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Sample records for intercellurar calcium signals

  1. Calcium Signaling and Neurodegeneration

    PubMed Central

    2010-01-01

    Neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and spinocerebellar ataxias (SCA) are very important both for fundamental science and for practical medicine. Despite extensive research into the causes of these diseases, clinical researchers have had very limited progress and, as of now, there is still no cure for any of these diseases. One of the main obstacles in the way of creating treatments for these disorders is the fact that their etiology and pathophysiology still remain unclear. This paper reviews results that support the so–called “calcium hypothesis of neurodegenerative diseases.” The calcium hypothesis states that the atrophic and degenerative processes in the neurons of AD, PD, ALS, HD, and SCA patients are accompanied by alterations in calcium homeostasis. Moreover, the calcium hypothesis states that this deregulation of calcium signaling is one of the early–stage and key processes in the pathogenesis of these diseases. Based on the results we reviewed, we conclude that the calcium channels and other proteins involved in the neuronal calcium signaling system are potential drug targets for AD, PD, ALS, HD, and SCA therapy. PMID:22649630

  2. Inositol trisphosphate and calcium signalling

    NASA Astrophysics Data System (ADS)

    Berridge, Michael J.

    1993-01-01

    Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.

  3. 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

  4. Theoretical aspects of calcium signaling

    NASA Astrophysics Data System (ADS)

    Pencea, Corneliu Stefan

    2001-08-01

    Experiments investigating intracellular calcium dynamics have revealed that calcium signals differentially affect a variety of intracellular processes, from fertilization and cell development and differentiation to subsequent cellular activity, ending with cell death. As an intracellular messenger, calcium transmits information within and between cells, thus regulating their activity. To control such a variety of processes, calcium signals have to be very flexible and also precisely regulated. The cell uses a calcium signaling ``toolkit'', where calcium ions can act in different contexts of space, amplitude and time. For different tasks, the cell selects the particular signal, or combination of signals, that triggers the appropriate physiological response. The physical foundations of such a versatile cellular signaling toolkit involving calcium are not completely understood, despite important experimental and theoretical progress made recently. The declared goal of this work is to investigate physical mechanisms on which the propagation of differential signals can be based. The dynamics of calcium near a cluster of inositol trisphosphate (IP3) activated calcium channels has been investigated analytically and numerically. Our work has demonstrated that clusters of different IP3 receptors can show similar bistable behavior, but differ in both the transient and long term dynamics. We have also investigated the conditions under which a calcium signal propagates between a pair of localized stores. We have shown that the propagation of the signal across a random distribution of such stores shows a percolation transition manifested in the shape of the wave front. More importantly, our work indicates that specific distribution of stores can be interpreted as calcium circuits that can perform important signal analyzing task, from unidirectional propagation and coincidence detection to a complete set of logic gates. We believe that phenomena like the ones described are

  5. Calcium signaling in taste cells.

    PubMed

    Medler, Kathryn F

    2015-09-01

    The sense of taste is a common ability shared by all organisms and is used to detect nutrients as well as potentially harmful compounds. Thus taste is critical to survival. Despite its importance, surprisingly little is known about the mechanisms generating and regulating responses to taste stimuli. All taste responses depend on calcium signals to generate appropriate responses which are relayed to the brain. Some taste cells have conventional synapses and rely on calcium influx through voltage-gated calcium channels. Other taste cells lack these synapses and depend on calcium release to formulate an output signal through a hemichannel. Beyond establishing these characteristics, few studies have focused on understanding how these calcium signals are formed. We identified multiple calcium clearance mechanisms that regulate calcium levels in taste cells as well as a calcium influx that contributes to maintaining appropriate calcium homeostasis in these cells. Multiple factors regulate the evoked taste signals with varying roles in different cell populations. Clearly, calcium signaling is a dynamic process in taste cells and is more complex than has previously been appreciated. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

  6. Revisiting intracellular calcium signaling semantics.

    PubMed

    Haiech, Jacques; Audran, Emilie; Fève, Marie; Ranjeva, Raoul; Kilhoffer, Marie-Claude

    2011-12-01

    Cells use intracellular free calcium concentration changes for signaling. Signal encoding occurs through both spatial and temporal modulation of the free calcium concentration. The encoded message is detected by an ensemble of intracellular sensors forming the family of calcium-binding proteins (CaBPs) which must faithfully translate the message using a new syntax that is recognized by the cell. The cell is home to a significant although limited number of genes coding for proteins involved in the signal encoding and decoding processes. In a cell, only a subset of this ensemble of genes is expressed, leading to a genetic regulation of the calcium signal pathways. Calmodulin (CaM), the most ubiquitous expressed intracellular calcium-binding protein, plays a major role in calcium signal translation. Similar to a hub, it is central to a large and finely tuned network, receiving information, integrating it and dispatching the cognate response. In this review, we examine the different steps starting with an external stimulus up to a cellular response, with special emphasis on CaM and the mechanism by which it decodes calcium signals and translates it into exquisitely coordinated cellular events. By this means, we will revisit the calcium signaling semantics, hoping that we will ease communication between scientists dealing with calcium signals in different biological systems and different domains.

  7. Calcium in plant defence-signalling pathways.

    PubMed

    Lecourieux, David; Ranjeva, Raoul; Pugin, Alain

    2006-01-01

    In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signalling pathways. Variations in the cytosolic concentration of Ca2+ ([Ca2+]cyt) couple a large array of signals and responses. Here we concentrate on calcium signalling in plant defence responses, particularly on the generation of the calcium signal and downstream calcium-dependent events participating in the establishment of defence responses with special reference to calcium-binding proteins.

  8. Calcium signalling in diabetes.

    PubMed

    Guerrero-Hernandez, Agustin; Verkhratsky, Alexei

    2014-11-01

    Molecular cascades responsible for Ca(2+) homeostasis and Ca(2+) signalling could be assembled in highly plastic toolkits that define physiological adaptation of cells to the environment and which are intimately involved in all types of cellular pathology. Control over Ca(2+) concentration in different cellular compartments is intimately linked to cell metabolism, because (i) ATP production requires low Ca(2+), (ii) Ca(2+) homeostatic systems consume ATP and (iii) Ca(2+) signals in mitochondria stimulate ATP synthesis being an essential part of excitation-metabolic coupling. The communication between the ER and mitochondria plays an important role in this metabolic fine tuning. In the insulin resistance state and diabetes this communication has been impaired leading to different disorders, for instance, diminished insulin production by pancreatic β cells, reduced heart and skeletal muscle contractility, reduced NO production by endothelial cells, increased glucose production by liver, increased lipolysis by adipose cells, reduced immune responses, reduced cognitive functions, among others. All these processes eventually trigger degenerative events resulting in overt diabetes due to reduction of pancreatic β cell mass, and different complications of diabetes, such as retinopathy, nephropathy, neuropathy, and different cardiovascular diseases.

  9. 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.

  10. 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

  11. 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.

  12. Calcium signaling in pluripotent stem cells.

    PubMed

    Apáti, Ágota; Pászty, Katalin; Erdei, Zsuzsa; Szebényi, Kornélia; Homolya, László; Sarkadi, Balázs

    2012-04-28

    Pluripotent stem cells represent a new source of biological material allowing the exploration of signaling phenomena during normal cell development and differentiation. Still, the calcium signaling pathways and intracellular calcium responses to various ligands or stress conditions have not been sufficiently explored as yet in embryonic or induced pluripotent stem cells and in their differentiated offspring. This is partly due to the special culturing conditions of these cell types, the rapid morphological and functional changes in heterogeneous cell populations during early differentiation, and methodological problems in cellular calcium measurements. In this paper, we review the currently available data in the literature on calcium signaling in pluripotent stem cells and discuss the potential shortcomings of these studies. Various assay methods are surveyed for obtaining reliable data both in undifferentiated embryonic stem cells and in specific, stem cell-derived human tissues. In this paper, we present the modulation of calcium signaling in human embryonic stem cells (hESC) and in their derivates; mesenchymal stem cell like (MSCl) cells and cardiac tissues using the fluorescent calcium indicator Fluo-4 and confocal microscopy. LPA, trypsin and angiotensin II were effective in inducing calcium signals both in HUES9 and MSCl cells. Histamine and thrombin induced calcium signal exclusively in the MSCl cells, while ATP was effective only in HUES9 cells. There was no calcium signal evoked by GABA, even at relatively high concentrations. In stem cell-derived cardiomyocytes a rapid increase in the beating rate and an increase of the calcium signal peaks could be observed after the addition of adrenaline, while verapamil led to a strong decrease in cellular calcium and stopped spontaneous contractions in a relaxed state.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Calcium signals in olfactory neurons.

    PubMed

    Tareilus, E; Noé, J; Breer, H

    1995-11-09

    Laser scanning confocal microscopy in combination with the fluorescent calcium indicators Fluo-3 and Fura-Red was employed to estimate the intracellular concentration of free calcium ions in individual olfactory receptor neurons and to monitor temporal and spatial changes in the Ca(2+)-level upon stimulation. The chemosensory cells responded to odorants with a significant increase in the calcium concentration, preferentially in the dendritic knob. Applying various stimulation paradigma, it was found that in a population of isolated cells, subsets of receptor neurons display distinct patterns of responsiveness.

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

    PubMed Central

    Verkhratsky, Alexei; Parpura, Vladimir

    2014-01-01

    Calcium as a divalent ion 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. 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, by neurotransmitters, by second messengers and/or mechanical stimulation, and as such is all-pervading pathway in physiology and pathophysiology of organisms. PMID:24291103

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. Calcium signaling in membrane repair

    PubMed Central

    Cheng, Xiping; Zhang, Xiaoli; Yu, Lu; Xu, Haoxing

    2015-01-01

    Resealing allows cells to mend damaged membranes rapidly when plasma membrane (PM) disruptions occur. Models of PM repair mechanisms include the “lipid-patch”, “endocytic removal”, and “macro-vesicle shedding” models, all of which postulate a dependence on local increases in intracellular Ca2+ at injury sites. Multiple calcium sensors, including synaptotagmin (Syt) VII, dysferlin, and apoptosis-linked gene-2 (ALG-2), are involved in PM resealing, suggesting that Ca2+ may regulate multiple steps of the repair process. Although earlier studies focused exclusively on external Ca2+, recent studies suggest that Ca2+ release from intracellular stores may also be important for PM resealing. Hence, depending on injury size and the type of injury, multiple sources of Ca2+ may be recruited to trigger and orchestrate repair processes. In this review, we discuss the mechanisms by which the resealing process is promoted by vesicular Ca2+ channels and Ca2+ sensors that accumulate at damage sites. PMID:26519113

  6. Calcium signaling in human pluripotent stem cells.

    PubMed

    Apáti, Ágota; Berecz, Tünde; Sarkadi, Balázs

    2016-03-01

    Human pluripotent stem cells provide new tools for developmental and pharmacological studies as well as for regenerative medicine applications. Calcium homeostasis and ligand-dependent calcium signaling are key components of major cellular responses, including cell proliferation, differentiation or apoptosis. Interestingly, these phenomena have not been characterized in detail as yet in pluripotent human cell sates. Here we review the methods applicable for studying both short- and long-term calcium responses, focusing on the expression of fluorescent calcium indicator proteins and imaging methods as applied in pluripotent human stem cells. We discuss the potential regulatory pathways involving calcium responses in hPS cells and compare these to the implicated pathways in mouse PS cells. A recent development in the stem cell field is the recognition of so called "naïve" states, resembling the earliest potential forms of stem cells during development, as well as the "fuzzy" stem cells, which may be alternative forms of pluripotent cell types, therefore we also discuss the potential role of calcium homeostasis in these PS cell types.

