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At least 811 records · Page 45Linked to original sources
PubMed2002

Integration of calcium signals by calmodulin in rat sensory neurons.

We have used the fluorescently labelled calmodulin TA-CaM to follow calmodulin activation during depolarization of adult rat sensory neurons. Calcium concentration was measured simultaneously using the low affinity indicator Oregon Green BAPTA 5N. TA-CaM fluorescence increased during a 200-ms depolarization but then continued to increase during the subsequent 500 ms, even though total cell calcium was falling at this time. In the next few seconds TA-CaM fluorescence fell, but to a new elevated level that was then maintained for several tens of seconds. During a train of depolarizations that evoked a series of largely independent calcium changes TA-CaM fluorescence was in contrast raised for the duration of the train and for many tens of seconds afterwards. The presence of a peptide corresponding to the calmodulin binding domain of myosin light chain kinase significantly increased the depolarization-induced TA-CaM fluorescence increase and slowed the subsequent fall of fluorescence. We interpret the slow recovery component of the TA-CaM signal as reflecting the slow dissociation of calcium--calmodulin--calmodulin binding protein complexes. Our results show that after brief electrical activity calmodulin's interaction with calmodulin binding proteins persists for approximately one minute.

Action Potentials↗
PubMed2004

Acetylcholine-induced calcium signalling in adrenaline- and noradrenaline-containing adrenal chromaffin cells.

Adrenal chromaffin cells secrete catecholamines in response to cholinergic receptor activation by acetylcholine (ACh). Characteristics of Ca(2+) transients induced by activation of nicotinic (nAChRs) and muscarinic (mAChRs) receptors were analyzed using Fura-2 fluorescent measurements on rat chromaffin cells. We first found two populations of chromaffin cells, which differently responded on AChR stimulation. In the first group (n-cells), consecutive ACh applications evoked persistent Ca(2+) transients, whereas desensitizing transients were observed in the other group (m-cells). The AChR agonists and antagonists precisely imitated or abolished the ACh action on n- and m-type cells, respectively. Cytochemical staining showed that n-cells contained adrenaline, whereas m-cells-noradrenaline. Thus, for the first time we found that nAChRs and mAChRs are differentially expressed in adrenergic and noradrenergic chromaffin cells, respectively. Our data suppose that chromaffin cells can be differentially regulated by incoming ACh signals and in such way release different substances-adrenaline and noradrenaline.

Acetylcholine↗
PubMed2004

Calcium signalling during cell interactions with bacterial pathogens.

The ability of a pathogenic microorganism to cause a disease is conditioned by its ability to colonise a given niche and implicates the expression of specific determinants, i.e. virulence factors, that allow the pathogen to adhere to or to invade epithelial cells. Diseases may be induced by bacteria that replicate extracellularly and alter the epithelial mucosa by producing toxins. Ca2+ signalling has been implicated in various steps of bacterial infection. Bacterial toxins can induce an increase in free cytosolic Ca2+ in host cells, itself required for the toxin-mediated effects. Such toxins, by diffusing in the extracellular media, can act at a distance from the site of infection and have a global effect on the integrity of the epithelium by promoting the expression of pro-inflammatory cytokines. Independent on toxins, bacteria can induce Ca2+ responses that play a role in cytoskeletal rearrangements required for cell binding or internalisation of the microorganism. In some instances, invasion of the epithelium may be followed by bacterial access to deeper tissue, dissemination to other organs, and sometimes persistence in host cells in a parasitic-like mode. Such strategies underline the pathogen abilities to control innate defence cells such as professional phagocytes, and may implicate the diversion of Ca(2+)-dependent cellular processes that normally result in killing of the ingested bacteria. Finally, bacterial pathogens can also induce the cell release of ATP, a Ca2+ agonist, that may expand bacterial cell signalling by a paracrine or autocrine route, leading to enhanced colonisation or enhanced host cell responses to the invading microorganism.

Animals↗
PubMed2002

FcepsilonRI cross-linking-induced actin assembly mediates calcium signalling in RBL-2H3 mast cells.

