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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 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

  4. 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

  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 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

  3. 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

  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. 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.

  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. 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

  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 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

  1. 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

  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 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

  7. 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

  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. 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

  14. 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

  15. 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.

  16. 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

  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. 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

  15. 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

  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. 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.

  7. 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

  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 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

  10. 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

  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. 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

  19. 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

  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. 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

  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. 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

  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. 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

  19. [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

  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.

  11. Effects of intracellular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors

    PubMed Central

    1975-01-01

    The calcium sequestering agent, EGTA, was injected into Limulus ventral photoreceptors. Before injection, the inward membrane current induced by a long stimulus had a large initial transient which declined to a smaller plateau. Iontophoretic injection of EGTA tended to prevent the decline from transient to plateau. Before injection the plateau response was a nonlinear function of light intensity. After EGTA injection the response-intensity curves tended to become linear. Before injection, bright lights lowered the sensitivity as determined with subsequent test flashes. EGTA injection decreased the light-induced changes in sensitivity. Ca-EGTA buffers having different levels of free calcium were pressure-injected into ventral photoreceptors; the higher the level of free calcium, the lower the sensitivity measured after injection. The effects of inotophoretic injection of EGTA were not mimicked by injection or similar amounts of sulfate and the effects of pressure injection of EGTA buffer solutions were not mimicked by injection of similar volumes of pH buffer or mannitol. The data are consistent with the hypothesis that light adaptation is mediated by a rise of the intracellular free calcium concentration. PMID:810540

  12. Blockade of intracellular actions of calcium may protect against ischaemic damage to the gerbil brain.

    PubMed Central

    Asano, T.; Ikegaki, I.; Satoh, S.; Mochizuki, D.; Hidaka, H.; Suzuki, Y.; Shibuya, M.; Sugita, K.

    1991-01-01

    1. The brain cytoprotective effects of a putative calcium-associated protein kinase inhibitor, HA1077, as well as a calcium entry blocker nicardipine were evaluated in models of cerebral ischaemia in Mongolian gerbils. Morphological changes characterizing delayed neuronal death of selectively vulnerable CA1 pyramidal neurones in the hippocampus of the Mongolian gerbil brain occurred 7 days after transient bilateral occlusion of the common carotid arteries. 2. A single injection of HA1077 (1 and 3 mg kg-1, i.p.) 5 min after the occlusion led to a dose-dependent protection of the CA1 neurones. Repeated administrations of HA1077 (1 and 3 mg kg-1, i.p., twice daily for 7 days post-ischaemia) revealed an increase in the number of normal cells, compared to findings with a single administration. 3. In contrast to HA1077, nicardipine (0.3 and 1 mg kg-1, i.p.) did not reduce neuronal degeneration. 4. HA1077 did not interact with the ion channel within which MK-801 binds, as determined by receptor binding. 5. The calcium ionophore, A23187, caused a tonic contraction in canine cerebral arterial strips. HA1077, but not nicardipine, relaxed the A23187-induced contraction, concentration-dependently. 6. These results suggest that blockade of the intracellular actions of calcium may provide protection against ischaemic damage in the brain. Images Figure 1 PMID:1912980

  13. cADP-ribose formation by blood platelets is not responsible for intracellular calcium mobilization.

    PubMed Central

    Ohlmann, P; Leray, C; Ravanat, C; Hallia, A; Cassel, D; Cazenave, J P; Gachet, C

    1998-01-01

    Human platelet CD38 is a multifunctional ectoenzyme catalysing the synthesis and hydrolysis of cADP-ribose (cADPR), a recently identified calcium-mobilizing agent that acts independently of D-myo-inositol 1,4,5-trisphosphate and is known to be expressed by human platelets. The present work shows that ADP-ribosyl cyclase activity is exclusively a membrane activity, of which the major part is located in plasma membranes and a small part in internal membranes. In broken cells, cyclase activity was insensitive to the presence of calcium and was not modulated by agonists such as thrombin or ADP, whereas in intact cells thrombin increased cADPR formation by 30%, an effect due to fusion of granules with the plasma membrane. In order to assess the role of cADPR as a calcium-mobilizing agent, vesicles were prepared from internal membranes and loaded with 45CaCl2. These vesicles were efficiently discharged by IP3 in a dose-dependent manner, but were not responsive to cADPR or ryanodine in the presence or absence of calmodulin. Thus cADPR is unlikely to play a role in intracellular calcium release in human blood platelets. PMID:9531481

  14. Modulation of bovine sperm signalling pathways: correlation between intracellular parameters and sperm capacitation and acrosome exocytosis.

    PubMed

    Pons-Rejraji, Hanae; Bailey, Janice L; Leclerc, Pierre

    2009-01-01

    In the present study, the viability, intracellular pH (pHi), cAMP ([cAMP]i), calcium concentration and protein phosphotyrosine content were evaluated in relation to the acrosomal and capacitation status of freshly ejaculated bull spermatozoa. These parameters were evaluated before and after incubation with the capacitation inducer heparin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), the phosphotyrosyl-protein phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate, and hydrogen peroxide. The results obtained were integrated to address the physiological interactions between the different signalling events affecting sperm capacitation and acrosome reaction. As expected, heparin promoted the expression of the 'B' pattern of chlortetracycline binding, increased pHi, [cAMP]i and the phosphotyrosine content of sperm proteins. The effects of heparin were enhanced by IBMX. Both PAO and sodium orthovanadate stimulated protein phosphotyrosine content and acrosomal exocytosis, although only PAO affected pH, Ca2+ and cAMP levels. Intracellular pH was increased while both Ca2+ and [cAMP]i were decreased. Physiological concentrations of H2O2 increased [cAMP]i and promoted acrosomal exocytosis. A significant positive correlation was found between sperm capacitation, protein phosphotyrosine content and stored Ca2+ concentration, whereas the acrosome reaction was correlated with pHi and Ca2+ concentration. This study presents the first global analysis of the major elements individually described during sperm capacitation and acrosome reaction signalling pathways, supported by statistical correlations. PMID:19383258

  15. Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

    PubMed Central

    2013-01-01

    Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis. PMID:23890218

  16. TRPM2: a multifunctional ion channel for calcium signalling

    PubMed Central

    Sumoza-Toledo, Adriana; Penner, Reinhold

    2011-01-01

    The transient potential receptor melastatin-2 (TRPM2) channel has emerged as an important Ca2+ signalling mechanism in a variety of cells, contributing to cellular functions that include cytokine production, insulin release, cell motility and cell death. Its ability to respond to reactive oxygen species has made TRPM2 a potential therapeutic target for chronic inflammation, neurodegenerative diseases, and oxidative stress-related pathologies. TRPM2 is a non-selective, calcium (Ca2+)-permeable cation channel of the melastatin-related transient receptor potential (TRPM) ion channel subfamily. It is activated by intracellular adenosine diphosphate ribose (ADPR) through a diphosphoribose hydrolase domain in its C-terminus and regulated through a variety of factors, including synergistic facilitation by [Ca2+]i, cyclic ADPR, H2O2, NAADP, and negative feedback regulation by AMP and permeating protons (pH). In addition to its role mediating Ca2+ influx into the cells, TRPM2 can also function as a lysosomal Ca2+ release channel, contributing to cell death. The physiological and pathophysiological context of ROS-mediated events makes TRPM2 a promising target for the development of therapeutic tools of inflammatory and degenerative diseases. PMID:21135052

  17. Characterization of calcium signals provoked by lysophosphatidylinositol in human microvascular endothelial cells.

    PubMed

    Al Suleimani, Y M; Hiley, C R

    2016-01-01

    The lipid molecule, lysophosphatidylinositol (LPI), is hypothesised to form part of a novel lipid signalling system that involves the G protein-coupled receptor GPR55 and distinct intracellular signalling cascades in endothelial cells. This work aimed to study the possible mechanisms involved in LPI-evoked cytosolic Ca(2+) mobilization in human brain microvascular endothelial cells. Changes in intracellular Ca(2+) concentrations were measured using cell population Ca(2+) assay. LPI evoked biphasic elevation of intracellular calcium concentration, a rapid phase and a sustained phase. The rapid phase was attenuated by the inhibitor of PLC (U 73122), inhibitor of IP(3) receptors, 2-APB and the depletor of endoplasmic reticulum Ca(2+) store, thapsigargin. The sustained phase, on the other hand, was enhanced by U 73122 and abolished by the RhoA kinase inhibitor, Y-27632. In conclusion, the Ca(2+) signal evoked by LPI is characterised by a rapid phase of Ca(2+) release from the endoplasmic reticulum, and requires activation of the PLC-IP(3) signalling pathway. The sustained phase mainly depends on RhoA kinase activation. LPI acts as novel lipid signalling molecule in endothelial cells, and elevation of cytosolic Ca(2+) triggered by it may present an important intracellular message required in gene expression and controlling of vascular tone. PMID:26596318

  18. Calcium signals in T lymphocytes from old mice.

    PubMed

    Miller, R A

    1996-01-01

    Mitogen-induced increases in free calcium ion concentration ([Ca]i) are a key element of the process by which T lymphocytes are induced to proliferate and differentiate into effector cells. T cells from old mice exhibit lower average rises in calcium concentration than T cells from young donors when stimulated with either mitogenic lectins or antibodies to the CD3 chains of the antigen receptor. The decline with age in calcium signal generation is largely attributable to a shift from naïve to memory T cells, in that memory T cells, from mice of any age, are more resistant to mitogen-induced changes in [Ca]i. The decline in calcium signal generation is likely to be functionally significant, since T cells isolated on the basis of poor calcium signals show diminished ability to produce and to respond to the growth factor IL-2. Con A induces a transient increase in uptake of radiolabeled calcium from extracellular sources, and the extent of this increase declines with age. Alterations in production of inositol tris-phosphate (IP3) seem not to contribute to age-related changes in calcium signal generation. T cells from old mice, and memory T cells from mice of any age, are relatively resistant to increases in [Ca]i even when these are induced by receptor-independent stimuli such as the calcium ionophore ionomycin. The ionomycin-resistance of memory T cells suggests that these cells may have an augmented ability to buffer changes in [Ca]i, perhaps by increased activity of the ATP-dependent plasma membrane calcium pump. It seems likely that age-related declines in calcium signal generation contribute to the functional immunodeficiency of old age. PMID:8761335

  19. Role of calcium in polycystic kidney disease: From signaling to pathology.

    PubMed

    Mangolini, Alessandra; de Stephanis, Lucia; Aguiari, Gianluca

    2016-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited monogenic kidney disease. Characterized by the development and growth of cysts that cause progressive kidney enlargement, it ultimately leads to end-stage renal disease. Approximately 85% of ADPKD cases are caused by mutations in the PKD1 gene, while mutations in the PKD2 gene account for the remaining 15% of cases. The PKD1 gene encodes for polycystin-1 (PC1), a large multi-functional membrane receptor protein able to regulate ion channel complexes, whereas polycystin-2 (PC2), encoded by the PKD2 gene, is an integral membrane protein that functions as a calcium-permeable cation channel, located mainly in the endoplasmic reticulum (ER). In the primary cilia of the epithelial cells, PC1 interacts with PC2 to form a polycystin complex that acts as a mechanosensor, regulating signaling pathways involved in the differentiation of kidney tubular epithelial cells. Despite progress in understanding the function of these proteins, the molecular mechanisms associated with the pathogenesis of ADPKD remain unclear. In this review we discuss how an imbalance between functional PC1 and PC2 proteins may disrupt calcium channel activities in the cilium, plasma membrane and ER, thereby altering intracellular calcium signaling and leading to the aberrant cell proliferation and apoptosis associated with the development and growth of renal cysts. Research in this field could lead to the discovery of new molecules able to rebalance intracellular calcium, thereby normalizing cell proliferation and reducing kidney cyst progression. PMID:26788466

  20. Cytosolic calcium homeostasis in fungi: Roles of plasma membrane transport and intracellular sequestration of calcium

    SciTech Connect

    Miller, A.J.; Vogg, G.; Sanders, D. )

    1990-12-01

    Cytosolic free calcium ((Ca{sup 2+}){sub c}) has been measured in the mycelial fungus Neurospora crassa with Ca{sup 2+} - selective microelectrodes. The mean value of (Ca{sup 2+}){sub c} is 92 {plus minus} 15 nM and it is insensitive to external pH values between 5.8 and 8.4. Simultaneous measurement of membrane potential enables the electrochemical potential difference for Ca{sup 2+} across the plasma membrane to be estimated as about {minus}60 kJmol{sup {minus}1} - a value that cannot be sustained either by a simple Ca{sup 2+} - ATPase, or, in alkaline conditions, by straightforward H{sup +}/Ca{sup 2+} exchange with a stoichiometric ratio of {lt}5 H{sup +}/Ca{sup 2+}. The authors propose that the most likely alternative mechanism of Ca{sup 2+} efflux is ATP-driven H{sup +}/Ca{sup 2+} exchange, with a stoichiometric ratio of at least 2 H{sup +}/Ca{sup 2+}. The increase in (Ca{sup 2+}){sub c} in the presence of CN{sup {minus}} at pH 8.4 is compared with {sup 45}Ca{sup 2+} influx under the same conditions. The proportion of entering Ca{sup 2+} remaining free in the cytosol is only 8 {times} 10{sup {minus}5}, and since the concentration of available chelation sites on Ca{sup 2+} binding proteins is unlikely to exceed 100 {mu}M, a major role for the fungal vacuole in short-term Ca{sup 2+} homeostasis is indicated. This notion is supported by the observation that cytosolic Ca{sup 2+} homeostasis is disrupted by a protonophore, which rapidly abolishes the driving force for Ca{sup 2+} uptake into fungal vacuoles.

  1. Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

    PubMed Central

    Hong, Jin Hee; Min, Cheol Hong; Jeong, Byeongha; Kojiya, Tomoyoshi; Morioka, Eri; Nagai, Takeharu; Ikeda, Masayuki; Lee, Kyoung J.

    2010-01-01

    Background Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of “Ca2+ spikes” (i.e., [Ca2+]c transients having a bandwidth of 10∼100 seconds) in SCN neurons, but it is unclear if these SCN Ca2+ spikes are related to the slow circadian rhythms. Methodology/Principal Findings We addressed this issue based on a Ca2+ indicator dye (fluo-4) and a protein Ca2+ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca2+ spikes in 18% of rat SCN cells in acute brain slices, but the Ca2+ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca2+ spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca2+ spikes was increased to 13∼14%. Conclusions/Significance Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca2+ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca2+ spiking activity is caused by the Ca2+ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca2+]c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca2+ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca2+ spikes in the function of SCN. PMID:20224788

  2. Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development

    NASA Astrophysics Data System (ADS)

    Sørhus, Elin; Incardona, John P.; Karlsen, Ørjan; Linbo, Tiffany; Sørensen, Lisbet; Nordtug, Trond; van der Meeren, Terje; Thorsen, Anders; Thorbjørnsen, Maja; Jentoft, Sissel; Edvardsen, Rolf B.; Meier, Sonnich

    2016-08-01

    Recent studies have shown that crude oil exposure affects cardiac development in fish by disrupting excitation-contraction (EC) coupling. We previously found that eggs of Atlantic haddock (Melanogrammus aeglefinus) bind dispersed oil droplets, potentially leading to more profound toxic effects from uptake of polycyclic aromatic hydrocarbons (PAHs). Using lower concentrations of dispersed crude oil (0.7–7 μg/L ∑PAH), here we exposed a broader range of developmental stages over both short and prolonged durations. We quantified effects on cardiac function and morphogenesis, characterized novel craniofacial defects, and examined the expression of genes encoding potential targets underlying cardiac and craniofacial defects. Because of oil droplet binding, a 24-hr exposure was sufficient to create severe cardiac and craniofacial abnormalities. The specific nature of the craniofacial abnormalities suggests that crude oil may target common craniofacial and cardiac precursor cells either directly or indirectly by affecting ion channels and intracellular calcium in particular. Furthermore, down-regulation of genes encoding specific components of the EC coupling machinery suggests that crude oil disrupts excitation-transcription coupling or normal feedback regulation of ion channels blocked by PAHs. These data support a unifying hypothesis whereby depletion of intracellular calcium pools by crude oil-derived PAHs disrupts several pathways critical for organogenesis in fish.

  3. Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development.

    PubMed

    Sørhus, Elin; Incardona, John P; Karlsen, Ørjan; Linbo, Tiffany; Sørensen, Lisbet; Nordtug, Trond; van der Meeren, Terje; Thorsen, Anders; Thorbjørnsen, Maja; Jentoft, Sissel; Edvardsen, Rolf B; Meier, Sonnich

    2016-01-01

    Recent studies have shown that crude oil exposure affects cardiac development in fish by disrupting excitation-contraction (EC) coupling. We previously found that eggs of Atlantic haddock (Melanogrammus aeglefinus) bind dispersed oil droplets, potentially leading to more profound toxic effects from uptake of polycyclic aromatic hydrocarbons (PAHs). Using lower concentrations of dispersed crude oil (0.7-7 μg/L ∑PAH), here we exposed a broader range of developmental stages over both short and prolonged durations. We quantified effects on cardiac function and morphogenesis, characterized novel craniofacial defects, and examined the expression of genes encoding potential targets underlying cardiac and craniofacial defects. Because of oil droplet binding, a 24-hr exposure was sufficient to create severe cardiac and craniofacial abnormalities. The specific nature of the craniofacial abnormalities suggests that crude oil may target common craniofacial and cardiac precursor cells either directly or indirectly by affecting ion channels and intracellular calcium in particular. Furthermore, down-regulation of genes encoding specific components of the EC coupling machinery suggests that crude oil disrupts excitation-transcription coupling or normal feedback regulation of ion channels blocked by PAHs. These data support a unifying hypothesis whereby depletion of intracellular calcium pools by crude oil-derived PAHs disrupts several pathways critical for organogenesis in fish. PMID:27506155

  4. Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development

    PubMed Central

    Sørhus, Elin; Incardona, John P.; Karlsen, Ørjan; Linbo, Tiffany; Sørensen, Lisbet; Nordtug, Trond; van der Meeren, Terje; Thorsen, Anders; Thorbjørnsen, Maja; Jentoft, Sissel; Edvardsen, Rolf B.; Meier, Sonnich

    2016-01-01

    Recent studies have shown that crude oil exposure affects cardiac development in fish by disrupting excitation-contraction (EC) coupling. We previously found that eggs of Atlantic haddock (Melanogrammus aeglefinus) bind dispersed oil droplets, potentially leading to more profound toxic effects from uptake of polycyclic aromatic hydrocarbons (PAHs). Using lower concentrations of dispersed crude oil (0.7–7 μg/L ∑PAH), here we exposed a broader range of developmental stages over both short and prolonged durations. We quantified effects on cardiac function and morphogenesis, characterized novel craniofacial defects, and examined the expression of genes encoding potential targets underlying cardiac and craniofacial defects. Because of oil droplet binding, a 24-hr exposure was sufficient to create severe cardiac and craniofacial abnormalities. The specific nature of the craniofacial abnormalities suggests that crude oil may target common craniofacial and cardiac precursor cells either directly or indirectly by affecting ion channels and intracellular calcium in particular. Furthermore, down-regulation of genes encoding specific components of the EC coupling machinery suggests that crude oil disrupts excitation-transcription coupling or normal feedback regulation of ion channels blocked by PAHs. These data support a unifying hypothesis whereby depletion of intracellular calcium pools by crude oil-derived PAHs disrupts several pathways critical for organogenesis in fish. PMID:27506155

  5. NADPH oxidase-2 inhibition restores contractility and intracellular calcium handling and reduces arrhythmogenicity in dystrophic cardiomyopathy

    PubMed Central

    Gonzalez, Daniel R.; Treuer, Adriana V.; Lamirault, Guillaume; Mayo, Vera; Cao, Yenong; Dulce, Raul A.

    2014-01-01

    Duchenne muscular dystrophy may affect cardiac muscle, producing a dystrophic cardiomyopathy in humans and the mdx mouse. We tested the hypothesis that oxidative stress participates in disrupting calcium handling and contractility in the mdx mouse with established cardiomyopathy. We found increased expression (fivefold) of the NADPH oxidase (NOX) 2 in the mdx hearts compared with wild type, along with increased superoxide production. Next, we tested the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility was decreased in mdx myocytes compared with wild type, and this was restored toward normal by pretreating with apocynin. In addition, the amplitude of evoked intracellular Ca2+ concentration transients that was diminished in mdx myocytes was also restored with NOX2 inhibition. Total sarcoplasmic reticulum (SR) Ca2+ content was reduced in mdx hearts and normalized by apocynin treatment. Additionally, NOX2 inhibition decreased the production of spontaneous diastolic calcium release events and decreased the SR calcium leak in mdx myocytes. In addition, nitric oxide (NO) synthase 1 (NOS-1) expression was increased eightfold in mdx hearts compared with wild type. Nevertheless, cardiac NO production was reduced. To test whether this paradox implied NOS-1 uncoupling, we treated cardiac myocytes with exogenous tetrahydrobioterin, along with the NOX inhibitor VAS2870. These agents restored NO production and phospholamban phosphorylation in mdx toward normal. Together, these results demonstrate that, in mdx hearts, NOX2 inhibition improves the SR calcium handling and contractility, partially by recoupling NOS-1. These findings reveal a new layer of nitroso-redox imbalance in dystrophic cardiomyopathy. PMID:25015966

  6. ER functions of oncogenes and tumor suppressors: Modulators of intracellular Ca(2+) signaling.

    PubMed

    Bittremieux, Mart; Parys, Jan B; Pinton, Paolo; Bultynck, Geert

    2016-06-01

    Intracellular Ca(2+) signals that arise from the endoplasmic reticulum (ER), the major intracellular Ca(2+)-storage organelle, impact several mitochondrial functions and dictate cell survival and cell death processes. Furthermore, alterations in Ca(2+) signaling in cancer cells promote survival and establish a high tolerance towards cell stress and damage, so that the on-going oncogenic stress does not result in the activation of cell death. Over the last years, the mechanisms underlying these oncogenic alterations in Ca(2+) signaling have started to emerge. An important aspect of this is the identification of several major oncogenes, including Bcl-2, Bcl-XL, Mcl-1, PKB/Akt, and Ras, and tumor suppressors, such as p53, PTEN, PML, BRCA1, and Beclin 1, as direct and critical regulators of Ca(2+)-transport systems located at the ER membranes, including IP3 receptors and SERCA Ca(2+) pumps. In this way, these proteins execute part of their function by controlling the ER-mitochondrial Ca(2+) fluxes, favoring either survival (oncogenes) or cell death (tumor suppressors). Oncogenic mutations, gene deletions or amplifications alter the expression and/or function of these proteins, thereby changing the delicate balance between oncogenes and tumor suppressors, impacting oncogenesis and favoring malignant cell function and behavior. In this review, we provided an integrated overview of the impact of the major oncogenes and tumor suppressors, often altered in cancer cells, on Ca(2+) signaling from the ER Ca(2+) stores. 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:26772784

  7. Agouti regulation of intracellular calcium: Role in the insulin resistance of viable yellow mice

    SciTech Connect

    Zemel, M.B.; Kim, J.H.; Woychik, R.P.; Michaud, E.J.; Hadwell, S.H.; Patel, I.R.; Wilkison, W.O.

    1995-05-23

    Several dominant mutations at the agouti locus in the mouse cause a syndrome of marked obesity, hyperinsulinemia, and insulin resistance. Although it is known that the agouti gene is expressed in an ectopic manner in these mutants, the precise mechanism by which the agouti gene product mediates these effects is unclear. Since intracellular Ca{sup 2+} is believed to play a role in mediating insulin action and dysregulation of Ca{sup 2+} flux is observed in diabetic animals and humans, we examined the status of intracellular Ca{sup 2+} in mice carrying the dominant agouti allele, viable yellow (A{sup vy}). We show here that in mice carrying this mutation, the intracellular free calcium concentration ([Ca{sup 2+}]{sub i}) is elevated in skeletal muscle, and the degree of elevation is closely correlated with the degree to which the mutant traits are expressed in individual animals. Moreover, we demonstrate that the agouti gene product is capable of inducing increased [Ca{sup 2+}]{sub i} in cultured and freshly isolated skeletal muscle myocytes from wild-type mice. Based on these findings, we present a model in which we propose that the agouti polypeptide promotes insulin resistance in mutant animals through its ability to increase [Ca{sup 2+}]{sub i}. 36 refs., 3 figs., 2 tabs.

  8. Intracellular calcium waves in bone cell networks under single cell nanoindentation.

    PubMed

    Guo, X Edward; Takai, Erica; Jiang, Xingyu; Xu, Qiaobing; Whitesides, George M; Yardley, James T; Hung, Clark T; Chow, Eugene M; Hantschel, Thomas; Costa, Kevin D

    2006-09-01

    In this study, bone cells were successfully cultured into a micropatterned network with dimensions close to that of in vivo osteocyte networks using microcontact printing and self-assembled monolyers (SAMs). The optimal geometric parameters for the formation of these networks were determined in terms of circle diameters and line widths. Bone cells patterned in these networks were also able to form gap junctions with each other, shown by immunofluorescent staining for the gap junction protein connexin 43, as well as the transfer of gap-junction permeable calcein-AM dye. We have demonstrated for the first time, that the intracellular calcium response of a single bone cell indented in this bone cell network, can be transmitted to neighboring bone cells through multiple calcium waves. Furthermore, the propagation of these calcium waves was diminished with increased cell separation distance. Thus, this study provides new experimental data that support the idea of osteocyte network memory of mechanical loading similar to memory in neural networks. PMID:17263256

  9. Combined analysis of intracellular calcium with dual excitation fluorescence photometry and imaging

    NASA Astrophysics Data System (ADS)

    Uttenweiler, Dietmar; Wojciechowski, Reinhold; Makabe, Makoto; Veigel, Claudia; Fink, Rainer H.

    1995-10-01

    We have developed an integrated microscopy system combining fast dual-excitation fluorescence photometry and digital image analysis with high spatial resolution, based mainly on standard components. With the combination of these well-established techniques in one setup it is possible to monitor intracellular calcium with both sufficiently high temporal and high spatial resolution on the same preparation for many biological applications. Our system consists of a commercially available dual-excitation photometric system, an attached ICCD camera, and a frame grabber board. With this integrated setup one can easily switch between the fast photometric mode and the imaging mode. We used the system to record Fura-2 calcium images (340/380 nm ratios), which were correlated with the faster spot measurements and were analyzed by means of image processing. As an example for its application we reconstructed caffeine-induced calcium transient released from the sarcoplasmic reticulum of isolated and permeabilized skeletal muscle fiber preparations. Such a combined technique will also be important for cellular studies using other fluorescence indicators. Additionally, the described system has an external trigger facility that enables combination with other cell physiological methods, e.g., electrophysiological techniques.

  10. Connexin 43 hemichannels and intracellular signaling in bone cells

    PubMed Central

    Plotkin, Lilian I.

