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Ca2+ Signals (ca2+ + signal)
Kinds of Ca2+ Signals Selected AbstractsBidirectional synaptic plasticity as a consequence of interdependent Ca2+ -controlled phosphorylation and dephosphorylation pathwaysEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2003Pablo D'Alcantara Abstract Postsynaptic Ca2+ signals of different amplitudes and durations are able to induce either long-lasting potentiation (LPT) or depression (LTD). The bidirectional character of synaptic plasticity may result at least in part from an increased or decreased responsiveness of the glutamatergic ,-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA-R) due to the modification of conductance and/or channel number, and controlled by the balance between the activities of phosphorylation and dephosphorylation pathways. AMPA-R depression can be induced by a long-lived Ca2+ signal of moderate amplitude favouring the activation of the dephosphorylation pathway, whereas a shorter but higher Ca2+ signal would induce AMPA-R potentiation resulting from the preferential activation of the phosphorylation pathway. Within the framework of a model involving calcium/calmodulin-dependent protein kinase II (CaMKII), calcineurin (PP2B) and type 1 protein phosphatase (PP1), we aimed at delineating the conditions allowing a biphasic U-shaped relationship between AMPA-R and Ca2+ signal amplitude, and thus bidirectional plasticity. Our theoretical analysis shows that such a property may be observed if the phosphorylation pathway: (i) displays higher cooperativity in its Ca2+ -dependence than the dephosphorylation pathway; (ii) displays a basal Ca2+ -independent activity; or (iii) is directly inhibited by the dephosphorylation pathway. Because the experimentally observed inactivation of CaMKII by PP1 accounts for this latter characteristic, we aimed at verifying whether a realistic model using reported parameters values can simulate the induction of either LTP or LTD, depending on the time and amplitude characteristics of the Ca2+ signal. Our simulations demonstrate that the experimentally observed bidirectional nature of Ca2+ -dependent synaptic plasticity could be the consequence of the PP1-mediated inactivation of CaMKII. [source] EP1 and EP4 Receptors Mediate Exocytosis Evoked by Prostaglandin E2 in Guinea-Pig Antral Mucous CellsEXPERIMENTAL PHYSIOLOGY, Issue 4 2001Atsuko Ohnishi Effects of prostaglandin E2 (PGE2) on exocytosis of mucin were studied in mucous cells isolated from guinea-pig antrum using video-microscopy. Stimulation with PGE2 elicited a sustained increase in the frequency of exocytotic events in a dose-dependent manner, which was under regulation by both Ca2+ and cAMP. Stimulation with a selective prostanoid EP4 receptor agonist (ONO-AEI-329, 10 ,M), which activates cAMP signals, elicited a sustained increase in the frequency of exocytotic events (30% of that evoked by 1 ,M PGE2). Stimulation with an EP1 agonist (17-P-T-PGE2, 1 ,M), which activates Ca2+ signals, increased the frequency of exocytotic events to a lesser extent (5% of that evoked by 1 ,M PGE2), while addition of an EP1 antagonist (ONO-8713, 10 ,M) decreased the frequency of exocytotic events (approximately 40% of that evoked by 1 ,M PGE2). However, addition of the EP1 agonist potentiated the frequency of exocytotic events evoked by the EP4 agonist or forskolin (which elevates cAMP levels) and increased the sensitivity of the exocytotic events to forskolin. These results suggest that the Ca2+ signal activated via the EP1 receptor potentiates the cAMP-regulated exocytotic events activated via the EP4 receptor during PGE2 stimulation, by increasing the sensitivity of the exocytotic response to cAMP. In conclusion, exocytotic events in PGE2 -stimulated antral mucous cells were regulated by interactions between EP1 and EP4 receptors. [source] Nucleotide-induced Ca2+ signaling in sustentacular supporting cells of the olfactory epitheliumGLIA, Issue 15 2008Thomas Hassenklöver Abstract Extracellular purines and pyrimidines are important signaling molecules acting via purinergic cell-surface receptors in neurons, glia, and glia-like cells such as sustentacular supporting cells (SCs) of the olfactory epithelium (OE). Here, we thoroughly characterize ATP-induced responses in SCs of the OE using functional Ca2+ imaging. The initial ATP-induced increase of the intracellular Ca2+ concentration [Ca2+]i always occurred in the apical part of SCs and subsequently propagated toward the basal lamina, indicating the occurrence of purinergic receptors in the apical part of SCs. The mean propagation velocity of the Ca2+ signal within SCs was 17.10 ± 1.02 ,m/s. ATP evoked increases in [Ca2+]i in both the presence and absence of extracellular Ca2+. Depletion of the intracellular Ca2+ stores abolished the responses. This shows that the ATP-induced [Ca2+]i increases were in large part, if not entirely, due to the activation of G protein-coupled receptors followed by Ca2+ mobilization from intracellular stores, suggesting an involvement of P2Y receptors. The order of potency of the applied purinergic agonists was UTP > ATP > ATP,S (with all others being only weakly active or inactive). The ATP-induced [Ca2+]i increases could be reduced by the purinergic antagonists PPADS and RB2, but not by suramin. Our findings suggest that extracellular nucleotides in the OE activate SCs via P2Y2/P2Y4 -like receptors and initiate a characteristic intraepithelial Ca2+ wave. © 2008 Wiley-Liss, Inc. [source] Molecular mechanisms supporting a paracrine role of GABA in rat adrenal medullary cellsTHE JOURNAL OF PHYSIOLOGY, Issue 20 2008Hidetada Matsuoka GABA is known to produce membrane depolarization and secretion in adrenal medullary (AM) cells in various species. However, whether the GABAergic system is intrinsic or extrinsic or both in the adrenal medulla and the role that GABA plays are controversial. Therefore, these issues were addressed by combining a biochemical and functional analysis. Glutamic acid decarboxylase (GAD), a GABA synthesizing enzyme, and vesicular GABA