5 2007Alexander Dityatev Abstract Extracellular matrix molecules,including chondroitin sulfate proteoglycans, hyaluronan, and tenascin-R,are enriched in perineuronal nets (PNs) associated with subsets of neurons in the brain and spinal cord. In the present study, we show that similar cell type-dependent extracellular matrix aggregates are formed in dissociated cell cultures prepared from early postnatal mouse hippocampus. Starting from the 5th day in culture, accumulations of lattice-like extracellular structures labeled with Wisteria floribunda agglutinin were detected at the cell surface of parvalbumin-expressing interneurons, which developed after 2,3 weeks into conspicuous PNs localized around synaptic contacts at somata and proximal dendrites, as well as around axon initial segments. Physiological recording and intracellular labeling of PN-expressing neurons revealed that these are large fast-spiking interneurons with morphological characteristics of basket cells. To study mechanisms of activity-dependent formation of PNs, we performed pharmacological analysis and found that blockade of action potentials, transmitter release, Ca2+ permeable AMPA subtype of glutamate receptors or L-type Ca2+ voltage-gated channels strongly decreased the extracellular accumulation of PN components in cultured neurons. Thus, we suggest that Ca2+ influx via AMPA receptors and L-type channels is necessary for activity-dependent formation of PNs. To study functions of chondroitin sulfate-rich PNs, we treated cultures with chondroitinase ABC that resulted in a prominent reduction of several major PN components. Removal of PNs did not affect the number and distribution of perisomatic GABAergic contacts but increased the excitability of interneurons in cultures, implicating the extracellular matrix of PNs in regulation of interneuronal activity. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Genetic and pharmacological studies of GluR5 modulation of inhibitory synaptic transmission in the anterior cingulate cortex of adult miceDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2007Long-Jun Wu Abstract In the anterior cingulate cortex (ACC), GluR5-containing kainate receptor mediated the small portion of excitatory postsynaptic current. However, little is known about its role in modulation of neurotransmitter release in this brain region. In the present study, we address this question by using selective GluR5 agonist and antagonist, as well as GluR5,/, mice. Our results showed that activation of GluR5 induced action potential-dependent GABA release, which is also required for the activation of voltage-dependent calcium channel and Ca2+ influx. The effect of GluR5 activation is selective to the GABAergic, but not glutamatergic synaptic transmission. Endogenous activation of GluR5 also enhanced GABA release to ACC pyramidal neurons and the corresponding postsynaptic tonic GABA current. Our results suggest the somatodendritic, but not presynaptic GluR5, in modulation of GABA release. The endogenous GluR5 activation and the subsequent tonic GABA current may play an inhibitory role in ACC-related brain functions. © 2006 Wiley Periodicals, Inc. Develop Neurobiol 67: 146,157, 2007. [source] Activation of a calcium entry pathway by sodium pyrithione in the bag cell neurons of AplysiaDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2004Ronald J. Knox Abstract The ability of sodium pyrithione (NaP), an agent that produces delayed neuropathy in some species, to alter neuronal physiology was accessed using ratiometric imaging of cytosolic free Ca2+ concentration ([Ca2+]i) in fura PE-filled cultured Aplysia bag cell neurons. Bath-application of NaP evoked a [Ca2+]i elevation in both somata and neurites with an EC50 of ,300 nM and a Hill coefficient of ,1. The response required the presence of external Ca2+, had an onset of 3,5 min, and generally reached a maximum within 30 min. 2-Methyl-sulfonylpyridine, a metabolite and close structural analog of NaP, did not elevate [Ca2+]i. Under whole-cell current-clamp recording, NaP produced a ,14 mV depolarization of resting membrane potential that was dependent on external Ca2+. These data suggested that NaP stimulates Ca2+ entry across the plasma membrane. To minimize the possibility that a change in cytosolic pH was the basis for NaP-induced Ca2+ entry, bag cell neuron intracellular pH was estimated with the dye 2,,7,-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester. Exposure of the neurons to NaP did not alter intracellular pH. The slow onset and sustained nature of the NaP response suggested that a cation exchange mechanism coupled either directly or indirectly to Ca2+ entry could underlie the phenomenon. However, neither ouabain, a Na+/K+ ATPase inhibitor, nor removal of extracellular Na+, which eliminates Na+/Ca2+ exchanger activity, altered the NaP-induced [Ca2+]i elevation. Finally, the