1. To determine the role of actin assembly in the Ca(2+) signalling of mast cells activated by cross-linking of FcepsilonRI, we examined the effects of cytochalasin D, an inhibitor of actin polymerization. 2. In the RBL-2H3 cells, F-actin content was increased by sensitization with anti-dinitrophenol (DNP) IgE. In these cells, cytochalasin D induced oscillatory increases in cytosolic Ca(2+) ([Ca(2+)](i)); these increase were inhibited by jasplakinolide, a stabilizer of actin filaments. 3. In the IgE-sensitized RBL-2H3 cells, DNP-human serum albumin (DNP-HSA) augmented actin assembly. DNP-HSA also increased the production of IP(3), [Ca(2+)](i) and degranulation. Cytochalasin D enhanced all of these DNP-HSA-induced effects. 4. In a Ca(2+)-free solution, DNP-HSA induced a transient increase in [Ca(2+)](i), and this increase was accelerated by cytochalasin D. After cessation of the DNP-HSA-induced Ca(2+) release, the re-addition of Ca(2+) induced a sustained increase in [Ca(2+)](i) through capacitative Ca(2+) entry (CCE), and this increase was enhanced by cytochalasin D. 5 The effect of cytochalasin D in enhancing the CCE activity was prevented by xestospongin C. 6. In contrast, neither the Ca(2+) release nor the CCE activation that was induced by thapsigargin was affected by cytochalasin D. 7. These results suggest that actin de-polymerization stimulates the FcepsilonRI-mediated signalling to augment the release of Ca(2+) from the endoplasmic reticulum in RBL-2H3 cells.

Actins↗
PubMed2003

Calcium signaling in plant cells in altered gravity.

Changes in the intracellular Ca2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus-response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80th, a review highlighting the performed research and the possible significance of such Ca2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface tension --> alterations in the physicochemical properties of the membrane --> changes in membrane permeability, ion transport, membrane-bound enzyme activity, etc. --> metabolism rearrangements --> physiological responses. An analysis of data available on biological effects of altered gravity at the cellular level allows one to conclude that microgravity environment appears to affect cytoskeleton, carbohydrate and lipid metabolism, cell wall biogenesis via changes in enzyme activity and protein expression, with involvement of regulatory Ca2+ messenger system. Changes in Ca2+ influx/efflux and possible pathways of Ca2+ signaling in plant cell biochemical regulation in altered gravity are discussed.

Calcium↗
PubMed2005

2,4-Toluene diisocyanate suppressed the calcium signaling of ligand gated ion channel receptors.

Toluene diisocyanate (TDI) is widely used as a chemical intermediate in the production of polyurethane. TDI-induced asthma is related to its disturbance of acetylcholine activity in most affected workers, but the relevant mechanisms are unclear. Toluene diamine (TDA) is the main metabolite of TDI. TDI and TDA have in common the basic toluene structure. Toluene is an abused solvent affecting neuronal signal transduction by influencing the function of ligand gated ion channel receptors, including nicotinic acetylcholine receptors (nAChR), P2X purinoceptors, [gamma]-aminobutyric acid type A (GABAA) receptors, etc. To understand the actions of TDI and TDA on ligand gated ion channels, we investigated their effects on the changes of cytosolic calcium concentration ([Ca2+]c) while stimulating nAChR in human neuroblastoma SH-SY5Y cells, P2 purinoceptors in PC12 cells, and GABAA receptors in bovine adrenal chromaffin cells. Our results showed that both TDI and TDA suppressed the [Ca2+]c rise induced by the potent nicotinic ligand, epibatidine, in human SH-SY5Y cells. Similar but stronger suppression of ATP-induced [Ca2+]c rise occurred in PC12 cells. TDI and TDA also partially suppressed the [Ca2+] c rise induced by GABA in bovine adrenal chromaffin cells. We conclude that TDI and TDA can act on ligand gated ion channel receptors. Our findings suggest that TDI and TDA might have some neurotoxicity that will need to be investigated.

Animals↗
PubMed2000

Calcium signaling induced by adhesion mediates protein tyrosine phosphorylation and is independent of pHi.

