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Sample records for intracellular calcium signalling

  1. Stochastic models of intracellular calcium signals

    NASA Astrophysics Data System (ADS)

    Rüdiger, Sten

    2014-01-01

    Cellular signaling operates in a noisy environment shaped by low molecular concentrations and cellular heterogeneity. For calcium release through intracellular channels-one of the most important cellular signaling mechanisms-feedback by liberated calcium endows fluctuations with critical functions in signal generation and formation. In this review it is first described, under which general conditions the environment makes stochasticity relevant, and which conditions allow approximating or deterministic equations. This analysis provides a framework, in which one can deduce an efficient hybrid description combining stochastic and deterministic evolution laws. Within the hybrid approach, Markov chains model gating of channels, while the concentrations of calcium and calcium binding molecules (buffers) are described by reaction-diffusion equations. The article further focuses on the spatial representation of subcellular calcium domains related to intracellular calcium channels. It presents analysis for single channels and clusters of channels and reviews the effects of buffers on the calcium release. For clustered channels, we discuss the application and validity of coarse-graining as well as approaches based on continuous gating variables (Fokker-Planck and chemical Langevin equations). Comparison with recent experiments substantiates the stochastic and spatial approach, identifies minimal requirements for a realistic modeling, and facilitates an understanding of collective channel behavior. At the end of the review, implications of stochastic and local modeling for the generation and properties of cell-wide release and the integration of calcium dynamics into cellular signaling models are discussed.

  2. Control of Intracellular Calcium Signaling as a Neuroprotective Strategy

    PubMed Central

    Duncan, R. Scott; Goad, Daryl L.; Grillo, Michael A.; Kaja, Simon; Payne, Andrew J.; Koulen, Peter

    2010-01-01

    Both acute and chronic degenerative diseases of the nervous system reduce the viability and function of neurons through changes in intracellular calcium signaling. In particular, pathological increases in the intracellular calcium concentration promote such pathogenesis. Disease involvement of numerous regulators of intracellular calcium signaling located on the plasma membrane and intracellular organelles has been documented. Diverse groups of chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified as potential neuroprotectants. The present review summarizes the discovery, mechanisms and biological activity of neuroprotective molecules targeting proteins that control intracellular calcium signaling to preserve or restore structure and function of the nervous system. Disease relevance, clinical applications and new technologies for the identification of such molecules are being discussed. PMID:20335972

  3. Calcium, channels, intracellular signaling and autoimmunity.

    PubMed

    Izquierdo, Jorge-Hernán; Bonilla-Abadía, Fabio; Cañas, Carlos A; Tobón, Gabriel J

    2014-01-01

    Calcium (Ca²⁺) is an important cation able to function as a second messenger in different cells of the immune system, particularly in B and T lymphocytes, macrophages and mastocytes, among others. Recent discoveries related to the entry of Ca²⁺ through the store-operated calcium entry (SOCE) has opened a new investigation area about the cell destiny regulated by Ca²⁺ especially in B and T lymphocytes. SOCE acts through calcium-release-activated calcium (CRAC) channels. The function of CRAC depends of two recently discovered regulators: the Ca²⁺ sensor in the endoplasmic reticulum or stromal interaction molecule (STIM-1) and one subunit of CRAC channels called Orai1. This review focuses on the role of Ca²⁺ signals in B and T lymphocytes functions, the signalling pathways leading to Ca²⁺ influx, and the relationship between Ca²⁺ signals and autoimmune diseases. PMID:24001934

  4. Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells.

    PubMed

    Péntek, Adrienn; Pászty, Katalin; Apáti, Ágota

    2016-01-01

    Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well. PMID:24482125

  5. A Dual Wavelength Microfluorimeter for Measuring Fast Intracellular Calcium Signals

    NASA Astrophysics Data System (ADS)

    Hogan, Perry M.; Besch, Stephen R.

    1995-06-01

    A dual excitation microfluorimeter is described for measuring rapidly changing, intracellular calcium signals. A spinning sector wheel is used in conjunction with a beam masking device to provide rapid, efficient switching between the 2 excitation wavelengths. Exposure intervals as short as 120 [mu]s can be achieved, yielding ratio samples at a rate of 6 kHz. Emission photons are collected using a photomultiplier tube operating in counting mode. When tested using FURA-2 as the calcium reporting dye, throughput noise in the system is demonstrated to be due to the statistical fluctuation inherent in photon counting. An example of the operation of the system, using a guinea pig cardiac myocyte, demonstrates that sufficient ratio data may be acquires to fully characterize the fastest components of the intracellular calcium signal.

  6. Evolution of the Calcium-Based Intracellular Signaling System.

    PubMed

    Marchadier, Elodie; Oates, Matt E; Fang, Hai; Donoghue, Philip C J; Hetherington, Alistair M; Gough, Julian

    2016-01-01

    To progress our understanding of molecular evolution from a collection of well-studied genes toward the level of the cell, we must consider whole systems. Here, we reveal the evolution of an important intracellular signaling system. The calcium-signaling toolkit is made up of different multidomain proteins that have undergone duplication, recombination, sequence divergence, and selection. The picture of evolution, considering the repertoire of proteins in the toolkit of both extant organisms and ancestors, is radically different from that of other systems. In eukaryotes, the repertoire increased in both abundance and diversity at a far greater rate than general genomic expansion. We describe how calcium-based intracellular signaling evolution differs not only in rate but in nature, and how this correlates with the disparity of plants and animals. PMID:27358427

  7. Evolution of the Calcium-Based Intracellular Signaling System

    PubMed Central

    Marchadier, Elodie; Oates, Matt E.; Fang, Hai; Donoghue, Philip C.J.; Hetherington, Alistair M.; Gough, Julian

    2016-01-01

    To progress our understanding of molecular evolution from a collection of well-studied genes toward the level of the cell, we must consider whole systems. Here, we reveal the evolution of an important intracellular signaling system. The calcium-signaling toolkit is made up of different multidomain proteins that have undergone duplication, recombination, sequence divergence, and selection. The picture of evolution, considering the repertoire of proteins in the toolkit of both extant organisms and ancestors, is radically different from that of other systems. In eukaryotes, the repertoire increased in both abundance and diversity at a far greater rate than general genomic expansion. We describe how calcium-based intracellular signaling evolution differs not only in rate but in nature, and how this correlates with the disparity of plants and animals. PMID:27358427

  8. Cyst formation following disruption of intracellular calcium signaling

    PubMed Central

    Kuo, Ivana Y.; DesRochers, Teresa M.; Kimmerling, Erica P.; Nguyen, Lily; Ehrlich, Barbara E.; Kaplan, David L.

    2014-01-01

    Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca2+) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca2+-activated Ca2+ channel of the endoplasmic reticulum. We hypothesize that Ca2+ signaling through PC2, or other intracellular Ca2+ channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca2+ signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca2+ signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca2+ transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca2+ signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca2+ signaling lead to ADPKD cysts. PMID:25228769

  9. Acidic calcium stores open for business: expanding the potential for intracellular Ca2+ signaling

    PubMed Central

    Patel, Sandip; Docampo, Roberto

    2010-01-01

    Changes in cytosolic calcium concentration are crucial for a variety of cellular processes in all cells. It has long been appreciated that calcium is stored and released from intracellular calcium stores such as the endoplasmic reticulum. However, emerging evidence indicates that calcium is also dynamically regulated by a seemingly disparate collection of acidic organelles. Here, we review the defining features of these acidic calcium stores and highlight recent progress in understanding the mechanisms of uptake and release of calcium from these stores. We also examine the nature of calcium buffering within the stores and summarize the physiological and patho-physiological significance of these ubiquitous organelles in calcium signaling. PMID:20303271

  10. Mitochondrial transporters as novel targets for intracellular calcium signaling.

    PubMed

    Satrústegui, Jorgina; Pardo, Beatriz; Del Arco, Araceli

    2007-01-01

    Ca(2+) signaling in mitochondria is important to tune mitochondrial function to a variety of extracellular stimuli. The main mechanism is Ca(2+) entry in mitochondria via the Ca(2+) uniporter followed by Ca(2+) activation of three dehydrogenases in the mitochondrial matrix. This results in increases in mitochondrial NADH/NAD ratios and ATP levels and increased substrate uptake by mitochondria. We review evidence gathered more than 20 years ago and recent work indicating that substrate uptake, mitochondrial NADH/NAD ratios, and ATP levels may be also activated in response to cytosolic Ca(2+) signals via a mechanism that does not require the entry of Ca(2+) in mitochondria, a mechanism depending on the activity of Ca(2+)-dependent mitochondrial carriers (CaMC). CaMCs fall into two groups, the aspartate-glutamate carriers (AGC) and the ATP-Mg/P(i) carriers, also named SCaMC (for short CaMC). The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle, and citrin, the liver AGC, is also a member of the urea cycle. Both types of CaMCs are activated by Ca(2+) in the intermembrane space and function together with the Ca(2+) uniporter in decoding the Ca(2+) signal into a mitochondrial response. PMID:17237342

  11. Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells

    PubMed Central

    Hwang, Jae Youn; Yoon, Chi Woo; Lim, Hae Gyun; Park, Jin Man; Yoon, Sangpil; Lee, Jungwoo; Shung, K. Kirk

    2016-01-01

    Extracellular matrix proteins such as fibronectin (FNT) play crucial roles in cell proliferation, adhesion, and migration. For better understanding of these associated cellular activities, various microscopic manipulation tools have been used to study their intracellular signaling pathways. Recently, it has appeared that acoustic tweezers may possess similar capabilities in the study. Therefore, we here demonstrate that our newly developed acoustic tweezers with a high-frequency lithium niobate ultrasonic transducer have potentials to study intracellular calcium signaling by FNT-binding to human breast cancer cells (SKBR-3). It is found that intracellular calcium elevations in SKBR-3 cells, initially occurring on the microbead-contacted spot and then eventually spreading over the entire cell, are elicited by attaching an acoustically trapped FNT-coated microbead. Interestingly, they are suppressed by either extracellular calcium elimination or phospholipase C (PLC) inhibition. Hence, this suggests that our acoustic tweezers may serve as an alternative tool in the study of intracellular signaling by FNT-binding activities. PMID:26150401

  12. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling

    PubMed Central

    Grant, Michael P.; Cavanaugh, Alice; Breitwieser, Gerda E.

    2015-01-01

    Calcium sensing receptors (CaSR) interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium. PMID:26317416

  13. Intracellular calcium signals display an avalanche-like behavior over multiple lengthscales

    PubMed Central

    Lopez, Lucía; Piegari, Estefanía; Sigaut, Lorena; Ponce Dawson, Silvina

    2012-01-01

    Many natural phenomena display “self-organized criticality” (SOC), (Bak et al., 1987). This refers to spatially extended systems for which patterns of activity characterized by different lengthscales can occur with a probability density that follows a power law with pattern size. Differently from power laws at phase transitions, systems displaying SOC do not need the tuning of an external parameter. Here we analyze intracellular calcium (Ca2+) signals, a key component of the signaling toolkit of almost any cell type. Ca2+ signals can either be spatially restricted (local) or propagate throughout the cell (global). Different models have suggested that the transition from local to global signals is similar to that of directed percolation. Directed percolation has been associated, in turn, to the appearance of SOC. In this paper we discuss these issues within the framework of simple models of Ca2+ signal propagation. We also analyze the size distribution of local signals (“puffs”) observed in immature Xenopus Laevis oocytes. The puff amplitude distribution obtained from observed local signals is not Gaussian with a noticeable fraction of large size events. The experimental distribution of puff areas in the spatio-temporal record of the image has a long tail that is approximately log-normal. The distribution can also be fitted with a power law relationship albeit with a smaller goodness of fit. The power law behavior is encountered within a simple model that includes some coupling among individual signals for a wide range of parameter values. An analysis of the model shows that a global elevation of the Ca2+ concentration plays a major role in determining whether the puff size distribution is long-tailed or not. This suggests that Ca2+-clearing from the cytosol is key to determine whether IP3-mediated Ca2+ signals can display a SOC-like behavior or not. PMID:22969730

  14. Intracellular calcium signals display an avalanche-like behavior over multiple lengthscales.

    PubMed

    Lopez, Lucía; Piegari, Estefanía; Sigaut, Lorena; Ponce Dawson, Silvina

    2012-01-01

    Many natural phenomena display "self-organized criticality" (SOC), (Bak et al., 1987). This refers to spatially extended systems for which patterns of activity characterized by different lengthscales can occur with a probability density that follows a power law with pattern size. Differently from power laws at phase transitions, systems displaying SOC do not need the tuning of an external parameter. Here we analyze intracellular calcium (Ca(2+)) signals, a key component of the signaling toolkit of almost any cell type. Ca(2+) signals can either be spatially restricted (local) or propagate throughout the cell (global). Different models have suggested that the transition from local to global signals is similar to that of directed percolation. Directed percolation has been associated, in turn, to the appearance of SOC. In this paper we discuss these issues within the framework of simple models of Ca(2+) signal propagation. We also analyze the size distribution of local signals ("puffs") observed in immature Xenopus Laevis oocytes. The puff amplitude distribution obtained from observed local signals is not Gaussian with a noticeable fraction of large size events. The experimental distribution of puff areas in the spatio-temporal record of the image has a long tail that is approximately log-normal. The distribution can also be fitted with a power law relationship albeit with a smaller goodness of fit. The power law behavior is encountered within a simple model that includes some coupling among individual signals for a wide range of parameter values. An analysis of the model shows that a global elevation of the Ca(2+) concentration plays a major role in determining whether the puff size distribution is long-tailed or not. This suggests that Ca(2+)-clearing from the cytosol is key to determine whether IP(3)-mediated Ca(2+) signals can display a SOC-like behavior or not. PMID:22969730

  15. Spatiotemporal properties of intracellular calcium signaling in osteocytic and osteoblastic cell networks under fluid flow.

    PubMed

    Jing, Da; Lu, X Lucas; Luo, Erping; Sajda, Paul; Leong, Pui L; Guo, X Edward

    2013-04-01

    Mechanical stimuli can trigger intracellular calcium (Ca(2+)) responses in osteocytes and osteoblasts. Successful construction of bone cell networks necessitates more elaborate and systematic analysis for the spatiotemporal properties of Ca(2+) signaling in the networks. In the present study, an unsupervised algorithm based on independent component analysis (ICA) was employed to extract the Ca(2+) signals of bone cells in the network. We demonstrated that the ICA-based technology could yield higher signal fidelity than the manual region of interest (ROI) method. Second, the spatiotemporal properties of Ca(2+) signaling in osteocyte-like MLO-Y4 and osteoblast-like MC3T3-E1 cell networks under laminar and steady fluid flow stimulation were systematically analyzed and compared. MLO-Y4 cells exhibited much more active Ca(2+) transients than MC3T3-E1 cells, evidenced by more Ca(2+) peaks, less time to the 1st peak and less time between the 1st and 2nd peaks. With respect to temporal properties, MLO-Y4 cells demonstrated higher spike rate and Ca(2+) oscillating frequency. The spatial intercellular synchronous activities of Ca(2+) signaling in MLO-Y4 cell networks were higher than those in MC3T3-E1 cell networks and also negatively correlated with the intercellular distance, revealing faster Ca(2+) wave propagation in MLO-Y4 cell networks. Our findings show that the unsupervised ICA-based technique results in more sensitive and quantitative signal extraction than traditional ROI analysis, with the potential to be widely employed in Ca(2+) signaling extraction in the cell networks. The present study also revealed a dramatic spatiotemporal difference in Ca(2+) signaling for osteocytic and osteoblastic cell networks in processing the mechanical stimulus. The higher intracellular Ca(2+) oscillatory behaviors and intercellular coordination of MLO-Y4 cells provided further evidences that osteocytes may behave as the major mechanical sensor in bone modeling and remodeling

  16. Prolonged Oxaliplatin Exposure Alters Intracellular Calcium Signaling: A New Mechanism To Explain Oxaliplatin-Associated Peripheral Neuropathy

    PubMed Central

    Schulze, Christin; McGowan, Margit; Jordt, Sven; Ehrlich, Barbara E

    2012-01-01

    Oxaliplatin is a platinum based cytotoxic agent commonly used to treat colorectal cancers. Despite its effectiveness, oxaliplatin administration is associated with the development of cold-induced peripheral neuropathy. This potentially permanent side effect is provoked by cold exposure and can range from mild and self limited to severe and debilitating. Even with tumor shrinkage, these painful side effects can force dose-reduction or discontinuation of treatment. Neither the mechanism of action of oxaliplatin nor that of cold-induced neuropathy is understood. Paclitaxel, an entirely different chemotherapeutic agent used to treat a variety of malignancies, also is associated with the development of peripheral neuropathy. Unlike oxaliplatin, neurotoxicity arising from paclitaxel treatment is better understood and was found to have profound effects on intracellular calcium signaling (1,2). In this study we examined the effects of oxaliplatin on calcium signaling pathways and found that acute exposure of either a neuroblastoma cell line or primary neurons with therapeutic concentrations of oxaliplatin had no effect on intracellular calcium signaling. We also found that cellular temperature sensors (TRP channels) were also not activated by oxaliplatin. Interestingly, prolonged exposure of oxaliplatin sensitized cells to subsequent stimuli and enhanced the magnitude of intracellular calcium responses. Taken together, our results suggest that acute oxaliplatin exposure will not induce abnormal calcium signaling but oxaliplatin-primed cells do exhibit enhanced sensitivity. These findings provide new insight to the mechanism behind oxaliplatin-induced neuropathy. PMID:21859566

  17. Hyperoside regulates the level of thymic stromal lymphopoietin through intracellular calcium signalling.

    PubMed

    Han, Na-Ra; Go, Ji-Hyun; Kim, Hyung-Min; Jeong, Hyun-Ja

    2014-07-01

    Hyperoside (HYP) is the principle active component of Crataegus pinnatifida. Thymic stromal lymphopoietin (TSLP) plays a vital role in the pathogenesis of allergic reactions. Here, we investigated how HYP regulates the levels of TSLP in a human mast cell line, HMC-1 cells. We analyzed the levels of TSLP by treatment with HYP in phorbol myristate acetate plus calcium ionophore A23187-stimulated HMC-1 cells with ELISA and a polymerase chain reaction analysis. We also analyzed the pathway that HYP regulates TSLP by measuring the level of fluorescent intracellular calcium and using a Western blot analysis. HYP decreased the level of intracellular calcium in stimulated HMC-1 cells. It also significantly decreased the production and mRNA expression of TSLP in stimulated HMC-1 cells. It significantly decreased the levels of receptor-interacting protein 2 and active caspase-1 in stimulated HMC-1 cells. HYP significantly decreased the translocation of NF-κB into the nucleus and degradation of IκBα in the cytoplasm in stimulated HMC-1 cells. Furthermore, it significantly decreased the production and mRNA expression of interleukin-1β and interleukin-6 in stimulated HMC-1 cells. Taken together, our findings establish HYP as a potential agent for the treatment of allergic reactions. PMID:24338918

  18. Modelling intracellular competition for calcium: kinetic and thermodynamic control of different molecular modes of signal decoding

    PubMed Central

    Antunes, Gabriela; Roque, Antonio C.; Simoes de Souza, Fabio M.

    2016-01-01

    Frequently, a common chemical entity triggers opposite cellular processes, which implies that the components of signalling networks must detect signals not only through their chemical natures, but also through their dynamic properties. To gain insights on the mechanisms of discrimination of the dynamic properties of cellular signals, we developed a computational stochastic model and investigated how three calcium ion (Ca2+)-dependent enzymes (adenylyl cyclase (AC), phosphodiesterase 1 (PDE1), and calcineurin (CaN)) differentially detect Ca2+ transients in a hippocampal dendritic spine. The balance among AC, PDE1 and CaN might determine the occurrence of opposite Ca2+-induced forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD). CaN is essential for LTD. AC and PDE1 regulate, indirectly, protein kinase A, which counteracts CaN during LTP. Stimulations of AC, PDE1 and CaN with artificial and physiological Ca2+ signals demonstrated that AC and CaN have Ca2+ requirements modulated dynamically by different properties of the signals used to stimulate them, because their interactions with Ca2+ often occur under kinetic control. Contrarily, PDE1 responds to the immediate amplitude of different Ca2+ transients and usually with the same Ca2+ requirements observed under steady state. Therefore, AC, PDE1 and CaN decode different dynamic properties of Ca2+ signals. PMID:27033299

  19. Modelling intracellular competition for calcium: kinetic and thermodynamic control of different molecular modes of signal decoding.

    PubMed

    Antunes, Gabriela; Roque, Antonio C; Simoes de Souza, Fabio M

    2016-01-01

    Frequently, a common chemical entity triggers opposite cellular processes, which implies that the components of signalling networks must detect signals not only through their chemical natures, but also through their dynamic properties. To gain insights on the mechanisms of discrimination of the dynamic properties of cellular signals, we developed a computational stochastic model and investigated how three calcium ion (Ca((2+)))-dependent enzymes (adenylyl cyclase (AC), phosphodiesterase 1 (PDE1), and calcineurin (CaN)) differentially detect Ca((2+)) transients in a hippocampal dendritic spine. The balance among AC, PDE1 and CaN might determine the occurrence of opposite Ca((2+))-induced forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD). CaN is essential for LTD. AC and PDE1 regulate, indirectly, protein kinase A, which counteracts CaN during LTP. Stimulations of AC, PDE1 and CaN with artificial and physiological Ca((2+)) signals demonstrated that AC and CaN have Ca((2+)) requirements modulated dynamically by different properties of the signals used to stimulate them, because their interactions with Ca((2+)) often occur under kinetic control. Contrarily, PDE1 responds to the immediate amplitude of different Ca((2+)) transients and usually with the same Ca((2+)) requirements observed under steady state. Therefore, AC, PDE1 and CaN decode different dynamic properties of Ca((2+)) signals. PMID:27033299

  20. THE ROLE OF INTRACELLULAR SODIUM (Na+) IN THE REGULATION OF CALCIUM (Ca2+)-MEDIATED SIGNALING AND TOXICITY

    PubMed Central

    Yu, Xian-Min; Groveman, Bradley R; Fang, Xiao-Qian; Lin, Shuang-Xiu

    2010-01-01

    It is known that activated N-methyl-D-aspartate receptors (NMDARs) are a major route of excessive calcium ion (Ca2+) entry in central neurons, which may activate degradative processes and thereby cause cell death. Therefore, NMDARs are now recognized to play a key role in the development of many diseases associated with injuries to the central nervous system (CNS). However, it remains a mystery how NMDAR activity is recruited in the cellular processes leading to excitotoxicity and how NMDAR activity can be controlled at a physiological level. The sodium ion (Na+) is the major cation in extracellular space. With its entry into the cell, Na+ can act as a critical intracellular second messenger that regulates many cellular functions. Recent data have shown that intracellular Na+ can be an important signaling factor underlying the up-regulation of NMDARs. While Ca2+ influx during the activation of NMDARs down-regulates NMDAR activity, Na+ influx provides an essential positive feedback mechanism to overcome Ca2+-induced inhibition and thereby potentiate both NMDAR activity and inward Ca2+ flow. Extensive investigations have been conducted to clarify mechanisms underlying Ca2+-mediated signaling. This review focuses on the roles of Na+ in the regulation of Ca2+-mediated NMDAR signaling and toxicity. PMID:21243124

  1. GPR120 promotes adipogenesis through intracellular calcium and extracellular signal-regulated kinase 1/2 signal pathway.

    PubMed

    Song, Tongxing; Zhou, Yuanfei; Peng, Jian; Tao, Ya-Xiong; Yang, Yang; Xu, Tao; Peng, Jie; Ren, Jiao; Xiang, Quanhang; Wei, Hongkui

    2016-10-15

    Numerous researches have demonstrated that GPR120 (also called FFAR4) exerts novel functions in insulin resistance and adipogenesis. However, the molecular mechanism of GPR120-mediated adipogenic differentiation is still unclear. This study was aimed to interpret the relevant function mechanism of GPR120 in the differentiation of 3T3-L1 adipocytes. The results showed that GPR120 expression was dramatically increased along with the adipogenic differentiation of 3T3-L1 adipocytes and the adipogenic ability was significantly inhibited in shGPR120-transfected cells. TUG-891, a selective agonist of GPR120, promoted the intracellular triglyceride accumulation in a dose-dependent manner and did not enhance adipogenesis in shGPR120-transfected cells. Markedly, TUG-891 increased the activation of PPARγ in a GPR120-dependent pathway as assessed by luciferase reporter assay. Furthermore, in the adipogenic differentiation process of 3T3-L1 adipocytes, TUG-891 increased the [Ca(2+)]i and phosphorylation level of ERK1/2. Pretreatment with inhibitors of either ERK1/2 (U0126) or [Ca(2+)]i (BAPTA-AM) notably attenuated the GPR120-mediated adipogenesis. These results show that GPR120 promotes adipogenesis by increasing PPARγ expression via [Ca(2+)]i and ERK1/2 signal pathway in 3T3-L1 adipocytes. PMID:27302893

  2. Intracellular Calcium Dysregulation: Implications for Alzheimer's Disease

    PubMed Central

    Magi, Simona; Castaldo, Pasqualina; Macrì, Maria Loredana; Maiolino, Marta; Matteucci, Alessandra; Bastioli, Guendalina; Gratteri, Santo; Lariccia, Vincenzo

    2016-01-01

    Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by progressive neuronal loss. AD is associated with aberrant processing of the amyloid precursor protein, which leads to the deposition of amyloid-β plaques within the brain. Together with plaques deposition, the hyperphosphorylation of the microtubules associated protein tau and the formation of intraneuronal neurofibrillary tangles are a typical neuropathological feature in AD brains. Cellular dysfunctions involving specific subcellular compartments, such as mitochondria and endoplasmic reticulum (ER), are emerging as crucial players in the pathogenesis of AD, as well as increased oxidative stress and dysregulation of calcium homeostasis. Specifically, dysregulation of intracellular calcium homeostasis has been suggested as a common proximal cause of neural dysfunction in AD. Aberrant calcium signaling has been considered a phenomenon mainly related to the dysfunction of intracellular calcium stores, which can occur in both neuronal and nonneuronal cells. This review reports the most recent findings on cellular mechanisms involved in the pathogenesis of AD, with main focus on the control of calcium homeostasis at both cytosolic and mitochondrial level. PMID:27340665

  3. Intracellular sphingosine releases calcium from lysosomes.

    PubMed

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. PMID:26613410

  4. Intracellular sphingosine releases calcium from lysosomes

    PubMed Central

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. DOI: http://dx.doi.org/10.7554/eLife.10616.001 PMID:26613410

  5. Regulation of Calcium signaling through spatial Organization

    NASA Astrophysics Data System (ADS)

    Ullah, Aman; Ullah, Ghanim; Machaca, Khalid; Jung, Peter

    2010-03-01

    Calcium waves and signals in oocytes are produced and sustained by the release of Ca^2+ from the Endoplasmic Reticulum (ER) through clustered release channels. Changes in the spatial organization of calcium signaling effectors regulate the spatiotemporal features of the calcium signal as is e.g. observed during oocyte maturation. We report here how specific changes in the clustering of the calcium release channels in conjunction with physiologic alterations of other signaling effectors can affect a) the sensitivity of the signaling machinery to external factors, b) the time course of global intracellular signals and c), the speed and propagation range of intracellular calcium waves.

  6. Signaling Microdomains Regulate Inositol 1,4,5-Trisphosphate-Mediated Intracellular Calcium Transients in Cultured Neurons

    PubMed Central

    Jacob, Simon N.; Choe, Chi-Un; Uhlen, Per; DeGray, Brenda; Yeckel, Mark F.; Ehrlich, Barbara E.

    2010-01-01

    Ca2+signals in neurons use specific temporal and spatial patterns to encode unambiguous information about crucial cellular functions. To understand the molecular basis for initiation and propagation of inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular Ca2+ signals, we correlated the subcellular distribution of components of the InsP3 pathway with measurements of agonist-induced intracellular Ca2+ transients in cultured rat hippocampal neurons and pheochromocytoma cells. We found specialized domains with high levels of phosphatidylinositol-4-phosphate kinase (PIPKIγ) and chromogranin B (CGB), proteins acting synergistically to increase InsP3 pumps in the plasma membrane (PMCA) and sarco-endoplasmic reticulum receptor (InsP3R) activity and sensitivity. In contrast, Ca2+ as well as buffers that antagonize the rise in intracellular Ca2+ were distributed uniformly. By pharmacologically blocking phosphatidylinositol-4-kinase and PIPKIγ or disrupting the CGB–InsP3R interaction by transfecting an interfering polypeptide fragment, we produced major changes in the initiation site and kinetics of the Ca2+signal. This study shows that a limited number of proteins can reassemble to form unique, spatially restricted signaling domains to generate distinctive signals in different regions of the same neuron. The finding that the subcellular location of initiation sites and protein microdomains was cell type specific will help to establish differences in spatiotemporal Ca2+signaling in different types of neurons. PMID:15772345

  7. SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells*

    PubMed Central

    Avet, Charlotte; Garrel, Ghislaine; Denoyelle, Chantal; Laverrière, Jean-Noël; Counis, Raymond; Cohen-Tannoudji, Joëlle; Simon, Violaine

    2013-01-01

    In mammals, the receptor of the neuropeptide gonadotropin-releasing hormone (GnRHR) is unique among the G protein-coupled receptor (GPCR) family because it lacks the carboxyl-terminal tail involved in GPCR desensitization. Therefore, mechanisms involved in the regulation of GnRHR signaling are currently poorly known. Here, using immunoprecipitation and GST pull-down experiments, we demonstrated that SET interacts with GnRHR and targets the first and third intracellular loops. We delineated, by site-directed mutagenesis, SET binding sites to the basic amino acids 66KRKK69 and 246RK247, located next to sequences required for receptor signaling. The impact of SET on GnRHR signaling was assessed by decreasing endogenous expression of SET with siRNA in gonadotrope cells. Using cAMP and calcium biosensors in gonadotrope living cells, we showed that SET knockdown specifically decreases GnRHR-mediated mobilization of intracellular cAMP, whereas it increases its intracellular calcium signaling. This suggests that SET influences signal transfer between GnRHR and G proteins to enhance GnRHR signaling to cAMP. Accordingly, complexing endogenous SET by introduction of the first intracellular loop of GnRHR in αT3-1 cells significantly reduced GnRHR activation of the cAMP pathway. Furthermore, decreasing SET expression prevented cAMP-mediated GnRH stimulation of Gnrhr promoter activity, highlighting a role of SET in gonadotropin-releasing hormone regulation of gene expression. In conclusion, we identified SET as the first direct interacting partner of mammalian GnRHR and showed that SET contributes to a switch of GnRHR signaling toward the cAMP pathway. PMID:23233674

  8. The Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria.

    PubMed

    Thai, Tiffany L; Yu, Ling; Galarza-Paez, Laura; Wu, Ming Ming; Lam, Ho Yin Colin; Bao, Hui Fang; Duke, Billie Jeanne; Al-Khalili, Otor; Ma, He-Ping; Liu, Bingchen; Eaton, Douglas C

    2015-11-27

    The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca(2+)]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca(2+)]i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca(2+)]i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca(2+)]i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca(2+)]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca(2+)]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca(2+)]i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca(2+)]i, creating [Ca(2+)]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca(2+)]i uptake destroyed the polarized response of ENaC to [Ca(2+)]i. Overall, our data suggest that ENaC is regulated by [Ca(2+)]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca(2+)]i sequestration. PMID:26451045

  9. Decoding of intracellular calcium spike trains

    NASA Astrophysics Data System (ADS)

    Prank, K.; Läer, L.; von zur Mühlen, A.; Brabant, G.; Schöfl, C.

    1998-04-01

    Cells respond to external signals, such as hormonal stimuli, by generating repetitive spikes in the intracellular free-calcium concentration ([Ca2+]i). These [Ca2+]i spikes, which can be modulated in their frequency and amplitude, regulate diverse cellular processes. Experimentally, [Ca2+]i can be assessed continuously in contrast to cellular responses represented by the activation of proteins. We propose a mathematical model that allows for the on-line decoding of [Ca2+]i spike trains into cellular responses represented by the activation of proteins.

  10. Pasteurella haemolytica A1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways

    PubMed Central

    Hsuan, S. L.; Kannan, M. S.; Jeyaseelan, S.; Prakash, Y. S.; Sieck, G. C.; Maheswaran, S. K.

    1998-01-01

    Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca2+ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+ in a concentration-dependent fashion by influx of extracellular Ca2+ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+ even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2 and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2 and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The

  11. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling.

    PubMed

    Stephen, Terri-Leigh; Higgs, Nathalie F; Sheehan, David F; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I Lorena; Kittler, Josef T

    2015-12-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca(2+). Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca(2+)-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca(2+) in astrocytic processes. Thus, the regulation of intracellular Ca(2+) signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca(2+) wave propagation, gliotransmission, and ultimately neuronal function. PMID:26631479

  12. Peroxisome is a reservoir of intracellular calcium.

    PubMed

    Raychaudhury, Bikramjit; Gupta, Shreedhara; Banerjee, Shouvik; Datta, Salil C

    2006-07-01

    We have examined fura 2-loaded purified peroxisomes under confocal microscope to prove that this mammalian organelle is a store of intracellular calcium pool. Presence of calcium channel and vanadate sensitive Ca(2+)-ATPase in the purified peroxisomal membrane has been demonstrated. We have further observed that machineries to maintain calcium pool in this mammalian organelle are impaired during infection caused by Leishmania donovani. Results reveal that peroxisomes have a merit to play a significant role in the metabolism of intracellular calcium. PMID:16713100

  13. Bimatoprost and prostaglandin F(2 alpha) selectively stimulate intracellular calcium signaling in different cat iris sphincter cells.

    PubMed

    Spada, Clayton S; Krauss, Achim H-P; Woodward, David F; Chen, June; Protzman, Charles E; Nieves, Amelia L; Wheeler, Larry A; Scott, David F; Sachs, George

    2005-01-01

    Bimatoprost is a synthetic analog of prostaglandin F(2 alpha) ethanolamide (prostamide F(2 alpha)), and shares a pharmacological profile consistent with that of the prostamides. Like prostaglandin F(2 alpha) carboxylic acid, bimatoprost potently lowers intraocular pressure in dogs, primates and humans. In order to distinguish its mechanism of action from prostaglandin F(2 alpha), fluorescence confocal microscopy was used to examine the effects of bimatoprost, prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) on calcium signaling in resident cells of digested cat iris sphincter, a tissue which exhibits contractile responses to both agonists. Constant superfusion conditions obviated effective conversion of bimatoprost. Serial challenge with 100 nM bimatoprost and prostaglandin F(2 alpha) consistently evoked responses in different cells within the same tissue preparation, whereas prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) elicited signaling responses in the same cells. Bimatoprost-sensitive cells were consistently re-stimulated with bimatoprost only, and prostaglandin F(2 alpha) sensitive cells could only be re-stimulated with prostaglandin F(2 alpha). The selective stimulation of different cells in the same cat iris sphincter preparation by bimatoprost and prostaglandin F(2 alpha), along with the complete absence of observed instances in which the same cells respond to both agonists, strongly suggests the involvement of distinct receptors for prostaglandin F(2 alpha) and bimatoprost. Further, prostaglandin F(2 alpha) but not bimatoprost potently stimulated calcium signaling in isolated human embryonic kidney cells stably transfected with the feline- and human-prostaglandin F(2 alpha) FP-receptor and in human dermal fibroblast cells, and only prostaglandin F(2 alpha) competed with radioligand binding in HEK-feFP cells. These studies provide further evidence for the existence of a bimatoprost-sensitive receptor that is distinct from

  14. Biosynthesis of B2-integrin, intracellular calcium signalling and functional responses of normal and CD18-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Higuchi, H; Nochi, H; Tamoto, K; Araiso, T; Noda, H; Kociba, G J

    1996-09-01

    1Biosynthesis of CD11/CD18 in bovine leucocytes, intracellular Ca2+ ([Ca2+]i) signalling, chemiluminescent responses and membrane fluidity of neutrophils and the effects of D-mannose on neutrophils from control heifers and a heifer with bovine leucocyte adhesion deficiency (BLAD) were measured. The synthesis of CD11/CD18 complex was clearly detected in leucocytes from a normal heifer, but not in a BLAD-affected heifer. The transient phase of increased [Ca2+]i was clearly detected in neutrophils from a heifer with BLAD stimulated with opsonised zymosan, aggregated bovine immunoglobulin G or concanavalin A, whereas the sustained phase was deficient or significantly decreased compared with control heifers. [Ca2+]i signalling of neutrophils from control heifers and a heifer with BLAD stimulated with phorbol myristate acetate via an 11b/CD18-independent pathway showed no transient phase, and the subsequent increase in [Ca2+]i was almost identical in neutrophils from affected and control heifers. [Ca2+]i concentration and chemiluminescent responses of neutrophils from a control heifer were clearly decreased by treatment with anti-CD18 and anti-IgG antibodies. No differences in membrane fluidity were detected between neutrophils derived from control and CD18-deficient cattle. D-mannose binds mainly to Fc rather than CD18 receptors, and decreased Agg-IgG induced [Ca2+]i and the chemiluminescent response of neutrophils. The [Ca2+]i responses and Agg-IgG induced chemiluminescent responses of neutrophils from control heifers and a BLAD-affected heifer were inhibited by D-mannose. The characteristic changes of [Ca2+]i signalling and functional responses of B2-integrin-deficient neutrophils were demonstrated. PMID:8880976

  15. The basal level of intracellular calcium gates the activation of phosphoinositide 3-kinase - Akt signaling by brain-derived neurotrophic factor in cortical neurons

    PubMed Central

    Zheng, Fei; Soellner, Deborah; Nunez, Joseph; Wang, Hongbing

    2008-01-01

    Brain derived neurotrophic factor (BDNF) mediates survival and neuroplasticity through the activation of phosphoinositide 3-kinase (PI3K)-Akt pathway. Although previous studies suggested the roles of MAPK, PLC-γ-mediated intra-cellular calcium ([Ca2+]i) increase, and extra-cellular calcium influx in regulating Akt activation, the cellular mechanisms are largely unknown. We demonstrated that sub-nanomolar BDNF significantly induced Akt activation in developing cortical neurons. The TrkB-dependent Akt phosphorylation at S473 and T308 required only PI3K, but not PLC and MAPK activity. Blocking NMDA receptors, L-type voltage-gated calcium channels, and chelating extra-cellular calcium by EGTA failed to block BDNF-induced Akt phosphorylation. In contrast, chelating [Ca2+]i by BAPTA-AM abolished Akt phosphorylation. Interestingly, sub-nanomolar BDNF did not stimulate [Ca2+]i increase under our culture conditions. Together with that NMDA- and membrane depolarization-induced [Ca2+]i increase did not activate Akt, we conclude that the basal level of [Ca2+]i gates BDNF function. Furthermore, inhibiting calmodulin by W13 suppressed Akt phosphorylation. On the other hand, inhibition of protein phosphatase 1 by okadaic acid and tautomycin rescued Akt phosphorylation in BAPTA- and W13-treated neurons. We further demonstrated that the phosphorylation of PDK1 did not correlate with Akt phosphorylation at T308. Our results suggested novel roles of basal [Ca2+]i, rather than activity-induced calcium elevation, in BDNF-Akt signaling. PMID:18485103

  16. A Novel Role of the L-Type Calcium Channel α1D Subunit as a Gatekeeper for Intracellular Zinc Signaling: Zinc Wave

    PubMed Central

    Yamasaki, Satoru; Hasegawa, Aiko; Hojyo, Shintaro; Ohashi, Wakana; Fukada, Toshiyuki; Nishida, Keigo; Hirano, Toshio

    2012-01-01

    Recent studies have shown that zinc ion (Zn) can behave as an intracellular signaling molecule. We previously demonstrated that mast cells stimulated through the high-affinity IgE receptor (FcεRI) rapidly release intracellular Zn from the endoplasmic reticulum (ER), and we named this phenomenon the “Zn wave”. However, the molecules responsible for releasing Zn and the roles of the Zn wave were elusive. Here we identified the pore-forming α1 subunit of the Cav1.3 (α1D) L-type calcium channel (LTCC) as the gatekeeper for the Zn wave. LTCC antagonists inhibited the Zn wave, and an agonist was sufficient to induce it. Notably, α1D was mainly localized to the ER rather than the plasma membrane in mast cells, and the Zn wave was impaired by α1D knockdown. We further found that the LTCC-mediated Zn wave positively controlled cytokine gene induction by enhancing the DNA-binding activity of NF- κB. Consistent with this finding, LTCC antagonists inhibited the cytokine-mediated delayed-type allergic reaction in mice without affecting the immediate-type allergic reaction. These findings indicated that the LTCC α1D subunit located on the ER membrane has a novel function as a gatekeeper for the Zn wave, which is involved in regulating NF-κB signaling and the delayed-type allergic reaction. PMID:22745805

  17. INTRACELLULAR SIGNALING AND DEVELOPMENTAL NEUROTOXICITY.

    EPA Science Inventory

    A book chapter in ?Molecular Toxicology: Transcriptional Targets? reviewed the role of intracellular signaling in the developmental neurotoxicity of environmental chemicals. This chapter covered a number of aspects including the development of the nervous system, role of intrace...