possibility that NaP gates a Ca2+ -permeable ion channel in the plasma membrane was examined. NaP did not appear to activate two major forms of bag cell neuron Ca2+ -permeable ion channels, as Ca2+ entry was unaffected by inhibition of voltage-gated Ca2+ channels using nifedipine or by inhibition of a voltage-dependent, nonselective cation channel using a high concentration of tetrodotoxin. In contrast, two potential store-operated Ca2+ entry current inhibitors, SKF-96365 and Ni2+, attenuated NaP-induced Ca2+ entry. We conclude that NaP activates a slow, persistent Ca2+ influx in Aplysia bag cell neurons. © 2004 Wiley Periodicals, Inc. J Neurobiol 411,423, 2004 [source] Econazole-induced Ca2+ fluxes and apoptosis in human oral cancer cellsDRUG DEVELOPMENT RESEARCH, Issue 4 2010Daih-Huang Kuo Abstract The effect of econazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability was explored in human oral cancer cells (OC2), using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations of >1,µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The econazole-induced Ca2+ influx was sensitive to blockade of aristolochic acid (phospholipase A2 inhibitor) and GF109203X (PKC inhibitor). In Ca2+ -free medium, after treatment with 1,µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 30,µM econazole failed to induce a [Ca2+]i rise. Inhibition of phospholipase C with 2,µM U73122 substantially suppressed econazole-induced [Ca2+]i rise. At concentrations of 5,70,µM econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50,µM econazole was enhanced by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,,N,-tetraacetic acid (BAPTA). The ERK MAPK inhibitor, PD98059 (10,µM), also enhanced 20,µM econazole-induced cell death. Propidium iodide staining data suggest that econazole induced apoptosis between concentrations of 10,70,µM. Collectively, in OC2 cells, econazole induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from phospholipase A2/PKC-regulated Ca2+ channels. Furthermore, econazole caused cell death appeared to be regulated by ERK MAPK. Drug Dev Res 71: 240,248, 2010. © 2010 Wiley-Liss, Inc. [source] Effect of capsaicin on Ca2+ fluxes in Madin-Darby canine renal tubular cellsDRUG DEVELOPMENT RESEARCH, Issue 2 2010Jeng-Hsien Yeh Abstract The effect of capsaicin, a transient receptor potential vanniloid-1 (TRPV1) receptor agonist, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether capsaicin changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+ -sensitive fluorescent dye. Capsaicin at concentrations between 10,100,µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 80% by removing extracellular Ca2+. Capsacin induced Mn2+ influx, leading to quench of fura-2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid and the non-selective Ca2+ entry blocker La3+, but not by store-operated Ca2+ channel blockers nifedipine, econazole, and SK&F96365, and protein kinase C/A modulators. In Ca2+ -free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished capsaicin-induced Ca2+ release. Conversely, pretreatment with capsaicin partly reduced thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter capsaicin-induced [Ca2+]i rise. The TRPV1 receptor antagonist capsazepine also induced significant Ca2+ entry and Ca2+ release. Collectively, in MDCK cells, capsaicin induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-regulated, La3+ -sensitive Ca2+ channels in a manner dissociated from stimulation of TRPV1 receptors. Drug Dev Res, 2009. © 2009 Wiley-Liss, Inc. [source] Nonylphenol-induced cytosolic Ca2+ elevation and death in renal tubular cellsDRUG DEVELOPMENT RESEARCH, Issue 5 2009Jeng-Yu Tsai Abstract Nonylphenol is an environmental endocrine disrupter. The effect of nonylphenol on intracellular free Ca2+ levels ([Ca2+]i) and viability in Madin-Darby canine kidney (MDCK) cells was explored. Nonylphenol increased [Ca2+]i in a concentration-dependent manner (EC50,0.8,,M). Nonylphenol-induced Mn2+ entry demonstrated Ca2+ influx and removal of extracellular Ca2+ partly decreased the [Ca2+]i rise. The [Ca2+]i rise was inhibited by the protein kinase C activator, phorbol 13-myristate acetate (PMA) but not by L-type Ca2+ channel blockers. In Ca2+ -free medium, nonylphenol-induced [Ca2+]i rise was partly inhibited by pretreatment with 1,,M thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Conversely, nonylphenol pretreatment abolished thapsigargin-induced Ca2+ release. Nonylphenol-induced Ca2+ release was unaltered by inhibition of phospholipase C. At concentrations of 5,100,,M, nonylphenol killed cells in a concentration-dependent manner. The cytotoxic effect of 100,,M nonylphenol was not affected by preventing [Ca2+]i rises with BAPTA/AM. Collectively, this study shows that nonylphenol induced [Ca2+]i increase in MDCK cells via evoking Ca2+ entry through protein kinase C-regulated Ca2+ channels, and