  7. 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. Copyright © 2013 Elsevier España, S.L. All rights reserved.

  8. 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

  9. Calcium binding proteins and calcium signaling in prokaryotes.

    PubMed

    Domínguez, Delfina C; Guragain, Manita; Patrauchan, Marianna

    2015-03-01

    With the continued increase of genomic information and computational analyses during the recent years, the number of newly discovered calcium binding proteins (CaBPs) in prokaryotic organisms has increased dramatically. These proteins contain sequences that closely resemble a variety of eukaryotic calcium (Ca(2+)) binding motifs including the canonical and pseudo EF-hand motifs, Ca(2+)-binding β-roll, Greek key motif and a novel putative Ca(2+)-binding domain, called the Big domain. Prokaryotic CaBPs have been implicated in diverse cellular activities such as division, development, motility, homeostasis, stress response, secretion, transport, signaling and host-pathogen interactions. However, the majority of these proteins are hypothetical, and only few of them have been studied functionally. The finding of many diverse CaBPs in prokaryotic genomes opens an exciting area of research to explore and define the role of Ca(2+) in organisms other than eukaryotes. This review presents the most recent developments in the field of CaBPs and novel advancements in the role of Ca(2+) in prokaryotes.

  10. Regulation of neurogenesis by calcium signaling

    PubMed Central

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

    2017-01-01

    Calcium (Ca2+) signaling has essential roles in the development of the nervous system from neural induction to the proliferation, migration, and differentiation of neural cells. Ca2+ signaling pathways are shaped by interactions among metabotropic signaling cascades, intracellular Ca2+ stores, ion channels, and a multitude of downstream effector proteins that activate specific genetic programs. The temporal and spatial dynamics of Ca2+ 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 Ca2+ signaling networks involved in neurogenesis. In this review, we assess the literature on the molecular and functional organization of Ca2+ 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 Ca2+ release-activated Ca2+ (CRAC) channels in these processes. PMID:27020657

  11. Another dimension to calcium signaling: a look at extracellular calcium.

    PubMed

    Hofer, Aldebaran M

    2005-03-01

    Cell biologists know the calcium ion best as a vital intracellular second messenger that governs countless cellular functions. However, the recent identification of cell-surface detectors for extracellular Ca(2+) has prompted consideration of whether Ca(2+) also functions as a signaling molecule in the extracellular milieu. The cast of Ca(2+) sensors includes the well-characterized extracellular-Ca(2+)-sensing receptor, a G-protein-coupled receptor originally isolated from the parathyroid gland. In addition, other receptors, channels and membrane proteins, such as gap junction hemichannels, metabotropic glutamate receptors, HERG K(+) channels and the receptor Notch, are all sensitive to external [Ca(2+)] fluctuations. A recently cloned Ca(2+) sensor (CAS) in Arabidopsis extends this concept to the plant kingdom. Emerging evidence indicates that [Ca(2+)] in the local microenvironment outside the cell undergoes alterations potentially sufficient to exert biological actions through these sensor proteins. The extracellular space might therefore constitute a much more dynamic Ca(2+) signaling compartment than previously appreciated.

  12. 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.

  13. Targeting calcium signaling in cancer therapy.

    PubMed

    Cui, Chaochu; Merritt, Robert; Fu, Liwu; Pan, Zui

    2017-01-01

    The intracellular calcium ions (Ca(2+)) act as second messenger to regulate gene transcription, cell proliferation, migration and death. Accumulating evidences have demonstrated that intracellular Ca(2+) homeostasis is altered in cancer cells and the alteration is involved in tumor initiation, angiogenesis, progression and metastasis. Targeting derailed Ca(2+) signaling for cancer therapy has become an emerging research area. This review summarizes some important Ca(2+) channels, transporters and Ca(2+)-ATPases, which have been reported to be altered in human cancer patients. It discusses the current research effort toward evaluation of the blockers, inhibitors or regulators for Ca(2+) channels/transporters or Ca(2+)-ATPase pumps as anti-cancer drugs. This review is also aimed to stimulate interest in, and support for research into the understanding of cellular mechanisms underlying the regulation of Ca(2+) signaling in different cancer cells, and to search for novel therapies to cure these malignancies by targeting Ca(2+) channels or transporters.

  14. Modeling the intracellular organization of calcium signaling.

    PubMed

    Dupont, Geneviève

    2014-01-01

    Calcium (Ca²⁺) is a key signaling ion that plays a fundamental role in many cellular processes in most types of tissues and organisms. The versatility of this signaling pathway is remarkable. Depending on the cell type and the stimulus, intracellular Ca²⁺ increases can last over different periods, as short spikes or more sustained signals. From a spatial point of view, they can be localized or invade the whole cell. Such a richness of behaviors is possible thanks to numerous exchange processes with the external medium or internal Ca²⁺ pools, mainly the endoplasmic or sarcoplasmic reticulum and mitochondria. These fluxes are also highly regulated. In order to get an accurate description of the spatiotemporal organization of Ca²⁺ signaling, it is useful to resort to modeling. Thus, each flux can be described by an appropriate kinetic expression. Ca²⁺ dynamics in a given cell type can then be simulated by a modular approach, consisting of the assembly of computational descriptions of the appropriate fluxes and regulations. Modeling can also be used to get insight into the mechanisms of decoding of the Ca²⁺ signals responsible for cellular responses. Cells can use frequency or amplitude coding, as well as take profit of Ca²⁺ oscillations to increase their sensitivity to small average Ca²⁺ increases. © 2014 Wiley Periodicals, Inc.

  15. 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

  16. Astrocyte calcium signalling orchestrates neuronal synchronization in organotypic hippocampal slices

    PubMed Central

    Sasaki, Takuya; Ishikawa, Tomoe; Abe, Reimi; Nakayama, Ryota; Asada, Akiko; Matsuki, Norio; Ikegaya, Yuji

    2014-01-01

    Astrocytes are thought to detect neuronal activity in the form of intracellular calcium elevations; thereby, astrocytes can regulate neuronal excitability and synaptic transmission. Little is known, however, about how the astrocyte calcium signal regulates the activity of neuronal populations. In this study, we addressed this issue using functional multineuron calcium imaging in hippocampal slice cultures. Under normal conditions, CA3 neuronal networks exhibited temporally correlated activity patterns, occasionally generating large synchronization among a subset of cells. The synchronized neuronal activity was correlated with astrocyte calcium events. Calcium buffering by an intracellular injection of a calcium chelator into multiple astrocytes reduced the synaptic strength of unitary transmission between pairs of surrounding pyramidal cells and caused desynchronization of the neuronal networks. Uncaging the calcium in the astrocytes increased the frequency of neuronal synchronization. These data suggest an essential role of the astrocyte calcium signal in the maintenance of basal neuronal function at the circuit level. PMID:24710057

  17. Store-operated calcium entry is essential for glial calcium signalling in CNS white matter.

    PubMed

    Papanikolaou, M; Lewis, A; Butt, A M

    2017-02-28

    'Calcium signalling' is the ubiquitous response of glial cells to multiple extracellular stimuli. The primary mechanism of glial calcium signalling is by release of calcium from intracellular stores of the endoplasmic reticulum (ER). Replenishment of ER Ca(2+) stores relies on store-operated calcium entry (SOCE). However, despite the importance of calcium signalling in glial cells, little is known about their mechanisms of SOCE. Here, we investigated SOCE in glia of the mouse optic nerve, a typical CNS white matter tract that comprises bundles of myelinated axons and the oligodendrocytes and astrocytes that support them. Using quantitative RT-PCR, we identified Orai1 channels, both Stim1 and Stim2, and the transient receptor potential M3 channel (TRPM3) as the primary channels for SOCE in the optic nerve, and their expression in both astrocytes and oligodendrocytes was demonstrated by immunolabelling of optic nerve sections and cultures. The functional importance of SOCE was demonstrated by fluo-4 calcium imaging on isolated intact optic nerves and optic nerve cultures. Removal of extracellular calcium ([Ca(2+)]o) resulted in a marked depletion of glial cytosolic calcium ([Ca(2+)]i), which recovered rapidly on restoration of [Ca(2+)]o via SOCE. 2-aminoethoxydiphenylborane (2APB) significantly decreased SOCE and severely attenuated ATP-mediated calcium signalling. The results provide evidence that Orai/Stim and TRPM3 are important components of the 'calcium toolkit' that underpins SOCE and the sustainability of calcium signalling in white matter glia.

  18. 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

  19. Calcium signal transmission in chick sensory neurones is diffusion based.

    PubMed

    Coatesworth, William; Bolsover, Stephen

    2008-03-01

    In many cells, the cytosol is an excitable medium through which calcium waves propagate by calcium induced calcium release (CICR). Many labs. have reported CICR in neurones subsequent to calcium influx through voltage gated channels. However, these have used long depolarizations. We have imaged calcium within chick sensory neurones following 50 ms depolarizations. Calcium signals travelled rapidly throughout the cell, such that changes at the cell centre were delayed by 24 ms compared to regions 3 microm from the plasma membrane. The nuclear envelope imposed a delay of 9 ms. A simple diffusion model with few unknowns gave good fits to the measured data, indicating that passive diffusion is responsible for signal transmission in these neurones. Simulations run without indicator dye did not reveal markedly different spatiotemporal dynamics, although concentration changes were larger. Simulations of calcium changes during action potentials revealed that large calcium transients occurring in the cytosol close to the nucleus are significantly attenuated by the nuclear envelope. Our results indicate that for the brief depolarisations that neurones will experience during normal signal processing calcium signals are transmitted by passive diffusion only. Diffusion is perfectly capable of transmitting the calcium signal into the interior of nerve cell bodies, and into the nucleoplasm.