transporter (VGAT) were expressed in rat AM cells at the mRNA and protein levels, and the adrenal medulla had no nerve fibre-like structures immunoreactive to an anti-GAD Ab. The double staining for VGAT and chromogranin A indicates that GABA was stored in chromaffin granules. The ,1, ,3, ,2/3, ,2 and , subunits of GABAA receptors were identified in AM cells at the mRNA and protein levels. Pharmacological properties of GABA-induced Cl, currents, immunoprecipitation experiments and immunocytochemistry indicated the expression of not only ,2-, but also ,-containing GABAA receptors, which have higher affinities for GABA and neurosteroids. Expression of GATs, which are involved in the clearance of GABA at GABAergic synapses, were conspicuously suppressed in the adrenal medulla, compared with expression levels of GABAA receptors. Increases in Ca2+ signal in AM cells evoked trans-synaptically by nerve stimulation were suppressed during the response to GABA, and this suppression was attributed to the shunt effect of the GABA-induced increase in conductance. Overall Ca2+ responses to electrical stimulation and GABA in AM cells were larger or smaller than those to electrical stimulation alone, depending on the frequency of stimulation. The results indicate that GABA functions as a paracrine in rat AM cells and this function may be supported by the suppression of GAT expression and the expression of not only ,2-, but also ,-GABAA receptors. [source] Ca2+ microdomains near plasma membrane Ca2+ channels: impact on cell functionTHE JOURNAL OF PHYSIOLOGY, Issue 13 2008Anant B. Parekh In eukaryotic cells, a rise in cytoplasmic Ca2+ can activate a plethora of responses that operate on time scales ranging from milliseconds to days. Inherent to the use of a promiscuous signal like Ca2+ is the problem of specificity: how can Ca2+ activate some responses but not others? We now know that the spatial profile of the Ca2+ signal is important Ca2+ does not simply rise uniformly throughout the cytoplasm upon stimulation but can reach very high levels locally, creating spatial gradients. The most fundamental local Ca2+ signal is the Ca2+ microdomain that develops rapidly near open plasmalemmal Ca2+ channels like voltage-gated L-type (Cav1.2) and store-operated CRAC channels. Recent work has revealed that Ca2+ microdomains arising from these channels are remarkably versatile in triggering a range of responses that differ enormously in both temporal and spatial profile. Here, I delineate basic features of Ca2+ microdomains and then describe how these highly local signals are used by Ca2+ -permeable channels to drive cellular responses. [source] Phosphorylation of inositol 1,4,5-triphosphate receptor 1 during in vitro maturation of porcine oocytesANIMAL SCIENCE JOURNAL, Issue 1 2010Junya ITO ABSTRACT During fertilization in mammalian species, a sperm-induced intracellular Ca2+ signal ([Ca2+]i) mediates both exit of meiosis and oocyte activation. Recently, we demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP3R1), the channel responsible for Ca2+ release and oscillations during fertilization, changed during maturation and fertilization. Therefore, we examined the expression and phosphorylation of IP3R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP3R1 protein did not change during maturation, although the phosphorylation status of the receptor, specifically at an MPM-2 epitope, did. We found that while at the beginning of maturation IP3R1 lacked MPM-2 immunoreactivity, it became MPM-2 reactive by 24 h and reached maximal reactivity by 36 h. Interestingly, the acquisition of MPM-2 reactivity coincided with the activation of p34cdc2 kinase and mitogen-activated protein kinase (MAPK), which are involved in meiotic progression. Following completion of maturation, inactivation of MAPK by U0126 did not affect IP3R1 phosphorylation, although inactivation of p34cdc2 kinase by roscovitine dramatically reduced IP3R1 phosphorylation. Neither inhibitor affected total expression of IP3R1. Altogether, our results show that IP3R1 undergoes dynamic phosphorylation during maturation and this might underlie the generation of oscillations at fertilization. [source] Antimuscarinic antibodies in primary Sjögren's syndrome reversibly inhibit the mechanism of fluid secretion by human submandibular salivary acinar cellsARTHRITIS & RHEUMATISM, Issue 4 2006L. J. Dawson Objective Sjögren's syndrome (SS) is an autoimmune condition affecting salivary glands, for which a clearly defined pathogenic autoantibody has yet to be identified. Autoantibodies that bind to the muscarinic M3 receptors (M3R), which regulate fluid secretion in salivary glands, have been proposed in this context. However, there are no previous data that directly show antisecretory activity. This study was undertaken to investigate and characterize the antisecretory activity of anti-M3R. Methods Microfluorimetric Ca2+ imaging and patch clamp electrophysiologic techniques were used to measure the secretagogue-evoked increase in [Ca2+]i and consequent activation of Ca2+ -dependent ion channels in individual mouse and human submandibular acinar cells. Together, these techniques form a sensitive bioassay that was used to determine whether IgG isolated from patients with primary SS and from control subjects has antisecretory activity. Results IgG (2 mg/ml) from patients with primary SS reduced the carbachol-evoked increase in [Ca2+]i in both mouse and human acinar cells by ,50%. IgG from control subjects had no effect on the Ca2+ signal. Furthermore, the inhibitory action of primary SS patient IgG on the Ca2+ signal was acutely reversible. We repeated our observations using rabbit serum containing antibodies raised against the second extracellular loop of M3R and found an identical pattern of acutely reversible inhibition. Anti-M3R,positive serum had no effect on Ca2+ -dependent ion channel activation evoked by the direct intracellular infusion of inositol 1,4,5-triphosphate. Conclusion These observations show for the first time that IgG from patients with primary SS contains autoantibodies