releasing Ca2+ from endoplasmic reticulum and other stores in a phospholipase C-independent manner. Nonylphenol also killed cells in a Ca2+ -independent fashion. Drug Dev Res, 2009. © 2009 Wiley-Liss, Inc. [source] Independent signaling pathways in ATP-evoked secretion of plasminogen and cytokines from microgliaDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001*Article first published online: 28 AUG 200, Kazuhide Inoue Abstract We investigated the action of ATP on the secretion of plasminogen, TNF-,, and IL-6 from microglia. ATP (10,100 ,M) stimulated the release of plasminogen from rat cultured microglia in a concentration-dependent manner with a peak response at 5,10 min after the stimulation. The release was dependent on extracellular Ca2+ and was blocked by pretreatment with oxidized ATP, a blocker of P2X7. UTP, an agonist of P2Y2, also stimulated the release of plasminogen from a subpopulation (about 20% of total cells) of cultured microglia. The release was also dependent on extracellular Ca2+, suggesting a role of stocker-operated calcium entry (SOC). ATP potently stimulated TNF-, release from 2 h after the stimulation with TNF-, mRNA expression in primary cultures of rat brain microglia. The TNF-, release was maximally elicited by 1 mM ATP and 2,- and 3,-O-(4-benzoylbenzoyl)-adenosine 5,-triphosphate (BzATP), a P2X7 selective agonist, suggesting the involvement of P2X7. This TNF-, release was correlated with a sustained Ca2+ influx. The release was inhibited by PD98059, an inhibitor of MEK1 which activates extracellular signal-regulated protein kinase (ERK), and SB203580, an inhibitor of p38 MAP kinase. However, both ERK and p38 were rapidly activated by ATP even in the absence of extracellular Ca2+. These results indicate that extracellular ATP triggers TNF-, release in rat microglia via P2X7 in a manner dependent on the sustained Ca2+ influx and via the ERK/p38 cascade independently of Ca2+ influx. ATP caused the mRNA expression and release of IL-6 in a concentration-dependent manner in MG-5. The physiological meaning of these independent release mechanisms is also discussed. Drug Dev. Res. 53:166,171, 2001. © 2001 Wiley-Liss, Inc. [source] Chalcones as potent antiplatelet agents and calcium channel blockersDRUG DEVELOPMENT RESEARCH, Issue 1 2001Chun-Nan Lin Abstract In an effort to continually develop potent antiplatelet agents with vasorelaxing and antiinflammatory actions, a novel series of antiinflammatory chalcones was continually screened to evaluate their antiplatelet and vasorelaxing effects. Their structure,activity relationships and mode of action were discussed and characterized. A novel series of antiinflammatory chalcones was studied on antiplatelet effect in rabbit washed platelets and human platelet-rich plasma (PRP) and vasorelaxing effect in rat thoracic aorta. Arachidonic acid-induced platelet aggregation was potently inhibited by almost all the chalcone derivatives and 13,15 also had a potent inhibitory effect on cyclooxygenase. The selective chalcones 12,16 tested in human PRP significantly inhibited secondary aggregation induced by adrenaline. In rat thoracic aorta, most of chalcones at high concentration significantly depressed the contractions induced by Ca2+ (1.9 mM) in high K+ (80 mM) medium and the phasic and tonic contractions caused by norepinephrine (3 ,M). In the rat thoracic aorta, the phenylephrine- and high K+ -induced 45Ca2+ influx were both inhibited by a selective chalcone derivative, 14. These results indicate that the antiplatelet actions of chalcones are mainly mediated through the suppression of cyclooxygenase activity and reduced thromboxane formation and their inhibitory effects on the contractile response caused by high K+ and norepinephrine in rat thoracic aorta are mainly due to inhibition of Ca2+ influx through both voltage-dependent and receptor-operated Ca2+ channels. Drug Dev. Res. 53:9,14, 2001. © 2001 Wiley-Liss, Inc. [source] Enhanced Calcium Influx in Hippocampal CA3 Neurons of Spontaneously Epileptic RatsEPILEPSIA, Issue 3 2001Hiroko Amano Summary: ,Purpose: The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence-like seizures and tonic convulsions. Our previous electrophysiologic studies have demonstrated that SER has abnormal excitability of hippocampal CA3 neurons, which shows a long-lasting depolarization shift by a single stimulation of mossy fibers, probably resulting from the Ca2+ channel abnormalities. The present study was performed to determine whether Ca2+ influx is actually enhanced in the CA3 area of SER. Methods: Hippocampal slices were prepared from normal Wistar rats and SER aged 11,16 weeks old, when the epileptic seizures had been observed, and loaded with fura-2AM. Intracellular Ca2+ concentration ([Ca2+]i) was monitored as the ratio of fluorescence intensities excited at wavelengths of 340 and 380 nm (RF340/F380) with