  18. Intracellular calcium dynamics dependent on defined microtopographical features of titanium.

    PubMed

    Staehlke, Susanne; Koertge, Andreas; Nebe, Barbara

    2015-04-01

    Detailed insights into the complex cellular behavior at the biomaterial interface are crucial for the improvement of implant surfaces with respect to their acceptance and integration. The cells perceive microtopographical features and, in consequence, rearrange their adhesion structures like the actin cytoskeleton and adaptor proteins. But little is known about whether these altered cellular phenotypes have consequences for intracellular calcium signaling and its dynamics. To elucidate if an artificial, geometrical microtopography influences calcium ion (Ca(2+)) mobilization in osteoblasts, human MG-63 cells were stained with the calcium dye Fluo 3-acetoxymethyl ester and set on defined silicon-titanium (Ti) arrays with regular pillar structures (P5, 5 × 5 × 5 μm) and compared with planar Ti. To induce an immediate calcium signal, cells were stimulated with adenosine 5'-triphosphate (ATP). Interestingly, osteoblasts on micropillars expressing a shortened actin cytoskeleton were hampered in their calcium mobilization potential in signal height as well duration. Even the basal level of the intracellular Ca(2+) concentration was reduced, which was accompanied by a disturbed fibronectin synthesis. The expression of the voltage-sensitive calcium channels Cav1.2, Cav1.3 (L-type) and Cav3.1, Cav3.2, Cav3.3 (T-type) as well as the signaling proteins phospho-AKT and phospho-GSK3α/β remained unaffected on pillars. The topography-dependent calcium dynamics observed here provide new insights into how topographical cues alter cell functions - via the intracellular Ca(2+) signaling. PMID:25678115

  19. Coexistence of amplitude and frequency modulations in intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    de Pittà, Maurizio; Volman, Vladislav; Levine, Herbert; Pioggia, Giovanni; de Rossi, Danilo; Ben-Jacob, Eshel

    2008-03-01

    The complex dynamics of intracellular calcium regulates cellular responses to information encoded in extracellular signals. Here we study the encoding of these external signals in the context of the Li-Rinzel model. We show that by control of biophysical parameters the information can be encoded in amplitude modulation (AM), frequency modulation (FM), or mixed (AM and FM) modulation. We briefly discuss the possible implications of this role of information encoding for astrocytes.

  20. Calcium signals and calcium channels in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Duncan, R. L.; Akanbi, K. A.; Farach-Carson, M. C.

    1998-01-01

    Calcium (Ca2+) channels are present in non-excitable as well as in excitable cells. In bone cells of the osteoblast lineage, Ca2+ channels play fundamental roles in cellular responses to external stimuli including both mechanical forces and hormonal signals. They are also proposed to modulate paracrine signaling between bone-forming osteoblasts and bone-resorbing osteoclasts at local sites of bone remodeling. Calcium signals are characterized by transient increases in intracellular Ca2+ levels that are associated with activation of intracellular signaling pathways that control cell behavior and phenotype, including patterns of gene expression. Development of Ca2+ signals is a tightly regulated cellular process that involves the concerted actions of plasma membrane and intracellular Ca2+ channels, along with Ca2+ pumps and exchangers. This review summarizes the current state of knowledge concerning the structure, function, and role of Ca2+ channels and Ca2+ signals in bone cells, focusing on the osteoblast.

  1. Calcium signaling in trypanosomatid parasites.

    PubMed

    Docampo, Roberto; Huang, Guozhong

    2015-03-01

    Calcium ion (Ca(2+)) is an important second messenger in trypanosomatids and essential for their survival although prolonged high intracellular Ca(2+) levels lead to cell death. As other eukaryotic cells, trypanosomes use two sources of Ca(2+) for generating signals: Ca(2+) release from intracellular stores and Ca(2+) entry across the plasma membrane. Ca(2+) release from intracellular stores is controlled by the inositol 1,4,5-trisphosphate receptor (IP3R) that is located in acidocalcisomes, acidic organelles that are the primary Ca(2+) reservoir in these cells. A plasma membrane Ca(2+)-ATPase controls the cytosolic Ca(2+) levels and a number of pumps and exchangers are responsible for Ca(2+) uptake and release from intracellular compartments. The trypanosomatid genomes contain a wide variety of signaling and regulatory proteins that bind Ca(2+) as well as many Ca(2+)-binding proteins that await further characterization. The mitochondrial Ca(2+) transporters of trypanosomatids have an important role in the regulation of cell bioenergetics and flagellar Ca(2+) appears to have roles in sensing the environment. In trypanosomatids in which an intracellular life cycle is present, Ca(2+) signaling is important for host cell invasion. PMID:25468729

  2. Rapid measurements of intracellular calcium using a fluorescence plate reader.

    PubMed

    Lin, K; Sadée, W; Quillan, J M

    1999-02-01

    Intracellular calcium is a universal second messenger that can serve as a broad-based measure of receptor activity. Recent developments in multi-well plate fluorescence readers facilitate measurement of intracellular free-calcium levels and reduce reliance on slower, more cumbersome or expensive data collection methods. In this report, we describe a rapid and sensitive method to assay intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from multi-well plates using a fluorometer equipped with on-line injectors. We examine the compatibility of visible-light excitable dyes Calcium Green-1 and Oregon Green 488 BAPTA-1. Using this assay, we were able to detect and quantify activity from muscarinic and beta-adrenergic receptors endogenous to HEK293 cells and detect calcium signals generated by activation of Gi-coupled recombinant mu-opioid and dopamine D2L receptors, and the Gs-coupled melanocortin subtype 4 (MC4) receptor. Fluorescence signals, stable in HEK293 cells, required the use of Oregon Green 488 BAPTA-1 and an inhibitor of organic anion transport in CHO cells. Under appropriate conditions, both cell types can be used to collect complete concentration-response data for a variety of receptors (including a recombinant muscarinic M1 receptor expressed in CHO cells) from a single plate of dye-loaded cells. PMID:10023544

  3. [Mitochondria, calcium homeostasis and calcium signaling].

    PubMed

    Zavodnik, I B

    2016-03-01

    Са2+ is a very important and versatile intracellular signal which controls numerous biochemical and physiological (pathophysiological) processes in the cell. Good evidence exists that mitochondria are sensors, decoders and regulators of calcium signaling. Precise regulation of calcium signaling in the cell involves numerous molecular targets, which induce and decode changes of Са2+ concentrations in the cell (pumps, channels, Са2+-binding proteins, Са2+-dependent enzymes, localized in the cytoplasm and organelles). Mitochondrial Са2+ uniporter accumulates excess of Са2+ in mitochondria, while Na+/Са2+- and H+/Са2+-antiporters extrude Са2+ in the cytoplasm. Mitochondrial Са2+ overloading results in formation of mitochondria permeability transition pores which play an important role in cell death under many pathological conditions. Mitochondria regulate Са2+ homeostasis and control important cellular functions such as metabolism, proliferation, survival. Identification of cellular and mitochondrial Ca2+ transporters and understanding their functional mechanisms open up new prospects for their using as therapeutic targets. PMID:27420625

  4. Monitoring the intracellular calcium response to a dynamic hypertonic environment

    NASA Astrophysics Data System (ADS)

    Huang, Xiaowen; Yue, Wanqing; Liu, Dandan; Yue, Jianbo; Li, Jiaqian; Sun, Dong; Yang, Mengsu; Wang, Zuankai

    2016-03-01

    The profiling of physiological response of cells to external stimuli at the single cell level is of importance. Traditional approaches to study cell responses are often limited by ensemble measurement, which is challenging to reveal the complex single cell behaviors under a dynamic environment. Here we report the development of a simple microfluidic device to investigate intracellular calcium response to dynamic hypertonic conditions at the single cell level in real-time. Interestingly, a dramatic elevation in the intracellular calcium signaling is found in both suspension cells (human leukemic cell line, HL-60) and adherent cells (lung cancer cell line, A549), which is ascribed to the exposure of cells to the hydrodynamic stress. We also demonstrate that the calcium response exhibits distinct single cell heterogeneity as well as cell-type-dependent responses to the same stimuli. Our study opens up a new tool for tracking cellular activity at the single cell level in real time for high throughput drug screening.

  5. The effect of the calcium-antagonist nitrendipine on intracellular calcium concentration in endothelial cells.

    PubMed Central

    Salameh, A.; Schomecker, G.; Breitkopf, K.; Dhein, S.; Klaus, W.

    1996-01-01

    1. Nitrendipine induces NO-release from coronary vascular endothelium presumably by activating endothelial NO-synthase. We have investigated whether this effect may be mediated by an influence on the intracellular calcium in endothelial cells. 2. Bovine aortic endothelial cells (BAEC) were incubated with Fura-2/AM (1 microM) for 30 min and Fura-2 fluorescence was measured at 510 nm in response to chopped excitation with both 340 and 380 nm. The ratio 340/380 nm (known to reflect changes in intracellular calcium) was calculated from these data. 3. Nitrendipine (0.1 to 100 microM) led to a significant, concentration-dependent, monophasic increase in [Ca2+]i in suspended BAEC by 11 +/- 2 nM (0.1 microM), 23 +/- 3 nM (1 microM), 34 +/- 4 nM (10 microM) and by 47 +/- 5 nM (100 microM) from a control levels of 118 +/- 10 nM. 4. This elevation of intracellular calcium was prevented by pretreatment of BAECs with gadolinium (100 microM) or by incubation with calcium free saline solution. In contrast, the application of 0.3 microM thapsigargin did not abolish the nitrendipine-induced calcium signal. In additional experiments it was shown that the nitrendipine-induced NO-release (as measured with the oxy-haemoglobin-method could also be inhibited by gadolinium and was absent in calcium-free solution. 5. Thus, nitrendipine elevates intracellular calcium in suspended BAECs in a concentration-dependent manner. This elevation is mainly due to a gadolinium-sensitive calcium influx from the extracellular space rather than a calcium release from intracellular stores. Images Figure 5 PMID:8864521

  6. Calcium Signaling Is Required for Erythroid Enucleation

    PubMed Central

    Russell, Sarah M.; Humbert, Patrick O.

    2016-01-01

    Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM) pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate. Together these functional studies highlight a critical role for calcium signaling in the regulation of erythroid enucleation. PMID:26731108

  7. Calcium Signaling Is Required for Erythroid Enucleation.

    PubMed

    Wölwer, Christina B; Pase, Luke B; Russell, Sarah M; Humbert, Patrick O

    2016-01-01

    Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM) pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate. Together these functional studies highlight a critical role for calcium signaling in the regulation of erythroid enucleation. PMID:26731108

  8. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  9. Altered Calcium Signaling Following Traumatic Brain Injury

    PubMed Central

    Weber, John T.

    2012-01-01

    Cell death and dysfunction after traumatic brain injury (TBI) is caused by a primary phase, related to direct mechanical disruption of the brain, and a secondary phase which consists of delayed events initiated at the time of the physical insult. Arguably, the calcium ion contributes greatly to the delayed cell damage and death after TBI. A large, sustained influx of calcium into cells can initiate cell death signaling cascades, through activation of several degradative enzymes, such as proteases and endonucleases. However, a sustained level of intracellular free calcium is not necessarily lethal, but the specific route of calcium entry may couple calcium directly to cell death pathways. Other sources of calcium, such as intracellular calcium stores, can also contribute to cell damage. In addition, calcium-mediated signal transduction pathways in neurons may be perturbed following injury. These latter types of alterations may contribute to abnormal physiology in neurons that do not necessarily die after a traumatic episode. This review provides an overview of experimental evidence that has led to our current understanding of the role of calcium signaling in death and dysfunction following TBI. PMID:22518104

  10. Intracellular Signal Modulation by Nanomaterials

    PubMed Central

    Hussain, Salik; Garantziotis, Stavros; Rodrigues-Lima, Fernando; Dupret, Jean-Marie; Baeza-Squiban, Armelle; Boland, Sonja

    2016-01-01

    A thorough understanding of the interactions of nanomaterials with biological systems and the resulting activation of signal transduction pathways is essential for the development of safe and consumer friendly nanotechnology. Here we present an overview of signaling pathways induced by nanomaterial exposures and describe the possible correlation of their physicochemical characteristics with biological outcomes. In addition to the hierarchical oxidative stress model and a review of the intrinsic and cell-mediated mechanisms of reactive Oxygen species (ROS) generating capacities of nanomaterials, we also discuss other oxidative stress dependent and independent cellular signaling pathways. Induction of the inflammasome, calcium signaling, and endoplasmic reticulum stress are reviewed. Furthermore, the uptake mechanisms can crucially affect the cytotoxicity of nanomaterials and membrane-dependent signaling pathways can be responsible for cellular effects of nanomaterials. Epigenetic regulation by nanomaterials effects of nanoparticle-protein interactions on cell signaling pathways, and the induction of various cell death modalities by nanomaterials are described. We describe the common trigger mechanisms shared by various nanomaterials to induce cell death pathways and describe the interplay of different modalities in orchestrating the final outcome after nanomaterial exposures. A better understanding of signal modulations induced by nanomaterials is not only essential for the synthesis and design of safer nanomaterials but will also help to discover potential nanomedical applications of these materials. Several biomedical applications based on the different signaling pathways induced by nanomaterials are already proposed and will certainly gain a great deal of attraction in the near future. PMID:24683030

  11. Intracellular signalling by C-peptide.

    PubMed

    Hills, Claire E; Brunskill, Nigel J

    2008-01-01

    C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na(+)/K(+) ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes. PMID:18382618

  12. Intracellular signalling during neutrophil recruitment.

    PubMed

    Mócsai, Attila; Walzog, Barbara; Lowell, Clifford A

    2015-08-01

    Recruitment of leucocytes such as neutrophils to the extravascular space is a critical step of the inflammation process and plays a major role in the development of various diseases including several cardiovascular diseases. Neutrophils themselves play a very active role in that process by sensing their environment and responding to the extracellular cues by adhesion and de-adhesion, cellular shape changes, chemotactic migration, and other effector functions of cell activation. Those responses are co-ordinated by a number of cell surface receptors and their complex intracellular signal transduction pathways. Here, we review neutrophil signal transduction processes critical for recruitment to the site of inflammation. The two key requirements for neutrophil recruitment are the establishment of appropriate chemoattractant gradients and the intrinsic ability of the cells to migrate along those gradients. We will first discuss signalling steps required for sensing extracellular chemoattractants such as chemokines and lipid mediators and the processes (e.g. PI3-kinase pathways) leading to the translation of extracellular chemoattractant gradients to polarized cellular responses. We will then discuss signal transduction by leucocyte adhesion receptors (e.g. tyrosine kinase pathways) which are critical for adhesion to, and migration through the vessel wall. Finally, additional neutrophil signalling pathways with an indirect effect on the neutrophil recruitment process, e.g. through modulation of the inflammatory environment, will be discussed. Mechanistic understanding of these pathways provide better understanding of the inflammation process and may point to novel therapeutic strategies for controlling excessive inflammation during infection or tissue damage. PMID:25998986

  13. Monitoring of intracellular free calcium in perfused rat liver.

    PubMed

    Ruttner, Z; Ligeti, L; Reinlib, L; Hines, K; McLaughlin, A C

    1993-06-01

    Fluorescent calcium indicators have been widely used to assess cytoplasmic calcium concentration in cells. To examine the role of calcium ions on different physiological functions (e.g. in case of liver; bile secretion, glucose metabolism, etc.) there is a need for whole organ studies. We have developed a technique to estimate intracellular free calcium changes in perfused rat liver. Krebs-Henseleit perfused livers were loaded with 7 microM or 35 microM Indo-1/AM. An area 3 mm in diameter and approximately 300 microns in depth was illuminated at 340 nm. Fluorescence was monitored with photomultiplier tubes at 3 wavelengths (400 nm for Ca-bound dye, 504 nm for free dye and 464 nm for NADH). The viability of liver preparations was assessed by measurement of the concentrations of lactate dehydrogenase and alanine aminotransferase in the effluent. Loading of the livers with 7 microM Indo-1/AM via the portal vein resulted in a 5-fold increase of fluorescence at 400 nm. However the dye 'leaked' out of the liver with a half-time of 18 min. Probenecid (a specific anion carrier blocker) inhibited loss of dye in a dose dependent fashion (2.5-10 mM). Transient calcium elevations were observed in response to vasopressin (5-50 nM) at physiological levels, ethanol (0.3-0.8 M) and the calcium ionophore, ionomycin. Certain limitations were apparent with this approach: (1) it was necessary to use an anion carrier blocker to maintain a relatively steady dye concentration; (2) endogenous NADH fluorescence interfered with the calcium signal; and (3) absolute values of calcium concentration could not be determined. PMID:8358770

  14. Intracellular calcium ions as regulators of renal tubular sodium transport.

    PubMed

    Windhager, E; Frindt, G; Yang, J M; Lee, C O

    1986-09-15

    This review addresses the putative role of intracellular calcium ions in the regulation of sodium transport by renal tubules. Cytoplasmic calcium-ion activities in proximal tubules of Necturus are less than 10(-7) M and can be increased by lowering the electrochemical potential gradient for sodium ions across the peritubular cell membrane, or by addition of quinidine or ionomycin to peritubular fluid. Whereas lowering of the peritubular Na concentration increases cytosolic [Ca++] and [H+], ionomycin, a calcium ionophore, raises intracellular [Ca++] without decreasing pHi. The intracellular calcium-ion level is maintained by transport processes in the plasma membrane and membranes of intracellular organelles, as well as by calcium-binding proteins. Calcium ions inhibit net transport of sodium by reducing the rate of sodium entry across the luminal cell membrane. In the collecting tubule this inhibition is caused, at least in part, by an indirect reduction in the activity of the amiloride-sensitive sodium channel. PMID:2430134

  15. TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

    PubMed Central

    Shambharkar, Prashant B.; Bittinger, Mark; Latario, Brian; Xiong, ZhaoHui; Bandyopadhyay, Somnath; Davis, Vanessa; Lin, Victor; Yang, Yi; Valdez, Reginald; Labow, Mark A.

    2015-01-01

    Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis. PMID:25996873

  16. Monitoring the intracellular calcium response to a dynamic hypertonic environment

    PubMed Central

    Huang, Xiaowen; Yue, Wanqing; Liu, Dandan; Yue, Jianbo; Li, Jiaqian; Sun, Dong; Yang, Mengsu; Wang, Zuankai

    2016-01-01

    The profiling of physiological response of cells to external stimuli at the single cell level is of importance. Traditional approaches to study cell responses are often limited by ensemble measurement, which is challenging to reveal the complex single cell behaviors under a dynamic environment. Here we report the development of a simple microfluidic device to investigate intracellular calcium response to dynamic hypertonic conditions at the single cell level in real-time. Interestingly, a dramatic elevation in the intracellular calcium signaling is found in both suspension cells (human leukemic cell line, HL-60) and adherent cells (lung cancer cell line, A549), which is ascribed to the exposure of cells to the hydrodynamic stress. We also demonstrate that the calcium response exhibits distinct single cell heterogeneity as well as cell-type-dependent responses to the same stimuli. Our study opens up a new tool for tracking cellular activity at the single cell level in real time for high throughput drug screening. PMID:27004604

  17. Calcium signaling and cytotoxicity.

    PubMed Central

    Kass, G E; Orrenius, S

    1999-01-01

    The divalent calcium cation Ca(2+) is used as a major signaling molecule during cell signal transduction to regulate energy output, cellular metabolism, and phenotype. The basis to the signaling role of Ca(2+) is an intricate network of cellular channels and transporters that allow a low resting concentration of Ca(2+) in the cytosol of the cell ([Ca(2+)]i) but that are also coupled to major dynamic and rapidly exchanging stores. This enables extracellular signals from hormones and growth factors to be transduced as [Ca(2+)]i spikes that are amplitude and frequency encoded. There is considerable evidence that a number of toxic environmental chemicals target these Ca(2+) signaling processes, alter them, and induce cell death by apoptosis. Two major pathways for apoptosis will be considered. The first one involves Ca(2+)-mediated expression of ligands that bind to and activate death receptors such as CD95 (Fas, APO-1). In the second pathway, Ca(2+) has a direct toxic effect and its primary targets include the mitochondria and the endoplasmic reticulum (ER). Mitochondria may respond to an apoptotic Ca(2+) signal by the selective release of cytochrome c or through enhanced production of reactive oxygen species and opening of an inner mitochondrial membrane pore. Toxic agents such as the environmental pollutant tributyltin or the natural plant product thapsigargin, which deplete the ER Ca(2+) stores, will induce as a direct result of this effect the opening of plasma membrane Ca(2+) channels and an ER stress response. In contrast, under some conditions, Ca(2+) signals may be cytoprotective and antagonize the apoptotic machinery. Images Figure 1 Figure 2 Figure 3 PMID:10229704

  18. Microheterogeneity of calcium signalling in dendrites.

    PubMed

    Pozzo-Miller, L D; Connor, J A; Andrews, S B

    2000-05-15

    Transient changes in the intracellular concentration of free Ca2+ ([Ca2+]i) originating from voltage- or ligand-gated influx and by ligand- or Ca2+-gated release from intracellular stores, trigger or modulate many fundamental neuronal processes, including neurotransmitter release and synaptic plasticity. Of the intracellular compartments involved in Ca2+ clearance, the endoplasmic reticulum (ER) has received the most attention because it expresses Ca2+ pumps and Ca2+ channels, thus endowing it with the potential to act as both an intracellular calcium sink and store. We review here our ongoing work on the role of calcium sequestration into, and release from, ER cisterns and the role that this plays in the generation and termination of free [Ca2+]i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. These studies have been approached by combining parallel microfluorometric measurements of free cytosolic [Ca2+]i transients with energy-dispersive X-ray microanalytical measurements of total Ca content within specific dendritic compartments at the electron microscopy level. Our observations support the emerging realization that specific subsets of dendritic ER cisterns provide spatial and temporal microheterogeneity of Ca2+ signalling, acting not only as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca2+ influx, but also as an intracellular Ca2+ source that can be mobilized by a signal cascade originating at activated synapses. PMID:10811724

  19. Use of photoproteins as intracellular calcium indicators.

    PubMed Central

    Blinks, J R

    1990-01-01

    The calcium-regulated photoproteins, of which aequorin is the best known, continue to be one of the most useful groups of intracellular Ca2+ indicators. They are self-contained bioluminescent systems that emit blue light in the presence of Ca2+ ions, can readily be purified intact, and are nontoxic when introduced into foreign cells. They have been used successfully as Ca2+ indicators in almost every kind of cell, but are most widely used in muscle cells because of their relative freedom from motion artifacts. Photoproteins have also been used in conjunction with microscopic image intensification to localize Ca2+ in cells. Their large molecular size makes them difficult to introduce into cells, but once there, they have the advantage of staying in the cytoplasm. Aequorin can be microinjected satisfactorily into single cells of almost any size, but a number of alternative methods for introducing photoproteins into cells have been developed in recent years. Disadvantages of the photoproteins for some applications include the nonlinear relation between [Ca2+] and light intensity, the modest speed with which they respond to sudden changes in [Ca2+], and the fact the Mg2+ antagonizes the effect of Ca2+. Native photoproteins consist of a mixture of isospecies, and there are differences in Ca2+ sensitivity and in kinetic properties--both among photoproteins and among the isospecies of a given photoprotein. The genes for several of the isospecies of aequorin have been cloned and expressed in E. coli. It seems reasonable to hope that genetic engineering techniques may soon make it possible to consider using, as Ca2+ indicators, rare isospecies or rare photoproteins that have optimal properties for particular applications. PMID:2190821

  20. Calcium signalling and calcium channels: evolution and general principles.

    PubMed

    Verkhratsky, Alexei; Parpura, Vladimir

    2014-09-15

    Calcium as a divalent cation was selected early in evolution as a signaling molecule to be used by both prokaryotes and eukaryotes. Its low cytosolic concentration likely reflects the initial concentration of this ion in the primordial soup/ocean as unicellular organisms were formed. As the concentration of calcium in the ocean subsequently increased, so did the diversity of homeostatic molecules handling calcium. This includes the plasma membrane channels that allowed the calcium entry, as well as extrusion mechanisms, i.e., exchangers and pumps. Further diversification occurred with the evolution of intracellular organelles, in particular the endoplasmic reticulum and mitochondria, which also contain channels, exchanger(s) and pumps to handle the homeostasis of calcium ions. Calcium signalling system, based around coordinated interactions of the above molecular entities, can be activated by the opening of voltage-gated channels, neurotransmitters, second messengers and/or mechanical stimulation, and as such is all-pervading pathway in physiology and pathophysiology of organisms. PMID:24291103

  1. The Analysis of Intracellular and Intercellular Calcium Signaling in Human Anterior Lens Capsule Epithelial Cells with Regard to Different Types and Stages of the Cataract

    PubMed Central

    Gosak, Marko; Markovič, Rene; Fajmut, Aleš; Marhl, Marko; Hawlina, Marko; Andjelić, Sofija

    2015-01-01

    In this work we investigated how modifications of the Ca2+ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca2+ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca2+ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca2+ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca2+ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca2+ activity. The presence of spontaneous activity was also found to affect the stimulated Ca2+ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca2+ signaling of

  2. Cilostazol Induces PGI2 Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells

    PubMed Central

    Hashimoto, Ayako

    2015-01-01

    Background Cilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is known as an anti-platelet drug and acts directly on platelets. Cilostazol has been shown to exhibit vascular protection in ischemic diseases. Although vascular endothelium-derived prostaglandin I2 (PGI2) plays an important role in vascular protection, it is unknown whether cilostazol directly stimulates PGI2 synthesis in endothelial cells. Here, we elucidate the mechanism of cilostazol-induced PGI2 stimulation in endothelial cells. Methods and Results Human aortic endothelial cells (HAECs) were stimulated with cilostazol and PGI2 accumulation in the culture media was measured. Cilostazol increased PGI2 synthesis via the arachidonic acid pathway. Cilostazol-induced intracellular calcium also promoted PGI2 synthesis via the inositol 1,4,5-trisphosphate receptor. Using RNAi, silencing of PDE3B abolished the induction effect of cilostazol on PGI2 synthesis and intracellular cAMP accumulation. Inhibition of the exchange protein, which was directly activated by cyclic AMP 1 (Epac-1) and its downstream signal the Ras-like small GTPase (Rap-1), abolished cilostazol-induced PGI2 synthesis, but this did not take place via protein kinase A (PKA). Inhibition of downstream signaling, such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) γ, and phospholipase C (PLC) ε, suppressed cilostazol-induced PGI2 synthesis. Conclusions The PDE3/Epac-1/Rap-1 signaling pathway plays an important role in cilostazol-induced PGI2 synthesis. Namely, stimulation of HAECs with cilostazol induces intracellular calcium elevation via the Rap-1/PLCε/IP3 pathway, along with MAPK activation via direct activation by Epac-1/Rap-1 and indirect activation by Epac-1/Rap-1/PI3Kγ, resulting in synergistically induced PGI2 synthesis. PMID:26181635

  3. Calcium-dependent intracellular signal pathways in primary cultured adipocytes and ANK3 gene variation in patients with bipolar disorder and healthy controls

    PubMed Central

    Hayashi, A; Le Gal, K; Södersten, K; Vizlin-Hodzic, D; Ågren, H; Funa, K

    2015-01-01

    Bipolar disorder (BD) is a chronic psychiatric disorder of public health importance affecting >1% of the Swedish population. Despite progress, patients still suffer from chronic mood switches with potential severe consequences. Thus, early detection, diagnosis and initiation of correct treatment are critical. Cultured adipocytes from 35 patients with BD and 38 healthy controls were analysed using signal pathway reporter assays, that is, protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)), Myc, Wnt and p53. The levels of activated target transcriptional factors were measured in adipocytes before and after stimulation with lithium and escitalopram. Variations were analysed in the loci of 25 different single-nucleotide polymorphisms (SNPs). Activation of intracellular signals in several pathways analysed were significantly higher in patients than in healthy controls upon drug stimulation, especially with escitalopram stimulation of PKC, JNK and Myc, as well as lithium-stimulated PKC, whereas no meaningful difference was observed before stimulation. Univariate analyses of contingency tables for 80 categorical SNP results versus diagnoses showed a significant link with the ANK3 gene (rs10761482; likelihood ratio χ2=4.63; P=0.031). In a multivariate ordinal logistic fit for diagnosis, a backward stepwise procedure selected ANK3 as the remaining significant predictor. Comparison of the escitalopram-stimulated PKC activity and the ANK3 genotype showed them to add their share of the diagnostic variance, with no interaction (15% of variance explained, P<0.002). The study is cross-sectional with no longitudinal follow-up. Cohorts are relatively small with no medication-free patients, and there are no ‘ill patient' controls. It takes 3 to 4 weeks of culture to expand adipocytes that may change epigenetic profiles but remove the possibility of medication effects

  4. Calcium-dependent intracellular signal pathways in primary cultured adipocytes and ANK3 gene variation in patients with bipolar disorder and healthy controls.

    PubMed

    Hayashi, A; Le Gal, K; Södersten, K; Vizlin-Hodzic, D; Ågren, H; Funa, K

    2015-08-01

    Bipolar disorder (BD) is a chronic psychiatric disorder of public health importance affecting >1% of the Swedish population. Despite progress, patients still suffer from chronic mood switches with potential severe consequences. Thus, early detection, diagnosis and initiation of correct treatment are critical. Cultured adipocytes from 35 patients with BD and 38 healthy controls were analysed using signal pathway reporter assays, that is, protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)), Myc, Wnt and p53. The levels of activated target transcriptional factors were measured in adipocytes before and after stimulation with lithium and escitalopram. Variations were analysed in the loci of 25 different single-nucleotide polymorphisms (SNPs). Activation of intracellular signals in several pathways analysed were significantly higher in patients than in healthy controls upon drug stimulation, especially with escitalopram stimulation of PKC, JNK and Myc, as well as lithium-stimulated PKC, whereas no meaningful difference was observed before stimulation. Univariate analyses of contingency tables for 80 categorical SNP results versus diagnoses showed a significant link with the ANK3 gene (rs10761482; likelihood ratio χ(2)=4.63; P=0.031). In a multivariate ordinal logistic fit for diagnosis, a backward stepwise procedure selected ANK3 as the remaining significant predictor. Comparison of the escitalopram-stimulated PKC activity and the ANK3 genotype showed them to add their share of the diagnostic variance, with no interaction (15% of variance explained, P<0.002). The study is cross-sectional with no longitudinal follow-up. Cohorts are relatively small with no medication-free patients, and there are no 'ill patient' controls. It takes 3 to 4 weeks of culture to expand adipocytes that may change epigenetic profiles but remove the possibility of medication effects

  5. Measurement of shear stress-mediated intracellular calcium dynamics in human dermal lymphatic endothelial cells

    PubMed Central

    Jafarnejad, M.; Cromer, W. E.; Kaunas, R. R.; Zhang, S. L.; Zawieja, D. C.

    2015-01-01

    The shear stress applied to lymphatic endothelial cells (LEC) by lymph flow changes dramatically under normal conditions as well as in response to disease conditions and immune reactions. In general, LEC are known to regulate the contraction frequency and strength of lymphatic pumping in response to shear stress. Intracellular calcium concentration ([Ca2+]i) is an important factor that regulates lymphatic contraction characteristics. In this study, we measured changes in the [Ca2+]i under different shear stress levels and determined the source of this calcium signal. Briefly, human dermal LEC were cultured in custom-made microchannels for 3 days before loading with 2 µM fura-2 AM, a ratiometric calcium dye to measure [Ca2+]i. Step changes in shear stress resulted in a rapid increase in [Ca2+]i followed by a gradual return to the basal level and sometimes below the initial baseline (45.2 ± 2.2 nM). The [Ca2+]i reached a peak at 126.2 ± 5.6 nM for 10 dyn/cm2 stimulus, whereas the peak was only 71.8 ± 5.4 nM for 1 dyn/cm2 stimulus, indicating that the calcium signal depends on the magnitude of shear stress. Removal of the extracellular calcium from the buffer or pharmocological blockade of calcium release-activated calcium (CRAC) channels significantly reduced the peak [Ca2+]i, demonstrating a role of extracellular calcium entry. Inhibition of endoplasmic reticulum (ER) calcium pumps showed the importance of intracellular calcium stores in the initiation of this signal. In conclusion, we demonstrated that the shear-mediated calcium signal is dependent on the magnitude of the shear and involves ER store calcium release and extracellular calcium entry. PMID:25617358

  6. Intracellular calcium levels can regulate Importin-dependent nuclear import

    SciTech Connect

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

    2014-07-18

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

  7. Abnormal Intracellular Calcium Signaling and SNARE-Dependent Exocytosis Contributes to SOD1G93A Astrocyte-Mediated Toxicity in Amyotrophic Lateral Sclerosis

    PubMed Central

    Kawamata, Hibiki; Ng, Seng Kah; Diaz, Natalia; Burstein, Suzanne; Morel, Lydie; Osgood, Alexandra; Sider, Brittany; Higashimori, Haruki; Haydon, Philip G.

    2014-01-01

    Motor neurons are progressively and predominantly degenerated in ALS, which is not only induced by multiple intrinsic pathways but also significantly influenced by the neighboring glial cells. In particular, astrocytes derived from the SOD1 mutant mouse model of ALS or from human familial or sporadic ALS patient brain tissue directly induce motor neuron death in culture; however, the mechanisms of pathological astroglial secretion remain unclear. Here we investigated abnormal calcium homeostasis and altered exocytosis in SOD1G93A astrocytes. We found that purinergic stimulation induces excess calcium release from the ER stores in SOD1G93A astrocytes, which results from the abnormal ER calcium accumulation and is independent of clearance mechanisms. Furthermore, pharmacological studies suggested that store-operated calcium entry (SOCE), a calcium refilling mechanism responsive to ER calcium depletion, is enhanced in SOD1G93A astrocytes. We found that oxidant-induced increased S-glutathionylation and calcium-independent puncta formation of the ER calcium sensor STIM1 underlies the abnormal SOCE response in SOD1G93A astrocytes. Enhanced SOCE contributes to ER calcium overload in SOD1G93A astrocytes and excess calcium release from the ER during ATP stimulation. In addition, ER calcium release induces elevated ATP release from SOD1G93A astrocytes, which can be inhibited by the overexpression of dominant-negative SNARE. Selective inhibition of exocytosis in SOD1G93A astrocytes significantly prevents astrocyte-mediated toxicity to motor neurons and delays disease onset in SOD1G93A mice. Our results characterize a novel mechanism responsible for calcium dysregulation in SOD1G93A astrocytes and provide the first in vivo evidence that astrocyte exocytosis contributes to the pathogenesis of ALS. PMID:24501372

  8. Mitochondrial dysfunction and intracellular calcium dysregulation in ALS

    PubMed Central

    Kawamata, Hibiki; Manfredi, Giovanni

    2010-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that affects the aging population. A progressive loss of motor neurons in the spinal cord and brain leads to muscle paralysis and death. As in other common neurodegenerative diseases, aging-related mitochondrial dysfunction is increasingly being considered among the pathogenic factors. Mitochondria are critical for cell survival: they provide energy to the cell, buffer intracellular calcium, and regulate apoptotic cell death. Whether mitochondrial abnormalities are a trigger or a consequence of the neurodegenerative process and the mechanisms whereby mitochondrial dysfunction contributes to disease are not clear yet. Calcium homeostasis is a major function of mitochondria in neurons, and there is ample evidence that intracellular calcium is dysregulated in ALS. The impact of mitochondrial dysfunction on intracellular calcium homeostasis and its role in motor neuron demise are intriguing issues that warrants in depth discussion. Clearly, unraveling the causal relationship between mitochondrial dysfunction, calcium dysregulation, and neuronal death is critical for the understanding of ALS pathogenesis. In this review, we will outline the current knowledge of various aspects of mitochondrial dysfunction in ALS, with a special emphasis on the role of these abnormalities on intracellular calcium handling. PMID:20493207

  9. Release of Intracellular Calcium Stores Facilitates Coxsackievirus Entry into Polarized Endothelial Cells

    PubMed Central

    Bozym, Rebecca A.; Morosky, Stefanie A.; Kim, Kwang S.; Cherry, Sara; Coyne, Carolyn B.