    2014-01-01

    Cell function and survival are controlled by intracellular signals, and modulated by surrounding cells and the extracellular environment. Connexin channels participate in these processes by mediating cell-to-cell communication. In bone cells, gap junction channels were detected in the early 1970s, and are present among bone resorbing osteoclasts, bone forming osteoblasts, and osteocytes - mature osteoblasts embedded in the mineralized matrix. These channels are composed mainly by Cx43, although the expression of other connexins (45, 46, and 37) has also been reported. It is now believed that undocked Cx43 hemichannels (connexons) formed in unopposed cell membranes facing the extracellular environment participate in the interaction of bone cells with the extracellular environment, and in their communication with neighboring cells. Thus, we and others demonstrated the presence of active hemichannels in osteoblastic and osteocytic cells. These hemichannels open in response to pharmacological and mechanical stimulation. In particular, preservation of the viability of osteoblasts and osteocytes by the anti-osteoporotic drugs bisphosphonates depends on Cx43 expression in vitro and in vivo, and is mediated by undocked hemichannels. Cx43 hemichannels are also required for the release of prostaglandins and ATP by osteocytes, and for cell survival induced by mechanical stimulation in vitro. Moreover, they are required for the anti-apoptotic effect of parathyroid hormone in osteoblastic cells. This review summarizes the current knowledge on the presence and function of undocked connexons, and the role of hemichannel regulation for the maintenance of bone cell viability and, potentially, bone health. PMID:24772090

  11. The role of calcium in hypoxia-induced signal transduction and gene expression.

    PubMed

    Seta, Karen A; Yuan, Yong; Spicer, Zachary; Lu, Gang; Bedard, James; Ferguson, Tsuneo K; Pathrose, Peterson; Cole-Strauss, Allyson; Kaufhold, Alexa; Millhorn, David E

    2004-01-01

    Mammalian cells require a constant supply of oxygen in order to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. Sophisticated mechanisms have therefore evolved which allow cells to respond and adapt to hypoxia. Specialized oxygen-sensing cells have the ability to detect changes in oxygen tension and transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in a wide variety of different organisms. An increase in intracellular calcium levels is a primary response of many cell types to hypoxia/ischemia. The response to hypoxia is complex and involves the regulation of multiple signaling pathways and coordinated expression of perhaps hundreds of genes. This review discusses the role of calcium in hypoxia-induced regulation of signal transduction pathways and gene expression. An understanding of the molecular events initiated by changes in intracellular calcium will lead to the development of therapeutic approaches toward the treatment of hypoxic/ischemic diseases and tumors. PMID:15261489

  12. Probing the intracellular calcium sensitivity of transmitter release during synaptic facilitation.

    PubMed

    Felmy, Felix; Neher, Erwin; Schneggenburger, Ralf

    2003-03-01

    In nerve terminals, residual Ca(2+) remaining from previous activity can cause facilitation of transmitter release by a mechanism that is still under debate. Here we show that the intracellular Ca(2+) sensitivity of transmitter release at the calyx of Held is largely unchanged during facilitation, which leaves an increased microdomain Ca(2+) signal as a possible mechanism for facilitation. We measured the Ca(2+) dependencies of facilitation, as well as of transmitter release, to estimate the required increment in microdomain Ca(2+). These measurements show that linear summation of residual and microdomain Ca(2+) accounts for only 30% of the observed facilitation. However, a small degree of supralinearity in the summation of intracellular Ca(2+) signals, which might be caused by saturation of cytosolic Ca(2+) buffer(s), is sufficient to explain facilitation at this CNS synapse. PMID:12628170

  13. Cellular Architecture Regulates Collective Calcium Signaling and Cell Contractility

    PubMed Central

    Hoying, James B.; Deymier, Pierre A.; Zhang, Donna D.; Wong, Pak Kin

    2016-01-01

    A key feature of multicellular systems is the ability of cells to function collectively in response to external stimuli. However, the mechanisms of intercellular cell signaling and their functional implications in diverse vascular structures are poorly understood. Using a combination of computational modeling and plasma lithography micropatterning, we investigate the roles of structural arrangement of endothelial cells in collective calcium signaling and cell contractility. Under histamine stimulation, endothelial cells in self-assembled and microengineered networks, but not individual cells and monolayers, exhibit calcium oscillations. Micropatterning, pharmacological inhibition, and computational modeling reveal that the calcium oscillation depends on the number of neighboring cells coupled via gap junctional intercellular communication, providing a mechanistic basis of the architecture-dependent calcium signaling. Furthermore, the calcium oscillation attenuates the histamine-induced cytoskeletal reorganization and cell contraction, resulting in differential cell responses in an architecture-dependent manner. Taken together, our results suggest that endothelial cells can sense and respond to chemical stimuli according to the vascular architecture via collective calcium signaling. PMID:27196735

  14. Lipid body accumulation alters calcium signaling dynamics in immune cells.

    PubMed

    Greineisen, William E; Speck, Mark; Shimoda, Lori M N; Sung, Carl; Phan, Nolwenn; Maaetoft-Udsen, Kristina; Stokes, Alexander J; Turner, Helen

    2014-09-01

    There is well-established variability in the numbers of lipid bodies (LB) in macrophages, eosinophils, and neutrophils. Similarly to the steatosis observed in adipocytes and hepatocytes during hyperinsulinemia and nutrient overload, immune cell LB hyper-accumulate in response to bacterial and parasitic infection and inflammatory presentations. Recently we described that hyperinsulinemia, both in vitro and in vivo, drives steatosis and phenotypic changes in primary and transformed mast cells and basophils. LB reach high numbers in these steatotic cytosols, and here we propose that they could dramatically impact the transcytoplasmic signaling pathways. We compared calcium release and influx responses at the population and single cell level in normal and steatotic model mast cells. At the population level, all aspects of FcɛRI-dependent calcium mobilization, as well as activation of calcium-dependent downstream signaling targets such as NFATC1 phosphorylation are suppressed. At the single cell level, we demonstrate that LB are both sources and sinks of calcium following FcɛRI cross-linking. Unbiased analysis of the impact of the presence of LB on the rate of trans-cytoplasmic calcium signals suggest that LB enrichment accelerates calcium propagation, which may reflect a Bernoulli effect. LB abundance thus impacts this fundamental signaling pathway and its downstream targets. PMID:25016314

  15. Lipid body accumulation alters calcium signaling dynamics in immune cells

    PubMed Central

    Greineisen, William E.; Speck, Mark; Shimoda, Lori M.N.; Sung, Carl; Phan, Nolwenn; Maaetoft-Udsen, Kristina; Stokes, Alexander J.; Turner, Helen

    2014-01-01

    Summary There is well-established variability in the numbers of lipid bodies (LB) in macrophages, eosinophils, and neutrophils. Similarly to the steatosis observed in adipocytes and hepatocytes during hyperinsulinemia and nutrient overload, immune cell LB hyper-accumulate in response to bacterial and parasitic infection and inflammatory presentations. Recently we described that hyperinsulinemia, both in vitro and in vivo, drives steatosis and phenotypic changes in primary and transformed mast cells and basophils. LB reach high numbers in these steatotic cytosols, and here we propose that they could dramatically impact the transcytoplasmic signaling pathways. We compared calcium release and influx responses at the population and single cell level in normal and steatotic model mast cells. At the population level, all aspects of FcεRI-dependent calcium mobilization, as well as activation of calcium-dependent downstream signalling targets such as NFATC1 phosphorylation are suppressed. At the single cell level, we demonstrate that LB are both sources and sinks of calcium following FcεRI cross-linking. Unbiased analysis of the impact of the presence of LB on the rate of trans-cytoplasmic calcium signals suggest that LB enrichment accelerates calcium propagation, which may reflect a Bernoulli effect. LB abundance thus impacts this fundamental signalling pathway and its downstream targets. PMID:25016314

  16. Nanoparticle PEBBLE Sensors for Quantitative Nanomolar Imaging of Intracellular Free Calcium Ions

    PubMed Central

    Si, Di; Epstein, Tamir; Lee, Yong-Eun Koo; Kopelman, Raoul

    2011-01-01

    Ca2+ is a universal second messenger and plays a major role in intracellular signaling, metabolism and a wide range of cellular processes. To date, one of the most successful approaches for intracellular Ca2+ measurement involves introduction of optically sensitive Ca2+ indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca2+ indicators for intracellular sensing and imaging has several limitations, such as nonratiometric measurement for the most sensitive indicators, cytotoxicity of the indicators, interference from non-specific binding caused by cellular biomacromolecules, challenging calibration and unwanted sequestration of the indicator molecules. These problems are minimized when the Ca2+ indicators are encapsulated inside porous and inert polyacrylamide nanoparticles. We present PEBBLE nanosensors encapsulated with rhodamine based Ca2+ fluorescence indicators. The here presented rhod-2 containing PEBBLEs show a stable sensing range at near-neutral pH (pH 6–9). Due to the protection of the PEBBLE matrix, the interference of protein non-specific binding to the indicator is minimal. The rhod-2 PEBBLEs give a nanomolar dynamic sensing range for both in-solution (Kd = 478 nM) and intracellular (Kd = 293 nM) measurements. These nanosensors are a useful quantitative tool for the measurement and imaging of the cytosolic nanomolar free Ca2+ levels. PMID:22122409

  17. Dopamine-induced oscillations of the pyloric pacemaker neuron rely on release of calcium from intracellular stores.

    PubMed

    Kadiri, Lolahon R; Kwan, Alex C; Webb, Watt W; Harris-Warrick, Ronald M

    2011-09-01

    Endogenously bursting neurons play central roles in many aspects of nervous system function, ranging from motor control to perception. The properties and bursting patterns generated by these neurons are subject to neuromodulation, which can alter cycle frequency and amplitude by modifying the properties of the neuron's ionic currents. In the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, the anterior burster (AB) neuron is a conditional oscillator in the presence of dopamine (DA) and other neuromodulators and serves as the pacemaker to drive rhythmic output from the pyloric network. We analyzed the mechanisms by which DA evokes bursting in the AB neuron. Previous work showed that DA-evoked bursting is critically dependent on external calcium (Harris-Warrick RM, Flamm RE. J Neurosci 7: 2113-2128, 1987). Using two-photon microscopy and calcium imaging, we show that DA evokes the release of calcium from intracellular stores well before the emergence of voltage oscillations. When this release from intracellular stores is blocked by antagonists of ryanodine or inositol trisphosphate (IP(3)) receptor channels, DA fails to evoke AB bursting. We further demonstrate that DA enhances the calcium-activated inward current, I(CAN), despite the fact that it significantly reduces voltage-activated calcium currents. This suggests that DA-induced release of calcium from intracellular stores activates I(CAN), which provides a depolarizing ramp current that underlies endogenous bursting in the AB neuron. PMID:21676929

  18. Antithrombotic activities of ferulic acid via intracellular cyclic nucleotide signaling.

    PubMed

    Hong, Qian; Ma, Zeng-Chun; Huang, Hao; Wang, Yu-Guang; Tan, Hong-Ling; Xiao, Cheng-Rong; Liang, Qian-De; Zhang, Han-Ting; Gao, Yue

    2016-04-15

    Ferulic acid (FA) produces protective effects against cardiovascular dysfunctions. However, the mechanisms of FA is still not known. Here we examined the antithrombotic effects of FA and its potential mechanisms. Anticoagulation assays and platelet aggregation was evaluated in vitro and in vivo. Thromboxane B2 (TXB2), cyclic adenosine monophosphate(cAMP), and cyclic guanosine monophosphate (cGMP) was determined using enzyme immunoassay kits. Nitric oxide (NO) production was measured using the Griess reaction. Protein expression was detected by Western blotting analysis. Oral administration of FA prevented death caused by pulmonary thrombosis and prolonged the tail bleeding and clotting time in mice,while, it did not alter the coagulation parameters, including the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). In addition, FA (50-200µM) dose-dependently inhibited platelet aggregation induced by various platelet agonists, including adenosine diphosphate (ADP), thrombin, collagen, arachidonic acid (AA), and U46619. Further, FA attenuated intracellular Ca(2)(+) mobilization and TXB2 production induced by the platelet agonists. FA increased the levels of cAMP and cGMP and phosphorylated vasodilator-stimulated phosphoprotein (VASP) while decreased phospho-MAPK (mitogen-activated protein kinase) and phosphodiesterase (PDE) in washed rat platelets, VASP is a substrate of cyclic nucleotide and PDE is an enzyme family responsible for hydrolysis of cAMP/cGMP. These results suggest that antithrombotic activities of FA may be regulated by inhibition of platelet aggregation, rather than through inhibiting the release of thromboplastin or formation of thrombin. The mechanism of this action may involve activation of cAMP and cGMP signaling. PMID:26948317

  19. IMPACT OF CELLULAR MICROENVIRONMENT AND MECHANICAL PERTURBATION ON CALCIUM SIGNALLING IN MENISCUS FIBROCHONDROCYTES

    PubMed Central

    Han, W.M.; Heo, S-J.; Driscoll, T.P.; Boggs, M.E.; Duncan, R.L.; Mauck, R.L.; Elliott, D.M.

    2015-01-01

    Mechanical signals regulate a multitude of cell functions and ultimately govern fibrous tissue growth, maintenance and repair. Such mechanotransduction processes often involve modulation of intracellular calcium concentration ([Ca2+]i). However, most studies interrogate these responses in cells in simplified culture systems, thereby removing potentially important inputs from the native extracellular microenvironment. The objective of this study was to test the hypothesis that the intracellular calcium response of meniscus fibrochondrocytes (MFCs) is dependent on both the microenvironmental context in which this perturbation is applied and on the tensile deformation. Using a custom micro-mechanical tester mounted on a confocal microscope, intracellular calcium activity in MFCs in response to incremental tissue strains (0, 3, 6 and 9 %) was monitored in situ (i.e., in the native tissues) on MFC-seeded aligned scaffolds and MFC-seeded silicone membranes. The [Ca2+]i regulation by MFCs within the native meniscus tissue microenvironment was considerably different from [Ca2+]i regulation by MFCs on either aligned nanofibrous scaffolds or flat silicone membranes. Additionally, increasing levels of tensile deformation resulted in a greater number of responding cells, both in situ and in vitro, while having no effects on temporal characteristics of [Ca2+]i signalling. Collectively, these findings have significant implications for mechanobiology of load-bearing fibrous tissues and their responses to injury and degeneration. In addition, from a tissue engineering perspective, the findings establish cellular benchmarks for maturing engineered constructs, where native tissue-like calcium mechano-regulation may be an important outcome parameter to achieve mechanical functionality comparable to native tissue. PMID:24908425

  20. Electrical slow waves in the mouse oviduct are dependent on extracellular and intracellular calcium sources

    PubMed Central

    Dixon, Rose Ellen; Britton, Fiona C.; Baker, Salah A.; Hennig, Grant W.; Rollings, Christina M.; Sanders, Kenton M.

    2011-01-01

    Spontaneous contractions of the myosalpinx are critical for oocyte transport along the oviduct. Slow waves, the electrical events that underlie myosalpinx contractions, are generated by a specialized network of pacemaker cells called oviduct interstitial cells of Cajal (ICC-OVI). The ionic basis of oviduct pacemaker activity is unknown. Intracellular recordings and Ca2+ imaging were performed to examine the role of extracellular and intracellular Ca2+ sources in slow wave generation. RT-PCR was performed to determine the transcriptional expression of Ca2+ channels. Molecular studies revealed most isoforms of L- and T-type calcium channels (Cav1.2,1.3,1.4,3.1,3.2,3.3) were expressed in myosalpinx. Reduction of extracellular Ca2+ concentration ([Ca2+]o) resulted in the abolition of slow waves and myosalpinx contractions without significantly affecting resting membrane potential (RMP). Spontaneous Ca2+ waves spread through ICC-OVI cells at a similar frequency to slow waves and were inhibited by reduced [Ca2+]o. Nifedipine depolarized RMP and inhibited slow waves; however, pacemaker activity returned when the membrane was repolarized with reduced extracellular K+ concentration ([K+]o). Ni2+ also depolarized RMP but failed to block slow waves. The importance of ryanodine and inositol 1,4,5 trisphosphate-sensitive stores were examined using ryanodine, tetracaine, caffeine, and 2-aminoethyl diphenylborinate. Results suggest that although both stores are involved in regulation of slow wave frequency, neither are exclusively essential. The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor cyclopiazonic acid inhibited pacemaker activity and Ca2+ waves suggesting that a functional SERCA pump is necessary for pacemaker activity. In conclusion, results from this study suggest that slow wave generation in the oviduct is voltage dependent, occurs in a membrane potential window, and is dependent on extracellular calcium and functional SERCA pumps. PMID:21881003

  1. The effects of thermal stimuli on intracellular calcium change and histamine releases in rat basophilic leukemia mast cells

    NASA Astrophysics Data System (ADS)

    Wu, Zu-Hui; Zhu, Dan; Chen, Ji-Yao; Zhou, Lu-Wei

    2012-05-01

    The effects of thermal stimuli on rat basophilic leukemia mast cells were studied. The cells in calcium-contained or calcium-free buffers were thermally stimulated in the temperature range of 25-60 °C. The corresponding calcium ion concentration in cells [Ca2+]i as well as the released histamine from cells was measured with fluorescence staining methods. The ruthenium red (RR), a block of membrane calcium channels (transient receptor potential family V (TRPV)), was used in experiments. Under the stimulus of 25-50 °C, no significant difference on [Ca2+]i was found between these three groups of the cells in calcium-contained buffer without or with RR and cells in calcium-free saline, indicating that the increased calcium in cytosol did not result from the extracellular buffer but came from the intracellular calcium stores. The [Ca2+]i continuously increased under the temperature of 50-60 °C, but the RR and calcium-free saline can obviously diminish the [Ca2+]i increase at these high temperatures, reflecting that the opening of the TRPV2 channels leads to a calcium influx resulting in the [Ca2+]i increment. The histamine release also became significant in these cases. Since the released histamine is a well-known mediator for the microcirculation promotion, the histamine release from mast cells could be one of the mechanisms of thermal therapy.

  2. RNA-induced silencing attenuates G protein-mediated calcium signals.

    PubMed

    Philip, Finly; Sahu, Shriya; Golebiewska, Urszula; Scarlata, Suzanne

    2016-05-01

    Phospholipase Cβ (PLCβ) is activated by G protein subunits in response to environmental stimuli to increase intracellular calcium. In cells, a significant portion of PLCβ is cytosolic, where it binds a protein complex required for efficient RNA-induced silencing called C3PO (component 3 promoter of RISC). Binding between C3PO and PLCβ raises the possibility that RNA silencing activity can affect the ability of PLCβ to mediate calcium signals. By use of human and rat neuronal cell lines (SK-N-SH and PC12), we show that overexpression of one of the main components of C3PO diminishes Ca(2+) release in response to Gαq/PLCβ stimulation by 30 to 40%. In untransfected SK-N-SH or PC12 cells, the introduction of siRNA(GAPDH) [small interfering RNA(glyceraldehyde 3-phosphate dehydrogenase)] reduces PLCβ-mediated calcium signals by ∼30%, but addition of siRNA(Hsp90) (heat shock protein 90) had little effect. Fluorescence imaging studies suggest an increase in PLCβ-C3PO association in cells treated with siRNA(GAPDH) but not siRNA(Hsp90). Taken together, our studies raise the possibility that Ca(2+) responses to extracellular stimuli can be modulated by components of the RNA silencing machinery.-Philip, F., Sahu, S., Golebiewska, U., Scarlata, S. RNA-induced silencing attenuates G protein-mediated calcium signals. PMID:26862135

  3. Cadmium Induces Apoptosis in Freshwater Crab Sinopotamon henanense through Activating Calcium Signal Transduction Pathway

    PubMed Central

    Wang, Jinxiang; Zhang, Pingping; Liu, Na; Wang, Qian; Luo, Jixian; Wang, Lan

    2015-01-01

    Calcium ion (Ca2+) is one of the key intracellular signals, which is implicated in the regulation of cell functions such as impregnation, cell proliferation, differentiation and death. Cadmium (Cd) is a toxic environmental pollutant that can disturb cell functions and even lead to cell death. Recently, we have found that Cd induced apoptosis in gill cells of the freshwater crab Sinopotamon henanense via caspase activation. In the present study, we further investigated the role of calcium signaling in the Cd-induced apoptosis in the animals. Our data showed that Cd triggered gill cell apoptosis which is evidenced by apoptotic DNA fragmentation, activations of caspases-3, -8 and -9 and the presence of apoptotic morphological features. Moreover, Cd elevated the intracellular concentration of Ca2+, the protein concentration of calmodulin (CaM) and the activity of Ca2+-ATPase in the gill cells of the crabs. Pretreatment of the animals with ethylene glycol-bis-(b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), Ca2+ chelator, inhibited Cd-induced activation of caspases-3, -8 and -9 as well as blocked the Cd-triggered apoptotic DNA fragmentation. The apoptotic morphological features were no longer observed in gill cells pretreated with the Ca2+ signaling inhibitors before Cd treatment. Our results indicate that Cd evokes gill cell apoptosis through activating Ca2+-CaM signaling transduction pathway. PMID:26714174

  4. Temporal protein expression pattern in intracellular signalling cascade during T-cell activation: a computational study.

    PubMed

    Ganguli, Piyali; Chowdhury, Saikat; Bhowmick, Rupa; Sarkar, Ram Rup

    2015-10-01

    Various T-cell co-receptor molecules and calcium channel CRAC play a pivotal role in the maintenance of cell's functional responses by regulating the production of effector molecules (mostly cytokines) that aids in immune clearance and also maintaining the cell in a functionally active state. Any defect in these co-receptor signalling pathways may lead to an altered expression pattern of the effector molecules. To study the propagation of such defects with time and their effect on the intracellular protein expression patterns, a comprehensive and largest pathway map of T-cell activation network is reconstructed manually. The entire pathway reactions are then translated using logical equations and simulated using the published time series microarray expression data as inputs. After validating the model, the effect of in silico knock down of co-receptor molecules on the expression patterns of their downstream proteins is studied and simultaneously the changes in the phenotypic behaviours of the T-cell population are predicted, which shows significant variations among the proteins expression and the signalling routes through which the response is propagated in the cytoplasm. This integrative computational approach serves as a valuable technique to study the changes in protein expression patterns and helps to predict variations in the cellular behaviour. PMID:26564978

  5. Calcium signaling is involved in ethanol-induced volume decrease and gap junction closure in cultured rat gastric mucosal cells.

    PubMed

    Mustonen, Harri; Kiviluoto, Tuula; Paimela, Hannu; Puolakkainen, Pauli; Kivilaakso, Eero

    2005-01-01

    it (from -23 +/- 5 to -11 +/- 3%; N = 9; P < 0.05). They also abolished the closure of gap junctions induced by ethanol (fluorescence recovery, 38 +/- 5% for BABTA and 30 +/- 4% for TMB-8 + lanthanum). We conclude that luminal ethanol opens basolateral calcium-dependent potassium selective channels with resultant shrinkage of the cells and blocks the intercellular gap junctions. These actions are mediated by intracellular calcium signaling. PMID:15712646

  6. Retinoic acid affects calcium signaling in adult molluscan neurons.

    PubMed

    Vesprini, Nicholas D; Dawson, Taylor F; Yuan, Ye; Bruce, Doug; Spencer, Gaynor E

    2015-01-01

    Retinoic acid, the active metabolite of vitamin A, is important for nervous system development, regeneration, as well as cognitive functions of the adult central nervous system. These central nervous system functions are all highly dependent on neuronal activity. Retinoic acid has previously been shown to induce changes in the firing properties and action potential waveforms of adult molluscan neurons in a dose- and isomer-dependent manner. In this study, we aimed to determine the cellular pathways by which retinoic acid might exert such effects, by testing the involvement of pathways previously shown to be affected by retinoic acid. We demonstrated that the ability of all-trans retinoic acid (atRA) to induce electrophysiological changes in cultured molluscan neurons was not prevented by inhibitors of protein synthesis, protein kinase A or phospholipase C. However, we showed that atRA was capable of rapidly reducing intracellular calcium levels in the same dose- and isomer-dependent manner as shown previously for changes in neuronal firing. Moreover, we also demonstrated that the transmembrane ion flux through voltage-gated calcium channels was rapidly modulated by retinoic acid. In particular, the peak current density was reduced and the inactivation rate was increased in the presence of atRA, over a similar time course as the changes in cell firing and reductions in intracellular calcium. These studies provide further evidence for the ability of atRA to induce rapid effects in mature neurons. PMID:25343782

  7. Vertical nanowire probes for intracellular signaling of living cells

    PubMed Central

    2014-01-01

    The single living cell action potential was measured in an intracellular mode by using a vertical nanoelectrode. For intracellular interfacing, Si nanowires were vertically grown in a controlled manner, and optimum conditions, such as diameter, length, and nanowire density, were determined by culturing cells on the nanowires. Vertical nanowire probes were then fabricated with a complimentary metal-oxide-semiconductor (CMOS) process including sequential deposition of the passivation and electrode layers on the nanowires, and a subsequent partial etching process. The fabricated nanowire probes had an approximately 60-nm diameter and were intracellular. These probes interfaced with a GH3 cell and measured the spontaneous action potential. It successfully measured the action potential, which rapidly reached a steady state with average peak amplitude of approximately 10 mV, duration of approximately 140 ms, and period of 0.9 Hz. PMID:24484729

  8. Vertical nanowire probes for intracellular signaling of living cells

    NASA Astrophysics Data System (ADS)

    Lee, Ki-Young; Kim, Ilsoo; Kim, So-Eun; Jeong, Du-Won; Kim, Ju-Jin; Rhim, Hyewhon; Ahn, Jae-Pyeong; Park, Seung-Han; Choi, Heon-Jin

    2014-02-01

    The single living cell action potential was measured in an intracellular mode by using a vertical nanoelectrode. For intracellular interfacing, Si nanowires were vertically grown in a controlled manner, and optimum conditions, such as diameter, length, and nanowire density, were determined by culturing cells on the nanowires. Vertical nanowire probes were then fabricated with a complimentary metal-oxide-semiconductor (CMOS) process including sequential deposition of the passivation and electrode layers on the nanowires, and a subsequent partial etching process. The fabricated nanowire probes had an approximately 60-nm diameter and were intracellular. These probes interfaced with a GH3 cell and measured the spontaneous action potential. It successfully measured the action potential, which rapidly reached a steady state with average peak amplitude of approximately 10 mV, duration of approximately 140 ms, and period of 0.9 Hz.

  9. Calcium Signaling in Oomycetes: An Evolutionary Perspective.

    PubMed

    Zheng, Limian; Mackrill, John J

    2016-01-01

    Oomycetes are a family of eukaryotic microbes that superficially resemble fungi, but which are phylogenetically distinct from them. These organisms cause major global economic losses to agriculture and fisheries, with representative pathogens being Phytophthora infestans, the cause of late potato blight and Saprolegnia diclina, the instigator of "cotton molds" in fish. As in all eukaryotes, cytoplasmic Ca(2+) is a key second messenger in oomycetes, regulating life-cycle transitions, controlling motility and chemotaxis and, in excess, leading to cell-death. Despite this, little is known about the molecular mechanisms regulating cytoplasmic Ca(2+) concentrations in these organisms. Consequently, this review analyzed the presence of candidate calcium channels encoded within the nine oomycete genomes that are currently available. This revealed key differences between oomycetes and other eukaryotes, in particular the expansion and loss of different channel families, and the presence of a phylum-specific group of proteins, termed the polycystic kidney disease tandem ryanodine receptor domain (PKDRR) channels. PMID:27092083

  10. Calcium and signal transduction in plants

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Reddy, A. S.

    1993-01-01

    Environmental and hormonal signals control diverse physiological processes in plants. The mechanisms by which plant cells perceive and transduce these signals are poorly understood. Understanding biochemical and molecular events involved in signal transduction pathways has become one of the most active areas of plant research. Research during the last 15 years has established that Ca2+ acts as a messenger in transducing external signals. The evidence in support of Ca2+ as a messenger is unequivocal and fulfills all the requirements of a messenger. The role of Ca2+ becomes even more important because it is the only messenger known so far in plants. Since our last review on the Ca2+ messenger system in 1987, there has been tremendous progress in elucidating various aspects of Ca(2+) -signaling pathways in plants. These include demonstration of signal-induced changes in cytosolic Ca2+, calmodulin and calmodulin-like proteins, identification of different Ca2+ channels, characterization of Ca(2+) -dependent protein kinases (CDPKs) both at the biochemical and molecular levels, evidence for the presence of calmodulin-dependent protein kinases, and increased evidence in support of the role of inositol phospholipids in the Ca(2+) -signaling system. Despite the progress in Ca2+ research in plants, it is still in its infancy and much more needs to be done to understand the precise mechanisms by which Ca2+ regulates a wide variety of physiological processes. The purpose of this review is to summarize some of these recent developments in Ca2+ research as it relates to signal transduction in plants.

  11. Transforming growth factor alpha treatment alters intracellular calcium levels in hair cells and protects them from ototoxic damage in vitro.

    PubMed

    Staecker, H; Dazert, S; Malgrange, B; Lefebvre, P P; Ryan, A F; Van de Water, T R

    1997-07-01

    To determine if transforming growth factor alpha (TGF alpha) pretreatment protects hair cells from aminoglycoside induced injury by modifying their intracellular calcium concentration, we assayed hair cell calcium levels in organ of Corti explants both before and after aminoglycoside (i.e. neomycin, 10(-3) M) exposure either with or without growth factor pretreatment. After TGF alpha (500 ng/ml) treatment, the intracellular calcium level of hair cells showed a five-fold increase as compared to the levels observed in the hair cells of control cultures. After ototoxin exposure, calcium levels in hair cells of control explants showed an increase relative to their baseline levels, while in the presence of growth factors pretreatment, hair cells showed a relative reduction in calcium levels. Pretreatment of organ of Corti explants afforded significant protection of hair cell stereocilia bundle morphology from ototoxic damage when compared to explants exposed to ototoxin alone. This study correlates a rise in hair cell calcium levels with the otoprotection of hair cells by TGF alpha in organ of Corti explants. PMID:9263032

  12. Mobilization of intracellular calcium by intracellular flash photolysis of caged dihydrosphingosine in cultured neonatal rat sensory neurones.

    PubMed

    Ayar, A; Thatcher, N M; Zehavi, U; Trentham, D R; Scott, R H

    1998-01-01

    The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 microM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 mM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 microM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82+/-13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11+/-3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 microM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 microM ryanodine or extracellular application of 10 microM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of

  13. Ionic osmolytes and intracellular calcium regulate tissue production in chondrocytes cultured in a 3D charged hydrogel.

    PubMed

    Farnsworth, Nikki L; Mead, Benjamin E; Antunez, Lorena R; Palmer, Amy E; Bryant, Stephanie J

    2014-11-01

    The goal of this study was to investigate the role of fixed negative charges in regulating cartilage-like tissue production by chondrocytes under static and dynamic three-dimensional culture, and to determine whether intracellular calcium ([Ca(2+)]i) is involved in mediating this response. Initial experiments using the 3D neutral hydrogel were conducted in static isotonic culture with ionic and non-ionic osmolytes added to the culture medium. Tissue production by bovine chondrocytes with non-ionic osmolytes was 1.9-fold greater than with ionic osmolytes, suggesting that the ionic nature of the osmolyte is an important regulator of tissue production. To investigate fixed negative charges, a 3D culture system containing encapsulated chondrocytes was employed based on a synthetic and neutral hydrogel platform within which negatively charged chondroitin sulfate was incorporated in a controlled manner. Incorporation of negative charges did not affect the mechanical properties of the hydrogel; however, intracellular ion concentration was elevated from the culture medium (330 mOsm) and estimated to be similar to that in ~400 mOsm culture medium. With dynamic loading, GAG synthesis decreased by 26% in neutral hydrogels cultured in 400mOsm medium, and increased by 26% in charged gels cultured in 330 mOsm. Treatment of chondrocyte-seeded hydrogels with the Ca(2+) chelator BAPTA-AM decreased GAG synthesis by 32-46% and was similar among all conditions, suggesting multiple roles for Ca(2+) mediated tissue production including with ionic osmolytes. In conclusion, findings from this study suggest that a dynamic ionic environment regulates tissue synthesis and points to [Ca(2+)]i signaling as a potential mediator. PMID:25128592

  14. Intracellular signaling pathways required for rat vascular smooth muscle cell migration. Interactions between basic fibroblast growth factor and platelet-derived growth factor.

    PubMed Central

    Bilato, C; Pauly, R R; Melillo, G; Monticone, R; Gorelick-Feldman, D; Gluzband, Y A; Sollott, S J; Ziman, B; Lakatta, E G; Crow, M T

    1995-01-01

    Intracellular signaling pathways activated by both PDGF and basic fibroblast growth factor (bFGF) have been implicated in the migration of vascular smooth muscle cells (VSMC), a key step in the pathogenesis of many vascular diseases. We demonstrate here that, while bFGF is a weak chemoattractant for VSMCs, it is required for the PDGF-directed migration of VSMCs and the activation of calcium/calmodulin-dependent protein kinase II (CamKinase II), an intracellular event that we have previously shown to be important in the regulation of VSMC migration. Neutralizing antibodies to bFGF caused a dramatic reduction in the size of the intracellular calcium transient normally seen after PDGF stimulation and inhibited both PDGF-directed VSMC migration and CamKinase II activation. Partially restoring the calcium transient with ionomycin restored migration and CamKinase II activation as did the forced expression of a mutant CamKinase II that had been "locked" in the active state by site-directed mutagenesis. These results suggest that bFGF links PDGF receptor stimulation to changes in intracellular calcium and CamKinase II activation, reinforcing the central role played by CamKinase II in regulating VSMC migration. Images PMID:7560082

  15. Intercellular calcium signaling in a gap junction-coupled cell network establishes asymmetric neuronal fates in C. elegans

    PubMed Central

    Schumacher, Jennifer A.; Hsieh, Yi-Wen; Chen, Shiuhwei; Pirri, Jennifer K.; Alkema, Mark J.; Li, Wen-Hong; Chang, Chieh; Chuang, Chiou-Fen

    2012-01-01

    The C. elegans left and right AWC olfactory neurons specify asymmetric subtypes, one default AWCOFF and one induced AWCON, through a stochastic, coordinated cell signaling event. Intercellular communication between AWCs and non-AWC neurons via a NSY-5 gap junction network coordinates AWC asymmetry. However, the nature of intercellular signaling across the network and how individual non-AWC cells in the network influence AWC asymmetry is not known. Here, we demonstrate that intercellular calcium signaling through the NSY-5 gap junction neural network coordinates a precise 1AWCON/1AWCOFF decision. We show that NSY-5 gap junctions in C. elegans cells mediate small molecule passage. We expressed vertebrate calcium-buffer proteins in groups of cells in the network to reduce intracellular calcium levels, thereby disrupting intercellular communication. We find that calcium in non-AWC cells of the network promotes the AWCON fate, in contrast to the autonomous role of calcium in AWCs to promote the AWCOFF fate. In addition, calcium in specific non-AWCs promotes AWCON side biases through NSY-5 gap junctions. Our results suggest a novel model in which calcium has dual roles within the NSY-5 network: autonomously promoting AWCOFF and non-autonomously promoting AWCON. PMID:23093425

  16. Calcium Signaling in Oomycetes: An Evolutionary Perspective

    PubMed Central

    Zheng, Limian; Mackrill, John J.