photometric devices. Results: High K+ (10,60 mM) applied to the bath for 2 min increased [Ca2+]i in hippocampal CA1, CA3, and dentate gyrus (DG) areas of both the normal rats and SER in a concentration-dependent manner. However, the high K+,induced increase in [Ca2+]i was significantly more pronounced in the CA3 area of the SER than in that of the normal animals, whereas there were no significant differences in high K+,induced increases of [Ca2+]i in CA1 or DG between the SER and controls. The high K+,induced increases in [Ca2+]i of CA1, CA3, and DG were inhibited by nifedipine (1,10 nM), a Ca2+ channel antagonist in both SER and controls. However, the inhibition of the high K+,induced increase in [Ca2+]i by nifedipine (1 nM) was significantly greater in the CA3 area of SER than that of controls. Conclusions: These findings suggest that Ca2+ influx through the L-type Ca2+ channels is much greater in the CA3 area of SER than in that of normal animals and is involved in the epileptic seizures of the SER. [source] Early cytoskeletal rearrangement during dendritic cell maturation enhances synapse formation and Ca2+ signaling in CD8+ T cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2004Marco Averbeck Abstract The interplay between dendritic cells (DC) and T cells is a dynamic process critically depending on DC maturation. Ca2+ influx is one of the initial events occurring during DC/T cell contacts. To determine how DC maturation influences DC/T cell contacts, time-lapse video microscopy was established using TCR-transgenic CD8+ T cells from P14 mice. DC maturation shifted DC/T cell contacts from short-lived interactions with transient Ca2+ influx in T cells to long-lasting interactions and sustained Ca2+ influx of 30,min and more. Follow-up of DC/T cell interactions after 2,h using confocal microscopy revealed that long-lasting Ca2+ responses in T cells were preferentially associated with the formation of an immunological synapse involving CD54 and H2-Kb at the DC/T cell interface. Such synapse formation preceded MHC or B7 up-regulation, since DC developed into potent Ca2+ stimulators 7,h after initiation of maturation. Instead, the enhanced capacity of 7,h-matured DC to induce sustained Ca2+ responses in CD8+ T cells is critically dependent on the polarization and rearrangement of the cytoskeleton, as shown by Clostridium difficile toxin B inhibitor experiments. These data indicate that already very early after receiving a maturation stimulus, DC display enhanced cytoskeletal activity resulting in the rapid formation of immunological synapses and effective CD8+ T cell stimulation. [source] Adenosine drives recycled vesicles to a slow-release pool at the mouse neuromuscular junctionEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2010Paula P. Perissinotti Abstract The effects of adenosine on neurotransmission have been widely studied by monitoring transmitter release. However, the effects of adenosine on vesicle recycling are still unknown. We used fluorescence microscopy of FM2-10-labeled synaptic vesicles in combination with intracellular recordings to examine whether adenosine regulates vesicle recycling during high-frequency stimulation at mouse neuromuscular junctions. The A1 adenosine receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine) increased the quantal content released during the first endplate potential, suggesting that vesicle exocytosis can be restricted by endogenous adenosine, which accordingly decreases the size of the recycling vesicle pool. Staining protocols designed to label specific vesicle pools that differ in their kinetics of release showed that all vesicles retrieved in the presence of 8-cyclopentyl-1,3-dipropylxanthine were recycled towards the fast-release pool, favoring its loading with FM2-10 and suggesting that endogenous adenosine promotes vesicle recycling towards the slow-release pool. In accordance with this effect, exogenous applied adenosine prevented the replenishment of the fast-release vesicle pool and, thus, hindered its loading with the dye. We had found that, during high-frequency stimulation, Ca2+ influx through L-type channels directs newly formed vesicles to a fast-release pool (Perissinotti et al., 2008). We demonstrated that adenosine did not prevent the effect of the L-type blocker on transmitter release. Therefore, activation of the A1 receptor promotes vesicle recycling towards the slow-release pool without a direct effect on the L-type channel. Further studies are necessary to elucidate the molecular mechanisms involved in the regulation of vesicle recycling by adenosine. [source] Impairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2010Shinji Matsumura Abstract Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of long-term potentiation (LTP), which can be triggered by N -methyl- d -aspartate (NMDA) receptor-mediated Ca2+ influx. We previously