Our goal was to evaluate early signaling events that occur as epithelial cells make initial contact with a substrate and to correlate them with phosphorylation. The corneal epithelium was chosen to study signaling events that occur with adhesion because it represents a simple system in which the tissue adheres to a basal lamina, is avascular, and is bathed by a tear film in which changes in the local environment are hypothesized to alter signaling. To perform these experiments we developed a novel adhesion assay to capture the changes in intracellular Ca(2+) and pH that occur as a cell makes its initial contact with a substrate. The first transient cytosolic Ca(2+) peak was detected only as the cell made contact with the substrate and was demonstrated using fluorimetric assays combined with live cell imaging. We demonstrated that this transient Ca(2+) peak always preceded a cytoplasmic alkalization. When the intracellular environment was modified, the initial response was altered. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N, N'N'-tetraacetic acid (BAPTA), an intracellular chelator, inhibited Ca(2+) mobilization, whereas benzamil altered the duration of the oscillations. Thapsigargin caused an initial Ca(2+) release followed by a long attenuated response. An inositol triphosphate analog induced a large initial response, whereas heparin inhibited Ca(2+) oscillations. Inhibitors of tyrosine phosphorylation did not alter the initial mobilization of cytosolic Ca(2) but clearance of cytosolic Ca(2+) was inhibited. Exposing corneal epithelial cells to BAPTA, benzamil, or thapsigargin also attenuated the phosphorylation of the focal adhesion protein paxillin. However, although heparin inhibited Ca(2+) oscillations, it did not alter phosphorylation of paxillin. These studies demonstrate that the initial contact that a cell makes with a substrate modulates the intracellular environment, and that changes in Ca(2+) mobilization can alter later signaling events such as the phosphorylation of specific adhesion proteins. These findings may have implications for wound repair and development.

Animals↗
PubMed2001

Strain-dependent differences in calcium signaling predict excitotoxicity in murine hippocampal neurons.

Commonly used inbred murine strains differ substantially in their vulnerability to excitotoxic insults. We investigated whether differences in dendritic Ca(2+) signaling could underlie the differential vulnerability of C57BL/6 (resistant to kainate excitotoxicity) and C57BL/10 strains (vulnerable). A striking difference was found in fine dendrite Ca(2+) responses after kainate exposure. Ca(2+) signals in distal dendrites were large in C57BL/10 neurons, and, if a threshold concentration of approximately 1.5 microm was reached, a region of sustained high Ca(2+) was established in the distal dendritic tree. This region then served as an initiation site for a degenerative cascade, producing high Ca(2+) levels that slowly spread to involve the entire neuron and led to cell death. Dendritic Ca(2+) signals in C57BL/6 neurons were much smaller and did not trigger these propagating secondary responses. Strain differences in dendritic Ca(2+) signaling were also evident after tetanic stimulation of Schaffer collaterals. Ca(2+) responses were much larger and peaked earlier in distal dendrites of C57BL/10 compared with those in C57BL/6. Neurons from both strains had similar membrane properties and responded to kainate with intense action potential firing. Degenerative Ca(2+) responses were seen in both strains if soma Ca(2+) could be sustained above 1.5 microm. The early phases of secondary Ca(2+) responses were attributable to Ca(2+) influx and were abolished rapidly by buffered zero Ca(2+) saline. Taken together, these data indicate that the substantial difference in Ca(2+) signals in fine distal dendrites and in the initiation of spreading secondary responses may underlie the selective vulnerability of these neurons to excitotoxic insults.

Action Potentials↗
PubMed2002

Altered mechanical properties and intracellular calcium signaling in cardiomyocytes from annexin 6 null-mutant mice.

Annexin 6 is one of a widely expressed family of calcium-binding proteins found in most mammalian tissues, including the heart. Several studies have implicated annexin 6 in the regulation of intracellular Ca2+ signaling, and it has been shown in vitro to act as a modulator of the sarcoplasmic reticulum Ca2+-release channel, cardiac L-type calcium channel, and Na+/Ca2+ exchanger. To investigate the role of annexin 6 in intact cardiomyocytes, we used mice containing a targeted disruption of the annexin 6 gene. Compared with controls, the myocytes of annexin 6 null-mutant mice demonstrated a significant increase in the rates of shortening and relengthening. Intracellular Ca2+ transients in fura-2-loaded cardiomyocytes induced by caffeine showed a normal baseline and amplitude, whereas the rate of decay was doubled in annexin 6-/- myocytes compared with control mice. These results show that annexin 6 knockout in the mouse leads to an increase in myocyte contractility and faster diastolic Ca2+ removal from the cytoplasm. In light of published findings showing annexin 6 to be down-regulated in end-stage heart failure, these results are consistent with a role for annexin 6 as a negative inotropic factor in the regulation of cardiomyocyte mechanics.

Animals↗
PubMed2006

Measurement and analysis of calcium signaling in heterogeneous cell cultures.