  20. 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

  1. Slow Calcium Signals after Tetanic Electrical Stimulation in Skeletal Myotubes

    PubMed Central

    Eltit, José M.; Hidalgo, Jorge; Liberona, José L.; Jaimovich, Enrique

    2004-01-01

    The fluorescent calcium signal from rat myotubes in culture was monitored after field-stimulation with tetanic protocols. After the calcium signal sensitive to ryanodine and associated to the excitation-contraction coupling, a second long-lasting calcium signal refractory to ryanodine was consistently found. The onset kinetics of this slow signal were slightly modified in nominally calcium-free medium, as were both the frequency and number of pulses during tetanus. No signal was detected in the presence of tetrodotoxin. The participation of the dihydropyridine receptor (DHPR) as the voltage sensor for this signal was assessed by treatment with agonist and antagonist dihydropyridines (Bay K 8644 and nifedipine), showing an enhanced and inhibitory response, respectively. In the dysgenic GLT cell line, which lacks the α1S subunit of the DHPR, the signal was absent. Transfection of these cells with the α1S subunit restored the slow signal. In myotubes, the inositol 1,4,5-trisphosphate (IP3) mass increase induced by a tetanus protocol preceded in time the slow calcium signal. Both an IP3 receptor blocker and a phospholipase C inhibitor (xestospongin C and U73122, respectively) dramatically inhibit this signal. Long-lasting, IP3-generated slow calcium signals appear to be a physiological response to activity-related fluctuations in membrane potential sensed by the DHPR. PMID:15111418

  2. Role of NAADP in Coordinating Spatiotemporal Aspects of Calcium Signalling

    NASA Astrophysics Data System (ADS)

    Churchill, Grant C.; Galione, Antony

    We outline the roles of two low molecular weight phosphorylated compounds as intracellular messengers in calcium signaling. These new intracellular messengers (cyclic ADP-ribose-cADPR and nicotinic acid adenine dinucleotide phosphate-NAADP) have been shown to regulate calcium signalling across the plant and animal kingdoms. A central question in cell biology is what are the mechanisms by which calcium ions, arguably most important and universal regulator of cell activation, can encode specificity. The hypothesis that we have been testing is that exist in cells multiple signalling molecules and pathways which give rise to different patterns of calcium signals leading to highly specific cellular responses. We discuss new information about the molecular components of these new Ca 2+ signalling pathways and their role in generating Ca 2+ signals.

  3. The Hepatitis B Virus X Protein Elevates Cytosolic Calcium Signals by Modulating Mitochondrial Calcium Uptake

    PubMed Central

    Yang, Bei

    2012-01-01

    Chronic hepatitis B virus (HBV) infections are associated with the development of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is thought to play an important role in the development of HBV-associated HCC. One fundamental HBx function is elevation of cytosolic calcium signals; this HBx activity has been linked to HBx stimulation of cell proliferation and transcription pathways, as well as HBV replication. Exactly how HBx elevates cytosolic calcium signals is not clear. The studies described here show that HBx stimulates calcium entry into cells, resulting in an increased plateau level of inositol 1,4,5-triphosphate (IP3)-linked calcium signals. This increased calcium plateau can be inhibited by blocking mitochondrial calcium uptake and store-operated calcium entry (SOCE). Blocking SOCE also reduced HBV replication. Finally, these studies also demonstrate that there is increased mitochondrial calcium uptake in HBx-expressing cells. Cumulatively, these studies suggest that HBx can increase mitochondrial calcium uptake and promote increased SOCE to sustain higher cytosolic calcium and stimulate HBV replication. PMID:22031934

  4. 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.

  5. 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.

  6. Spindle function in Xenopus oocytes involves possible nanodomain calcium signaling

    PubMed Central

    Li, Ruizhen; Leblanc, Julie; He, Kevin; Liu, X. Johné

    2016-01-01

    Intracellular calcium transients are a universal phenomenon at fertilization and are required for egg activation, but the exact role of Ca2+ in second-polar-body emission remains unknown. On the other hand, similar calcium transients have not been demonstrated during oocyte maturation, and yet, manipulating intracellular calcium levels interferes with first-polar-body emission in mice and frogs. To determine the precise role of calcium signaling in polar body formation, we used live-cell imaging coupled with temporally precise intracellular calcium buffering. We found that BAPTA-based calcium chelators cause immediate depolymerization of spindle microtubules in meiosis I and meiosis II. Surprisingly, EGTA at similar or higher intracellular concentrations had no effect on spindle function or polar body emission. Using two calcium probes containing permutated GFP and the calcium sensor calmodulin (Lck-GCaMP3 and GCaMP3), we demonstrated enrichment of the probes at the spindle but failed to detect calcium increase during oocyte maturation at the spindle or elsewhere. Finally, endogenous calmodulin was found to colocalize with spindle microtubules throughout all stages of meiosis. Our results—most important, the different sensitivities of the spindle to BAPTA and EGTA—suggest that meiotic spindle function in frog oocytes requires highly localized, or nanodomain, calcium signaling. PMID:27582389

  7. Calcium Signaling in Interstitial Cells: Focus on Telocytes

    PubMed Central

    Radu, Beatrice Mihaela; Banciu, Adela; Banciu, Daniel Dumitru; Radu, Mihai; Cretoiu, Dragos; Cretoiu, Sanda Maria

    2017-01-01

    In this review, we describe the current knowledge on calcium signaling pathways in interstitial cells with a special focus on interstitial cells of Cajal (ICCs), interstitial Cajal-like cells (ICLCs), and telocytes. In detail, we present the generation of Ca2+ oscillations, the inositol triphosphate (IP3)/Ca2+ signaling pathway and modulation exerted by cytokines and vasoactive agents on calcium signaling in interstitial cells. We discuss the physiology and alterations of calcium signaling in interstitial cells, and in particular in telocytes. We describe the physiological contribution of calcium signaling in interstitial cells to the pacemaking activity (e.g., intestinal, urinary, uterine or vascular pacemaking activity) and to the reproductive function. We also present the pathological contribution of calcium signaling in interstitial cells to the aortic valve calcification or intestinal inflammation. Moreover, we summarize the current knowledge of the role played by calcium signaling in telocytes in the uterine, cardiac and urinary physiology, and also in various pathologies, including immune response, uterine and cardiac pathologies. PMID:28208829

  8. Calcium Signaling in Interstitial Cells: Focus on Telocytes.

    PubMed

    Radu, Beatrice Mihaela; Banciu, Adela; Banciu, Daniel Dumitru; Radu, Mihai; Cretoiu, Dragos; Cretoiu, Sanda Maria

    2017-02-13

    In this review, we describe the current knowledge on calcium signaling pathways in interstitial cells with a special focus on interstitial cells of Cajal (ICCs), interstitial Cajal-like cells (ICLCs), and telocytes. In detail, we present the generation of Ca(2+) oscillations, the inositol triphosphate (IP₃)/Ca(2+) signaling pathway and modulation exerted by cytokines and vasoactive agents on calcium signaling in interstitial cells. We discuss the physiology and alterations of calcium signaling in interstitial cells, and in particular in telocytes. We describe the physiological contribution of calcium signaling in interstitial cells to the pacemaking activity (e.g., intestinal, urinary, uterine or vascular pacemaking activity) and to the reproductive function. We also present the pathological contribution of calcium signaling in interstitial cells to the aortic valve calcification or intestinal inflammation. Moreover, we summarize the current knowledge of the role played by calcium signaling in telocytes in the uterine, cardiac and urinary physiology, and also in various pathologies, including immune response, uterine and cardiac pathologies.

  9. Calcium Buildup in Young Arteries May Signal Heart Attack Risk

    MedlinePlus

    ... html Calcium Buildup in Young Arteries May Signal Heart Attack Risk Even small amount in 30s, 40s appears ... their arteries are already at risk of a heart attack, a new study finds. Among those 32 to ...

  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. 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

  12. 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

  13. Calcium signaling in skeletal muscle development, maintenance and regeneration.

    PubMed

    Tu, Michelle K; Levin, Jacqueline B; Hamilton, Andrew M; Borodinsky, Laura N

    2016-03-01

    Skeletal muscle-specific stem cells are pivotal for tissue development and regeneration. Muscle plasticity, inherent in these processes, is also essential for daily life activities. Great advances and efforts have been made in understanding the function of the skeletal muscle-dedicated stem cells, called muscle satellite cells, and the specific signaling mechanisms that activate them for recruitment in the repair of the injured muscle. Elucidating these signaling mechanisms may contribute to devising therapies for muscular injury or disease. Here we review the studies that have contributed to our understanding of how calcium signaling regulates skeletal muscle development, homeostasis and regeneration, with a focus on the calcium dynamics and calcium-dependent effectors that participate in these processes.

  14. Astrocyte calcium elevations: properties, propagation, and effects on brain signaling.

    PubMed

    Fiacco, Todd A; McCarthy, Ken D

    2006-11-15

    The possibility that astrocytes are involved in brain signaling began to emerge in the late 1970s, when it was first shown that astroglia in vitro possess numerous receptors for neurotransmitters. It was later demonstrated that cultured astroglia and astrocytes in situ respond to neurotransmitters with increases in intracellular second messengers, including cyclic AMP and calcium. Astrocyte calcium responses have since been extensively studied both in culture and in intact tissue. We continue to gather information regarding the various compounds able to trigger astrocyte calcium increases, as well as the mechanisms involved in their initiation, propagation as a calcium wave within and between astrocytes, and effects on signaling within the brain. This review will focus on each of these aspects of astrocyte calcium regulation, and attempt to sort out which effects are more likely to occur in developmental, pathological, and physiological conditions. While we have come far in our understanding of the properties or potential of astrocytes' ability to signal to neurons using our array of pharmacological tools, we still understand very little regarding the level of involvement of astrocyte signaling in normal brain physiology.

  15. The role of Noise for Intracellular Calcium Signaling

    NASA Astrophysics Data System (ADS)

    Jung, Peter

    2003-03-01

    Calcium signaling is one of the most important and common cellular signaling mechanisms. Calcium signals turn on the wound response in epithelia cells (e.g. the cornea) and brain tissue, play an important role for metabolic processes in liver and pancreas, signal the heart muscle to contract, and are important players in learning and memory. Binding of agonist to receptors in the cell membrane can trigger the release of Ca^2+ from internal stores through small patches of release channels and the formation of intracellular, spatiotemporal calcium patterns that can be observed by using fluorescent markers. What makes these patterns so interesting from the biologic as well as the nonlinear dynamics perspective is that active elements (the release channels) are distributed discretely in small patches (about 100nm in size) that are typically 2mm apart. Processes on this scale are subject to large fluctuations that can dominate the overall calcium signal on a cellular and tissue scale depending on physiologic parameters. Pattern formation in such systems, with discretely distributed active sites and fluctuations poses new challenges that researchers have started to address only in the last years. Recent results in computational modeling of these processes from the elementary release process to the cellular level, are put into context with experimental findings. We focus on the effects of receptor clustering in the context of the cellular Ca^2+ signaling capability.

  16. 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.

  17. 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.

  18. 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.