demonstrated that Fyn kinase-mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F-KI mice with a knock-in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F-KI mice among the major phosphorylation sites. There was no difference in the Ca2+ response to glutamate and sensitivity to NMDA receptor antagonists between naive wild-type and Y1472F-KI mice, and the Ca2+ response to glutamate was attenuated in the Y1472F-KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F-KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase C, at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild-type and Y1472F-KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor-mediated CaMKII signaling in Y1472F-KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain. [source] AMPA and metabotropic glutamate receptors cooperatively generate inspiratory-like depolarization in mouse respiratory neurons in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2008Ryland W. Pace Abstract Excitatory transmission mediated by AMPA receptors is critical for respiratory rhythm generation. However, the role of AMPA receptors has not been fully explored. Here we tested the functional role of AMPA receptors in inspiratory neurons of the neonatal mouse preBötzinger complex (preBötC) using an in vitro slice model that retains active respiratory function. Immediately before and during inspiration, preBötC neurons displayed envelopes of depolarization, dubbed inspiratory drive potentials, that required AMPA receptors but largely depended on the Ca2+ -activated non-specific cation current (ICAN). We showed that AMPA receptor-mediated depolarization opened voltage-gated Ca2+ channels to directly evoke ICAN. Inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca2+ release also evoked ICAN. Inositol 1,4,5-trisphosphate receptors acted downstream of group I metabotropic glutamate receptor activity but, here too, AMPA receptor-mediated Ca2+ influx was essential to trigger the metabotropic glutamate receptor contribution to inspiratory drive potential generation. This study helps to elucidate the role of excitatory transmission in respiratory rhythm generation in vitro. AMPA receptors in preBötC neurons initiate convergent signaling pathways that evoke post-synaptic ICAN, which underlies inspiratory drive potentials. The coupling of AMPA receptors with ICAN suggests that latent burst-generating intrinsic conductances are recruited by excitatory synaptic interactions among preBötC neurons in the context of respiratory network activity in vitro, exemplifying a rhythmogenic mechanism based on emergent properties of the network. [source] Multiple functions of GABAA and GABAB receptors during pattern processing in the zebrafish olfactory bulbEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2008Rico Tabor Abstract ,-Aminobutyric acid (GABA)ergic synapses are thought to play pivotal roles in the processing of activity patterns in the olfactory bulb (OB), but their functions have been difficult to study during odor responses in the intact system. We pharmacologically manipulated GABAA and GABAB receptors in the OB of zebrafish and analysed the effects on odor responses of the output neurons, the mitral cells (MCs), by electrophysiological recordings and temporally deconvolved two-photon Ca2+ imaging. The blockade of GABAB receptors enhanced presynaptic Ca2+ influx into afferent axon terminals, and changed the amplitude and time course of a subset of MC responses, indicating that GABAB receptors have a modulatory influence on OB output activity. The blockade of GABAA receptors induced epileptiform firing, enhanced excitatory responses and abolished fast oscillations in the local field potential. Moreover, the topological reorganization and decorrelation of MC activity patterns during the initial phase of the response was perturbed. These results indicate that GABAA receptor-containing circuits participate in the balance of excitation and inhibition, the regulation of total OB output activity, the synchronization of odor-dependent neuronal ensembles, and the reorganization of odor-encoding activity patterns. GABAA and GABAB receptors are therefore differentially involved in multiple functions of neuronal circuits in the OB. [source] NCS-1 differentially regulates growth cone and somata calcium channels in Lymnaea neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2008Kwokyin Hui Abstract Local voltage-gated calcium channels, which regulate intracellular Ca2+ levels by allowing Ca2+ influx, play an important role in guiding and shaping growth cones, and in regulating the outgrowth and branching of neurites. Therefore, elucidating the mechanisms that regulate the biophysical properties of whole-cell calcium currents in the growth cones and somata of growing neurons is important to improving our understanding of neuronal development and regeneration. In this