    2010-01-01

    Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers. PMID:20949071

  10. Micropatterning C2C12 myotubes for orderly recording of intracellular calcium transients.

    PubMed

    Takayama, Yuzo; Wagatsuma, Akira; Hoshino, Takayuki; Mabuchi, Kunihiko

    2013-01-01

    Reconstruction of skeletal muscle myotubes in vitro using myogenic cell lines have been widely carried out to study functional properties and disease-related biological changes of myotubes, such as intracellular calcium dynamics. However, the analysis of biological signals in isolated single myotubes or interactions among several myotubes is quite difficult problem because of the randomness in size, morphology and orientation of differentiated myotubes cultured on a conventional tissue culture dish. In the present study, we attempted to form uniform-size myotubes and detect intracellular calcium dynamics from the fabricated myotubes. We modified surfaces of culture dishes using a poly(-dimethylsiloxane) (PDMS) stamp and a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to form line patterns for myotube formation. We could form uniform-size and -orientation C2C12 myotubes and detect intracellular calcium dynamics from it. This simple method would be a useful for studying properties in myotubes with specific sizes and morphologies. PMID:24111271

  11. Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients

    PubMed Central

    Espino, Javier; Mediero, Matías; Lozano, Graciela M; Bejarano, Ignacio; Ortiz, Águeda; García, Juan F; Pariente, José A; Rodríguez, Ana B

    2009-01-01

    Background Asthenozoospermia is one of the most common findings present in infertile males characterized by reduced or absent sperm motility, but its aetiology remains unknown in most cases. In addition, calcium is one of the most important ions regulating sperm motility. In this study we have investigated the progesterone-evoked intracellular calcium signal in ejaculated spermatozoa from men with normospermia or asthenozoospermia. Methods Human ejaculates were obtained from healthy volunteers and asthenospermic men by masturbation after 4–5 days of abstinence. For determination of cytosolic free calcium concentration, spermatozoa were loaded with the fluorescent ratiometric calcium indicator Fura-2. Results Treatment of spermatozoa from normospermic men with 20 micromolar progesterone plus 1 micromolar thapsigargin in a calcium free medium induced a typical transient increase in cytosolic free calcium concentration due to calcium release from internal stores. Similar results were obtained when spermatozoa were stimulated with progesterone alone. Subsequent addition of calcium to the external medium evoked a sustained elevation in cytosolic free calcium concentration indicative of capacitative calcium entry. However, when progesterone plus thapsigargin were administered to spermatozoa from patients with asthenozoospermia, calcium signal and subsequent calcium entry was much smaller compared to normospermic patients. As expected, pretreatment of normospermic spermatozoa with both the anti-progesterone receptor c262 antibody and with progesterone receptor antagonist RU-38486 decreased the calcium release induced by progesterone. Treatment of spermatozoa with cytochalasin D or jasplakinolide decreased the calcium entry evoked by depletion of internal calcium stores in normospermic patients, whereas these treatments proved to be ineffective at modifying the calcium entry in patients with asthenozoospermia. Conclusion Our results suggest that spermatozoa from

  12. Intracellular signaling and hepatocellular carcinoma.

    PubMed

    Iakova, Polina; Timchenko, Lubov; Timchenko, Nikolai A

    2011-02-01

    Liver cancer is the fifth most common cancer and the third most common cause of cancer related death in the world. The recent development of new techniques for the investigations of global change in the gene expression, signaling pathways and wide genome binding has provided novel information for the mechanisms underlying liver cancer progression. Although these studies identified gene expression signatures in hepatocellular carcinoma, the early steps of the development of hepatocellular carcinomas (HCC) are not well understood. The development of HCC is a multistep process which includes the progressive alterations of gene expression leading to the increased proliferation and to liver cancer. This review summarizes recent progress in the identification of the key steps of the development of HCC with the focus on early events of carcinogenesis and on the role of translational and epigenetic alterations in the development of HCC. Quiescent stage of the liver is supported by several tumor suppressor proteins including p53, Rb and C/EBPα. Studies with chemical models of liver carcinogenesis and with human HCC have shown that the elevation of gankyrin is responsible for the elimination of these three proteins at early steps of carcinogenesis. Later stages of progression of the liver cancer are associated with alterations in many signaling pathways including translation which leads to epigenetic silencing/activation of many genes. Particularly, recent reports suggest a critical role of histone deacetylase 1, HDAC1, in the development of HCC through the interactions with transcription factors such as C/EBP family proteins. PMID:20850540

  13. Diffusive spatio-temporal noise in a first-passage time model for intracellular calcium release

    NASA Astrophysics Data System (ADS)

    Flegg, Mark B.; Rüdiger, Sten; Erban, Radek

    2013-04-01

    The intracellular release of calcium from the endoplasmic reticulum is controlled by ion channels. The resulting calcium signals exhibit a rich spatio-temporal signature, which originates at least partly from microscopic fluctuations. While stochasticity in the gating transition of ion channels has been incorporated into many models, the distribution of calcium is usually described by deterministic reaction-diffusion equations. Here we test the validity of the latter modeling approach by using two different models to calculate the frequency of localized calcium signals (calcium puffs) from clustered IP3 receptor channels. The complexity of the full calcium system is here limited to the basic opening mechanism of the ion channels and, in the mathematical reduction simplifies to the calculation of a first passage time. Two models are then studied: (i) a hybrid model, where channel gating is treated stochastically, while calcium concentration is deterministic and (ii) a fully stochastic model with noisy channel gating and Brownian calcium ion motion. The second model utilises the recently developed two-regime method [M. B. Flegg, S. J. Chapman, and R. Erban, "The two-regime method for optimizing stochastic reaction-diffusion simulations," J. R. Soc., Interface 9, 859-868 (2012)], 10.1098/rsif.2011.0574 in order to simulate a large domain with precision required only near the Ca2+ absorbing channels. The expected time for a first channel opening that results in a calcium puff event is calculated. It is found that for a large diffusion constant, predictions of the interpuff time are significantly overestimated using the model (i) with a deterministic non-spatial calcium variable. It is thus demonstrated that the presence of diffusive noise in local concentrations of intracellular Ca2+ ions can substantially influence the occurrence of calcium signals. The presented approach and results may also be relevant for other cell-physiological first-passage time problems with

  14. Stochastic hybrid modeling of intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    Choi, TaiJung; Maurya, Mano Ram; Tartakovsky, Daniel M.; Subramaniam, Shankar

    2010-10-01

    Deterministic models of biochemical processes at the subcellular level might become inadequate when a cascade of chemical reactions is induced by a few molecules. Inherent randomness of such phenomena calls for the use of stochastic simulations. However, being computationally intensive, such simulations become infeasible for large and complex reaction networks. To improve their computational efficiency in handling these networks, we present a hybrid approach, in which slow reactions and fluxes are handled through exact stochastic simulation and their fast counterparts are treated partially deterministically through chemical Langevin equation. The classification of reactions as fast or slow is accompanied by the assumption that in the time-scale of fast reactions, slow reactions do not occur and hence do not affect the probability of the state. Our new approach also handles reactions with complex rate expressions such as Michaelis-Menten kinetics. Fluxes which cannot be modeled explicitly through reactions, such as flux of Ca2+ from endoplasmic reticulum to the cytosol through inositol 1,4,5-trisphosphate receptor channels, are handled deterministically. The proposed hybrid algorithm is used to model the regulation of the dynamics of cytosolic calcium ions in mouse macrophage RAW 264.7 cells. At relatively large number of molecules, the response characteristics obtained with the stochastic and deterministic simulations coincide, which validates our approach in the limit of large numbers. At low doses, the response characteristics of some key chemical species, such as levels of cytosolic calcium, predicted with stochastic simulations, differ quantitatively from their deterministic counterparts. These observations are ubiquitous throughout dose response, sensitivity, and gene-knockdown response analyses. While the relative differences between the peak-heights of the cytosolic [Ca2+] time-courses obtained from stochastic (mean of 16 realizations) and deterministic

  15. Stem Cells and Calcium Signaling

    PubMed Central

    Tonelli, Fernanda M.P.; Santos, Anderson K.; Gomes, Dawidson A.; da Silva, Saulo L.; Gomes, Katia N.; Ladeira, Luiz O.

    2014-01-01

    The increasing interest in stem cell research is linked to the promise of developing treatments for many lifethreatening, debilitating diseases, and for cell replacement therapies. However, performing these therapeutic innovations with safety will only be possible when an accurate knowledge about the molecular signals that promote the desired cell fate is reached. Among these signals are transient changes in intracellular Ca2+ concentration [Ca2+]i. Acting as an intracellular messenger, Ca2+ has a key role in cell signaling pathways in various differentiation stages of stem cells. The aim of this chapter is to present a broad overview of various moments in which Ca2+-mediated signaling is essential for the maintenance of stem cells and for promoting their development and differentiation, also focusing on their therapeutic potential. PMID:22453975

  16. Calcium Signaling in the Liver

    PubMed Central

    Amaya, Maria Jimena; Nathanson, Michael H.

    2014-01-01

    Intracellular free Ca2+ ([Ca2+]i) is a highly versatile second messenger that regulates a wide range of functions in every type of cell and tissue. To achieve this versatility, the Ca2+ signaling system operates in a variety of ways to regulate cellular processes that function over a wide dynamic range. This is particularly well exemplified for Ca2+ signals in the liver, which modulate diverse and specialized functions such as bile secretion, glucose metabolism, cell proliferation, and apoptosis. These Ca2+ signals are organized to control distinct cellular processes through tight spatial and temporal coordination of [Ca2+]i signals, both within and between cells. This article will review the machinery responsible for the formation of Ca2+ signals in the liver, the types of subcellular, cellular, and intercellular signals that occur, the physiological role of Ca2+ signaling in the liver, and the role of Ca2+ signaling in liver disease. PMID:23720295

  17. Measuring intracellular calcium dynamics of HeLa cells exposed to nitric oxide by microplate fluorescence reader

    NASA Astrophysics Data System (ADS)

    Huang, Yimei; Chen, Jiangxu; Yang, Hongqin; Zheng, Liqin; Wang, Yuhua; Li, Hui; Xie, Shusen

    2012-12-01

    Nitric oxide (NO) has been reported to have the ability to promote or inhibit the proliferation and metastasis of cancer cells. It appears to have an effect on inducing calcium transient, which participates in essential cellular signaling in the physiological and pathological processes. Our work was intended to study the effects of exogenous NO on intracellular calcium dynamics of HeLa cells with Fluo-3, a calcium fluorescent indicator by microplate fluorescence reader. The results showed that after NO donor was injected into the wells, intracellular Ca2+ fluorescence intensity increased significantly compared with that of control group. Furthermore, the calcium transient activated by NO was mainly due to the calcium release from intracellular calcium stores. These would be helpful to further recognize the role of NO involved in cancer cell proliferation and metastasis.

  18. Arrhythmogenic consequences of intracellular calcium waves.

    PubMed

    Xie, Lai-Hua; Weiss, James N

    2009-09-01

    Intracellular Ca(2+) (Ca(i)(2+)) waves are known to cause delayed afterdepolarizations (DADs), which have been associated with arrhythmias in cardiac disease states such as heart failure, catecholaminergic polymorphic ventricular tachycardia, and digitalis toxicity. Here we show that, in addition to DADs, Ca(i)(2+) waves also have other consequences relevant to arrhythmogenesis, including subcellular spatially discordant alternans (SDA, in which the amplitude of the local Ca(i)(2+) transient alternates out of phase in different regions of the same cell), sudden repolarization changes promoting the dispersion of refractoriness, and early afterdepolarizations (EADs). Ca(i)(2+) was imaged using a charge-coupled device-based system in fluo-4 AM-loaded isolated rabbit ventricular myocytes paced at constant or incrementally increasing rates, using either field stimulation, current clamp, or action potential (AP) clamp. Ca(i)(2+) waves were induced by Bay K 8644 (50 nM) + isoproterenol (100 nM), or low temperature. When pacing was initiated during a spontaneous Ca(i)(2+) wave, SDA occurred abruptly and persisted during pacing. Similarly, during rapid pacing, SDA typically arose suddenly from spatially concordant alternans, due to an abrupt phase reversal of the subcellular Ca(i)(2+) transient in a region of the myocyte. Ca(i)(2+) waves could be visualized interspersed with AP-triggered Ca(i)(2+) transients, producing a rich variety of subcellular Ca(i)(2+) transient patterns. With free-running APs, complex Ca(i)(2+) release patterns were associated with DADs, EADs, and sudden changes in AP duration. These findings link Ca(i)(2+) waves directly to a variety of arrhythmogenic phenomena relevant to the intact heart. PMID:19561309

  19. Effect of ticlopidine ex vivo on platelet intracellular calcium mobilization

    SciTech Connect

    Derian, C.K.; Friedman, P.A.

    1988-04-01

    The antiplatelet compound ticlopidine exerts its potent inhibitory activity through an as yet undetermined mechanism(s). The goal of this study was to determine the effect, if any, of ticlopidine ex vivo on platelet calcium mobilization. Ticlopidine inhibited ADP-induced platelet aggregation by 50-80%. In the presence of 1 mM EGTA, ticlopidine inhibited ADP- and thrombin-stimulated increases in (Ca2+)i in fura-2 loaded platelets. We evaluated further the effect of ticlopidine on calcium mobilization by examining both agonist-stimulated formation of inositol trisphosphate in intact platelets and the ability of inositol trisphosphate to release /sup 45/Ca from intracellular sites in permeabilized cells. We show here that while ticlopidine significantly affected agonist-induced intracellular calcium mobilization in intact platelets, the drug was without effect on agonist-stimulated formation of inositol trisphosphate in intact platelets and on inositol trisphosphate-induced /sup 45/Ca release in saponin-permeabilized platelets. Our study demonstrates that ticlopidine exerts at least part of its effect via inhibition of intracellular calcium mobilization but that its site of action remains to be determined.

  20. Vesicular demyelination induced by raised intracellular calcium.

    PubMed

    Smith, K J; Hall, S M; Schauf, C L

    1985-11-01

    myelin vesiculation. We conclude that vesicular demyelination can be initiated in vital Schwann cells by a raised intracellular Ca2+ concentration. Such demyelination does not necessarily lead to Schwann cell death. The possible relevance of the findings to vesicular demyelinating neuropathies is discussed, and a hypothesis regarding the mechanism of demyelination is advanced. PMID:3003255

  1. Optogenetic control of intracellular signaling pathways

    PubMed Central

    Zhang, Kai; Cui, Bianxiao

    2014-01-01

    Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, though useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open up exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways, and discuss future prospects for the field, including integration of new genetic approaches into optogenetics. PMID:25529484

  2. Calcium signaling and cell proliferation.

    PubMed

    Pinto, Mauro Cunha Xavier; Kihara, Alexandre Hiroaki; Goulart, Vânia A M; Tonelli, Fernanda M P; Gomes, Katia N; Ulrich, Henning; Resende, Rodrigo R

    2015-11-01

    Cell proliferation is orchestrated through diverse proteins related to calcium (Ca(2+)) signaling inside the cell. Cellular Ca(2+) influx that occurs first by various mechanisms at the plasma membrane, is then followed by absorption of Ca(2+) ions by mitochondria and endoplasmic reticulum, and, finally, there is a connection of calcium stores to the nucleus. Experimental evidence indicates that the fluctuation of Ca(2+) from the endoplasmic reticulum provides a pivotal and physiological role for cell proliferation. Ca(2+) depletion in the endoplasmatic reticulum triggers Ca(2+) influx across the plasma membrane in an phenomenon called store-operated calcium entries (SOCEs). SOCE is activated through a complex interplay between a Ca(2+) sensor, denominated STIM, localized in the endoplasmic reticulum and a Ca(2+) channel at the cell membrane, denominated Orai. The interplay between STIM and Orai proteins with cell membrane receptors and their role in cell proliferation is discussed in this review. PMID:26275497

  3. Calcium Signaling in Smooth Muscle

    PubMed Central

    Hill-Eubanks, David C.; Werner, Matthias E.; Heppner, Thomas J.; Nelson, Mark T.

    2011-01-01

    Changes in intracellular Ca2+ are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca2+ signal is a reflection of the source of Ca2+ (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca2+ entry may be detected optically as graded elevations in intracellular Ca2+, junctional Ca2+ transients, Ca2+ flashes, or Ca2+ sparklets, whereas release of Ca2+ from intracellular stores may manifest as Ca2+ sparks, Ca2+ puffs, or Ca2+ waves. These diverse Ca2+ signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression). PMID:21709182

  4. Short-range intercellular calcium signaling in bone.

    PubMed

    Jørgensen, Niklas Rye

    2005-01-01

    The regulation of bone turnover is a complex and finely tuned process. Many factors regulate bone remodeling, including hormones, growth factors, cytokines etc. However, little is known about the signals coupling bone formation to bone resorption, and how mechanical forces are translated into biological effects in bone. Intercellular calcium waves are increases in intracellular calcium concentration in single cells, subsequently propagating to adjacent cells, and can be a possible mechanism for the coupling of bone formation to bone resorption. The aim of the present studies was to investigate whether bone cells are capable of communicating via intercellular calcium signals, and determine by which mechanisms the cells propagate the signals. First, we found that osteoblastic cells can propagate intercellular calcium transients upon mechanical stimulation, and that there are two principally different mechanisms for this propagation. One mechanism involves the secretion of a nucleotide, possibly ATP, acting in an autocrine action to purinergic P2Y2 receptors on the neighboring cells, leading to intracellular IP3 generation and subsequent release of calcium from intracellular stores. The other mechanism involves the passage of a small messenger through gap junctions to the cytoplasm of the neighboring cells, inducing depolarization of the plasma membrane with subsequent opening of membrane bound voltage-operated calcium channels. Next, we found that osteoblasts can propagate these signals to osteoclasts as well. We demonstrated that paracrine action of ATP was responsible for the wave propagation, but now the purinergic P2X7 receptor was involved. Thus, the studies demonstrate that calcium signals can be propagated not only among osteoblasts, but also between osteoblasts and osteoclasts in response to mechanical stimulation. Thus, intercellular calcium signaling can be a mechanism by which mechanical stimuli on bone are translated into biological signals in bone cells

  5. Implications of purinergic receptor-mediated intracellular calcium transients in neural differentiation

    PubMed Central

    2013-01-01

    Purinergic receptors participate, in almost every cell type, in controlling metabolic activities and many physiological functions including signal transmission, proliferation and differentiation. While most of P2Y receptors induce transient elevations of intracellular calcium concentration by activation of intracellular calcium pools and forward these signals as waves which can also be transmitted into neighboring cells, P2X receptors produce calcium spikes which also include activation of voltage-operating calcium channels. P2Y and P2X receptors induce calcium transients that activate transcription factors responsible for the progress of differentiation through mediators including calmodulin and calcineurin. Expression of P2X2 as well as of P2X7 receptors increases in differentiating neurons and glial cells, respectively. Gene expression silencing assays indicate that these receptors are important for the progress of differentiation and neuronal or glial fate determination. Metabotropic receptors, mostly P2Y1 and P2Y2 subtypes, act on embryonic cells or cells at the neural progenitor stage by inducing proliferation as well as by regulation of neural differentiation through NFAT translocation. The scope of this review is to discuss the roles of purinergic receptor-induced calcium spike and wave activity and its codification in neurodevelopmental and neurodifferentiation processes. PMID:23414261

  6. Calcium influx, but not intracellular calcium release, supports PACAP-mediated ERK activation in HEK PAC1 receptor cells.

    PubMed

    May, Victor; Clason, Todd A; Buttolph, Thomas R; Girard, Beatrice M; Parsons, Rodney L

    2014-11-01

    In HEK cells expressing GFP-tagged PAC1Hop1 receptors, PACAP augments ERK phosphorylation through two parallel pathways: one through PACAP/PAC1 receptor internalization/endosome MEK/ERK signaling and the other through PLC/DAG/PKC activation. We examined whether elevation of intracellular calcium ([Ca(2+)]i) was required for either of the PACAP/PAC1 receptor-mediated ERK activation mechanisms. The PACAP (25 nM)-induced elevation of [Ca(2+)]i was greater with cells maintained in Ca(2+)-containing than in Ca(2+)-deficient solution, suggesting that both calcium release from internal stores and calcium influx contributed to the rise in [Ca(2+)]i. A thapsigargin-induced increase in [Ca(2+)]i also was greater with calcium in the external solution. OAG, the cell permeable analogue of DAG, increased [Ca(2+)]i, but only in Ca(2+)-containing solution. Decreasing external calcium or depleting internal calcium stores did not block PACAP-induced PAC1 receptor internalization. Omission of calcium from the external solution, but not thapsigargin pretreatment, significantly blunted PACAP-stimulated ERK phosphorylation. The PKC inhibitor BimI decreased PACAP-mediated ERK activation in both Ca(2+)-containing or Ca(2+)-deficient solutions. In contrast, following Pitstop 2 pretreatment to block endocytic mechanisms, PACAP activated ERK only when calcium was present in the external solution. We conclude that the endosome signaling pathway is largely calcium-independent whereas calcium influx appears necessary for the PLC/DAG/PKC component of PACAP-induced ERK activation. PMID:24723666

  7. Estimating the biophysical properties of neurons with intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    Ye, Jingxin; Rozdeba, Paul J.; Morone, Uriel I.; Daou, Arij; Abarbanel, Henry D. I.

    2014-06-01

    We investigate the dynamics of a conductance-based neuron model coupled to a model of intracellular calcium uptake and release by the endoplasmic reticulum. The intracellular calcium dynamics occur on a time scale that is orders of magnitude slower than voltage spiking behavior. Coupling these mechanisms sets the stage for the appearance of chaotic dynamics, which we observe within certain ranges of model parameter values. We then explore the question of whether one can, using observed voltage data alone, estimate the states and parameters of the voltage plus calcium (V+Ca) dynamics model. We find the answer is negative. Indeed, we show that voltage plus another observed quantity must be known to allow the estimation to be accurate. We show that observing both the voltage time course V (t) and the intracellular Ca time course will permit accurate estimation, and from the estimated model state, accurate prediction after observations are completed. This sets the stage for how one will be able to use a more detailed model of V+Ca dynamics in neuron activity in the analysis of experimental data on individual neurons as well as functional networks in which the nodes (neurons) have these biophysical properties.

  8. Intracellular free calcium concentration and calcium transport in human erythrocytes of lead-exposed workers

    SciTech Connect

    Quintanar-Escorza, M.A.; Gonzalez-Martinez, M.T.; Navarro, L.; Maldonado, M.; Arevalo, B.; Calderon-Salinas, J.V. . E-mail: jcalder@cinvestav.mx

    2007-04-01

    Erythrocytes are the route of lead distribution to organs and tissues. The effect of lead on calcium homeostasis in human erythrocytes and other excitable cells is not known. In the present work we studied the effect of lead intoxication on the uptake and efflux (measured as (Ca{sup 2+}-Mg{sup 2+})-ATPase activity) of calcium were studied in erythrocytes obtained from lead-exposed workers. Blood samples were taken from 15 workers exposed to lead (blood lead concentration 74.4 {+-} 21.9 {mu}g/dl) and 15 non-exposed workers (9.9 {+-} 2 {mu}g/dl). In erythrocytes of lead-exposed workers, the intracellular free calcium was 79 {+-} 13 nM, a significantly higher concentration (ANOVA, P < 0.01) than the one detected in control (30 {+-} 9 nM). The enhanced intracellular free calcium was associated with a higher osmotic fragility and with important modifications in erythrocytes shape. The high intracellular free calcium in lead-exposed workers was also related to a 100% increase in calcium incorporation and to 50% reduction of (Ca{sup 2+}-Mg{sup 2+})-ATPase activity. Lipid peroxidation was 1.7-fold higher in erythrocytes of lead-exposed workers as compared with control. The alteration on calcium equilibrium in erythrocytes is discussed in light of the toxicological effects in lead-exposed workers.

  9. Understanding anomalous delays in a model of intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    Harvey, Emily; Kirk, Vivien; Osinga, Hinke M.; Sneyd, James; Wechselberger, Martin

    2010-12-01

    In many cell types, oscillations in the concentration of free intracellular calcium ions are used to control a variety of cellular functions. It has been suggested [J. Sneyd et al., "A method for determining the dependence of calcium oscillations on inositol trisphosphate oscillations," Proc. Natl. Acad. Sci. U.S.A. 103, 1675-1680 (2006)] that the mechanisms underlying the generation and control of such oscillations can be determined by means of a simple experiment, whereby a single exogenous pulse of inositol trisphosphate (IP3) is applied to the cell. However, more detailed mathematical investigations [M. Domijan et al., "Dynamical probing of the mechanisms underlying calcium oscillations," J. Nonlinear Sci. 16, 483-506 (2006)] have shown that this is not necessarily always true, and that the experimental data are more difficult to interpret than first thought. Here, we use geometric singular perturbation techniques to study the dynamics of models that make different assumptions about the mechanisms underlying the calcium oscillations. In particular, we show how recently developed canard theory for singularly perturbed systems with three or more slow variables [M. Wechselberger, "A propos de canards (Apropos canards)," Preprint, 2010] applies to these calcium models and how the presence of a curve of folded singularities and corresponding canards can result in anomalous delays in the response of these models to a pulse of IP3.

  10. Calcium Signaling and Meiotic Exit at Fertilization in Xenopus Egg

    PubMed Central

    Tokmakov, Alexander A.; Stefanov, Vasily E.; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

    2014-01-01

    Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation. PMID:25322156

  11. Regulation of neurogenesis by calcium signaling.

    PubMed

    Toth, Anna B; Shum, Andrew K; Prakriya, Murali

    2016-03-01

    Calcium (Ca(2+)) signaling has essential roles in the development of the nervous system from neural induction to the proliferation, migration, and differentiation of neural cells. Ca(2+) signaling pathways are shaped by interactions among metabotropic signaling cascades, intracellular Ca(2+) stores, ion channels, and a multitude of downstream effector proteins that activate specific genetic programs. The temporal and spatial dynamics of Ca(2+) signals are widely presumed to control the highly diverse yet specific genetic programs that establish the complex structures of the adult nervous system. Progress in the last two decades has led to significant advances in our understanding of the functional architecture of Ca(2+) signaling networks involved in neurogenesis. In this review, we assess the literature on the molecular and functional organization of Ca(2+) signaling networks in the developing nervous system and its impact on neural induction, gene expression, proliferation, migration, and differentiation. Particular emphasis is placed on the growing evidence for the involvement of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels in these processes. PMID:27020657

  12. Systematical bifurcation analysis of an intracellular calcium oscillation model.

    PubMed

    Liu, Xijun; Li, Xiang

    2016-07-01

    As a very important second messenger, Ca(2+) plays the role of adjusting various cellular physiological processes through calcium oscillations. In this paper, a further theoretical study is conducted to explore the kinetic behavior of the calcium signals based on a mathematical model. At first, the causes behind the appearance and disappearance of calcium oscillations are strictly verified from the theoretical level and a comparative analysis between the improved model and the original model is also made. Then, it is found that with the increase of relaxation time, the second bifurcation point of the system moves towards the increasing direction of the stimulus intensity and the oscillation interval displays gradual increase. It is also found that under given stimulus intensity, with the relaxation time getting longer, both the peak value and the period of the calcium oscillations display significant increase. Combining the results from the comparative analysis between the improved model and the original model with the results from the analysis of the relaxation time, it shows that the calcium pump activity exerts a direct impact on the calcium oscillation interval. Finally, the calcium leakage item is introduced into the improved model and it is found that as the calcium leakage increases, the two Hopf bifurcation points of the system both move towards the decreasing direction of the stimulus intensity and the oscillation interval gradually narrows down. The study also shows that under given stimulus intensity, as the calcium leakage increases, the peak value of the calcium oscillations displays slow increase and the oscillation period displays gradual decline. PMID:27172874

  13. Glutamate-induced intracellular calcium oscillations in astrocytes with confocal microscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Yuan; Zhou, Wei; Liu, Xiuli; Zhu, Geng; Wu, Yuxiang; Luo, Qingming

    2006-02-01

    Changes in the intracellular Ca 2+ concentration ([Ca 2+]i) play a crucial role involved in the modulation of signal transduction, development, and plasticity in the CNS. Glial cells can respond to various stimuli with an increase in [Ca 2+]i. In this paper, we used confocal microscopy to study calcium transient induced by glutamate in cultured astrocytes. Firstly, 100 μM glutamate induced long-time intracellular calcium oscillations in astrocytes and only a single spike under calcium-free solution. When the concentration of glutamate decreased to 1 μM, only a single spike could be induced. It shows that intracellular calcium oscillations depend on agonist concentration and extracellular Ca 2+. Secondly, we investigated amplitude of responses under different stimulation. The amplitude of initial peak induced by 100 μM glutamate decreased in Ca 2+-free condition, whereas the duration of kinetics was prolonged. But both the amplitude and area of a single spike induced by 1 μM Glu decreased in Ca 2+-free condition. The results show that areaof peak is more accurate than amplitude to display transients of [Ca 2+]i. All results above suggest that astrocytes are not passive, they display diverse temporal and spatial increases in [Ca 2+]i in response to a variety of stimuli. These [Ca 2+]i increases provide a possible means for information coding.

  14. Regulation of PKC Mediated Signaling by Calcium during Visceral Leishmaniasis

    PubMed Central

    Roy, Nivedita; Chakraborty, Supriya; Paul Chowdhury, Bidisha; Banerjee, Sayantan; Halder, Kuntal; Majumder, Saikat; Majumdar, Subrata; Sen, Parimal C.

    2014-01-01

    Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca2+ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis. PMID:25329062

  15. Regulation of PKC mediated signaling by calcium during visceral leishmaniasis.

    PubMed

    Roy, Nivedita; Chakraborty, Supriya; Paul Chowdhury, Bidisha; Banerjee, Sayantan; Halder, Kuntal; Majumder, Saikat; Majumdar, Subrata; Sen, Parimal C

    2014-01-01

    Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca2+ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis. PMID:25329062

  16. Calcium signalling and Alzheimer's disease.

    PubMed

    Berridge, Michael J

    2011-07-01

    New insights into how Ca(2+) regulates learning and memory have begun to provide clues as to how the amyloid-dependent remodelling of neuronal Ca(2+) signalling pathways can disrupt the mechanisms of learning and memory in Alzheimer's disease (AD). The calcium hypothesis of AD proposes that activation of the amyloidogenic pathway remodels the neuronal Ca(2+) signalling pathways responsible for cognition by enhancing the entry of Ca(2+) and/or the release of internal Ca(2+) by ryanodine receptors or InsP(3) receptors. The specific proposal is that Ca(2+) signalling remodelling results in a persistent elevation in the level of Ca(2+) that constantly erases newly acquired memories by enhancing the mechanism of long-term depression (LTD). Neurons can still form memories through the process of LTP, but this stored information is rapidly removed by the persistent activation of LTD. Further dysregulation in Ca(2+) signalling will then go on to induce the neurodegeneration that characterizes the later stages of dementia. PMID:21184278

  17. Towards the Physics of Calcium Signalling in Plants

    PubMed Central

    Vaz Martins, Teresa; Evans, Matthew J.; Woolfenden, Hugh C.; Morris, Richard J.

    2013-01-01

    Calcium is an abundant element with a wide variety of important roles within cells. Calcium ions are inter- and intra-cellular messengers that are involved in numerous signalling pathways. Fluctuating compartment-specific calcium ion concentrations can lead to localised and even plant-wide oscillations that can regulate downstream events. Understanding the mechanisms that give rise to these complex patterns that vary both in space and time can be challenging, even in cases for which individual components have been identified. Taking a systems biology approach, mathematical and computational techniques can be employed to produce models that recapitulate experimental observations and capture our current understanding of the system. Useful models make novel predictions that can be investigated and falsified experimentally. This review brings together recent work on the modelling of calcium signalling in plants, from the scale of ion channels through to plant-wide responses to external stimuli. Some in silico results that have informed later experiments are highlighted. PMID:27137393

  18. Role of intracellular calcium in cellular volume regulation

    SciTech Connect

    Wong, S.M.; Chase, H.S. Jr.

    1986-06-01

    We investigated the role of intracellular calcium in epithelial cell volume regulation using cells isolated from the toad urinary bladder. A suspension of cells was prepared by treatment of the bladder with collagenase followed by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid. The cells retained their ion-transporting capabilities: ouabain (1 mM) and amiloride (10 microM) inhibited cellular uptake of /sup 86/Rb and /sup 22/Na, respectively. Using a Coulter counter to measure cellular volume, we found that we could swell cells either by reducing the extracellular osmolality or by adding the permeant solute urea (45 mM) isosmotically. Under both conditions, cells first swelled and then returned to their base-line volume, in spite of the continued presence of the stimulus to swell. Volume regulation was inhibited when cells were swelled at low extracellular (Ca) (100 nM) and was retarded in cells preloaded with the calcium buffer quin 2. Swelling increased the intracellular free calcium concentration ((Ca)i), as measured by quin 2 fluorescence: (Ca)i increased 35 +/- 9 nM (n = 6) after hypotonic swelling and 42 +/- 3 nM (n = 3) after urea swelling. Reducing extracellular (Ca) to less than 100 nM prevented the swelling-induced increase in (Ca)i, suggesting that the source of the increase in (Ca)i was extracellular. This result was confirmed in measurements of cellular uptake of 45Ca: the rate of uptake was significantly higher in swollen cells compared with control (1.1 +/- 0.2 vs. 0.4 +/- 0.1 fmol . cell-1 X 5 min-1). Our experiments provide the first demonstration that cellular swelling increases (Ca)i. This increase is likely to play a critical role in cellular volume regulation.

  19. Imaging atrial arrhythmic intracellular calcium in intact heart

    PubMed Central

    Xie, Wenjun; Santulli, Gaetano; Guo, Xiaoxiao; Gao, Melanie; Chen, Bi-Xing; Marks, Andrew R.

    2014-01-01

    Abnormalities in intracellular Ca2+ signaling have been proposed to play an essential role in the pathophysiology of atrial arrhythmias. However, a direct observation of intracellular Ca2+ in atrial myocytes during atrial arrhythmias is lacking. Here, we have developed an ex vivo model of simultaneous Ca2+ imaging and electrocardiographic recording in cardiac atria. Using this system we were able to record atrial arrhythmic intracellular Ca2+ activities. Our results indicate that atrial arrhythmias can be tightly linked to intracellular Ca2+ waves and Ca2+ alternans. Moreover, we applied this strategy to analyze Ca2+ signals in the hearts of WT and knock-in mice harboring a ‘leaky’ type 2 ryanodine receptor (RyR2-R2474S). We showed that sarcoplasmic reticulum (SR) Ca2+ leak increases the susceptibility to Ca2+ alternans and Ca2+ waves increasing the incidence of atrial arrhythmias. Reduction of SR Ca2+ leak via RyR2 by acute treatment with S107 reduced both Ca2+ alternans and Ca2+ waves, and prevented atrial arrhythmias. PMID:24041536

  20. Calcium Signals from the Vacuole

    PubMed Central

    Schönknecht, Gerald

    2013-01-01

    The vacuole is by far the largest intracellular Ca2+ store in most plant cells. Here, the current knowledge about the molecular mechanisms of vacuolar Ca2+ release and Ca2+ uptake is summarized, and how different vacuolar Ca2+ channels and Ca2+ pumps may contribute to Ca2+ signaling in plant cells is discussed. To provide a phylogenetic perspective, the distribution of potential vacuolar Ca2+ transporters is compared for different clades of photosynthetic eukaryotes. There are several candidates for vacuolar Ca2+ channels that could elicit cytosolic [Ca2+] transients. Typical second messengers, such as InsP3 and cADPR, seem to trigger vacuolar Ca2+ release, but the molecular mechanism of this Ca2+ release still awaits elucidation. Some vacuolar Ca2+ channels have been identified on a molecular level, the voltage-dependent SV/TPC1 channel, and recently two cyclic-nucleotide-gated cation channels. However, their function in Ca2+ signaling still has to be demonstrated. Ca2+ pumps in addition to establishing long-term Ca2+ homeostasis can shape cytosolic [Ca2+] transients by limiting their amplitude and duration, and may thus affect Ca2+ signaling. PMID:27137394

  1. Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli

    PubMed Central

    Jin, Xingjian; Mohieldin, Ashraf M.; Muntean, Brian S.; Green, Jill A.; Shah, Jagesh V.; Mykytyn, Kirk; Nauli, Surya M.