capable of damaging saliva production and contributing to xerostomia. The unusual but not unprecedented acute reversibility of the effects of anti-M3 autoantibodies is the subject of further research. [source] Dynamical analysis of the calcium signaling pathway in cardiac myocytes based on logarithmic sensitivity analysisBIOTECHNOLOGY JOURNAL, Issue 5 2008Tae-Hwan Kim Abstract Many cellular functions are regulated by the Ca2+ signal which contains specific information in the form of frequency, amplitude, and duration of the oscillatory dynamics. Any alterations or dysfunctions of components in the calcium signaling pathway of cardiac myocytes may lead to a diverse range of cardiac diseases including hypertrophy and heart failure. In this study, we have investigated the hidden dynamics of the intracellular Ca2+ signaling and the functional roles of its regulatory mechanism through in silico simulations and parameter sensitivity analysis based on an experimentally verified mathematical model. It was revealed that the Ca2+ dynamics of cardiac myocytes are determined by the balance among various system parameters. Moreover, it was found through the parameter sensitivity analysis that the self-oscillatory Ca2+ dynamics are most sensitive to the Ca2+ leakage rate of the sarcolemmal membrane and the maximum rate of NCX, suggesting that these two components have dominant effects on circulating the cytosolic Ca2+. [source] Inhibitory effects of N -acetylcysteine on the functional responses of human eosinophils in vitroCLINICAL & EXPERIMENTAL ALLERGY, Issue 5 2007M. Martinez-Losa Summary Background Oxidative stress appears to be relevant in the pathogenesis of inflammation in allergic diseases like bronchial asthma. Eosinophils are oxidant-sensitive cells considered as key effectors in allergic inflammation. Objective The aim of this work was to study the effects of the clinically used antioxidant N -acetyl- l -cysteine (NAC) on the functional responses of human-isolated eosinophils. Methods Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of NAC were investigated on the generation of reactive oxygen species (chemiluminescence and flow cytometry), Ca2+ signal (fluorimetry), intracellular glutathione (GSH; flow cytometry), p47phox,p67phox translocation (Western blot) and eosinophil cationic protein (ECP) release (radioimmunoassay). Results NAC (0.1,1 mm) inhibited the extracellular generation of oxygen species induced by N -formyl- l -methionyl- l -leucyl- l -phenylalanine (fMLP) and eotaxin (in the presence of IL-5) with ,logIC50 values of 3.61±0.03 and 3.36±0.09, respectively. Also, the intracellular generation of hydrogen peroxide was virtually abolished by NAC (0.5,1 mm). NAC (1 mm) did not alter the fMLP-induced Ca2+ signal but augmented the eosinophil content of reduced GSH and inhibited p47phox,p67phox translocation. NAC inhibited the release of ECP (,90% inhibition at 1 mm) from fMLP-activated eosinophils. Conclusion Inhibition by NAC of human eosinophil functions in vitro is potentially useful in the treatment of allergic inflammation. [source] Acute atrial arrhythmogenesis in murine hearts following enhanced extracellular Ca2+ entry depends on intracellular Ca2+ storesACTA PHYSIOLOGICA, Issue 2 2010Y. Zhang Abstract Aim:, To investigate the effect of increases in extracellular Ca2+ entry produced by the L-type Ca2+ channel agonist FPL-64176 (FPL) upon acute atrial arrhythmogenesis in intact Langendorff-perfused mouse hearts and its dependence upon diastolic Ca2+ release from sarcoplasmic reticular Ca2+ stores. Methods:, Confocal microscope studies of Fluo-3 fluorescence in isolated atrial myocytes were performed in parallel with electrophysiological examination of Langendorff-perfused mouse hearts. Results:, Atrial myocytes stimulated at 1 Hz and exposed to FPL (0.1 ,m) initially showed (<10 min) frequent, often multiple, diastolic peaks following the evoked Ca2+ transients whose amplitudes remained close to control values. With continued pacing (>10 min) this reverted to a regular pattern of evoked transients with increased amplitudes but in which diastolic peaks were absent. Higher FPL concentrations (1.0 ,m) produced sustained and irregular patterns of cytosolic Ca2+ activity, independent of pacing. Nifedipine (0.5 ,m), and caffeine (1.0 mm) and cyclopiazonic acid (CPA) (0.15 ,m) pre-treatments respectively produced immediate and gradual reductions in the F/F0 peaks. Such nifedipine and caffeine, or CPA pre-treatments, abolished, or reduced, the effects of 0.1 and 1.0 ,m FPL on cytosolic Ca2+ signals. FPL (1.0 ,m) increased the incidence of atrial tachycardia and fibrillation in intact Langendorff-perfused hearts without altering atrial effective refractory periods. These effects were inhibited by nifedipine and caffeine, and reduced by CPA. Conclusion:, Enhanced extracellular Ca2+ entry exerts acute atrial arrhythmogenic effects that is nevertheless dependent upon diastolic Ca2+ release. These findings complement reports that associate established, chronic, atrial arrhythmogenesis with decreased overall inward Ca2+ current. [source] Modulation of calcium signalling by intracellular organelles seen with targeted aequorinsACTA PHYSIOLOGICA, Issue 1 2009M. T. Alonso Abstract The cytosolic Ca2+ signals that trigger cell responses occur either as localized domains of high Ca2+ concentration or as propagating Ca2+ waves. Cytoplasmic organelles, taking up or releasing Ca2+ to the cytosol, shape the cytosolic signals. On the other hand, Ca2+ concentration inside organelles is also important in physiology and pathophysiology. Comprehensive study of these matters requires to measure [Ca2+] inside organelles and at the relevant cytosolic domains. Aequorins, the best-known chemiluminescent Ca2+ probes, are excellent for this end as they do not require stressing illumination, have a large dynamic range and a sharp Ca2+ -dependence, can be targeted to the appropriate location and engineered to have the proper Ca2+ affinity. Using this methodology, we have evidenced the existence in chromaffin cells of functional units