    2016-01-01

    Oomycetes are a family of eukaryotic microbes that superficially resemble fungi, but which are phylogenetically distinct from them. These organisms cause major global economic losses to agriculture and fisheries, with representative pathogens being Phytophthora infestans, the cause of late potato blight and Saprolegnia diclina, the instigator of “cotton molds” in fish. As in all eukaryotes, cytoplasmic Ca2+ is a key second messenger in oomycetes, regulating life-cycle transitions, controlling motility and chemotaxis and, in excess, leading to cell-death. Despite this, little is known about the molecular mechanisms regulating cytoplasmic Ca2+ concentrations in these organisms. Consequently, this review analyzed the presence of candidate calcium channels encoded within the nine oomycete genomes that are currently available. This revealed key differences between oomycetes and other eukaryotes, in particular the expansion and loss of different channel families, and the presence of a phylum-specific group of proteins, termed the polycystic kidney disease tandem ryanodine receptor domain (PKDRR) channels. PMID:27092083

  17. Regulation of Angiogenic Functions by Angiopoietins through Calcium-Dependent Signaling Pathways

    PubMed Central

    Pafumi, Irene; Favia, Annarita; Gambara, Guido; Papacci, Francesca; Ziparo, Elio; Palombi, Fioretta; Filippini, Antonio

    2015-01-01

    Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca2+ signaling, antagonizing IP3 with 2-APB or cADPR with 8Br-cADPR, was found to modulate in vitro angiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by “scratch assay,” was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery involved in the complex interplay regulating angiogenic processes showing that IP3- and cADPR-induced Ca2+ release specifically regulates distinct Angs-mediated angiogenic steps. PMID:26146638

  18. Contribution of calcium ions and hydrogen ions to the signal transduction chain in phytochrome-mediated spore germination. [Onoclea sensibilis L

    SciTech Connect

    Wayne, R.

    1985-01-01

    Red light stimulates germination in the spores of Onoclea sensibilis L. Phytochrome is confirmed to be the photoreceptor pigment in the germination response by demonstrating red-far-red photoreversibility. External Ca/sup 2 +/ is required for this response with a threshold at a submicromolar concentration. Red light stimulates an increase in the total concentration of intracellular calcium in the spores as determined by atomic absorption spectroscopy. Subsequent exposure to far-red light inhibits the red light-induced increase in intracellular calcium. The majority of the increase occurs 5 minutes after the onset of irradiation. The calcium-antagonist, La/sup 3 +/ inhibits both germination and the red light-induced increase in intracellular calcium. Using /sup 31/P-nuclear magnetic resonance spectroscopy, the author tested the hypothesis that a sustained increase in intracellular pH contributes to the signal transduction chain. He never detected a red light-induced increase in intracellular pH or a change in portion efflux. An artificially induced change in intracellular pH of greater than 1 pH unit (5.8-7.2) has no effect on germination. Although the intracellular pH can be varied in magnitude greater than it would be expected to change if it were acting as an intracellular signal, germination of Onoclea spores is independent of intracellular pH in this range. These data indicate that a sustained increase in intracellular pH does not contribute to the single transduction chain phytochrome-mediated fern spore germination. Therefore, Ca/sup 2 +/, but not pH, contributes to the signal transduction chain in phytochrome-mediated fern spore germination.

  19. Intracellular calcium and Na+-Ca2+ exchange current in isolated toad pacemaker cells

    PubMed Central

    Ju, Yue-Kun; Allen, David G

    1998-01-01

    Single pacemaker cells were isolated from the sinus venosus of cane toad (Bufo marinus) in order to study the mechanisms involved in the spontaneous firing rate of action potentials. Intracellular calcium concentration ([Ca2+]i) was measured with indo-1 to determine whether [Ca2+]i influenced firing rate. A rapid transient rise of [Ca2+]i was recorded together with each spontaneous action potential. [Ca2+]i at the peak of systole was 655 ± 64 nm and the minimum at the end of diastole was 195 ± 15 nm. Reduction of extracellular Ca2+ concentration from 2 to 0.5 mm caused a reduction in both systolic and diastolic [Ca2+]i and the spontaneous firing rate also gradually declined. Application of the acetoxymethyl (AM) ester of BAPTA (10 μm), in order to increase intracellular calcium buffering, caused a decline in systolic and diastolic [Ca2+]i. The firing rate declined progressively until the cells stopped firing after 10–15 min. At the time that firing ceased, the diastolic [Ca2+]i had declined by 141 ± 38 nm. In the presence of ryanodine (2 μm), which interferes with Ca2+ release from the sarcoplasmic reticulum, the systolic and diastolic [Ca2+]i both declined and the firing rate decreased until the cells stopped firing. At quiescence diastolic [Ca2+]i had declined by 93 ± 20 nm. Exposure of the cells to Na+-free solution caused a rise in [Ca2+]i which exceeded the systolic level after 4.8 ± 0.3 s. This rise is consistent with Ca2+ entry on a Na+-Ca2+ exchanger. Rapid application of caffeine (10–20 mm) to cells clamped at −60 mV caused a rapid increase in [Ca2+]i which then spontaneously declined. An inward current with a similar time course to that of [Ca2+]i was also generated. Application of Ni2+ (5 mm) or 2,4-dichlorobenzamil (25 μm) reduced the amplitude of the inward current produced by caffeine by 96 ± 1 % and 74 ± 10 %, respectively. In a Na+-free solution the caffeine-induced current was reduced by 93 ± 7 %. Under a variety of circumstances

  20. Glycolytic inhibition: effects on diastolic relaxation and intracellular calcium handling in hypertrophied rat ventricular myocytes.

    PubMed Central

    Kagaya, Y; Weinberg, E O; Ito, N; Mochizuki, T; Barry, W H; Lorell, B H

    1995-01-01

    We tested the hypothesis that glycolytic inhibition by 2-deoxyglucose causes greater impairment of diastolic relaxation and intracellular calcium handling in well-oxygenated hypertrophied adult rat myocytes compared with control myocytes. We simultaneously measured cell motion and intracellular free calcium concentration ([Ca2+]i) with indo-1 in isolated paced myocytes from aortic-banded rats and sham-operated rats. There was no difference in either the end-diastolic or peak-systolic [Ca2+]i between control and hypertrophied myocytes (97 +/- 18 vs. 105 +/- 15 nM, 467 +/- 92 vs. 556 +/- 67 nM, respectively). Myocytes were first superfused with oxygenated Hepes-buffered solution containing 1.2 mM CaCl2, 5.6 mM glucose, and 5 mM acetate, and paced at 3 Hz at 36 degrees C. Exposure to 20 mM 2-deoxyglucose as substitution of glucose for 15 min caused an upward shift of end-diastolic cell position in both control (n = 5) and hypertrophied myocytes (n = 10) (P < 0.001 vs. baseline), indicating an impaired extent of relaxation. Hypertrophied myocytes, however, showed a greater upward shift in end-diastolic cell position and slowing of relaxation compared with control myocytes (delta 144 +/- 28 vs. 55 +/- 15% of baseline diastolic position, P < 0.02). Exposure to 2-deoxyglucose increased end-diastolic [Ca2+]i in both groups (P < 0.001 vs. baseline), but there was no difference between hypertrophied and control myocytes (218 +/- 38 vs. 183 +/- 29 nM, respectively). The effects of 2-deoxyglucose were corroborated in isolated oxygenated perfused hearts in which glycolytic inhibition which caused severe elevation of isovolumic diastolic pressure and prolongation of relaxation in the hypertrophied hearts compared with controls. In summary, the inhibition of the glycolytic pathway impairs diastolic relaxation to a greater extent in hypertrophied myocytes than in control myocytes even in well-oxygenated conditions. The severe impairment of diastolic relaxation induced by 2

  1. Root zone calcium can modulate GA induced tuberization signal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present study was conducted to investigate the possible relationship between root zone calcium and GA3 concentration in tuberization signal. For this purpose, we developed a system utilizing in vitro propagated potato plantlets and pure silica sand that allows precise control of root zone chemic...

  2. Differences in intracellular calcium dynamics cause differences in α-granule secretion and phosphatidylserine expression in platelets adhering on glass and TiO2.

    PubMed

    Gupta, Swati; Donati, Alessia; Reviakine, Ilya

    2016-06-01

    In this study, the activation of purified human platelets due to their adhesion on glass and TiO2 in the absence of extracellular calcium was investigated. Differences in α-granule secretion between platelets adhering on the two surfaces were detected by examining the expression and secretion of the α-granule markers P-selectin (CD62P) and β-thromboglobulin. Similarly, differences in the expression of phosphatidylserine (PS), and in the activation of the major integrin GPIIb/IIIa, on the surfaces of the adhering platelets, were also observed. While all of these activation markers were expressed in platelets adhering on glass, the surface markers were not expressed in platelets adhering on TiO2, and β-thromboglobulin secretion levels were substantially reduced. Differences in marker expression and secretion correlated with differences in the intracellular calcium dynamics. Calcium ionophore treatment triggered α-granule secretion and PS expression in TiO2-adhering platelets but had no effect on the activation of GPIIb/IIIa. These results demonstrate specificity in the way surfaces of artificial materials activate platelets, link differences in the intracellular calcium dynamics observed in the platelets adhering on the two surfaces to the differences in some of the platelet responses (α-granule secretion and PS expression), but also highlight the involvement of synergistic, calcium-independent pathways in platelet activation. The ability to control activation in surface-adhering platelets makes this an attractive model system for studying platelet signaling pathways and for tissue engineering applications. PMID:27124595

  3. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization.

    PubMed

    Bates, Ryan C; Fees, Colby P; Holland, William L; Winger, Courtney C; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J

    2014-02-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca](i)). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca](i) and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca](i), PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca](i) release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca](i) release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  4. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading

    PubMed Central

    Jing, Da; Baik, Andrew D.; Lu, X. Lucas; Zhou, Bin; Lai, Xiaohan; Wang, Liyun; Luo, Erping; Guo, X. Edward

    2014-01-01

    Osteocytes have been hypothesized to be the major mechanosensors in bone. How in situ osteocytes respond to mechanical stimuli is still unclear because of technical difficulties. In vitro studies have shown that osteocytes exhibited unique calcium (Ca2+) oscillations to fluid shear. However, whether this mechanotransduction phenomenon holds for in situ osteocytes embedded within a mineralized bone matrix under dynamic loading remains unknown. Using a novel synchronized loading/imaging technique, we successfully visualized in real time and quantified Ca2+ responses in osteocytes and bone surface cells in situ under controlled dynamic loading on intact mouse tibia. The resultant fluid-induced shear stress on the osteocyte in the lacunocanalicular system (LCS) was also quantified. Osteocytes, but not surface cells, displayed repetitive Ca2+ spikes in response to dynamic loading, with spike frequency and magnitude dependent on load magnitude, tissue strain, and shear stress in the LCS. The Ca2+ oscillations were significantly reduced by endoplasmic reticulum (ER) depletion and P2 purinergic receptor (P2R)/phospholipase C (PLC) inhibition. This study provides direct evidence that osteocytes respond to in situ mechanical loading by Ca2+ oscillations, which are dependent on the P2R/PLC/inositol trisphosphate/ER pathway. This study develops a novel approach in skeletal mechanobiology and also advances our fundamental knowledge of bone mechanotransduction.—Jing, D., Baik, A. D., Lu, X. L., Zhou, B., Lai, X., Wang, L., Luo, E., Guo, X. E. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading. PMID:24347610

  5. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

    PubMed Central

    Bates, Ryan C.; Fees, Colby P.; Holland, William L.; Winger, Courtney C.; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J.

    2014-01-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC- γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca]i). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 minute after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca]i and other fertilization events. As compared to 14 other lipids, PA strongly bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca]i, PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca]i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca]i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  6. LOX-1 regulates estrogenesis via intracellular calcium release from bovine granulosa cells.

    PubMed

    Weitzel, J M; Vernunft, A; Krüger, B; Plinski, C; Viergutz, T

    2014-01-01

    Estradiol produced by ovarian granulosa cells triggers the luteinizing hormone surge which in turn initiates ovulation in female mammals. Disturbances in estradiol production from granulosa cells are a major reason for reproductive dysfunctions in dairy cows. Endogenous estradiol production might be altered by reactive oxygen species (ROS) such as oxidized low-density lipoprotein (ox-LDL). Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a receptor of ox-LDL, leads to increased estrogenesis in granulosa cells. This activity is mediated by calcium release from endoplasmic reticulum (ER)-dependent and ER-independent calcium pools. Inhibition of the LOX-1 signal transduction pathway is followed by mitochondrial alterations. The membrane potential ΔΨ increases and the ROS production decreases in mitochondria after blocking LOX-1. Our data indicate that blocking the LOX-1 receptor signal pathway might be a promising way to improve steroid hormone concentrations in metabolically highly active female mammals and, therefore, to defend against reproductive dysfunctions in humans and animals. PMID:24115745

  7. Platelet Activating Factor Enhances Synaptic Vesicle Exocytosis Via PKC, Elevated Intracellular Calcium, and Modulation of Synapsin 1 Dynamics and Phosphorylation

    PubMed Central

    Hammond, Jennetta W.; Lu, Shao-Ming; Gelbard, Harris A.

    2016-01-01

    Platelet activating factor (PAF) is an inflammatory phospholipid signaling molecule implicated in synaptic plasticity, learning and memory and neurotoxicity during neuroinflammation. However, little is known about the intracellular mechanisms mediating PAF’s physiological or pathological effects on synaptic facilitation. We show here that PAF receptors are localized at the synapse. Using fluorescent reporters of presynaptic activity we show that a non-hydrolysable analog of PAF (cPAF) enhances synaptic vesicle release from individual presynaptic boutons by increasing the size or release of the readily releasable pool and the exocytosis rate of the total recycling pool. cPAF also activates previously silent boutons resulting in vesicle release from a larger number of terminals. The underlying mechanism involves elevated calcium within presynaptic boutons and protein kinase C activation. Furthermore, cPAF increases synapsin I phosphorylation at sites 1 and 3, and increases dispersion of synapsin I from the presynaptic compartment during stimulation, freeing synaptic vesicles for subsequent release. These findings provide a conceptual framework for how PAF, regardless of its cellular origin, can modulate synapses during normal and pathologic synaptic activity. PMID:26778968

  8. Constant change: dynamic regulation of membrane transport by calcium signalling networks keeps plants in tune with their environment.

    PubMed

    Kleist, Thomas J; Luan, Sheng

    2016-03-01

    Despite substantial variation and irregularities in their environment, plants must conform to spatiotemporal demands on the molecular composition of their cytosol. Cell membranes are the major interface between organisms and their environment and the basis for controlling the contents and intracellular organization of the cell. Membrane transport proteins (MTPs) govern the flow of molecules across membranes, and their activities are closely monitored and regulated by cell signalling networks. By continuously adjusting MTP activities, plants can mitigate the effects of environmental perturbations, but effective implementation of this strategy is reliant on precise coordination among transport systems that reside in distinct cell types and membranes. Here, we examine the role of calcium signalling in the coordination of membrane transport, with an emphasis on potassium transport. Potassium is an exceptionally abundant and mobile ion in plants, and plant potassium transport has been intensively studied for decades. Classic and recent studies have underscored the importance of calcium in plant environmental responses and membrane transport regulation. In reviewing recent advances in our understanding of the coding and decoding of calcium signals, we highlight established and emerging roles of calcium signalling in coordinating membrane transport among multiple subcellular locations and distinct transport systems in plants, drawing examples from the CBL-CIPK signalling network. By synthesizing classical studies and recent findings, we aim to provide timely insights on the role of calcium signalling networks in the modulation of membrane transport and its importance in plant environmental responses. PMID:26139029

  9. Intracellular calcium and its sodium-independent regulation in voltage-clamped snail neurones.

    PubMed Central

    Kennedy, H J; Thomas, R C

    1995-01-01

    1. We have used both Ca(2+)-sensitive microelectrodes and fura-2 to measure the intracellular free calcium ion concentration ([Ca2+]i or its negative log, pCai) of snail neurones voltage clamped to -50 or -60 mV. Using Ca(2+)-sensitive microelectrodes, [Ca2+]i was found to be approximately 174 nM and pCai, 6.76 +/- 0.09 (mean +/- S.E.M.; n = 11); using fura-2, [Ca2+]i was approximately 40 nM and pCai, 7.44 +/- 0.06 (mean +/- S.E.M., n = 10). 2. Depolarizations (1-20 s) caused an increase in [Ca2+]i which was abolished by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i was due to Ca2+ influx through voltage-activated Ca2+ channels. 3. Caffeine (10-20 mM) caused an increase in [Ca2+]i in the presence or absence of extracellular Ca2+. The effects of caffeine on [Ca2+]i could be prevented by ryanodine. 4. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a small increase in resting [Ca2+]i and slowed the rate of recovery from Ca2+ loads following 20 s depolarizations. 5. Neither replacement of extracellular sodium with N-methyl-D-glucamine (NMDG), nor loading the cells with intracellular sodium, had any effect on resting [Ca2+]i or the rate of recovery of [Ca2+]i following depolarizations. 6. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCmP) caused a small gradual rise in resting [Ca2+]i. Removal of extracellular sodium during exposure to CCmP had no further effect on [Ca2+]i. 7. Intracellular orthovanadate caused an increase in resting [Ca2+]i and prevented the full recovery of [Ca2+]i following small Ca2+ loads, but removal of extracellular sodium did not cause a rise in [Ca2+]i. We conclude that there is no Na(+)-Ca2+ exchanger present in the cell body of these neurones and that [Ca2+]i is maintained by an ATP-dependent Ca2+ pump. Images Figure 1 PMID:7623274

  10. Gαq/11-mediated intracellular calcium responses to retrograde flow in endothelial cells.

    PubMed

    Melchior, Benoît; Frangos, John A

    2012-08-15

    Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow

  11. The intracellular calcium increase at fertilization in Urechis caupo oocytes: activation without waves.

    PubMed

    Stephano, J L; Gould, M C

    1997-11-01

    The intracellular Ca2+ (Cai) increase at fertilization of the marine worm Urechis caupo (Echiura) was studied with conventional and confocal epifluorescence microscopy in oocytes microinjected with calcium green dextran or dually labeled with the calcium-insensitive dye tetramethylrhodamine dextran. Calcium green fluorescence was also measured with a photomultiplier system while the oocyte membrane potential was recorded and manipulated. The results show that Cai rises simultaneously around the oocyte cortex and peaks slightly later in the nucleoplasm. The Cai rise coincides with the initiation of the fertilization potential and we conclude that it is due primarily to external Ca2+ entering through the voltage-gated Ca2+ action potential channels that open during the fertilization potential because: (1) current clamping the oocyte membrane potential to positive values in the absence of sperm produces a similar Cai increase, (2) external Ca2+ is required, (3) and the confocal images are consistent with this mechanism. External application of sperm acrosomal peptide (P23) also caused a Cai increase that was inhibited in the presence of CoCl2. Cai and pHi (measured with BCECF dextran) were manipulated in experiments employing microinjection of BAPTA (to chelate Cai), external application of NH4Cl (to increase pHi) and CoCl2 (to block Ca2+ channels), and fertilization of eggs in pH 7 seawater (Cai increase without pHi increase). The results showed that increases in both Cai and pHi are required for GVBD; neither alone is sufficient. However, although nuclear and cytoplasmic Ca2+ levels tended to parallel each other in oocytes fertilized at pH 7, and during the initial Cai response in oocytes fertilized at pH 8, there was a disproportionate fluorescence increase in the nucleoplasm of the latter prior to GVBD which could not be explained by any artifact we tested, suggesting there may be a selective increase in nuclear Ca2+ associated with GVBD. Finally

  12. The Calcium Signaling Toolkit of the Apicomplexan Parasites Toxoplasma gondii and Plasmodium spp

    PubMed Central

    Lourido, Sebastian; Moreno, Silvia N.J.

    2015-01-01

    Apicomplexan parasites have complex life cycles, frequently split between different hosts and reliant on rapid responses as the parasites react to changing environmental conditions. Calcium ion (Ca2+) signaling is consequently essential for the cellular and developmental changes that support apicomplexan parasitism. Apicomplexan genomes reveal a rich repertoire of genes involved in calcium signaling, although many of the genes responsible for observed physiological changes remain unknown. There is evidence, for example, for the presence of a nifedipine-sensitive calcium entry mechanism in Toxoplasma, but the molecular components involved in Ca2+ entry in both Toxoplasma and Plasmodium, have not been identified. The major calcium stores are the endoplasmic reticulum (ER), the acidocalcisomes, and the plant-like vacuole in Toxoplasma, or the food vacuole in Plasmodium spp. Pharmacological evidence suggests that Ca2+ release from intracellular stores may be mediated by inositol 1,4,5-trisphosphate (IP3) or cyclic ADP ribose (cADPR) although there is no molecular evidence for the presence of receptors for these second messengers in the parasites. Several Ca2+-ATPases are present in apicomplexans and a putative mitochondrial Ca2+/H+ exchanger has been identified. Apicomplexan genomes contain numerous genes encoding Ca2+-binding proteins, with the notable expansion of calcium-dependent protein kinases (CDPKs), whose study has revealed novel roles in gliding motility, microneme secretion, host cell invasion and egress, and parasite differentiation. Microneme secretion has also been shown to depend on the C2 domain containing protein DOC2 in both Plasmodium spp. and Toxoplasma, providing further evidence for the complex transduction of Ca2+ signals in these organisms. The characterization of these pathways could lead to the discovery of novel drug targets and to a better understanding of the role of Ca2+ in these parasites. PMID:25605521

  13. Spatiotemporal Intracellular Nitric Oxide Signaling Captured Using Internalized, Near-Infrared Fluorescent Carbon Nanotube Nanosensors

    PubMed Central

    2015-01-01

    Fluorescent nanosensor probes have suffered from limited molecular recognition and a dearth of strategies for spatial-temporal operation in cell culture. In this work, we spatially imaged the dynamics of nitric oxide (NO) signaling, important in numerous pathologies and physiological functions, using intracellular near-infrared fluorescent single-walled carbon nanotubes. The observed spatial-temporal NO signaling gradients clarify and refine the existing paradigm of NO signaling based on averaged local concentrations. This work enables the study of transient intracellular phenomena associated with signaling and therapeutics. PMID:25029087

  14. Plastid-nucleus communication involves calcium-modulated MAPK signalling.

    PubMed

    Guo, Hailong; Feng, Peiqiang; Chi, Wei; Sun, Xuwu; Xu, Xiumei; Li, Yuan; Ren, Dongtao; Lu, Congming; David Rochaix, Jean; Leister, Dario; Zhang, Lixin

    2016-01-01

    Chloroplast retrograde signals play important roles in coordinating the plastid and nuclear gene expression and are critical for proper chloroplast biogenesis and for maintaining optimal chloroplast functions in response to environmental changes in plants. Until now, the signals and the mechanisms for retrograde signalling remain poorly understood. Here we identify factors that allow the nucleus to perceive stress conditions in the chloroplast and to respond accordingly by inducing or repressing specific nuclear genes encoding plastid proteins. We show that ABI4, which is known to repress the LHCB genes during retrograde signalling, is activated through phosphorylation by the MAP kinases MPK3/MPK6 and the activity of these kinases is regulated through 14-3-3ω-mediated Ca(2+)-dependent scaffolding depending on the chloroplast calcium sensor protein CAS. These findings uncover an additional mechanism in which chloroplast-modulated Ca(2+) signalling controls the MAPK pathway for the activation of critical components of the retrograde signalling chain. PMID:27399341

  15. Calcium-binding proteins and development

    NASA Technical Reports Server (NTRS)

    Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

  16. The Impact of Vitamin D3 Supplementation on Mechanisms of Cell Calcium Signaling in Chronic Kidney Disease

    PubMed Central

    Lajdova, Ingrid; Spustova, Viera; Oksa, Adrian; Kaderjakova, Zuzana; Chorvat, Dusan; Morvova, Marcela; Sikurova, Libusa; Marcek Chorvatova, Alzbeta

    2015-01-01

    Intracellular calcium concentration in peripheral blood mononuclear cells (PBMCs) of patients with chronic kidney disease (CKD) is significantly increased, and the regulatory mechanisms maintaining cellular calcium homeostasis are impaired. The purpose of this study was to examine the effect of vitamin D3 on predominant regulatory mechanisms of cell calcium homeostasis. The study involved 16 CKD stages 2-3 patients with vitamin D deficiency treated with cholecalciferol 7000–14000 IU/week for 6 months. The regulatory mechanisms of calcium signaling were studied in PBMCs and red blood cells. After vitamin D3 supplementation, serum concentration of 25(OH)D3 increased (P < 0.001) and [Ca2+]i decreased (P < 0.001). The differences in [Ca2+]i were inversely related to differences in 25(OH)D3 concentration (P < 0.01). Vitamin D3 supplementation decreased the calcium entry through calcium release activated calcium (CRAC) channels and purinergic P2X7 channels. The function of P2X7 receptors was changed in comparison with their baseline status, and the expression of these receptors was reduced. There was no effect of vitamin D3 on P2X7 pores and activity of plasma membrane Ca2+-ATPases. Vitamin D3 supplementation had a beneficial effect on [Ca2+]i decreasing calcium entry via CRAC and P2X7 channels and reducing P2X7 receptors expression. PMID:26064953

  17. Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics

    PubMed Central

    Helassa, Nordine; Zhang, Xiao-hua; Conte, Ianina; Scaringi, John; Esposito, Elric; Bradley, Jonathan; Carter, Thomas; Ogden, David; Morad, Martin; Török, Katalin

    2015-01-01

    Faithful reporting of temporal patterns of intracellular Ca2+ dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca2+ signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca2+ affinities and accelerated kinetics by weakening the Ca2+-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca2+ decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca2+ dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca2+ at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca2+ kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes. PMID:26527405

  18. Trimeric intracellular cation channels and sarcoplasmic/endoplasmic reticulum calcium homeostasis.

    PubMed

    Zhou, Xinyu; Lin, Peihui; Yamazaki, Daiju; Park, Ki Ho; Komazaki, Shinji; Chen, S R Wayne; Takeshima, Hiroshi; Ma, Jianjie

    2014-02-14

    Trimeric intracellular cation channels (TRIC) represents a novel class of trimeric intracellular cation channels. Two TRIC isoforms have been identified in both the human and the mouse genomes: TRIC-A, a subtype predominantly expressed in the sarcoplasmic reticulum (SR) of muscle cells, and TRIC-B, a ubiquitous subtype expressed in the endoplasmic reticulum (ER) of all tissues. Genetic ablation of either TRIC-A or TRIC-B leads to compromised K(+) permeation and Ca(2+) release across the SR/ER membrane, supporting the hypothesis that TRIC channels provide a counter balancing K(+) flux that reduces SR/ER membrane depolarization for maintenance of the electrochemical gradient that drives SR/ER Ca(2+) release. TRIC-A and TRIC-B seem to have differential functions in Ca(2+) signaling in excitable and nonexcitable cells. Tric-a(-/-) mice display defective Ca(2+) sparks and spontaneous transient outward currents in arterial smooth muscle and develop hypertension, in addition to skeletal muscle dysfunction. Knockout of TRIC-B results in abnormal IP3 receptor-mediated Ca(2+) release in airway epithelial cells, respiratory defects, and neonatal lethality. Double knockout mice lacking both TRIC-A and TRIC-B show embryonic lethality as a result of cardiac arrest. Such an aggravated lethality indicates that TRIC-A and TRIC-B share complementary physiological functions in Ca(2+) signaling in embryonic cardiomyocytes. Tric-a(-/-) and Tric-b(+/-) mice are viable and susceptible to stress-induced heart failure. Recent evidence suggests that TRIC-A directly modulates the function of the cardiac ryanodine receptor 2 Ca(2+) release channel, which in turn controls store-overload-induced Ca(2+) release from the SR. Thus, the TRIC channels, in addition to providing a countercurrent for SR/ER Ca(2+) release, may also function as accessory proteins that directly modulate the ryanodine receptor/IP3 receptor channel functions. PMID:24526676

  19. Intracellular calcium levels are differentially regulated in T lymphocytes triggered by anti-CD2 and anti-CD3 monoclonal antibodies.

    PubMed

    Spinozzi, F; Agea, E; Bistoni, O; Belia, S; Travetti, A; Gerli, R; Muscat, C; Bertotto, A

    1995-03-01

    Antigen and/or mitogen-driven T-cell activation is mediated by a rise in intracellular free Ca2+, as second messenger. A regulatory key role for this process is represented by membrane-associated [Ca2+/Mg2+] ATP-ase that is mainly devoted to extrusion of intracellular ion excess. In the present study we have investigated the kinetics of CA2+ fluxes in both resting and already activated (Jurkat T-cell line) T lymphocytes after CD3 and CD2 (T11(2) and T11(3)) triggering and focused our attention on plasma membrane [Ca2+/Mg2+] ATP-ase activity. In both resting T cells and Jurkat cell line, the CD2 stimulation was able to determine a rise in intracellular free Ca2+ higher than that observed after CD3 triggering. In addition, this calcium signal was independent of negative feedback control exerted by [Ca2+/Mg2+] ATP-ase, as well as of IP3 generation. Thus the CD2 molecular system may, together with cell-adhesion properties, act as an amplifier of Ca2+ signals that, if delivered in the context of other molecular systems, such as CD3 or MHC class II antigens, are essentially devoted to the polyclonal co-stimulatory recruitment of a larger cellular repertoire. PMID:7662514

  20. Sodium-Calcium Exchanger 1 Regulates Epithelial Cell Migration via Calcium-dependent Extracellular Signal-regulated Kinase Signaling*

    PubMed Central

    Balasubramaniam, Sona Lakshme; Gopalakrishnapillai, Anilkumar; Gangadharan, Vimal; Duncan, Randall L.; Barwe, Sonali P.