study, taking advantage of the large size of the pedal A (PeA) neurons in Lymnaea stagnalis, we compared the biophysical properties of somata and growth cone whole-cell calcium channel currents using Ba2+ and Ca2+ as current carriers. We found that somata and growth cone currents exhibit similar high-voltage activation properties. However, Ba2+ and Ca2+ currents in growth cones and somata are differentially affected by a dominant-negative peptide containing the C-terminal amino acid sequence of neuronal calcium sensor-1 (NCS-1). The peptide selectively reduces the peak and sustained components of current densities and the slope conductance in growth cones, and shifts the reversal potential of the growth cone currents to more hyperpolarized voltages. In contrast, the peptide had no significant effect on the somata calcium channels. Thus, we conclude that NCS-1 differentially modulates Ca2+ currents in the somata and growth cones of regenerating neurons, and may serve as a key regulator to facilitate the growth cone calcium channel activity. [source] Depolarization promotes GAD 65-mediated GABA synthesis by a post-translational mechanism in neural stem cell-derived neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2008Nidhi Gakhar-Koppole Abstract Neuronal activity regulates neurogenesis and neuronal differentiation in the mammalian brain. The commencement of neurotransmitter expression establishes the neuronal phenotype and enables the formation of functional connectivity between neurons. In addition, release of neurotransmitters from differentiating neurons may modulate the behaviour of neural precursors. Here, we show that neuronal activity regulates ,-aminobutyric acid (GABA) expression in neurons generated from stem cells of the striatum and adult subventricular zone (SVZ). Differentiating neurons display spontaneous Ca2+ events, which are voltage-gated calcium channel (VGCC) dependent. Depolarization increases both the frequency of Ca2+ transients and the amount of Ca2+ influx in differentiating neurons. We show that depolarization-dependent GABA expression is regulated by the amplitude and not by the frequency of Ca2+ influx. Brief activation of VGCCs leads to Ca2+ influx that in turn promotes a rapid expression of GABA. Depolarization-dependent GABA expression does not require changes in gene expression. Instead, it involves cAMP-dependent protein kinase (PKA) and Ca2+ and phospholipid-dependent protein kinase (PKC) signalling. Activity increases the number of glutamic acid decarboxylase (GAD) 65-immunoreactive neurons in a PKA-dependent manner, without altering the expression of GAD 65, suggesting that depolarization promotes recruitment of GAD 65 by a post-translational mechanism. In line with this, depolarization does not permanently increase the expression of GABA in neurons derived from neural stem cells of the embryonic striatum, cortex and adult SVZ. Thus, neuronal activity does not merely accelerate neuronal differentiation but it may alter the mechanism of GABA synthesis in newly generated neurons. [source] Proteolytic cleavage of the voltage-gated Ca2+ channel ,2, subunit: structural and functional featuresEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007Arturo Andrade Abstract By mediating depolarization-induced Ca2+ influx, high-voltage-activated Ca2+ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (,1) and three auxiliary (,2,, , and ,) subunits. The ,2, subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, ,2, must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular ,2 from its transmembrane , domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature ,2,, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of ,2, was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane. [source] Negative cross-talk between presynaptic adenosine and acetylcholine receptorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2006A. V. Shakirzyanova Abstract Functional interactions between presynaptic adenosine and acetylcholine (ACh) autoreceptors were studied at the frog neuromuscular junction by recording miniature end-plate potentials (MEPPs) during bath or local application of agonists. The frequency of MEPPs was reduced by adenosine acting on presynaptic adenosine A1 receptors (EC50 = 1.1 µm) or by carbachol acting on muscarinic M2 receptors (EC50 = 1.8 µm). However, carbachol did not produce the depressant effect when it was applied after the action of adenosine had reached its maximum. This phenomenon implied that the negative cross-talk (occlusion) had occurred between A1 and M2 receptors. Moreover, the occlusion was receptor-specific as ATP applied in the presence of adenosine continued to depress MEPP frequency. Muscarinic antagonists [atropine or 1-[[2-[(diethylamino)methyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one) (AFDX-116)] had no effect on the inhibitory