composed by three closely interrelated elements: (1) plasma membrane Ca2+ channels, (2) subplasmalemmal endoplasmic reticulum and (3) mitochondria. These Ca2+ -signalling triads optimize Ca2+ microdomains for secretion and prevent propagation of the Ca2+ wave towards the cell core. Oscillatory cytosolic Ca2+ signals originate also oscillations of mitochondrial Ca2+ in several cell types. The nuclear envelope slows down the propagation of the Ca2+ wave to the nucleus and filters high frequencies. On the other hand, inositol-trisphosphate may produce direct release of Ca2+ to the nucleoplasm in GH3 pituitary cells, thus providing mechanisms for selective nuclear signalling. Aequorins emitting at different wavelengths, prepared by fusion either with green or red fluorescent protein, permit simultaneous and independent monitorization of the Ca2+ signals in different subcellular domains within the same cell. [source] Regulation of early response genes in pancreatic acinar cells: external calcium and nuclear calcium signalling aspectsACTA PHYSIOLOGICA, Issue 1 2009N. Fedirko Abstract Nuclear calcium signalling has been an important topic of investigation for many years and some aspects have been the subject of debate. Our data from isolated nuclei suggest that the nuclear pore complexes (NPCs) are open even after depletion of the Ca2+ store in the nuclear envelope (NE). The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P3 receptors [Ins(1,4,5)P3Rs], most likely on both sides of the NE and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P3Rs by Ins(1,4,5)P3. We have also investigated the possible consequences of nuclear calcium signals: the role of Ca2+ in the regulation of immediate early genes (IEG): c-fos, c-myc and c-jun in pancreatic acinar cells. Stimulation with Ca2+ -mobilizing agonists induced significant increases in levels of expression. Cholecystokinin (CCK) (10 nm) evoked a substantial rise in the expression levels, highly dependent on external Ca2+: the IEG expression level was lowest in Ca2+ -free solution, increased at the physiological level of 1 mm [Ca2+]o and was maximal at 10 mm [Ca2+]o, i.e.: 102 ± 22% and 163 ± 15% for c-fos; c-myc ,73 ± 13% and 106 ± 24%; c-jun ,49 ± 8% and 59 ± 9% at 1 and 10 mm of extracellular Ca2+ respectively. A low CCK concentration (10 pm) induced a small increase in expression. We conclude that extracellular Ca2+ together with nuclear Ca2+ signals induced by CCK play important roles in the induction of IEG expression. [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] Visualization of stochastic Ca2+ signals in the formed somites during the early segmentation period in intact, normally developing zebrafish embryosDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2009Christina F. Leung Localized Ca2+ signals were consistently visualized in the formed somites of intact zebrafish embryos during the early segmentation period. Unlike the regular process of somitogenesis, these signals were stochastic in nature with respect to time and location. They did, however, occur predominantly at the medial and lateral boundaries within the formed somites. Embryos were treated with modulators of [Ca2+]i to explore the signal generation mechanism and possible developmental function of the stochastic transients. Blocking elements in the phosphoinositol pathway eliminated the stochastic signals but had no obvious effect, stochastic or otherwise, on the formed somites. Such treatments did, however, result in the subsequently formed somites being longer in the mediolateral dimension. Targeted uncaging of buffer (diazo-2) or Ca2+ (NP-ethyleneglycoltetraacetic acid [EGTA]) in the presomitic mesoderm, resulted in a regular mediolateral lengthening and shortening, respectively, of subsequently formed somites. These data suggest a requirement for IP3 receptor-mediated Ca2+ release during convergence cell movements in the presomitic mesoderm, which appears to have a distinct function from that of the IP3 receptor-mediated stochastic Ca2+ signaling in the formed somites. [source] The patterns of spontaneous Ca2+ signals generated by ventral spinal neurons in vitro show time-dependent refinementEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2009Sara Sibilla Abstract Embryonic spinal neurons maintained in organotypic slice culture are known to mimic certain maturation-dependent signalling changes. With such a model we investigated, in embryonic mouse spinal segments, the age-dependent spatio-temporal control of intracellular Ca2+ signalling generated by neuronal populations in ventral circuits and its relation with electrical activity. We used Ca2+ imaging to monitor areas located within the ventral spinal horn at 1 and 2 weeks of in vitro growth. Primitive patterns of spontaneous neuronal Ca2+ transients (detected at 1 week) were typically synchronous. Remarkably, such transients originated from widespread propagating waves that became organized into large-scale rhythmic bursts. These activities were associated with the generation of synaptically mediated inward currents under whole-cell patch-clamp. Such patterns disappeared during longer culture of spinal segments: at 2 weeks in culture, only a subset of ventral neurons displayed spontaneous, asynchronous and repetitive Ca2+ oscillations dissociated from background synaptic activity. We observed that the emergence of oscillations was a restricted phenomenon arising together with the transformation of ventral network electrophysiological bursting into asynchronous synaptic discharges. This change was accompanied by the appearance of discrete calbindin immunoreactivity against an unchanged background of calretinin-positive cells. It is attractive to assume that periodic oscillations of Ca2+ confer a summative ability to these cells to shape the plasticity of local circuits through different changes (phasic or tonic) in intracellular Ca2+. [source] Chronic interleukin-6 alters the level of synaptic proteins in hippocampus in culture and in vivoEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2007Elly J. F. Vereyken Abstract