  19. Role of calcium signaling in epithelial bicarbonate secretion.

    PubMed

    Jung, Jinsei; Lee, Min Goo

    2014-06-01

    Transepithelial bicarbonate secretion plays a key role in the maintenance of fluid and protein secretion from epithelial cells and the protection of the epithelial cell surface from various pathogens. Epithelial bicarbonate secretion is mainly under the control of cAMP and calcium signaling. While the physiological roles and molecular mechanisms of cAMP-induced bicarbonate secretion are relatively well defined, those induced by calcium signaling remain poorly understood in most epithelia. The present review summarizes the current status of knowledge on the role of calcium signaling in epithelial bicarbonate secretion. Specifically, this review introduces how cytosolic calcium signaling can increase bicarbonate secretion by regulating membrane transport proteins and how it synergizes with cAMP-induced mechanisms in epithelial cells. In addition, tissue-specific variations in the pancreas, salivary glands, intestines, bile ducts, and airways are discussed. We hope that the present report will stimulate further research into this important topic. These studies will provide the basis for future medicines for a wide spectrum of epithelial disorders including cystic fibrosis, Sjögren's syndrome, and chronic pancreatitis.

  20. Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors.

    PubMed

    van Koppen, C J; Meyer zu Heringdorf, D; Alemany, R; Jakobs, K H

    2001-04-27

    Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.

  1. [Electrophysiology and calcium signalling in human bronchial smooth muscle].

    PubMed

    Marthan, R; Hyvelin, J M; Roux, E; Savineau, J P

    1999-01-01

    Recently, cells isolated from airways have been used to characterize precisely the electrophysiological properties of this smooth muscle and to describe the changes in cytosolic calcium concentration ([Ca2+]i) occurring upon agonist stimulation. Although most studies have produced consistent results in terms of types of ion channel and pathways of calcium signalling implicated in the mechanical activity of airways, there are differences according to (i) the site along the bronchial tree (trachea vs. bronchi); (ii) the proliferating status of the cells (freshly isolated vs. cultured) and (iii) the species (human vs. animals). With regard to the electrophysiological properties of airway smooth muscle, the contribution to [Ca2+]i rise of Ca2+ influx through L-type voltage-dependent calcium channels depends on the balance between depolarization related to non-specific cation channel and/or chloride channel activation and hyperpolarization related to activation of a variety of potassium channels. Most of the above-mentioned channels appear to be controlled, directly or indirectly, by agonists in human bronchial smooth muscle. With regard to calcium signalling, the pattern of agonist-induced [Ca2+]i responses, the so-called [Ca2+]i oscillations, has been observed recently in freshly isolated airway smooth muscle cells. The role and the calcium sources involved in these oscillations in human bronchial smooth muscle are currently being investigated.

  2. 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

  3. 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

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

    PubMed Central

    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 Ca2+-dependent scaffolding depending on the chloroplast calcium sensor protein CAS. These findings uncover an additional mechanism in which chloroplast-modulated Ca2+ signalling controls the MAPK pathway for the activation of critical components of the retrograde signalling chain. PMID:27399341

  5. [Neurotransmitters, calcium signalling and neuronal communication].

    PubMed

    Eguiagaray, J G; Egea, J; Bravo-Cordero, J J; García, A G

    2004-04-01

    In this article we show some recent findings that constitute a great progress in the molecular knowledge of synaptic dynamics. To communicate, neurons use a code that includes electrical (action potentials) and chemical signals (neurotransmitters, neuromodulators). At the moment a great variety of molecules are known, whose neurotransmitter function in brain and the peripheral nervous system are out of question. Monoamines like acetylcholine, dopamine, noradrenaline, adrenaline, histamine, serotonin, glutamate, aspartate, glycine, ATP and GABA are good examples. Opioid neuropeptides, vasoactive intestinal peptide (VIP), neurokinines (substance P), somatostatin, neurotensin, neuropeptide Y, cholecystokinine, vasopressin or oxitocin have been related to the control of the stress response, sexual behaviour, food intake, pain, learning and memory, qualities that are also related to nitric oxide (NO). A great part of the molecular structure of the secretory machinery is known to be responsible for fast neurotransmitter release at the synapse, in response to action potentials. Proteins like sinaptobrevin (located in the membrane of the synaptic vesicle), sintaxin and SNAP-25 (both located at the presynaptic plasma membrane) constitute a trimeric complex which is responsible of the vesicular docking at the active sites for exocytosis. From this strategic location, vesicles release their neurotransmitter within few milliseconds, when the action potential invades the nerve terminal and activates the opening of the different subtypes of voltage-dependent Ca2+ channels. The asymmetric geographical distribution of each type of channel, in different neurons, rose the hypothesis that Ca2+ that enters through each subtype of channel is compartmentalised, thus favouring the generation of Ca2+ microdomains, in the cytosol and the nucleus, involved in different cellular functions. This great biochemical synaptic heterogeneity is facilitating the selection of many biological targets

  6. 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

  7. Systematic Characterization of Dynamic Parameters of Intracellular Calcium Signals

    PubMed Central

    Mackay, Laurent; Mikolajewicz, Nicholas; Komarova, Svetlana V.; Khadra, Anmar

    2016-01-01

    Dynamic processes, such as intracellular calcium signaling, are hallmark of cellular biology. As real-time imaging modalities become widespread, a need for analytical tools to reliably characterize time-series data without prior knowledge of the nature of the recordings becomes more pressing. The goal of this study is to develop a signal-processing algorithm for MATLAB that autonomously computes the parameters characterizing prominent single transient responses (TR) and/or multi-peaks responses (MPR). The algorithm corrects for signal contamination and decomposes experimental recordings into contributions from drift, TRs, and MPRs. It subsequently provides numerical estimates for the following parameters: time of onset after stimulus application, activation time (time for signal to increase from 10 to 90% of peak), and amplitude of response. It also provides characterization of the (i) TRs by quantifying their area under the curve (AUC), response duration (time between 1/2 amplitude on ascent and descent of the transient), and decay constant of the exponential decay region of the deactivation phase of the response, and (ii) MPRs by quantifying the number of peaks, mean peak magnitude, mean periodicity, standard deviation of periodicity, oscillatory persistence (time between first and last discernable peak), and duty cycle (fraction of period during which system is active) for all the peaks in the signal, as well as coherent oscillations (i.e., deterministic spikes). We demonstrate that the signal detection performance of this algorithm is in agreement with user-mediated detection and that parameter estimates obtained manually and algorithmically are correlated. We then apply this algorithm to study how metabolic acidosis affects purinergic (P2) receptor-mediated calcium signaling in osteoclast precursor cells. Our results reveal that acidosis significantly attenuates the amplitude and AUC calcium responses at high ATP concentrations. Collectively, our data

  8. Calcium Efflux Systems in Stress Signaling and Adaptation in Plants

    PubMed Central

    Bose, Jayakumar; Pottosin, Igor I.; Shabala, Stanislav S.; Palmgren, Michael G.; Shabala, Sergey

    2011-01-01

    Transient cytosolic calcium ([Ca2+]cyt) elevation is an ubiquitous denominator of the signaling network when plants are exposed to literally every known abiotic and biotic stress. These stress-induced [Ca2+]cyt elevations vary in magnitude, frequency, and shape, depending on the severity of the stress as well the type of stress experienced. This creates a unique stress-specific calcium “signature” that is then decoded by signal transduction networks. While most published papers have been focused predominantly on the role of Ca2+ influx mechanisms to shaping [Ca2+]cyt signatures, restoration of the basal [Ca2+]cyt levels is impossible without both cytosolic Ca2+ buffering and efficient Ca2+ efflux mechanisms removing excess Ca2+ from cytosol, to reload Ca2+ stores and to terminate Ca2+ signaling. This is the topic of the current review. The molecular identity of two major types of Ca2+ efflux systems, Ca2+-ATPase pumps and Ca2+/H+ exchangers, is described, and their regulatory modes are analyzed in detail. The spatial and temporal organization of calcium signaling networks is described, and the importance of existence of intracellular calcium microdomains is discussed. Experimental evidence for the role of Ca2+ efflux systems in plant responses to a range of abiotic and biotic factors is summarized. Contribution of Ca2+-ATPase pumps and Ca2+/H+ exchangers in shaping [Ca2+]cyt signatures is then modeled by using a four-component model (plasma- and endo-membrane-based Ca2+-permeable channels and efflux systems) taking into account the cytosolic Ca2+ buffering. It is concluded that physiologically relevant variations in the activity of Ca2+-ATPase pumps and Ca2+/H+ exchangers are sufficient to fully describe all the reported experimental evidence and determine the shape of [Ca2+]cyt signatures in response to environmental stimuli, emphasizing the crucial role these active efflux systems play in plant adaptive responses to environment. PMID:22639615

  9. Can calcium signaling be harnessed for cancer immunotherapy?

    PubMed

    Rooke, Ronald

    2014-10-01

    Experimental evidence shows the importance of the immune system in controlling tumor appearance and growth. Immunotherapy is defined as the treatment of a disease by inducing, enhancing or suppressing an immune response. In the context of cancer treatment, it involves breaking tolerance to a cancer-specific self-antigen and/or enhancing the existing anti-tumor immune response, be it specific or not. Part of the complexity in developing such treatment is that cancers are selected to escape adaptive or innate immune responses. These escape mechanisms are numerous and they may cumulate in one cancer. Moreover, different cancers of a same type may present different combinations of escape mechanisms. The limited success of immunotherapeutics in the clinic as stand-alone products may in part be explained by the fact that most of them only activate one facet of the immune response. It is important to identify novel methods to broaden the efficacy of immunotherapeutics. Calcium signaling is central to numerous cellular processes, leading to immune responses, cancer growth and apoptosis induced by cancer treatments. Calcium signaling in cancer therapy and control will be integrated to current cancer immunotherapy approaches. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

  10. Calcium Signaling in Synapse-to-Nucleus Communication

    PubMed Central

    Hagenston, Anna M.; Bading, Hilmar

    2011-01-01

    Changes in the intracellular concentration of calcium ions in neurons are involved in neurite growth, development, and remodeling, regulation of neuronal excitability, increases and decreases in the strength of synaptic connections, and the activation of survival and programmed cell death pathways. An important aspect of the signals that trigger these processes is that they are frequently initiated in the form of glutamatergic neurotransmission within dendritic trees, while their completion involves specific changes in the patterns of genes expressed within neuronal nuclei. Accordingly, two prominent aims of research concerned with calcium signaling in neurons are determination of the mechanisms governing information conveyance between synapse and nucleus, and discovery of the rules dictating translation of specific patterns of inputs into appropriate and specific transcriptional responses. In this article, we present an overview of the avenues by which glutamatergic excitation of dendrites may be communicated to the neuronal nucleus and the primary calcium-dependent signaling pathways by which synaptic activity can invoke changes in neuronal gene expression programs. PMID:21791697

  11. 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

  12. 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

  13. 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

  14. Tastants evoke cAMP signal in taste buds that is independent of calcium signaling.

    PubMed

    Trubey, Kristina R; Culpepper, Schartess; Maruyama, Yutaka; Kinnamon, Sue C; Chaudhari, Nirupa

    2006-08-01

    We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by Ca2+. Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular Ca2+. Specifically, 5 microM thapsigargin or 3 microM A-23187 (calcium ionophore), both of which increase intracellular Ca2+ concentration ([Ca2+]i), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular Ca2+, suggesting that it is dependent on Ca2+ entry rather than release from stores. With immunofluorescence microscopy, we show that the calcium-stimulated AC8 is principally expressed in taste cells that also express phospholipase Cbeta2 (i.e., cells that elevate [Ca2+]i in response to sweet, bitter, or umami stimuli). Taste transduction for sucrose is known to result in an elevation of both cAMP and calcium in taste buds. Thus we tested whether the cAMP increase in response to sucrose is a downstream consequence of calcium elevation. Even under conditions of depletion of stored and extracellular calcium, the cAMP response to sucrose stimulation persists in taste cells. The cAMP signal in response to monosodium glutamate stimulation is similarly unperturbed by calcium depletion. Our results suggest that tastant-evoked cAMP signals are not simply a secondary consequence of calcium modulation. Instead, cAMP and released Ca2+ may represent independent second messenger signals downstream of taste receptors.