    2015-01-01

    Na+/Ca2+ exchanger-1 (NCX1) is a major calcium extrusion mechanism in renal epithelial cells enabling the efflux of one Ca2+ ion and the influx of three Na+ ions. The gradient for this exchange activity is provided by Na,K-ATPase, a hetero-oligomer consisting of a catalytic α-subunit and a regulatory β-subunit (Na,K-β) that also functions as a motility and tumor suppressor. We showed earlier that mice with heart-specific ablation (KO) of Na,K-β had a specific reduction in NCX1 protein and were ouabain-insensitive. Here, we demonstrate that Na,K-β associates with NCX1 and regulates its localization to the cell surface. Madin-Darby canine kidney cells with Na,K-β knockdown have reduced NCX1 protein and function accompanied by 2.1-fold increase in free intracellular calcium and a corresponding increase in the rate of cell migration. Increased intracellular calcium up-regulated ERK1/2 via calmodulin-dependent activation of PI3K. Both myosin light chain kinase and Rho-associated kinase acted as mediators of ERK1/2-dependent migration. Restoring NCX1 expression in β-KD cells reduced migration rate and ERK1/2 activation, suggesting that NCX1 functions downstream of Na,K-β in regulating cell migration. In parallel, inhibition of NCX1 by KB-R7943 in Madin-Darby canine kidney cells, LLC-PK1, and human primary renal epithelial cells (HREpiC) increased ERK1/2 activation and cell migration. This increased migration was associated with high myosin light chain phosphorylation by PI3K/ERK-dependent mechanism in HREpiC cells. These data confirm the role of NCX1 activity in regulating renal epithelial cell migration. PMID:25770213

  1. Role of intracellular calcium and sodium in light adaptation in the retina of the honey bee drone (Apis mellifera, L)

    PubMed Central

    1976-01-01

    In the honey bee drone, the decrease in sensitivity to light of a retinula cell exposed to background illumination was found to be accurately reflected by the difference in amplitude between the initial transient depolarization and the lowest steady depolarization evoked by the background light. It is shown that both the decrease in sensitivity to light and the accompanying drop in potential from the transient to the plateau can be prevented by injecting EGTA intracellularly. A decrease in duration and amplitude of responses to short test flashes such as observed immediately after illumination was found to occur too when Ca or Na, but not K, Li, or Mg injected into dark-adapted retinula cells. Injection of EGTA into a retinula cell maintained a steady state of light adaptation, was found to cause an increase in amplitude and duration of the response to a short test flash, thus producing the effects of dark adaptation. It is suggested that, in the retina of the honey bee drone, an increase in intracellular calcium concentration plays a central role in light adaptation and that an increase in intracellular sodium concentration, resulting from the influx of sodium ions during the responses to light, could lead to this increase in intracellular free calcium. PMID:818341

  2. Modulation of the intracellular calcium concentration in photoreceptor terminals by a presynaptic metabotropic glutamate receptor

    PubMed Central

    Koulen, Peter; Kuhn, Rainer; Wässle, Heinz; Brandstätter, Johann Helmut

    1999-01-01

    Fast excitatory neurotransmission in the central nervous system is mediated through glutamate acting on ionotropic glutamate receptors. However, glutamate acting on metabotropic glutamate receptors (mGluRs) can also exert an inhibitory action. Here, we report by immunocytochemistry and physiology, to our knowledge, the first glutamate receptor to be found in terminals of photoreceptors in the mammalian retina—the group III metabotropic glutamate receptor mGluR8. Glutamate is the transmitter of photoreceptors, and thus mGluR8 functions as an autoreceptor. Activation of mGluR8 by the group III mGluR agonists l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate, or by glutamate itself, evokes a decrease in the intracellular calcium ion concentration ([Ca2+]i) in isolated photoreceptors. This effect is blocked by the group III mGluR antagonists (RS)-α-methyl-4-phosphonophenylglycine and (RS)-α-methylserine-O-phosphate. Agonists for other classes of glutamate receptors—n-methyl-d-aspartic acid, quisqualic acid, kainic acid, or (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid—have no effect on the [Ca2+]i in isolated photoreceptors. The down-regulation of the [Ca2+]i in photoreceptors by mGluR8 provides evidence for an inhibitory feedback loop at the photoreceptor synapse in the mammalian retina. This negative feedback may be a mechanism for the fine adjustment of the light-regulated release of glutamate from photoreceptors and may serve as a safety device against excitotoxic levels of release at this tonic synapse. Such a mechanism may provide a model for feedback inhibition in other parts of the central nervous system. PMID:10449793

  3. Sulfur mustard-induced increase in intracellular calcium: A mechanism of mustard toxicity

    SciTech Connect

    Ray, R.; Majerus, B.J.; Munavalli, G.S.; Petrali, J.P.

    1993-05-13

    The effect of sulfur mustard SM, bis-(2-chloroethyl) sulfide on intracellular free Ca2+ concentration (Ca2+)i was studied in vitro using the clonal mouse neuroblastoma-rat glioma hybrid NG108-15 and primary normal human epidermal keratinocyte (NHEK) cell culture models. SM depletes cellular glutathione (GSH) and thus may inhibit GSH-dependent Ca2+-ATPase (Ca2+ pump), leading to a high (Ca2+) and consequent cellular toxicity. Following 0.3 mM SM exposure, GSH levels decreased 20-34% between 1-6 hr in NG108-15 cells. SM increased (Ca2+)i, measured using the Ca2+-specific fluorescent probe Fluo-3 AM, in both NG108-15 cells (1030% between 2-6 hr) and NHEK (23-30% between 0.5-3 hr) . Depletion of cellular GSH by buthionine sulfoximine (1 mM), a specific GSH biosynthesis inhibitor, also increased Ca2+, (88% at 1 hr) in NHEK, suggesting that GSH depletion may lead to increased (Ca2+)i. Calcium, localized cytochemically with antimony, accumulated in increased amounts around mitochondria and endoplasmic reticula, in the cytosol, and in particular in the euchromatin regions of the nucleus beginning at 6 hr after 0.3 mM SM exposure of NG108-15 cells. Cell membrane integrity examined with the fluorescent membrane probe calcein AM was unaffected through 6 hr following 1 mM SM exposure; and cell viability (NG108-15 cells) measured by trypan blue exclusion was >80% of control through 9 hr following 0.3 mM SM exposure.

  4. Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro.

    PubMed

    Dobrinski, I; Smith, T T; Suarez, S S; Ball, B A

    1997-04-01

    Interaction of equine spermatozoa with oviductal epithelial cells (OEC) prolongs sperm viability and maintains low intracellular calcium concentration ([Ca2+]i) in spermatozoa. Experiments were designed to investigate 1) whether release of spermatozoa from OEC in vitro is associated with elevated [Ca2+]i and 2) whether soluble products from OEC or direct membrane contact between spermatozoa and OEC mediates the effects of OEC on sperm [Ca2+]i. In the first experiment, changes in [Ca2+]i in spermatozoa loaded with indo-1 acetoxymethylester were determined in motile spermatozoa released from OEC monolayers after 4 h of culture compared to [Ca2+]i in spermatozoa still attached to OEC. In addition, [Ca2+]i was determined in spermatozoa incubated with OEC-conditioned medium for 6 h compared to that in spermatozoa incubated in control medium. [Ca2+]i was higher in motile spermatozoa released from OEC than in spermatozoa still attached to OEC after 4 h of incubation. Incubation in OEC-conditioned medium resulted in lower sperm [Ca2+]i only at 4 h of incubation, but not at 0.5, 2, or 6 h of incubation. In the second experiment, a suspension of apical plasma membrane vesicles (AMV) isolated from isthmic oviductal epithelium was used to study the specific effect of sperm contact with OEC membranes on sperm viability, capacitation, and [Ca2+]i. Direct membrane contact between spermatozoa and AMV prolonged sperm viability, delayed capacitation, and maintained low [Ca2+]i in spermatozoa. These results indicated that membrane contact between equine spermatozoa and OEC is required to maintain low [Ca2+]i, delay capacitation, and prolong viability of spermatozoa in vitro. Modulation of capacitation rate for spermatozoa stored in the isthmic sperm reservoir might ensure the availability of a competent sperm population at the time of fertilization. PMID:9096866

  5. Insulin attenuates intracellular calcium responses and cell contraction caused by vasoactive agents.

    PubMed

    Inishi, Y; Okuda, T; Arakawa, T; Kurokawa, K

    1994-05-01

    In the present study, we examined the effects of insulin and insulin-like growth factor I (IGF-I) on cultured rat mesangial cell responses to vasoactive agents. Intracellular calcium concentration ([Ca2+]i) was measured with the Fura-2 method in suspended mesangial cells. Pretreatment of mesangial cells with insulin (from 0.05 to 5 micrograms/ml) attenuated Ca2+ transients by platelet activating factor (PAF) in a dose dependent and a time dependent manner. Insulin also attenuated sustained elevation of [Ca2+]i elicited by PAF. Basal [Ca2+]i was not affected by insulin pretreatment. Since the effective dose of insulin (0.5 microgram/ml or higher) is much higher than the physiological concentration, the effects of insulin may be via IGF-I receptor. Indeed, IGF-I (50 ng/ml) similarly attenuated [Ca2+]i responses to PAF. Moreover, insulin pretreatment attenuated [Ca2+]i responses evoked by angiotensin II (Ang II) and endothelin-1. In addition, the pretreatment with insulin or IGF-I inhibited mesangial cell contraction in response to Ang II. The suppression of [Ca2+]i responses to vasoactive agents by insulin was abolished when extracellular Ca2+ was removed. These data suggest that insulin, probably via IGF-I receptor, attenuates [Ca2+]i responses and cell contraction of mesangial cells induced by vasoactive agents. It is likely that the change in Ca2+ influx from outside to inside the cell underlie the effect of insulin. The modification of mesangial cell function through IGF-I receptor may play a role in the regulation of glomerular hemodynamics. PMID:8072243

  6. Intracellular influx of calcium induced by quartz particles in alveolar macrophages.

    PubMed

    Tian, Feng; Zhu, Tong; Shang, Yu

    2010-01-15

    Historical studies report that cellular injury and silicosis are related to cytosolic free calcium (Ca2+). Moreover, reactive oxygen species (ROS) have been linked to cellular injury. However, the detail mechanism of the increase in [Ca2+]i and the relationship between [Ca2+]i and ROS production remains unknown. Quartz particle has been found to increase [Ca2+]i and activate the generation of ROS. Our hypothesis is that [Ca2+]i increase induced by quartz particle is from extracellular Ca2+ through the Ca2+ channel, and [Ca2+]i increase is believed to activate ROS production. In order to examine this hypothesis, we treated rat alveolar macrophages with quartz (SiO2) particles and used laser scanning confocal microscopy to measure [Ca2+]i and the fluorescence intensity of ROS. Time- and dose-dependent increases in [Ca2+]I and ROS in macrophages as well as cell viability were observed. Through chelating extracellular Ca2+ with ethylene glycol tetraacetic acid and releasing intracellular Ca2+ with thapsigargin, we found that 72.7% of the [Ca2+]i increase was due to the influx of Ca2+ from the extracellular environment, via Ca2+ channels in the plasma membrane. By adding mannitol to scavenge hydroxyl radicals (OH(.)), and removing surface iron from the quartz particles to reduce OH(.) generation, we observed a reduced level of ROS generation, whereas the increase in [Ca2+]i was unaffected. When using EGTA to reduce [Ca2+]i, we observed a decrease in ROS production. This study suggests that the [Ca2+]i influx was independent of OH(.) production, and the [Ca2+]i increase resulted in ROS production. These results further indicate that there is a strong relationship between cytosolic free Ca2+ content and cellular injury as well as silica exposure. PMID:19835900

  7. Intravital imaging reveals p53-dependent cancer cell death induced by phototherapy via calcium signaling

    PubMed Central

    Missiroli, Sonia; Poletti, Federica; Ramirez, Fabian Galindo; Morciano, Giampaolo; Morganti, Claudia; Pandolfi, Pier Paolo; Mammano, Fabio; Pinton, Paolo

    2015-01-01

    One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca2+). In the present study, we established conditions that allow the in vivo detection of Ca2+ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca2+ concentrations and, consequently, an increase in cell death in a p53-dependent pathway. PMID:25544762

  8. Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel.

    PubMed

    Parkinson, Katie; Baines, Abigail E; Keller, Thomas; Gruenheit, Nicole; Bragg, Laricia; North, R Alan; Thompson, Christopher R L

    2014-01-01

    Rab GTPases play key roles in the delivery, docking and fusion of intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase activity is controlled is poorly understood. Here we describe a mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. We show that activation of P2XA, an intracellular ion channel localized to the Dictyostelium discoideum contractile vacuole system, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Vacuole fusion and Rab11a downregulation require the activity of CnrF, an EF-hand-containing Rab GAP found in a complex with Rab11a and P2XA. CnrF Rab GAP activity for Rab11a is enhanced by the presence of calcium and the EF-hand domain. These findings suggest that P2XA activation results in vacuolar calcium release, which triggers activation of CnrF Rab GAP activity and subsequent downregulation of Rab11a to allow vacuole fusion. PMID:24335649

  9. Intracellular sensing of microbes and danger signals by the inflammasomes.

    PubMed

    Horvath, Gabor L; Schrum, Jacob E; De Nardo, Christine M; Latz, Eicke

    2011-09-01

    The cells of the innate immune system mobilize a coordinated immune response towards invading microbes and after disturbances in tissue homeostasis. These immune responses typically lead to infection control and tissue repair. Exaggerated or uncontrolled immune responses, however, can also induce acute of chronic inflammatory pathologies that are characteristic for many common diseases such as sepsis, arthritis, atherosclerosis, or Alzheimer's disease. In recent years, the concerted efforts of many scientists have uncovered numerous mechanisms by which immune cells detect foreign or changed self-substances that appear in infections or during tissue damage. These substances stimulate signaling receptors, which leads to cellular activation and the induction of effector mechanisms. Here, we review the role of inflammasomes, a family of signaling molecules that form multi-molecular signaling platforms and activate inflammatory caspases and interleukin-1β cytokines. PMID:21884172

  10. Intracellular sensing of microbes and danger signals by the inflammasomes

    PubMed Central

    Horvath, Gabor L.; Schrum, Jacob E.; De Nardo, Christine M.

    2013-01-01

    Summary The cells of the innate immune system mobilize a coordinated immune response towards invading microbes and after disturbances in tissue homeostasis. These immune responses typically lead to infection control and tissue repair. Exaggerated or uncontrolled immune responses, however, can also induce acute of chronic inflammatory pathologies that are characteristic for many common diseases such as sepsis, arthritis, atherosclerosis or Alzheimer’s disease. In recent years the concerted efforts of many scientists have uncovered numerous mechanisms by which immune cells detect foreign or changed self-substances that appear in infections or during tissue damage. These substances stimulate signaling receptors, which leads to cellular activation and the induction of effector mechanisms. Here, we review the role of inflammasomes, a family of signaling molecules that form multi-molecular signaling platforms and activate inflammatory caspases and IL-1β cytokines. PMID:21884172

  11. Calcium signaling phenomena in heart diseases: a perspective.

    PubMed

    Chakraborti, Sajal; Das, Sudip; Kar, Pulak; Ghosh, Biswarup; Samanta, Krishna; Kolley, Saurav; Ghosh, Samarendranath; Roy, Soumitra; Chakraborti, Tapati

    2007-04-01

    Ca(2+) is a major intracellular messenger and nature has evolved multiple mechanisms to regulate free intracellular (Ca(2+))(i) level in situ. The Ca(2+) signal inducing contraction in cardiac muscle originates from two sources. Ca(2+) enters the cell through voltage dependent Ca(2+) channels. This Ca(2+) binds to and activates Ca(2+) release channels (ryanodine receptors) of the sarcoplasmic reticulum (SR) through a Ca(2+) induced Ca(2+) release (CICR) process. Entry of Ca(2+) with each contraction requires an equal amount of Ca(2+) extrusion within a single heartbeat to maintain Ca(2+) homeostasis and to ensure relaxation. Cardiac Ca(2+) extrusion mechanisms are mainly contributed by Na(+)/Ca(2+) exchanger and ATP dependent Ca(2+) pump (Ca(2+)-ATPase). These transport systems are important determinants of (Ca(2+))(i) level and cardiac contractility. Altered intracellular Ca(2+) handling importantly contributes to impaired contractility in heart failure. Chronic hyperactivity of the beta-adrenergic signaling pathway results in PKA-hyperphosphorylation of the cardiac RyR/intracellular Ca(2+) release channels. Numerous signaling molecules have been implicated in the development of hypertrophy and failure, including the beta-adrenergic receptor, protein kinase C, Gq, and the down stream effectors such as mitogen activated protein kinases pathways, and the Ca(2+) regulated phosphatase calcineurin. A number of signaling pathways have now been identified that may be key regulators of changes in myocardial structure and function in response to mutations in structural components of the cardiomyocytes. Myocardial structure and signal transduction are now merging into a common field of research that will lead to a more complete understanding of the molecular mechanisms that underlie heart diseases. Recent progress in molecular cardiology makes it possible to envision a new therapeutic approach to heart failure (HF), targeting key molecules involved in intracellular Ca(2

  12. Dendritic signal transmission induced by intracellular charge inhomogeneities

    NASA Astrophysics Data System (ADS)

    Lazarevich, Ivan A.; Kazantsev, Victor B.

    2013-12-01

    Signal propagation in neuronal dendrites represents the basis for interneuron communication and information processing in the brain. Here we take into account charge inhomogeneities arising in the vicinity of ion channels in cytoplasm and obtain a modified cable equation. We show that charge inhomogeneities acting on a millisecond time scale can lead to the appearance of propagating waves with wavelengths of hundreds of micrometers. They correspond to a certain frequency band predicting the appearance of resonant properties in brain neuron signaling. We also show that membrane potential in spiny dendrites obeys the modified cable equation suggesting a crucial role of the spines in dendritic subthreshold resonance.

  13. Protective effect of nifedipine against cytotoxicity and intracellular calcium alterations induced by acetaminophen in rat hepatocyte cultures.

    PubMed

    Ellouk-Achard, S; Mawet, E; Thibault, N; Dutertre-Catella, H; Thevenin, M; Claude, J R

    1995-01-01

    Alteration of calcium homeostasis has been proposed to play a major role in cell necrosis induced by a variety of chemical agents such as acetaminophen (APAP). In this study, a potential protective effect of the dihydropyridine calcium channel blocking agent, nifedipine, was investigated in vitro on acetaminophen-induced hepatocyte damage. Rat hepatocytes were exposed during 20 hours to various concentrations of APAP (0.50 to 4.00 mM). The following metabolic and functional parameters were investigated: -lactate dehydrogenase (LDH) release as an indicator of plasma membrane integrity, -cell viability evaluated by the colorimetric MTT assay, and intracellular calcium concentration as evaluated by two fluorimetric methods: a scanning laser cytometer using indo-1-AM as fluorescent probe and a fluorescence plate reader using fluo-3-AM as calcium indicator. Incubation of hepatocytes with APAP alone in the range 0.50 to 4.00mM resulted in a dose-response relationship with regard to LDH release (243% to 750% of control) and to the loss of cell viability (0 to 67% of control). Moreover these results were correlated with a significant increase in cytosolic calcium content (189 to 406 nM). Nifedipine treatment prior to APAP exposure, partially prevented LDH release, the plasma membrane blebbing, and thereby the loss of viability. In addition, intracellular calcium level progressively returned within the limits of the control values with increasing concentrations of nifedipine. It can be concluded that, in vitro conditions, nifedipine pretreatment exhibits a preventive effect against acetaminophen hepatocyte injury. PMID:7497906

  14. Detection of light-induced changes of intracellular ionized calcium concentration in Limulus ventral photoreceptors using arsenazo III

    PubMed Central

    Brown, J. E.; Brown, P. K.; Pinto, L. H.

    1977-01-01

    1. The metallochromic indicator dye, arsenazo III, was injected intracellularly into Limulus ventral photoreceptor cells to concentrations greater than 1 mM. 2. The absorption spectrum (450-750 nm) of the dye in single dark-adapted cells was measured by a scanning microspectrophotometer. When a cell was light-adapted, the absorption of the dye changed; the difference spectrum had two maxima at about 610 and 660 nm, a broad minimum at about 540 nm and an isosbestic point at about 585 nm. 3. When intracellular calcium concentration was raised in dark-adapted cells previously injected with arsenazo III, the difference spectum had two maxima at about 610 and 660 nm, a broad minimum at about 530 nm and an isosbestic point at about 585 nm. The injection of Mg2+ into dark-adapted cells previously injected with the dye induced a difference spectrum that had a single maximum at about 620 nm. Also, decreasing the intracellular pH of cells previously injected with the dye induced a difference spectrum that had a minimum at about 620 nm. The evidence suggests that there is a rise of intracellular ionized calcium when a Limulus ventral photoreceptor is light-adapted. 4. The intracellular calcium concentration, [Ca2+]1, in light-adapted photoreceptors was estimated to reach at least 10-4 M by compaing the light-induced difference spectra measured in ventral photoreceptors with a standard curve determined in microcuvettes containing 2mM arsenazo III in 400 mM-KCl, 1 mM-MgCl2 and 25 mM MOPS at pH 7·0. 5. In cells injected to less than 3 mM arsenazo III, light induced a transient decrease in optical transmission at 660 nm (T660). This decrease in T660 indicates that illumination of a ventral photoreceptor normally causes a transient increase of [Ca2+]1. 6. Arsenazo III was found to be sensitive, selective and rapid enough to measure light-induced changes of intracellular ionized calcium in Limulus ventral photoreceptor cells. PMID:17732

  15. Intracellular autocrine VEGF signaling promotes EBDC cell proliferation, which can be inhibited by Apatinib.

    PubMed

    Peng, Sui; Zhang, Yanyan; Peng, Hong; Ke, Zunfu; Xu, Lixia; Su, Tianhong; Tsung, Allan; Tohme, Samer; Huang, Hai; Zhang, Qiuyang; Lencioni, Riccardo; Zeng, Zhirong; Peng, Baogang; Chen, Minhu; Kuang, Ming

    2016-04-10

    Tumor cells produce vascular endothelial growth factor (VEGF) which can interact with membrane or cytoplasmic VEGF receptors (VEGFRs) to promote cell growth. We aimed to investigate the role of extracellular/intracellular autocrine VEGF signaling and Apatinib, a highly selective VEGFR2 inhibitor, in extrahepatic bile duct cancer (EBDC). We found conditioned medium or recombinant human VEGF treatment promoted EBDC cell proliferation through a phospholipase C-γ1-dependent pathway. This pro-proliferative effect was diminished by VEGF, VEGFR1 or VEGFR2 neutralizing antibodies, but more significantly suppressed by intracellular VEGFR inhibitor. The rhVEGF induced intracellular VEGF signaling by promoting nuclear accumulation of pVEGFR1/2 and enhancing VEGF promoter activity, mRNA and protein expression. Internal VEGFR2 inhibitor Apatinib significantly inhibited intracellular VEGF signaling, suppressed cell proliferation in vitro and delayed xenograft tumor growth in vivo, while anti-VEGF antibody Bevacizumab showed no effect. Clinically, overexpression of pVEGFR1 and pVEGFR2 was significantly correlated with poorer overall survival (P = .007 and P = .020, respectively). In conclusion, the intracellular autocrine VEGF loop plays a predominant role in VEGF-induced cell proliferation. Apatinib is an effective intracellular VEGF pathway blocker that presents a great therapeutic potential in EBDC. PMID:26805764

  16. Application of atomic force microscopy for studying intracellular signalization in neurons

    NASA Astrophysics Data System (ADS)

    Ankudinov, A. V.; Khalisov, M. M.; Penniyainen, V. A.; Podzorova, S. A.; Krylov, B. V.

    2015-10-01

    The first attempt is made at applying the method of atomic force microscopy (AFM) for determining the molecular mechanisms of intracellular signalization with the participation of Na+, K+-ATPhase playing an important role of a signal transductor (amplifier). The AFM method combined with the organotypic cultivation makes it possible to obtain quantitative information on the Young moduli of living neurons and cells subjected to the action of very low concentrations of ouabain. This substance is known to trigger in this case the intracellular signalization processes by transferring a molecular signal to the genome of a cell. The cell response is manifested in a sharp intensification of protein synthesis accompanied by a rearrangement of the cytoskeleton and activation of enzyme signal pathways in a cytosol. AFM measurements of the images of the cell surface relief are performed using the PeakForce quantitative nanomechanical properties mapping PeakForce QNM mode. The Young moduli of control neurons and of sensory neurons under the action of ouabain are measured simultaneously. It is found that the activation of the signal-transducing function of Na+, K+-ATPhase triggers intracellular signal cascades, which increase the cell stiffness. The application of the AFM method in further studies of the mechanisms of intracellular molecular processes appears as promising. Its combination with inhibitory analysis will clarify the role of individual molecules (e.g., a number of ferments) in regulation of growth and development of living organisms.

  17. Calcium signaling as a mediator of cell energy demand and a trigger to cell death.

    PubMed

    Bhosale, Gauri; Sharpe, Jenny A; Sundier, Stephanie Y; Duchen, Michael R

    2015-09-01

    Calcium signaling is pivotal to a host of physiological pathways. A rise in calcium concentration almost invariably signals an increased cellular energy demand. Consistent with this, calcium signals mediate a number of pathways that together serve to balance energy supply and demand. In pathological states, calcium signals can precipitate mitochondrial injury and cell death, especially when coupled to energy depletion and oxidative or nitrosative stress. This review explores the mechanisms that couple cell signaling pathways to metabolic regulation or to cell death. The significance of these pathways is exemplified by pathological case studies, such as those showing loss of mitochondrial calcium uptake 1 in patients and ischemia/reperfusion injury. PMID:26375864

  18. Calcium signaling as a mediator of cell energy demand and a trigger to cell death

    PubMed Central

    Bhosale, Gauri; Sharpe, Jenny A.; Sundier, Stephanie Y.

    2015-01-01

    Calcium signaling is pivotal to a host of physiological pathways. A rise in calcium concentration almost invariably signals an increased cellular energy demand. Consistent with this, calcium signals mediate a number of pathways that together serve to balance energy supply and demand. In pathological states, calcium signals can precipitate mitochondrial injury and cell death, especially when coupled to energy depletion and oxidative or nitrosative stress. This review explores the mechanisms that couple cell signaling pathways to metabolic regulation or to cell death. The significance of these pathways is exemplified by pathological case studies, such as those showing loss of mitochondrial calcium uptake 1 in patients and ischemia/reperfusion injury. PMID:26375864

  19. Calcium signaling and the secretory activity of bile duct epithelia.

    PubMed

    Amaya, Maria Jimena; Nathanson, Michael H

    2014-06-01

    Cytosolic calcium (Cai(2+)) is a second messenger that is important for the regulation of secretion in many types of tissues. Bile duct epithelial cells, or cholangiocytes, are polarized epithelia that line the biliary tree in liver and are responsible for secretion of bicarbonate and other solutes into bile. Cai(2+) signaling plays an important role in the regulation of secretion by cholangiocytes, and this review discusses the machinery involved in the formation of Ca(2+) signals in cholangiocytes, along with the evidence that these signals regulate ductular secretion. Finally, this review discusses the evidence that impairments in cholangiocyte Ca(2+) signaling play a primary role in the pathogenesis of cholestatic disorders, in which hepatic bile secretion is impaired. PMID:24612866

  20. Ion channels and calcium signaling in motile cilia

    PubMed Central

    Doerner, Julia F; Delling, Markus; Clapham, David E

    2015-01-01

    The beating of motile cilia generates fluid flow over epithelia in brain ventricles, airways, and Fallopian tubes. Here, we patch clamp single motile cilia of mammalian ependymal cells and examine their potential function as a calcium signaling compartment. Resting motile cilia calcium concentration ([Ca2+] ~170 nM) is only slightly elevated over cytoplasmic [Ca2+] (~100 nM) at steady state. Ca2+ changes that arise in the cytoplasm rapidly equilibrate in motile cilia. We measured CaV1 voltage-gated calcium channels in ependymal cells, but these channels are not specifically enriched in motile cilia. Membrane depolarization increases ciliary [Ca2+], but only marginally alters cilia beating and cilia-driven fluid velocity within short (~1 min) time frames. We conclude that beating of ependymal motile cilia is not tightly regulated by voltage-gated calcium channels, unlike that of well-studied motile cilia and flagella in protists, such as Paramecia and Chlamydomonas. DOI: http://dx.doi.org/10.7554/eLife.11066.001 PMID:26650848

  1. Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells.

    PubMed

    Simões-Wüst, A P; Grãos, M; Duarte, C B; Brenneisen, R; Hamburger, M; Mennet, M; Ramos, M H; Schnelle, M; Wächter, R; Worel, A M; von Mandach, U

    2010-10-01

    The use of preparations from Bryophyllum pinnatum in tocolysis is supported by both clinical (retrospective comparative studies) and experimental (using uterus strips) evidence. We studied here the effect of B. pinnatum juice on the response of cultured human myometrial cells to stimulation by oxytocin, a hormone known to be involved in the control of uterine contractions by increasing the intracellular free calcium concentration ([Ca2+]i). In this work, [Ca2+]i was measured online during stimulation of human myometrial cells (hTERT-C3 and M11) with oxytocin, which had been pre-incubated in the absence or in the presence of B. pinnatum juice. Since no functional voltage-gated Ca2+ channels could be detected in these myometrial cells, the effect of B. pinnatum juice was as well studied in SH-SY5Y neuroblastoma cells, which are known to have such channels and can be depolarised with KCl. B. pinnatum juice prevented the oxytocin-induced increase in [Ca2+]i in hTERT-C3 human myometrial cells in a dose-dependent manner, achieving a ca. 80% inhibition at a 2% concentration. Comparable results were obtained with M11 human primary myometrial cells. In hTERT-C3 cells, prevention of the oxytocin-induced increase in [Ca2+]i was independent of the extracellular Ca2+ concentration and of voltage-dependent Ca2+-channels. B. pinnatum juice delayed, but did not prevent the depolarization-induced increase in [Ca2+]i in SH-SY5Y cells. Taken together, the data suggest a specific and concentration-dependent effect of B. pinnatum juice on the oxytocin signalling pathway, which seems to corroborate its use in tocolysis. Such a specific mechanism would explain the rare and minor side-effects in tocolysis with B. pinnatum as well as its high therapeutic index. PMID:20381326

  2. Responsiveness of vomeronasal cells to a newt peptide pheromone, sodefrin as monitored by changes of intracellular calcium concentrations.