There is now considerable evidence that the level of expression of the proinflammatory cytokine, interleukin-6 (IL-6), is increased in the central nervous system (CNS) during neuroinflammatory conditions such as occurs in neurological disorders and in disease and injury. However, our understanding of the consequences of increased expression of IL-6 on the CNS is still limited, especially with respect to the developing nervous system, which is known to be particularly vulnerable to environmental factors. To address this issue, we investigated the properties of cultured hippocampal neurons exposed chronically to IL-6 during the main period of morphological and physiological development, which occurs during the first 2 weeks of culture. IL-6 was tested at 500 U/mL, considered to reflect a pathophysiologic concentration. The morphological features of neuronal development in the control and IL-6-treated cultures appeared similar. However, Western blot analysis showed a significant reduction in the level of Group-II metabotropic receptors (mGluR2/3) and L-type Ca2+ channels in the IL-6-treated cultures. A similar reduction in mGluR2/3 and L-type Ca2+ channel protein was observed in transgenic mice that over-express IL-6 in the CNS through astrocyte production starting early in development. Analysis of Ca2+ signals produced by spontaneous synaptic network activity in the hippocampal cultures and effects of a mGluR2/3 agonist and antagonist showed that the reduced levels of mGluR2/3 impact on the functional properties of hippocampal synaptic network activity. These results have important implications relative to the mechanisms responsible for altered CNS function during conditions associated with increased levels of IL-6 in the CNS. [source] The Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006G. T. Macleod Abstract Voltage-gated Ca2+ channels in nerve terminals open in response to action potentials and admit Ca2+, the trigger for neurotransmitter release. The cacophony gene encodes the primary presynaptic voltage-gated Ca2+ channel in Drosophila motor-nerve terminals. The cacts2 mutant allele of cacophony is associated with paralysis and reduced neurotransmission at non-permissive temperatures but the basis for the neurotransmission deficit has not been established. The cacts2 mutation occurs in the cytoplasmic carboxyl tail of the ,1 -subunit, not within the pore-forming trans-membrane domains, making it difficult to predict the mutation's impact. We applied a Ca2+ -imaging technique at motor-nerve terminals of mutant larvae to test the hypothesis that the neurotransmission deficit is a result of impaired Ca2+ entry. Presynaptic Ca2+ signals evoked by single and multiple action potentials showed a temperature-dependent reduction. The amplitude of the reduction was sufficient to account for the neurotransmission deficit, indicating that the site of the cacts2 mutation plays a role in Ca2+ channel activity. As the mutation occurs in a motif conserved in mammalian high-voltage-activated Ca2+ channels, we used a heterologous expression system to probe the effect of this mutation on channel function. The mutation was introduced into rat Cav2.1 channels expressed in human embryonic kidney cells. Patch-clamp analysis of mutant channels at the physiological temperature of 37 °C showed much faster inactivation rates than for wild-type channels, demonstrating that the integrity of this motif is critical for normal Cav2.1 channel inactivation. [source] Bidirectional synaptic plasticity as a consequence of interdependent Ca2+ -controlled phosphorylation and dephosphorylation pathwaysEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2003Pablo D'Alcantara Abstract Postsynaptic Ca2+ signals of different amplitudes and durations are able to induce either long-lasting potentiation (LPT) or depression (LTD). The bidirectional character of synaptic plasticity may result at least in part from an increased or decreased responsiveness of the glutamatergic ,-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA-R) due to the modification of conductance and/or channel number, and controlled by the balance between the activities of phosphorylation and dephosphorylation pathways. AMPA-R depression can be induced by a long-lived Ca2+ signal of moderate amplitude favouring the activation of the dephosphorylation pathway, whereas a shorter but higher Ca2+ signal would induce AMPA-R potentiation resulting from the preferential activation of the phosphorylation pathway. Within the framework of a model involving calcium/calmodulin-dependent protein kinase II (CaMKII), calcineurin (PP2B) and type 1 protein phosphatase (PP1), we aimed at delineating the conditions allowing a biphasic U-shaped relationship between AMPA-R and Ca2+ signal amplitude, and thus bidirectional plasticity. Our theoretical analysis shows that such a property may be observed if the phosphorylation pathway: (i) displays higher cooperativity in its Ca2+ -dependence than the dephosphorylation pathway; (ii) displays a basal Ca2+ -independent activity; or (iii) is directly inhibited by the dephosphorylation pathway. Because the experimentally observed inactivation of CaMKII by PP1 accounts for this latter characteristic, we aimed at verifying whether a realistic model using reported parameters values can simulate the induction of either LTP or LTD, depending on the time and amplitude characteristics of the Ca2+ signal. Our simulations demonstrate that the experimentally observed bidirectional nature of Ca2+ -dependent synaptic plasticity could be the consequence of the PP1-mediated inactivation of CaMKII. [source] Intracellular Ca2+ responses and cell volume regulation upon cholinergic and purinergic stimulation in an immortalized salivary cell lineEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 3 2010Marit H. Aure Aure MH, Røed A, Kanli Galtung H. Intracellular Ca2+responses and cell volume regulation upon cholinergic and purinergic stimulation in an immortalized salivary cell line. Eur J Oral Sci 2010; 118: 237,244. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci The water channel aquaporin 5 (AQP5) seems to play a key role in salivary fluid secretion and appears to be critical in the cell volume regulation of acinar cells. Recently, the cation channel transient potential vanilloid receptor 4 (TRPV4) was shown to be functionally connected to AQP5 and also to cell volume regulation in salivary glands. We used the Simian virus 40 (SV40) immortalized cell line SMG C10 from the rat submandibular salivary gland to investigate the effect of ATP and the neurotransmitter analogue carbachol on Ca2+ signalling and cell volume regulation, as well as the involvement of TRPV4 in the responses. We used fura-2-AM imaging, cell volume measurements, and western blotting. Both carbachol and ATP increased the concentration of intracellular Ca2+, but no volume changes could be measured. Inhibition of TRPV4 with ruthenium red impaired both ATP- and carbachol-stimulated Ca2+ signals. Peak Ca2+ signalling during hyposmotic exposure was significantly decreased following inhibition of TRPV4, while the cells' ability to volume regulate appeared to be unaffected. These results show that in the SMG C10 cells, simulation of nervous stimulation did not induce cell swelling, although the cells had intact volume regulatory mechanisms. Furthermore, even though Ca2+ signals were not needed for this volume regulation, TRPV4 seems to play a role during ATP and carbachol stimulation. [source] EP1 and EP4 Receptors Mediate Exocytosis Evoked by Prostaglandin E2 in Guinea-Pig Antral Mucous CellsEXPERIMENTAL PHYSIOLOGY, Issue 4 2001Atsuko Ohnishi Effects of prostaglandin E2 (PGE2) on exocytosis of mucin were studied in mucous cells isolated from guinea-pig antrum using video-microscopy. Stimulation with PGE2 elicited a sustained increase in the frequency of exocytotic events in a dose-dependent manner, which was under regulation by both Ca2+ and cAMP. Stimulation with a selective prostanoid EP4 receptor agonist (ONO-AEI-329, 10 ,M), which activates cAMP signals, elicited a sustained increase in the frequency of exocytotic events (30% of that evoked by 1 ,M PGE2). Stimulation with an EP1 agonist (17-P-T-PGE2, 1 ,M), which activates Ca2+ signals, increased the frequency of exocytotic events to a lesser extent (5% of that evoked by 1 ,M PGE2), while addition of an EP1 antagonist (ONO-8713, 10 ,M) decreased the frequency of exocytotic events (approximately 40% of that evoked by 1 ,M PGE2). However, addition of the EP1 agonist potentiated the frequency of exocytotic events evoked by the EP4 agonist or forskolin (which elevates cAMP levels) and increased the sensitivity of the exocytotic events to forskolin. These results suggest that the Ca2+ signal activated via the EP1 receptor potentiates the cAMP-regulated exocytotic events activated via the EP4 receptor during PGE2 stimulation, by increasing the sensitivity of the exocytotic response to cAMP. In conclusion, exocytotic events in PGE2 -stimulated antral mucous cells were regulated by interactions between EP1 and EP4 receptors. [source] Effects of prolactin on intracellular calcium concentration and cell proliferation in human glioma cellsGLIA, Issue 3 2002Thomas Ducret Abstract Prolactin (PRL) has several physiological effects on peripheral tissues and the brain. This hormone acts via its membrane receptor (PRL-R) to induce cell differentiation or proliferation. Using reverse transcription,polymerase chain reaction (RT-PCR) combined with Southern blot analysis, we detected PRL-R transcripts in a human glioma cell line (U87-MG) and in primary cultured human glioblastoma cells. These transcripts were deleted or not in their extracellular domains. We examined the effects of PRL on intracellular free Ca2+ concentration ([Ca2+]i) in these cells in order to improve our understanding of the PRL transduction mechanism, which is still poorly documented. [Ca2+]i was measured by microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. Spatiotemporal aspects of PRL-induced Ca2+ signals were investigated using high-speed fluo-3 confocal imaging. We found that physiological concentrations (0.4,4 nM) of PRL-stimulated Ca2+ entry and intracellular Ca2+ mobilization via a tyrosine kinase,dependent mechanism. The two types of Ca2+ responses observed were distinguishable by their kinetics: one showing a slow (type I) and the other a fast (type II) increase in [Ca2+]i. The amplitude of PRL-induced Ca2+ increases may be sufficient to provoke several physiological responses, such as stimulating proliferation. Furthermore, PRL induced a dose-dependent increase in [3H]thymidine incorporation levels and in cellular growth and survival, detected by the MTT method. These data indicate that PRL induced mitogenesis of human glioma cells. GLIA 38:200,214, 2002. © 2002 Wiley-Liss, Inc. [source] Mitochondria and Ca2+ signalingJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2000Emil C. Toescu Abstract Mitochondria play a central role in cell homeostasis. Amongst others, one of the important functions of mitochondria is to integrate its metabolic response with one of the major signaling pathways - the Ca2+ signaling. Mitochondria are capable to sense the levels of cytosolic Ca2+ and generate mitochondrial Ca2+ responses. Specific mechanisms for both Ca2+ uptake and Ca2+ release exist in the mitochondrial membranes. In turn, the mitochondrial Ca2+ signals are able to produce changes in the mitochondrial function and metabolism, which provide the required level of functional integration. This essay reviews briefly the current available information regarding the mitochondrial Ca2+ transport systems and some of the functional consequences of mitochondrial Ca2+ uptake [source] Mitochondria and aging: a role for the permeability transition?AGING CELL, Issue 1 2004M. Crompton Summary When mitochondria are subjected to oxidative stress and relatively high [Ca2+], they undergo a ,permeability transition' in which the inner membrane becomes freely permeable to low-molecular-weight solutes. This phenomenon reflects reversible deformation of the adenine nucleotide translocase, the loss of