  15. The use of flow cytometry to examine calcium signalling by TRPV1 in mixed cell populations.

    PubMed

    Assas, Bakri M; Abdulaal, Wesam H; Wakid, Majed H; Zakai, Haytham A; Miyan, J; Pennock, J L

    2017-03-31

    Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation. Multi parameter staining alongside Indo-1 AM to demonstrate calcium mobilization allows the study of real time calcium signaling in a complex environment.

  16. Nuclear calcium sensors reveal that repetition of trains of synaptic stimuli boosts nuclear calcium signaling in CA1 pyramidal neurons.

    PubMed

    Bengtson, C Peter; Freitag, H Eckehard; Weislogel, Jan-Marek; Bading, Hilmar

    2010-12-15

    Nuclear calcium is a key signal in the dialogue between synapse and nucleus that controls the genomic responses required for persistent adaptations, including memory and acquired neuroprotection. The amplitude and duration of nuclear calcium transients specify activity-induced transcriptional changes. However, the precise relationship between synaptic input and nuclear calcium output is unknown. Here, we used stereotaxic delivery to the rat brain of recombinant adeno-associated viruses encoding nuclear-targeted calcium sensors to assess nuclear calcium transients in CA1 pyramidal neurons after stimulation of the Schaffer collaterals. We show that in acute hippocampal slices, a burst of synaptic activity elicits a nuclear calcium signal with a regenerative component at above-threshold stimulation intensities. Using classical stimulation paradigms (i.e., high-frequency stimulation (HFS) and θ burst stimulation (TBS)) to induce early LTP (E-LTP) and transcription-dependent late LTP (L-LTP), we found that the magnitude of nuclear calcium signals and the number of action potentials activated by synaptic stimulation trains are greatly amplified by their repetition. Nuclear calcium signals and action potential generation were reduced by blockade of either NMDA receptors or L-type voltage-gated calcium channels, but not by procedures that lead to internal calcium store depletion or by blockade of metabotropic glutamate receptors. These findings identify a repetition-induced switch in nuclear calcium signaling that correlates with the transition from E-LTP to L-LTP, and may explain why the transcription-dependent phase of L-LTP is not induced by a single HFS or TBS but requires repeated trains of activity. Recombinant, nuclear-targeted indicators may prove useful for further analysis of nuclear calcium signaling in vivo.

  17. Nuclear Calcium Sensors Reveal that Repetition of Trains of Synaptic Stimuli Boosts Nuclear Calcium Signaling in CA1 Pyramidal Neurons

    PubMed Central

    Bengtson, C. Peter; Freitag, H. Eckehard; Weislogel, Jan-Marek; Bading, Hilmar

    2010-01-01

    Nuclear calcium is a key signal in the dialogue between synapse and nucleus that controls the genomic responses required for persistent adaptations, including memory and acquired neuroprotection. The amplitude and duration of nuclear calcium transients specify activity-induced transcriptional changes. However, the precise relationship between synaptic input and nuclear calcium output is unknown. Here, we used stereotaxic delivery to the rat brain of recombinant adeno-associated viruses encoding nuclear-targeted calcium sensors to assess nuclear calcium transients in CA1 pyramidal neurons after stimulation of the Schaffer collaterals. We show that in acute hippocampal slices, a burst of synaptic activity elicits a nuclear calcium signal with a regenerative component at above-threshold stimulation intensities. Using classical stimulation paradigms (i.e., high-frequency stimulation (HFS) and θ burst stimulation (TBS)) to induce early LTP (E-LTP) and transcription-dependent late LTP (L-LTP), we found that the magnitude of nuclear calcium signals and the number of action potentials activated by synaptic stimulation trains are greatly amplified by their repetition. Nuclear calcium signals and action potential generation were reduced by blockade of either NMDA receptors or L-type voltage-gated calcium channels, but not by procedures that lead to internal calcium store depletion or by blockade of metabotropic glutamate receptors. These findings identify a repetition-induced switch in nuclear calcium signaling that correlates with the transition from E-LTP to L-LTP, and may explain why the transcription-dependent phase of L-LTP is not induced by a single HFS or TBS but requires repeated trains of activity. Recombinant, nuclear-targeted indicators may prove useful for further analysis of nuclear calcium signaling in vivo. PMID:21156150

  18. Inhibition of Paclitaxel-induced Decreases in Calcium Signaling*

    PubMed Central

    Benbow, Jennifer H.; Mann, Taylor; Keeler, Camille; Fan, Chengpeng; Hodsdon, Michael E.; Lolis, Elias; DeGray, Brenda; Ehrlich, Barbara E.

    2012-01-01

    Peripheral neuropathy is one of the most severe and irreversible side effects caused by treatment from several chemotherapeutic drugs, including paclitaxel (Taxol®) and vincristine. Strategies are needed that inhibit this unwanted side effect without altering the chemotherapeutic action of these drugs. We previously identified two proteins in the cellular pathway that lead to Taxol-induced peripheral neuropathy, neuronal calcium sensor-1 (NCS-1) and calpain. Prolonged treatment with Taxol induces activation of calpain, degradation of NCS-1, and loss of intracellular calcium signaling. This paper has focused on understanding the molecular basis for prevention of peripheral neuropathy by testing the effects of addition of two candidate compounds to the existing chemotherapeutic drug regime: lithium and ibudilast. We found that the co-administration of either lithium or ibudilast to neuroblastoma cells that were treated with Taxol or vincristine inhibited activation of calpain and the reductions in NCS-1 levels and calcium signaling associated with these chemotherapeutic drugs. The ability of Taxol to alter microtubule formation was unchanged by the addition of either candidate compound. These results allow us to suggest that it is possible to prevent the unnecessary and irreversible damage caused by chemotherapeutic drugs while still maintaining therapeutic efficacy. Specifically, the addition of either lithium or ibudilast to existing chemotherapy treatment protocols has the potential to prevent chemotherapy-induced peripheral neuropathy. PMID:22988235

  19. Distinct cellular states determine calcium signaling response.

    PubMed

    Yao, Jason; Pilko, Anna; Wollman, Roy

    2016-12-15

    The heterogeneity in mammalian cells signaling response is largely a result of pre-existing cell-to-cell variability. It is unknown whether cell-to-cell variability rises from biochemical stochastic fluctuations or distinct cellular states. Here, we utilize calcium response to adenosine trisphosphate as a model for investigating the structure of heterogeneity within a population of cells and analyze whether distinct cellular response states coexist. We use a functional definition of cellular state that is based on a mechanistic dynamical systems model of calcium signaling. Using Bayesian parameter inference, we obtain high confidence parameter value distributions for several hundred cells, each fitted individually. Clustering the inferred parameter distributions revealed three major distinct cellular states within the population. The existence of distinct cellular states raises the possibility that the observed variability in response is a result of structured heterogeneity between cells. The inferred parameter distribution predicts, and experiments confirm that variability in IP3R response explains the majority of calcium heterogeneity. Our work shows how mechanistic models and single-cell parameter fitting can uncover hidden population structure and demonstrate the need for parameter inference at the single-cell level. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  20. Localized intracellular calcium signaling in muscle: calcium sparks and calcium quarks.

    PubMed

    Niggli, E

    1999-01-01

    Subcellularly localized Ca2+ signals in cardiac and skeletal muscle have recently been identified as elementary Ca2+ signaling events. The signals, termed Ca2+ sparks and Ca2+ quarks, represent openings of Ca2+ release channels located in the membrane of the sarcoplasmic reticulum (SR). In cardiac muscle, the revolutionary discovery of Ca2+ sparks has allowed the development of a fundamentally different concept for the amplification of Ca2+ signals by Ca(2+)-induced Ca2+ release. In such a system, a graded amplification of the triggering Ca2+ signal entering the myocyte via L-type Ca2+ channels is accomplished by a recruitment process whereby individual SR Ca2+ release units are locally controlled by L-type Ca2+ channels. In skeletal muscle, the initial SR Ca2+ release is governed by voltage-sensors but subsequently activates additional Ca2+ sparks by Ca(2+)-induced Ca2+ release from the SR. Results from studies on elementary Ca2+ release events will improve our knowledge of muscle Ca2+ signaling at all levels of complexity, from the molecule to normal cellular function, and from the regulation of cardiac and skeletal muscle force to the pathophysiology of excitation-contraction coupling.

  1. Novel Approach for Generation of Low Calcium Reagents for Investigations of Heavy Metal Effects on Calcium Signaling

    PubMed Central

    Niu, Katelyn Y.; Noyes, Nathanial; Abrams, Thomas W.