    2013-01-01

    Primary cilia with a diameter of ~200 nm have been implicated in development and disease. Calcium signaling within a primary cilium has never been directly visualized and has therefore remained a speculation. Fluid-shear stress and dopamine receptor type-5 (DR5) agonist are among the few stimuli that require cilia for intracellular calcium signal transduction. However, it is not known if these stimuli initiate calcium signaling within the cilium, or if the calcium signal originates in the cytoplasm. Using an integrated single-cell imaging technique, we demonstrate for the first time that calcium signaling triggered by fluid-shear stress initiates in the primary cilium and can be distinguished from the subsequent cytosolic calcium response through the ryanodine receptor. Importantly, this flow-induced calcium signaling depends on the ciliary polycystin-2 calcium channel. While DR5-specific agonist induces calcium signaling mainly in the cilioplasm via ciliary CaV1.2, thrombin specifically induces cytosolic calcium signaling through the IP3 receptor. Furthermore, a non-specific calcium ionophore triggers both ciliary and cytosolic calcium responses. We suggest that cilia not only act as sensory organelles but also function as calcium signaling compartments. Cilium-dependent signaling can spread to the cytoplasm or be contained within the cilioplasm. Our study also provides the first model to understand signaling within the cilioplasm of a living cell. PMID:24104765

  2. Plant organellar calcium signalling: an emerging field.

    PubMed

    Stael, Simon; Wurzinger, Bernhard; Mair, Andrea; Mehlmer, Norbert; Vothknecht, Ute C; Teige, Markus

    2012-02-01

    This review provides a comprehensive overview of the established and emerging roles that organelles play in calcium signalling. The function of calcium as a secondary messenger in signal transduction networks is well documented in all eukaryotic organisms, but so far existing reviews have hardly addressed the role of organelles in calcium signalling, except for the nucleus. Therefore, a brief overview on the main calcium stores in plants-the vacuole, the endoplasmic reticulum, and the apoplast-is provided and knowledge on the regulation of calcium concentrations in different cellular compartments is summarized. The main focus of the review will be the calcium handling properties of chloroplasts, mitochondria, and peroxisomes. Recently, it became clear that these organelles not only undergo calcium regulation themselves, but are able to influence the Ca(2+) signalling pathways of the cytoplasm and the entire cell. Furthermore, the relevance of recent discoveries in the animal field for the regulation of organellar calcium signals will be discussed and conclusions will be drawn regarding potential homologous mechanisms in plant cells. Finally, a short overview on bacterial calcium signalling is included to provide some ideas on the question where this typically eukaryotic signalling mechanism could have originated from during evolution. PMID:22200666

  3. Plant organellar calcium signalling: an emerging field

    PubMed Central

    Stael, Simon; Wurzinger, Bernhard; Mair, Andrea; Mehlmer, Norbert; Vothknecht, Ute C.; Teige, Markus

    2014-01-01

    This review provides a comprehensive overview of the established and emerging roles that organelles play in calcium signalling. The function of calcium as a secondary messenger in signal transduction networks is well documented in all eukaryotic organisms, but so far existing reviews have hardly addressed the role of organelles in calcium signalling, except for the nucleus. Therefore, a brief overview on the main calcium stores in plants—the vacuole, the endoplasmic reticulum, and the apoplast—is provided and knowledge on the regulation of calcium concentrations in different cellular compartments is summarized. The main focus of the review will be the calcium handling properties of chloroplasts, mitochondria, and peroxisomes. Recently, it became clear that these organelles not only undergo calcium regulation themselves, but are able to influence the Ca2+ signalling pathways of the cytoplasm and the entire cell. Furthermore, the relevance of recent discoveries in the animal field for the regulation of organellar calcium signals will be discussed and conclusions will be drawn regarding potential homologous mechanisms in plant cells. Finally, a short overview on bacterial calcium signalling is included to provide some ideas on the question where this typically eukaryotic signalling mechanism could have originated from during evolution. PMID:22200666

  4. Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

    PubMed Central

    Sundaramoorthy, Pasupathi; Sim, Jae Jun; Jang, Yeong-Su; Mishra, Siddhartha Kumar; Jeong, Keun-Yeong; Mander, Poonam; Chul, Oh Byung; Shim, Won-Sik; Oh, Seung Hyun; Nam, Ky-Youb; Kim, Hwan Mook

    2015-01-01

    Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca2+ (iCa2+) levels for a prolonged period of time. Ca2+ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca2+ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca2+ supplementation to patient undergoing treatment for metastatic cancer. PMID:25629974

  5. Ultrafine particles cause cytoskeletal dysfunctions in macrophages: role of intracellular calcium

    PubMed Central

    Möller, Winfried; Brown, David M; Kreyling, Wolfgang G; Stone, Vicki

    2005-01-01

    Background Particulate air pollution is reported to cause adverse health effects in susceptible individuals. Since most of these particles are derived form combustion processes, the primary composition product is carbon with a very small diameter (ultrafine, less than 100 nm in diameter). Besides the induction of reactive oxygen species and inflammation, ultrafine particles (UFP) can cause intracellular calcium transients and suppression of defense mechanisms of alveolar macrophages, such as impaired migration or phagocytosis. Methods In this study the role of intracellular calcium transients caused by UFP was studied on cytoskeleton related functions in J774A.1 macrophages. Different types of fine and ultrafine carbon black particles (CB and ufCB, respectively), such as elemental carbon (EC90), commercial carbon (Printex 90), diesel particulate matter (DEP) and urban dust (UD), were investigated. Phagosome transport mechanisms and mechanical cytoskeletal integrity were studied by cytomagnetometry and cell viability was studied by fluorescence microscopy. Macrophages were exposed in vitro with 100 and 320 μg UFP/ml/million cells for 4 hours in serum free medium. Calcium antagonists Verapamil, BAPTA-AM and W-7 were used to block calcium channels in the membrane, to chelate intracellular calcium or to inhibit the calmodulin signaling pathways, respectively. Results Impaired phagosome transport and increased cytoskeletal stiffness occurred at EC90 and P90 concentrations of 100 μg/ml/million cells and above, but not with DEP or UD. Verapamil and W-7, but not BAPTA-AM inhibited the cytoskeletal dysfunctions caused by EC90 or P90. Additionally the presence of 5% serum or 1% bovine serum albumin (BSA) suppressed the cytoskeletal dysfunctions. Cell viability showed similar results, where co-culture of ufCB together with Verapamil, W-7, FCS or BSA produced less cell dead compared to the particles only. PMID:16202162

  6. Calcium signaling in plant cells in microgravity

    NASA Astrophysics Data System (ADS)

    Kordyum, E.

    Changes in the intracellular Ca 2 + 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 eighties, a review highlighting the performed research and the possible significance of such Ca 2 + 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 presumably 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 Ca 2 + ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca 2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravis ensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane

  7. Fluorescence Ratio Imaging Of Dynamic Intracellular Signals

    NASA Astrophysics Data System (ADS)

    Harootunian, Alec T.; Kao, J. P.; Tsien, Roger Y.

    1989-12-01

    Traditional biochemical assays of cellular messengers require grinding up thousands or millions of cells for each data point. Such destructive measurements use up large amounts of tissue, have poor time resolution, and cannot assess heterogeneity between individual cells or dynamic spatial localizations. Recent technical advances now enable important ionic signals to be continuously imaged inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+. Binding of these ions shifts the fluorescence excitation spectrum of the corresponding indicator. The ratio of excitation amplitudes at two wavelengths measures the free ion concentration while canceling out intensity variations due to nonuniform cell thickness or dye content. By rapidly alternating between the two ion-sensitive excitation wavelengths, a fluorescence microscope equipped with a low-light television camera and digital image processor can produce dynamic images of intracellular messenger levels. In many populations of cells traditionally assumed to be homogeneous, we find that neighboring individual cells can differ enormously in their cytosolic Ca2+ response to agonist stimulation, some ignoring the stimulus, others raising cytosolic Ca2+ transiently, others showing oscillations. Oscillations have been speculated to be important as a basis for frequency-coding of oscillations. Oscillations have been speculated to be important as a basis for frequency-coding of graded inputs; we are investigating the mechanism of their generation using light flashes to generate pulses of intracellular messengers. Spatial gradients of cytosolic Ca t+ within single cells have been observed in embryos during fertilization and development, neurons exposed to electrical or drug stimulation and in cytotoxic T lymphocytes during killing of target

  8. Application of confocal microscopy on glutamate-induced intracellular calcium transient in neurons

    NASA Astrophysics Data System (ADS)

    Zhu, Geng; Zhou, Wei; Zhang, Yuan; Liu, Xiuli; Wu, Yuxiang; Luo, Qingming

    2006-02-01

    Intracellular calcium, as an important second messenger, plays a significant role in cell signaling transduction and metabolism. Glutamate can induce the intracellular calcium transient through triggering diverse signaling pathways. To test the effect of glutamate to neurons, we loaded Fluo-3/Am in cultured rat hippocampal neurons, and then acquired two-dimensional fluorescent image by confocal microscopy and the analyzed fluorescent intensity. In cultured neurons, we observed two types of neurons that have different morphology: bipolar-type and pyramidal-type. Inducing [Ca 2+] i transient by glutamate, we found the amplitude and time constant of the response curves of bipolar neurons are larger than those of pyramidal neurons. Further, we induced [Ca 2+] ii transient under different concentrations of glutamate. Two different types of kinetic of the [Ca 2+] i transient have been found, corresponded to the two kinds of neuron. The amplitude of [Ca 2+] i transient increased when applying higher concentration of glutamate in pyramidal neurons; while it decreased in bipolar ones. Responses of neurons bathing in calcium-free extracellular solution to glutamate were different from those bathing in normal solution. [Ca 2+] i transient of pyramidal neurons caused by any concentration were totally blocked; while [Ca 2+] i transient in bipolar neurons caused by high concentration of glutamate (500μM) were partly inhibited. All of the phenomena suggest that different types of cultured hippocampal neurons may have different mechanism of the response to glutamate.

  9. In Vivo Epithelial Wound Repair Requires Mobilization of Endogenous Intracellular and Extracellular Calcium*

    PubMed Central

    Aihara, Eitaro; Hentz, Courtney L.; Korman, Abraham M.; Perry, Nicholas P. J.; Prasad, Vikram; Shull, Gary E.; Montrose, Marshall H.

    2013-01-01

    We report that a localized intracellular and extracellular Ca2+ mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca2+-sensitive protein (yellow cameleon 3.0) report that intracellular Ca2+ selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca2+ increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca2+ increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca2+ mobilization. Indomethacin and verapamil also inhibit the luminal Ca2+ increase. Intracellular Ca2+ chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca2+ increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N′-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca2+ and unevenly inhibits late-phase intracellular Ca2+ mobilization. Both modes of Ca2+ chelation slow gastric repair. In plasma membrane Ca-ATPase 1+/− mice, but not plasma membrane Ca-ATPase 4−/− mice, there is slowed epithelial repair and a diminished gastric surface Ca2+ increase. We conclude that endogenous Ca2+, mobilized by signaling pathways and transmembrane Ca2+ transport, causes increased Ca2+ levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo. PMID:24121509

  10. Calcium signaling mediates cold sensing in insect tissues

    PubMed Central

    Teets, Nicholas M.; Yi, Shu-Xia; Lee, Richard E.; Denlinger, David L.

    2013-01-01

    The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms. PMID:23671084

  11. Extracellular calcium sensing and extracellular calcium signaling

    NASA Technical Reports Server (NTRS)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)

    2001-01-01

    , localized changes in Ca(o)(2+) within the ECF can originate from several mechanisms, including fluxes of calcium ions into or out of cellular or extracellular stores or across epithelium that absorb or secrete Ca(2+). In any event, the CaR and other receptors/sensors for Ca(o)(2+) and probably for other extracellular ions represent versatile regulators of numerous cellular functions and may serve as important therapeutic targets.

  12. Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells.

    PubMed

    Tomić, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S

    2015-12-01

    TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. PMID:26453278

  13. Origins of intracellular calcium mobilization evoked by infrared laser stimulation

    NASA Astrophysics Data System (ADS)

    Olsovsky, Cory A.; Tolstykh, Gleb P.; Ibey, Bennett L.; Beier, Hope T.

    2015-03-01

    Cellular delivery of pulsed IR laser energy has been shown to stimulate action potentials in neurons. The mechanism for this stimulation is not completely understood. Certain hypotheses suggest the rise in temperature from IR exposure could activate temperature- or pressure-sensitive channels, or create pores in the cellular outer membrane. Studies using intensity-based Ca2+-responsive dyes show changes in Ca2+ levels after various IR stimulation parameters; however, determination of the origin of this signal proved difficult. An influx of larger, typically plasma-membrane-impermeant ions has been demonstrated, which suggests that Ca2+ may originate from the external solution. However, activation of intracellular signaling pathways, possibly indicating a more complex role of increasing Ca2+ concentration, has also been shown. By usingCa2+ sensitive dye Fura-2 and a high-speed ratiometric imaging system that rapidly alternates the excitation wavelengths, we have quantified the Ca2+ mobilization in terms of influx from the external solution and efflux from intracellular organelles. CHO-K1 cells, which lack voltage-gated Ca2+ channels, and NG-108 neuroblastoma cells, which do not produce action potentials in an early undifferentiated state, are used to determine the origin of the Ca2+ signals and investigate the role these mechanisms may play in IR neural stimulation.

  14. A physiologic signaling role for the γ-secretase-derived intracellular fragment of APP

    PubMed Central

    Leissring, Malcolm A.; Murphy, M. Paul; Mead, Tonya R.; Akbari, Yama; Sugarman, Michael C.; Jannatipour, Mehrdad; Anliker, Brigitte; Müller, Ulrike; Saftig, Paul; De Strooper, Bart; Wolfe, Michael S.; Golde, Todd E.; LaFerla, Frank M.

    2002-01-01

    Presenilins mediate an unusual intramembranous proteolytic activity known as γ-secretase, two substrates of which are the Notch receptor (Notch) and the β-amyloid precursor protein (APP). γ-Secretase-mediated cleavage of APP, like that of Notch, yields an intracellular fragment [APP intracellular domain (AICD)] that forms a transcriptively active complex. We now demonstrate a functional role for AICD in regulating phosphoinositide-mediated calcium signaling. Genetic ablation of the presenilins or pharmacological inhibition of γ-secretase activity (and thereby AICD production) attenuated calcium signaling in a dose-dependent and reversible manner through a mechanism involving the modulation of endoplasmic reticulum calcium stores. Cells lacking APP (and hence AICD) exhibited similar calcium signaling deficits, and—notably—these disturbances could be reversed by transfection with APP constructs containing an intact AICD, but not by constructs lacking this domain. Our findings indicate that the AICD regulates phosphoinositide-mediated calcium signaling through a γ-secretase-dependent signaling pathway, suggesting that the intramembranous proteolysis of APP may play a signaling role analogous to that of Notch. PMID:11917117

  15. Role of Calcium Signaling in B Cell Activation and Biology.

    PubMed

    Baba, Yoshihiro; Kurosaki, Tomohiro

    2016-01-01

    Increase in intracellular levels of calcium ions (Ca2+) is one of the key triggering signals for the development of B cell response to the antigen. The diverse Ca2+ signals finely controlled by multiple factors participate in the regulation of gene expression, B cell development, and effector functions. B cell receptor (BCR)-initiated Ca2+ mobilization is sourced from two pathways: one is the release of Ca2+ from the intracellular stores, endoplasmic reticulum (ER), and other is the prolonged influx of extracellular Ca2+ induced by depleting the stores via store-operated calcium entry (SOCE) and calcium release-activated calcium (CRAC) channels. The identification of stromal interaction molecule 1(STIM1), the ER Ca2+ sensor, and Orai1, a key subunit of the CRAC channel pore, has now provided the tools to understand the mode of Ca2+ influx regulation and physiological relevance. Herein, we discuss our current understanding of the molecular mechanisms underlying BCR-triggered Ca2+ signaling as well as its contribution to the B cell biological processes and diseases. PMID:26369772

  16. Presynaptic Calcium Signalling in Cerebellar Mossy Fibres

    PubMed Central

    Thomsen, Louiza B.; Jörntell, Henrik; Midtgaard, Jens

    2009-01-01

    Whole-cell recordings were obtained from mossy fibre terminals in adult turtles in order to characterize the basic membrane properties. Calcium imaging of presynaptic calcium signals was carried out in order to analyse calcium dynamics and presynaptic GABA B inhibition. A tetrodotoxin (TTX)-sensitive fast Na+ spike faithfully followed repetitive depolarizing pulses with little change in spike duration or amplitude, while a strong outward rectification dominated responses to long-lasting depolarizations. High-threshold calcium spikes were uncovered following addition of potassium channel blockers. Calcium imaging using Calcium-Green dextran revealed a stimulus-evoked all-or-none TTX-sensitive calcium signal in simple and complex rosettes. All compartments of a complex rosette were activated during electrical activation of the mossy fibre, while individual simple and complex rosettes along an axon appeared to be isolated from one another in terms of calcium signalling. CGP55845 application showed that GABA B receptors mediated presynaptic inhibition of the calcium signal over the entire firing frequency range of mossy fibres. A paired-pulse depression of the calcium signal lasting more than 1 s affected burst firing in mossy fibres; this paired-pulse depression was reduced by GABA B antagonists. While our results indicated that a presynaptic rosette electrophysiologically functioned as a unit, topical GABA application showed that calcium signals in the branches of complex rosettes could be modulated locally, suggesting that cerebellar glomeruli may be dynamically sub-compartmentalized due to ongoing inhibition mediated by Golgi cells. This could provide a fine-grained control of mossy fibre-granule cell information transfer and synaptic plasticity within a mossy fibre rosette. PMID:20162034

  17. Measurement of intracellular calcium gradients in single living cells using optical sectioning microscopy

    NASA Astrophysics Data System (ADS)

    Yelamarty, Rao V.; Cheung, Joseph Y.

    1992-06-01

    Intracellular free calcium has been recognized as a regulator of many cellular processes and plays a key role in mediating actions of many drugs. To elucidate subcellular spatial calcium changes throughout the cell in three dimensions (3-D), optical sectioning microscopy was applied using digital imaging coupled fluorescence microscopy. The cell was loaded with a fluorescent indicator, fura-2, and a stack of sectional fluorescent images were acquired, digitized and finally stored on-line for post image analysis. Each sectional image was then deconvolved, to remove contaminating light signals from adjacent planes, using the Nearest Neighboring Deconvolution Algorithm (NNDA) and the overall imaging system's empirical Point Spread Function (PSF) that is measured with a 0.25 micrometers fluorescent bead. Using this technique, we measured that the addition of growth factors caused a 2 - 3 fold increase (1) in nuclear calcium compared to cytosolic calcium in blood cells and (2) in both nuclear and cytosolic calcium in liver cells. Such spatial information, which is important in understanding subcellular processes, would not be possible to measure with other methods.

  18. Ultrastructural localization of intracellular calcium stores by a new cytochemical method.

    PubMed

    Poenie, M; Epel, D

    1987-09-01

    We describe a new cytochemical method for ultrastructural localization of intracellular calcium stores. This method uses fluoride ions for in situ precipitation of intracellular calcium during fixation. Comparisons made using oxalate, antimonate, or fluoride showed that fluoride was clearly superior for intracellular calcium localization in eggs of the sea urchin Strongylocentrotus purpuratus. Whereas oxalate generally gave no intracellular precipitate and antimonate gave copious but random precipitate, three prominent calcium stores were detected using fluoride: the tubular endoplasmic reticulum, the cortical granules, and large, clear, acidic vesicles of unknown function. The mitochondria of these eggs generally showed no detectable calcium deposits. X-ray spectra confirmed the presence of calcium in the fluoride precipitates, although in some cases magnesium was also detected. Rat skeletal muscle and sea urchin sperm were used to test the reliability of the fluoride method for calcium localization. In rat skeletal muscle, most fluoride precipitate was confined to the sarcoplasmic reticulum. Using sea urchin sperm, which transport calcium into the mitochondria after exposure to egg jelly to induce the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria contain no detectable calcium-containing precipitate. Within 4 min after induction of the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria displayed many foci of calcium-containing precipitate. The use of fluoride for intracellular calcium localization therefore appears to be a substantial improvement over previous cytochemical methods. PMID:3611737

  19. Lead Poisoning Disturbs Oligodendrocytes Differentiation Involved in Decreased Expression of NCX3 Inducing Intracellular Calcium Overload

    PubMed Central

    Ma, Teng; Wu, Xiyan; Cai, Qiyan; Wang, Yun; Xiao, Lan; Tian, Yanping; Li, Hongli

    2015-01-01

    Lead (Pb) poisoning has always been a serious health concern, as it permanently damages the central nervous system. Chronic Pb accumulation in the human body disturbs oligodendrocytes (OLs) differentiation, resulting in dysmyelination, but the molecular mechanism remains unknown. In this study, Pb at 1 μM inhibits OLs precursor cells (OPCs) differentiation via decreasing the expression of Olig 2, CNPase proteins in vitro. Moreover, Pb treatment inhibits the sodium/calcium exchanger 3 (NCX3) mRNA expression, one of the major means of calcium (Ca2+) extrusion at the plasma membrane during OPCs differentiation. Also addition of KB-R7943, NCX3 inhibitor, to simulate Pb toxicity, resulted in decreased myelin basic protein (MBP) expression and cell branching. Ca2+ response trace with Pb and KB-R7943 treatment did not drop down in the same recovery time as the control, which elevated intracellular Ca2+ concentration reducing MBP expression. In contrast, over-expression of NCX3 in Pb exposed OPCs displayed significant increase MBP fluorescence signal in positive regions and CNPase expression, which recovered OPCs differentiation to counterbalance Pb toxicity. In conclusion, Pb exposure disturbs OLs differentiation via affecting the function of NCX3 by inducing intracellular calcium overload. PMID:26287169

  20. The calcium-signaling toolkit: Updates needed.

    PubMed

    Dubois, Charlotte; Prevarskaya, Natalia; Vanden Abeele, Fabien

    2016-06-01

    Here, we review the role of Ca(2+) in apoptosis, namely that ER Ca(2+) depletion or a sustained elevation of cytosolic or mitochondrial Ca(2+) concentration are sufficient to trigger apoptosis. These concepts have emerged by the use of ER stressor agents that decrease the ER Ca(2+) pool by inhibiting SERCA pumps. However, aside from their well-known actions on Ca(2+) homeostasis disruption leading to apoptosis, new evidence show that some ER Ca(2+) modulators have significant implications in other Ca(2+)-mediated or Ca(2+)-independent pathways determining cell fate suggesting a more complex regulation of apoptosis by intracellular Ca(2+). Here, we discuss the crucial interplay between Ca(2+) mediated apoptosis, the Unfold Protein Response and autophagy determining cell fate, and the molecular compounds that have been used to depict these pathways. This review of the literature clearly shows the need for new inhibitors that do not interfere concomitantly with autophagy and Ca(2+) signaling. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. PMID:26658643

  1. The dysregulation of intracellular calcium in Alzheimer disease.

    PubMed

    Supnet, Charlene; Bezprozvanny, Ilya

    2010-02-01

    Alzheimer disease (AD) is the most common neurodegenerative disorder worldwide and is at present, incurable. The accumulation of toxic amyloid-beta (Abeta) peptide aggregates in AD brain are thought to trigger the extensive synaptic loss and neurodegeneration linked to cognitive decline, an idea that underlies the 'amyloid hypothesis' of AD etiology in both the familal (FAD) and sporadic forms of the disease. Mutations causing FAD also result in the dysregulation of neuronal calcium (Ca2+) handling and may contribute to AD pathogenesis, an idea termed the 'calcium hypothesis' of AD. In particular, Ca2+ dysregulation by the endoplasmic reticulum (ER) in AD mouse models results in augmented cytosolic Ca2+ levels which can trigger signalling cascades that are detrimental to neuronal function and health. However, there is growing evidence to suggest that not all forms of Ca2+ dysregulation in AD neurons are harmful and some of them instead may be compensatory. These changes may help modulate neuronal excitability and slow AD pathology, especially in the early stages of the disease. Clearly, a better understanding of how dysregulation of neuronal Ca2+ handling contributes to neurodegeneration and neuroprotection in AD is needed as Ca2+ signalling modulators are targets of great interest as potential AD therapeutics. PMID:20080301

  2. [Role of endoplasmic reticulum-plasma membrane junctions in intracellular calcium homeostasis and cardiovascular disease].

    PubMed

    Zhao, Ming; Jia, Hang-Huan; Xu, Man; Yu, Xiao-Jiang; Liu, Long-Zhu; Zang, Wei-Jin

    2016-08-25

    Calcium overload is one of the important mechanisms of cardiovascular disease. Endoplasmic reticulum is an important organelle which regulates intracellular calcium homeostasis by uptake, storage and mobilization of calcium. So it plays a critical role in regulation of intracellular calcium homeostasis. Endoplasmic reticulum, which is widely distributed in cytoplasm, has a large number of membrane junction sites. Recent studies have reported that these junction sites are distributed on plasma membrane and organelle membranes (mitochondria, lysosomes, Golgi apparatus, etc.), separately. They could form complexes to regulate calcium transport. In this review, we briefly outlined the recent research progresses of endoplasmic reticulum-plasma membrane junctions in intracellular calcium homeostasis and cardiovascular disease, which may offer a new strategy for prevention and treatment of cardiovascular disease. PMID:27546511

  3. Axotomy Depletes Intracellular Calcium Stores in Primary Sensory Neurons

    PubMed Central

    Rigaud, Marcel; Gemes, Geza; Weyker, Paul D.; Cruikshank, James M.; Kawano, Takashi; Wu, Hsiang-En; Hogan, Quinn H.

    2010-01-01

    Background The cellular mechanisms of neuropathic pain are inadequately understood. Previous investigations have revealed disrupted Ca2+ signaling in primary sensory neurons after injury. We therefore examined the effect of injury on intracellular Ca2+ stores of the endoplasmic reticulum, which critically regulate the Ca2+ signal and neuronal function. Methods Intracellular Ca2+ levels were measured with Fura-2 or mag-Fura-2 microfluorometry in axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent L4 neurons isolated from hyperalgesic rats following L5 spinal nerve ligation, compared to neurons from control animals. Results Endoplasmic reticulum Ca2+ stores released by the ryanodine-receptor agonist caffeine decreased by 46% in axotomized small neurons. This effect persisted in Ca2+-free bath solution that removes the contribution of store-operated membrane Ca2+ channels, and after blockade of both the mitochondrial, sarco-endoplasmic Ca2+-ATPase, and the plasma membrane Ca2+ ATPase pathways. Ca2+ released by the sarco-endoplasmic Ca2+-ATPase blocker thapsigargin and by the Ca2+-ionophore ionomycin was also diminished by 25% and 41%, respectively. In contrast to control neurons, Ca2+ stores in axotomized neurons were not expanded by neuronal activation by K+ depolarization, and the proportionate rate of refilling by sarco-endoplasmic Ca2+-ATPase was normal. Luminal Ca2+ concentration was also reduced by 38% in axotomized neurons in permeabilized neurons. The adjacent neurons of the L4 dorsal root ganglia showed modest and inconsistent changes after L5 spinal nerve ligation. Conclusions Painful nerve injury leads to diminished releasable endoplasmic reticulum Ca2+ stores and a reduced luminal Ca2+ concentration. Depletion of Ca2+ stores may contribute to the pathogenesis of neuropathic pain. PMID:19602958

  4. Intracellular calcium and cyclic nucleotide levels modulate neurite guidance by microtopographical substrate features.

    PubMed

    Li, Shufeng; Tuft, Bradley; Xu, Linjing; Polacco, Marc; Clarke, Joseph C; Guymon, C Allan; Hansen, Marlan R

    2016-08-01

    Micro- and nanoscale surface features have emerged as potential tools to direct neurite growth into close proximity with next generation neural prosthesis electrodes. However, the signaling events underlying the ability of growth cones to respond to topographical features remain largely unknown. Accordingly, this study probes the influence of [Ca(2+) ]i and cyclic nucleotide levels on the ability of neurites from spiral ganglion neurons (SGNs) to precisely track topographical micropatterns. Photopolymerization and photomasking were used to generate micropatterned methacrylate polymer substrates. Dissociated SGN cultures were plated on the micropatterned surfaces. Calcium influx and release from internal stores were manipulated by elevating extracellular K(+) , maintenance in calcium-free media, or bath application of various calcium channel blockers. Cyclic nucleotide activity was increased by application of cpt-cAMP or 8-Br-cGMP. Elevation of [Ca(2+) ]i by treatment of cultures with elevated potassium reduced neurite alignment to physical microfeatures. Maintenance of cultures in Ca(2+) -free medium or treatment with the non-selective voltage-gated calcium channel blocker cadmium or L-type Ca(2+) channel blocker nifedipine did not signficantly alter SGN neurite alignment. By contrast, ryanodine or xestospongin C, which block release of internal calcium stores via ryanodine-sensitive channels or inositol-1,4,5-trisphosphate receptors respectively, each significantly decreased neurite alignment. Cpt-cAMP significantly reduced neurite alignment while 8-Br-cGMP significantly enhanced neurite alignment. Manipulation of [Ca(2+) ]i or cAMP levels significantly disrupts neurite guidance while elevation of cGMP levels increases neurite alignment. The results suggest intracellular signaling pathways similar to those recruited by chemotactic cues are involved in neurite guidance by topographical features. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2037

  5. Nucleoplasmic calcium signaling and cell proliferation: calcium signaling in the nucleus.

    PubMed

    Resende, Rodrigo R; Andrade, Lidia M; Oliveira, Andre G; Guimarães, Erika S; Guatimosim, Silvia; Leite, M Fatima

    2013-01-01

    Calcium (Ca2+) is an essential signal transduction element involved in the regulation of several cellular activities and it is required at various key stages of the cell cycle. Intracellular Ca2+ is crucial for the orderly cell cycle progression and plays a vital role in the regulation of cell proliferation. Recently, it was demonstrated by in vitro and in vivo studies that nucleoplasmic Ca2+ regulates cell growth. Even though the mechanism by which nuclear Ca2+ regulates cell proliferation is not completely understood, there are reports demonstrating that activation of tyrosine kinase receptors (RTKs) leads to translocation of RTKs to the nucleus to generate localized nuclear Ca2+ signaling which are believed to modulate cell proliferation. Moreover, nuclear Ca2+ regulates the expression of genes involved in cell growth. This review will describe the nuclear Ca2+ signaling machinery and its role in cell proliferation. Additionally, the potential role of nuclear Ca2+ as a target in cancer therapy will be discussed. PMID:23433362

  6. Resveratrol reduces intracellular free calcium concentration in rat ventricular myocytes.

    PubMed

    Liu, Zheng; Zhang, Li-Ping; Ma, Hui-Jie; Wang, Chuan; Li, Ming; Wang, Qing-Shan

    2005-10-25

    Resveratrol (trans-3, 4', 5-trihydroxy stilbene), a phytoalexin found in grape skins and red wine, has been reported to have a wide range of biological and pharmacological properties. It has been speculated that resveratrol may have cardioprotective activity. The objective of our study was to investigate the effects of resveratrol on intracellular calcium concentration ([Ca(2+)](i)) in rat ventricular myocytes. [Ca(2+)](i) was detected by laser scanning confocal microscopy. The results showed that resveratrol (15~60 mumol/L) reduced [Ca(2+)](i) in normal and Ca(2+)-free Tyrode's solution in a concentration-dependent manner. The effects of resveratrol on [Ca(2+)](i) in normal Tyrode's solution was partially inhibited by pretreatment with sodium orthovanadate (Na3VO4, 1.0 mmol/L, P<0.01), an inhibitor of protein tyrosine phosphatase, or L-type Ca(2+) channel agonist Bay K8644 (10 mumol/L, P<0.05), but could not be antagonized by NO synthase inhibitor L-NAME (1.0 mmol/L). Resveratrol also markedly inhibited the ryanodine-induced [Ca(2+)](i) increase in Ca(2+)-free Tyrode's solution (P<0.01). When Ca(2+) waves were produced by increasing extracellular Ca(2+) concentration from 1 to 10 mmol/L, resveratrol (60 mumol/L) could reduce the velocity and duration of propagating waves, and block the propagating waves of elevated [Ca(2+)](i). These results suggest that resveratrol may reduce the [Ca(2+)](i) in isolated rat ventricular myocytes. The inhibition of voltage-dependent Ca(2+) channel and tyrosine kinase, and alleviation of Ca(2+) release from sarcoplasmic reticulum (SR) are possibly involved in the effects of resveratrol on rat ventricular myocytes. These findings could help explain the protective activity of resveratrol against cardiovascular disease. PMID:16220198

  7. Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

    PubMed

    Stefano, G B; Prevot, V; Beauvillain, J C; Fimiani, C; Welters, I; Cadet, P; Breton, C; Pestel, J; Salzet, M; Bilfinger, T V

    1999-10-01

    We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium. PMID:10490972

  8. Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes.

    PubMed

    Wei, Jinhong; Sun, Junqing; Xu, Hao; Shi, Liang; Sun, Lijun; Zhang, Jianbao

    2015-03-01

    Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15 Hz, 50 Hz, 75 Hz and 100 Hz) and at a flux density of 2 mT. Intracellular calcium concentration ([Ca(2+)]i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10 mM) was carried out to verify the function of the cardiac Na(+)/Ca(2+) exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca(2+)-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca(2+)]i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes. PMID:24499289

  9. Calcium signalling remodelling and disease.

    PubMed

    Berridge, Michael J

    2012-04-01

    A wide range of Ca2+ signalling systems deliver the spatial and temporal Ca2+ signals necessary to control the specific functions of different cell types. Release of Ca2+ by InsP3 (inositol 1,4,5-trisphosphate) plays a central role in many of these signalling systems. Ongoing transcriptional processes maintain the integrity and stability of these cell-specific signalling systems. However, these homoeostatic systems are highly plastic and can undergo a process of phenotypic remodelling, resulting in the Ca2+ signals being set either too high or too low. Such subtle dysregulation of Ca2+ signals have been linked to some of the major diseases in humans such as cardiac disease, schizophrenia, bipolar disorder and Alzheimer's disease. PMID:22435804

  10. Calcium Oscillations

    PubMed Central

    Dupont, Geneviève; Combettes, Laurent; Bird, Gary S.; Putney, James W.