its native gating properties and the stabilization of the deformed state by cyclophilin-D. The permeability transition may be a factor in cell dysfunction associated with aging. This can manifest in a number of ways ranging, in the most severe, from impaired energy transduction and compromised viability to more subtle influences on the propagation of Ca2+ signals. This article critically examines data relevant to this issue. [source] Cav1 L-type Ca2+ channel signaling complexes in neuronsJOURNAL OF NEUROCHEMISTRY, Issue 3 2008Irina Calin-Jageman Abstract Cav1 L-type Ca2+ channels play crucial and diverse roles in the nervous system. The pre- and post-synaptic functions of Cav1 channels not only depend on their intrinsic biophysical properties but also their dynamic regulation by a host of cellular influences. These include protein kinases and phosphatases, G-protein coupled receptors, scaffolding proteins, and Ca2+ -binding proteins. The cytoplasmic domains of the main pore forming ,1 subunit of Cav1 offer a number of binding sites for these modulators, permitting fast and localized regulation of Ca2+ entry. Through effects on Cav1 gating, localization, and coupling to effectors, protein modulators are efficiently positioned to adjust Cav1 Ca2+ signals that control neuronal excitability, synaptic plasticity, and gene expression. [source] Calcium channel subtypes differentially regulate fusion pore stability and expansionJOURNAL OF NEUROCHEMISTRY, Issue 4 2007Alvaro O. Ardiles Abstract Various studies have focused in the relative contribution of different voltage-activated Ca2+ channels (VACC) to total transmitter release. However, how Ca2+ entry through a given VACC subtype defines the pattern of individual exocytotic events remains unknown. To address this question, we have used amperometry in bovine chromaffin cells. L, N, and P/Q channels were individually or jointly blocked with furnidipine, ,-conotoxin GVIA, ,-agatoxin IVA, or ,-conotoxin MVIIC. The three channel types contributed similarly to cytosolic Ca2+ signals induced by 70 mmol/L K+. However, they exhibited different contributions to the frequency of exocytotic events and they were shown to differently regulate the final steps of the exocytosis. When compared with the other VACC subtypes, Ca2+ entry through P/Q channels effectively induced exocytosis, it decreased fusion pore stability and accelerated its expansion. Conversely, Ca2+ entry through N channels was less efficient in inducing exocytotic events, also slowing fusion pore expansion. Finally, Ca2+ entry through L channels inefficiently induced exocytosis, and the individual blockade of this channel significantly modified fusion pore dynamics. The distance between a given VACC subtype and the release sites could account for the differential effects of the distinct VACC on the fusion pore dynamics. [source] NSF binds calcium to regulate its interaction with AMPA receptor subunit GluR2JOURNAL OF NEUROCHEMISTRY, Issue 6 2007Jonathan G. Hanley Abstract N -ethylmaleimide-sensitive fusion protein (NSF) is essential for numerous Ca2+ -triggered vesicle trafficking events. It functions as a molecular chaperone to regulate trafficking protein complexes such as the soluble NSF attachment protein (SNAP) receptor complex and the ,-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-protein interacting with C-kinase (PICK1) complex. AMPAR trafficking is fundamental to processes of synaptic plasticity, which may underlie learning and memory. Changes in synaptic strength brought about by AMPAR trafficking are triggered by a post-synaptic influx of Ca2+, which may have numerous molecular targets including PICK1. NSF binds AMPAR subunit glutamate receptor subunit 2 (GluR2) and functions to maintain receptors at the synapse. In this study, it was showed that NSF is a Ca2+ -binding protein and that GluR2,NSF interactions are inhibited by the presence of 15 ,mol/L Ca2+. NSF Ca2+ -binding is reciprocally inhibited by the presence of GluR2 C-terminus. Mutant of NSF that binds Ca2+ with reduced affinity and binds GluR2 with reduced sensitivity to Ca2+ was identied. In addition, the interaction of ,SNAP with PICK1 is sensitive to Ca2+. This study demonstrates that the GluR2-NSF-,SNAP-PICK1 complex is regulated directly by Ca2+, allowing for the transduction of Ca2+ signals into concerted alterations in protein,protein interactions to bring about changes in AMPAR trafficking during synaptic plasticity. [source] Altered distribution of mitochondria impairs calcium homeostasis in rat hippocampal neurons in cultureJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Guang Jian Wang Abstract The specificity of Ca2+ signals is conferred in part by limiting changes in cytosolic Ca2+ to subcellular domains. Mitochondria play a major role in regulating Ca2+ in neurons and may participate in its spatial localization. We examined the effects of changes in the distribution of mitochondria on NMDA-induced Ca2+ increases. Hippocampal cultures were treated with the microtubule-destabilizing agent vinblastine, which caused the mitochondria to aggregate and migrate towards one side of the neuron. This treatment did not appear to decrease the energy status of mitochondria, as indicated by a normal membrane potential and pH gradient across the inner membrane. Moreover, electron microscopy showed that vinblastine treatment altered the distribution but not the ultrastructure of mitochondria. NMDA (200 µm, 1 min) evoked a greater increase in cytosolic Ca2+ in vinblastine-treated cells than in untreated cells. This increase did not result from impaired Ca2+ efflux, enhanced Ca2+ influx, opening of the mitochondrial permeability transition pore or altered function of endoplasmic reticulum Ca2+ stores. Ca2+ uptake into mitochondria was reduced by 53% in vinblastine-treated cells, as reported by mitochondrially targeted aequorin. Thus, the distribution of mitochondria maintained by microtubules is critical for buffering Ca2+ influx. A subset of mitochondria close to a Ca2+ source may preferentially regulate Ca2+ microdomains, set the threshold