action of adenosine and adenosine antagonists [8-(p -sulfophenyl)theophylline (8-SPT) or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX)] had no effect on the action of carbachol. These data suggested that membrane,delimited interactions did not occur between A1 and M2 receptors. Both carbachol and adenosine similarly inhibited quantal release triggered by high potassium, ionomycin or sucrose. These results indicated a convergence of intracellular pathways activated by M2 and A1 receptors to a common presynaptic effector located downstream of Ca2+ influx. We propose that the negative cross-talk between two major autoreceptors could take place during intense synaptic activity and thereby attenuate the presynaptic inhibitory effects of ACh and adenosine. [source] AMPA receptor-mediated presynaptic inhibition at cerebellar GABAergic synapses: a characterization of molecular mechanismsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2004Shin'Ichiro Satake Abstract A major subtype of glutamate receptors, AMPA receptors (AMPARs), are generally thought to mediate excitation at mammalian central synapses via the ionotropic action of ligand-gated channel opening. It has recently emerged, however, that synaptic activation of AMPARs by glutamate released from the climbing fibre input elicits not only postsynaptic excitation but also presynaptic inhibition of GABAergic transmission onto Purkinje cells in the cerebellar cortex. Although presynaptic inhibition is critical for information processing at central synapses, the molecular mechanisms by which AMPARs take part in such actions are not known. This study therefore aimed at further examining the properties of AMPAR-mediated presynaptic inhibition at GABAergic synapses in the rat cerebellum. Our data provide evidence that the climbing fibre-induced inhibition of GABA release from interneurons depends on AMPAR-mediated activation of GTP-binding proteins coupled with down-regulation of presynaptic voltage-dependent Ca2+ channels. A Gi/o -protein inhibitor, N-ethylmaleimide, selectively abolished the AMPAR-mediated presynaptic inhibition at cerebellar GABAergic synapses but did not affect AMPAR-mediated excitatory actions on Purkinje cells. Furthermore, both Gi/o -coupled receptor agonists, baclofen and DCG-IV, and the P/Q-type calcium channel blocker ,-agatoxin IVA markedly occluded the AMPAR-mediated inhibition of GABAergic transmission. Conversely, AMPAR activation inhibited action potential-triggered Ca2+ influx into individual axonal boutons of cerebellar GABAergic interneurons. By suppressing the inhibitory inputs to Purkinje cells, the AMPAR-mediated presynaptic inhibition could thus provide a feed-forward mechanism for the information flow from the cerebellar cortex. [source] SK channels and the varieties of slow after-hyperpolarizations in neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2003Fivos Vogalis Abstract Action potentials and associated Ca2+ influx can be followed by slow after-hyperpolarizations (sAHPs) caused by a voltage-insensitive, Ca2+ -dependent K+ current. Slow AHPs are a widespread phenomenon in mammalian (including human) neurons and are present in both peripheral and central nervous systems. Although, the molecular identity of ion channels responsible for common membrane potential mechanisms has been largely determined, the nature of the channels that underlie the sAHPs in neurons, both in the brain and in the periphery, remains unresolved. This short review discusses why there is no clear molecular candidate for sAHPs. [source] Dynamics of Ca2+ and Na+ in the dendrites of mouse cerebellar Purkinje cells evoked by parallel fibre stimulationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2003Akinori Kuruma Abstract Ca2+ and Na+ play important roles in neurons, such as in synaptic plasticity. Their concentrations in neurons change dynamically in response to synaptic inputs, but their kinetics have not been compared directly. Here, we show the mechanisms and dynamics of Ca2+ and Na+ transients by simultaneous monitoring in Purkinje cell dendrites in mouse cerebellar slices. High frequency parallel fibre stimulation (50 Hz, 3,50-times) depolarized Purkinje cells, and Ca2+ transients were observed at the anatomically expected sites. The magnitude of the Ca2+ transients increased linearly with increasing numbers of parallel fibre inputs. With 50 stimuli, Ca2+ transients lasted for seconds, and the peak [Ca2+] reached ,100 µm, which was much higher than that reported previously, although it was still confined to a part of the dendrite. In contrast, Na+ transients were sustained for tens of seconds and diffused away from the stimulated site. Pharmacological interventions revealed that Na+ influx through ,-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and Ca2+ influx through P-type Ca channels were essential players, that AMPA receptors