    2011-01-01

    Calcium signaling results from a complex interplay between activation and inactivation of intracellular and extracellular calcium permeable channels. This complexity is obvious from the pattern of calcium signals observed with modest, physiological concentrations of calcium-mobilizing agonists, which typically present as sequential regenerative discharges of stored calcium, a process referred to as calcium oscillations. In this review, we discuss recent advances in understanding the underlying mechanism of calcium oscillations through the power of mathematical modeling. We also summarize recent findings on the role of calcium entry through store-operated channels in sustaining calcium oscillations and in the mechanism by which calcium oscillations couple to downstream effectors. PMID:21421924

  11. Azelnidipine prevents cardiac dysfunction in streptozotocin-diabetic rats by reducing intracellular calcium accumulation, oxidative stress and apoptosis

    PubMed Central

    2011-01-01

    Background Numerous evidences suggest that diabetic heart is characterized by compromised ventricular contraction and prolonged relaxation attributable to multiple causative factors including calcium accumulation, oxidative stress and apoptosis. Therapeutic interventions to prevent calcium accumulation and oxidative stress could be therefore helpful in improving the cardiac function under diabetic condition. Methods This study was designed to examine the effect of long-acting calcium channel blocker (CCB), Azelnidipine (AZL) on contractile dysfunction, intracellular calcium (Ca2+) cycling proteins, stress-activated signaling molecules and apoptosis on cardiomyocytes in diabetes. Adult male Wistar rats were made diabetic by a single intraperitoneal (IP) injection of streptozotocin (STZ). Contractile functions were traced from live diabetic rats to isolated individual cardiomyocytes including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR90), maximal velocity of shortening/relengthening (± dL/dt) and intracellular Ca2+ fluorescence. Results Diabetic heart showed significantly depressed PS, ± dL/dt, prolonged TPS, TR90 and intracellular Ca2+ clearing and showed an elevated resting intracellular Ca2+. AZL itself exhibited little effect on myocyte mechanics but it significantly alleviated STZ-induced myocyte contractile dysfunction. Diabetes increased the levels of superoxide, enhanced expression of the cardiac damage markers like troponin I, p67phox NADPH oxidase subunit, restored the levels of the mitochondrial superoxide dismutase (Mn-SOD), calcium regulatory proteins RyR2 and SERCA2a, and suppressed the levels of the anti-apoptotic Bcl-2 protein. All of these STZ-induced alterations were reconciled by AZL treatment. Conclusion Collectively, the data suggest beneficial effect of AZL in diabetic cardiomyopathy via altering intracellular Ca2+ handling proteins and preventing apoptosis by its antioxidant property. PMID:22054019

  12. Regionalized calcium signaling in zebrafish fertilization.

    PubMed

    Sharma, Dipika; Kinsey, William H

    2008-01-01

    Fertilization involves an initial, highly localized signal delivered by the sperm, which becomes amplified by a signal transduction cascade to impact the entire oocyte cytoplasm. The zebrafish oocyte presents a unique opportunity to study this process since fertilization always occurs at the micropyle, allowing the investigator to image the earliest steps in the oocyte activation process. The objective of the present study was to characterize the amplification of the sperm-induced calcium transient in the zebrafish oocyte and test the role of Fyn kinase in this process. Confocal fluorescence microscopy revealed that the sperm-induced calcium transient was composed of two elements, one of which was unique to the oocyte cortex and a second, slower transient that occurred in the central cytoplasm of the oocyte. The cortical transient was initiated immediately deep to the micropyle, became amplified at the animal pole, and progressed peripherally through the oocyte cortex. This was followed by a slower transient that occurred in the central cytoplasm of the oocyte. Several lines of evidence indicate that calcium release in these two compartments may be regulated differently. The calcium transient in the oocyte cortex is highly sensitive to inhibition by Fyn-SH2 domain containing fusion proteins, while the central cytoplasmic transient is relatively resistant to this treatment. Oocytes stimulated by injection of a soluble extract prepared from zebrafish sperm respond only with a cortical calcium transient initiated at the micropyle, while oocytes stimulated parthenogenetically by hypotonic shock exhibit a defective cortical transient but a normal transient in the central cytoplasm. Analysis of the subcellular distribution of Fyn kinase and the IP3 receptor reveal that these important signaling components are highly enriched in the oocyte cortex, a factor which may facilitate a faster propagation of the calcium transient in this compartment. In summary, analysis of

  13. Activation of NADPH oxidase 1 increases intracellular calcium and migration of smooth muscle cells.

    PubMed

    Zimmerman, Matthew C; Takapoo, Maysam; Jagadeesha, Dammanahalli K; Stanic, Bojana; Banfi, Botond; Bhalla, Ramesh C; Miller, Francis J

    2011-09-01

    Redox-dependent migration and proliferation of vascular smooth muscle cells (SMCs) are central events in the development of vascular proliferative diseases; however, the underlying intracellular signaling mechanisms are not fully understood. We tested the hypothesis that activation of Nox1 NADPH oxidase modulates intracellular calcium ([Ca(2+)](i)) levels. Using cultured SMCs from wild-type and Nox1 null mice, we confirmed that thrombin-dependent generation of reactive oxygen species requires Nox1. Thrombin rapidly increased [Ca(2+)](i), as measured by fura-2 fluorescence ratio imaging, in wild-type but not Nox1 null SMCs. The increase in [Ca(2+)](i) in wild-type SMCs was inhibited by antisense to Nox1 and restored by expression of Nox1 in Nox1 null SMCs. Investigation into potential mechanisms by which Nox1 modulates [Ca(2+)](i) showed that thrombin-induced inositol triphosphate generation and thapsigargin-induced intracellular calcium mobilization were similar in wild-type and Nox1 null SMCs. To examine the effects of Nox1 on Ca(2+) entry, cells were either bathed in Ca(2+)-free medium or exposed to dihydropyridines to block L-type Ca(2+) channel activity. Treatment with nifedipine or removal of extracellular Ca(2+) reduced the thrombin-mediated increase of [Ca(2+)](i) in wild-type SMCs, whereas the response in Nox1 null SMCs was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases, restored the thrombin-induced increase of [Ca(2+)](i) in Nox1 null SMCs. Migration of SMCs was impaired with deficiency of Nox1 and restored with expression of Nox1 or the addition of sodium vanadate. In summary, we conclude that Nox1 NADPH oxidase modulates Ca(2+) mobilization in SMCs, in part through regulation of Ca(2+) influx, to thereby promote cell migration. PMID:21810651

  14. Store-operated channels regulate intracellular calcium in mammalian rods.

    PubMed

    Molnar, Tünde; Barabas, Peter; Birnbaumer, Lutz; Punzo, Claudio; Kefalov, Vladimir; Križaj, David

    2012-08-01

    Exposure to daylight closes cyclic nucleotide-gated (CNG) and voltage-operated Ca(2+) -permeable channels in mammalian rods. The consequent lowering of the cytosolic calcium concentration ([Ca(2+)](i)), if protracted, can contribute to light-induced damage and apoptosis in these cells. We here report that mouse rods are protected against prolonged lowering of [Ca(2+)](i) by store-operated Ca(2+) entry (SOCE). Ca(2+) stores were depleted in Ca(2+)-free saline supplemented with the endoplasmic reticulum (ER) sequestration blocker cyclopiazonic acid. Store depletion elicited [Ca(2+)](i) signals that exceeded baseline [Ca(2+)](i) by 5.9 ± 0.7-fold and were antagonized by an inhibitory cocktail containing 2-APB, SKF 96365 and Gd(3+). Cation influx through SOCE channels was sufficient to elicit a secondary activation of L-type voltage-operated Ca2+ entry. We also found that TRPC1, the type 1 canonical mammalian homologue of the Drosophila photoreceptor TRP channel, is predominantly expressed within the outer nuclear layer of the retina. Rod loss in Pde6b(rdl) (rd1), Chx10/Kip1(-/-rdl) and Elovl4(TG2) dystrophic models was associated with ∼70% reduction in Trpc1 mRNA content whereas Trpc1 mRNA levels in rodless cone-full Nrl(-/-) retinas were decreased by ∼50%. Genetic ablation of TRPC1 channels, however, had no effect on SOCE, the sensitivity of the rod phototransduction cascade or synaptic transmission at rod and cone synapses. Thus, we localized two new mechanisms, SOCE and TRPC1, to mammalian rods and characterized the contribution of SOCE to Ca(2+) homeostasis. By preventing the cytosolic [Ca(2+)](i) from dropping too low under sustained saturating light conditions, these signalling pathways may protect Ca(2+)-dependent mechanisms within the ER and the cytosol without affecting normal rod function. PMID:22674725

  15. Coincident signalling between the Gi/Go-coupled delta-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells.

    PubMed

    Yeo, A; Samways, D S; Fowler, C E; Gunn-Moore, F; Henderson, G

    2001-03-01

    In SH-SY5Y cells, activation of delta-opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 microM) did not alter the intracellular free Ca(2+) concentration [Ca(2+)](i). However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca(2+)](i). The DPDPE-evoked increase in [Ca(2+)](i) was abolished when the carbachol-sensitive intracellular Ca(2+) store was emptied. There was a marked difference between the concentration-response relationship for the elevation of [Ca(2+)](i) by carbachol (EC(50) 13 microM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta-opioid receptor-mediated elevation of [Ca(2+)] (EC(50) 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca(2+)](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta-opioid receptor activation did not stimulate phospholipase C. Furthermore, delta-opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the delta-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca(2+)](i) in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed delta-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca(2+) release signalling pathway at a step after the activation of phospholipase C. PMID:11259487

  16. Presenilins and calcium signaling – systems biology to the rescue

    PubMed Central

    Bezprozvanny, Ilya

    2016-01-01

    Mutations in presenilins result in familial Alzheimer’s disease (FAD). Presenilins encode a catalytic subunit of γ-secretase complex, and FAD mutations in presenilins alter γ-secretase activity. Many FAD mutations in presenilins also affect intracellular calcium signaling. To explain these results it was proposed that presenilins encode endoplasmic reticulum (ER) calcium leak channels, and that this function is disrupted by FAD mutations. This hypothesis has been controversial. Two recent reports provide new evidence for the calcium leak channel hypothesis. One group reported the presence of putative ion-conduction pore in the high resolution crystal structure of bacterial presenilin homologue PSH1. Another group identified an essential role of presenilins in mediating ER calcium leak in unbiased cell-based screen for calcium homeostasis modulators. These results should enable the field to move forward and to focus on exploring connections between FAD mutations in presenilins, changes in γ-secretase and ER Ca2+ leak functions and development of the disease. PMID:23838181

  17. Astrocyte calcium signaling: the third wave.

    PubMed

    Bazargani, Narges; Attwell, David

    2016-02-01

    The discovery that transient elevations of calcium concentration occur in astrocytes, and release 'gliotransmitters' which act on neurons and vascular smooth muscle, led to the idea that astrocytes are powerful regulators of neuronal spiking, synaptic plasticity and brain blood flow. These findings were challenged by a second wave of reports that astrocyte calcium transients did not mediate functions attributed to gliotransmitters and were too slow to generate blood flow increases. Remarkably, the tide has now turned again: the most important calcium transients occur in fine astrocyte processes not resolved in earlier studies, and new mechanisms have been discovered by which astrocyte [Ca(2+)]i is raised and exerts its effects. Here we review how this third wave of discoveries has changed our understanding of astrocyte calcium signaling and its consequences for neuronal function. PMID:26814587

  18. Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent.

    PubMed

    Szöllösi, J; Feuerstein, B G; Vereb, G; Pershadsingh, H A; Marton, L J

    1991-07-01

    Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells. PMID:1657394

  19. Modulation of iron metabolism by iron chelation regulates intracellular calcium and increases sensitivity to doxorubicin

    PubMed Central

    Yalcintepe, Leman; Halis, Emre

    2016-01-01

    Increased intracellular iron levels can both promote cell proliferation and death, as such; iron has a “two-sided effect” in the delicate balance of human health. Though the role of iron in the development of cancer remains unclear, investigations of iron chelators as anti-tumor agents have revealed promising results. Here, we investigated the influence of iron and desferrioxamine (DFO), the iron chelating agent on intracellular calcium in a human leukemia cell line, K562. Iron uptake is associated with increased reactive oxygen species (ROS) generation. Therefore, we showed that iron also caused dose-dependent ROS generation in K562 cells. The measurement of intracellular calcium was determined using Furo-2 with a fluorescence spectrophotometer. The iron delivery process to the cytoplasmic iron pool was examined by monitoring the fluorescence of cells loaded with calcein-acetoxymethyl. Our data showed that iron increased intracellular calcium, and this response was 8 times higher when cells were incubated with DFO. K562 cells with DFO caused a 3.5 times increase of intracellular calcium in the presence of doxorubicin (DOX). In conclusion, DFO induces intracellular calcium and increases their sensitivity to DOX, a chemotherapeutic agent. PMID:26773173

  20. Continuous Fluorescence Imaging of Intracellular Calcium by Use of Ion-Selective Nanospheres with Adjustable Spectra.

    PubMed

    Yang, Chenye; Qin, Yu; Jiang, Dechen; Chen, Hong-Yuan

    2016-08-10

    Continuous fluorescence imaging of intracellular ions in various spectral ranges is important for biological studies. In this paper, fluorescent calcium-selective nanospheres, including calix[4]arene-functionalized bodipy (CBDP) or 9-(diethylamino)-5-[(2-octyldecyl)imino]benzo[a]phenoxazine (ETH 5350) as the chromoionophore, were prepared to demonstrate intracellular calcium imaging in visible or near-IR regions, respectively. The fluorescence of the nanospheres was controlled by the chromoionophore, and thus the spectral range for detection was adjustable by choosing the proper chromoionophore. The response time of the nanospheres to calcium was typically 1 s, which allowed accurate measurement of intracellular calcium. These nanospheres were loaded into cells through free endocytosis and exhibited fluorescence for 24 h, and their intensity was correlated with the elevation of intracellular calcium upon stimulation. The successful demonstration of calcium imaging by use of ion-selective nanospheres within two spectral ranges in 24 h supported that these nanospheres could be applied for continuous imaging of intracellular ions with adjustable spectra. PMID:27408988

  1. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

    PubMed Central

    Hasan, Md. Ashraful; Ahn, Won-Gyun

    2016-01-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca2+]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca2+]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca2+]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca2+]i in human neutrophils was observed. In Ca2+-free buffer, NAC- and cysteine-induced [Ca2+]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca2+]i in human neutrophils occur through Ca2+ influx. NAC- and cysteine-induced [Ca2+]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na+-free HEPES, both NAC and cysteine induced a marked increase in [Ca2+]i in human neutrophils, arguing against the possibility that Na+-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca2+]i increasing activity. Our results show that NAC and cysteine induce [Ca2+]i increase through Ca2+ influx in human neutrophils via SKF96365- and ruthenium red-dependent way. PMID:27610031

  2. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils.

    PubMed

    Hasan, Md Ashraful; Ahn, Won-Gyun; Song, Dong-Keun

    2016-09-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca(2+) signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca(2+)]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca(2+)]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca(2+)]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca(2+)]i in human neutrophils was observed. In Ca(2+)-free buffer, NAC- and cysteine-induced [Ca(2+)]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca(2+)]i in human neutrophils occur through Ca(2+) influx. NAC- and cysteine-induced [Ca(2+)]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na(+)-free HEPES, both NAC and cysteine induced a marked increase in [Ca(2+)]i in human neutrophils, arguing against the possibility that Na(+)-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca(2+)]i increasing activity. Our results show that NAC and cysteine induce [Ca(2+)]i increase through Ca(2+) influx in human neutrophils via SKF96365- and ruthenium red-dependent way. PMID:27610031

  3. Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels.

    PubMed

    Yang, Xinhui; Jiang, Jiangtao; Yang, Xinyan; Han, Jichun; Zheng, Qiusheng

    2016-07-01

    Licochalcone A (LCA) has been reported to significantly inhibit cell proliferation, increase reactive oxygen species (ROS) levels, and induce apoptosis of T24 human bladder cancer cells via mitochondria and endoplasmic reticulum (ER) stress-triggered signaling pathways. Based on these findings, the present study aimed to investigate the mechanisms by which LCA induces apoptosis of T24 cells. Cultured T24 cells were treated with LCA, and cell viability was measured using the sulforhodamine B assay. Apoptosis was detected by flow cytometry with Annexin V/propidium iodide staining, and by fluorescent microscopy with Hoechst 33258 staining. The levels of intracellular free calcium ions were determined using Fluo-3 AM dye marker. Intracellular ROS levels were assessed using the 2',7'-dichlorodihydrofluorescein diacetate probe assay. The mitochondrial membrane potential was measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide. Furthermore, the mRNA expression levels of B‑cell lymphoma (Bcl)‑extra large, Bcl‑2‑associated X protein, Bcl‑2‑interacting mediator of cell death, apoptotic protease activating factor‑1 (Apaf‑1), calpain 2, cysteinyl aspartate specific proteinase (caspase)‑3, caspase‑4 and caspase‑9 were determined using reverse transcription semiquantitative and quantitative polymerase chain reaction analyses. Treatment with LCA inhibited proliferation and induced apoptosis of T24 cells, and increased intracellular Ca2+ levels and ROS production. Furthermore, LCA induced mitochondrial dysfunction, decreased mitochondrial membrane potential, and increased the mRNA expression levels of Apaf‑1, caspase‑9 and caspase‑3. Exposure of T24 cells to LCA also triggered calpain 2 and caspase‑4 activation, resulting in apoptosis. These findings indicated that LCA increased intracellular Ca2+ levels, which may be associated with mitochondrial dysfunction. In addition, the ER stress pathway may be

  4. Calcium Signaling in Lacrimal Glands

    PubMed Central

    Putney, James W.; Bird, Gary S.

    2014-01-01

    Lacrimal glands provide the important function of lubricating and protecting the ocular surface. Failure of proper lacrimal gland function results in a number of debilitating dry eye diseases. Lacrimal glands secrete lipids, mucins, proteins, salts and water and these secretions are at least partially regulated by neurotransmitter-mediated cell signaling. The predominant signaling mechanism for lacrimal secretion involves activation of phospholipase C, generation of the Ca2+-mobilizing messenger, IP3, and release of Ca2+ stored in the endoplasmic reticulum. The loss of Ca2+ from the endoplasmic reticulum then triggers a process known as store-operated Ca2+ entry, involving a Ca2+ sensor in the endoplasmic reticulum, STIM1, which activates plasma membrane store-operated channels comprised of Orai subunits. Recent studies with deletions of the channel subunit, Orai1, confirm the important role of SOCE in both fluid and protein secretion in lacrimal glands, both in vivo and in vitro. PMID:24507443

  5. Regulating Intracellular Calcium in Plants: From Molecular Genetics to Physiology

    SciTech Connect

    Heven Sze

    2008-06-22

    To grow, develop, adapt, and reproduce, plants have evolved mechanisms to regulate the uptake, translocation and sorting of calcium ions into different cells and subcellular compartments. Yet how plants accomplish this remarkable feat is still poorly understood. The spatial and temporal changes in intracellular [Ca2+] during growth and during responses to hormonal and environmental stimuli indicate that Ca2+ influx and efflux transporters are diverse and tightly regulated in plants. The specific goals were to determine the biological roles of multiple Ca pumps (ECAs) in the model plant Arabidopsis thaliana. We had pioneered the use of K616 yeast strain to functionally express plant Ca pumps, and demonstrated two distinct types of Ca pumps in plants (Sze et al., 2000. Annu Rev Plant Biol. 51,433). ACA2 represented one type that was auto-inhibited by the N-terminal region and stimulated by calmodulin. ECA1 represented another type that was not sensitive to calmodulin and phylogenetically distinct from ACAs. The goal to determine the biological roles of multiple ECA-type Ca pumps in Arabidopsis has been accomplished. Although we demonstrated ECA1 was a Ca pump by functional expression in yeast, the in vivo roles of ECAs was unclear. A few highlights are described. ECA1 and/or ECA4 are Ca/Mn pumps localized to the ER and are highly expressed in all cell types. Using homozygous T-DNA insertional mutants of eca1, we demonstrated that the ER-bound ECA1 supports growth and confers tolerance of plants growing on medium low in Ca or containing toxic levels of Mn. This is the first genetic study to determine the in vivo function of a Ca pump in plants. A phylogenetically distinct ECA3 is also a Ca/Mn pump that is localized to endosome, such as post-Golgi compartments. Although it is expressed at lower levels than ECA1, eca3 mutants are impaired in Ca-dependent root growth and in pollen tube elongation. Increased secretion of wall proteins in mutants suggests that Ca and Mn

  6. Resveratrol and Calcium Signaling: Molecular Mechanisms and Clinical Relevance

    PubMed Central

    McCalley, Audrey E.; Kaja, Simon; Payne, Andrew J.; Koulen, Peter

    2014-01-01

    Resveratrol is a naturally occurring compound contributing to cellular defense mechanisms in plants. Its use as a nutritional component and/or supplement in a number of diseases, disorders, and syndromes such as chronic diseases of the central nervous system, cancer, inflammatory diseases, diabetes, and cardiovascular diseases has prompted great interest in the underlying molecular mechanisms of action. The present review focuses on resveratrol, specifically its isomer trans-resveratrol, and its effects on intracellular calcium signaling mechanisms. As resveratrol's mechanisms of action are likely pleiotropic, its effects and interactions with key signaling proteins controlling cellular calcium homeostasis are reviewed and discussed. The clinical relevance of resveratrol's actions on excitable cells, transformed or cancer cells, immune cells and retinal pigment epithelial cells are contrasted with a review of the molecular mechanisms affecting calcium signaling proteins on the plasma membrane, cytoplasm, endoplasmic reticulum, and mitochondria. The present review emphasizes the correlation between molecular mechanisms of action that have recently been identified for resveratrol and their clinical implications. PMID:24905603

  7. Simulations of intracellular calcium release dynamics in response to a high-intensity, ultrashort electric pulse

    NASA Astrophysics Data System (ADS)

    Joshi, R. P.; Nguyen, A.; Sridhara, V.; Hu, Q.; Nuccitelli, R.; Beebe, S. J.; Kolb, J.; Schoenbach, K. H.

    2007-04-01

    Numerical simulations for electrically induced, intracellular calcium release from the endoplasmic reticulum are reported. A two-step model is used for self-consistency. Distributed electrical circuit representation coupled with the Smoluchowski equation yields the ER membrane nanoporation for calcium outflow based on a numerical simulation. This is combined with the continuum Li-Rinzel model and drift diffusion for calcium dynamics. Our results are shown to be in agreement with reported calcium release data. A modest increase (rough doubling) of the cellular calcium is predicted in the absence of extra-cellular calcium. In particular, the applied field of 15kV/cm with 60ns pulse duration makes for a strong comparison. No oscillations are predicted and the net recovery period of about 5min are both in agreement with published experimental results. A quantitative explanation for the lack of such oscillatory behavior, based on the density dependent calcium fluxes, is also provided.

  8. Calcium signaling in UV-induced damage

    NASA Astrophysics Data System (ADS)

    Sun, Dan; Zhang, Su-juan; Li, Yuan-yuan; Qu, Ying; Ren, Zhao-Yu

    2007-05-01

    Hepa1-6 cells were irradiated with UV and incubated for varying periods of time. [Ca 2+] i (intracellular calcium concentration) of UV-irradiated cell was measured by ratio fluorescence imaging system. The comet assay was used to determine DNA damage. During the UVB-irradiation, [Ca 2+] i had an ascending tendency from 0.88 J/m2 to 92.4J/m2. Comet assay instant test indicated that when the irradiation dosage was above 0.88J/m2, DNA damage was observed. Even after approximate 2 h of incubation, DNA damage was still not detected by 0.88J/m2 of UVB irradiation. During UVA-irradiation, the elevation of [Ca 2+] i was not dose-dependent in a range of 1200 J/m2-6000J/m2 and DNA damage was not observed by comet assay. These results suggested that several intracellular UV receptors might induce [Ca 2+] i rising by absorption of the UV energy. Just [Ca 2+] i rising can't induce DNA damage certainly, it is very likely that the breakdown of calcium steady state induces DNA damage.u

  9. Glutamate Induces Calcium Waves in Cultured Astrocytes: Long-Range Glial Signaling

    NASA Astrophysics Data System (ADS)

    Cornell-Bell, Ann H.; Finkbeiner, Steven M.; Cooper, Mark S.; Smith, Stephen J.

    1990-01-01

    The finding that astrocytes possess glutamate-sensitive ion channels hinted at a previously unrecognized signaling role for these cells. Now it is reported that cultured hippocampal astrocytes can respond to glutamate with a prompt and oscillatory elevation of cytoplasmic free calcium, visible through use of the fluorescent calcium indicator fluo-3. Two types of glutamate receptor-one preferring quisqualate and releasing calcium from intracellular stores and the other preferring kainate and promoting surface-membrane calcium influx-appear to be involved. Moreover, glutamate-induced increases in cytoplasmic free calcium frequently propagate as waves within the cytoplasm of individual astrocytes and between adjacent astrocytes in confluent cultures. These propagating waves of calcium suggest that networks of astrocytes may constitute a long-range signaling system within the brain.

  10. Mechanisms of intracellular calcium homeostasis in developing and mature bovine corpora lutea.

    PubMed

    Wright, Marietta F; Bowdridge, Elizabeth; McDermott, Erica L; Richardson, Samuel; Scheidler, James; Syed, Qaisar; Bush, Taylor; Inskeep, E Keith; Flores, Jorge A

    2014-03-01

    Although calcium (Ca(2+)) is accepted as an intracellular mediator of prostaglandin F2 alpha (PGF2alpha) actions on luteal cells, studies defining mechanisms of Ca(2+) homeostasis in bovine corpora lutea (CL) are lacking. The increase in intracellular Ca(2+) concentration ([Ca(2+)]i) induced by PGF2alpha in steroidogenic cells from mature CL is greater than in those isolated from developing CL. Our hypothesis is that differences in signal transduction associated with developing and mature CL contribute to the increased efficacy of PGF2alpha to induce a Ca(2+) signal capable of inducing regression in mature CL. To test this hypothesis, major genes participating in Ca(2+) homeostasis in the bovine CL were identified, and expression of mRNA, protein, or activity, in the case of phospholipase Cbeta (PLCbeta), in developing and mature bovine CL was compared. In addition, we examined the contribution of external and internal Ca(2+) to the PGF2alpha stimulated rise in [Ca(2+)]i in LLCs isolated from developing and mature bovine CL. Three differences were identified in mechanisms of calcium homeostasis between developing and mature CL, which could account for the lesser increase in [Ca(2+)]i in response to PGF2alpha in developing than in mature CL. First, there were lower concentrations of inositol 1,4,5-trisphosphate (IP3) after similar PGF2alpha challenge, indicating reduced phospholipase C beta (PLCbeta) activity, in developing than mature CL. Second, there was an increased expression of sorcin (SRI) in developing than in mature CL. This cytoplasmic Ca(2+) binding protein modulates the endoplasmic reticulum (ER) Ca(2+) release channel, ryanodine receptor (RyR), to be in the closed configuration. Third, there was greater expression of ATP2A2 or SERCA, which causes calcium reuptake into the ER, in developing than in mature CL. Developmental differences in expression detected in whole CL were confirmed by Western blots using protein samples from steroidogenic cells

  11. Arsenite promotes apoptosis and dysfunction in microvascular endothelial cells via an alteration of intracellular calcium homeostasis.

    PubMed

    Suriyo, Tawit; Watcharasit, Piyajit; Thiantanawat, Apinya; Satayavivad, Jutamaad

    2012-04-01

    Vascular endothelium has been considered as a target for arsenic-induced cardiovascular toxicity. The present study demonstrated that arsenite caused slow and sustained elevation of intracellular free calcium levels ([Ca2+]i) in HMEC-1, a human microvessel-derived endothelial cell line, in a concentration-dependent manner. Pretreatment with U-73122 (a specific PLC inhibitor) or 2-APB (a specific IP3 receptor antagonist) attenuated this effect, suggesting that PLC/IP3 signaling cascade is involved in arsenite-induced elevation of [Ca2+]i. Cytotoxic concentrations of arsenite (5 and 10 μM) significantly enhanced endothelial nitric oxide synthase (eNOS) phosphorylation, nitric oxide (NO) production and apoptosis after 24-h exposure. Additionally, 2-APB attenuated eNOS phosphorylation and apoptosis induced by arsenite, indicating that Ca2+ -mediated eNOS activation participates in arsenite-induced endothelial cell apoptosis. Moreover, we also found that non-apoptotic concentrations of arsenite (0.5 and 1 μM) dramatically mitigated thrombin-induced rapid transient rise of [Ca2+]i, eNOS phosphorylation and NO production, suggesting functional disruption of endothelial by arsenite, and these effects occurred without an alteration of PLC-β1 and thrombin receptor levels. Altogether, the results reveal that arsenite induces apoptotic cell death and endothelial dysfunction as indicated by the reduction of thrombin responses, particularly related to an alteration of intracellular Ca2+ homeostasis. PMID:22244921

  12. Detection and measurement of the intracellular calcium variation in follicular cells.

    PubMed

    Herrera-Navarro, Ana M; Terol-Villalobos, Iván R; Jiménez-Hernández, Hugo; Peregrina-Barreto, Hayde; Gonzalez-Barboza, José-Joel

    2014-01-01

    This work presents a new method for measuring the variation of intracellular calcium in follicular cells. The proposal consists in two stages: (i) the detection of the cell's nuclei and (ii) the analysis of the fluorescence variations. The first stage is performed via watershed modified transformation, where the process of labeling is controlled. The detection process uses the contours of the cells as descriptors, where they are enhanced with a morphological filter that homogenizes the luminance variation of the image. In the second stage, the fluorescence variations are modeled as an exponential decreasing function, where the fluorescence variations are highly correlated with the changes of intracellular free Ca(2+). Additionally, it is introduced a new morphological called medium reconstruction process, which helps to enhance the data for the modeling process. This filter exploits the undermodeling and overmodeling properties of reconstruction operators, such that it preserves the structure of the original signal. Finally, an experimental process shows evidence of the capabilities of the proposal. PMID:25342958

  13. Detection and Measurement of the Intracellular Calcium Variation in Follicular Cells

    PubMed Central

    Herrera-Navarro, Ana M.; Terol-Villalobos, Iván R.; Jiménez-Hernández, Hugo; Peregrina-Barreto, Hayde; Gonzalez-Barboza, José-Joel

    2014-01-01

    This work presents a new method for measuring the variation of intracellular calcium in follicular cells. The proposal consists in two stages: (i) the detection of the cell's nuclei and (ii) the analysis of the fluorescence variations. The first stage is performed via watershed modified transformation, where the process of labeling is controlled. The detection process uses the contours of the cells as descriptors, where they are enhanced with a morphological filter that homogenizes the luminance variation of the image. In the second stage, the fluorescence variations are modeled as an exponential decreasing function, where the fluorescence variations are highly correlated with the changes of intracellular free Ca2+. Additionally, it is introduced a new morphological called medium reconstruction process, which helps to enhance the data for the modeling process. This filter exploits the undermodeling and overmodeling properties of reconstruction operators, such that it preserves the structure of the original signal. Finally, an experimental process shows evidence of the capabilities of the proposal. PMID:25342958

  14. Extrinsic periodic information interpolates between monostable and bistable states in intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    Lin, Ling; Duan, Wei-Long

    2015-06-01

    Extrinsic periodic information including physiological cyclical and circadian replacement would affect inevitably a real cell, in this paper we investigate the effect of extrinsic periodic information on intracellular calcium dynamics by means of second-order algorithm for stochastic simulation colored noises. By simulating time evolutions and stationary probability distribution of intracellular Ca2+ concentrations, the results show: (i) intracellular calcium oscillation between cytosol and calcium store shows synchronous and anti-synchronous oscillation as intensity and frequency of extrinsic periodic information vary; (ii) extrinsic periodic information interpolates stability from bistable state → monostable state → bistable state → monostable state as frequency of extrinsic periodic information increases; (iii) extrinsic periodic information interpolates stability from monostable state → bistable state as intensity of extrinsic periodic information increases.

  15. C-Peptide and its intracellular signaling.

    PubMed

    Hills, Claire E; Brunskill, Nigel J

    2009-01-01

    Although long believed to be inert, C-peptide has now been shown to have definite biological effects both in vitro and in vivo in diabetic animals and in patients with type 1 diabetes. These effects point to a protective action of C-peptide against the development of diabetic microvascular complications. Underpinning these observations is undisputed evidence of C-peptide binding to a variety of cell types at physiologically relevant concentrations, and the downstream stimulation of multiple cell signaling pathways and gene transcription via the activation of numerous transcription factors. These pathways affect such fundamental cellular processes as re-absorptive and/or secretory phenotype, migration, growth, and survival. Whilst the receptor remains to be identified, experimental data points strongly to the existence of a specific G-protein-coupled receptor for C-peptide. Of the cell types studied so far, kidney tubular cells express the highest number of C-peptide binding sites. Accordingly, C-peptide exerts major effects on the function of these cells, and in the context of diabetic nephropathy appears to antagonise the pathophysiological effects of major disease mediators such as TGFbeta1 and TNFalpha. Therefore, based on its cellular activity profile C-peptide appears well positioned for development as a therapeutic tool to treat microvascular complications in type 1 diabetes. PMID:20039003

  16. Intracellular calcium affects prestin's voltage operating point indirectly via turgor-induced membrane tension

    NASA Astrophysics Data System (ADS)

    Song, Lei; Santos-Sacchi, Joseph

    2015-12-01

    Recent identification of a calmodulin binding site within prestin's C-terminus indicates that calcium can significantly alter prestin's operating voltage range as gauged by the Boltzmann parameter Vh (Keller et al., J. Neuroscience, 2014). We reasoned that those experiments may have identified the molecular substrate for the protein's tension sensitivity. In an effort to understand how this may happen, we evaluated the effects of turgor pressure on such shifts produced by calcium. We find that the shifts are induced by calcium's ability to reduce turgor pressure during whole cell voltage clamp recording. Clamping turgor pressure to 1kPa, the cell's normal intracellular pressure, completely counters the calcium effect. Furthermore, following unrestrained shifts, collapsing the cells abolishes induced shifts. We conclude that calcium does not work by direct action on prestin's conformational state. The possibility remains that calcium interaction with prestin alters water movements within the cell, possibly via its anion transport function.

  17. Intracellular calcium levels as screening tool for nanoparticle toxicity

    PubMed Central

    Meindl, Claudia; Kueznik, Tatjana; Bösch, Martina; Roblegg, Eva; Fröhlich, Eleonore

    2015-01-01

    The use of engineered nano-sized materials led to revolutionary developments in many industrial applications and in the medical field. These materials, however, also may cause cytotoxicity. In addition to size, surface properties and shape were identified as relevant parameters for cell damage. Cell damage may occur as disruption of membrane integrity, induction of apoptosis and by organelle damage. Generation of oxidative stress may serve as an indicator for cytotoxicity. Effects occurring upon short contact of particles with cells, for instance in the systemic blood circulation, could be identified according to increases of intracellular [Ca2+] levels, which are caused by variety of toxic stimuli. Negatively charged, neutral and positively charged polystyrene particles of different sizes were used to study the role of size and surface properties on viability, membrane disruption, apoptosis, lysosome function, intracellular [Ca2+] levels and generation of oxidative stress. Silica particles served to test this hypothesis. Twenty nm polystyrene particles as well as 12 nm and 40 nm silica particles caused membrane damage and apoptosis with no preference of the surface charge. Only 20 nm plain and amine functionalized polystyrene particles cause oxidative stress and only the plain particles lysosomal damage. A potential role of surface charge was identified for 200 nm polystyrene particles, where only the amidine particles caused lysosomal damage. Increases in intracellular [Ca2+] levels and cytotoxicity after 24 h was often linked but determination of intracellular [Ca2+] levels could serve to characterize further the type of membrane damage. © 2015 The Authors. Journal of Applied Toxicology Published by John Wiley & Sons Ltd. Nano-sized materials may cause cytotoxicity. Negatively charged, neutral and positively charged polystyrene particles of different sizes and silica nanoparticles were used to study the role of size and surface properties on viability, membrane

  18. Streptococcus pneumoniae Infection of Host Epithelial Cells via Polymeric Immunoglobulin Receptor Transiently Induces Calcium Release from Intracellular Stores*

    PubMed Central

    Asmat, Tauseef M.; Agarwal, Vaibhav; Räth, Susann; Hildebrandt, Jan-Peter; Hammerschmidt, Sven

    2011-01-01

    The pneumococcal surface protein C (PspC) is a major adhesin of Streptococcus pneumoniae (pneumococci) that interacts in a human-specific manner with the ectodomain of the human polymeric immunoglobulin receptor (pIgR) produced by respiratory epithelial cells. This interaction promotes bacterial colonization and bacterial internalization by initiating host signal transduction cascades. Here, we examined alterations of intracellular calcium ([Ca2+]i) levels in epithelial cells during host cell infections with pneumococci via the PspC-hpIgR mechanism. The release of [Ca2+]i from intracellular stores in host cells was significantly increased by wild-type pneumococci but not by PspC-deficient pneumococci. The increase in [Ca2+]i was dependent on phospholipase C as pretreatment of cells with a phospholipase C-specific inhibitor U73122 abolished the increase in [Ca2+]i. In addition, we demonstrated the effect of [Ca2+]i on pneumococcal internalization by epithelial cells. Uptake of pneumococci was significantly increased after pretreatment of epithelial cells with the cell-permeable calcium chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid-tetraacetoxymethyl ester or use of EGTA as an extracellular Ca2+-chelating agent. In contrast, thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ATPase, which increases [Ca2+]i in a sustained fashion, significantly reduced pIgR-mediated pneumococcal invasion. Importantly, pneumococcal adherence to pIgR-expressing cells was not altered in the presence of inhibitors as demonstrated by immunofluorescence microscopy. In conclusion, these results demonstrate that pneumococcal infections induce mobilization of [Ca2+]i from intracellular stores. This may constitute a defense response of host cells as the experimental reduction of intracellular calcium levels facilitates pneumococcal internalization by pIgR-expressing cells, whereas elevated calcium levels diminished bacterial internalization by host epithelial

  19. T-Type voltage-sensitive calcium channels mediate mechanically-induced intracellular calcium oscillations in osteocytes by regulating endoplasmic reticulum calcium dynamics.

    PubMed

    Brown, Genevieve N; Leong, Pui L; Guo, X Edward

    2016-07-01

    One of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular calcium (Ca(2+)), and osteocytes in particular exhibit robust oscillations in Ca(2+) when subjected to loading. Previous studies implicate roles for both the endoplasmic reticulum (ER) and T-Type voltage-sensitive calcium channels (VSCC) in these responses, but their interactions or relative contributions have not been studied. By observing Ca(2+) dynamics in the cytosol (Ca(2+)cyt) and the ER (Ca(2+)ER), the focus of this study was to explore the role of the ER and T-Type channels in Ca(2+) signaling in bone cells. We demonstrate that inhibition of T-Type VSCC in osteocytes significantly reduces the number of Ca(2+)cyt responses and affects Ca(2+)ER depletion dynamics. Simultaneous observation of Ca(2+) exchange among these spaces revealed high synchrony between rises in Ca(2+)cyt and depressions in Ca(2+)ER, and this synchrony was significantly reduced by challenging T-Type VSCC. We further confirmed that this effect was mediated directly through the ER and not through store-operated Ca(2+) entry (SOCE) pathways. Taken together, our data suggests that T-Type VSCC facilitate the recovery of Ca(2+)ER in osteocytes to sustain mechanically-induced Ca(2+) oscillations, uncovering a new mechanism underlying the behavior of osteocytes as mechanosensors. PMID:27108342

  20. The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation

    PubMed Central

    Boonrungsiman, Suwimon; Gentleman, Eileen; Carzaniga, Raffaella; Evans, Nicholas D.; McComb, David W.; Porter, Alexandra E.; Stevens, Molly M.

    2012-01-01

    Mineralization is a ubiquitous process in the animal kingdom and is fundamental to human development and health. Dysfunctional or aberrant mineralization leads to a variety of medical problems, and so an understanding of these processes is essential to their mitigation. Osteoblasts create the nano-composite structure of bone by secreting a collagenous extracellular matrix (ECM) on which apatite crystals subsequently form. However, despite their requisite function in building bone and decades of observations describing intracellular calcium phosphate, the precise role osteoblasts play in mediating bone apatite formation remains largely unknown. To better understand the relationship between intracellular and extracellular mineralization, we combined a sample-preparation method that simultaneously preserved mineral, ions, and ECM with nano-analytical electron microscopy techniques to examine osteoblasts in an in vitro model of bone formation. We identified calcium phosphate both within osteoblast mitochondrial granules and intracellular vesicles that transported material to the ECM. Moreover, we observed calcium-containing vesicles conjoining mitochondria, which also contained calcium, suggesting a storage and transport mechanism. Our observations further highlight the important relationship between intracellular calcium phosphate in osteoblasts and their role in mineralizing the ECM. These observations may have important implications in deciphering both how normal bone forms and in understanding pathological mineralization. PMID:22879397

  1. Astroglial calcium signaling displays short-term plasticity and adjusts synaptic efficacy

    PubMed Central

    Sibille, Jérémie; Zapata, Jonathan; Teillon, Jérémie; Rouach, Nathalie

    2015-01-01

    Astrocytes are dynamic signaling brain elements able to sense neuronal inputs and to respond by complex calcium signals, which are thought to represent their excitability. Such signaling has been proposed to modulate, or not, neuronal activities ranging from basal synaptic transmission to epileptiform discharges. However, whether calcium signaling in astrocytes exhibits activity-dependent changes and acutely modulates short-term synaptic plasticity is currently unclear. We here show, using dual recordings of astroglial calcium signals and synaptic transmission, that calcium signaling in astrocytes displays, concomitantly to excitatory synapses, short-term plasticity in response to prolonged repetitive and tetanic stimulations of Schaffer collaterals. We also found that acute inhibition of calcium signaling in astrocytes by intracellular calcium chelation rapidly potentiates excitatory synaptic transmission and short-term plasticity of Shaffer collateral CA1 synapses, i.e., paired-pulse facilitation and responses to tetanic and prolonged repetitive stimulation. These data reveal that calcium signaling of astrocytes is plastic and down-regulates basal transmission and short-term plasticity of hippocampal CA1 glutamatergic synapses. PMID:26074766

  2. Chelation of intracellular calcium blocks insulin action in the adipocyte

    SciTech Connect

    Pershadsingh, H.A.; Shade, D.L.; Delfert, D.M.; McDonald, J.M.