High-content imaging platforms capable of studying kinetic responses at a single-cell level have elevated kinetic recording techniques from labor-intensive low-throughput experiments to potential high-throughput screening assays. We have applied this technology to the investigation of heterogeneous cell cultures derived from primary neural tissue. The neuronal cultures mature into a coupled network and display spontaneous oscillations in intracellular calcium, which can be modified by the addition of pharmacological agents. We have developed algorithms to perform Fourier analysis and quantify both the degree of synchronization and the effects of modulators on the oscillations. Functional and phenotypic experiments can be combined using this approach. We have used post-hoc immunolabeling to identify subpopulations of cells in cocultures and to dissect the calcium responses of these cells from the population response. The combination of these techniques represents a powerful tool for drug discovery.

Algorithms↗
PubMed2003

Calcium signaling to nucleus via store-operated system during cell cycle in retinal neuroepithelium.

Intracellular Ca(2+) is a regulatory signal for cell proliferation. To reveal Ca(2+) signal dynamics during cell cycle, we applied Ca(2+) fluorescence imaging to the neural retina of chick embryo, where the soma changes its position during the cell cycle. Purinoceptors were stimulated to cause Ca(2+) release from Ca(2+) stores, since the purinoceptor activation promotes DNA synthesis. Ca(2+) rises occurred in the nucleoplasm of cells at around S-phase. The soma of S-phase cell is located in the inner layer of the retinal neuroepithelium and issues an outer process, which extends to the ventricular surface. Fluorescent probes for endoplasmic reticulum (ER) showed that the ERs in the outer process and the nuclear envelope (NE) or peri-nuclear ER formed the Ca(2+) store. Depletion of the Ca(2+) store induced capacitative Ca(2+) entry (CCE), which caused Ca(2+) rises in the terminal of outer process and soma. The store-operated Ca(2+) signaling declined in M-phase cells and postmitotic cells (retinal ganglion cells (RGCs)) with the loss of the outer process. These results suggest that the Ca(2+) signaling to nucleus via the store-operated system including the ERs in the outer process is crucial for the cell cycle progression in the retinal neuroepithelium.

Adenosine Triphosphate↗
PubMed2000

Calcium signalling in the guidance of nerve growth by netrin-1.

Pathfinding by growing axons in the developing nervous system is guided by diffusible or bound factors that attract or repel the axonal growth cone. The cytoplasmic signalling mechanisms that trigger the responses of the growth cone to guidance factors are mostly unknown. Previous studies have shown that the level and temporal patterns of cytoplasmic Ca2+ can regulate the rate of growth-cone extension in vitro and in vivo. Here we report that Ca2+ also mediates the turning behaviour of the growth cones of cultured Xenopus neurons that are induced by an extracellular gradient of netrin-1, an established diffusible guidance factor in vivo. The netrin-1-induced turning response depends on Ca2+ influx through plasma membrane Ca2+ channels, as well as Ca2+-induced Ca2+ release from cytoplasmic stores. Reduction of Ca2+ signals by blocking either of these two Ca2+ sources converted the netrin-1-induced response from attraction to repulsion. Activation of Ca2+-induced Ca2+ release from internal stores with a gradient of ryanodine in the absence of netrin-1 was sufficient to trigger either attractive or repulsive responses, depending on the ryanodine concentration used. These results support the model that cytoplasmic Ca2+ signals mediate growth-cone guidance by netrin-1, and different patterns of Ca2+ elevation trigger attractive and repulsive turning responses.

Animals↗
PubMed2002

Mechanisms regulating expression of the tumor necrosis factor-related light gene. Role of calcium-signaling pathway in the transcriptional control.

LIGHT (TNFSF14) is a newly identified tumor necrosis factor superfamily member involved in the regulation of immune responses by control of activation, maturation, and survival of immune effector cells. Despite the immunological relevance of the LIGHT protein, little knowledge is available as to how light gene expression is regulated. In T-lymphocytes, most LIGHT surface expression and transcript accumulation occurs after T cell activation. In this study, we have shown that these events are blocked at the transcriptional level by cyclosporin A, an immuno-suppressive drug. Besides, we identified a role for Ca2+ -signaling pathways and NFAT transcription factors in T cell activation-induced LIGHT expression. To further investigate this process, we have identified, cloned, and characterized a 2.1-kilobase 5'-flanking DNA genomic fragment from the human light gene. We have shown the transcriptional activity of the herein-identified minimal 5' regulatory region of human light gene parallels the endogenous expression of light in T cells. Moreover, we demonstrated that induced LIGHT promoter activity can be equally blocked by cyclosporin A treatment or dominant negative NFAT overexpression and further identified by site-directed mutagenesis and electrophoretic mobility supershift analysis of a NFAT transcription factor binding site within the human light minimal promoter. Finally, Sp1 and Ets1 binding sites were identified and shown to regulate light basal promoter activity. Thus, the present study establishes a molecular basis to further understand the mechanisms governing human light gene expression and, consequently, could potentially lead to novel therapeutic manipulations that control the signaling cascade, resulting in LIGHT production in conditions characterized by immunopathologic activation of T cells.