for Ca2+ -induced toxicity and participate in local ATP production. [source] Correlation of visinin-like-protein-1 expression with clinicopathological features in squamous cell carcinoma of the esophagusMOLECULAR CARCINOGENESIS, Issue 8 2006Carla Wickborn Abstract EF-hand Ca2+ -sensor proteins are key molecules for transducing Ca2+ signals into physiological answers and changes in cytosolic Ca2+ concentration control a variety of cellular responses, including proliferation, migration, and differentiation, which are relevant for tumor progression. The Ca2+ -sensor visinin-like protein-1 (VILIP-1) has recently attracted major interest due to its putative tumor suppressor function. Whereas VILIP-1 is expressed in normal skin, it is downregulated in skin tumors in a murine tumor model. The aim of this study was to investigate the expression of the Ca2+ -sensor VILIP-1 in squamous cell carcinoma of the esophagus and to correlate expression levels with clinicopathological features of the tumor. We examined VILIP-1 expression in 54 specimens of esophageal squamous cell carcinomas and 24 normal esophagus tissues, with immunohistochemical staining and immunofluorescence co-staining techniques. VILIP-1 expression was completely lost or significantly reduced in esophageal tumor tissue compared with normal squamous epithelium. Correlation with clinicopathological features indicated that there was significantly less VILIP-1 expression in lymph node positive (N,=,1) versus lymph node negative (N,=,0) tumors (P,=,0.002). Although there was no significant difference between highly (G1), moderately (G2) and poorly differentiated (G3) tumors (P,=,0.177), VILIP-1 expression in tumors is significantly correlated with the depth of tumor invasion (P,=,0.028 between T1, T2, T3, and T4). In contrast, co-staining with the proliferation marker Ki-67 indicated no significant correlation with proliferation rates in tumors (Ki-67 index of the tumor). In summary, the expression of the Ca2+ -sensor VILIP-1 was found to be lost during development of squamous cell carcinoma of the esophagus. The protein expression level significantly correlates with invasive features, such as depth of tumor invasion and local lymph node metastasis, but not with proliferation rate of tumor cells. © 2006 Wiley-Liss, Inc. [source] Leukocyte Pyruvate Kinase Expression is Reduced in Normal Human Pregnancy but not in Pre-eclampsiaAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 2 2010Yi Xu Citation Xu Y, Madsen-Bouterse SA, Romero R, Hassan S, Mittal P, Elfline M, Zhu A, Petty HR. Leukocyte pyruvate kinase expression is reduced in normal human pregnancy but not in pre-eclampsia. Am J Reprod Immunol 2010; 64: 137,151 Problem, Emerging evidence suggests that metabolism influences immune cell signaling and immunoregulation. To examine the immunoregulatory role of glycolysis in pregnancy, we evaluated the properties of pyruvate kinase in leukocytes from non-pregnant women and those with normal pregnancy and pre-eclampsia. Method of study, We evaluated pyruvate kinase expression in lymphocytes and neutrophils from non-pregnant, pregnant, and pre-eclampsia patients using fluorescence microscopy and flow cytometry. Leukocyte pyruvate kinase activity and pyruvate concentrations were also evaluated. To study pyruvate's effect on signaling, we labeled Jurkat T cells with Ca2+ dyes and measured cell responses in the presence of agents influencing intracellular pyruvate. Results, The expression of pyruvate kinase is reduced in lymphocytes and neutrophils from normal pregnant women in comparison with those of non-pregnant women and pre-eclampsia patients. Similarly, the activity of pyruvate kinase and the intracellular pyruvate concentration are reduced in leukocytes of normal pregnant women in comparison with non-pregnant women and women with pre-eclampsia. Using Jurkat cells as a model of leukocyte signaling, we have shown that perturbations of intracellular pyruvate influence Ca2+ signals. Conclusion, Normal pregnancy is characterized by reduced pyruvate kinase expression within lymphocytes and neutrophils. We speculate that reduced pyruvate kinase expression modifies immune cell responses due to reduced pyruvate concentrations. [source] The molecular architecture of the arachidonate-regulated Ca2+ -selective ARC channel is a pentameric assembly of Orai1 and Orai3 subunitsTHE JOURNAL OF PHYSIOLOGY, Issue 17 2009Olivier Mignen The activation of Ca2+ entry is a critical component of agonist-induced cytosolic Ca2+ signals in non-excitable cells. Although a variety of different channels may be involved in such entry, the recent identification of the STIM and Orai proteins has focused attention on the channels in which these proteins play a key role. To date, two distinct highly Ca2+ -selective STIM1-regulated and Orai-based channels have been identified , the store-operated CRAC channels and the store-independent arachidonic acid activated ARC channels. In contrast to the CRAC channels, where the channel pore is composed of only Orai1 subunits, both Orai1 and Orai3 subunits are essential components of the ARC channel pore. Using an approach involving the co-expression of a dominant-negative Orai1 monomer along with different preassembled concatenated Orai1 constructs, we recently demonstrated that the functional CRAC channel pore is formed by a homotetrameric assembly of Orai1 subunits. Here, we use a similar approach to demonstrate that the functional ARC channel pore is a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits. Expression of concatenated pentameric constructs with this stoichiometry results in the appearance of large currents that display all the key biophysical and pharmacological features of the endogenous ARC channels. They also replicate the essential regulatory characteristics of native ARC channels including specific activation by low concentrations of arachidonic acid, complete independence of store depletion, and an absolute requirement for the pool of STIM1 that constitutively resides in the plasma membrane. [source] | |||||||||||||||||||||||||