    1987-02-01

    The hypothesis that intracellular Ca/sup 2 +/ is an essential component of the intracellular mechanism of insulin action in the adipocyte was evaluated. Cells were loaded with the Ca/sup 2 +/ chelator quin-2, by preincubating them with quin-2 AM, the tetrakis(acetoxymethyl) ester of quin-2. Quin-2 loading inhibited insulin-stimulated glucose transport without affecting basal activity. The ability of insulin to stimulate glucose uptake in quin-2-loaded cells could be partially restored by preincubating cells with buffer supplemented with 1.2 mM CaCl/sub 2/ and the Ca/sup 2 +/ ionophore A23187. These conditions had no effect on basal activity and omission of CaCl/sub 2/ from the buffer prevented the restoration of insulin-stimulated glucose uptake by A23187. Quin-2 loading also inhibited insulin-stimulated glucose oxidation and the ability of insulin to inhibit cAMP-stimulated lipolysis without affecting their basal activities. Incubation of cells with 100 ..mu..M quin-2 or quin-2 AM had no effect on intracellular ATP concentration or the specific binding of /sup 125/I=labeled insulin to adipocytes. These findings suggest that intracellular Ca/sup 2 +/ is an essential component in the coupling of the insulin-activated receptor complex to cellular physiological/metabolic machinery. Furthermore, differing quin-2 AM dose-response profiles suggest the presence of dual Ca/sup 2 +/-dependent pathways in the adipocyte. One involves insulin stimulation of glucose transport and oxidation, whereas the other involves the antilipolytic action of insulin.

  3. Nicotinic acid modulates intracellular calcium concentration and disassembles the cytoskeleton

    PubMed Central

    LI, JIEJING; LI, YANXI; ZHANG, PENGHUI; NIU, HUA; SHI, YU

    2014-01-01

    Nicotinic acid (NA), a member of the vitamin B family, is well known for its functions in the treatment and prevention of atherosclerosis due to decreasing plasma levels of low-density lipoprotein cholesterol. In recent years, the major side effect of NA, cutaneous flushing, has also attracted extensive attention. However, the effects of NA in other aspects of physiology or cell biology have remained elusive. The present study provided evidence that high concentrations of NA were able to first reduce and later elevate intracellular [Ca2+] in the NIH3T3 cell line. The reduction of the intracellular Ca2+ concentration was achieved within the initial 10 sec, and was preceded by a gradual elevation of intracellular [Ca2+]. Notably, marked accumulation of opaque materials in the perinuclear region was observed in NIH3T3 cells treated with 70 mM NA. Further analysis revealed that treatment with 70 mM NA for 1 h disassembled the microtubule and F-actin cytoskeleton systems and resulted in β-tubulin degradation in an ubiquitin-proteasome-dependent manner. These data indicated that high concentrations of NA disrupted cytoskeleton structures, which may have contributed to minus end (nucleus region) to plus end (cell membrane region)-directed transport processes and resulted in the deposition of material in the perinuclear region. Artificially increasing [Ca2+] adding CaCl2 to the culture media effected the disassembly of F-actin, while it had no apparent effect on microtubules. These results suggested that the disruption of the cytoskeleton systems was not entirely due to the NA-induced elevation of [Ca2+]. Finally, microinjection of NA into xenopus embryos blocked the transport of melanosomes to the peripheral cellular area. In conclusion, the present study indicated that NA disassembles F-actin and microtubule systems, thereby blocking cytoskeleton-dependent intracellular transport. PMID:25241762

  4. Nicotinic acid modulates intracellular calcium concentration and disassembles the cytoskeleton.

    PubMed

    Li, Jiejing; Li, Yanxi; Zhang, Penghui; Niu, Hua; Shi, Yu

    2014-12-01

    Nicotinic acid (NA), a member of the vitamin B family, is well known for its functions in the treatment and prevention of atherosclerosis due to decreasing plasma levels of low-density lipoprotein cholesterol. In recent years, the major side effect of NA, cutaneous flushing, has also attracted extensive attention. However, the effects of NA in other aspects of physiology or cell biology have remained elusive. The present study provided evidence that high concentrations of NA were able to first reduce and later elevate intracellular [Ca2+] in the NIH3T3 cell line. The reduction of the intracellular Ca2+ concentration was achieved within the initial 10 sec, and was preceded by a gradual elevation of intracellular [Ca2+]. Notably, marked accumulation of opaque materials in the perinuclear region was observed in NIH3T3 cells treated with 70 mM NA. Further analysis revealed that treatment with 70 mM NA for 1 h disassembled the microtubule and F‑actin cytoskeleton systems and resulted in β‑tubulin degradation in an ubiquitin‑proteasome-dependent manner. These data indicated that high concentrations of NA disrupted cytoskeleton structures, which may have contributed to minus end (nucleus region) to plus end (cell membrane region)-directed transport processes and resulted in the deposition of material in the perinuclear region. Artificially increasing [Ca2+] adding CaCl2 to the culture media effected the disassembly of F‑actin, while it had no apparent effect on microtubules. These results suggested that the disruption of the cytoskeleton systems was not entirely due to the NA-induced elevation of [Ca2+]. Finally, microinjection of NA into xenopus embryos blocked the transport of melanosomes to the peripheral cellular area. In conclusion, the present study indicated that NA disassembles F‑actin and microtubule systems, thereby blocking cytoskeleton-dependent intracellular transport. PMID:25241762

  5. Intracellular calcium rise is not a necessary step for the stimulated actin polymerization

    SciTech Connect

    Yassin, R.

    1986-03-01

    Stimulation of rabbit peritoneal neutrophils by many chemotactic (formyl Methionyl-Leucyl-Phenylalanine (fMLP), Leukotriene B/sub 4/ (LTB/sub 4/)) and non-chemotactic (phorbol 12-myristate, 13-acetate (PMA), platelet activating factor (PAF), and the calcium ionophore A23187) factors produces rapid and dose dependent increases in the amount of actin associated with the cytoskeleton. The stimulated increase in cytoskeletal actin does not appear to require a rise in the intracellular concentration of free calcium. The increase in cytoskeletal actin produced by A23187 is transient and does not depend on the presence of calcium in the suspending medium. In the presence of extracellular calcium, the effect of the ionophore is biphasic with respect to concentration. The increases in actin association with cytoskeletal produced by fMLP, LTB/sub 4/, and A23187 but not by PMA, are inhibited by hyperosmolarity and pertussis toxin pretreatment. On the other hand, the addition of hyperosmolarity or pertussis toxin has small effect on the rise in the intracellular calcium produced by A23187. The results presented here suggest that an increase in the intracellular concentration of free calcium is not necessary for the stimulated increases in cytoskeletal actin.

  6. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    PubMed

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  7. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

    PubMed Central

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  8. Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla.

    PubMed

    Schnell, Christian; Negm, Mahmoud; Driehaus, Johannes; Scheller, Anja; Hülsmann, Swen

    2016-06-01

    The neuronal activity in the respiratory network of the ventrolateral medulla strongly depends on a variety of different neuromodulators. Since the respiratory activity generated by neurons in the pre-Bötzinger complex (preBötC) is stabilized by astrocytes, we investigated potential effects of the neuromodulator norepinephrine (NE) on the astrocytic calcium signaling in the ventral respiratory group. In acutely isolated brainstem slices from wild type mice (postnatal day 1-10) we performed calcium imaging experiments using Oregon Green 488 BAPTA-1 AM as a calcium indicator dye. Astrocytes in the preBötC, which were identified by their unique intracellular calcium rise after the reduction of the extracellular K(+) concentration, showed calcium rises in response to norepinephrine. These calcium signals persisted after blockade of neuronal activity by tetrodotoxin (TTX) indicating that they were independent of neuronal activity. Furthermore, application of the endoplasmic reticulum calcium pump blocker cyclopiazonic acid (CPA) diminished norepinephrine-induced calcium signals. This results could be confirmed using transgenic mice with astrocyte specific expression of GCaMP3. Thus, norepinephrine might, apart from acting directly on neurons, influence and modulate respiratory network activity via the modulation of astroglial calcium signaling. PMID:26514085

  9. Fast Kinetics of Calcium Signaling and Sensor Design

    PubMed Central

    Tang, Shen; Reddish, Florence; Zhuo, You; Yang, Jenny J.

    2015-01-01

    Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change. PMID:26151819

  10. Calcium signaling in cancer and vitamin D.

    PubMed

    Sergeev, Igor N

    2005-10-01

    Calcium signals induced by the Ca(2+) regulatory hormone 1,25(OH)(2)D(3) may determine the fate of the cancer cell. We have shown that, in breast cancer cell lines, 1,25(OH)(2)D(3) induces a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) by depleting the endoplasmic reticulum (ER) Ca(2+) stores via inositol 1,4,5-trisphosphate receptor/Ca(2+) release channel and activating Ca(2+) entry from the extracellular space via voltage-insensitive Ca(2+) channels. In normal cells, 1,25(OH)(2)D(3) triggered a transient Ca(2+) response via activation of voltage-dependent Ca(2+) channels, which were absent in breast cancer cells. The normal cells, but not breast cancer cells, expressed the Ca(2+) binding/buffering protein calbindin-D(28k) and were capable of buffering [Ca(2+)](i) increases induced by a mobilizer of the ER Ca(2+) stores, thapsigargin, or a Ca(2+) ionophore, ionomycin. The 1,25(OH)(2)D(3)-induced sustained increase in [Ca(2+)](i) in breast cancer cells was associated with induction of apoptotic cell death, whereas the transient [Ca(2+)](i) increase in normal cells was not. The forced expression of calbindin-D(28k) in cytosol or increase in the cytosolic Ca(2+) buffering capacity with the cell-permeant Ca(2+) buffer BAPTA prevented induction of apoptosis with 1,25(OH)(2)D(3) in cancer cells. The sustained increase in [Ca(2+)](i) in breast cancer cells was associated with activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the fluorogenic peptide substrates. Selective inhibition of the Ca(2+) binding sites of mu-calpain decreased apoptotic indices in the cancer cells treated with 1,25(OH)(2)D(3), thapsigargin, or ionomycin. The mu-calpain activation preceded expression/activation of caspase-12, and calpain was required for activation/cleavage of caspase-12. Certain non-calcemic vitamin D analogs (e.g., EB 1089) triggered a sustained

  11. The atrazine metabolite diaminochlorotriazine suppresses LH release from murine LβT2 cells by suppressing GnRH-induced intracellular calcium transients

    PubMed Central

    Dooley, Gregory P.; Tjalkens, Ronald B.; Hanneman, William H.

    2013-01-01

    The primary metabolite of the herbicide atrazine (ATRA), diaminochlorotriazine (DACT), has been suggested to cause disruption in the hypothalamic-pituitary-gonadal axis leading to inhibition of luteinizing hormone (LH) release. DACT is a reactive electrophile known to form covalent protein adducts both in vitro and in vivo following ATRA exposure and maybe targeting proteins involved in GnRH-induced calcium signaling and subsequent LH release. To test this hypothesis, LβT2 pituitary cells were exposed to 300 μM DACT for 24 hrs and examined by fluorescence microscopy for GnRH-induced changes in intracellular calcium and LH release. LβT2 cells exposed to DACT had markedly diminished GnRH-induced intracellular calcium transients and a significant decreased LH release in response to GnRH. DACT appeared to cause a selective decrease in caffeine-sensitive ryanodine receptor-operated calcium stores in LβT2 cells, rather than in thapsigargin-sensitive ER calcium stores. This sensitivity correlated with the formation of covalent protein adducts by DACT, as determined by mass spectrometry. ERp57 was identified by mass spectrometry as a target of DACT adduction in the ER that could potentially mediate the effects of DACT on inhibition of GnRH-induced calcium signaling and inhibition of LH release. Intracellular calcium responses to GnRH and release of LH were restored in DACT-treated cells with the addition of a calcium ionophore (A23187). These data suggest that DACT forms adducts on proteins involved in calcium handling within the ER and that dysfunction in this critical signaling system is associated with loss of normal sensitivity to GnRH and subsequent decreased release of LH. PMID:24052811

  12. [Roles of intracellular calcium and monomeric G-proteins in regulating exocytosis of human neutrophils].

    PubMed

    Zhu, Ying; Wang, Jun-Han; Wu, Jian-Min; Xu, Tao; Zhang, Chun-Guang

    2003-12-25

    Neutrophils play a major role in host defense against microbial infection. There are some clues indicate that neutrophils may also play a role in the pathophysiology of the airway obstruction in chronic asthma. We studied the roles of intracellular calcium and GTP gamma S in the regulation of neutrophils exocytosis using pipette perfusion and membrane capacitance measurement technique in whole cell patch clamp configuration. The results showed that the membrane capacitance increase induced by calcium revealed a biphasic process. The first phase occurred when the calcium level was between 0.2-14 micromol/L with a plateau amplitude of 1.23 pF and a calcium EC50 of 1.1 micromol/L. This phase might correspond to the release of the tertiary granules. The second phase occurred when the calcium concentration was between 20-70 micromol/L with a plateau increment of 6.36 pF, the calcium EC50 being about 33 micromol/L. This phase might represent the release of the primary and secondary granules. Intracellular calcium also simultaneously increased the exocytotic rate and the eventual extent in neutrophils. On the other hand, GTP gamma S can increase the exocytotic rate in a dose-dependent manner but had no effect on the eventual extent of membrane capacitance increment (>6 pF) if the cell was stimulated for a long period (>20 min). GTP gamma S (ranging from 20 to 100 micromol/L) induced the neutrophils to release all four types of the granules at very low intracellular calcium level. PMID:14695488

  13. Ryanodine receptors selectively contribute to the formation of taste-evoked calcium signals in mouse taste cells

    PubMed Central

    Rebello, Michelle R.; Medler, Kathryn F.

    2010-01-01

    The peripheral taste system uses multiple signaling pathways to transduce a stimulus into an output signal that activates afferent neurons. All of these signaling pathways depend on transient increases in intracellular calcium but the current understanding of these calcium signals is not well-developed. Using molecular and physiological techniques, this study establishes that ryanodine receptors (RyRs), specifically isoform 1, are expressed in taste cells and that their physiological function differs among cell types employing different signaling pathways. RyR1 contributes to some taste-evoked signals that rely on calcium release from internal stores but can also supplement the calcium signal that is initiated by opening VGCCs. In taste cells expressing both signaling pathways, RyR1 contributes to the depolarization-induced calcium signal but not to the calcium signal that depends on calcium release from stores. These data suggest that RyR1 is an important regulator of calcium signaling and that its physiological role in taste cells is dictated by the nature of the calcium signaling mechanisms expressed. PMID:20955474

  14. Role of time delay on intracellular calcium dynamics driven by non-Gaussian noises

    PubMed Central

    Duan, Wei-Long; Zeng, Chunhua

    2016-01-01

    Effect of time delay (τ) on intracellular calcium dynamics with non-Gaussian noises in transmission processes of intracellular Ca2+ is studied by means of second-order stochastic Runge-Kutta type algorithm. By simulating and analyzing time series, normalized autocorrelation function, and characteristic correlation time of cytosolic and calcium store’s Ca2+ concentration, the results exhibit: (i) intracellular calcium dynamics’s time coherence disappears and stability strengthens as τ → 0.1s; (ii) for the case of τ < 0.1s, the normalized autocorrelation functions of cytosolic and calcium store’s Ca2+ concentration show damped motion when τ is very short, but they trend to a level line as τ → 0.1s, and for the case of τ > 0.1s, they show different variation as τ increases, the former changes from underdamped motion to a level line, but the latter changes from damped motion to underdamped motion; and (iii) at the moderate value of time delay, reverse resonance occurs both in cytosol and calcium store. PMID:27121687

  15. Role of time delay on intracellular calcium dynamics driven by non-Gaussian noises.

    PubMed

    Duan, Wei-Long; Zeng, Chunhua

    2016-01-01

    Effect of time delay (τ) on intracellular calcium dynamics with non-Gaussian noises in transmission processes of intracellular Ca(2+) is studied by means of second-order stochastic Runge-Kutta type algorithm. By simulating and analyzing time series, normalized autocorrelation function, and characteristic correlation time of cytosolic and calcium store's Ca(2+) concentration, the results exhibit: (i) intracellular calcium dynamics's time coherence disappears and stability strengthens as τ → 0.1s; (ii) for the case of τ < 0.1s, the normalized autocorrelation functions of cytosolic and calcium store's Ca(2+) concentration show damped motion when τ is very short, but they trend to a level line as τ → 0.1s, and for the case of τ > 0.1s, they show different variation as τ increases, the former changes from underdamped motion to a level line, but the latter changes from damped motion to underdamped motion; and (iii) at the moderate value of time delay, reverse resonance occurs both in cytosol and calcium store. PMID:27121687

  16. Role of time delay on intracellular calcium dynamics driven by non-Gaussian noises

    NASA Astrophysics Data System (ADS)

    Duan, Wei-Long; Zeng, Chunhua

    2016-04-01

    Effect of time delay (τ) on intracellular calcium dynamics with non-Gaussian noises in transmission processes of intracellular Ca2+ is studied by means of second-order stochastic Runge-Kutta type algorithm. By simulating and analyzing time series, normalized autocorrelation function, and characteristic correlation time of cytosolic and calcium store’s Ca2+ concentration, the results exhibit: (i) intracellular calcium dynamics’s time coherence disappears and stability strengthens as τ → 0.1s (ii) for the case of τ < 0.1s, the normalized autocorrelation functions of cytosolic and calcium store’s Ca2+ concentration show damped motion when τ is very short, but they trend to a level line as τ → 0.1s, and for the case of τ > 0.1s, they show different variation as τ increases, the former changes from underdamped motion to a level line, but the latter changes from damped motion to underdamped motion; and (iii) at the moderate value of time delay, reverse resonance occurs both in cytosol and calcium store.

  17. STIM proteins: dynamic calcium signal transducers

    PubMed Central

    Soboloff, Jonathan; Rothberg, Brad S.; Madesh, Muniswamy; Gill, Donald L.

    2012-01-01

    Stromal interaction molecule (STIM) proteins function in cells as dynamic coordinators of cellular calcium (Ca2+) signals. Spanning the endoplasmic reticulum (ER) membrane, they sense tiny changes in the levels of Ca2+ stored within the ER lumen. As ER Ca2+ is released to generate primary Ca2+ signals, STIM proteins undergo an intricate activation reaction and rapidly translocate into junctions formed between the ER and the plasma membrane. There, STIM proteins tether and activate the highly Ca2+-selective Orai channels to mediate finely controlled Ca2+ signals and to homeostatically balance cellular Ca2+. Details are emerging on the remarkable organization within these STIM-induced junctional microdomains and the identification of new regulators and alternative target proteins for STIM. PMID:22914293

  18. Intracellular localization of the calcium- and calmodulin antagonist fendiline.

    PubMed

    Weyhenmeyer, R; Gross, M; Maurer-Schultze, B

    1989-02-01

    Microautoradiograpic studies of mouse heart sections were performed to evaluate intracellular localization of the antianginal drug fendiline (Sensit). 15 microCi 3H-fendiline/g b. w. (0.5 mg/kg b.w.) were injected intravenously. 10 min p.a. the heart was removed and either frozen in acetone/dry ice or, after perfusion in situ with dextran 40 and formaldehyde, fixed in formaldehyde. Frozen, paraffin-embedded, and semithin epoxy resin sections were prepared and coated with Ilford K2 emulsion. After 7 to 30 days exposure and development in amidol silver grains were counted above cells, nuclei and extracellular space. The results show that fendiline is able to enter myocardial cells. PMID:2730688

  19. Cannabidiol induces intracellular calcium elevation and cytotoxicity in oligodendrocytes.

    PubMed

    Mato, Susana; Victoria Sánchez-Gómez, María; Matute, Carlos

    2010-11-01

    Heavy marijuana use has been linked to white matter histological alterations. However, the impact of cannabis constituents on oligodendroglial pathophysiology remains poorly understood. Here, we investigated the in vitro effects of cannabidiol, the main nonpsychoactive marijuana component, on oligodendrocytes. Exposure to cannabidiol induced an intracellular Ca(2+) rise in optic nerve oligodendrocytes that was not primarily mediated by entry from the extracellular space, nor by interactions with ryanodine or IP(3) receptors. Application of the mitochondrial protonophore carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 1 μM) completely prevented subsequent cannabidiol-induced Ca(2+) responses. Conversely, the increase in cytosolic Ca(2+) levels elicited by FCCP was reduced after previous exposure to cannabidiol, further suggesting that the mitochondria acts as the source of cannabidiol-evoked Ca(2+) rise in oligodendrocytes. n addition, brief exposure to cannabidiol (100 nM-10 μM) led to a concentration-dependent decrease of oligodendroglial viability that was not prevented by antagonists of CB(1), CB(2), vanilloid, A(2A) or PPARγ receptors, but was instead reduced in the absence of extracellular Ca(2+). The oligodendrotoxic effect of cannabidiol was partially blocked by inhibitors of caspase-3, -8 and -9, PARP-1 and calpains, suggesting the activation of caspase-dependent and -independent death pathways. Cannabidiol also elicited a concentration-dependent alteration of mitochondrial membrane potential, and an increase in reactive oxygen species (ROS) that was reduced in the absence of extracellular Ca(2+). Finally, cannabidiol-induced cytotoxicity was partially prevented by the ROS scavenger trolox. Together, these results suggest that cannabidiol causes intracellular Ca(2+) dysregulation which can lead to oligodendrocytes demise. PMID:20645411

  20. Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

    PubMed

    Berk, B C; Corson, M A; Peterson, T E; Tseng, H

    1995-12-01

    Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow. PMID:8666584

  1. Depletion of intracellular calcium stores activates a calcium conducting nonselective cation current in mouse pancreatic acinar cells.

    PubMed

    Krause, E; Pfeiffer, F; Schmid, A; Schulz, I

    1996-12-20

    Receptor-mediated Ca2+ release from inositol (1,4,5)-trisphosphate (IP3)-sensitive Ca2+ stores causes "capacitative calcium entry" in many cell types (Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12; Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624). We used patch-clamp and fluorescence techniques in isolated mouse pancreatic acinar cells to identify ion currents and cytosolic calcium concentrations under conditions in which intracellular Ca2+ stores were emptied. We found that depletion of Ca2+ stores activated a calcium-release-activated nonselective cation current (ICRANC) which did not discriminate between monovalent cations. ICRANC possessed a significant conductance for Ca2+ and Ba2+. It was not inhibited by La3+, Gd3+, Co2+, or Cd2+ but was completely abolished by flufenamic acid or genistein. In whole cell and cell-attached recordings, a 40-45 pS nonselective cation channel was identified which was activated by Ca2+ store depletion. Calcium entry as detected by single cell fluorescence measurements with fluo-3 or fura-2, showed the same pharmacological properties as ICRANC. We conclude that in mouse pancreatic acinar cells 40-45 pS nonselective cation channels serve as a pathway for capacitative Ca2+ entry. This entry pathway differs from the previously described ICRAC (Hoth, M., and Penner, R. (1992) Nature 355, 353-356) in its ion-selectivity, pharmacological profile, and single-channel conductance. PMID:8955076

  2. Neocortical GABA release at high intracellular sodium and low extracellular calcium: an anti-seizure mechanism.

    PubMed

    Rassner, Michael P; Moser, Andreas; Follo, Marie; Joseph, Kevin; van Velthoven-Wurster, Vera; Feuerstein, Thomas J

    2016-04-01

    In epilepsy, the GABA and glutamate balance may be disrupted and a transient decrease in extracellular calcium occurs before and during a seizure. Flow Cytometry based fluorescence activated particle sorting experiments quantified synaptosomes from human neocortical tissue, from both epileptic and non-epileptic patients (27.7% vs. 36.9% GABAergic synaptosomes, respectively). Transporter-mediated release of GABA in human and rat neocortical synaptosomes was measured using the superfusion technique for the measurement of endogenous GABA. GABA release was evoked by either a sodium channel activator or a sodium/potassium-ATPase inhibitor when exocytosis was possible or prevented, and when the sodium/calcium exchanger was active or inhibited. The transporter-mediated release of GABA is because of elevated intracellular sodium. A reduction in the extracellular calcium increased this release (in both non-epileptic and epileptic, except Rasmussen encephalitis, synaptosomes). The inverse was seen during calcium doubling. In humans, GABA release was not affected by exocytosis inhibition, that is, it was solely transporter-mediated. However, in rat synaptosomes, an increase in GABA release at zero calcium was only exhibited when the exocytosis was prevented. The absence of calcium amplified the sodium/calcium exchanger activity, leading to elevated intracellular sodium, which, together with the stimulation-evoked intracellular sodium increment, enhanced GABA transporter reversal. Sodium/calcium exchange inhibitors diminished GABA release. Thus, an important seizure-induced extracellular calcium reduction might trigger a transporter- and sodium/calcium exchanger-related anti-seizure mechanism by augmenting transporter-mediated GABA release, a mechanism absent in rats. Uniquely, the additional increase in GABA release because of calcium-withdrawal dwindled during the course of illness in Rasmussen encephalitis. Seizures cause high Na(+) influx through action potentials. A

  3. Effects of Staphylococcus aureus-hemolysin A on calcium signalling in immortalized human airway epithelial cells.

    PubMed

    Eichstaedt, Stefanie; Gäbler, Karoline; Below, Sabine; Müller, Christian; Kohler, Christian; Engelmann, Susanne; Hildebrandt, Petra; Völker, Uwe; Hecker, Michael; Hildebrandt, Jan-Peter

    2009-02-01

    Part of the innate defence of bronchial epithelia against bacterial colonization is secretion of salt and water which generally depends on coordinated actions of receptor-mediated cAMP- and calcium signalling. The hypothesis that Staphylococcus aureus-virulence factors interfere with endogenous signals in host cells was tested by measuring agonist-mediated changes in [Ca(2+)](i) in S9 cells upon pre-incubation with bacterial secretory products. S9 cells responded to mAChR-activation with calcium release from intracellular stores and capacitative calcium influx. Treatment of cells with culture supernatants of S. aureus (COL) or with recombinant alpha-hemolysin (Hla) resulted in time- and concentration-dependent changes in [Ca(2+)](i). High concentrations of Hla (2000 ng/ml) resulted in elevations in [Ca(2+)](i) elicited by accelerated calcium influx. A general Hla-mediated permeabilization of S9 cell membranes to small molecules, however, did not occur. Lower concentrations of Hla (200 ng/ml) induced a reduction in [Ca(2+)](i)-levels during the sustained plateau phase of receptor-mediated calcium signalling which was abolished by pre-incubation of cells with carboxyeosin, an inhibitor of the plasma membrane calcium-ATPase. This indicates that low concentrations of Hla change calcium signalling by accelerating pump-driven extrusion of Ca(2+) ions. In vivo, such a mechanism may result in attenuation of calcium-mediated cellular defence functions and facilitation of bacterial adherence to the bronchial epithelium. PMID:18922576

  4. Collective Calcium Signaling of Defective Multicellular Networks

    NASA Astrophysics Data System (ADS)

    Potter, Garrett; Sun, Bo

    2015-03-01

    A communicating multicellular network processes environmental cues into collective cellular dynamics. We have previously demonstrated that, when excited by extracellular ATP, fibroblast monolayers generate correlated calcium dynamics modulated by both the stimuli and gap junction communication between the cells. However, just as a well-connected neural network may be compromised by abnormal neurons, a tissue monolayer can also be defective with cancer cells, which typically have down regulated gap junctions. To understand the collective cellular dynamics in a defective multicellular network we have studied the calcium signaling of co-cultured breast cancer cells and fibroblast cells in various concentrations of ATP delivered through microfluidic devices. Our results demonstrate that cancer cells respond faster, generate singular spikes, and are more synchronous across all stimuli concentrations. Additionally, fibroblast cells exhibit persistent calcium oscillations that increase in regularity with greater stimuli. To interpret these results we quantitatively analyzed the immunostaining of purigenic receptors and gap junction channels. The results confirm our hypothesis that collective dynamics are mainly determined by the availability of gap junction communications.

  5. Diquafosol promotes corneal epithelial healing via intracellular calcium-mediated ERK activation.

    PubMed

    Byun, Yong-Soo; Yoo, Young-Sik; Kwon, Ji-Young; Joo, Jong-Soo; Lim, Sung-A; Whang, Woong-Joo; Mok, Jee-Won; Choi, Jun-Sub; Joo, Choun-Ki

    2016-02-01

    Diquafosol is known as a purinergic P2Y2 receptor (P2Y2R) agonist that stimulates water and mucin secretion from conjunctival epithelial cells and goblet cells, leading to tear film stability in dry eye. However, its effect on corneal epithelial healing has not yet been elucidated. The aim of the present study was to evaluate the effect of diquafosol on corneal epithelial healing in vivo and on P2Y2R-related downstream signaling pathways in vitro. We administered 3% diquafosol ophthalmic solution on 3 mm-diameter epithelial defects made in rat corneas and assessed the wound closure over time. Corneal epithelial healing was significantly accelerated in diquafosol-treated eyes compared to control eyes at 12 and 24 h. During wound healing, P2Y2R staining appeared stronger in the re-epithelized margin near the wound defect. To evaluate whether diquafosol stimulates epidermal growth factor receptor/extracellular-signal-regulated kinase (EGFR/ERK)-related cell proliferation and migration, simian virus 40-transfected human corneal epithelial (THCE) cells were used for in vitro experiments. Cell proliferation was accelerated by diquafosol at concentrations from 20 to 200 μM during 48 h, but inhibited at concentrations over 2000 μM. The intracellular calcium ([Ca(2+)]i) elevation was measured in diquafosol (100 μM)-stimulated cells using Fluo-4/AM ([Ca(2+)]i indicator). [Ca(2+)]i elevation was observed in diquafosol-stimulated cells regardless of the presence of calcium in media, and suramin pretreatment inhibited the calcium response. The effect of diquafosol on phosphorylation of EGFR, ERK and Akt, and cell migration was determined by western blotting and in vitro cell migration assay. Diquafosol induced phosphorylation of EGFR at 2 min post-stimulation, and phosphorylation of ERK at 5 min post-stimulation. Phosphorylation of ERK was attenuated in cells pretreated with suramin or BAPTA/AM ([Ca(2+)]i chelator), and partially with AG1478 (EGFR inhibitor

  6. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  7. Physiology and Pathology of Calcium Signaling in the Brain

    PubMed Central

    Kawamoto, Elisa Mitiko; Vivar, Carmen; Camandola, Simonetta

    2012-01-01

    Calcium (Ca2+) plays fundamental and diversified roles in neuronal plasticity. As second messenger of many signaling pathways, Ca2+ as been shown to regulate neuronal gene expression, energy production, membrane excitability, synaptogenesis, synaptic transmission, and other processes underlying learning and memory and cell survival. The flexibility of Ca2+ signaling is achieved by modifying cytosolic Ca2+ concentrations via regulated opening of plasma membrane and subcellular Ca2+ sensitive channels. The spatiotemporal patterns of intracellular Ca2+ signals, and the ultimate cellular biological outcome, are also dependent upon termination mechanism, such as Ca2+ buffering, extracellular extrusion, and intra-organelle sequestration. Because of the central role played by Ca2+ in neuronal physiology, it is not surprising that even modest impairments of Ca2+ homeostasis result in profound functional alterations. Despite their heterogeneous etiology neurodegenerative disorders, as well as the healthy aging process, are all characterized by disruption of Ca2+ homeostasis and signaling. In this review we provide an overview of the main types of neuronal Ca2+ channels and their role in neuronal plasticity. We will also discuss the participation of Ca2+ signaling in neuronal aging and degeneration. PMID:22518105

  8. Calcium-Mediated Abiotic Stress Signaling in Roots.

    PubMed

    Wilkins, Katie A; Matthus, Elsa; Swarbreck, Stéphanie M; Davies, Julia M

    2016-01-01

    Roots are subjected to a range of abiotic stresses as they forage for water and nutrients. Cytosolic free calcium is a common second messenger in the signaling of abiotic stress. In addition, roots take up calcium both as a nutrient and to stimulate exocytosis in growth. For calcium to fulfill its multiple roles must require strict spatio-temporal regulation of its uptake and efflux across the plasma membrane, its buffering in the cytosol and its sequestration or release from internal stores. This prompts the question of how specificity of signaling output can be achieved against the background of calcium's other uses. Threats to agriculture such as salinity, water availability and hypoxia are signaled through calcium. Nutrient deficiency is also emerging as a stress that is signaled through cytosolic free calcium, with progress in potassium, nitrate and boron deficiency signaling now being made. Heavy metals have the capacity to trigger or modulate root calcium signaling depending on their dose and their capacity to catalyze production of hydroxyl radicals. Mechanical stress and cold stress can both trigger an increase in root cytosolic free calcium, with the possibility of membrane deformation playing a part in initiating the calcium signal. This review addresses progress in identifying the calcium transporting proteins (particularly channels such as annexins and cyclic nucleotide-gated channels) that effect stress-induced calcium increases in roots and explores links to reactive oxygen species, lipid signaling, and the unfolded protein response. PMID:27621742

  9. Intracellular calcium channels: inositol-1,4,5-trisphosphate receptors

    PubMed Central

    Fedorenko, Olena A.; Popugaeva, Elena; Enomoto, Masahiro; Stathopulos, Peter B.; Ikura, Mitsuhiko; Bezprozvanny, Ilya

    2014-01-01

    The inositol-1,4,5-trisphosphate receptors (InsP3Rs) are the major intracellular Ca2+-release channels in cells. Activity of InsP3Rs is essential for elementary and global Ca2+ events in the cell. There are three InsP3Rs isoforms that are present in mammalian cells. In this review review we will focus primarily on InsP3R type 1. The InsP3R1 is a predominant isoform in neurons and it is most extensively studied isoform. Combination of biophysical and structural methods revealed key mechanisms of InsP3R function and modulation. Cell biological and biochemical studies lead to identification of a large number of InsP3R-binding proteins. InsP3Rs are involved in the regulation of numerous physiological processes, including learning and memory, proliferation, differentiation, development and cell death. Malfunction of InsP3R1 play a role in a number of neurodegenerative disorders and other disease states. InsP3Rs represent a potentially valuable drug target for treatment of these disorders and for modulating activity of neurons and other cells. Future studies will provide better understanding of physiological functions of InsP3Rs in health and disease. PMID:24300389

  10. Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release.

    PubMed

    Woodier, Jason; Rainbow, Richard D; Stewart, Alan J; Pitt, Samantha J

    2015-07-10

    Aberrant Zn(2+) homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn(2+) modulates cardiac ryanodine receptor gating and Ca(2+) dynamics in isolated cardiomyocytes. We reveal that Zn(2+) is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn(2+) concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca(2+). At concentrations of free Zn(2+) >1 nm, Zn(2+) is the main activating ligand, and the dependence on Ca(2+) is removed. Zn(2+) is therefore a higher affinity activator of RyR2 than Ca(2+). Millimolar levels of free Zn(2+) were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn(2+) modulates both the frequency and amplitude of Ca(2+) waves in a concentration-dependent manner and that physiological levels of Zn(2+) elicit Ca(2+) release in the absence of activating levels of cytosolic Ca(2+). This highlights a new role for intracellular Zn(2+) in shaping Ca(2+) dynamics in cardiomyocytes through modulation of RyR2 gating. PMID:26041778

  11. Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release*

    PubMed Central

    Woodier, Jason; Rainbow, Richard D.; Stewart, Alan J.; Pitt, Samantha J.

    2015-01-01

    Aberrant Zn2+ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn2+ modulates cardiac ryanodine receptor gating and Ca2+ dynamics in isolated cardiomyocytes. We reveal that Zn2+ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn2+ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca2+. At concentrations of free Zn2+ >1 nm, Zn2+ is the main activating ligand, and the dependence on Ca2+ is removed. Zn2+ is therefore a higher affinity activator of RyR2 than Ca2+. Millimolar levels of free Zn2+ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn2+ modulates both the frequency and amplitude of Ca2+ waves in a concentration-dependent manner and that physiological levels of Zn2+ elicit Ca2+ release in the absence of activating levels of cytosolic Ca2+. This highlights a new role for intracellular Zn2+ in shaping Ca2+ dynamics in cardiomyocytes through modulation of RyR2 gating. PMID:26041778

  12. The role of PIP2 and the IP3/DAG pathway in intracellular calcium release and cell survival during nanosecond electric pulse exposures

    NASA Astrophysics Data System (ADS)

    Steelman, Zachary A.; Tolstykh, Gleb P.; Estlack, Larry E.; Roth, Caleb C.; Ibey, Bennett L.

    2015-03-01

    Phosphatidylinositol4,5-biphosphate (PIP2) is a membrane phospholipid of particular importance in cell-signaling pathways. Hydrolysis of PIP2 releases inositol-1,4,5-triphosphate (IP3) from the membrane, activating IP3 receptors on the smooth endoplasmic reticulum (ER) and facilitating a release of intracellular calcium stores and activation of protein kinase C (PKC). Recent studies suggest that nanosecond pulsed electric fields (nsPEF) cause depletion of PIP2 in the cellular membrane, activating the IP3 signaling pathway. However, the exact mechanism(s) causing this observed depletion of PIP2 are unknown. Complicating the matter, nsPEF create nanopores in the plasma membrane, allowing calcium to enter the cell and thus causing an increase in intracellular calcium. While elevated intracellular calcium can cause activation of phospholipase C (PLC) (a known catalyst of PIP2 hydrolysis), PIP2 depletion has been shown to occur in the absence of both extracellular and intracellular calcium. These observations have led to the hypothesis that the high electric field itself may be playing a direct role in the hydrolysis of PIP2 from the plasma membrane. To support this hypothesis, we used edelfosine to block PLC and prevent activation of the IP3/DAG pathway in Chinese Hamster Ovarian (CHO) cells prior to applying nsPEF. Fluorescence microscopy was used to monitor intracellular calcium bursts during nsPEF, while MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survivability assays were utilized to determine whether edelfosine improved cell survival during nsPEF exposure. This work is critical to refine the role of PIP2 in the cellular response to nsPEF, and also to determine the fundamental biological effects of high electric field exposures.