    2014-01-01

    Lead exposure can cause learning disabilities, memory loss and severe damage to the nervous system. However, the exact mechanism by which lead causes learning disabilities is not fully understood. The effects of lead on calcium-regulated signaling pathways are difficult to study biochemically; with the traditional method of controlling the free calcium concentration with EGTA, the exact concentrations of free lead and calcium ions in solution are interdependent and prone to error because EGTA also buffers lead. In our approach, we first reduced the free calcium concentration in the solution using calcium-binding resins before adding lead to buffers. The solution was sequentially treated with Chelex-100 ion exchange resin, followed by immobilized BAPTA resin. The final concentration of free calcium in the solution was measured with Fluo-3 indicator. Our protocol successfully produced buffers with free calcium levels below 15 nM, which is substantially below threshold for activation of calcium-dependent enzymes in signaling pathways (which is typically a few hundred nanomolar calcium, when determined in vitro). This method provides an improved approach to study the effect of heavy metals on calcium-stimulated signaling pathways. PMID:22504007

  2. Lymphocyte adhesion-dependent calcium signaling in human endothelial cells

    PubMed Central

    1995-01-01

    Vascular endothelial cells (ECs) can undergo dramatic phenotypic and functional alterations in response to humoral and cellular stimuli. These changes promote endothelial participation in the inflammatory response through active recruitment of immune effector cells, increased vascular permeability, and alteration in vascular tone. In an attempt to define early events in lymphocyte-mediated EC signaling, we investigated cytosolic-free calcium (Ca2+) changes in single, Fluo-3- labeled human umbilical vein ECs (HUVECs), using an ACAS interactive laser cytometer. Of all lymphocyte subsets tested, allogeneic CD3-, CD56+ natural killer (NK) cells uniquely elicited oscillatory EC Ca2+ signals in cytokine (interleukin [IL]-1- or tumor necrosis factor [TNF])-treated ECs. The induction of these signals required avid intercellular adhesion, consisted of both Ca2+ mobilization and extracellular influx, and was associated with EC inositol phosphate (IP) generation. Simultaneous recording of NK and EC Ca2+ signals using two-color fluorescence detection revealed that, upon adhesion, NK cells flux prior to EC. Lymphocyte Ca2+ buffering with 1,2-bis-5-methyl-amino- phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate (MAPTAM) demonstrated that lymphocyte fluxes are, in fact, prerequisites for the adhesion- dependent EC signals. mAb studies indicate that the beta 2 integrin- intercellular adhesion molecule (ICAM)-1 adhesion pathway is critically involved. However, ICAM-1 antisense oligonucleotide inhibition of IL-1- mediated ICAM-1 hyperinduction had no effect on EC Ca2+ signaling in lymphocyte-EC conjugates, indicating that additional cytokine-induced EC alteration is required. These experiments combine features of lymphocyte-endothelial interactions, intercellular adhesion, EC cytokine activation and transmembrane signaling. The results implicate the IP/Ca2+ second messenger pathway in EC outside-in signaling induced by cytotoxic lymphocytes, and suggest that these signals may play a

  3. 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.

  4. Practical aspects of measuring intracellular calcium signals with fluorescent indicators.

    PubMed

    Kao, Joseph P Y; Li, Gong; Auston, Darryl A

    2010-01-01

    The use of fluorescent indicators for monitoring calcium (Ca(2+)) signals and for measuring Ca(2+) concentration ([Ca(2+)]) in living cells is described. The following topics are covered in detail: (1) ratiometric and nonratiometric fluorescent indicators and the principles underlying their use, (2) techniques for loading Ca(2+) indicators and Ca(2+) buffers into living cells, (3) calibration of indicator fluorescence intensity measurements to yield values of intracellular [Ca(2+)], (4) analysis of nonratiometric fluorescence intensity data and caveats relating to their interpretation, (5) techniques for manipulating intracellular and extracellular [Ca(2+)], and (6) the use of fluorescent indicators to monitor Ca(2+) signals in mitochondria. The chapter aims to present these fundamental topics in a manner that is practically useful and intuitively accessible. The origins of key mathematical equations used in the article are outlined in two appendices.

  5. Location matters: somatic and dendritic SK channels answer to distinct calcium signals.

    PubMed

    Rudolph, Stephanie; Thanawala, Monica S

    2015-07-01

    Voltage-dependent calcium channels (VDCCs) couple neuronal activity to diverse intracellular signals with exquisite spatiotemporal specificity. Using calcium imaging and electrophysiology, Jones and Stuart (J Neurosci 33: 19396-19405, 2013) examined the intimate relationship between distinct types of VDCCs and small-conductance calcium-activated potassium (SK) channels that contribute to the compartmentalized control of excitability in the soma and dendrites of cortical pyramidal neurons. Here we discuss the importance of calcium domains for signal specificity, explore the possible functions and mechanisms for local control of SK channels, and highlight technical considerations for the optical detection of calcium signals.

  6. New concepts in calcium-sensing receptor pharmacology and signalling.

    PubMed

    Ward, Donald T; Riccardi, Daniela

    2012-01-01

    The calcium-sensing receptor (CaR) is the key controller of extracellular calcium (Ca(2+)(o)) homeostasis via its regulation of parathyroid hormone (PTH) secretion and renal Ca(2+) reabsorption. The CaR-selective calcimimetic drug Cinacalcet stimulates the CaR to suppress PTH secretion in chronic kidney disease and represents the world's first clinically available receptor positive allosteric modulator (PAM). Negative CaR allosteric modulators (NAMs), known as calcilytics, can increase PTH secretion and are being investigated as possible bone anabolic treatments against age-related osteoporosis. Here we address the current state of development and clinical use of a series of positive and negative CaR modulators. In addition, clinical CaR mutations and transgenic mice carrying tissue-specific CaR deletions have provided a novel understanding of the relative functional importance of CaR in both calciotropic tissues and those elsewhere in the body. The development of CaR-selective modulators and signalling reagents have provided us with a more detailed appreciation of how the CaR signals in vivo. Thus, both of these areas of CaR research will be reviewed. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  7. Spatial characteristics to calcium signalling; the calcium wave as a basic unit in plant cell calcium signalling

    PubMed Central

    Malhó, R.

    1998-01-01

    Many signals that modify plant cell growth and development initiate changes in cytoplasmic Ca2+. The subsequent movement of Ca2+ in the cytoplasm is thought to take place via waves of free Ca2+. These waves may be initiated at defined regions of the cell and movement requires release from a reticulated endoplasmic reticulum and the vacuole. The mechanism of wave propagation is outlined and the possible basis of repetitive reticulum wave formation, Ca2+ oscillations and capacitative Ca2+ signalling is discussed. Evidence for the presence of Ca2+ waves in plant cells is outlined, and from studies on raphides it is suggested that the capabilities for capacitative Ca2+ signalling are also present. The paper finishes with an outline of the possible interrelation between Ca2+ waves and organelles and describes the intercellular movement of Ca2+ waves and the relevance of such information communication to plant development.

  8. Criticality in intracellular calcium signaling in cardiac myocytes.

    PubMed

    Nivala, Michael; Ko, Christopher Y; Nivala, Melissa; Weiss, James N; Qu, Zhilin

    2012-06-06

    Calcium (Ca) is a ubiquitous second messenger that regulates many biological functions. The elementary events of local Ca signaling are Ca sparks, which occur randomly in time and space, and integrate to produce global signaling events such as intra- and intercellular Ca waves and whole-cell Ca oscillations. Despite extensive experimental characterization in many systems, the transition from local random to global synchronous events is still poorly understood. Here we show that criticality, a ubiquitous dynamical phenomenon in nature, is responsible for the transition from local to global Ca signaling. We demonstrate this first in a computational model of Ca signaling in a cardiac myocyte and then experimentally in mouse ventricular myocytes, complemented by a theoretical agent-based model to delineate the underlying dynamics. We show that the interaction between the Ca release units via Ca-induced Ca release causes self-organization of Ca spark clusters. When the coupling between Ca release units is weak, the cluster-size distribution is exponential. As the interactions become strong, the cluster-size distribution changes to a power-law distribution, which is characteristic of criticality in thermodynamic and complex nonlinear systems, and facilitates the formation and propagation of Ca waves and whole-cell Ca oscillations. Our findings illustrate how criticality is harnessed by a biological cell to regulate Ca signaling via self-organization of random subcellular events into cellular-scale oscillations, and provide a general theoretical framework for the transition from local Ca signaling to global Ca signaling in biological cells.

  9. 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

  10. 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.

  11. Deconvolution of calcium fluorescent indicator signal from AFM cantilever reflection.

    PubMed

    Lopez-Ayon, G Monserratt; Oliver, David J; Grutter, Peter H; Komarova, Svetlana V

    2012-08-01

    Atomic force microscopy (AFM) can be combined with fluorescence microscopy to measure the changes in intracellular calcium levels (indicated by fluorescence of Ca²⁺ sensitive dye fluo-4) in response to mechanical stimulation performed by AFM. Mechanical stimulation using AFM is associated with cantilever movement, which may interfere with the fluorescence signal. The motion of the AFM cantilever with respect to the sample resulted in changes of the reflection of light back to the sample and a subsequent variation in the fluorescence intensity, which was not related to changes in intracellular Ca²⁺ levels. When global Ca²⁺ responses to a single stimulation were assessed, the interference of reflected light with the fluorescent signal was minimal. However, in experiments where local repetitive stimulations were performed, reflection artifacts, correlated with cantilever motion, represented a significant component of the fluorescent signal. We developed a protocol to correct the fluorescence traces for reflection artifacts, as well as photobleaching. An added benefit of our method is that the cantilever reflection in the fluorescence recordings can be used for precise temporal correlation of the AFM and fluorescence measurements.

  12. Endoplasmic reticulum-mitochondria calcium signaling in hepatic metabolic diseases.

    PubMed

    Rieusset, Jennifer

    2017-06-01

    The liver plays a central role in glucose homeostasis, and both metabolic inflexibility and insulin resistance predispose to the development of hepatic metabolic diseases. Mitochondria and endoplasmic reticulum (ER), which play a key role in the control of hepatic metabolism, also interact at contact points defined as mitochondria-associated membranes (MAM), in order to exchange metabolites and calcium (Ca(2+)) and regulate cellular homeostasis and signaling. Here, we overview the role of the liver in the control of glucose homeostasis, mainly focusing on the independent involvement of mitochondria, ER and Ca(2+) signaling in both healthy and pathological contexts. Then we focus on recent data highlighting MAM as important hubs for hormone and nutrient signaling in the liver, thus adapting mitochondria physiology and cellular metabolism to energy availability. Lastly, we discuss how chronic ER-mitochondria miscommunication could participate to hepatic metabolic diseases, pointing MAM interface as a potential therapeutic target for metabolic disorders. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Calcium Signaling Regulates Trafficking of Familial Hypocalciuric Hypercalcemia (FHH) Mutants of the Calcium Sensing Receptor

    PubMed Central

    Grant, Michael P.; Stepanchick, Ann

    2012-01-01

    Calcium-sensing receptors (CaSRs) regulate systemic Ca2+ homeostasis. Loss-of-function mutations cause familial benign hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). FHH/NSHPT mutations can reduce trafficking of CaSRs to the plasma membrane. CaSR signaling is potentiated by agonist-driven anterograde CaSR trafficking, leading to a new steady state level of plasma membrane CaSR, which is maintained, with minimal functional desensitization, as long as extracellular Ca2+ is elevated. This requirement for CaSR signaling to drive CaSR trafficking to the plasma membrane led us to reconsider the mechanism(s) contributing to dysregulated trafficking of FHH/NSHPT mutants. We simultaneously monitored dynamic changes in plasma membrane levels of CaSR and intracellular Ca2+, using a chimeric CaSR construct, which allowed explicit tracking of plasma membrane levels of mutant or wild-type CaSRs in the presence of nonchimeric partners. Expression of mutants alone revealed severe defects in plasma membrane targeting and Ca2+ signaling, which were substantially rescued by coexpression with wild-type CaSR. Biasing toward heterodimerization of wild-type and FHH/NSHPT mutants revealed that intracellular Ca2+ oscillations were insufficient to rescue plasma membrane targeting. Coexpression of the nonfunctional mutant E297K with the truncation CaSRΔ868 robustly rescued trafficking and Ca2+ signaling, whereas coexpression of distinct FHH/NSHPT mutants rescued neither trafficking nor signaling. Our study suggests that rescue of FHH/NSHPT mutants requires a steady state intracellular Ca2+ response when extracellular Ca2+ is elevated and argues that Ca2+ signaling by wild-type CaSRs rescues FHH mutant trafficking to the plasma membrane. PMID:23077345

  14. Modulation of calcium signalling by the endoplasmic reticulum in Carassius neurons.

    PubMed

    Lukyanets, Igor A; Lukyanetz, Elena A

    2013-04-19

    It is known that endoplasmic reticulum (ER), being a calcium store participates in the regulation of intracellular calcium concentration. Ca-ATPase of the ER is one of the crucial agents providing the calcium-accumulating function of this intracellular structure. We studied the role of the ER in modulation of calcium signalling in Carassius neurons using a Ca2+-imaging technique. We tested the role of the ER in the maintenance of a steady state calcium level in the cytoplasm and in modulation of Ca2+ transients evoked by cell depolarizations. The ER calcium stores were depleted using inhibitors of ER Ca-ATPase, which provided blocking of Ca2+ uptake by the ER. Our experiments firstly showed that the ER can significantly modulate the characteristics of intracellular calcium signals in Carassius neurons during their activity. These findings also indicate that the ER modulates the shape of Ca2+ signals rather than the basal level of intracellular Ca2+ in these neurons.