    PubMed

    Iwata, Takeo; Nakada, Tomoaki; Toyoda, Fumiyo; Yada, Toshihiko; Shioda, Seiji; Kikuyama, Sakae

    2013-07-01

    A peptide pheromone of the red-bellied male newt, sodefrin was tested for its ability to increase intracellular concentrations of Ca(2+) ([Ca(2+)]i) in the dissociated vomeronasal (VN) cells of females by means of calcium imaging system. The pheromone elicited a marked elevation of [Ca(2+)]i in a small population of VN cells from sexually developed females. The population of cells exhibiting sodefrin-induced elevation of [Ca(2+)]i increased concentration-dependently. A pheromone of a different species was ineffective in this respect. The VN cells from non-reproductive females or from reproductive males scarcely responded to sodefrin in terms of elevating [Ca(2+)]i. In the cells from hypophysectomized and ovariectomized females, the sodefrin-inducible increase of [Ca(2+)]i never occurred. The cells from the operated newts supplemented with prolactin and estradiol exhibited [Ca(2+)]i responses to sodefrin with a high incidence. Thus, sex- and hormone-dependency as well as species-specificity of the responsiveness of the VN cells to sodefrin was evidenced at the cellular level. Subsequently, possibility of involvement of phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3) and/or PLC-diacylglycerol (DAG)-protein kinase C (PKC) pathways in increasing [Ca(2+)]i in VN cells in response to sodefrin was explored using pharmacological approaches. The results indicated that PLC is involved in generating the Ca(2+) signal in all sodefrin-responsive VN cells, whereas IP3 in approximately 50% of the cells and DAG-PKC in the remaining cells. In the latter case, the increase of [Ca(2+)]i was postulated to be induced by the influx of Ca(2+) through the L-type channel. The significance of the finding is discussed. PMID:23619348

  3. Calmodulin Binds to Extracellular Sites on the Plasma Membrane of Plant Cells and Elicits a Rise in Intracellular Calcium Concentration*S⃞

    PubMed Central

    Wang, Qinli; Chen, Bo; Liu, Peng; Zheng, Maozhong; Wang, Yuqing; Cui, Sujuan; Sun, Daye; Fang, Xiaohong; Liu, Chun-Ming; Lucas, William J.; Lin, Jinxing

    2009-01-01

    Calmodulin (CaM) is a highly conserved intracellular calcium sensor. In plants, CaM also appears to be present in the apoplasm, and application of exogenous CaM has been shown to influence a number of physiological functions as a polypeptide signal; however, the existence and localization of its corresponding apoplasmic binding sites remain controversial. To identify the site(s) of action, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection at the surface of plant cells. Using this approach, we show that QD-CaM binds selectively to sites on the outer surface of the plasma membrane, which was further confirmed by high resolution transmission electron microscopy. Measurements of Ca2+ fluxes across the plasma membrane, using ion-selective microelectrodes, demonstrated that exogenous CaM induces a net influx into protoplasts. Consistent with these flux studies, calcium-green-dextran and FRET experiments confirmed that applied CaM/QD-CaM elicited an increase in cytoplasmic Ca2+ levels. These results support the hypothesis that apoplasmic CaM can act as a signaling agent. These findings are discussed in terms of CaM acting as an apoplasmic peptide ligand to mediate transmembrane signaling in the plant kingdom. PMID:19254956

  4. A new, nongenomic estrogen action: the rapid release of intracellular calcium.

    PubMed

    Morley, P; Whitfield, J F; Vanderhyden, B C; Tsang, B K; Schwartz, J L

    1992-09-01

    We have investigated the effects of steroids on the intracellular calcium ion concentration [Ca2+]i in chicken granulosa cells obtained from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 100 +/- 5 nM. There was an immediate (i.e. less than 5 sec) 4- to 8-fold increase in [Ca2+]i in all of the 76 cells examined after the addition of 10(-7) M estradiol-17 bdta. Estradiol-17 beta was effective between 10(-10)-10(-6) M. Estradiol-17 alpha, estrone, and estriol (10(-8)-10(-6) M) were as effective as estradiol-17 beta, but the progestins, pregnenolone, and progesterone, and the androgens, testosterone, androstenedione, or 5 alpha-dihydrotestosterone were ineffective at concentrations up to 10(-5) M. The prompt estradiol-17 beta-induced [Ca2+]i spike was not affected by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating them with the Ca2+ channel blockers lanthanum (1 mM), cobalt (5 mM), methoxyverapamil (D600; 50 microM), or nifedipine (20 microM). The estrogen-triggered [Ca2+]i surge was also not affected by pretreating the cells with the conventional estrogen receptor antagonist tamoxifen (10(-5) M), or the RNA and protein synthesis inhibitors actinomycin D (1 microgram/ml) and cycloheximide (1 microgram/ml), but was abolished by pretreating the cells with inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) and U-73,122 (2.5 microM). The closely related, but inactive, compound U-73,343 (1 microM) did not affect the estrogen-triggered [Ca2+]i surge. Estradiol-17 beta (10(-7) M), but not progesterone (10(-5) M), also triggered a large [Ca2+]i surge in pig granulosa cells, which, like the [Ca2+]i surge in chicken granulosa cells, was almost immediate, transient, and unaffected by incubation in Ca(2+)-free medium or pretreatment with methoxyverapamil (D600; 50 microM), lanthanum (1 mM), or

  5. Detection and Quantification of Intracellular Signaling Using FRET-Based Biosensors and High Content Imaging.

    PubMed

    Halls, Michelle L; Poole, Daniel P; Ellisdon, Andrew M; Nowell, Cameron J; Canals, Meritxell

    2015-01-01

    Förster resonance energy transfer (FRET) biosensors represent invaluable tools to detect the spatiotemporal context of second messenger production and intracellular signaling that cannot be attained using traditional methods. Here, we describe a detailed protocol for the use of high content imaging in combination with FRET biosensors to assess second messenger production and intracellular signaling in a time-effective manner. We use four different FRET biosensors to measure cAMP levels, kinase (ERK and PKC), and GTPase activity. Importantly, we provide the protocols to express and measure these sensors in a variety of model cell lines and primary dorsal root ganglia neurons. PMID:26260599

  6. Involvement of intracellular calcium in anaerobic gene expression and survival of maize seedlings.

    PubMed Central

    Subbaiah, C C; Zhang, J; Sachs, M M

    1994-01-01

    Ca-mediated processes are known to be involved in transducing many developmental, hormonal, and environmental cues in plant cells. In this study, the role of Ca in the perception of anoxic stress signals by maize (Zea mays L. cv B73) roots was assessed by studying the effect of various Ca antagonists on the induction of alcohol dehydrogenase (ADH) and sucrose synthase mRNA as well as ADH activity under anoxia. The effect of these compounds on the poststress recovery of the seedlings was also monitored. Ruthenium red (RR), an inhibitor of organellar Ca fluxes, repressed the anoxic activation of the alcohol dehydrogenase1 and shrunken1 genes as measured by their transcript levels as well as ADH activity. Furthermore, RR-treated seedlings could not recover even after only 2 h of flooding, in contrast to untreated B73 seedlings that survived 72 h of submergence. Ca, when supplied along with RR, allowed normal anoxic gene expression and also prevented the RR-imposed death of the seedlings from short-term anoxia. Ca (45Ca) fluxes were measured in maize roots to elucidate the mode of action of RR. RR abolished anoxia-stimulated 45Ca influx into maize roots but did not affect aerobic Ca2+ uptake, unlike a few other antagonists that blocked both the aerobic and anoxic fluxes. However, Ca uptake across the plasma membrane was not necessary for the adaptive response to anoxia, since chelation of extracellular Ca by ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid did not affect the induction of ADH activity or poststress survival of flooded seedlings. The data suggest that RR may act on one of the intracellular stores of Ca and the Ca mobilized from this source is a physiological transducer of anoxic stress signals in maize roots. PMID:7518090

  7. Intracellular Calcium Gradients in Single Living Cells: Measurement and Analysis by Optical and Digital Techniques

    NASA Astrophysics Data System (ADS)

    Yelamarty, Rao Viswanadha

    Intracellular calcium (Ca^{2+ }) has been considered as a regulator of many cellular processes. In addition, Ca^{2+ } also plays a key role in mediating actions of many hormones, growth factors, and drugs. This thesis describes two general approaches, digital video and photomultiplier (PMT) based fluorescence microscopic systems, to measure such Ca^{2+} changes throughout the cell. They reveal the heterogeneous spatial and fast temporal changes of Ca^{2+} within a single isolated living cell. In order to measure spatial Ca^ {2+} in three dimensions (3-D), optical section microscopy (OSM) coupled to digital video imaging is introduced. With this approach, an increase in nuclear Ca^{2+} compared to cytosolic Ca^{2+} is detected in human erythroblasts and rat hepatocytes under the addition of growth factors: erythropoietin and epidermal growth factor respectively. In addition, the primary effect of non growth-promoting hormone vasopressin, raise in cytosolic Ca^{2+}, is also observed. These observations are the first to underscore the importance of nuclear Ca^{2+} increase in cell growth and differentiation. On the other hand, to track fast Ca^ {2+} transients (mesc) during excitation -contraction (EC) cycle and then examine alterations in Ca^{2+} transients in healthy and diseased (hypertensive) heart cells, a PMT based system is implemented. Significant alterations in Ca^{2+} transients in hypertensive heart cells were observed. This finding is compatible with the clinical finding that patients with hypertensive cardiomyopathy suffer a lack of adequate relaxation. Finally, to correlate the Ca^{2+} dynamics in an EC cycle with mechanical activity, a hybrid optical digital processor was developed. The performance of the hybrid processor is analyzed and applied simultaneously with the PMT based system. The mechanical contraction and relaxation of a single cardiac cell closely paralleled that of Ca^{2+} dynamics during an EC cycle. In summary, this thesis illustrates

  8. Intracellular influx of calcium induced by quartz particles in alveolar macrophages

    SciTech Connect

    Feng Tian; Tong Zhu; Yu Shang

    2010-01-15

    Historical studies report that cellular injury and silicosis are related to cytosolic free calcium (Ca{sup 2+}). Moreover, reactive oxygen species (ROS) have been linked to cellular injury. However, the detail mechanism of the increase in [Ca{sup 2+}]{sub i} and the relationship between [Ca{sup 2+}]{sub i} and ROS production remains unknown. Quartz particle has been found to increase [Ca{sup 2+}]{sub i} and activate the generation of ROS. Our hypothesis is that [Ca{sup 2+}]{sub i} increase induced by quartz particle is from extracellular Ca{sup 2+} through the Ca{sup 2+} channel, and [Ca{sup 2+}]{sub i} increase is believed to activate ROS production. In order to examine this hypothesis, we treated rat alveolar macrophages with quartz (SiO{sub 2}) particles and used laser scanning confocal microscopy to measure [Ca{sup 2+}]{sub i} and the fluorescence intensity of ROS. Time- and dose-dependent increases in [Ca{sup 2+}]{sub I} and ROS in macrophages as well as cell viability were observed. Through chelating extracellular Ca{sup 2+} with ethylene glycol tetraacetic acid and releasing intracellular Ca{sup 2+} with thapsigargin, we found that 72.7% of the [Ca{sup 2+}]{sub i} increase was due to the influx of Ca{sup 2+} from the extracellular environment, via Ca{sup 2+} channels in the plasma membrane. By adding mannitol to scavenge hydroxyl radicals (OH.), and removing surface iron from the quartz particles to reduce OH. generation, we observed a reduced level of ROS generation, whereas the increase in [Ca{sup 2+}]{sub i} was unaffected. When using EGTA to reduce [Ca{sup 2+}]{sub i}, we observed a decrease in ROS production. This study suggests that the [Ca{sup 2+}]{sub i} influx was independent of OH. production, and the [Ca{sup 2+}]{sub i} increase resulted in ROS production. These results further indicate that there is a strong relationship between cytosolic free Ca{sup 2+} content and cellular injury as well as silica exposure.

  9. When Neurons Encounter Nanoobjects: Spotlight on Calcium Signalling

    PubMed Central

    Lovisolo, Davide; Gilardino, Alessandra; Ruffinatti, Federico Alessandro

    2014-01-01

    Nanosized objects are increasingly present in everyday life and in specialized technological applications. In recent years, as a consequence of concern about their potential adverse effects, intense research effort has led to a better understanding of the physicochemical properties that underlie their biocompatibility or potential toxicity, setting the basis for a rational approach to their use in the different fields of application. Among the functional parameters that can be perturbed by interaction between nanoparticles (NPs) and living structures, calcium homeostasis is one of the key players and has been actively investigated. One of the most relevant biological targets is represented by the nervous system (NS), since it has been shown that these objects can access the NS through several pathways; moreover, engineered nanoparticles are increasingly developed to be used for imaging and drug delivery in the NS. In neurons, calcium homeostasis is tightly regulated through a complex set of mechanisms controlling both calcium increases and recovery to the basal levels, and even minor perturbations can have severe consequences on neuronal viability and function, such as excitability and synaptic transmission. In this review, we will focus on the available knowledge about the effects of NPs on the mechanisms controlling calcium signalling and homeostasis in neurons. We have taken into account the data related to environmental NPs, and, in more detail, studies employing engineered NPs, since their more strictly controlled chemical and physical properties allow a better understanding of the relevant parameters that determine the biological responses they elicit. The literature on this specific subject is all quite recent, and we have based the review on the data present in papers dealing strictly with nanoparticles and calcium signals in neuronal cells; while they presently amount to about 20 papers, and no related review is available, the field is rapidly growing and

  10. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators

    SciTech Connect

    Noah, T.L.; Paradiso, A.M.; Madden, M.C.; McKinnon, K.P.; Devlin, R.B. )

    1991-11-01

    Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. The authors therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of their data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

  11. Depletion of calcium stores regulates calcium influx and signal transmission in rod photoreceptors

    PubMed Central

    Szikra, Tamas; Cusato, Karen; Thoreson, Wallace B; Barabas, Peter; Bartoletti, Theodore M; Krizaj, David

    2008-01-01

    Tonic synapses are specialized for sustained calcium entry and transmitter release, allowing them to operate in a graded fashion over a wide dynamic range. We identified a novel plasma membrane calcium entry mechanism that extends the range of rod photoreceptor signalling into light-adapted conditions. The mechanism, which shares molecular and physiological characteristics with store-operated calcium entry (SOCE), is required to maintain baseline [Ca2+]i in rod inner segments and synaptic terminals. Sustained Ca2+ entry into rod cytosol is augmented by store depletion, blocked by La3+ and Gd3+ and suppressed by organic antagonists MRS-1845 and SKF-96365. Store depletion and the subsequent Ca2+ influx directly stimulated exocytosis in terminals of light-adapted rods loaded with the activity-dependent dye FM1–43. Moreover, SOCE blockers suppressed rod-mediated synaptic inputs to horizontal cells without affecting presynaptic voltage-operated Ca2+ entry. Silencing of TRPC1 expression with small interference RNA disrupted SOCE in rods, but had no effect on cone Ca2+ signalling. Rods were immunopositive for TRPC1 whereas cone inner segments immunostained with TRPC6 channel antibodies. Thus, SOCE modulates Ca2+ homeostasis and light-evoked neurotransmission at the rod photoreceptor synapse mediated by TRPC1. PMID:18755743

  12. [Low-dose radiation effects and intracellular signaling pathways].

    PubMed

    Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami

    2006-10-01

    Accumulated evidence has shown that exposure to low-dose radiation, especially doses less than 0.1 Gy, induces observable effects on mammalian cells. However, the underlying molecular mechanisms have not yet been clarified. Recently, it has been shown that low-dose radiation stimulates growth factor receptor, which results in a sequential activation of the mitogen-activated protein kinase pathway. In addition to the activation of the membrane-bound pathways, it is becoming evident that nuclear pathways are also activated by low-dose radiation. Ionizing radiation has detrimental effects on chromatin structure, since radiation-induced DNA double-strand breaks result in discontinuity of nucleosomes. Recently, it has been shown that ATM protein, the product of the ATM gene mutated in ataxia-telangiectasia, recognizes alteration in the chromatin structure, and it is activated through intermolecular autophosphorylation at serine 1981. Using antibodies against phosphorylated ATM, we found that the activated and phosphorylated ATM protein is detected as discrete foci in the nucleus between doses of 10 mGy and 1 Gy. Interestingly, the size of the foci induced by low-dose radiation was equivalent to the foci induced by high-dose radiation. These results indicate that the initial signal is amplified through foci growth, and cells evolve a system by which they can respond to a small number of DNA double-strand breaks. From these results, it can be concluded that low-dose radiation is sensed both in the membrane and in the nucleus, and activation of multiple signal transduction pathways could be involved in manifestations of low-dose effects. PMID:17016017

  13. Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway

    PubMed Central

    Lee, Eunkyung; Choi, So-Young; Yang, Jae-Ho

    2016-01-01

    Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway. PMID:27382356

  14. Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway.

    PubMed

    Lee, Eunkyung; Choi, So-Young; Yang, Jae-Ho; Lee, Youn Ju

    2016-07-01

    Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway. PMID:27382356

  15. Role of the JAKs/STATs pathway in the intracellular calcium changes induced by interleukin-6 in hippocampal neurons.

    PubMed

    Orellana, D I; Quintanilla, R A; Gonzalez-Billault, C; Maccioni, R B

    2005-11-01

    Recent studies show that inflammation has an active role in the onset of neurodegenerative diseases. It is known that in response to extracellular insults microglia and/or astrocytes produce inflammatory agents. These contribute to the neuropathological events in the aging process and neuronal degeneration. Interleukin-6 (IL-6) has been involved in the pathogenesis of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Here, we show that IL-6 treatment of rat hippocampal neurons increases the calcium influx via NMDA-receptor, an effect that is prevented by the specific NMDA receptor antagonist MK-801 (dizocilpine). We also show that this calcium influx is mediated by the JAKs/STATs pathway, since the inhibitor of JAKs/STATs pathway, JAK 3 inhibitor, blocks calcium influx even in the presence of IL-6. This increase in calcium signal was dependent on external sources, since this signal was not observed in the presence of EGTA. Additional studies indicate that the increase in cytosolic calcium induces tau protein hyperphosphorylation, as revealed by using specific antibodies against Alzheimer phosphoepitopes. This anomalous tau hyperphosphorylation was dependent on both the JAKs/STATs pathway and NMDA receptor. These results suggest that IL-6 would induce a cascade of molecular events that produce a calcium influx through NMDA receptors, mediated by the JAKs/STATs pathway, which subsequently modifies the tau hyperphosphorylation patterns. PMID:16371324

  16. Kinetic insulation as an effective mechanism for achieving pathway specificity in intracellular signaling networks

    PubMed Central

    Behar, Marcelo; Dohlman, Henrik G.; Elston, Timothy C.

    2007-01-01

    Intracellular signaling pathways that share common components often elicit distinct physiological responses. In most cases, the biochemical mechanisms responsible for this signal specificity remain poorly understood. Protein scaffolds and cross-inhibition have been proposed as strategies to prevent unwanted cross-talk. Here, we report a mechanism for signal specificity termed “kinetic insulation.” In this approach signals are selectively transmitted through the appropriate pathway based on their temporal profile. In particular, we demonstrate how pathway architectures downstream of a common component can be designed to efficiently separate transient signals from signals that increase slowly over time. Furthermore, we demonstrate that upstream signaling proteins can generate the appropriate input to the common pathway component regardless of the temporal profile of the external stimulus. Our results suggest that multilevel signaling cascades may have evolved to modulate the temporal profile of pathway activity so that stimulus information can be efficiently encoded and transmitted while ensuring signal specificity. PMID:17913886

  17. Intracellular calcium dynamics permit a Purkinje neuron model to perform toggle and gain computations upon its inputs

    PubMed Central

    Forrest, Michael D.

    2014-01-01

    Without synaptic input, Purkinje neurons can spontaneously fire in a repeating trimodal pattern that consists of tonic spiking, bursting and quiescence. Climbing fiber input (CF) switches Purkinje neurons out of the trimodal firing pattern and toggles them between a tonic firing and a quiescent state, while setting the gain of their response to Parallel Fiber (PF) input. The basis to this transition is unclear. We investigate it using a biophysical Purkinje cell model under conditions of CF and PF input. The model can replicate these toggle and gain functions, dependent upon a novel account of intracellular calcium dynamics that we hypothesize to be applicable in real Purkinje cells. PMID:25191262

  18. Effect of vanadate on ATP-induced increase in intracellular calcium ion levels in human umbilical vein endothelial cells.

    PubMed

    Nejime, Namie; Tada, Yukari; Kagota, Satomi; Kubota, Yoko; Shibuichi, Ikuo; Shinoda, Yuki; Yamamoto, Tomohiro; Watanabe, Yasuo; Shinozuka, Kazumasa

    2010-01-01

    We investigated the effect of ammonium vanadate (vanadate) on ATP-induced increases in intracellular calcium ion level ([Ca(2+)](i)) of human umbilical vein endothelial cells (HUVEC) by fluorescence confocal microscopic imaging using the Ca(2+)-sensitive probe Calcium Green 1/AM. The ATP analogue 2-methylthio-ATP (2meS-ATP), at 10 microM, significantly increased the [Ca(2+)](i) of HUVEC, and this was abolished by 1 microM thapsigargin (a calcium pump inhibitor), whereas extracellular free calcium had no effect. Vanadate at 10 microM also significantly increased the [Ca(2+)](i) of HUVEC, which was abolished by 1 microM thapsigargin. However, vanadate at 1 microM did not exert such a significant effect. We thus examined the influence of < or =1 microM vanadate for 24 h on 2meS-ATP-induced increase in [Ca(2+)](i). Vanadate significantly reduced the action of 2meS-ATP at 1 microM but not at 0.1 microM. Endogenously released ATP is known to induce various actions on endothelial cells. The present results suggest that vanadate exerts a regulatory influence on the function of vascular endothelial cells. PMID:20522978

  19. Intracellular calcium disturbances induced by arsenic and its methylated derivatives in relation to genomic damage and apoptosis induction.

    PubMed

    Florea, Ana-Maria; Yamoah, Ebenezer N; Dopp, Elke

    2005-06-01

    Arsenic and its methylated derivatives are contaminants of air, water, and food and are known as toxicants and carcinogens. Arsenic compounds are also being used as cancer chemotherapeutic agents. In humans, inorganic arsenic is metabolically methylated to mono-, di-, and trimethylated forms. Recent findings suggest that the methylation reactions represent a toxification rather than a detoxification pathway. In recent years, the correlation between arsenic exposure, cytotoxicity and genotoxicity, mutagenicity, and tumor promotion has been established, as well as the association of arsenic exposure with perturbation of physiologic processes, generation of reactive oxygen species, DNA damage, and apoptosis induction. Trivalent forms of arsenic have been found to induce apoptosis in several cellular systems with involvement of membrane-bound cell death receptors, activation of caspases, release of calcium stores, and changes of the intracellular glutathione level. It is well known that calcium ion deregulation plays a critical role in apoptotic cell death. A calcium increase in the nuclei might lead to toxic effects in the cell. In this review, we highlight the relationship between induced disturbances of calcium homeostasis, genomic damage, and apoptotic cell death caused by arsenic and its organic derivatives. PMID:15929885

  20. Oxidized low density lipoprotein (LDL) and platelet intracellular calcium: interaction with nitric oxide.

    PubMed

    Zuliani, V; Tommasol, R; Gaino, S; Degan, M; Cominacini, L; Davoli, A; Lechi, C; Lechi, A; Minuz, P

    1998-01-01

    The present study tested the effects of ox-low density lipoprotein (LDL) on nitric oxide (NO)-dependent decrease in agonist-stimulated [Ca2+]i. The effects of ox-LDL on platelet aggregation were also evaluated. Platelets loaded with FURA 2 AM (2 micromol/litre) were incubated with NO-donors for 2-10 min to obtain a 40-50% reduction in \\[Ca2+]i and with NO-donors plus ox-LDL (100 microg of protein/ml). Thrombin (0.03 U/ml) was used as an agonist. In some experiments 8-Br-cGMP (0.5-1 mmol/l) was used to investigate the NO-dependent intraplatelet signalling system. Slightly oxidized LDL was obtained by leaving native LDL in the light at room temperature for at least 7 days. Ox-LDL did not cause any increase in thrombin-induced [Ca2+] (control: 215.4 +/- 44.3 nmol/l, ox-LDL 223.4 +/- 35.3 nmol/l, M +/- SEM; n = 8) and platelet aggregation (control: 78.7 +/- 4.9% , ox-LDL: 78.9 +/- 4.2% , n = 12). Ox-LDL antagonized the effects of NO-donors on platelet [Ca2+]i (NO-donor: 137.4 +/- 22.1 nmol/l, NO + ox-LDL: 177.3 +/- 27.6 nmol/l, n = 11; P < 0.001) and platelet aggregation (NO-donor: 15.4 +/- 3.4% , NO + ox-LDL: 28.9 +/- 3.8%, n = 24; P < 0.001). Ox-LDL did not affect the inhibitory activities of 8-Br-cGMP on platelet aggregation (8-Br-cGMP: 22.0 +/- 8.5%, 8-Br-cGMP + ox-LDL: 19.3 +/- 7.8%, n = 5) and platelet [Ca2+]i . In conclusion, slightly oxidized LDL does not directly activate platelets and does not i affect the intracellular NO-dependent signalling system. The present results suggest that LDL reduces the antiplatelet activity of NO mainly by preventing its biological effects. PMID:16793716

  1. Signaling in cells and organisms - calcium holds the line.

    PubMed

    Steinhorst, Leonie; Kudla, Jörg

    2014-12-01

    Previous research has established calcium (Ca(2+)) and reactive oxygen species (ROS) as important cellular second messengers in eukaryotes. Recently, the occurrence of cell-to-cell moving Ca(2+) and ROS waves was reported in plants. This was paralleled by the discovery of long-distance wound-activated surface potential changes (WASPs) that require the function of putatively Ca(2+)-releasing glutamate receptor-like channels (GLRs) in Arabidopsis. Although the functional interconnection of Ca(2+)-dependent phosphorylation and ROS waves via NADPH oxidase activation has been clearly established, potential further interconnections between these long-distance signaling processes are less clear. In this review we cover emerging concepts and existing open questions that interconnect cellular and global signaling via Ca(2+), ROS and WASPs. PMID:25195171

  2. Effects of hypercapnia on membrane potential and intracellular calcium in rat carotid body type I cells.

    PubMed Central

    Buckler, K J; Vaughan-Jones, R D

    1994-01-01

    1. An acid-induced rise in the intracellular calcium concentration ([Ca2+]i) of type I cells is thought to play a vital role in pH/PCO2 chemoreception by the carotid body. In this present study we have investigated the cause of this rise in [Ca2+]i in enzymatically isolated, neonatal rat type I cells. 2. The rise in [Ca2+]i induced by a hypercapnic acidosis was inhibited in Ca(2+)-free media, and by 2 mM Ni2+. Acidosis also increased Mn2+ permeability. The rise in [Ca2+]i is dependent, therefore, upon a Ca2+ influx from the external medium. 3. The acid-induced rise in [Ca2+]i was attenuated by both nicardipine and methoxyverapamil (D600), suggesting a role for L-type Ca2+ channels. 4. Acidosis depolarized type I cells and often (approximately 50% of cells) induced action potentials. These effects coincided with a rise in [Ca2+]i. When membrane depolarization was prevented by a voltage clamp, acidosis failed to evoke a rise in [Ca2+]i. The acid-induced rise in [Ca2+]i is a consequence, therefore, of membrane depolarization. 5. Acidosis decreased the resting membrane conductance of type I cells. The reversal potential of the acid-sensitive current was about -75 mV. 6. A depolarization (30 mM [K+]o)-induced rise in [Ca2+]i was blocked by either the removal of extracellular Ca2+ or the presence of 2 mM Ni2+, and was also substantially inhibited by nicardipine. Under voltage-clamp conditions, [Ca2+]i displayed a bell-shaped dependence on membrane potential. Depolarization raises [Ca2+]i, therefore, through voltage-operated Ca2+ channels. 7. Caffeine (10 mM) induced only a small rise in [Ca2+]i (< 10% of that induced by 30 mM extracellular K+). Ca(2+)-induced Ca2+ release is unlikely, therefore, to contribute greatly to the rise in [Ca2+]i induced by depolarization. 8. Although the replacement of extracellular Na+ with N-methyl-D-glucamine (NMG), but not Li+, inhibited the acid-induced rise in [Ca2+]i, this was due to membrane hyperpolarization and not to the inhibition

  3. Modification of bursting in a Helix neuron by drugs influencing intracellular regulation of calcium level.

    PubMed

    Salánki, J; Budai, D; Véró, M

    1983-01-01

    The effect of ruthenium red, caffein and EGTA (ethyleneglycol tetraacetic acid) influencing intracellular Ca2+ level as well as that of pH-lowering was investigated on identified RPal neuron of Helix pomatia characterized by bimodal pacemaker (bursting) activity. Drugs were applied both extracellularly and intracellularly. Intracellular injection was performed from micropipettes by pressure. It was found that intracellular injection of ruthenium red, caffein, EGTA and pH-lowering caused immediate short hyperpolarization and suspension of bursting. The effect of caffein and lowering of pH was biphasic, hyperpolarization was followed by an increase of spiking. Following EGTA injection the amplitudes of interburst hyperpolarizing waves decreased, and prolongation of spikes occurred. Extracellular application of ruthenium red caused slight depolarization, while caffein produced mainly effects that were similar to those of the intracellular injection. Adding EGTA into the bath resulted in cessation of bursting, and later on also spike generation was blocked. All these effects could be eliminated by washing. It is concluded that Ca-influx during spiking cannot be considered as a single factor in maintaining bursting activity, nevertheless, intracellular binding and liberation of Ca depending on the cell metabolism should also be taken into consideration as a possible mechanism of burst regulation. PMID:6198869

  4. The effect of photoinitiators on intracellular AKT signaling pathway in tissue engineering application

    PubMed Central

    Xu, Leyuan; Sheybani, Natasha; Yeudall, W. Andrew; Yang, Hu

    2015-01-01

    Free-radical photopolymerization initiated by photoinitiators is an important method to make tissue engineering scaffolds. To advance understanding of photoinitiator cytocompatibility, we examined three photoinitiators including 2,2-dimethoxy-2-phenylacetophenone (DMPA), Irgacure 2959 (I-2959), and eosin Y photoinitiating system (EY) in terms of their effects on viability of HN4 cells and expression levels of intracellular AKT and its phosphorylated form p-AKT. Our results show that the photoinitiators and their UV-exposed counterparts affect intracellular AKT signaling, which can be used in conjunction with cell viability for cytocompatibility assessment of photoinitiators. PMID:25709809

  5. Cadmium-Induced Apoptosis in Primary Rat Cerebral Cortical Neurons Culture Is Mediated by a Calcium Signaling Pathway

    PubMed Central

    Xu, Hui; Sun, Ya; Hu, Fei-fei; Bian, Jian-chun; Liu, Xue-zhong; Gu, Jian-hong; Liu, Zong-ping

    2013-01-01

    Cadmium (Cd) is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca2+]i) homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of [Ca2+]i and inhibition of Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities. This Cd-induced elevation of [Ca2+]i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca2+ is involved. In addition, we observed elevation of reactive oxygen species (ROS) levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III), depletion of mitochondrial membrane potential (ΔΨm), and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca2+ chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca2+ homeostasis, by compromising ATPases activities and ER-regulated Ca2+, and this elevation in Ca2+ triggers the activation of the Ca2+-mitochondria apoptotic signaling pathway. This

  6. Characteristics of calcium signaling in astrocytes induced by photostimulation with femtosecond laser

    NASA Astrophysics Data System (ADS)

    Zhao, Yuan; Zhang, Yuan; Zhou, Wei; Liu, Xiuli; Zeng, Shaoqun; Luo, Qingming

    2010-05-01

    Astrocytes have been identified to actively contribute to brain functions through Ca2+ signaling, serving as a bridge to communicate with neurons and other brain cells. However, conventional stimulation techniques are hard to apply to delicate investigations on astrocytes. Our group previously reported photostimulation with a femtosecond laser to evoke astrocytic calcium (Ca2+) waves, providing a noninvasive and efficient approach with highly precise targeting. In this work, detailed characteristics of astrocytic Ca2+ signaling induced by photostimulation are presented. In a purified astrocytic culture, after the illumination of a femtosecond laser onto one cell, a Ca2+ wave throughout the network with reduced speed is induced, and intracellular Ca2+ oscillations are observed. The intercellular propagation is pharmacologically confirmed to be mainly mediated by ATP through P2Y receptors. Different patterns of Ca2+ elevations with increased amplitude in the stimulated astrocyte are discovered by varying the femtosecond laser power, which is correspondingly followed by broader intercellular waves. These indicate that the strength of photogenerated Ca2+ signaling in astrocytes has a positive relationship with the stimulating laser power. Therefore, distinct Ca2+ signaling is feasibly available for specific studies on astrocytes by employing precisely controlled photostimulation.