Base Sequence↗
PubMedDate not supplied

Modulation of intracellular calcium signaling and mitochondrial function in cultured osteoblastic cells by dexamethasone and celecoxib during mechanical stimulation.

OBJECTIVE: Evaluation of potentially therapeutically relevant effects of dexamethasone and celecoxib on crucial parameters of bone physiology during and following mechanical stimulation in cultured osteoblasts. METHODS: An in vitro mechanical stimulation model based on the rat osteogenic cell line UMR-106 was developed to investigate glucocorticoid (dexamethasone) and selective COX-2 inhibitor (celecoxib) induced changes in the intracellular calcium concentration ([Ca2+]i) and mitochondrial membrane potential (deltapsi(m)). Microfluorometric techniques were applied to monitor [Ca2+]i (Fura-2 AM) and deltapsi(m) (rhodamine 123) online as the main parameters of the actual cellular metabolism. RESULTS: Basal [Ca2+] was found to be 92.2 +/- 3.7 nM and increased tip to 711 +/- 27 nM during mechanical stimulation under controlled conditions. Addition of 100 nM dexamethasone or 10 microM celecoxib for 24 h suppressed the increase in [Ca2+]i significantly to 530 +/- 33 nM and 546 +/- 39 nM, respectively. Dexamethasone significantly reduced, but celecoxib significantly increased the spread velocity of the mechanically induced intracellular calcium wave. Furthermore, the effects induced by dexamethasone were amplified during the inhibition of gap junction coupling and diminished following enlarged gap junction coupling. In contrast, the modulation of gap junction coupling exerted only a minor influence on the celecoxib-induced effects. Short-term application of dexamethasone (5 min) caused significantly reduced mechanically induced depolarization of the mitochondrial membrane, but long-term application (24 h) did not. In contrast, only the long-term application (24 h) of celecoxib caused such depolarization. CONCLUSION: The observed effects of dexamethasone and celecoxib on mechanically induced changes in [Ca2+] and deltapsi(m) are suggested to result from short-term changes in membrane characteristics and long-term changes in protein synthesis. This indicates an influence of these drugs on cell-to-cell communication and metabolism that may be therapeutically relevant.

Animals↗
PubMed2006

Effect of gamma-secretase inhibitors on muscarinic receptor-mediated calcium signaling in human salivary epithelial cells.

Altered intracellular Ca(2+) signaling has been observed in cells derived from Alzheimer's disease patients, and a possible link between gamma-secretase activity and the content of intracellular Ca(2+) stores has been suggested. To test this hypothesis we studied the effects of several gamma-secretase inhibitors on muscarinic receptor-mediated intracellular calcium release in the human salivary gland cell line HSG. Although several inhibitors in the peptide aldehyde class partially blocked carbachol-induced Ca(2+) transients, these effects did not appear to be due to gamma-secretase inhibition, and overall we found no evidence that inhibition of gamma-secretase activity had any significant effect on agonist-induced intracellular calcium release in HSG cells. In complementary experiments with presenilin-null cells we found that the reconstitution of gamma-secretase activity by transfection with wild-type presenilin 1 likewise had no significant effect on thapsigargin-induced Ca(2+) release. In a test of the specific hypothesis that the level of APP intracellular domain (AICD), the intracellular fragment of the beta-amyloid precursor protein (APP) resulting from gamma-secretase cleavage, can modulate the Ca(2+) content of the endoplasmic reticulum, we were unable to demonstrate any effect of APP small interfering RNA on the magnitude of carbachol-induced intracellular calcium release in HSG cells. Together our data cast considerable doubt on the hypothesis that there is a direct link between gamma-secretase activity and the content of intracellular Ca(2+) stores.

Amyloid Precursor Protein Secretases↗