  13. Involvement of aberrant calcium signalling in herpetic neuralgia.

    PubMed

    Warwick, Rebekah A; Hanani, Menachem

    2016-03-01

    Alpha-herpesviruses, herpes simplex viruses (HSV) and varicella zoster virus (VZV), are pathogens of the peripheral nervous system. After primary infection, these viruses establish latency within sensory ganglia, while retaining the ability to reactivate. Reactivation of VZV results in herpes zoster, a condition characterized by skin lesions that leads to post-herpetic neuralgia. Recurrent reactivations of HSV, which cause mucocutaneous lesions, may also result in neuralgia. During reactivation of alpha-herpesviruses, satellite glial cells (SGCs), which surround neurons in sensory ganglia, become infected with the replicating virus. SGCs are known to contribute to neuropathic pain in a variety of animal pain models. Here we investigated how infection of short-term cultures of mouse trigeminal ganglia with HSV-1 affects communication between SGCs and neurons, and how this altered communication may increase neuronal excitability, thus contributing to herpetic neuralgia. Mechanical stimulation of single neurons or SGCs resulted in intercellular calcium waves, which were larger in cultures infected with HSV-1. Two differences were observed between control and HSV-1 infected cultures that could account for this augmentation. Firstly, HSV-1 infection induced cell fusion among SGCs and neurons, which would facilitate the spread of calcium signals over farther distances. Secondly, using calcium imaging and intracellular electrical recordings, we found that neurons in the HSV-1 infected cultures exhibited augmented influx of calcium upon depolarization. These virally induced changes may not only cause more neurons in the sensory ganglia to fire action potentials, but may also increase neurotransmitter release at the presynaptic terminals in the spinal cord. They are therefore likely to be contributing factors to herpetic neuralgia. PMID:26684187

  14. Inhibitor of apoptosis proteins as intracellular signaling intermediates.

    PubMed

    Kocab, Andrew J; Duckett, Colin S

    2016-01-01

    Inhibitor of apoptosis (IAP) proteins have often been considered inhibitors of cell death due to early reports that described their ability to directly bind and inhibit caspases, the primary factors that implement apoptosis. However, a greater understanding is evolving regarding the vital roles played by IAPs as transduction intermediates in a diverse set of signaling cascades associated with functions ranging from the innate immune response to cell migration to cell-cycle regulation. In this review, we discuss the functions of IAPs in signaling, focusing primarily on the cellular IAP (c-IAP) proteins. The c-IAPs are important components in tumor necrosis factor receptor superfamily signaling cascades, which include activation of the NF-κB transcription factor family. As these receptors modulate cell proliferation and cell death, the involvement of the c-IAPs in these pathways provides an additional means of controlling cellular fate beyond simply inhibiting caspase activity. Additionally, IAP-binding proteins, such as Smac and caspases, which have been described as having cell death-independent roles, may affect c-IAP activity in intracellular signaling. Collectively, the multi-faceted functions and complex regulation of the c-IAPs illustrate their importance as intracellular signaling intermediates. PMID:26462035

  15. In Vivo Detection of Intracellular Signaling Pathways in Developing Thymocytes

    PubMed Central

    Zúñiga-Pflücker, Juan Carlos

    2000-01-01

    Information regarding the intracellular signaling processes that occur during the development of T cells has largely been obtained with the use of transgenic mouse models, which although providing invaluable information are time consuming and costly. To this end, we have developed a novel system that facilitates the In Vivo analysis of signal transduction pathways during T-lymphocyte development. This approach uses reporter-plasmids for the detection of intracellular signals mediated by the mitogen-activated protein kinase or cyclic AMP-dependent protein kinase. Reporter-plasmids are transfected into thymocytes in fetal thymic organ culture by accelerated DNA/particle bombardment (gene gun), and the activation of a signaling pathway is determined in the form of a standard luciferase assay. Importantly, this powerful technique preserves the structural integrity of the thymus, and will provide an invaluable tool to study how thymocytes respond to normal environmental stimuli encountered during differentiation within the thymic milieu. Thus, this method allows for the monitoring of signals that occur in a biological time frame, such as during differentiation, and within the natural environment of differentiating cells. PMID:11293810

  16. Scrophularia orientalis extract induces calcium signaling and apoptosis in neuroblastoma cells.

    PubMed

    Lange, Ingo; Moschny, Julia; Tamanyan, Kamilla; Khutsishvili, Manana; Atha, Daniel; Borris, Robert P; Koomoa, Dana-Lynn

    2016-04-01

    Effective neuroblastoma (NB) treatments are still limited despite treatment options available today. Therefore, this study attempted to identify novel plant extracts that have anticancer effects. Cytotoxicity and increased intracellular calcium levels were determined using the Sulforhodamine B (SRB) assay and Fluo4-AM (acetoxymethyl) staining and fluorescence microscopy in NB cells in order to screen a library of plant extracts. The current study examined the anticancer effects of a dichloromethane extract from Scrophularia orientalis L. (Scrophulariaceae), a plant that has been used in Traditional Chinese Medicine. This extract contained highly potent agents that significantly reduced cell survival and increased calcium levels in NB cells. Further analysis revealed that cell death induced by this extract was associated with intracellular calcium release, opening of the MPTP, caspase 3- and PARP-cleavage suggesting that this extract induced aberrant calcium signaling that resulted in apoptosis via the mitochondrial pathway. Therefore, agents from Scrophularia orientalis may have the potential to lead to new chemo-therapeutic anticancer drugs. Furthermore, targeting intracellular calcium signaling may be a novel strategy to develop more effective treatments for NB. PMID:26848085

  17. Scrophularia orientalis extract induces calcium signaling and apoptosis in neuroblastoma cells

    PubMed Central

    LANGE, INGO; MOSCHNY, JULIA; TAMANYAN, KAMILLA; KHUTSISHVILI, MANANA; ATHA, DANIEL; BORRIS, ROBERT P.; KOOMOA, DANA-LYNN

    2016-01-01

    Effective neuroblastoma (NB) treatments are still limited despite treatment options available today. Therefore, this study attempted to identify novel plant extracts that have anticancer effects. Cytotoxicity and increased intracellular calcium levels were determined using the Sulforhodamine B (SRB) assay and Fluo4-AM (acetoxymethyl) staining and fluorescence microscopy in NB cells in order to screen a library of plant extracts. The current study examined the anticancer effects of a dichloromethane extract from Scrophularia orientalis L. (Scrophulariaceae), a plant that has been used in Traditional Chinese Medicine. This extract contained highly potent agents that significantly reduced cell survival and increased calcium levels in NB cells. Further analysis revealed that cell death induced by this extract was associated with intracellular calcium release, opening of the MPTP, caspase 3- and PARP-cleavage suggesting that this extract induced aberrant calcium signaling that resulted in apoptosis via the mitochondrial pathway. Therefore, agents from Scrophularia orientalis may have the potential to lead to new chemo therapeutic anticancer drugs. Furthermore, targeting intracellular calcium signaling may be a novel strategy to develop more effective treatments for NB. PMID:26848085

  18. Calcium-Mediated Abiotic Stress Signaling in Roots

    PubMed Central

    Wilkins, Katie A.; Matthus, Elsa; Swarbreck, Stéphanie M.; Davies, Julia M.

    2016-01-01

    Roots are subjected to a range of abiotic stresses as they forage for water and nutrients. Cytosolic free calcium is a common second messenger in the signaling of abiotic stress. In addition, roots take up calcium both as a nutrient and to stimulate exocytosis in growth. For calcium to fulfill its multiple roles must require strict spatio-temporal regulation of its uptake and efflux across the plasma membrane, its buffering in the cytosol and its sequestration or release from internal stores. This prompts the question of how specificity of signaling output can be achieved against the background of calcium’s other uses. Threats to agriculture such as salinity, water availability and hypoxia are signaled through calcium. Nutrient deficiency is also emerging as a stress that is signaled through cytosolic free calcium, with progress in potassium, nitrate and boron deficiency signaling now being made. Heavy metals have the capacity to trigger or modulate root calcium signaling depending on their dose and their capacity to catalyze production of hydroxyl radicals. Mechanical stress and cold stress can both trigger an increase in root cytosolic free calcium, with the possibility of membrane deformation playing a part in initiating the calcium signal. This review addresses progress in identifying the calcium transporting proteins (particularly channels such as annexins and cyclic nucleotide-gated channels) that effect stress-induced calcium increases in roots and explores links to reactive oxygen species, lipid signaling, and the unfolded protein response. PMID:27621742

  19. Calcium fluxes in mouse mammary tissue in vitro: intracellular and extracellular calcium pools

    PubMed Central

    Neville, M. C.; Peaker, M.

    1982-01-01

    1. The total Ca content of the mammary gland increased from about 2 to 12 μmole/g tissue during the transition from pregnancy to lactation in the mouse. In tissue from lactating mice at least two thirds of the total Ca exchanged with external Ca in 6 hr. There was little non-exchangeable Ca in tissues from pregnant mice. 2. At 37 °C the time courses of influx and efflux of 45Ca in lactating tissues could be analysed by assuming three exponential components with rate constants of about 0·3, 0·06 and 0·005 min-1 and containing, respectively, 1·7, 1·5 and 4·7 μmole 45Ca/g tissue at the steady state. 3. The rapidly effluxing component showed the time- and temperature-dependence characteristic of bulk-phase-limited diffusion through the extracellular space. The diffusion coefficient was about one quarter of the self-diffusion coefficient of Ca in aqueous solution, consistent with a tortuosity factor of about 2. A portion of the Ca in this component was displaced by La3+. The amount remaining in the presence of 3 mm-La3+ was close to that expected for free extracellular Ca. The rapid component was therefore interpreted as originating from an extracellular compartment containing both free and bound Ca. 4. The rate of efflux of the intermediate component was slowed by a factor of ten when the temperature was decreased from 37 to 0 °C giving a Q10 of 2·7, expected for membrane transport. The slow component present at 37 °C was not displaced by EGTA or La3+, suggesting that it is not localized extracellularly. It was not apparent in the 0 °C efflux curves. 5. The biphasic time course of uptake of ionophore (A23187)-releasable 45Ca in particulate fractions obtained by homogenization and centrifugation of tissues which had been incubated with the isotope was consistent with the hypothesis that the two slower components of 45Ca flux originate from intracellular compartments. Mitochondrial uptake probably did not contribute significantly to Ca exchange in these

  20. Calcium signaling during reproduction and biotrophic fungal interactions in plants.

    PubMed

    Chen, Junyi; Gutjahr, Caroline; Bleckmann, Andrea; Dresselhaus, Thomas

    2015-04-01

    Many recent studies have indicated that cellular communications during plant reproduction, fungal invasion, and defense involve identical or similar molecular players and mechanisms. Indeed, pollen tube invasion and sperm release shares many common features with infection of plant tissue by fungi and oomycetes, as a tip-growing intruder needs to communicate with the receptive cells to gain access into a cell and tissue. Depending on the compatibility between cells, interactions may result in defense, invasion, growth support, or cell death. Plant cells stimulated by both pollen tubes and fungal hyphae secrete, for example, small cysteine-rich proteins and receptor-like kinases are activated leading to intracellular signaling events such as the production of reactive oxygen species (ROS) and the generation of calcium (Ca(2+)) transients. The ubiquitous and versatile second messenger Ca(2+) thereafter plays a central and crucial role in modulating numerous downstream signaling processes. In stimulated cells, it elicits both fast and slow cellular responses depending on the shape, frequency, amplitude, and duration of the Ca(2+) transients. The various Ca(2+) signatures are transduced into cellular information via a battery of Ca(2+)-binding proteins. In this review, we focus on Ca(2+) signaling and discuss its occurrence during plant reproduction and interactions of plant cells with biotrophic filamentous microbes. The participation of Ca(2+) in ROS signaling pathways is also discussed. PMID:25660409

  1. Roles of Intracellular Cyclic AMP Signal Transduction in the Capacitation and Subsequent Hyperactivation of Mouse and Boar Spermatozoa

    PubMed Central

    HARAYAMA, Hiroshi

    2013-01-01

    It is not until accomplishment of a variety of molecular changes during the transit through the female reproductive tract that mammalian spermatozoa are capable of exhibiting highly activated motility with asymmetric whiplash beating of the flagella (hyperactivation) and undergoing acrosomal exocytosis in the head (acrosome reaction). These molecular changes of the spermatozoa are collectively termed capacitation and promoted by bicarbonate, calcium and cholesterol acceptors. Such capacitation-promoting factors can stimulate intracellular cyclic AMP (cAMP) signal transduction in the spermatozoa. Meanwhile, hyperactivation and the acrosome reaction are essential to sperm fertilization with oocytes and are apparently triggered by a sufficient increase of intracellular Ca2+ in the sperm flagellum and head, respectively. Thus, it is necessary to investigate the relationship between cAMP signal transduction and calcium signaling cascades in the spermatozoa for the purpose of understanding the molecular basis of capacitation. In this review, I cover updated insights regarding intracellular cAMP signal transduction, the acrosome reaction and flagellar motility in mammalian spermatozoa and then account for possible roles of intracellular cAMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa. PMID:24162806

  2. Fatty Acid Signaling: The New Function of Intracellular Lipases

    PubMed Central

    Papackova, Zuzana; Cahova, Monika

    2015-01-01

    Until recently, intracellular triacylglycerols (TAG) stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed. PMID:25674855

  3. Calcium signaling and secretion in cholangiocytes

    PubMed Central

    Guerra, Mateus T.; Nathanson, Michael H.

    2015-01-01

    Alcoholic hepatitis affects up to one-third of individuals who abuse alcohol and can be associated with high mortality. Although this disorder is characterized by hepatocellular damage, steatosis and neutrophil infiltration, recent evidence suggests that cholestasis or impaired bile secretion may be a frequent occurrence as well. Bile secretion results from the concerted activity of hepatocytes and cholangiocytes, the epithelial cells that line the bile ducts. Hepatocytes secrete bile acids and conjugated products into the bile canaliculi, which then are modified by cholangiocytes through secretion of bicarbonate and water to give rise to the final secreted bile. Here the molecular mechanisms regulating bile secretion in cholangiocytes are reviewed. Moreover, we discuss how the expression of intracellular Ca2+ channels might be regulated in cholangiocytes, plus evidence that components of the Ca2+ signaling machinery are altered in a range of cholestatic diseases of the bile ducts. PMID:26100660

  4. Calcium signaling and secretion in cholangiocytes.

    PubMed

    Guerra, Mateus T; Nathanson, Michael H

    2015-07-01

    Alcoholic hepatitis affects up to one-third of individuals who abuse alcohol and can be associated with high mortality. Although this disorder is characterized by hepatocellular damage, steatosis and neutrophil infiltration, recent evidence suggests that cholestasis or impaired bile secretion may be a frequent occurrence as well. Bile secretion results from the concerted activity of hepatocytes and cholangiocytes, the epithelial cells that line the bile ducts. Hepatocytes secrete bile acids and conjugated products into the bile canaliculi, which then are modified by cholangiocytes through secretion of bicarbonate and water to give rise to the final secreted bile. Here the molecular mechanisms regulating bile secretion in cholangiocytes are reviewed. Moreover, we discuss how the expression of intracellular Ca(2+) channels might be regulated in cholangiocytes, plus evidence that components of the Ca(2+) signaling machinery are altered in a range of cholestatic diseases of the bile ducts. PMID:26100660

  5. Ryanodine receptors, a family of intracellular calcium ion channels, are expressed throughout early vertebrate development

    PubMed Central

    2011-01-01

    Background Calcium signals ([Ca2+]i) direct many aspects of embryo development but their regulation is not well characterised. Ryanodine receptors (RyRs) are a family of intracellular Ca2+ release channels that control the flux of Ca2+ from internal stores into the cytosol. RyRs are primarily known for their role in excitation-contraction coupling in adult striated muscle and ryr gene mutations are implicated in several human diseases. Current evidence suggests that RyRs do not have a major role to play prior to organogenesis but regulate tissue differentiation. Findings The sequences of the five zebrafish ryr genes were confirmed, their evolutionary relationship established and the primary sequences compared to other vertebrates, including humans. RyRs are differentially expressed in slow (ryr1a), fast (ryr3) and both types (ryr1b) of developing skeletal muscle. There are two ryr2 genes (ryr2a and ryr2b) which are expressed exclusively in developing CNS and cardiac tissue, respectively. In addition, ryr3 and ryr2a mRNA is detectable in the initial stages of development, prior to embryonic axis formation. Conclusions Our work reveals that zebrafish ryr genes are differentially expressed throughout the developing embryo from cleavage onwards. The data suggests that RyR-regulated Ca2+ signals are associated with several aspects of embryonic development, from organogenesis through to the differentiation of the musculoskeletal, cardiovascular and nervous system. These studies will facilitate further work to explore the developmental function of RyRs in each of these tissue types. PMID:22168922

  6. Complexity of calcium signaling in synaptic spines

    PubMed Central

    Franks, Kevin M.; Sejnowski, Terrence J.

    2010-01-01

    Summary Long-term potentiation and long-term depression are thought to be cellular mechanisms contributing to learning and memory. Although the physiological phenomena have been well characterized, little consensus of their underlying molecular mechanisms has emerged. One reason for this may be the under-appreciated complexity of the signaling pathways that can arise if key signaling molecules are discretely localized within the synapse. Recent findings suggest an unanticipated degree of structural organization at the synapse, and improved methods in cellular imaging of living tissue have provided much-needed information about the intracellular dynamics of Ca2+, thought to be critical for both LTP and LTD. In this review, we briefly summarize some of these developments, and show that a more complete understanding of cellular signaling depends on the successful integration of traditional biochemistry and molecular biology with the spatial and temporal details of synaptic ultrastructure. Biophysically realistic computer simulations can have an important role in bridging these disciplines. PMID:12447978

  7. Dietary calcium attenuates platelet aggregation and intracellular Ca2+ mobilization in spontaneously hypertensive rats

    NASA Technical Reports Server (NTRS)

    Otsuka, K.; Watanabe, M.; Yue, Q.; McCarron, D. A.; Hatton, D.

    1997-01-01

    Spontaneously hypertensive rats (SHR) are known to be blood pressure sensitive to dietary calcium. The effects of dietary calcium on platelet aggregation and intracellular Ca2+ mobilization were assessed by turbidimetric methods and fura-2 methods, respectively, in washed platelets of SHR. Ca2+ ATPase activity was examined in aortic membrane fractions. Six weeks of dietary calcium supplementation attenuated the increase of systolic blood pressure (SBP 199 +/- 16 v 170 +/- 9 mm Hg, P < .001) and thrombin-induced platelet aggregation (84.5 +/- 3.7 v 73.7 +/- 7.4%, P < .004) at 9 weeks of age. The ionomycin-induced intracellular calcium ([Ca2+]i) peak in the absence of external Ca2+, which reflects [Ca2+]i storage size, and thrombin-evoked [Ca2+]i release from [Ca2+]i storage were decreased by 2.0% Ca diet (472 +/- 55 v 370 +/- 23 nmol/L, P < .001, 339 +/- 29 v 278 +/- 33 nmol/L, P < .002). In addition, SBP was positively correlated with platelet aggregation (r = 0.703, P = .0088), thrombin-evoked [Ca2+]i (r = 0.739, P = .0044), and ionomycin-induced [Ca2+]i (r = 0.591, P = .0415), respectively. However, there was no significant effect of dietary calcium on Ca2+-ATPase activity in aortic membranes. These results suggest that dietary calcium supplementation had a beneficial effect on platelets of SHR by attenuating [Ca2+]i mobilization from [Ca2+]i storage. The hypotensive effect of dietary calcium might be associated with attenuated [Ca2+]i mobilization in SHR.

  8. Intestinal Stem Cells: Got Calcium?

    PubMed

    Nászai, Máté; Cordero, Julia B

    2016-02-01

    Calcium ions are well-known intracellular signalling molecules. A new study identifies local cytoplasmic calcium as a central integrator of metabolic and proliferative signals in Drosophila intestinal stem cells. PMID:26859268

  9. Modeling intracellular signaling underlying striatal function in health and disease.

    PubMed

    Nair, Anu G; Gutierrez-Arenas, Omar; Eriksson, Olivia; Jauhiainen, Alexandra; Blackwell, Kim T; Kotaleski, Jeanette H

    2014-01-01

    Striatum, which is the input nucleus of the basal ganglia, integrates cortical and thalamic glutamatergic inputs with dopaminergic afferents from the substantia nigra pars compacta. The combination of dopamine and glutamate strongly modulates molecular and cellular properties of striatal neurons and the strength of corticostriatal synapses. These actions are performed via intracellular signaling networks, containing several intertwined feedback loops. Understanding the role of dopamine and other neuromodulators requires the development of quantitative dynamical models for describing the intracellular signaling, in order to provide precise unambiguous descriptions and quantitative predictions. Building such models requires integration of data from multiple data sources containing information regarding the molecular interactions, the strength of these interactions, and the subcellular localization of the molecules. Due to the uncertainty, variability, and sparseness of these data, parameter estimation techniques are critical for inferring or constraining the unknown parameters, and sensitivity analysis evaluates which parameters are most critical for a given observed macroscopic behavior. Here, we briefly review the modeling approaches and tools that have been used to investigate biochemical signaling in the striatum, along with some of the models built around striatum. We also suggest a future direction for the development of such models from the, now becoming abundant, high-throughput data. PMID:24560149

  10. Modeling intracellular signaling underlying striatal function in health and disease

    PubMed Central

    Nair, Anu G; Gutierrez-Arenas, Omar; Eriksson, Olivia; Jauhiainen, Alexandra; Blackwell, Kim T; Kotaleski, Jeanette Hellgren

    2014-01-01

    Striatum, which is the input nucleus of the basal ganglia, integrates cortical and thalamic glutamatergic inputs with dopaminergic afferents from the substantia nigra pars compacta. The combination of dopamine and glutamate strongly modulates molecular and cellular properties of striatal neurons and the strength of corticostriatal synapses. These actions are performed via intracellular signaling networks, containing several intertwined feedback loops. Understanding the role of dopamine and other neuromodulators requires the development of quantitative dynamical models for describing the intracellular signaling, in order to provide precise unambiguous descriptions and quantitative predictions. Building such models requires integration of data from multiple data sources containing information regarding the molecular interactions, the strength of these interactions, and the subcellular localization of the molecules. Due to the uncertainty, variability, and sparseness of these data, parameter estimation techniques are critical for inferring or constraining the unknown parameters, and sensitivity analysis evaluates which parameters are most critical for a given observed macroscopic behavior. Here, we briefly review the modeling approaches and tools that have been used to investigate biochemical signaling in the striatum, along with some of the models built around striatum. We also suggest a future direction for the development of such models from the, now becoming abundant, high-throughput data. PMID:24560149

  11. Intracellular Mono-ADP-Ribosylation in Signaling and Disease

    PubMed Central

    Bütepage, Mareike; Eckei, Laura; Verheugd, Patricia; Lüscher, Bernhard

    2015-01-01

    A key process in the regulation of protein activities and thus cellular signaling pathways is the modification of proteins by post-translational mechanisms. Knowledge about the enzymes (writers and erasers) that attach and remove post-translational modifications, the targets that are modified and the functional consequences elicited by specific modifications, is crucial for understanding cell biological processes. Moreover detailed knowledge about these mechanisms and pathways helps to elucidate the molecular causes of various diseases and in defining potential targets for therapeutic approaches. Intracellular adenosine diphosphate (ADP)-ribosylation refers to the nicotinamide adenine dinucleotide (NAD+)-dependent modification of proteins with ADP-ribose and is catalyzed by enzymes of the ARTD (ADP-ribosyltransferase diphtheria toxin like, also known as PARP) family as well as some members of the Sirtuin family. Poly-ADP-ribosylation is relatively well understood with inhibitors being used as anti-cancer agents. However, the majority of ARTD enzymes and the ADP-ribosylating Sirtuins are restricted to catalyzing mono-ADP-ribosylation. Although writers, readers and erasers of intracellular mono-ADP-ribosylation have been identified only recently, it is becoming more and more evident that this reversible post-translational modification is capable of modulating key intracellular processes and signaling pathways. These include signal transduction mechanisms, stress pathways associated with the endoplasmic reticulum and stress granules, and chromatin-associated processes such as transcription and DNA repair. We hypothesize that mono-ADP-ribosylation controls, through these different pathways, the development of cancer and infectious diseases. PMID:26426055

  12. Nesfatin-1 increases intracellular calcium concentration by protein kinase C activation in cultured rat dorsal root ganglion neurons.

    PubMed

    Ozcan, Mete; Gok, Zeynep Betul; Kacar, Emine; Serhatlioglu, Ihsan; Kelestimur, Haluk

    2016-04-21

    Nesfatin-1 is a recently identified anorexigenic hypothalamic polypeptide derived from the posttranslational processing of nucleobindin 2 (NUCB2). Several studies have indicated that this neuropeptide may be participated in somatosensory and visceral transmission including pain signals in addition to energy metabolism. The aim of this study was to explore the possible role of nesfatin-1 in the transmission of peripheral neural signals by investigating the effects of nesfatin-1 on intracellular free calcium levels ([Ca(2+)]i) in cultured neonatal rat dorsal root ganglion (DRG) neurons. The effects of nesfatin-1 on [Ca(2+)]i in DRG neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1-or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of nesfatin-1 on [Ca(2+)]i and role of the protein kinase C (PKC)-mediated pathway in nesfatin-1 effect were assessed. Nesfatin-1 elevated [Ca(2+)]i in cultured DRG neurons. The response was prevented by pretreating the cells with pertussis toxin. The protein kinase C inhibitor chelerythrine chloride suppressed nesfatin-1-induced rise in [Ca(2+)]i. The result shows that nesfatin-1 interacts with a G protein-coupled receptor, leading to an increase of [Ca(2+)]i, which is linked to protein kinase C activation in cultured rat DRG neurons. PMID:26975784

  13. ATP-Evoked Intracellular Ca(2+) Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea.

    PubMed

    Horváth, T; Polony, G; Fekete, Á; Aller, M; Halmos, G; Lendvai, B; Heinrich, A; Sperlágh, B; Vizi, E S; Zelles, T

    2016-02-01

    Hearing and its protection is regulated by ATP-evoked Ca(2+) signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca(2+) imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca(2+) signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca(2+)]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca(2+) transients in all three cell types, showing desensitization. Inhibiting the Ca(2+) signaling of the ionotropic P2X (omission of extracellular Ca(2+)) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca(2+) stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca(2+)-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca(2+) leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca(2+) signaling in these cells. Differences in Ca(2+) homeostasis and ATP-induced Ca(2+) signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology. PMID:26801171

  14. Elevated Intracellular Calcium Increases Ferritin H Expression Through an NFAT-Independent Posttranscriptional Mechanism Involving mRNA Stabilization

    PubMed Central

    MacKenzie, Elizabeth L.; Tsuji, Yoshiaki

    2009-01-01

    An increase in intracellular Ca2+ is one of the initiating events in T cell activation. A calcium-mediated signaling cascade in T cells involves activation of calcineurin and the dephosphorylation and translocation of Nuclear Factor of Activated T-cells (NFAT), resulting in the transcriptional activation of target genes such as IL-2. In the present study, we found that increased intracellular calcium leads to induction of the antioxidant protein ferritin H. We previously reported that the ferritin H gene is transcriptionally activated under oxidative stress conditions through an antioxidant responsive element (ARE). The facts that the ferritin H ARE contains a composite AP1 site, and that NFAT collaborates with AP1 transcription factors, led us to test whether calcium-activated NFAT is involved in the ferritin H induction through the ARE. Treatment of Jurkat T cells with the calcium ionophore, ionomycin, increased ferritin H mRNA and protein expression. Though NFAT translocated to the nucleus and bound a consensus NFAT sequence located in the IL-2 promoter following ionomycin treatment, it did not activate ferritin H transcription despite the presence of a putative NFAT binding sequence in the ferritin H ARE. In addition, the calcineurin inhibitor cyclosporin A treatment blocked ionomycin-mediated NFAT nuclear translocation but failed to abrogate the increase in ferritin H mRNA. Analysis of mRNA stability following actinomycin D treatment revealed that ionomycin prolongs ferritin H mRNA half-life. Taken together, these results suggest that ionomycin-mediated induction of ferritin H may occur in an NFAT-independent manner but through posttranscriptional stabilization of the ferritin H mRNA. PMID:18076382

  15. Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS.

    PubMed

    Borges-Pereira, Lucas; Budu, Alexandre; McKnight, Ciara A; Moore, Christina A; Vella, Stephen A; Hortua Triana, Miryam A; Liu, Jing; Garcia, Celia R S; Pace, Douglas A; Moreno, Silvia N J

    2015-11-01

    Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca(2+) oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca(2+) enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca(2+) changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca(2+) oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca(2+) influx. This is the first study showing, in real time, Ca(2+) signals preceding egress and their direct link with motility, an essential virulence trait. PMID:26374900

  16. Raised intracellular free calcium within the lens causes opacification and cellular uncoupling in the frog.

    PubMed Central

    Jacob, T J

    1983-01-01

    Ion-sensitive micro-electrodes were used to measure the levels of intracellular free Ca2+ within the intact amphibian lens. The free [Ca2+] was found to constitute 0.4% of the total lens calcium. The pCa measured at the anterior lens surface was found to 6.59, while that at the posterior was 5.70. An 8-fold anterior/posterior Ca2+ gradient thus exists along the optical axis. The intracellular free Ca2+ could be manipulated by incubating the lens in high-Ca2+ or cA2+-free EGTA Ringer solutions. Raising the intracellular free Ca2+ to 0.22 mM caused lens opacification and cellular uncoupling; the coupling ratio was reduced from 1 in control to 0.41 in high Ca2+. Images Fig. 3 PMID:6604808

  17. Calcium-Sensing Receptor: A Key Target for Extracellular Calcium Signaling in Neurons

    PubMed Central

    Jones, Brian L.; Smith, Stephen M.

    2016-01-01

    Though both clinicians and scientists have long recognized the influence of extracellular calcium on the function of muscle and nervous tissue, recent insights reveal that the mechanisms allowing changes in extracellular calcium to alter cellular excitability have been incompletely understood. For many years the effects of calcium on neuronal signaling were explained only in terms of calcium entry through voltage-gated calcium channels and biophysical charge screening. More recently however, it has been recognized that the calcium-sensing receptor is prevalent in the nervous system and regulates synaptic transmission and neuronal activity via multiple signaling pathways. Here we review the multiplicity of mechanisms by which changes in extracellular calcium alter neuronal signaling and propose that multiple mechanisms are required to describe the full range of experimental observations. PMID:27065884

  18. Methotrexate loaded self stabilized calcium phosphate nanoparticles: a novel inorganic carrier for intracellular drug delivery.

    PubMed

    Mukesh, Ukawala; Kulkarni, Vijay; Tushar, Rajyaguru; Murthy, R S R

    2009-02-01

    Calcium phosphate is considered as a potential biomaterial for drug and gene delivery because of its excellent features. In this study, we reported the formulation and characterization of calcium phosphate nanoparticle containing anticancer drug, methotrexate (MTX). Calcium phosphate nanoparticles containing MTX (CaPi-MTX) were prepared by reverse micelles technique. CaPi-MTX nanoparticles of average size 262 +/- 47.64 nm with entrapment efficiency of 58.04 +/- 4.09% were obtained. The IR spectrum of CaPi-MTX showed characteristics of composite formation of hydroxyapatite with MTX. X-RD analysis revealed that, CaPi-MTX nanoparticles were crystalline and in hydroxyapatite form. TEM studies showed that CaPi-MTX nanoparticles were spherical in shape. In vitro release study of CaPi-MTX nanoparticles showed slow release of MTX at physiological pH (pH 7.4) while > 90% release was observed within 3-4 hours at endosomal pH (pH 5.5 and pH 6.0). Confocal microscopy was performed using CHO cell lines, showed intracellular localization of FITC-Dextran loaded calcium phosphate nanoparticles. Results indicate that prepared CaPi-MTX nanoparticles could serve the purpose for intracellular drug delivery. PMID:20055112

  19. Calcium Binding to PICK1 is Essential for the Intracellular Retention of AMPA Receptors Underlying LTD

    PubMed Central

    Citri, Ami; Bhattacharyya, Samarjit; Ma, Cong; Morishita, Wade; Fang, Scarlett; Rizo, Josep; Malenka, Robert C.

    2010-01-01

    NMDA receptor (NMDAR)-dependent LTD in the hippocampus is mediated primarily by the calcium-dependent removal of AMPA receptors (AMPARs) from the postsynaptic density. The AMPAR-binding, PDZ and BAR domain containing protein PICK1 has been implicated in the regulation of AMPAR trafficking underlying several forms of synaptic plasticity. Using a strategy involving shRNA-mediated knockdown of PICK1 and its replacement with recombinant PICK1, we performed a detailed structure-function analysis of the role of PICK1 in hippocampal synaptic plasticity and the underlying NMDAR-induced AMPAR trafficking. We found that PICK1 is not necessary for maintenance of the basal synaptic complement of AMPARs or expression of either mGluR-LTD or NMDAR-dependent LTP. Rather, PICK1 function is specific to NMDAR-dependent LTD and the underlying AMPAR trafficking. Furthermore, while PICK1 does not regulate the initial phase of NMDAR-induced AMPAR endocytosis, it is required for intracellular retention of internalized AMPARs. Detailed biophysical analysis of an N-terminal acidic motif indicated that it is involved in intramolecular electrostatic interactions that are disrupted by calcium. Mutations that interfered with the calcium-induced structural changes in PICK1 precluded LTD and the underlying NMDAR-induced intracellular retention of AMPARs. These findings support a model whereby calcium-induced modification of PICK1 structure is critical for its function in the retention of internalized AMPARs that underlies the expression of hippocampal NMDAR-dependent LTD. PMID:21147983

  20. Ca2+ Content and Expression of an Acidocalcisomal Calcium Pump Are Elevated in Intracellular Forms of Trypanosoma cruzi

    PubMed Central

    Lu, Hong-Gang; Zhong, Li; de Souza, Wanderley; Benchimol, Marlene; Moreno, Silvia; Docampo, Roberto

    1998-01-01

    The survival of a eukaryotic protozoan as an obligate parasite in the interior of a eukaryotic host cell implies its adaptation to an environment with a very different ionic composition from that of its extracellular habitat. This is particularly important in the case of Ca2+, the intracellular concentration of which is 3 orders of magnitude lower than the extracellular value. Ca2+ entry across the plasma membrane is a widely recognized mechanism for Ca2+ signaling, needed for a number of intracellular processes, and obviously, it would be restricted in the case of intracellular parasites. Here we show that Trypanosoma cruzi amastigotes possess a higher Ca2+ content than the extracellular stages of the parasite. This correlates with the higher expression of a calcium pump, the gene for which was cloned and sequenced. The deduced protein product (Tca1) of this gene has a calculated molecular mass of 121,141 Da and exhibits 34 to 38% identity with vacuolar Ca2+-ATPases of Saccharomyces cerevisiae and Dictyostelium discoideum, respectively. The tca1 gene suppresses the Ca2+ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca2+ accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that Tca1 colocalizes with the vacuolar H+-ATPase to the plasma membrane and to intracellular vacuoles of T. cruzi. These vacuoles were shown to have the same size and distribution as the calcium-containing vacuoles identified by the potassium pyroantimoniate-osmium technique and as the electron-dense vacuoles observed in whole unfixed parasites by transmission electron microscopy and identified in a previous work (D. A. Scott, R. Docampo, J. A. Dvorak, S. Shi, and R. D. Leapman, J. Biol. Chem. 272:28020–28029, 1997) as being acidic and possessing a high calcium content (i.e., acidocalcisomes). Together, these results suggest that acidocalcisomes are distinct from other previously recognized organelles present in these parasites

  1. Spatiotemporal calcium signaling in a Drosophila melanogaster cell line stably expressing a Drosophila muscarinic acetylcholine receptor.

    PubMed

    Cordova, D; Delpech, V Raymond; Sattelle, D B; Rauh, J J

    2003-11-01

    A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca(2+)](i)) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca(2+)](i) transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx. PMID:12827518

  2. Elevation of intracellular calcium levels in spiral ganglion cells by trimethyltin.

    PubMed

    Fechter, L D; Liu, Y

    1995-11-01

    The neurotoxicant, trimethyltin (TMT) produces cochlear impairment at far lower dose levels and far more rapidly than it does central nervous system effects. The initial effects of TMT in the cochlea, in vivo, are consistent with disruption of the inner hair cell type-1 spiral ganglion cell synapse although it is uncertain whether the effect is on presynaptic and/or postsynaptic units. This synapse is believed to be an excitatory glutamatergic one, providing the possibility that TMT could induce an excitotoxic process resulting in elevations in intracellular calcium ([Ca2+]i). The objective of this study was to determine whether TMT had direct toxic effects on the postsynaptic spiral ganglion cells studied in primary culture and to identify the role of extracellular calcium in such an effect. The marker of interest was the effect of this agent on [Ca2+]i levels as determined using quantitation of the fluorescent calcium dye, Fura-2. TMT did induce a marked and sustained elevation in [Ca2+]i level in the spiral ganglion cells that appeared to have a rapid initial phase and a slower saturating phase. Studies performed using calcium-free medium showed that elevation of [Ca2+]i in spiral ganglion cells by TMT was attenuated but not entirely blocked. Further, the L-type calcium channel blocker, nifedipine, was able to inhibit the initial increase in [Ca2+]i, suggesting that at least this phase of the TMT effect was mediated by calcium channels, although nifedipine had no significant effect on the time to reach the maximal [Ca2+]i level. Parallel control experiments performed using application of exogenous glutamate and depolarizing K+ concentrations also produced elevation in [Ca2+]i levels. The data indicate that TMT elevates [Ca2+]i in isolated spiral ganglion cells both by increasing extracellular uptake via Ca2+ channels and also by releasing Ca2+ from intracellular stores. Thus TMT ototoxicity appears to include a direct postsynaptic toxic event. PMID:8647712

  3. Quantifying bursting neuron activity from calcium signals using blind deconvolution.

    PubMed

    Park, In Jun; Bobkov, Yuriy V; Ache, Barry W; Principe, Jose C

    2013-09-15

    Advances in calcium imaging have enabled studies of the dynamic activity of both individual neurons and neuronal assemblies. However, challenges, such as unknown nonlinearities in the spike-calcium relationship, noise, and the often relatively low temporal resolution of the calcium signal compared to the time-scale of spike generation, restrict the accurate estimation of action potentials from the calcium signal. Complex neuronal discharge, such as the activity demonstrated by bursting and rhythmically active neurons, represents an even greater challenge for reconstructing spike trains based on calcium signals. We propose a method using blind calcium signal deconvolution based on an information-theoretic approach. This model is meant to maximise the output entropy of a nonlinear filter where the nonlinearity is defined by the cumulative distribution function of the spike signal. We tested our maximum entropy (ME) algorithm using bursting olfactory receptor neurons (bORNs) of the lobster olfactory organ. The advantage of the ME algorithm is that the filter can be trained online based only on the statistics of the spike signal, without any assumptions regarding the unknown transfer function characterizing the relation between the spike and calcium signal. We show that the ME method is able to more accurately reconstruct the timing of the first and last spikes of a burst compared to other methods and that it improves the temporal precision fivefold compared to direct timing resolution of calcium signal. PMID:23711821

  4. Calcium signaling in plant cells in altered gravity

    NASA Astrophysics Data System (ADS)

    Kordyum, E. L.