  7. Redox Regulation of Intracellular Zinc: Molecular Signaling in the Life and Death of Neurons

    PubMed Central

    Aizenman, Elias

    2011-01-01

    Abstract Zn2+ has emerged as a major regulator of neuronal physiology, as well as an important signaling agent in neural injury. The intracellular concentration of this metal is tightly regulated through the actions of Zn2+ transporters and the thiol-rich metal binding protein metallothionein, closely linking the redox status of the cell to cellular availability of Zn2+. Accordingly, oxidative and nitrosative stress during ischemic injury leads to an accumulation of neuronal free Zn2+ and the activation of several downstream cell death processes. While this Zn2+ rise is an established signaling event in neuronal cell death, recent evidence suggests that a transient, sublethal accumulation of free Zn2+ can also play a critical role in neuroprotective pathways activated during ischemic preconditioning. Thus, redox-sensitive proteins, like metallothioneins, may play a critical role in determining neuronal cell fate by regulating the localization and concentration of intracellular free Zn2+. Antioxid. Redox Signal. 15, 2249–2263. PMID:20849376

  8. Neurodegeneration in Alzheimer Disease: Role of Amyloid Precursor Protein and Presenilin 1 Intracellular Signaling

    PubMed Central

    Nizzari, Mario; Thellung, Stefano; Corsaro, Alessandro; Villa, Valentina; Pagano, Aldo; Porcile, Carola; Russo, Claudio; Florio, Tullio

    2012-01-01

    Alzheimer disease (AD) is a heterogeneous neurodegenerative disorder characterized by (1) progressive loss of synapses and neurons, (2) intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3) amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2). The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration. PMID:22496686

  9. Light generation of intracellular Ca2+ signals by a genetically encoded protein BACCS

    PubMed Central

    Ishii, Tomohiro; Sato, Koji; Kakumoto, Toshiyuki; Miura, Shigenori; Touhara, Kazushige; Takeuchi, Shoji; Nakata, Takao

    2015-01-01

    Ca2+ signals are highly regulated in a spatiotemporal manner in numerous cellular physiological events. Here we report a genetically engineered blue light-activated Ca2+ channel switch (BACCS), as an optogenetic tool for generating Ca2+ signals. BACCS opens Ca2+-selective ORAI ion channels in response to light. A BACCS variant, dmBACCS2, combined with Drosophila Orai, elevates the Ca2+ concentration more rapidly, such that Ca2+ elevation in mammalian cells is observed within 1 s on light exposure. Using BACCSs, we successfully control cellular events including NFAT-mediated gene expression. In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses. Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca2+. Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca2+ signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies. PMID:26282514

  10. The non-excitable smooth muscle: Calcium signaling and phenotypic switching during vascular disease

    PubMed Central

    House, Suzanne J.; Potier, Marie; Bisaillon, Jonathan; Singer, Harold A.

    2008-01-01

    Calcium (Ca2+) is a highly versatile second messenger that controls vascular smooth muscle cell (VSMC) contraction, proliferation, and migration. By means of Ca2+ permeable channels, Ca2+ pumps and channels conducting other ions such as potassium and chloride, VSMC keep intracellular Ca2+ levels under tight control. In healthy quiescent contractile VSMC, two important components of the Ca2+ signaling pathways that regulate VSMC contraction are the plasma membrane voltage-operated Ca2+ channel of the high voltage-activated type (L-type) and the sarcoplasmic reticulum Ca2+ release channel, Ryanodine Receptor (RyR). Injury to the vessel wall is accompanied by VSMC phenotype switch from a contractile quiescent to a proliferative motile phenotype (synthetic phenotype) and by alteration of many components of VSMC Ca2+ signaling pathways. Specifically, this switch that culminates in a VSMC phenotype reminiscent of a non-excitable cell is characterized by loss of L-type channels expression and increased expression of the low voltage-activated (T-type) Ca2+ channels and the canonical transient receptor potential (TRPC) channels. The expression levels of intracellular Ca2+ release channels, pumps and Ca2+-activated proteins are also altered: the proliferative VSMC lose the RyR3 and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase isoform 2a pump and reciprocally regulate isoforms of the ca2+/calmodulin-dependent protein kinase II. This review focuses on the changes in expression of Ca2+ signaling proteins associated with VSMC proliferation both in vitro and in vivo. The physiological implications of the altered expression of these Ca2+ signaling molecules, their contribution to VSMC dysfunction during vascular disease and their potential as targets for drug therapy will be discussed. PMID:18365243

  11. Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum

    PubMed Central

    Echevarría, Wihelma; Leite, M. Fatima; Guerra, Mateus T.; Zipfel, Warren R.; Nathanson, Michael H.

    2013-01-01

    Calcium is a second messenger in virtually all cells and tissues1. Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP3) receptors, and the nuclear component of InsP3-mediated calcium signals begins in its locality. Stimulation of these receptors with a little InsP3 results in small calcium signals that are initiated in this region of the nucleus. Localized release of calcium in the nucleus causes nuclear protein kinase C (PKC) to translocate to the region of the nuclear envelope, whereas release of calcium in the cytosol induces translocation of cytosolic PKC to the plasma membrane. Our findings show that the nucleus contains a nucleoplasmic reticulum with the capacity to regulate calcium signals in localized subnuclear regions. The presence of such machinery provides a potential mechanism by which calcium can simultaneously regulate many independent processes in the nucleus. PMID:12717445

  12. Effects of ethanol on neurotransmitter release and intracellular free calcium in PC12 cells

    SciTech Connect

    Rabe, C.S.; Weight, F.F.

    1988-02-01

    The effect of ethanol on muscarine-stimulated release of l-(/sup 3/H)norepinephrine ((/sup 3/H)NE) was studied using the rat pheochromocytoma cell line, PC12. At concentrations of 25 mM and above, ethanol produced a dose-dependent inhibition of muscarine-stimulated release of (/sup 3/H)NE. The inhibition of muscarine-stimulated transmitter release occurred in the absence of any detectable effect of ethanol on (/sup 3/H)NE uptake or on muscarinic binding to the cells. However, ethanol produced an inhibition of muscarine-stimulated elevation of intracellular free Ca++ which corresponded with the inhibition of transmitter release. At concentrations greater than 100 mM, ethanol produced an increase in the basal release of (/sup 3/H)NE. Intracellular free Ca++ also was increased by ethanol concentrations greater than 100 mM. The elevation of basal transmitter release and intracellular free Ca++ by concentrations of ethanol greater than 100 mM occurred independently of the inhibition by ethanol of muscarine-stimulated elevation of intracellular free Ca++ and transmitter secretion. These results suggest that the effects of ethanol on neurotransmitter release are associated with the effects of ethanol on intracellular free Ca++.

  13. (Z)3,4,5,4‧-trans-tetramethoxystilbene, a new analogue of resveratrol, inhibits gefitinb-resistant non-small cell lung cancer via selectively elevating intracellular calcium level

    NASA Astrophysics Data System (ADS)

    Fan, Xing-Xing; Yao, Xiao-Jun; Xu, Su Wei; Wong, Vincent Kam-Wai; He, Jian-Xing; Ding, Jian; Xue, Wei-Wei; Mujtaba, Tahira; Michelangeli, Francesco; Huang, Min; Huang, Jun; Xiao, Da-Kai; Jiang, Ze-Bo; Zhou, Yan-Ling; Kin-Ting Kam, Richard; Liu, Liang; Lai-Han Leung, Elaine

    2015-11-01

    Calcium is a second messenger which is required for regulation of many cellular processes. However, excessive elevation or prolonged activation of calcium signaling would lead to cell death. As such, selectively regulating calcium signaling could be an alternative approach for anti-cancer therapy. Recently, we have identified an effective analogue of resveratrol, (Z)3,4,5,4‧-trans-tetramethoxystilbene (TMS) which selectively elevated the intracellular calcium level in gefitinib-resistant (G-R) non-small-cell lung cancer (NSCLC) cells. TMS exhibited significant inhibitory effect on G-R NSCLC cells, but not other NSCLC cells and normal lung epithelial cells. The phosphorylation and activation of EGFR were inhibited by TMS in G-R cells. TMS induced caspase-independent apoptosis and autophagy by directly binding to SERCA and causing endoplasmic reticulum (ER) stress and AMPK activation. Proteomics analysis also further confirmed that mTOR pathway, which is the downstream of AMPK, was significantly suppressed by TMS. JNK, the cross-linker of ER stress and mTOR pathway was significantly activated by TMS. In addition, the inhibition of JNK activation can partially block the effect of TMS. Taken together, TMS showed promising anti-cancer activity by mediating calcium signaling pathway and inducing apoptosis as well as autophagy in G-R NSCLC cells, providing strategy in designing multi-targeting drug for treating G-R patients.

  14. (Z)3,4,5,4'-trans-tetramethoxystilbene, a new analogue of resveratrol, inhibits gefitinb-resistant non-small cell lung cancer via selectively elevating intracellular calcium level.

    PubMed

    Fan, Xing-Xing; Yao, Xiao-Jun; Xu, Su Wei; Wong, Vincent Kam-Wai; He, Jian-Xing; Ding, Jian; Xue, Wei-Wei; Mujtaba, Tahira; Michelangeli, Francesco; Huang, Min; Huang, Jun; Xiao, Da-Kai; Jiang, Ze-Bo; Zhou, Yan-Ling; Kam, Richard Kin-Ting; Liu, Liang; Leung, Elaine Lai-Han

    2015-01-01

    Calcium is a second messenger which is required for regulation of many cellular processes. However, excessive elevation or prolonged activation of calcium signaling would lead to cell death. As such, selectively regulating calcium signaling could be an alternative approach for anti-cancer therapy. Recently, we have identified an effective analogue of resveratrol, (Z)3,4,5,4'-trans-tetramethoxystilbene (TMS) which selectively elevated the intracellular calcium level in gefitinib-resistant (G-R) non-small-cell lung cancer (NSCLC) cells. TMS exhibited significant inhibitory effect on G-R NSCLC cells, but not other NSCLC cells and normal lung epithelial cells. The phosphorylation and activation of EGFR were inhibited by TMS in G-R cells. TMS induced caspase-independent apoptosis and autophagy by directly binding to SERCA and causing endoplasmic reticulum (ER) stress and AMPK activation. Proteomics analysis also further confirmed that mTOR pathway, which is the downstream of AMPK, was significantly suppressed by TMS. JNK, the cross-linker of ER stress and mTOR pathway was significantly activated by TMS. In addition, the inhibition of JNK activation can partially block the effect of TMS. Taken together, TMS showed promising anti-cancer activity by mediating calcium signaling pathway and inducing apoptosis as well as autophagy in G-R NSCLC cells, providing strategy in designing multi-targeting drug for treating G-R patients. PMID:26542098

  15. (Z)3,4,5,4′-trans-tetramethoxystilbene, a new analogue of resveratrol, inhibits gefitinb-resistant non-small cell lung cancer via selectively elevating intracellular calcium level

    PubMed Central

    Fan, Xing-Xing; Yao, Xiao-Jun; Xu, Su Wei; Wong, Vincent Kam-Wai; He, Jian-Xing; Ding, Jian; Xue, Wei-Wei; Mujtaba, Tahira; Michelangeli, Francesco; Huang, Min; Huang, Jun; Xiao, Da-Kai; Jiang, Ze-Bo; Zhou, Yan-Ling; Kin-Ting Kam, Richard; Liu, Liang; Lai-Han Leung, Elaine

    2015-01-01

    Calcium is a second messenger which is required for regulation of many cellular processes. However, excessive elevation or prolonged activation of calcium signaling would lead to cell death. As such, selectively regulating calcium signaling could be an alternative approach for anti-cancer therapy. Recently, we have identified an effective analogue of resveratrol, (Z)3,4,5,4′-trans-tetramethoxystilbene (TMS) which selectively elevated the intracellular calcium level in gefitinib-resistant (G-R) non-small-cell lung cancer (NSCLC) cells. TMS exhibited significant inhibitory effect on G-R NSCLC cells, but not other NSCLC cells and normal lung epithelial cells. The phosphorylation and activation of EGFR were inhibited by TMS in G-R cells. TMS induced caspase-independent apoptosis and autophagy by directly binding to SERCA and causing endoplasmic reticulum (ER) stress and AMPK activation. Proteomics analysis also further confirmed that mTOR pathway, which is the downstream of AMPK, was significantly suppressed by TMS. JNK, the cross-linker of ER stress and mTOR pathway was significantly activated by TMS. In addition, the inhibition of JNK activation can partially block the effect of TMS. Taken together, TMS showed promising anti-cancer activity by mediating calcium signaling pathway and inducing apoptosis as well as autophagy in G-R NSCLC cells, providing strategy in designing multi-targeting drug for treating G-R patients. PMID:26542098

  16. Effect of intracellular magnesium on calcium extrusion by the plasma membrane calcium pump of intact human red cells.

    PubMed Central

    Raftos, J E; Lew, V L

    1995-01-01

    1. The effect of varying the concentration of intracellular magnesium on the Ca(2+)-saturated Ca(2+)-extrusion rate through the Ca2+ pump (phi max) was investigated in human red blood cells with the aid of the divalent cation ionophore A23187. The aim was to characterize the [Mg2+]i dependence of the Ca2+ pump in the intact cell. 2. The initial experimental protocol consisted of applying a high ionophore concentration to obtain rapid sequential Mg2+ and [45Ca]CaCl2 equilibration, prior to measuring phi max at constant internal [MgT]i by either the Co2+ block method or by ionophore removal. With this protocol, competition between Ca2+ and Mg2+ through the ionophore prevented Ca2+ equilibration at high [Mg2+]o. To provide rapid and comparable Ca2+ loads and maintain intracellular ATP within normal levels it was necessary to separate the Mg2+ and the Ca2+ loading-extrusion stages by an intermediate ionophore and external Mg2+ removal step, and to use different metabolic substrates during Mg2+ loading (glucose) and Ca2+ loading-extrusion (inosine) periods. 3. Intracellular Co2+ was found to sustain Ca2+ extrusion by the pump at subphysiological [Mg2+]i. Ionophore removal was therefore used to estimate the [Mg2+]i dependence of the pump at levels below [MgT]i (approximately 2 mmol (340 g Hb)-1), whereas both ionophore removal and Co2+ block were used for higher [MgT]i levels. 4. [Mg2+]i was computed from measured [MgT]i using known cytoplasmic Mg(2+)-buffering data. The phi max of the Ca2+ pump increased hyperbolically with [Mg2+]i. The Michaelis parameter (K 1/2) of activation was 0.12 +/- 0.04 mmol (1 cell water)-1 (mean +/- S.E.M.). Increasing [MgT]i and [Mg2+]i to 9 mmol (340 g Hb)-1 and 2.6 mmol (1 cell water)-1, respectively, failed to cause significant inhibition of the phi max of the Ca2+ pump. 5. The results suggest that within the physiological and pathophysiological range of [Mg2+]i, from 0.3 mmol (1 cell water)-1 in the oxygenated state to 1.2 mmol (1 cell

  17. Structural Reconstruction of Protein-Protein Complexes Involved in Intracellular Signaling.

    PubMed

    Kirsch, Klára; Sok, Péter; Reményi, Attila

    2016-01-01

    Signaling complexes within the cell convert extracellular cues into physiological outcomes. Their assembly involves signaling enzymes, allosteric regulators and scaffold proteins that often contain long stretches of disordered protein regions, display multi-domain architectures, and binding affinity between individual components is low. These features are indispensable for their central roles as dynamic information processing hubs, on the other hand they also make reconstruction of structurally homogeneous complex samples highly challenging. In this present chapter we discuss protein machinery which influences extracellular signal reception, intracellular pathway activity, and cytoskeletal or transcriptional activity. PMID:27165334

  18. An atmospheric-pressure cold plasma leads to apoptosis in Saccharomyces cerevisiae by accumulating intracellular reactive oxygen species and calcium

    NASA Astrophysics Data System (ADS)

    Ma, R. N.; Feng, H. Q.; Liang, Y. D.; Zhang, Q.; Tian, Y.; Su, B.; Zhang, J.; Fang, J.

    2013-07-01

    A non-thermal plasma is known to induce apoptosis of various cells but the mechanism is not yet clear. A eukaryotic model organism Saccharomyces cerevisiaewas used to investigate the cellular and biochemical regulations of cell apoptosis and cell cycle after an atmospheric-pressure cold plasma treatment. More importantly, intracellular calcium (Ca2+) was first involved in monitoring the process of plasma-induced apoptosis in this study. We analysed the cell apoptosis and cell cycle by flow cytometry and observed the changes in intracellular reactive oxygen species (ROS) and Ca2+ concentration, cell mitochondrial membrane potential (Δψm) as well as nuclear DNA morphology via fluorescence staining assay. All experimental results indicated that plasma-generated ROS leads to the accumulation of intracellular ROS and Ca2+ that ultimately contribute to apoptosis associated with cell cycle arrest at G1 phase through depolarization of Δψm and fragmenting nuclear DNA. This work provides a novel insight into the physical and biological mechanism of apoptosis induced by a plasma which could benefit for promoting the development of plasmas applied to cancer therapy.

  19. Two-pore channels: Regulation by NAADP and customized roles in triggering calcium signals.

    PubMed

    Patel, Sandip; Marchant, Jonathan S; Brailoiu, Eugen

    2010-06-01

    NAADP is a potent regulator of cytosolic calcium levels. Much evidence suggests that NAADP activates a novel channel located on an acidic (lysosomal-like) calcium store, the mobilisation of which results in further calcium release from the endoplasmic reticulum. Here, we discuss the recent identification of a family of poorly characterized ion channels (the two-pore channels) as endo-lysosomal NAADP receptors. The generation of calcium signals by these channels is likened to those evoked by depolarisation during excitation-contraction coupling in muscle. We discuss the idea that two-pore channels can mediate a trigger release of calcium which is then amplified by calcium-induced calcium release from the endoplasmic reticulum. This is similar to the activation of voltage-sensitive calcium channels and subsequent mobilisation of sarcoplasmic reticulum calcium stores in cardiac tissue. We suggest that two-pore channels may physically interact with ryanodine receptors to account for more direct release of calcium from the endoplasmic reticulum in analogy with the conformational coupling of voltage-sensitive calcium channels and ryanodine receptors in skeletal muscle. Interaction of two-pore channels with other calcium release channels likely occurs between stores "trans-chatter" and possibly within the same store "cis-chatter". We also speculate that trafficking of two-pore channels through the endo-lysosomal system facilitates interactions with calcium entry channels. Strategic placing of two-pore channels thus provides a versatile means of generating spatiotemporally complex cellular calcium signals. PMID:20621760

  20. Molecular Basis of Calcium Signaling in Lymphocytes: STIM and ORAI

    PubMed Central

    Hogan, Patrick G.; Lewis, Richard S.; Rao, Anjana

    2010-01-01

    Ca2+ entry into cells of the peripheral immune system occurs through highly Ca2+-selective channels known as CRAC (calcium release-activated calcium) channels. CRAC channels are a very well-characterized example of store-operated Ca2+ channels, so designated because they open when the endoplasmic reticulum (ER) Ca2+ store becomes depleted. Physiologically, Ca2+ is released from the ER lumen into the cytoplasm when activated receptors couple to phospholipase C and trigger production of the second messenger inositol 1,4,5-trisphosphate (IP3). IP3 binds to IP3 receptors in the ER membrane and activates Ca2+ release. The proteins STIM and ORAI were discovered through limited and genome-wide RNAi screens, respectively, performed in Drosophila cells and focused on identifying modulators of store-operated Ca2+ entry. STIM1 and STIM2 sense the depletion of ER Ca2+ stores, whereas ORAI1 is a pore subunit of the CRAC channel. In this review, we discuss selected aspects of Ca2+ signaling in cells of the immune system, focusing on the roles of STIM and ORAI proteins in store-operated Ca2+ entry. PMID:20307213

  1. Functional inhibition of β-catenin-mediatedWnt signaling by intracellular VHHantibodies

    PubMed Central

    Newnham, Laura E; Wright, Michael J; Holdsworth, Gill; Kostarelos, Kostas; Robinson, Martyn K; Rabbitts, Terence H; Lawson, Alastair D

    2015-01-01

    The Wnt signaling pathway is of central importance in embryogenesis, development and adult tissue homeostasis, and dysregulation of this pathway is associated with cancer and other diseases. Despite the developmental and potential therapeutic significance of this pathway, many aspects of Wnt signaling, including the control of the master transcriptional co-activator β-catenin, remain poorly understood. In order to explore this aspect, a diverse immune llama VHH phagemid library was constructed and panned against β-catenin. VHH antibody fragments from the library were expressed intracellularly, and a number of antibodies were shown to possess function-modifying intracellular activity in a luciferase-based Wnt signaling HEK293 reporter bioassay. Further characterization of one such VHH (named LL3) confirmed that it bound endogenous β-catenin, and that it inhibited the Wnt signaling pathway downstream of the destruction complex, while production of a control Ala-substituted complementarity-determining region (CDR)3 mutant demonstrated that the inhibition of β-catenin activity by the parent intracellular antibody was dependent on the specific CDR sequence of the antibody. PMID:25524068

  2. Intracellular Ca(2+) signaling in endothelial cells by the angiogenesis inhibitors endostatin and angiostatin.

    PubMed

    Jiang, L; Jha, V; Dhanabal, M; Sukhatme, V P; Alper, S L

    2001-05-01

    Intracellular signaling mechanisms by the angiogenesis inhibitors endostatin and angiostatin remain poorly understood. We have found that endostatin (2 microg/ml) and angiostatin (5 microg/ml) elicited transient, approximately threefold increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Acute exposure to angiostatin or endostatin nearly abolished subsequent endothelial [Ca(2+)](i) responses to carbachol or to thapsigargin; conversely, thapsigargin attenuated the Ca(2+) signal elicited by endostatin. The phospholipase C inhibitor U-73122 and the inositol trisphosphate (IP(3)) receptor inhibitor xestospongin C both inhibited endostatin-induced elevation in [Ca(2+)](i), and endostatin rapidly elevated endothelial cell IP(3) levels. Pertussis toxin and SB-220025 modestly inhibited the endostatin-induced Ca(2+) signal. Removal of extracellular Ca(2+) inhibited the endostatin-induced rise in [Ca(2+)](i), as did a subset of Ca(2+)-entry inhibitors. Peak Ca(2+) responses to endostatin and angiostatin in endothelial cells exceeded those in epithelial cells and were minimal in NIH/3T3 cells. Overnight pretreatment of endothelial cells with endostatin reduced the subsequent acute elevation in [Ca(2+)](i) in response to vascular endothelial growth factor or to fibroblast growth factor by approximately 70%. Intracellular Ca(2+) signaling may initiate or mediate some of the cellular actions of endostatin and angiostatin. PMID:11287327

  3. Antagonizing amyloid-β/calcium-sensing receptor signaling in human astrocytes and neurons: a key to halt Alzheimer's disease progression?

    PubMed Central

    Dal Prà, Ilaria; Chiarini, Anna; Armato, Ubaldo

    2015-01-01

    Astrocytes’ roles in late-onset Alzheimer's disease (LOAD) promotion are important, since they survive soluble or fibrillar amyloid-β peptides (Aβs) neurotoxic effects, undergo alterations of intracellular and intercellular Ca2+ signaling and gliotransmitters release via the Aβ/α7-nAChR (α7-nicotinic acetylcholine receptor) signaling, and overproduce/oversecrete newly synthesized Aβ42 oligomers, NO, and VEGF-A via the Aβ/CaSR (calcium-sensing receptor) signaling. Recently, it was suggested that the NMDAR (N-methyl-D-aspartate receptor) inhibitor nitromemantine would block the synapse-destroying effects of Aβ/α7-nAChR signaling. Yet, this and the progressive extracellular accrual and spreading of Aβ42 oligomers would be stopped well upstream by NPS 2143, an allosteric CaSR antagonist (calcilytic). PMID:25883618

  4. Parvalbumin overexpression alters immune-mediated increases in intracellular calcium, and delays disease onset in a transgenic model of familial amyotrophic lateral sclerosis

    NASA Technical Reports Server (NTRS)

    Beers, D. R.; Ho, B. K.; Siklos, L.; Alexianu, M. E.; Mosier, D. R.; Mohamed, A. H.; Otsuka, Y.; Kozovska, M. E.; McAlhany, R. E.; Smith, R. G.; Appel, S. H.

    2001-01-01

    Intracellular calcium is increased in vulnerable spinal motoneurons in immune-mediated as well as transgenic models of amyotrophic lateral sclerosis (ALS). To determine whether intracellular calcium levels are influenced by the calcium-binding protein parvalbumin, we developed transgenic mice overexpressing parvalbumin in spinal motoneurons. ALS immunoglobulins increased intracellular calcium and spontaneous transmitter release at motoneuron terminals in control animals, but not in parvalbumin overexpressing transgenic mice. Parvalbumin transgenic mice interbred with mutant SOD1 (mSOD1) transgenic mice, an animal model of familial ALS, had significantly reduced motoneuron loss, and had delayed disease onset (17%) and prolonged survival (11%) when compared with mice with only the mSOD1 transgene. These results affirm the importance of the calcium binding protein parvalbumin in altering calcium homeostasis in motoneurons. The increased motoneuron parvalbumin can significantly attenuate the immune-mediated increases in calcium and to a lesser extent compensate for the mSOD1-mediated 'toxic-gain-of-function' in transgenic mice.

  5. Biphasic Role of Calcium in Mouse Sperm Capacitation Signaling Pathways

    PubMed Central

    Alvau, Antonio; Escoffier, Jessica; Krapf, Dario; Sánchez-Cárdenas, Claudia; Salicioni, Ana M.; Darszon, Alberto; Visconti, Pablo E.

    2016-01-01

    Mammalian sperm acquire fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with up-regulation of a cAMP-dependent pathway, changes in intracellular pH, intracellular Ca2+ and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca2+ ions have a biphasic role in the regulation of cAMP-dependent signaling. Media without added Ca2+ salts (nominal zero Ca2+) still contain micromolar concentrations of this ion. Sperm incubated in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca2+. However, chelation of the extracellular Ca2+ traces by EGTA induced both cAMP-dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca2+ media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca2+ ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin. Interestingly, contrary to wild type mouse sperm, sperm from CatSper1 KO mice underwent PKA activation and an increase in tyrosine phosphorylation upon incubation in nominal zero Ca2+ media. Therefore, sperm lacking Catsper Ca2+ channels behave as wild-type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca2+ involved in the regulation of cAMP-dependent and tyrosine phosphorylation pathways required for sperm capacitation. PMID:25597298

  6. Calcium signaling triggered by ouabain protects the embryonic kidney from adverse developmental programming.

    PubMed

    Khodus, Georgiy R; Kruusmägi, Markus; Li, Juan; Liu, Xiao-Li; Aperia, Anita

    2011-09-01

    The kidney is extraordinarily sensitive to adverse fetal programming. Malnutrition, the most common form of developmental challenge, retards formation of the kidney's functional units, the nephrons. The resulting low nephron endowment increases susceptibility to renal injury and disease. Using explanted rat embryonic kidneys, we found that the sodium-potassium-adenosine triphosphatase (Na, K-ATPase) ligand ouabain triggers, via the Na, K-ATPase/ inositol 1,4,5-trisphosphate receptor signalosome, a calcium-nuclear factor-kappa B (NF-κB) signal that protects kidney development from adverse effects of malnutrition. Serum deprivation resulted in severe retardation of nephron formation and robust increase in apoptotic rate, but in ouabain-exposed kidneys, no adverse effects of serum deprivation were observed. Depletion of intracellular calcium stores and inhibition of NF-κB activity abolished the rescuing effect of ouabain. Proof of principle that ouabain rescues development of embryonic kidneys exposed to malnutrition was obtained from studies on pregnant rats given low-protein diets and treated with ouabain or vehicle throughout pregnancy. PMID:21424905

  7. Extracellular group A Streptococcus induces keratinocyte apoptosis by dysregulating calcium signalling.

    PubMed

    Cywes Bentley, Colette; Hakansson, Anders; Christianson, Jennifer; Wessels, Michael R

    2005-07-01

    Group A Streptococcus (GAS) colonizes the oropharynx and damaged skin. To cause local infection or severe invasive syndromes the bacteria must gain access into deeper tissues. Host cell death may facilitate this process. GAS internalization has been identified to induce apoptosis. We now report an alternate mechanism of GAS-mediated apoptosis of primary human keratinocytes, initiated by extracellular GAS and involving dysregulation of intracellular calcium to produce endoplasmic reticulum stress. Two bacterial virulence factors are required for effective induction of apoptosis by extracellular GAS: (i) hyaluronic acid capsule that inhibits bacterial internalization and (ii) secreted cytolysin, streptolysin O (SLO), that forms transmembrane pores that permit extracellular calcium influx into the cytosol. Induction of keratinocyte apoptosis by wild-type GAS was accompanied by cell detachment and loss of epithelial integrity, a phenomenon not observed with GAS deficient in capsule or SLO. We propose that cell signalling initiated by extracellular GAS compromises the epithelial barrier by inducing premature keratinocyte differentiation and apoptosis, thereby facilitating GAS invasion of deeper tissues. PMID:15953027

  8. Ethanol's effects on neurotransmitter release and intracellular free calcium in PC12 cells

    SciTech Connect

    Rabe, C.S.; Weight, F.F.

    1988-01-01

    The effect of ethanol on muscarine-stimulated release of (/sup 3/H)NE was studied using the rat pheochromocytoma cell line, PC12. At concentrations of 25 mM and above, ethanol produced a dose dependent inhibition of muscarine-stimulated release of (/sup 3/H)NE. The inhibition of muscarine-stimulated transmitter release occurred in the absence of any effect of ethanol on (/sup 3/H)NE uptake, metabolism or on muscarinic binding to the cells. However, ethanol produced an inhibition of muscarine-stimulated elevation of intracellular free Ca2+ which corresponded with the inhibition of transmitter release. At concentrations greater than 100 mM, ethanol produced both a stimulation of the release of (/sup 3/H)NE as well as an increase in intracellular free Ca2+. The increase in basal transmitter release and intracellular free Ca2+ occurred independent of the inhibition by ethanol of muscarine-stimulated elevation of intracellular free Ca2+ or transmitter section. These results demonstrate the relationship of the effects of ethanol on cellular free Ca2+ and neurotransmitter release.