did not operate as a Ca2+ influx pathway and that Ca2+ release from intracellular stores through inositol trisphosphate receptors or ryanodine receptors did not contribute greatly to the large Ca2+ transients. [source] Phosphorylation of voltage-gated ion channels in rat olfactory receptor neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2001Christian H. Wetzel Abstract In olfactory receptor neurons (ORNs), ligand,odorant receptor interactions cause G protein-mediated activation of adenylate cyclase and a subsequent increase in concentration of the intracellular messenger cAMP. Odorant-evoked elevation in cAMP is thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca2+ influx, leading to membrane depolarization and the generation of action potentials. Our data show that in freshly dissociated rat ORNs, odorant-induced elevation in cAMP also activates cAMP-dependent protein kinase (PKA), which is then able to phosphorylate various protein targets in the olfactory signal transduction pathway, specifically voltage-gated sodium and calcium channels. The presence of PKI (PKA inhibitor peptide) blocked the modulatory action of cAMP on voltage-gated ion channels. By modulating the input/output properties of the sensory neurons, this mechanism could take part in the complex adaptation process in odorant perception. In addition, we found modulation of voltage-gated sodium and calcium channel currents by 5-hydroxytryptamine and the dopamine D1 receptor agonist SKF 38393. These findings suggest that in situ ORNs might also be a target for efferent modulation. [source] Differential induction of LTP and LTD is not determined solely by instantaneous calcium concentration: an essential involvement of a temporal factorEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2001Tomoyuki Mizuno Abstract Two opposite types of synaptic plasticity in the CA1 hippocampus, long-term potentiation (LTP) and long-term depression (LTD), require postsynaptic Ca2+ elevation. To explain these apparently contradictory phenomena, the current view assumes that a moderate postsynaptic increase in Ca2+ leads to LTD, whereas a large increase leads to LTP. No detailed study has so far been attempted to investigate whether the instantaneous Ca2+ elevation level differentially induces LTP or LTD. We therefore used low-frequency (1 Hz) stimulation of Schaffer collateral/commissural fibers in rat hippocampal slices, during a Mg2+ -free period, as the conditioning stimulus to investigate this. This allowed low-frequency afferent stimulation to cause a postsynaptic Ca2+ influx because the voltage-dependent block of N -methyl- d -aspartate (NMDA) receptor-channels by Mg2+ was removed. When delivered during the Mg2+ -free period, a single pulse, as well as 2,600 pulses, induced LTP that was occluded with tetanus-induced LTP. To decrease the Ca2+ influx, ,-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors were completely blocked by the addition of 10 µm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the conditioning medium, in which 1 Hz afferent stimuli (1,600 pulses) induced less LTP and never induced LTD. To further reduce the Ca2+ influx, NMDA receptors were partially blocked with d -(,)-2-amino-5-phosphonopentanoic acid (d -AP5). A small number of 1 Hz stimuli, however, never induced LTD. Only when the conditioning stimuli exceeded 200 pulses was LTD induced. The present findings provide definitive evidence that protracted conditioning is a prerequisite for the induction of LTD. Thus, not only the amplitude but also the duration of postsynaptic Ca2+ elevation could be essential factors for differentially inducing LTP or LTD. [source] A modulatory role for protein phosphatase 2B (calcineurin) in the regulation of Ca2+ entryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2000J. Russell Burley Abstract The Ca2+/calmodulin-dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+ -dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+ -channel activity, phosphatase expression was altered in NG108-15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2B,3. Relative to mock-transfected (wild-type) controls, cells overexpressing calcineurin showed dramatically reduced high-voltage-activated Ca2+ currents which were recoverable by the inclusion of 1 ,m FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high-voltage-activated Ca2+ currents were larger relative to controls. Additionally in these cells, low-voltage-activated currents were significantly reduced. Analysis of high-voltage-activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large-scale Ca2+ influx across the cell membrane, Ca2+ -dependent inactivation of high-voltage-activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 m m BAPTA or 1 ,m FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage-operated Ca2+ channels. [source] | |||||||||||||||||||||||||||||||||||||