    2003-10-01

    Changes in the intracellular Ca 2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca 2+ 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 80 th, a review highlighting the performed research and the possible significance of such Ca 2+ 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 Ca 2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca 2+ 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

  5. 5-HT2B receptor-mediated calcium release from ryanodine-sensitive intracellular stores in human pulmonary artery endothelial cells.

    PubMed Central

    Ullmer, C.; Boddeke, H. G.; Schmuck, K.; Lübbert, H.

    1996-01-01

    1. We have characterized the 5-hydroxytryptamine (5-HT)-induced calcium signalling in endothelial cells from the human pulmonary artery. Using RT-PCR we show, that of all cloned G-protein coupled 5-HT receptors, these cells express only 5-HT1D beta, 5-HT2B and little 5-HT4 receptor mRNA. 2. In endothelial cells 5-HT inhibits the formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) via 5-HT1D beta receptors but fails to activate phosphoinositide (PI) turnover. However, the latter pathway is strongly activated by histamine. 3. Despite the lack of detectable inositol phosphate (IP) formation in human pulmonary artery endothelial cells, 5-HT (pD2 = 5.82 +/- 0.06, n = 6) or the selective 5-HT2 agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (pD2 = 5.66 +/- 0.03, n = 7) elicited transient calcium signals comparable to those evoked by histamine (pD2 = 6.44 +/- 0.01, n = 7). Since 5-HT2A and 5-HT2C receptor mRNAs are not detectable in pulmonary artery endothelial cells, activation of 5-HT2B receptors is responsible for the transient calcium release. The calcium transients are independent of the inhibition of adenylate cyclase, since DOI does not stimulate 5-HT1D beta receptors. 4. Both, the 5-HT- and histamine-stimulated calcium signals were also observed when the cells were placed in calcium-free medium. This indicates that 5-HT triggers calcium release from intracellular stores. 5. Heparin is an inhibitor of the IP3-activated calcium release channels on the endoplasmic reticulum. Intracellular infusion of heparin through patch pipettes in voltage clamp experiments failed to block 5-HT-induced calcium signals, whereas it abolished the histamine response. This supports the conclusion that the 5-HT-induced calcium release is independent of IP3 formation. 6. Unlike the histamine response, the 5-HT response was sensitive to micromolar concentrations of ryanodine and, to a lesser extent, ruthenium red. This implies that 5-HT2B receptors trigger calcium

  6. Intracellular calcium in cardiac myocytes: calcium transients measured using fluorescence imaging.

    PubMed

    Cannell, M B; Berlin, J R; Lederer, W J

    1987-01-01

    We have examined the distribution of Ca2+ in voltage-clamped cardiac myocytes under resting conditions and during the Ca2+ transient. We find that the resting Ca2+ level in a quiescent rat myocyte bathed in 1 mM extracellular Ca is relatively low (between 60 and 100 nM) and uniform. At the peak of the Ca2+ transient, Ca2+ can rise to a level as high as 600 nM to 1.0 microM. Furthermore, the magnitude of the Ca2+ transient is dependent on the size of the membrane depolarization. There is good agreement between measurements made using video imaging and those made using a photomultiplier tube for the value of intracellular Ca2+ at the peak of the Ca2+ transient and for the subsequent slow changes in intracellular Ca2+. On repolarization, intracellular Ca2+ falls with a half-time of approximately 100 ms. The uniform distribution of Ca2+ reported in the Ca2+ images of myocytes at rest and at the peak of the Ca2+ transient under normal conditions is in contrast to what is observed during "Ca2+ overload" when subcellular regions of elevated Ca2+ are observed to propagate along the cell. Thus, the measurement of [Ca2+]i in cardiac myocytes with fura-2 has already yielded important new information that was not available using other techniques to measure [Ca2+]i in cardiac ventricular muscle. PMID:3505361

  7. Propagation of Intracellular Ca2+ Signals in Aged Exocrine Cells.

    PubMed

    Martin-Cano, Francisco E; Camello-Almaraz, Cristina; Macías, Jesús González; Pozo, Maria J; Camello, Pedro J

    2016-02-01

    There is little information on the effects of aging in the propagation of calcium signals and its underlying mechanisms. We studied the effects of aging on propagation of Ca(2+) signals in pancreatic acinar cells. Fura-2 loaded cells isolated from young (3-4 months old) and aged (24 months old) mouse responded to acetylcholine (ACh) and cholecystokinin (CCK) with a polarized Ca(2+) response initiated at the secretory pole before spreading to the basal one. Aging slowed down the propagation of the response to ACh but enhanced the velocity of the CCK response. This pattern can be explained by the age-induced depolarization of mitochondria, because it can be reproduced in young cells by mitochondrial inhibitors. Aging also increased the role of acidic stores in the CCK signal, as judged by the folimycin-induced suppression of the polarization in aged but not in young cells. The involvement of ryanodine receptors in the ACh response was also enhanced, as indicated by the loss of polarization after the treatment with 8Br-cyclic ADP ribose. Therefore, we conclude that aging modifies differentially the propagation of ACh and CCK-evoked Ca(2+) signals through mitochondrial depolarization and changes in the role of the acidic Ca(2+) stores and ryanodine receptors in the initiation of the signals. PMID:25805851

  8. Molecular mechanisms of corticotropin-releasing factor receptor-induced calcium signaling.

    PubMed

    Gutknecht, Eric; Van der Linden, Ilse; Van Kolen, Kristof; Verhoeven, Kim F C; Vauquelin, Georges; Dautzenberg, Frank M

    2009-03-01

    The molecular mechanisms governing calcium signal transduction of corticotropin-releasing factor (CRF) receptors CRF(1) and CRF(2(a)) stably expressed in human embryonic kidney (HEK) 293 cells were investigated. Calcium signaling strictly depended on intracellular calcium sources, and this is the first study to establish a prominent contribution of the three major G-protein families to CRF receptor-mediated calcium signaling. Overexpression of Galpha(q/11) and Galpha(16) led to leftward shifts of the agonist concentration-response curves. Blockade of Galpha(q/11) proteins by the small interfering RNA (siRNA) technology partially reduced agonist-mediated calcium responses in CRF(1)- and CRF(2(a))-expressing HEK293 cells, thereby proving a contribution of the G(q) protein family. A small but significant inhibition of calcium signaling was recorded by pharmacological inhibition of G(i/o) proteins with pertussis toxin treatment. This effect was mediated by direct binding of Gbetagamma subunits to phospholipase C. G(i/o) inhibition also elevated cAMP responses in CRF receptor-overexpressing HEK293 cells and in Y79 retinoblastoma cells endogenously expressing human CRF(1) and CRF(2(a)) receptors, thereby demonstrating natural coupling of G(i) proteins to both CRF receptors. The strongest reduction of CRF receptor-mediated calcium mobilization was noted when blocking the G(s) signaling protein either by cholera toxin or by siRNA. It is noteworthy that simultaneous inhibition of two G-proteins shed light on the additive effects of G(s) and G(q) on the calcium signaling and, hence, that they act in parallel. On the other hand, G(i) coupling required prior G(s) activation. PMID:19098121

  9. MicroRNA-30 family members regulate calcium/calcineurin signaling in podocytes

    PubMed Central

    Wu, Junnan; Zheng, Chunxia; Wang, Xiao; Yun, Shifeng; Zhao, Yue; Liu, Lin; Lu, Yuqiu; Ye, Yuting; Zhu, Xiaodong; Zhang, Changming; Shi, Shaolin; Liu, Zhihong

    2015-01-01

    Calcium/calcineurin signaling is critical for normal cellular physiology. Abnormalities in this pathway cause many diseases, including podocytopathy; therefore, understanding the mechanisms that underlie the regulation of calcium/calcineurin signaling is essential. Here, we showed that critical components of calcium/calcineurin signaling, including TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3, are the targets of the microRNA-30 family (miR-30s). We found that these 5 genes are highly expressed as mRNA, but the level of the proteins is low in normal podocytes. Conversely, protein levels were markedly elevated in podocytes from rats treated with puromycin aminonucleoside (PAN) and from patients with focal segmental glomerulosclerosis (FSGS). In both FSGS patients and PAN-treated rats, miR-30s were downregulated in podocytes. In cultured podocytes, PAN or a miR-30 sponge increased TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 expression; calcium influx; intracellular Ca2+ concentration; and calcineurin activity. Moreover, NFATC3 nuclear translocation, synaptopodin degradation, integrin β3 (ITGB3) activation, and actin fiber loss, which are downstream of calcium/calcineurin signaling, were induced by miR-30 reduction but blocked by the calcineurin inhibitor FK506. Podocyte-specific expression of the miR-30 sponge in mice increased calcium/calcineurin pathway component protein expression and calcineurin activity. The mice developed podocyte foot process effacement and proteinuria, which were prevented by FK506. miR-30s also regulated calcium/calcineurin signaling in cardiomyocytes. Together, our results identify miR-30s as essential regulators of calcium/calcineurin signaling. PMID:26436650

  10. The versatility of mitochondrial calcium signals: from stimulation of cell metabolism to induction of cell death

    PubMed Central

    Rimessi, Alessandro; Giorgi, Carlotta; Pinton, Paolo; Rizzuto, Rosario

    2008-01-01

    Both the contribution of mitochondria to intracellular calcium (Ca2+) signalling and the role of mitochondrial Ca2+ uptake in shaping the cytoplasmic response and controlling mitochondrial function are areas of intense investigation. These studies rely on the appropriate use of emerging techniques coupled with judicious data interpretation to a large extent. The development of targeted probes based on the molecular engineering of luminescent proteins has allowed the specific measurement of Ca2+ concentration ([Ca2+]) and adenosine trisphosphate concentration ([ATP]) in intracellular organelles or cytoplasmic subdomains. This approach has given novel information on different aspects of mitochondrial homeostasis. PMID:18573473

  11. Intracellular calcium store filling by an L-type calcium current in the basolateral amygdala at subthreshold membrane potentials

    PubMed Central

    Power, John M; Sah, Pankaj

    2005-01-01

    The long-term changes that underlie learning and memory are activated by rises in intracellular Ca2+ that activate a number of signalling pathways and trigger changes in gene transcription. Ca2+ rises due to influx via L-type voltage-dependent Ca2+ channels (L-VDCCs) and release from intracellular Ca2+ stores have been consistently implicated in the biochemical cascades that underlie the final changes in memory formation. Here, we show that pyramidal neurones in the basolateral amygdala express an L-VDCC that is active at resting membrane potentials. Subthreshold depolarization of neurones either by current injection or summating synaptic potentials led to a sustained rise in cytosolic Ca2+ that was blocked by the dihydropyridine nicardipine. Activation of metabotropic receptors released Ca2+ from intracellular Ca2+ stores. At hyperpolarized potentials, metabotropic-evoked store release ran down with repeated stimulation. Depolarization of cells to −50 mV, or maintaining them at the resting membrane potential, restored release from intracellular Ca2+ stores, an effect that was blocked by nicardipine. These results show that Ca2+ influx via a low-voltage-activated L-type Ca2+ current refills inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores, and maintains Ca2+ release and wave generation by metabotropic receptor activation. PMID:15550460

  12. Effects of mechanical signaling on plant cell cytosolic calcium.

    PubMed Central

    Haley, A; Russell, A J; Wood, N; Allan, A C; Knight, M; Campbell, A K; Trewavas, A J

    1995-01-01

    Mechanical signals are important influences on the development and morphology of higher plants. Using tobacco transformed with the Ca(2+)-sensitive luminescent protein aequorin, we recently reported the effects of mechanical signals of touch and wind on the luminescence and thus intracellular calcium of young seedlings. When mesophyll protoplasts are isolated from these transgenic tobacco plants and mechanically stimulated by swirling them in solution, cytoplasmic Ca2+ increases immediately and transiently up to 10 microM, and these transients are unaffected by an excess of EGTA in the medium. The size of the transient effect is related to the strength of swirling. Epidermal strips isolated from transgenic tobacco leaves and containing only viable guard cells and trichomes also respond to the strength of swirling in solution and can increase their cytoplasmic Ca2+ transiently up to 10 microM. Finally, the moss Physcomitrella patens containing recombinant aequorin exhibits transient increases in cytoplasmic Ca2+ up to 5 microM when swirled in solution. This effect is strongly inhibited by ruthenium red. Our data indicate that the effect of mechanical stimulation can be found in a number of different cell types and in a lower plant as well as tobacco and suggest that mechanoperception and the resulting increase in cytoplasmic Ca2+ may be widespread. PMID:11536690

  13. Regulation of gamma T-cell antigen receptor expression by intracellular calcium in acute lymphoblastic leukemia cell line DND41.

    PubMed

    Peralta-Zaragoza, O; Martínez-Valdez, H; Madrid-Marina, V

    1996-01-01

    The calcium ionophore, ionomycin, promotes an increase of intracellular calcium and regulates mRNA expression of gamma/delta-TcR gene in human T lymphocytes. The mechanism of this regulation is not yet clear. Thus, the regulation by intracellular calcium requires elucidation. We studied the gamma-TcR gene expression in acute lymphoblastic leukemia cell line DND41 (CD4- CD8-) by Northern blot and flow cytometric analysis. The mRNA levels of gamma-TcR increased by ionomycin, anti-CD3, and with TPA. TPA had an antagonistic effect to both ionomycin and anti-CD3. Also, TPA inhibits the increased intracellular calcium promoted by ionomycin but not the increase promoted by anti-CD3 and ionomycin. Our results suggest that intracellular calcium induces mRNA and protein expression of gamma-TcR chain. This effect is antagonized by protein kinase C-activation. Thus, we conclude that the target cells of the differential regulation on gamma-TcR mRNA expression by intracellular calcium modulators are the CD4- CD8- cells, and this is due to cytosolic calcium mobilization. PMID:8854386

  14. THE INTRACELLULAR DISTRIBUTION OF CALCIUM IN THE MUCOSA OF THE AVIAN SHELL GLAND

    PubMed Central

    Hohman, Wayne; Schraer, Harald

    1966-01-01

    The intracellular distribution of calcium has been studied in the mucosa of the avian shell gland, a tissue which transports large quantities of calcium during discrete time intervals. Ca45 was administered to hens either in a single dose followed by sacrifice 5 min later or in repeated doses over an extended period followed by sacrifice 2 hr or 24 hr after the last injection. Subcellular fractions were isolated by differential centrifugation and analyzed for Ca45. The Ca45 was located principally in the particulate fractions; the concentration (CPM Ca45/mg N) was highest in the mitochondrial fraction. Comparisons of (1) the Ca45 distribution in shell gland cells with that of liver cells, (2) the alterations which occur due to the phase of the egg laying cycle, (3) the effects due to the time elapsed since the last injection of Ca45, and (4) the Ca45 distribution of the short term experiments with that of the long term experiments revealed that the mitochondrial fraction of the shell gland appeared to be active in the movement of calcium. The microsomal fraction showed increased values in CPM Ca45/mg N when calcification was occurring, which may indicate that the subcellular components of this fraction have a role in calcium transport. The nuclear and supernatant fractions did not seem to be involved in the transport process. The implications of these results concerning the manner by which calcium may be controlled on a cellular level in this system are discussed. PMID:5968974

  15. Calcium influx affects intracellular transport and membrane repair following nanosecond pulsed electric field exposure

    NASA Astrophysics Data System (ADS)

    Thompson, Gary Lee; Roth, Caleb C.; Dalzell, Danielle R.; Kuipers, Marjorie; Ibey, Bennett L.

    2014-05-01

    The cellular response to subtle membrane damage following exposure to nanosecond pulsed electric fields (nsPEF) is not well understood. Recent work has shown that when cells are exposed to nsPEF, ion permeable nanopores (<2 nm) are created in the plasma membrane in contrast to larger diameter pores (>2 nm) created by longer micro- and millisecond duration pulses. Nanoporation of the plasma membrane by nsPEF has been shown to cause a transient increase in intracellular calcium concentration within milliseconds after exposure. Our research objective is to determine the impact of nsPEF on calcium-dependent structural and repair systems in mammalian cells. Chinese hamster ovary (CHO-K1) cells were exposed in the presence and absence of calcium ions in the outside buffer to either 1 or 20, 600-ns duration electrical pulses at 16.2 kV/cm, and pore size was determined using propidium iodide and calcium green. Membrane organization was observed with morphological changes and increases in FM1-43 fluorescence. Migration of lysosomes, implicated in membrane repair, was followed using confocal microscopy of red fluorescent protein-tagged LAMP1. Microtubule structure was imaged using mEmerald-tubulin. We found that at high 600-ns PEF dosage, calcium-induced membrane restructuring and microtubule depolymerization coincide with interruption of membrane repair via lysosomal exocytosis.

  16. Overexpression of Sly41 suppresses COPII vesicle–tethering deficiencies by elevating intracellular calcium levels

    PubMed Central

    Mukherjee, Indrani; Barlowe, Charles

    2016-01-01

    SLY41 was identified as a multicopy suppressor of loss of Ypt1, a Rab GTPase essential for COPII vesicle tethering at the Golgi complex. SLY41 encodes a polytopic membrane protein with homology to a class of solute transporter proteins, but how overexpression suppresses vesicle-tethering deficiencies is not known. Here we show that Sly41 is efficiently packaged into COPII vesicles and actively cycles between the ER and Golgi compartments. SLY41 displays synthetic negative genetic interactions with PMR1, which encodes the major Golgi-localized Ca2+/Mn2+ transporter and suggests that Sly41 influences cellular Ca2+ and Mn2+ homeostasis. Experiments using the calcium probe aequorin to measure intracellular Ca2+ concentrations in live cells reveal that Sly41 overexpression significantly increases cytosolic calcium levels. Although specific substrates of the Sly41 transporter were not identified, our findings indicate that localized overexpression of Sly41 to the early secretory pathway elevates cytosolic calcium levels to suppress vesicle-tethering mutants. In vitro SNARE cross-linking assays were used to directly monitor the influence of Ca2+ on tethering and fusion of COPII vesicles with Golgi membranes. Strikingly, calcium at suppressive concentrations stimulated SNARE-dependent membrane fusion when vesicle-tethering activity was reduced. These results show that calcium positively regulates the SNARE-dependent fusion stage of ER–Golgi transport. PMID:27030673

  17. Trichloroethylene-mediated cytotoxicity in human epidermal keratinocytes is mediated by the rapid accumulation of intracellular calcium: Interception by naringenin.

    PubMed

    Ali, F; Khan, A Q; Khan, R; Sultana, S

    2016-02-01

    Industrial solvents pose a significant threat to the humankind. The mechanisms of their toxicity still remain in debate. Trichloroethylene (TCE) is a widespread industrial solvent responsible for severe liver dysfunction, cutaneous toxicity in occupationally exposed humans. We utilized an in vitro system of human epidermal keratinocyte (HaCaT) cells in this study to avoid complex cell and extracellular interactions. We report the cytotoxicity of organic solvent TCE in HaCaT and its reversal by a natural flavanone, naringenin (Nar). The cytotoxicity was attributed to the rapid intracellular free calcium (Ca(2+)) release, which might lead to the elevation of protein kinase C along with robust free radical generation, instability due to energy depletion, and sensitization of intracellular stress signal transducer nuclear factor κB. These effects were actually seen to induce significant amount of genomic DNA fragmentation. Furthermore, all these effects of TCE were effectively reversed by the treatment of Nar, a natural flavanone. Our studies identify intracellular Ca as a unique target used by organic solvents in the cytotoxicity and highlight the Ca(2+) ion stabilizer properties of Nar. PMID:25855085

  18. [Effects of coriaria lactone on the concentration of intracellular free calcium of rat hippocampal neurons].

    PubMed

    Lai, Xiaohui; Zhang, Qin; Zhou, Dong

    2008-08-01

    We instituted an investigation to elucidate the role of Ca2+ and calcium channels in epileptogenesis and to analyze the mechanism by which coriaria lactone (CL) regulates intracellular Ca2+ concentration. The hippocampal neurons of Sprague-Dawley rats (post natal days 7 to 14) were acutely isolated and loaded with calcium-sensitive fluorescent indicator Fluo-3/AM. Intracellular calcium concentration ([Ca2+]i) changes were measured using laser scanning confocal microscopy. The study included five groups, namely the CL group, the NiCl2 plus CL group, the Nifedipine plus CL group, the NiCl2+ Nifedipine plus CL group, and the control group. The results indicated that 20 microl/ml CL induced a significant increase of [Ca2+]i in hippocampal neurons when compared to the control (P < 0.01), the mean fluorescent intensity of intracellular calcium displaying an increase from 5.46 +/- 2.37 to 34.03 +/- 3.45. Although the increase of relative intracellular fluorescent intensity was delayed by 3 or 4 minutes in the NiCl2 plus CL group, the Nifedipine plus CL group, and the NiCl2+ Nifedipine plus CL group, yet the use to 20 microl/ml CL in these 3 groups caused a significant ascending level of the fluorescent intensities (from 3.94 +/- 1.75 to 30.18 +/- 4.22; from 3.38 +/- 1.11 to 36.39 +/- 3.97; from 3.05 +/- 1.02 to 28.05 +/- 2.71), and the effect was comparable to that observed in the CL group (P > 0.05). So CL can increase [Ca2+]i in acutely isolated rat hippocampal neurons. This effect can be delayed but can not be completely blocked by NiCl2 and Nifedipine. These findings indicate that CL can increase [Ca2+]i by other means besides T- and L-type voltage-gated calcium channels, and that CL can increase the excitability of neurons and play a role in the epileptogenesis process. PMID:18788307

  19. A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells.

    PubMed

    Delles, C; Haller, T; Dietl, P

    1995-08-01

    1. The whole-cell patch clamp technique and fluorescence microscopy with the Ca2+ indicators fura-2 and fluo-3 were used to measure the whole-cell current and the free intracellular Ca2+ concentration ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells. 2. In a Ca(2+)-free bath solution, thapsigargin (TG) caused a transient increase of [Ca2+]i. Subsequent addition of Ca2+ caused a long lasting elevation of [Ca2+]i. 3. In a Ca(2+)-free bath solution, extracellular application of TG, ATP or ionomycin, or intracellular application of inositol 1,4,5-trisphosphate (IP3), caused a small but significant inward current (Iin) and a transient outward Ca(2+)-dependent K+ current (IK(Ca)), consistent with intracellular Ca2+ release. Subsequent addition of Ca2+ induced a prominent Iin with a current density of -4.2 +/- 0.7 pA pF-1. This Iin was unaffected by inositol 1,3,4,5-tetrakisphosphate (IP4). 4. Na+ replacement by mannitol, N-methyl-D-glucamine+ (NMG+), aminomethylidin-trimethanol+ (Tris+) or choline+ reduced Iin by 54, 65, 52 and 56%, respectively. This indicates an apparent Ca2+ selectivity over Na+ of 26:1. Iin was, however, unaffected by replacing Cl- with gluconate- or by the K+ channel blocker charybdotoxin (CTX). 5. Iin was completely blocked by La3+ (IC50 = 0.77 microM). Consistently, La3+ completely reversed the TG-induced elevation of [Ca2+]i. SK&F 96365 (1-[3-(4-methoxyphenyl)-propoxyl]-1-(4-methoxy-phenyl)-ethyl-1H-im idazole) HCl did not inhibit the TG-induced Iin. It did, however, exhibit a biphasic effect on [Ca2+]i, consisting of an initial Ca2+ decay and a subsequent Ca2+ elevation. La3+ completely reversed the SK&F 96365-induced elevation of [Ca2+]i. 6. In the absence of Na+, Iin was dependent on the bath Ca2+ concentration (EC50 = 1.02 mM). Ca2+ replacement by Ba2+ or Mn2+ resulted in a reduction of Iin by 95 and 94%, respectively. 7. From these experiments we conclude that Ca2+ release from intracellular Ca2+ stores, induced by different independent

  20. Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer's disease.

    PubMed

    Haas, Laura T; Salazar, Santiago V; Kostylev, Mikhail A; Um, Ji Won; Kaufman, Adam C; Strittmatter, Stephen M

    2016-02-01

    Alzheimer's disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer's disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer's disease transgenes or by human Alzheimer's disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp-Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer's disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer's disease pathogenesis, and the complex is a potential target for disease-modifying intervention. PMID:26667279

  1. Calcium Signaling in Intact Dorsal Root Ganglia

    PubMed Central

    Gemes, Geza; Rigaud, Marcel; Koopmeiners, Andrew S.; Poroli, Mark J.; Zoga, Vasiliki; Hogan, Quinn H.

    2013-01-01

    Background Ca2+ is the dominant second messenger in primary sensory neurons. In addition, disrupted Ca2+ signaling is a prominent feature in pain models involving peripheral nerve injury. Standard cytoplasmic Ca2+ recording techniques use high K+ or field stimulation and dissociated neurons. To compare findings in intact dorsal root ganglia, we used a method of simultaneous electrophysiologic and microfluorimetric recording. Methods Dissociated neurons were loaded by bath-applied Fura-2-AM and subjected to field stimulation. Alternatively, we adapted a technique in which neuronal somata of intact ganglia were loaded with Fura-2 through an intracellular microelectrode that provided simultaneous membrane potential recording during activation by action potentials (APs) conducted from attached dorsal roots. Results Field stimulation at levels necessary to activate neurons generated bath pH changes through electrolysis and failed to predictably drive neurons with AP trains. In the intact ganglion technique, single APs produced measurable Ca2+ transients that were fourfold larger in presumed nociceptive C-type neurons than in nonnociceptive Aβ-type neurons. Unitary Ca2+ transients summated during AP trains, forming transients with amplitudes that were highly dependent on stimulation frequency. Each neuron was tuned to a preferred frequency at which transient amplitude was maximal. Transients predominantly exhibited monoexponential recovery and had sustained plateaus during recovery only with trains of more than 100 APs. Nerve injury decreased Ca2+ transients in C-type neurons, but increased transients in Aβ-type neurons. Conclusions Refined observation of Ca2+ signaling is possible through natural activation by conducted APs in undissociated sensory neurons and reveals features distinct to neuronal types and injury state. PMID:20526180

  2. Raised Intracellular Calcium Contributes to Ischemia-Induced Depression of Evoked Synaptic Transmission

    PubMed Central

    Jalini, Shirin; Ye, Hui; Tonkikh, Alexander A.; Charlton, Milton P.; Carlen, Peter L.

    2016-01-01

    Oxygen-glucose deprivation (OGD) leads to depression of evoked synaptic transmission, for which the mechanisms remain unclear. We hypothesized that increased presynaptic [Ca2+]i during transient OGD contributes to the depression of evoked field excitatory postsynaptic potentials (fEPSPs). Additionally, we hypothesized that increased buffering of intracellular calcium would shorten electrophysiological recovery after transient ischemia. Mouse hippocampal slices were exposed to 2 to 8 min of OGD. fEPSPs evoked by Schaffer collateral stimulation were recorded in the stratum radiatum, and whole cell current or voltage clamp recordings were performed in CA1 neurons. Transient ischemia led to increased presynaptic [Ca2+]i, (shown by calcium imaging), increased spontaneous miniature EPSP/Cs, and depressed evoked fEPSPs, partially mediated by adenosine. Buffering of intracellular Ca2+ during OGD by membrane-permeant chelators (BAPTA-AM or EGTA-AM) partially prevented fEPSP depression and promoted faster electrophysiological recovery when the OGD challenge was stopped. The blocker of BK channels, charybdotoxin (ChTX), also prevented fEPSP depression, but did not accelerate post-ischemic recovery. These results suggest that OGD leads to elevated presynaptic [Ca2+]i, which reduces evoked transmitter release; this effect can be reversed by increased intracellular Ca2+ buffering which also speeds recovery. PMID:26934214

  3. Changes in Intracellular Free Calcium Concentration during Illumination of Invertebrate Photoreceptors

    PubMed Central

    Brown, J. E.; Blinks, J. R.

    1974-01-01

    Aequorin, which luminesces in the presence of calcium, was injected into photoreceptor cells of Limulus ventral eye. A bright light stimulus elicited a large increase in aequorin luminescence, the aequorin response, indicating a rise of intracellular calcium ion concentration, Cai. The aequorin response reached a maximum after the peak of the electrical response of the photoreceptor, decayed during a prolonged stimulus, and returned to an undetectable level in the dark. Reduction of Cao reduced the amplitude of the aequorin response by a factor no greater than 3. Raising Cao increased the amplitude of the aequorin response. The aequorin response became smaller when membrane voltage was clamped to successively more positive values. These results indicate that the stimulus-induced rise of Cai may be due in part to a light-induced influx of Ca and in part to release of Ca from an intracellular store. Our findings are consistent with the hypothesis that a rise in Cai is a step in the sequence of events underlying light-adaptation in Limulus ventral photoreceptors. Aequorin was also injected into photoreceptors of Balanus. The aequorin responses were similar to those recorded from Limulus cells in all but two ways: (a) A large sustained aequorin luminescence was measured during a prolonged stimulus, and (b) removal of extracellular calcium reduced the aequorin response to an undetectable level. PMID:4155426

  4. Plasmodesmata dynamics are coordinated by intracellular signaling pathways

    PubMed Central

    Brunkard, Jacob O.; Runkel, Anne M.; Zambryski, Patricia C.

    2013-01-01

    Membrane-lined channels called plasmodesmata (PD) connect the cytoplasts of adjacent plant cells across the cell wall, permitting intercellular movement of small molecules, proteins, and RNA. Recent genetic screens for mutants with altered PD transport identified genes suggesting that chloroplasts play crucial roles in coordinating PD transport. Complementing this discovery, studies manipulating expression of PD-localized proteins imply that changes in PD transport strongly impact chloroplast biology. Ongoing efforts to find genes that control root and stomatal development reveal the critical role of PD in enforcing tissue patterning, and newly discovered PD-localized proteins show that PD influence development, intracellular signaling, and defense against pathogens. Together, these studies demonstrate that PD function and formation are tightly integrated with plant physiology. PMID:23978390

  5. Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium

    PubMed Central

    Melikov, Kamran; Hara, Ann; Yamoah, Kwabena; Zaitseva, Elena; Zaitsev, Eugene; Chernomordik, Leonid V.

    2015-01-01

    Understanding the mechanism of entry of cationic peptides such as nona-arginine (R9) into cells remains an important challenge to their use as efficient drug-delivery vehicles. At nanomolar to low micromolar R9 concentrations and at physiological temperature, peptide entry involves endocytosis. In contrast, at a concentration ≥10 μM, R9 induces a very effective non-endocytic entry pathway specific for cationic peptides. We found that a similar entry pathway is induced at 1–2 μM concentrations of R9 if peptide application is accompanied by a rapid temperature drop to 15°C. Both at physiological and at sub-physiological temperatures, this entry mechanism was inhibited by depletion of the intracellular ATP pool. Intriguingly, we found that R9 at 10–20 μM and 37°C induces repetitive spikes in intracellular Ca2+ concentration. This Ca2+ signalling correlated with the efficiency of the peptide entry. Pre-loading cells with the Ca2+ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) inhibited both Ca2+ spikes and peptide entry, suggesting that an increase in intracellular Ca2+ precedes and is required for peptide entry. One of the hallmarks of Ca2+ signalling is a transient cell-surface exposure of phosphatidylserine (PS), a lipid normally residing only in the inner leaflet of the plasma membrane. Blocking the accessible PS with the PS-binding domain of lactadherin strongly inhibited non-endocytic R9 entry, suggesting the importance of PS externalization in this process. To conclude, we uncovered a novel mechanistic link between calcium signalling and entry of cationic peptides. This finding will enhance our understanding of the properties of plasma membrane and guide development of future drug-delivery vehicles. PMID:26272944

  6. Activation of PAC1 Receptors in Rat Cerebellar Granule Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through N-Type Calcium Channels

    PubMed Central

    Basille-Dugay, Magali; Vaudry, Hubert; Fournier, Alain; Gonzalez, Bruno; Vaudry, David

    2013-01-01

    High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca2+]i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca2+ response. Preincubation of granule cells with the Ca2+ ATPase inhibitor, thapsigargin, or the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca2+]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca2+ channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca2+]i increase. Preincubation of granule neurons with the N-type Ca2+ channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca2+]i response, whereas the L-type Ca2+ channel blocker, nifedipine, and the P- and Q-type Ca2+ channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca2+]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca2+ channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes. PMID:23675369

  7. Activation of PAC1 Receptors in Rat Cerebellar Granule Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through N-Type Calcium Channels.

    PubMed

    Basille-Dugay, Magali; Vaudry, Hubert; Fournier, Alain; Gonzalez, Bruno; Vaudry, David

    2013-01-01

    High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca(2+)]i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca(2+) response. Preincubation of granule cells with the Ca(2+) ATPase inhibitor, thapsigargin, or the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca(2+)]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca(2+) channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca(2+)]i increase. Preincubation of granule neurons with the N-type Ca(2+) channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca(2+)]i response, whereas the L-type Ca(2+) channel blocker, nifedipine, and the P- and Q-type Ca(2+) channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca(2+)]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca(2+) channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes. PMID:23675369

  8. CatSper and the relationship of hyperactivated motility to intracellular calcium and pH kinetics in equine sperm.

    PubMed

    Loux, Shavahn C; Crawford, Kristin R; Ing, Nancy H; González-Fernández, Lauro; Macías-García, Beatriz; Love, Charles C; Varner, Dickson D; Velez, Isabel C; Choi, Young Ho; Hinrichs, Katrin

    2013-11-01

    In vitro fertilization does not occur readily in the horse. This may be related to failure of equine sperm to initiate hyperactivated motility, as treating with procaine to induce hyperactivation increases fertilization rates. In mice, hyperactivated motility requires a sperm-specific pH-gated calcium channel (CatSper); therefore, we investigated this channel in equine sperm. Motility was assessed by computer-assisted sperm motility analysis and changes in intracellular pH and calcium were assessed using fluorescent probes. Increasing intracellular pH induced a rise in intracellular calcium, which was inhibited by the known CatSper blocker mibefradil, supporting the presence of a pH-gated calcium channel, presumably CatSper. Hyperactivation was associated with moderately increased intracellular pH, but appeared inversely related to increases in intracellular calcium. In calcium-deficient medium, high-pH treatment induced motility loss, consistent with influx of sodium through open CatSper channels in the absence of environmental calcium. However, sperm treated with procaine in calcium-deficient medium both maintained motility and underwent hyperactivation, suggesting that procaine did not act via opening of the CatSper channel. CATSPER1 mRNA was identified in equine sperm by PCR, and CATSPER1 protein was localized to the principal piece on immunocytochemistry. Analysis of the predicted equine CATSPER1 protein revealed species-specific differences in structure in the pH-sensor region. We conclude that the CatSper channel is present in equine sperm but that the relationship of hyperactivated motility to calcium influx is weak. Procaine does not appear to act via CatSper in equine sperm, and its initial hyperactivating action is not dependent upon external calcium influx. PMID:24048572

  9. The Role of Intracellular Calcium for the Development and Treatment of Neuroblastoma

    PubMed Central

    Satheesh, Noothan Jyothi; Büsselberg, Dietrich

    2015-01-01

    Neuroblastoma is the second most common paediatric cancer. It develops from undifferentiated simpatico-adrenal lineage cells and is mostly sporadic; however, the aetiology behind the development of neuroblastoma is still not fully understood. Intracellular calcium ([Ca2+]i) is a secondary messenger which regulates numerous cellular processes and, therefore, its concentration is tightly regulated. This review focuses on the role of [Ca2+]i in differentiation, apoptosis and proliferation in neuroblastoma. It describes the mechanisms by which [Ca2+]i is regulated and how it modulates intracellular pathways. Furthermore, the importance of [Ca2+]i for the function of anti-cancer drugs is illuminated in this review as [Ca2+]i could be a target to improve the outcome of anti-cancer treatment in neuroblastoma. Overall, modulations of [Ca2+]i could be a key target to induce apoptosis in cancer cells leading to a more efficient and effective treatment of neuroblastoma. PMID:26010602

  10. Calcium/calmodulin-mediated signal network in plants

    NASA Technical Reports Server (NTRS)

    Yang, Tianbao; Poovaiah, B. W.

    2003-01-01

    Various extracellular stimuli elicit specific calcium signatures that can be recognized by different calcium sensors. Calmodulin, the predominant calcium receptor, is one of the best-characterized calcium sensors in eukaryotes. In recent years, completion of the Arabidopsis genome project and advances in functional genomics have helped to identify and characterize numerous calmodulin-binding proteins in plants. There are some similarities in Ca(2+)/calmodulin-mediated signaling in plants and animals. However, plants possess multiple calmodulin genes and many calmodulin target proteins, including unique protein kinases and transcription factors. Some of these proteins are likely to act as "hubs" during calcium signal transduction. Hence, a better understanding of the function of these calmodulin target proteins should help in deciphering the Ca(2+)/calmodulin-mediated signal network and its role in plant growth, development and response to environmental stimuli.