  9. Regulation of intracellular calcium is closely linked to glucose metabolism in J774 macrophages.

    PubMed

    Darbha, S; Marchase, R B

    1996-10-01

    The effects of 2-deoxy-D-glucose (2dGlc) and glucose deprivation were investigated in the J774 murine macrophage-like cell line. 2dGlc addition or glucose deprivation for 4 min led to an inhibition in the transient increase in cytoplasmic free Ca2+ ([Ca2+]i) that otherwise occurs in response to three different agonists: IgG, ATP and platelet activating factor. This inhibition was preceded by a partial release of Ca2+ from intracellular, thapsigargin-sensitive stores. In contrast, the transition from 5 to 30 mM glucose caused a decrease in [Ca2+]i and a corresponding increase in thapsigargin-sensitive sequestered Ca2+. The effects of an alternate glycolytic inhibitor, NaF, and a mitochondrial inhibitor, rotenone, were also tested. These inhibitors caused neither a release of Ca2+ from intracellular stores nor an inhibition in any of the agonist responses. The capacitative influx of extracellular Ca2+ following depletion of intracellular stores was also found to be selectively inhibited by the prior addition of 2dGlc or with glucose deprivation. In addition, when an elevated plateau of [Ca2+]i was established by the irreversible depletion of intracellular Ca2+ stores, the addition of 2dGlc caused a decrease in the on-going capacitative entry of Ca2+. PMID:8939356

  10. Neuronal calcium signaling, mitochondrial dysfunction and Alzheimer’s disease

    PubMed Central

    Supnet, Charlene; Bezprozvanny, Ilya

    2016-01-01

    Alzheimer disease (AD) is the most common neurodegenerative disorder that affects millions of ageing people worldwide. AD is characterized by extensive synaptic and neuronal loss which lead to impaired memory and cognitive decline. The cause of pathology in AD is not completely understood and no effective therapy so far has been developed. The accumulation of toxic amyloid-beta 42 (Aβ42) peptide oligomers and aggregates in AD brain has been proposed to be primarily responsible for the pathology of the disease, an idea dubbed ‘amyloid hypothesis’ of AD etiology. In addition to increase in Aβ42 levels, disturbances in neuronal calcium (Ca2+) signaling and alterations in expression levels of Ca2+ signaling proteins have been observed in animal models of familial AD and in studies of postmortem brain samples from sporadic AD patients. Based on these evidence ‘Ca2+ hypothesis of AD’ has been proposed. In particular, familal AD has been linked with enhanced Ca2+ release from the endoplasmic reticulum (ER) and elevated cytosolic Ca2+ levels. The augmented cytosolic Ca2+ levels can trigger signaling cascades that affect synaptic stability and function and can be detrimental to neuronal health, such as Ca2+-dependent phosphatase calcineurin and Ca2+-dependent proteases calpains. Here we review the latest results supporting ‘Ca2+ hypothesis’ of AD pathogenesis. We further argue that over long period of time supranormal cytosolic Ca2+ signaling can impaire mitochondrial function in AD neurons. We conclude that inhibitors and stablizers of neuronal Ca2+ signaling and mitochondrial function may have a therapeutic potential for treatment of AD. We discuss latest and planned AD therapeutic trials of agents targeting Ca2+ channels and mitochodria. PMID:20413848

  11. alpha-Difluoromethylornithine alters calcium signaling in platelet-derived growth factor-stimulated A172 brain tumor cells in culture.

    PubMed

    Feuerstein, B G; Szöllösi, J; Basu, H S; Marton, L J

    1992-12-15

    alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, inhibits the growth of brain tumor cell lines and is undergoing clinical trials as a treatment for brain tumors. Platelet-derived growth factor (PDGF) is thought to regulate the growth and development of precursors of both normal and neoplastic astrocytic cells; calcium signaling is thought to play a role in the transduction of PDGF signals. Using laser fluorescence image cytometry, flow cytometry, and spectrofluorometry, we studied the effect of DFMO on the calcium signals induced by PDGF in A172 human glioblastoma cells. Four days of treatment with 5 mM DFMO substantially shortened PDGF-induced calcium signals. The effect was reversed more than 10 h but less than 24 h after putrescine treatment, even though polyamines were repleted 4 h after putrescine and spermidine were added. DFMO did not substantially affect intracellular calcium release or the timing of the opening and closing of plasma membrane calcium channels. These findings support the notion that calcium signaling may be a target for inhibitors of polyamine metabolism. PMID:1458466

  12. Calcium and protein phosphorylation in the transduction of gravity signal in corn roots

    NASA Technical Reports Server (NTRS)

    Friedmann, M.; Poovaiah, B. W.

    1991-01-01

    The involvement of calcium and protein phosphorylation in the transduction of gravity signal was studied using corn roots of a light-insensitive variety (Zea mays L., cv. Patriot). The gravitropic response was calcium-dependent. Horizontal placement of roots preloaded with 32P for three minutes resulted in changes in protein phosphorylation of polypeptides of 32 and 35 kD. Calcium depletion resulted in decreased phosphorylation of these phosphoproteins and replenishment of calcium restored the phosphorylation.

  13. Simplest relationship between local field potential and intracellular signals in layered neural tissue

    NASA Astrophysics Data System (ADS)

    Chizhov, Anton V.; Sanchez-Aguilera, Alberto; Rodrigues, Serafim; de la Prida, Liset Menendez

    2015-12-01

    The relationship between the extracellularly measured electric field potential resulting from synaptic activity in an ensemble of neurons and intracellular signals in these neurons is an important but still open question. Based on a model neuron with a cylindrical dendrite and lumped soma, we derive a formula that substantiates a proportionality between the local field potential and the total somatic transmembrane current that emerges from the difference between the somatic and dendritic membrane potentials. The formula is tested by intra- and extracellular recordings of evoked synaptic responses in hippocampal slices. Additionally, the contribution of different membrane currents to the field potential is demonstrated in a two-population mean-field model. Our formalism, which allows for a simple estimation of unknown dendritic currents directly from somatic measurements, provides an interpretation of the local field potential in terms of intracellularly measurable synaptic signals. It is also applicable to the study of cortical activity using two-compartment neuronal population models.

  14. Simplest relationship between local field potential and intracellular signals in layered neural tissue.

    PubMed

    Chizhov, Anton V; Sanchez-Aguilera, Alberto; Rodrigues, Serafim; de la Prida, Liset Menendez

    2015-12-01

    The relationship between the extracellularly measured electric field potential resulting from synaptic activity in an ensemble of neurons and intracellular signals in these neurons is an important but still open question. Based on a model neuron with a cylindrical dendrite and lumped soma, we derive a formula that substantiates a proportionality between the local field potential and the total somatic transmembrane current that emerges from the difference between the somatic and dendritic membrane potentials. The formula is tested by intra- and extracellular recordings of evoked synaptic responses in hippocampal slices. Additionally, the contribution of different membrane currents to the field potential is demonstrated in a two-population mean-field model. Our formalism, which allows for a simple estimation of unknown dendritic currents directly from somatic measurements, provides an interpretation of the local field potential in terms of intracellularly measurable synaptic signals. It is also applicable to the study of cortical activity using two-compartment neuronal population models. PMID:26764724

  15. Extracellular Calcium Has Multiple Targets to Control Cell Proliferation.

    PubMed

    Capiod, Thierry

    2016-01-01

    Calcium channels and the two G-protein coupled receptors sensing extracellular calcium, calcium-sensing receptor (CaSR) and GPRC6a, are the two main means by which extracellular calcium can signal to cells and regulate many cellular processes including cell proliferation, migration and invasion of tumoral cells. Many intracellular signaling pathways are sensitive to cytosolic calcium rises and conversely intracellular signaling pathways can modulate calcium channel expression and activity. Calcium channels are undoubtedly involved in the former while the CaSR and GPRC6a are most likely to interfere with the latter. As for neurotransmitters, calcium ions use plasma membrane channels and GPCR to trigger cytosolic free calcium concentration rises and intracellular signaling and regulatory pathways activation. Calcium sensing GPCR, CaSR and GPRC6a, allow a supplemental degree of control and as for metabotropic receptors, they not only modulate calcium channel expression but they may also control calcium-dependent K+ channels. The multiplicity of intracellular signaling pathways involved, their sensitivity to local and global intracellular calcium increase and to CaSR and GPRC6a stimulation, the presence of membrane signalplex, all this confers the cells the plasticity they need to convert the effects of extracellular calcium into complex physiological responses and therefore determine their fate. PMID:27161228

  16. Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design

    SciTech Connect

    Akerboom, Jasper; Velez Rivera, Jonathan D.; Rodriguez Guilbe, María M.; Alfaro Malavé, Elisa C.; Hernandez, Hector H.; Tian, Lin; Hires, S. Andrew; Marvin, Jonathan S.; Looger, Loren L.; Schreiter, Eric R.

    2009-03-16

    The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca{sup 2+}-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.

  17. Aroclor 1254, a developmental neurotoxicant, alters energy metabolism- and intracellular signaling-associated protein networks in rat cerebellum and hippocampus

    SciTech Connect

    Kodavanti, Prasada Rao S.; Osorio, Cristina; Royland, Joyce E.; Ramabhadran, Ram; Alzate, Oscar

    2011-11-15

    The vast literature on the mode of action of polychlorinated biphenyls (PCBs) indicates that PCBs are a unique model for understanding the mechanisms of toxicity of environmental mixtures of persistent chemicals. PCBs have been shown to adversely affect psychomotor function and learning and memory in humans. Although the molecular mechanisms for PCB effects are unclear, several studies indicate that the disruption of Ca{sup 2+}-mediated signal transduction plays significant roles in PCB-induced developmental neurotoxicity. Culminating events in signal transduction pathways include the regulation of gene and protein expression, which affects the growth and function of the nervous system. Our previous studies showed changes in gene expression related to signal transduction and neuronal growth. In this study, protein expression following developmental exposure to PCB is examined. Pregnant rats (Long Evans) were dosed with 0.0 or 6.0 mg/kg/day of Aroclor-1254 from gestation day 6 through postnatal day (PND) 21, and the cerebellum and hippocampus from PND14 animals were analyzed to determine Aroclor 1254-induced differential protein expression. Two proteins were found to be differentially expressed in the cerebellum following PCB exposure while 18 proteins were differentially expressed in the hippocampus. These proteins are related to energy metabolism in mitochondria (ATP synthase, sub unit {beta} (ATP5B), creatine kinase, and malate dehydrogenase), calcium signaling (voltage-dependent anion-selective channel protein 1 (VDAC1) and ryanodine receptor type II (RyR2)), and growth of the nervous system (dihydropyrimidinase-related protein 4 (DPYSL4), valosin-containing protein (VCP)). Results suggest that Aroclor 1254-like persistent chemicals may alter energy metabolism and intracellular signaling, which might result in developmental neurotoxicity. -- Highlights: Black-Right-Pointing-Pointer We performed brain proteomic analysis of rats exposed to the neurotoxicant

  18. Shock wave irradiations avoiding fluid flow evoke intracellular Ca2+ signaling

    NASA Astrophysics Data System (ADS)

    Takahashi, Toru; Tsukamoto, Akira; Tada, Shigeru

    Shock wave irradiation accelerates therapeutic effects including angiogenesis. One mechanism underlying those effects is cellular responses evoked by shock wave irradiation. Fluid flow is one of major physical phenomena induced by shock wave irradiation. Cellular responses evoked by fluid flow are similar to those evoked by shock wave irradiation. Thus, fluid flow could be responsible for cellular responses evoked by shock wave irradiation. However, it is obscure whether fluid flow is required for the cellular responses evoked by shock wave irradiation. In this study, intracellular Ca2 + signaling was observed in cells seeded in down-sized chambers. In the down-sized chambers, fluid flow was supposed to be suppressed because size of chambers (6 mm in diameter, 1 mm in thickness) was analogous to size of shock wave focus region (3mm in diameter). Dynamics of polystyrene microbeads suspended in the chambers were visualized with a CCD camera and analyzed with a particle image velocimetry (PIV) method to quantify fluid flow in the chamber. As a result, shock wave irradiation evoked intracellular Ca2 + signaling. However, fluid flow was not observed in the chamber due to shock wave irradiation. Thus, it was suggested that physical mechanics, not fluid flow, are further required for evoking intracellular Ca2 + signaling following to shock wave irradiation.

  19. Caveats and limitations of plate reader-based high-throughput kinetic measurements of intracellular calcium levels

    SciTech Connect

    Heusinkveld, Harm J.; Westerink, Remco H.S.

    2011-08-15

    Calcium plays a crucial role in virtually all cellular processes, including neurotransmission. The intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) is therefore an important readout in neurotoxicological and neuropharmacological studies. Consequently, there is an increasing demand for high-throughput measurements of [Ca{sup 2+}]{sub i}, e.g. using multi-well microplate readers, in hazard characterization, human risk assessment and drug development. However, changes in [Ca{sup 2+}]{sub i} are highly dynamic, thereby creating challenges for high-throughput measurements. Nonetheless, several protocols are now available for real-time kinetic measurement of [Ca{sup 2+}]{sub i} in plate reader systems, though the results of such plate reader-based measurements have been questioned. In view of the increasing use of plate reader systems for measurements of [Ca{sup 2+}]{sub i} a careful evaluation of current technologies is warranted. We therefore performed an extensive set of experiments, using two cell lines (PC12 and B35) and two fluorescent calcium-sensitive dyes (Fluo-4 and Fura-2), for comparison of a linear plate reader system with single cell fluorescence microscopy. Our data demonstrate that the use of plate reader systems for high-throughput real-time kinetic measurements of [Ca{sup 2+}]{sub i} is associated with many pitfalls and limitations, including erroneous sustained increases in fluorescence, limited sensitivity and lack of single cell resolution. Additionally, our data demonstrate that probenecid, which is often used to prevent dye leakage, effectively inhibits the depolarization-evoked increase in [Ca{sup 2+}]{sub i}. Overall, the data indicate that the use of current plate reader-based strategies for high-throughput real-time kinetic measurements of [Ca{sup 2+}]{sub i} is associated with caveats and limitations that require further investigation. - Research Highlights: > The use of plate readers for high-throughput screening of intracellular

  20. 17β-estradiol rapidly activates calcium release from intracellular stores via the GPR30 pathway and MAPK phosphorylation in osteocyte-like MLO-Y4 cells.

    PubMed

    Ren, Jian; Wu, Jun Hua

    2012-05-01

    Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E(2)) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E(2) elevated [Ca(2+)]( i ) and increased Ca(2+) oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E(2) mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E(2) activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E(2) induces the non-genomic responses Ca(2+) release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E(2) responses. PMID:22392527

  1. Calcium ion, an intracellular messenger of light adaptation, also participates in excitation of Limulus photoreceptors.

    PubMed Central

    Bolsover, S R; Brown, J E

    1985-01-01

    Photoreceptor cells of Limulus ventral eyes were bathed in artificial sea water (ASW) that contained 10 mM-EGTA and no added Ca2+ (EGTA-ASW). Test flashes elicited responses that increased to a maximum size within 10 min in EGTA-ASW but did not change further when dark-adapted cells were bathed for an additional 35 min in this solution. Light responses progressively declined from this maximum size if the cells were repetitively illuminated in EGTA-ASW. In this state of reduced responsiveness, response amplitudes were further reduced by intracellular ionophoretic injection of EGTA; response amplitudes were increased by intracellular ionophoretic injection of Ca2+. Both of these findings are opposite to what is normally observed for cells bathed in ASW. Also, after repetitive illumination in EGTA-ASW, both the slope of the response versus intensity relationship became steeper and light responses often had a delayed increase in amplitude. The light responses and the response versus intensity relation returned to normal when the bathing medium was changed back to ASW containing 10 mM-Ca2+. The light-induced rise in luminescence recorded from photoreceptors injected with the photoprotein aequorin (the 'aequorin response') declined by at most 50% after dark-adapted photoreceptors were bathed in EGTA-ASW for 45 min. However, the aequorin response progressively declined by 98% if cells were repetitively illuminated while bathed in EGTA-ASW. The total intracellular Ca content of whole end-organs was measured by atomic absorption spectroscopy. Total intracellular Ca content did not change significantly while photoreceptors were bathed in EGTA-ASW even after repetitive illumination. We suggest that cytosolic Ca2+ is required by one or more steps in the mechanisms that link rhodopsin isomerization to both (i) an increase in the conductance of the cell membrane to Na+ and (ii) a release of Ca2+ from a light-labile store. PMID:3928878

  2. Roles of mitochondria and temperature in the control of intracellular calcium in adult rat sensory neurons.

    PubMed

    Kang, S H; Carl, A; McHugh, J M; Goff, H R; Kenyon, J L

    2008-04-01

    We recorded Ca2+ current and intracellular Ca2+ ([Ca2+](i)) in isolated adult rat dorsal root ganglion (DRG) neurons at 20 and 30 degrees C. In neurons bathed in tetraethylammonium and dialyzed with cesium, warming reduced resting [Ca2+](i) from 87 to 49 nM and the time constant of the decay of [Ca2+](i) transients (tau(r)) from 1.3 to 0.99s (Q(10)=1.4). The Buffer Index, the ratio between Ca2+ influx and Delta[Ca2+](i) (f I(ca)d(t)/Delta[Ca2+]i) , increased two- to threefold with warming. Neither inhibition of the plasma membrane Ca2+ -ATPase by intracellular sodium orthovanadate nor inhibition of Ca2+ uptake by the endoplasmic reticulum by thapsigargin plus ryanodine were necessary for the effects of warming on these parameters. In contrast, inhibition of the mitochondrial Ca2+ uniporter by intracellular ruthenium red largely reversed the effects of warming. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 500 nM) increased resting [Ca2+](i) at 30 degrees C. Ten millimolar intracellular sodium prolonged the recovery of [Ca2+](i) transients to 10-40s. This effect was reversed by an inhibitor of mitochondrial Na(+)/Ca2+ -exchange (CGP 37157, 10 microM). Thus, mitochondrial Ca2+ uptake is necessary for the temperature-dependent increase in Ca2+ buffering and mitochondrial Ca2+ fluxes contribute to the control of [Ca2+](i) between 50 and 150 nM at 30 degrees C. PMID:17716728

  3. Caffeine inhibits inositol trisphosphate-mediated liberation of intracellular calcium in Xenopus oocytes.

    PubMed Central

    Parker, I; Ivorra, I

    1991-01-01

    1. Voltage-clamp recording of Ca(2+)-activated chloride currents in Xenopus oocytes was used to study the effects of caffeine on the liberation of intracellular Ca2+ induced by photo-release of inositol 1,4,5-trisphosphate (InsP3) from caged InsP3. Bath application of caffeine, at concentrations between 0.1 and 10 mM, reduced or abolished the current evoked by photo-release of InsP3 and by microinjection of InsP3. 2. Caffeine did not appreciably reduce currents evoked by injection of Ca2+ into oocytes, whereas measurements using the Ca2+ indicator Rhod-2 showed that it instead inhibited the liberation of Ca2+ by InsP3. 3. Caffeine increased the threshold amount of InsP3 required to evoke a current response and proportionally reduced the currents evoked by suprathreshold levels of InsP3. 4. Theophylline and 3-isobutyl-1-methylxanthine (IBMX) were much less potent than caffeine, and few changes were seen in the InsP3 responses following application of forskolin or intracellular injection of cyclic AMP. Thus, inhibition of InsP3 responses by caffeine does not arise through inhibition of phosphodiesterase enzymes. 5. Even at high (10 mM) concentrations, caffeine did not itself elicit any clear Ca(2+)-activated current. It is therefore unlikely that inhibition of the InsP3 responses arise because caffeine itself liberates Ca2+ from intracellular stores. 6. The site of action of caffeine is intracellular, because injections of caffeine into the oocyte strongly inhibited responses to InsP3, whereas local extracellular applications of similar amounts were almost without effect. PMID:1844813

  4. The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction

    PubMed Central

    Picas, Laura; Gaits-Iacovoni, Frederique; Goud, Bruno

    2016-01-01

    Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction. PMID:27092250

  5. A quantitative comparison of the effects of intracellular calcium injection and light adaptation on the photoresponse of Limulus ventral photoreceptors

    PubMed Central

    1977-01-01

    Calcium ions were iontophoretically injected into ventral photoreceptors of Limulus by passing current between two intracellular pipettes. Changes in sensitivity and photoresponse time course were measured for both light adaptation and Ca++ injection. We found for some photoreceptors that there was no significant difference in the photoresponse time course for desensitization produced by light adaptation or by Ca++ injection. In other photoreceptors, the time delay of photoresponse for Ca++ injection was slightly longer than for light adaptation. The variability of threshold response amplitude and time delay decreases when the photoreceptor is desensitized by either light adaptation or Ca++ injection. The peak amplitude versus log stimulus intensity relationships for controls, light adaptation, and Ca++ injection all could be described very closely by a single template curve shifted along the log intensity axis. A 40- to 50-fold change in sensitivity is associated with a 2-fold change in photoresponse time delay for both light adaptation and Ca++ injection. PMID:591913

  6. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens. PMID:26667172

  7. Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

    PubMed

    Bergeron, John J M; Di Guglielmo, Gianni M; Dahan, Sophie; Dominguez, Michel; Posner, Barry I

    2016-06-01

    Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated. PMID:27023845

  8. Modulation of intracellular calcium waves and triggered activities by mitochondrial ca flux in mouse cardiomyocytes.

    PubMed

    Zhao, Zhenghang; Gordan, Richard; Wen, Hairuo; Fefelova, Nadezhda; Zang, Wei-Jin; Xie, Lai-Hua

    2013-01-01

    Recent studies have suggested that mitochondria may play important roles in the Ca(2+) homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca(2+) flux can regulate the generation of Ca(2+) waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca(2+) (Cai (2+)) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and CaWs were induced in the presence of high (4 mM) external Ca(2+) (Cao (2+)). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai (2+) levels even after depletion of SR Ca(2+) in the absence of Cao (2+) , suggesting Ca(2+) release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m ) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m ) or Ru360 (a mitochondrial Ca(2+) uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca(2+) uniporter activator kaempferol. Our results suggest that mitochondrial Ca(2+) release and uptake exquisitely control the local Ca(2+) level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis. PMID:24348912

  9. Enhancement of calcium signalling dynamics and stability by delayed modulation of the plasma-membrane calcium-ATPase in human T cells

    PubMed Central

    Bautista, Diana M; Hoth, Markus; Lewis, Richard S

    2002-01-01

    In addition to its homeostatic role of maintaining low resting levels of intracellular calcium ([Ca2+]i), the plasma-membrane calcium-ATPase (PMCA) may actively contribute to the generation of complex Ca2+ signals. We have investigated the role of the PMCA in shaping Ca2+ signals in Jurkat human leukaemic T cells using single-cell voltage-clamp and calcium-imaging techniques. Crosslinking the T-cell receptor with the monoclonal antibody OKT3 induces a biphasic elevation in [Ca2+]i consisting of a rapid overshoot to a level > 1 μM, followed by a slow decay to a plateau of ≈0.5 μM. A similar overshoot was triggered by a constant level of Ca2+ influx through calcium-release-activated Ca2+ (CRAC) channels in thapsigargin-treated cells, due to a delayed increase in the rate of Ca2+ clearance by the PMCA. Following a rise in [Ca2+]i, PMCA activity increased in two phases: a rapid increase followed by a further calcium-dependent increase of up to approximately fivefold over 10-60 s, termed modulation. After the return of [Ca2+]i to baseline levels, the PMCA recovered slowly from modulation (τ ≈4 min), effectively retaining a ‘memory’ of the previous [Ca2+]i elevation. Using a Michaelis-Menten model with appropriate corrections for cytoplasmic Ca2+ buffering, we found that modulation extended the dynamic range of PMCA activity by increasing both the maximal pump rate and Ca2+ sensitivity (reduction of KM). A simple flux model shows how pump modulation and its reversal produce the initial overshoot of the biphasic [Ca2+]i response. The modulation of PMCA activity enhanced the stability of Ca2+ signalling by adjusting the efflux rate to match influx through CRAC channels, even at high [Ca2+]i levels that saturate the transport sites and would otherwise render the cell defenceless against additional Ca2+ influx. At the same time, the delay in modulation enables small Ca2+ fluxes to transiently elevate [Ca2+]i, thus enhancing Ca2+ signalling dynamics. PMID:12068047

  10. Host Intracellular Signaling Events and Pro-inflammatory Cytokine Production in African Trypanosomiasis

    PubMed Central

    Kuriakose, Shiby M.; Singh, Rani; Uzonna, Jude E.

    2016-01-01

    Pathogens, such as bacteria, viruses, and parasites, possess specific molecules or proteins that are recognized by several host innate immune receptors, leading to the activation of several intracellular signaling molecules and pathways. The magnitude and quality of these events significantly affect the outcome of infection. African trypanosomes, including Trypanosoma congolense, are capable of manipulating the host immune response, including the activity of macrophages, which are the key immune cells that contribute to the immunopathogenesis of African trypanosomiasis. Although it is known that immune hyperactivation and excessive pro-inflammatory cytokine production are the hallmarks of African trypanosomiasis, the mechanisms through which these events are triggered are poorly defined. However, it is known that macrophages may play a significant role in these processes, because phagocytosis of trypanosomes by macrophages initiates intracellular signal transduction cascades that lead to the release of pro-inflammatory cytokines and alteration in cell function. This review highlights recent progress in our understanding of the innate immune receptors, signaling pathways, and transcription factors involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. It will reveal the existence of complex signaling events through which the parasite modulates the host immune response, thus identifying novel targets that could aid in designing strategies to effectively control the disease. PMID:27242788

  11. Host Intracellular Signaling Events and Pro-inflammatory Cytokine Production in African Trypanosomiasis.

    PubMed

    Kuriakose, Shiby M; Singh, Rani; Uzonna, Jude E

    2016-01-01

    Pathogens, such as bacteria, viruses, and parasites, possess specific molecules or proteins that are recognized by several host innate immune receptors, leading to the activation of several intracellular signaling molecules and pathways. The magnitude and quality of these events significantly affect the outcome of infection. African trypanosomes, including Trypanosoma congolense, are capable of manipulating the host immune response, including the activity of macrophages, which are the key immune cells that contribute to the immunopathogenesis of African trypanosomiasis. Although it is known that immune hyperactivation and excessive pro-inflammatory cytokine production are the hallmarks of African trypanosomiasis, the mechanisms through which these events are triggered are poorly defined. However, it is known that macrophages may play a significant role in these processes, because phagocytosis of trypanosomes by macrophages initiates intracellular signal transduction cascades that lead to the release of pro-inflammatory cytokines and alteration in cell function. This review highlights recent progress in our understanding of the innate immune receptors, signaling pathways, and transcription factors involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. It will reveal the existence of complex signaling events through which the parasite modulates the host immune response, thus identifying novel targets that could aid in designing strategies to effectively control the disease. PMID:27242788

  12. Developmental axon stretch stimulates neuron growth while maintaining normal electrical activity, intracellular calcium flux, and somatic morphology

    PubMed Central

    Loverde, Joseph R.; Pfister, Bryan J.

    2015-01-01

    Elongation of nerve fibers intuitively occurs throughout mammalian development, and is synchronized with expansion of the growing body. While most tissue systems enlarge through mitosis and differentiation, elongation of nerve fibers is remarkably unique. The emerging paradigm suggests that axons undergo stretch as contiguous tissues enlarge between the proximal and distal segments of spanning nerve fibers. While stretch is distinct from growth, tension is a known stimulus which regulates the growth of axons. Here, we hypothesized that the axon stretch-growth process may be a natural form of injury, whereby regenerative processes fortify elongating axons in order to prevent disconnection. Harnessing the live imaging capability of our axon stretch-growth bioreactors, we assessed neurons both during and following stretch for biomarkers associated with injury. Utilizing whole-cell patch clamp recording, we found no evidence of changes in spontaneous action potential activity or degradation of elicited action potentials during real-time axon stretch at strains of up to 18% applied over 5 min. Unlike traumatic axonal injury, functional calcium imaging of the soma revealed no shifts in free intracellular calcium during axon stretch. Finally, the cross-sectional areas of nuclei and cytoplasms were normal, with no evidence of chromatolysis following week-long stretch-growth limited to the lower of 25% strain or 3 mm total daily stretch. The neuronal growth cascade coupled to stretch was concluded to be independent of the changes in membrane potential, action potential generation, or calcium flux associated with traumatic injury. While axon stretch-growth is likely to share overlap with regenerative processes, we conclude that developmental stretch is a distinct stimulus from traumatic axon injury. PMID:26379492

  13. Intracellular distributions and putative functions of calcium-binding proteins in the bullfrog vestibular otolith organs

    NASA Technical Reports Server (NTRS)

    Baird, R. A.; Steyger, P. S.; Schuff, N. R.

    1997-01-01

    Hair cells in the bullfrog vestibular otolith organs were immunolabeled by monoclonal and polyclonal antisera against calbindin (CaB), calmodulin (CaM), calretinin (CaR), and parvalbumin (PA). S-100, previously shown to immunolabel striolar hair cells in fish vestibular organs, only weakly immunolabeled hair cells in the bullfrog vestibular otolith organs. Immunolabeling was not detected in supporting cells. With the exception of CaR, myelinated axons and unmyelinated nerve terminals were immunolabeled by all of the above antisera. Immunolabeling was seen in all saccular hair cells, although hair cells at the macular margins were immunolabeled more intensely for CaB, CaM, and PA than more centrally located hair cells. As the macula margins are known to be a growth zone, this labeling pattern suggests that marginal hair cells up-regulate their calcium-binding proteins during hair cell development. In the utriculus, immunolabeling for CaM and PA was generally restricted to striolar hair cells. CaR immunolabeling was restricted to the stereociliary array. Immunolabeling for other calcium-binding proteins was generally seen in both the cell body and hair bundles of hair cells, although this labeling was often localized to the stereociliary array and the apical portion of the cell body. CaM and PA immunolabeling in the stereociliary array in saccular and utricular striolar cells suggests a functional role for these proteins in mechanoelectric transduction and adaptation.

  14. Intracellular ionized calcium concentration in muscles from humans with malignant hyperthermia.

    PubMed

    López, J R; Alamo, L; Caputo, C; Wikinski, J; Ledezma, D

    1985-06-01

    Ca2+ selective microelectrodes have been used to determine the free myoplasmic [Ca2+] in human skeletal muscle obtained from patients who had developed early signs associated with malignant hyperthermia (MH) during anesthesia. Intercostal muscle biopsies were performed under local anesthesia in four MH patients 15 days to 4 months after developing the MH crisis and in three control subjects. We used only microelectrodes that showed a Nernstian response between pCa3 and pCa7 (30.5 mV per decade at 37 degrees C). Membrane resting potential (V(m)) and calcium potential (V(Ca)) were obtained from superficial fibers. The free cytosolic [Ca2+] was 0.39 +/- 0.1 microM (mean +/- SEM, n = 18) in muscle fibers obtained from malignant hyperthermic patients, whereas in control subjects it was 0.11 +/- 0.02 microM (n = 10). These results suggest that this syndrome might be related to an abnormally high myoplasmic free resting calcium concentration, probably due to a defective function of the plasma membrane or the sarcoplasmic reticulum. PMID:16758579

  15. Differential Calcium Signaling Mediated by Voltage-Gated Calcium Channels in Rat Retinal Ganglion Cells and Their Unmyelinated Axons

    PubMed Central

    Sargoy, Allison; Sun, Xiaoping

    2014-01-01

    Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury. PMID:24416240

  16. Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells.

    PubMed

    Brandt, Benjamin; Munemasa, Shintaro; Wang, Cun; Nguyen, Desiree; Yong, Taiming; Yang, Paul G; Poretsky, Elly; Belknap, Thomas F; Waadt, Rainer; Alemán, Fernando; Schroeder, Julian I

    2015-01-01

    A central question is how specificity in cellular responses to the eukaryotic second messenger Ca(2+) is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca(2+)-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca(2+)-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca(2+)-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca(2+)-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca(2+)-dependent and Ca(2+)-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca(2+)-signaling on a cellular, genetic, and biochemical level. PMID:26192964

  17. Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells

    PubMed Central

    Brandt, Benjamin; Munemasa, Shintaro; Wang, Cun; Nguyen, Desiree; Yong, Taiming; Yang, Paul G; Poretsky, Elly; Belknap, Thomas F; Waadt, Rainer; Alemán, Fernando; Schroeder, Julian I

    2015-01-01

    A central question is how specificity in cellular responses to the eukaryotic second messenger Ca2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca2+-dependent and Ca2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca2+-signaling on a cellular, genetic, and biochemical level. DOI: http://dx.doi.org/10.7554/eLife.03599.001 PMID:26192964

  18. Sex and age dependent effects of androgens on glutamate-induced cell death and intracellular calcium regulation in the developing hippocampus

    PubMed Central

    Zup, Susan L.; Edwards, N. Shalon; McCarthy, Margaret M.