  15. 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.

  16. 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.

  17. A tidal wave of signals: calcium and ROS at the forefront of rapid systemic signaling.

    PubMed

    Gilroy, Simon; Suzuki, Nobuhiro; Miller, Gad; Choi, Won-Gyu; Toyota, Masatsugu; Devireddy, Amith R; Mittler, Ron

    2014-10-01

    Systemic signaling pathways enable multicellular organisms to prepare all of their tissues and cells to an upcoming challenge that may initially only be sensed by a few local cells. They are activated in plants in response to different stimuli including mechanical injury, pathogen infection, and abiotic stresses. Key to the mobilization of systemic signals in higher plants are cell-to-cell communication events that have thus far been mostly unstudied. The recent identification of systemically propagating calcium (Ca(2+)) and reactive oxygen species (ROS) waves in plants has unraveled a new and exciting cell-to-cell communication pathway that, together with electric signals, could provide a working model demonstrating how plant cells transmit long-distance signals via cell-to-cell communication mechanisms. Here, we summarize recent findings on the ROS and Ca(2+) waves and outline a possible model for their integration.

  18. 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

  19. Calcium signaling in plant cells in altered gravity.

    PubMed

    Kordyum, E L

    2003-01-01

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

  20. Novel vistas of calcium-mediated signalling in the thalamus.

    PubMed

    Pape, Hans-Christian; Munsch, Thomas; Budde, Thomas

    2004-05-01

    Traditionally, the role of calcium ions (Ca(2+)) in thalamic neurons has been viewed as that of electrical charge carriers. Recent experimental findings in thalamic cells have only begun to unravel a highly complex Ca(2+) signalling network that exploits extra- and intracellular Ca(2+) sources. In thalamocortical relay neurons, interactions between T-type Ca(2+) channel activation, Ca(2+)-dependent regulation of adenylyl cyclase activity and the hyperpolarization-activated cation current ( I(h)) regulate oscillatory burst firing during periods of sleep and generalized epilepsy, while a functional triad between Ca(2+) influx through high-voltage-activated (most likely L-type) Ca(2+) channels, Ca(2+)-induced Ca(2+) release via ryanodine receptors (RyRs) and a repolarizing mechanism (possibly via K(+) channels of the BK(Ca) type) supports tonic spike firing as required during wakefulness. The mechanisms seem to be located mostly at dendritic and somatic sites, respectively. One functional compartment involving local GABAergic interneurons in certain thalamic relay nuclei is the glomerulus, in which the dendritic release of GABA is regulated by Ca(2+) influx via canonical transient receptor potential channels (TRPC), thereby presumably enabling transmitters of extrathalamic input systems that are coupled to phospholipase C (PLC)-activating receptors to control feed-forward inhibition in the thalamus. Functional interplay between T-type Ca(2+) channels in dendrites and the A-type K(+) current controls burst firing, contributing to the range of oscillatory activity observed in these interneurons. GABAergic neurons in the reticular thalamic (RT) nucleus recruit a specific set of Ca(2+)-dependent mechanisms for the generation of rhythmic burst firing, of which a particular T-type Ca(2+) channel in the dendritic membrane, the Ca(2+)-dependent activation of non-specific cation channels ( I(CAN)) and of K(+) channels (SK(Ca) type) are key players. Glial Ca(2+) signalling in

  1. Plants, endosymbionts and parasites: Abscisic acid and calcium signaling.

    PubMed

    Nagamune, Kisaburo; Xiong, Liming; Chini, Eduardo; Sibley, L David

    2008-01-01

    It was recently discovered that the protozoan parasite, Toxoplasma gondii produces and uses the plant hormone, abscisic acid (ABA), for communication. Following intracellular replication, ABA production influences the timing of parasite egress from the host cell. This density-dependent signal may serve to coordinate exit from the host cell in a synchronous manner by triggering calcium-dependent activation of motility. In the absence of ABA production, parasites undergo differentiation to the semidormant, tissue cyst. The pathway for ABA production in T. gondii may be derived from a relict endosymbiont, acquired by ingestion of a red algal cell. Although the parasite has lost the capacity for photosynthesis, the plant-like nature of this signaling pathway may be exploited to develop new drugs. In support of this idea, an inhibitor of ABA biosynthesis protected mice against lethal infection with T. gondii. Here, we compare the role of ABA in parasites to its activities in plants, where it is know to control development and stress responses.

  2. Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent.

    PubMed

    Davis, F M; Azimi, I; Faville, R A; Peters, A A; Jalink, K; Putney, J W; Goodhill, G J; Thompson, E W; Roberts-Thomson, S J; Monteith, G R

    2014-05-01

    Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of

  3. Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent

    PubMed Central

    Davis, Felicity M.; Azimi, Iman; Faville, Richard A.; Peters, Amelia A.; Jalink, Kees; Putney, James W.; Goodhill, Geoffrey J.; Thompson, Erik W.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.

    2014-01-01

    Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. While intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium

  4. Nicotine elicits prolonged calcium signaling along ventral hippocampal axons.

    PubMed

    Zhong, Chongbo; Talmage, David A; Role, Lorna W

    2013-01-01

    Presynaptic nicotinic acetylcholine receptors (nAChRs) have long been implicated in the modulation of CNS circuits. We previously reported that brief exposure to low concentrations of nicotine induced sustained potentiation of glutamatergic transmission at ventral hippocampal (vHipp)-striatal synapses. Here, we exploited nAChR subtype-selective antagonists and agonists and α7*nAChR knockout mutant mice (α7-/-) to elucidate the signaling mechanisms underlying nAChR-mediated modulation of synaptic transmission. Using a combination of micro-slices culture from WT and α7-/-mice, calcium imaging, and immuno-histochemical techniques, we found that nicotine elicits localized and oscillatory increases in intracellular Ca(2+) along vHipp axons that persists for up to 30 minutes. The sustained phase of the nicotine-induced Ca(2+) response was blocked by α-BgTx but not by DHβE and was mimicked by α7*nAChR agonists but not by non-α7*nAChR agonists. In vHipp slices from α7-/- mice, nicotine elicited only transient increases of axonal Ca(2+) signals and did not activate CaMKII. The sustained phase of the nicotine-induced Ca(2+) response required localized activation of CaMKII, phospholipase C, and IP3 receptor mediated Ca(2+)-induced Ca(2+) release (CICR). In conclusion, activation of presynaptic nAChRs by nicotine elicits Ca(2+) influx into the presynaptic axons, the sustained phase of the nicotine-induced Ca(2+) response requires that axonal α7*nAChR activate a downstream signaling network in the vHipp axons.

  5. 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

  6. Characterization of postsynaptic calcium signals in the pyramidal neurons of anterior cingulate cortex.

    PubMed

    Li, Xu-Hui; Song, Qian; Chen, Tao; Zhuo, Min

    2017-01-01

    Calcium signaling is critical for synaptic transmission and plasticity. N-methyl-D-aspartic acid (NMDA) receptors play a key role in synaptic potentiation in the anterior cingulate cortex. Most previous studies of calcium signaling focus on hippocampal neurons, little is known about the activity-induced calcium signals in the anterior cingulate cortex. In the present study, we show that NMDA receptor-mediated postsynaptic calcium signals induced by different synaptic stimulation in anterior cingulate cortex pyramidal neurons. Single and multi-action potentials evoked significant suprathreshold Ca(2+) increases in somas and spines. Both NMDA receptors and voltage-gated calcium channels contributed to this increase. Postsynaptic Ca(2+)signals were induced by puff-application of glutamate, and a NMDA receptor antagonist AP5 blocked these signals in both somas and spines. Finally, long-term potentiation inducing protocols triggered postsynaptic Ca(2+) influx, and these influx were NMDA receptor dependent. Our results provide the first study of calcium signals in the anterior cingulate cortex and demonstrate that NMDA receptors play important roles in postsynaptic calcium signals in anterior cingulate cortex pyramidal neurons.

  7. Characterization of postsynaptic calcium signals in the pyramidal neurons of anterior cingulate cortex

    PubMed Central

    Li, Xu-Hui; Song, Qian; Chen, Tao

    2017-01-01

    Calcium signaling is critical for synaptic transmission and plasticity. N-methyl-D-aspartic acid (NMDA) receptors play a key role in synaptic potentiation in the anterior cingulate cortex. Most previous studies of calcium signaling focus on hippocampal neurons, little is known about the activity-induced calcium signals in the anterior cingulate cortex. In the present study, we show that NMDA receptor-mediated postsynaptic calcium signals induced by different synaptic stimulation in anterior cingulate cortex pyramidal neurons. Single and multi-action potentials evoked significant suprathreshold Ca2+ increases in somas and spines. Both NMDA receptors and voltage-gated calcium channels contributed to this increase. Postsynaptic Ca2+signals were induced by puff-application of glutamate, and a NMDA receptor antagonist AP5 blocked these signals in both somas and spines. Finally, long-term potentiation inducing protocols triggered postsynaptic Ca2+ influx, and these influx were NMDA receptor dependent. Our results provide the first study of calcium signals in the anterior cingulate cortex and demonstrate that NMDA receptors play important roles in postsynaptic calcium signals in anterior cingulate cortex pyramidal neurons. PMID:28726541

  8. 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

  9. 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.