    2014-01-01

    Hippocampal neurons must maintain control over cytosolic calcium levels, especially during development, as excitation and calcium flux is necessary for proper growth and function. But excessive calcium can lead to excitotoxic cell death. Previous work suggests that neonatal male and female hippocampal neurons regulate cytosolic calcium differently, thereby leading to differential susceptibility to excitotoxic damage. Hippocampal neurons are also exposed to gonadal hormones during development and express high levels of androgen receptors. Androgens have both neuroprotective and neurotoxic effects in adults and developing animals. The present study sought to examine the effect of androgen on cell survival after an excitatory stimulus in the developing hippocampus, and whether androgen mediated calcium regulation was the governing mechanism. We observed that glutamate did not induce robust or sexually dimorphic apoptosis in cultured hippocampal neurons at an early neonatal time point, but did five days later – only in males. Further, pretreatment with the androgen dihydrotestosterone (DHT) protected males from apoptosis during this time, but had no effect on females. Calcium imaging of sex specific cultures revealed that DHT decreased the peak of intracellular calcium induced by glutamate, but only in males. To determine a possible mechanism for this androgen neuroprotection and calcium regulation, we quantified three calcium regulatory proteins, plasma membrane calcium ATPase1 (PMCA1), sodium/calcium exchanger1 (NCX1), and the sarco/endoplasmic reticulum calcium ATPase 2 (SERCA2). Surprisingly, there was no sex difference in the level of any of the three proteins. Treatment with DHT significantly decreased PMCA1 and NCX1, but increased SERCA2 protein levels in very young animals but not at a later timepoint. Taken together, these data suggest a complex interaction of sex, hormones, calcium regulation and developmental age; however androgens acting during the first

  19. Characterization of NAADP-mediated calcium signaling in human spermatozoa

    SciTech Connect

    Sánchez-Tusie, A.A.; Vasudevan, S.R.; Churchill, G.C.; Nishigaki, T.; Treviño, C.L.

    2014-01-10

    Highlights: •Human sperm cells synthesize NAADP. •NAADP-AM mediates [Ca{sup 2+}]{sub i} increases in human sperm in the absence of [Ca{sup 2+}]{sub o}. •Human sperm have two acidic compartments located in the head and midpiece. -- Abstract: Ca{sup 2+} signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca{sup 2+}-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca{sup 2+} signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca{sup 2+} and pH. Ca{sup 2+} fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca{sup 2+}] increases in human sperm even in the absence of extracellular Ca{sup 2+}. Using LysoTracker®, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-L-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker®, suggesting that these stores are the targets of NAADP action.

  20. Biphasic modulation by mGlu5 receptors of TRPV1-mediated intracellular calcium elevation in sensory neurons contributes to heat sensitivity

    PubMed Central

    Masuoka, T; Nakamura, T; Kudo, M; Yoshida, J; Takaoka, Y; Kato, N; Ishibashi, T; Imaizumi, N; Nishio, M

    2015-01-01

    Background and Purpose Elevation of glutamate, an excitatory amino acid, during inflammation and injury plays a crucial role in the reception and transmission of sensory information via ionotropic and metabotropic receptors. This study aimed to investigate the mechanisms underlying the biphasic effects of metabotropic glutamate mGlu5 receptor activation on responses to noxious heat. Experimental Approach We assessed the effects of intraplantar quisqualate, a non-selective glutamate receptor agonist, on heat and mechanical pain behaviours in mice. In addition, the effects of quisqualate on the intracellular calcium response and on membrane currents mediated by TRPV1 channels, were examined in cultured dorsal root ganglion neurons from mice. Key Results Activation of mGlu5 receptors in hind paw transiently increased, then decreased, the response to noxious heat. In sensory neurons, activation of mGlu5 receptors potentiated TRPV1-mediated intracellular calcium elevation, while terminating activation of mGlu5 receptors depressed it. TRPV1-induced currents were potentiated by activation of mGlu5 receptors under voltage clamp conditions and these disappeared after washout. However, voltage-gated calcium currents were inhibited by the mGlu5 receptor agonist, even after washout. Conclusions and Implications These results suggest that, in sensory neurons, mGlu5 receptors biphasically modulate TRPV1-mediated intracellular calcium response via transient potentiation of TRPV1 channel-induced currents and persistent inhibition of voltage-gated calcium currents, contributing to heat hyper- and hypoalgesia. PMID:25297838

  1. Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation

    PubMed Central

    Colinet, Anne-Sophie; Sengottaiyan, Palanivelu; Deschamps, Antoine; Colsoul, Marie-Lise; Thines, Louise; Demaegd, Didier; Duchêne, Marie-Clémence; Foulquier, François; Hols, Pascal; Morsomme, Pierre

    2016-01-01

    Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca2+ exchangers. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca2+ uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca2+/H+ antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn2+ to the cells. PMID:27075443

  2. Tuning cell migration: contractility as an integrator of intracellular signals from multiple cues

    PubMed Central

    Bordeleau, Francois; Reinhart-King, Cynthia A.

    2016-01-01

    There has been immense progress in our understanding of the factors driving cell migration in both two-dimensional and three-dimensional microenvironments over the years. However, it is becoming increasingly evident that even though most cells share many of the same signaling molecules, they rarely respond in the same way to migration cues. To add to the complexity, cells are generally exposed to multiple cues simultaneously, in the form of growth factors and/or physical cues from the matrix. Understanding the mechanisms that modulate the intracellular signals triggered by multiple cues remains a challenge. Here, we will focus on the molecular mechanism involved in modulating cell migration, with a specific focus on how cell contractility can mediate the crosstalk between signaling initiated at cell-matrix adhesions and growth factor receptors. PMID:27508074

  3. Tuning cell migration: contractility as an integrator of intracellular signals from multiple cues.

    PubMed

    Bordeleau, Francois; Reinhart-King, Cynthia A

    2016-01-01

    There has been immense progress in our understanding of the factors driving cell migration in both two-dimensional and three-dimensional microenvironments over the years. However, it is becoming increasingly evident that even though most cells share many of the same signaling molecules, they rarely respond in the same way to migration cues. To add to the complexity, cells are generally exposed to multiple cues simultaneously, in the form of growth factors and/or physical cues from the matrix. Understanding the mechanisms that modulate the intracellular signals triggered by multiple cues remains a challenge. Here, we will focus on the molecular mechanism involved in modulating cell migration, with a specific focus on how cell contractility can mediate the crosstalk between signaling initiated at cell-matrix adhesions and growth factor receptors. PMID:27508074

  4. The PHR proteins: intracellular signaling hubs in neuronal development and axon degeneration.

    PubMed

    Grill, Brock; Murphey, Rodney K; Borgen, Melissa A

    2016-01-01

    During development, a coordinated and integrated series of events must be accomplished in order to generate functional neural circuits. Axons must navigate toward target cells, build synaptic connections, and terminate outgrowth. The PHR proteins (consisting of mammalian Phr1/MYCBP2, Drosophila Highwire and C. elegans RPM-1) function in each of these events in development. Here, we review PHR function across species, as well as the myriad of signaling pathways PHR proteins regulate. These findings collectively suggest that the PHR proteins are intracellular signaling hubs, a concept we explore in depth. Consistent with prominent developmental functions, genetic links have begun to emerge between PHR signaling networks and neurodevelopmental disorders, such as autism, schizophrenia and intellectual disability. Finally, we discuss the recent and important finding that PHR proteins regulate axon degeneration, which has further heightened interest in this fascinating group of molecules. PMID:27008623

  5. Calcium signaling orchestrates glioblastoma development: Facts and conjunctures.

    PubMed

    Leclerc, Catherine; Haeich, Jacques; Aulestia, Francisco J; Kilhoffer, Marie-Claude; Miller, Andrew L; Néant, Isabelle; Webb, Sarah E; Schaeffer, Etienne; Junier, Marie-Pierre; Chneiweiss, Hervé; Moreau, Marc

    2016-06-01

    While it is a relatively rare disease, glioblastoma multiform (GBM) is one of the more deadly adult cancers. Following current interventions, the tumor is never eliminated whatever the treatment performed; whether it is radiotherapy, chemotherapy, or surgery. One hypothesis to explain this poor outcome is the "cancer stem cell" hypothesis. This concept proposes that a minority of cells within the tumor mass share many of the properties of adult neural stem cells and it is these that are responsible for the growth of the tumor and its resistance to existing therapies. Accumulating evidence suggests that Ca(2+) might also be an important positive regulator of tumorigenesis in GBM, in processes involving quiescence, maintenance, proliferation, or migration. Glioblastoma tumors are generally thought to develop by co-opting pathways that are involved in the formation of an organ. We propose that the cells initiating the tumor, and subsequently the cells of the tumor mass, must hijack the different checkpoints that evolution has selected in order to prevent the pathological development of an organ. In this article, two main points are discussed. (i) The first is the establishment of a so-called "cellular society," which is required to create a favorable microenvironment. (ii) The second is that GBM can be considered to be an organism, which fights to survive and develop. Since GBM evolves in a limited space, its only chance of development is to overcome the evolutionary checkpoints. For example, the deregulation of the normal Ca(2+) signaling elements contributes to the progression of the disease. Thus, by manipulating the Ca(2+) signaling, the GBM cells might not be killed, but might be reprogrammed toward a new fate that is either easy to cure or that has no aberrant functioning. 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:26826650

  6. Effects of low-level laser exposure on calcium channels and intracellular release in cultured astrocytes

    NASA Astrophysics Data System (ADS)

    Mang, Thomas S.; Maneshi, Mohammed M.; Shucard, David W.; Hua, Susan; Sachs, Frederick

    2016-03-01

    Prompted by a study of traumatic brain injury (TBI) in a model system of cultured astrocytes, we discovered that low level laser illumination (LLL) at 660nm elevates the level of intracellular Ca2+. The coherence of the illumination was not essential since incoherent red light also worked. For cells bathed in low Ca2+ saline so that influx was suppressed, the Ca2+ level rose with no significant latency following illumination and consistent with a slow leak of Ca2+ from storage such as from the endoplasmic reticulum and/or mitochondria. When the cells were bathed in normal Ca2+ saline, the internal Ca2+ rose, but with a latency of about 17 seconds from the beginning of illumination. Pharmacologic studies with ryanodine inhibited the light effect. Testing the cells with fluid shear stress as used in the TBI model showed that mechanically induced elevation of cell Ca2+ was unaffected by illumination.

  7. Lipofundin® MCT/LCT 20% increase left ventricular systolic pressure in an ex vivo rat heart model via increase of intracellular calcium level

    PubMed Central

    Kim, Yeon A; Han, Jeong Yeol; Jin, Sangkyu; Ok, Seong-Ho; Lee, Heon-Keun; Chung, Young-Kyun

    2016-01-01

    Background Lipid emulsions have been used to treat various drug toxicities and for total parenteral nutrition therapy. Their usefulness has also been confirmed in patients with local anesthetic-induced cardiac toxicity. The purpose of this study was to measure the hemodynamic and composition effects of lipid emulsions and to elucidate the mechanism associated with changes in intracellular calcium levels in myocardiocytes. Methods We measured hemodynamic effects using a digital analysis system after Intralipid® and Lipofundin® MCT/LCT were infused into hearts hanging in a Langendorff perfusion system. We measured the effects of the lipid emulsions on intracellular calcium levels in H9c2 cells by confocal microscopy. Results Infusion of Lipofundin® MCT/LCT 20% (1 ml/kg) resulted in a significant increase in left ventricular systolic pressure compared to that after infusing modified Krebs-Henseleit solution (1 ml/kg) (P = 0.003, 95% confidence interval [CI], 2.4–12.5). Lipofundin® MCT/LCT 20% had a more positive inotropic effect than that of Intralipid® 20% (P = 0.009, 95% CI, 1.4–11.6). Both lipid emulsion treatments increased intracellular calcium levels. Lipofundin® MCT/LCT (0.01%) increased intracellular calcium level more than that of 0.01% Intralipid® (P < 0.05, 95% CI, 0.0–1.9). Conclusions These two lipid emulsions had different inotropic effects depending on their triglyceride component. The inotropic effect of lipid emulsions could be related with intracellular calcium level. PMID:26885303

  8. Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells.

    PubMed

    Centuori, Sara M; Gomes, Cecil J; Trujillo, Jesse; Borg, Jamie; Brownlee, Joshua; Putnam, Charles W; Martinez, Jesse D

    2016-07-01

    Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects. PMID:27086143

  9. Effect of intracellular injection of inositol trisphosphate on cytosolic calcium and membrane currents in Aplysia neurons.

    PubMed

    Levy, S

    1992-06-01

    Pacemaker cells of Aplysia californica display a regular bursting that results from a complex interplay of Ca(2+)-mediated conductances and a continuous influx and extrusion of Ca2+. The effect of the second messenger 1,4,5-inositol trisphosphate (InsP3) on intracellular free Ca2+ concentration (Cai) regulation and electrical properties was investigated in identified neurons of the abdominal ganglion (R15, L2-L4, L6). Double-barreled Ca-selective microelectrodes were used to pressure inject InsP3 and measure Cai at the same point. Brief injection of InsP3 resulted in an average increase of Cai of 9.2 +/- 10.0 microM (+/- SE; n = 14) that decayed in about 1 min. The InsP3-induced elevation of Cai increased in a dose-dependent manner and saturated when large amounts of InsP3 were injected. The InsP3-induced Cai increase was the result of mobilization from intracellular stores; Cai could be repeatedly mobilized by InsP3 in cells superfused with 0 Ca artificial seawater for more than 60 min. Following multiple injections of InsP3, there was no evidence of immediate inhibition or facilitation. the spatial nature of the InsP3-induced Cai increase was investigated by moving the double-barreled Ca-selective microelectrode tip in a stepwise manner relative to the membrane surface. The largest InsP3-induced Cai increases were measured in an area 0-80 microns from the membrane surface; some cells had their largest InsP3-induced Cai increase some 120-160 microns away from the membrane. Injection of InsP3 in a bursting neuron induced an immediate train of action potentials followed by membrane hyperpolarization and a decrease in the burst frequency. Injection of InsP3 in voltage-clamped cells induced a biphasic response: a rapid inward current followed by a more prolonged outward current; the temporal overlap of the currents was depth dependent. Injection of InsP3 or Ca2+ from a double-barreled injecting electrode induced currents that were different in waveform and time course

  10. Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger.

    PubMed

    Saini, Harjot K; Dhalla, Naranjan S

    2006-12-01

    Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the

  11. Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2.

    PubMed Central

    Blatter, L. A.; Wier, W. G.

    1990-01-01

    We studied intracellular binding and possible compartmentalization of the fluorescent Ca2+ indicators, indo-1 and fura-2, in single mammalian cardiac ventricular cells that had been loaded with indo-1 and fura-2 by exposure to the acetoxymethylester form of the indicators (indo-1/AM and fura-2/AM). Techniques similar to those used in experiments on fluorescence recovery after photobleaching (FRAP) were used. It was assumed that reversible binding in myoplasm would be evident as slowed recovery of fluorescence after photobleaching, and that irreversible binding of the indicators to immobile myoplasmic sites (or "compartmentalization" in organelles) would be evident as incomplete recovery. Through the use of a mask, one half of a cell was exposed to high-intensity ultraviolet (UV) light to bleach the indo-1 or fura-2 in only that part of the cell. Upon removal of the mask and termination of the high-intensity UV illumination, fluorescence recovered in the bleached half of the cell, indicating diffusion of indo-1 and fura-2. Mathematical modeling of the diffusional redistribution of the indicators indicated that in these cells the apparent diffusion coefficient for indo-1 is 1.57 x 10(-7) cm2 s-1 (SD 0.48 x 10(-7) cm2 s-1; n = 5 cells, 21 degrees C), and for fura-2 is 3.19 x 10(-7) cm2 s-1 (SD 1.85 x 10(-7) cm2 s-1; n = 6 cells, 21 degrees C). These values are approximately 6 and 3, respectively, times smaller than those expected for free diffusion in the myoplasm. In the bleached half of the cell the recovered level of fluorescence never reached the final level in the half not exposed to UV light. The extent of incomplete recovery was variable amongst the cells. Our analysis indicated that, under the conditions we used, approximately one-third of the intracellular dye is not diffusible in the myoplasm. Images FIGURE 1 FIGURE 2 PMID:2275965

  12. Maturation of intracellular calcium homeostasis in sheep pulmonary arterial smooth muscle cells.

    PubMed

    Goyal, Ravi; Creel, Kara D; Chavis, Erica; Smith, Gregory D; Longo, Lawrence D; Wilson, Sean M

    2008-11-01

    Cytosolic Ca(2+) signaling dynamics are important to pulmonary arterial reactivity, and alterations are implicated in pulmonary vascular disorders. Yet, adaptations in cellular Ca(2+) homeostasis and receptor-mediated Ca(2+) signaling with maturation from fetal to adult life in pulmonary arterial smooth muscle cells (PASMCs) are not known. The present study tested the hypothesis that cytosolic Ca(2+) homeostasis and receptor-generated Ca(2+) signaling adapt with maturation in sheep PASMCs. Digitalized fluorescence microscopy was performed using isolated PASMCs from fetal and adult sheep that were loaded with the Ca(2+) indicator fura 2. The results show that basal cytosolic and sarcoplasmic reticulum Ca(2+) levels are attained before birth. Similarly, Ca(2+) efflux pathways from the cytosol and basal as well as capacitative Ca(2+) entry (CCE) are also developed before birth. However, receptor-mediated Ca(2+) signaling adapts with maturation. Prominently, serotonin stimulation elicited Ca(2+) elevations in very few fetal compared with adult PASMCs; in contrast, phenylephrine elevated Ca(2+) in a similar percentage of fetal and adult PASMCs. Serotonin and phenylephrine elicited Ca(2+) increases of a similar magnitude in reactive cells of fetus and adult, supporting the assertion that inositol trisphosphate signaling is intact. Caffeine and ATP elevated Ca(2+) in equivalent numbers of fetal and adult PASMCs. However, the caffeine-induced cytosolic Ca(2+) increase was significantly greater in fetal PASMCs, whereas the ATP-elicited increase was greater in adult cells. Overall, the results of this study demonstrate selective adaptations in receptor-mediated Ca(2+) signaling, but not in cellular Ca(2+) homeostasis. PMID:18776056

  13. The adaptor protein CIN85 assembles intracellular signaling clusters for B cell activation.

    PubMed

    Kühn, Julius; Wong, Leo E; Pirkuliyeva, Sona; Schulz, Kathrin; Schwiegk, Claudia; Fünfgeld, Kevser Gencalp; Keppler, Selina; Batista, Facundo D; Urlaub, Henning; Habeck, Michael; Becker, Stefan; Griesinger, Christian; Wienands, Jürgen

    2016-01-01

    The adaptor molecule Cbl-interacting protein of 85 kD (CIN85) regulates signaling from a number of cell surface receptors, such as growth factor receptors and antigen receptors on lymphocytes. Because of its multidomain structure, CIN85 is thought to act as a classical adaptor protein that connects functionally distinct components of a given signaling pathway through diverse protein domains. However, we found that in B lymphocytes, CIN85 functions to oligomerize SLP-65, which is the central effector protein of the B cell receptor (BCR). Therefore, CIN85 trimerizes through a carboxyl-terminal, coiled-coil domain. The multiple Src homology 3 (SH3) domains of trimeric CIN85 molecules associated with multiple SLP-65 molecules, which recruited further CIN85 trimers, thereby perpetuating the oligomerization process. Formation of this oligomeric signaling complex in resting B cells rendered the cells poised for the efficient initiation of intracellular signaling upon BCR stimulation. Our data suggest that the functionality of signaling cascades does not rely solely on the qualitative linkage of their various components but requires a critical number of effectors to become concentrated in signaling complexes. PMID:27353366

  14. Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling1[OPEN

    PubMed Central

    2016-01-01

    Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area. PMID:27208308

  15. Calcium signalling mediated by the 9 acetylcholine receptor in a cochlear cell line from the immortomouse.

    PubMed

    Jagger, D J; Griesinger, C B; Rivolta, M N; Holley, M C; Ashmore, J F

    2000-08-15

    1. We have investigated the characteristics of the alpha9 acetylcholine receptor (alpha9AChR) expressed in hair cell precursors in an immortalized cell line UB/OC-2 developed from the organ of Corti of the transgenic H-2Kb-tsA58 mouse (the Immortomouse) using both calcium imaging and whole-cell recording. 2. Ratiometric measurements of fura-2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 microM ACh. The calcium increase was seen in 66 % of the cells grown at 39 degrees C in differentiated conditions. A sm aller fraction (34%) of cells grown at 33 degrees C in proliferative con ditions responded. 3. Caffeine (10mM) elevated cell calcium. In the ab sence of caffeine, the majority of imaged cells responded only once to A Ch presentations. Pretreatment with caffeine ingibited all calcium respo nses to ACh. 4. In whole-cell tight-seal recordings 10 microM ACh activa ted inward current was dependent on the extracellular calcium concentrat ion with an estimated PCa/PNa of 80 for the alpha9 receptor at physiological calcium levels. 5 . The data indicate that ACh activates a calcium-permeable channel alpha 9AChR in UB/OC-2 cells and that the channel has a significantly higher c alcium permeability than other AChRs. The results indicate that the alp ha9AChR may be able to elevate intracellular calcium levels in hair cell s both directly and via store release. PMID:11011664

  16. Simultaneous Optical Mapping of Intracellular Free Calcium and Action Potentials from Langendorff Perfused Hearts

    PubMed Central

    Salama, Guy; Hwang, Seong-min

    2015-01-01

    The cardiac action potential (AP) controls the rise and fall of intracellular free Ca2+ (Cai), and thus the amplitude and kinetics of force generation. Besides excitation-contraction coupling, the reverse process where Cai influences the AP through Cai-dependent ionic currents has been implicated as the mechanism underlying QT alternans and cardiac arrhythmias in heart failure, ischemia/reperfusion, cardiac myopathy, myocardial infarction, congenital and drug-induced long QT syndrome, and ventricular fibrillation. The development of dual optical mapping at high spatial and temporal resolution provides a powerful tool to investigate the role of Cai anomalies in eliciting cardiac arrhythmias. This unit describes experimental protocols to map APs and Cai transients from perfused hearts by labeling the heart with two fluorescent dyes, one to measure transmembrane potential (Vm), the other Cai transients. High spatial and temporal resolution is achieved by selecting Vm and Cai probes with the same excitation but different emission wavelengths, to avoid cross-talk and mechanical components. PMID:19575468

  17. A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm.

    PubMed

    Romarowski, Ana; Sánchez-Cárdenas, Claudia; Ramírez-Gómez, Héctor V; Puga Molina, Lis del C; Treviño, Claudia L; Hernández-Cruz, Arturo; Darszon, Alberto; Buffone, Mariano G

    2016-03-01

    During capacitation, sperm acquire the ability to undergo the acrosome reaction (AR), an essential step in fertilization. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AR. An increase in intracellular Ca(2+) ([Ca(2+)]i) is necessary for AR to occur. In this study, we investigated the spatiotemporal correlation between the changes in [Ca(2+)]i and AR in single mouse spermatozoa in response to progesterone. We found that progesterone stimulates an [Ca(2+)]i increase in five different patterns: gradual increase, oscillatory, late transitory, immediate transitory, and sustained. We also observed that the [Ca(2+)]i increase promoted by progesterone starts at either the flagellum or the head. We validated the use of FM4-64 as an indicator for the occurrence of the AR by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, we have simultaneously visualized the rise in [Ca(2+)]i and the process of exocytosis in response to progesterone and found that only a specific transitory increase in [Ca(2+)]i originating in the sperm head promotes the initiation of AR. PMID:26819478

  18. Fc receptor-mediated phagocytosis, superoxide production and calcium signaling of beta 2 integrin-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Sawada, C; Higuchi, H; Teraoka, H; Yamaguchi, M

    1997-01-01

    Fc receptor for immunoglobulin G-mediated phagocytosis, superoxide production and intracellular calcium ([Ca2+]i) signaling of complement receptor type 3 (CR3)-deficient neutrophils from a heifer with leukocyte adhesion deficiency (BLAD) were compared to those of control heifers. The mean phagocytic activity of IgG-coated yeasts and aggregated bovine IgG (Agg-IgG)-induced superoxide production of CR3-deficient neutrophils were 10% and 77.9%, respectively, of those of control neutrophils. The [Ca2+]i signals in CR3-deficient neutrophils stimulated with Agg-IgG or concanavalin A were different with mean peak [Ca2+]i concentrations of 78% and 41.9%, respectively, of those of control neutrophils. These findings suggest that Fc receptor-mediated neutrophil functions are closely dependent on the presence of CR3 (CD11b/CD18) on the neutrophil cell surfaces. PMID:9343828

  19. Enhanced Proliferation of Porcine Bone Marrow Mesenchymal Stem Cells Induced by Extracellular Calcium is Associated with the Activation of the Calcium-Sensing Receptor and ERK Signaling Pathway

    PubMed Central

    Ye, Jingjing; Ai, Wei; Zhang, Fenglin; Zhu, Xiaotong; Shu, Gang; Wang, Lina; Gao, Ping; Xi, Qianyun; Zhang, YongLiang; Jiang, Qingyan; Wang, Songbo

    2016-01-01

    Porcine bone marrow mesenchymal stem cells (pBMSCs) have the potential for application in regenerative medicine. This study aims to investigate the effects of extracellular calcium ([Ca2+]o) on pBMSCs proliferation and to explore the possible underlying mechanisms. The results demonstrated that 4 mM [Ca2+]o significantly promoted pBMSCs proliferation by reducing the G0/G1 phase cell percentage and by increasing the S phase cell proportion and the proliferation index of pBMSCs. Accordingly, [Ca2+]o stimulated the expression levels of proliferative genes such as cyclin A2, cyclin D1/3, cyclin E2, and PCNA and inhibited the expression of p21. In addition, [Ca2+]o resulted in a significant elevation of intracellular calcium and an increased ratio of p-ERK/ERK. However, inhibition of calcium-sensing receptor (CaSR) by its antagonist NPS2143 abolished the aforementioned effects of [Ca2+]o. Moreover, [Ca2+]o-induced promotion of pBMSCs proliferation, the changes of proliferative genes expression levels, and the activation of ERK1/2 signaling pathway were effectively blocked by U0126, a selective ERK kinase inhibitor. In conclusion, our findings provided evidence that the enhanced pBMSCs proliferation in response to [Ca2+]o was associated with the activation of CaSR and ERK1/2 signaling pathway, which may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair. PMID:27123007

  20. Intracellular calcium changes induced by the endozepine triakontatetraneuropeptide in human polymorphonuclear leukocytes: role of protein kinase C and effect of calcium channel blockers

    PubMed Central

    Marino, Franca; Cosentino, Marco; Ferrari, Marco; Cattaneo, Simona; Frigo, Giuseppina; Fietta, Anna M; Lecchini, Sergio; Frigo, Gian Mario

    2004-01-01

    Background The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca++]i) changes followed by activation in human polymorphonuclear leukocytes (PMNs). The present study was undertaken to investigate the role of protein kinase (PK) C in the modulation of the response to TTN by human PMNs, and to examine the pharmacology of TTN-induced Ca++ entry through the plasma membrane of these cells. Results The PKC activator 12-O-tetradecanoylphorbol-13-acetate (PMA) concentration-dependently inhibited TTN-induced [Ca++]i rise, and this effect was reverted by the PKC inhibitors rottlerin (partially) and Ro 32-0432 (completely). PMA also inhibited TTN-induced IL-8 mRNA expression. In the absence of PMA, however, rottlerin (but not Ro 32-0432) per se partially inhibited TTN-induced [Ca++]i rise. The response of [Ca++]i to TTN was also sensitive to mibefradil and flunarizine (T-type Ca++-channel blockers), but not to nifedipine, verapamil (L-type) or ω-conotoxin GVIA (N-type). In agreement with this observation, PCR analysis showed the expression in human PMNs of the mRNA for all the α1 subunits of T-type Ca++ channels (namely, α1G, α1H, and α1I). Conclusions In human PMNs TTN activates PKC-modulated pathways leading to Ca++ entry possibly through T-type Ca++ channels. PMID:15228623

  1. Walnut extracts protect cultured microglia against LPS-induced neurotoxicity via modulation of intracellular calcium concentration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Walnuts are rich in omega-3 fatty acids, alpha-linolenic acid (ALA) and linoleic acid (LA), as compared to other edible plants. Previously, our laboratory had demonstrated that dietary walnut supplementation in aged animals enhanced protective signaling pathways, altered membrane microstructures, an...

  2. Calcium Mediates Glomerular Filtration through Calcineurin and mTORC2/Akt Signaling

    PubMed Central

    Bracken, Christina; Matthews, Douglas; O'Brien, Stephen; Schiavi, Susan; Wawersik, Stefan

    2011-01-01

    Alterations to the structure of the glomerular filtration barrier lead to effacement of podocyte foot processes, leakage of albumin, and the development of proteinuria. To better understand the signaling pathways involved in the response of the glomerular filtration barrier to injury, we studied freshly isolated rat glomeruli, which allows for the monitoring and pharmacologic manipulation of early signaling events. Administration of protamine sulfate rapidly damaged the isolated glomeruli, resulting in foot process effacement and albumin leakage. Inhibition of calcium channels and chelation of extracellular calcium reduced protamine sulfate-induced damage, suggesting that calcium signaling plays a critical role in the initial stages of glomerular injury. Calcineurin inhibitors (FK506 and cyclosporine A) and the cathepsin L inhibitor E64 all inhibited protamine sulfate-mediated barrier changes, which suggests that calcium signaling acts, in part, through calcineurin- and cathepsin L-dependent cleavage of synaptopodin, a regulator of actin dynamics. The mTOR inhibitor rapamycin also protected glomeruli, demonstrating that calcium signaling has additional calcineurin-independent components. Furthermore, activation of Akt through mTOR had a direct role on glomerular barrier integrity, and activation of calcium channels mediated this process, likely independent of phosphoinositide 3-kinase. Taken together, these results demonstrate the importance of calcium and related signaling pathways in the structure and function of the glomerular filtration barrier. PMID:21784900

  3. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    SciTech Connect

    Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

    2011-09-09

    Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  4. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  5. Inhibition of Cav3.2 T-type Calcium Channels by Its Intracellular I-II Loop.

    PubMed

    Monteil, Arnaud; Chausson, Patrick; Boutourlinsky, Katia; Mezghrani, Alexandre; Huc-Brandt, Sylvaine; Blesneac, Iulia; Bidaud, Isabelle; Lemmers, Céline; Leresche, Nathalie; Lambert, Régis C; Lory, Philippe

    2015-06-26

    Voltage-dependent calcium channels (Cav) of the T-type family (Cav3.1, Cav3.2, and Cav3.3) are activated by low threshold membrane depolarization and contribute greatly to neuronal network excitability. Enhanced T-type channel activity, especially Cav3.2, contributes to disease states, including absence epilepsy. Interestingly, the intracellular loop connecting domains I and II (I-II loop) of Cav3.2 channels is implicated in the control of both surface expression and channel gating, indicating