  10. Dynamic kisspeptin receptor trafficking modulates kisspeptin-mediated calcium signaling.

    PubMed

    Min, Le; Soltis, Kathleen; Reis, Ana Claudia S; Xu, Shuyun; Kuohung, Wendy; Jain, Manisha; Carroll, Rona S; Kaiser, Ursula B

    2014-01-01

    Kisspeptin receptor (KISS1R) signaling plays a critical role in the regulation of reproduction. We investigated the role of kisspeptin-stimulated KISS1R internalization, recycling, and degradation in the modulation of KISS1R signaling. Kisspeptin stimulation of Chinese hamster ovary or GT1-7 cells expressing KISS1R resulted in a biphasic increase in intracellular Ca(2+) ([Ca(2+)]i), with a rapid acute increase followed by a more sustained second phase. In contrast, stimulation of the TRH receptor, another Gq/11-coupled receptor, resulted in a much smaller second-phase [Ca(2+)]i response. The KISS1R-mediated second-phase [Ca(2+)]i response was abolished by removal of kisspeptin from cell culture medium. Notably, the second-phase [Ca(2+)]i response was also inhibited by dynasore, brefeldin A, and phenylarsine oxide, which inhibit receptor internalization and recycling, suggesting that KISS1R trafficking contributes to the sustained [Ca(2+)]i response. We further demonstrated that KISS1R undergoes dynamic ligand-dependent and -independent recycling. We next investigated the fate of the internalized kisspeptin-KISS1R complex. Most internalized kisspeptin was released extracellularly in degraded form within 1 hour, suggesting rapid processing of the internalized kisspeptin-KISS1R complex. Using a biotinylation assay, we demonstrated that degradation of cell surface KISS1R was much slower than that of the internalized ligand, suggesting dissociated processing of the internalized kisspeptin-KISS1R complex. Taken together, our results suggest that the sustained calcium response to kisspeptin is dependent on the continued presence of extracellular ligand and is the result of dynamic KISS1R trafficking.

  11. 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

  12. Peptidoglycan Induces the Production of Interleukin-8 via Calcium Signaling in Human Gingival Epithelium

    PubMed Central

    Son, Aran; Hong, Jeong Hee

    2015-01-01

    The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells. PMID:25605997

  13. A genetically targeted optical sensor to monitor calcium signals in astrocyte processes

    PubMed Central

    Shigetomi, Eiji; Kracun, Sebastian; Sofroniew, Michael V; Khakh, Baljit S

    2010-01-01

    Calcium signaling is studied as a potential form of astrocyte excitability that may control astrocyte involvement in synaptic and cerebrovascular regulation. Fundamental questions remain unanswered about astrocyte calcium signaling, as current methods can not resolve calcium in small volume compartments, such as near the cell membrane and in distal cell processes. We modified the genetically encoded calcium sensor GCaMP2 with a membrane-tethering domain, Lck, increasing the level of Lck-GCaMP2 near the plasma membrane tenfold as compared with conventional GCaMP2. Using Lck-GCaMP2 in rat hippocampal astrocyte-neuron cocultures, we measured near-membrane calcium signals that were evoked pharmacologically or by single action potential–mediated neurotransmitter release. Moreover, we identified highly localized and frequent spontaneous calcium signals in astrocyte somata and processes that conventional GCaMP2 failed to detect. Lck-GCaMP2 acts as a genetically targeted calcium sensor for monitoring calcium signals in previously inaccessible parts of astrocytes, including fine processes. PMID:20495558

  14. Opposing roles of synaptic and extrasynaptic NMDA receptors in neuronal calcium signalling and BDNF gene regulation.

    PubMed

    Vanhoutte, Peter; Bading, Hilmar

    2003-06-01

    Neuronal responses to electrical activity-induced calcium signals are specified by the localization of the calcium entry site and the spatial properties of the calcium transient. Calcium flux through NMDA receptors located in the synapse initiates changes in synaptic efficacy and promotes pro-survival events, whereas calcium flux through extrasynaptic NMDA receptors is coupled to cell death pathways. The dialogue between the synaptic NMDA receptors and the nucleus is also modulated by extrasynaptic NMDA receptors, which shut down activity of CRE-binding protein (CREB) and antagonize the increase in brain-derived neurotrophic factor (BDNF) expression induced by synaptic NMDA receptors. The specification of the biological response by the localization of the receptor activated is a new concept in neuronal calcium signalling that can explain many of the opposing roles of NMDA receptors.

  15. 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.

  16. Calcium signalling in human neutrophil cell lines is not affected by low-frequency electromagnetic fields.

    PubMed

    Golbach, Lieke A; Philippi, John G M; Cuppen, Jan J M; Savelkoul, Huub F J; Verburg-van Kemenade, B M Lidy

    2015-09-01

    We are increasingly exposed to low-frequency electromagnetic fields (LF EMFs) by electrical devices and power lines, but if and how these fields interact with living cells remains a matter of debate. This study aimed to investigate the potential effect of LF EMF exposure on calcium signalling in neutrophils. In neutrophilic granulocytes, activation of G-protein coupled receptors leads to efflux of calcium from calcium stores and influx of extracellular calcium via specialised calcium channels. The cytoplasmic rise of calcium induces cytoskeleton rearrangements, modified gene expression patterns, and cell migration. If LF EMF modulates intracellular calcium signalling, this will influence cellular behaviour and may eventually lead to health problems. We found that calcium mobilisation upon chemotactic stimulation was not altered after a short 30 min or long-term LF EMF exposure in human neutrophil-like cell lines HL-60 or PLB-985. Neither of the two investigated wave forms (Immunent and 50 Hz sine wave) at three magnetic flux densities (5 μT, 300 μT, and 500 μT) altered calcium signalling in vitro. Gene-expression patterns of calcium-signalling related genes also did not show any significant changes after exposure. Furthermore, analysis of the phenotypical appearance of microvilli by scanning electron microscopy revealed no alterations induced by LF EMF exposure. The findings above indicate that exposure to 50 Hz sinusoidal or Immunent LF EMF will not affect calcium signalling in neutrophils in vitro. © 2015 Wiley Periodicals, Inc.

  17. Calcium

    MedlinePlus

    ... You'll also find calcium in broccoli and dark green, leafy vegetables (especially collard and turnip greens, ... can enjoy good sources of calcium such as dark green, leafy vegetables, broccoli, chickpeas, and calcium-fortified ...

  18. 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.

  19. 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.

  20. ETHANOL ALTERS CALCIUM SIGNALING IN AXONAL GROWTH CONES

    PubMed Central

    Mah, Stephanie J.; Fleck, Mark W.

    2011-01-01

    Calcium (Ca2+) channels are sensitive to ethanol and Ca2+ signaling is a critical regulator of axonal growth and guidance. Effects of acute and chronic exposure to ethanol (22, 43, or 87 mM) on voltage-gated Ca2+ channels (VGCCs) in whole cells, and KCl-induced Ca2+ transients in axonal growth cones, were examined using dissociated hippocampal cultures. Whole-cell patch-clamp analysis in neurons with newly-formed axons (Stage 3) revealed that rapidly inactivating, low-voltage activated (LVA) and non-inactivating, high-voltage activated (HVA) currents were both inhibited in a dose-dependent manner by acute ethanol, with relatively greater inhibition of HVA currents. When assessed by Fluo-4-AM imaging, baseline fluorescence and Ca2+ response to ethanol in Stage 3 neurons was similar compared to neurons without axons, but peak Ca2+ transient amplitudes in response to bath-applied KCl were greater in Stage 3 neurons and were decreased by acute ethanol. The amplitude of Ca2+ transients elicited specifically in axonal growth cones by focal application of KCl was also inhibited by acute exposure to moderate-to-high concentrations of ethanol (43 or 87 mM), whereas a lower concentration (22 mM) had no effect. When 43 or 87 mM ethanol was present continuously in the medium, KCl-evoked Ca2+ transient amplitudes were also reduced in growth cones. In contrast, Ca2+ transients were increased by continuous exposure to 22 mM ethanol. Visualization using a fluorescent dihydropyridine analog revealed that neurons continuously exposed to ethanol expressed increased amounts of L-type Ca2+ channels, with greater increases in axonal growth cones than cell bodies. Thus, acute ethanol reduces Ca2+ current and KCl-induced Ca2+ responses in whole cells and axonal growth cones, respectively, and chronic exposure is also generally inhibitory despite apparent up-regulation of L-type channel expression. These results are consistent with a role for altered growth cone Ca2+ signaling in abnormal

  1. Impact of Calcium Signaling during Infection of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

    PubMed Central

    Asmat, Tauseef M.; Tenenbaum, Tobias; Jonsson, Ann-Beth

    2014-01-01

    The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells. PMID:25464500

  2. Isolated plant nuclei as mechanical and thermal sensors involved in calcium signalling.

    PubMed

    Xiong, Tou Cheu; Jauneau, Alain; Ranjeva, Raoul; Mazars, Christian

    2004-10-01

    Calcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calcium signals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and to convert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits a single calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequency reflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. The stretch- and the temperature-activated processes differ by their sensitivity to pharmacological drugs known to affect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gauge physical parameters of their environment. This might have a profound influence on the functioning of calcium-dependent processes known to control a large array of molecular events in the nucleus.

  3. SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification

    PubMed Central

    Schumacher, Jennifer A.; Wang, Xiaohong; Merrill, Sean A.; Millington, Grethel; Bayne, Brittany; Jorgensen, Erik M.; Chuang, Chiou-Fen

    2016-01-01

    The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons. PMID:26771544

  4. Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels.

    PubMed

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

    Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca(2+)]E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca(2+)]I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca(2+)]E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca(2+)]I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca(2+)]I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca(2+)]I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs.

  5. Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling.

    PubMed

    Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Pergel, Enikő; Bősze, Zsuzsanna; Bender, Balázs; Vajdovich, Péter; Tóvári, József; Homolya, László; Szakács, Gergely; Héja, László; Enyedi, Ágnes; Sarkadi, Balázs; Apáti, Ágota; Orbán, Tamás I

    2015-08-03

    In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na(+)/Ca(2+) exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.

  6. Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling

    PubMed Central

    Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Pergel, Enikő; Bősze, Zsuzsanna; Bender, Balázs; Vajdovich, Péter; Tóvári, József; Homolya, László; Szakács, Gergely; Héja, László; Enyedi, Ágnes; Sarkadi, Balázs; Apáti, Ágota; Orbán, Tamás I.

    2015-01-01

    In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na+/Ca2+ exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies. PMID:26234466

  7. Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo.

    PubMed

    Yang, Helen H; St-Pierre, François; Sun, Xulu; Ding, Xiaozhe; Lin, Michael Z; Clandinin, Thomas R

    2016-06-30

    A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise.

  8. Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish.

    PubMed

    Yoo, Sa Kan; Freisinger, Christina M; LeBert, Danny C; Huttenlocher, Anna

    2012-10-15

    Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H(2)O(2) at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immediate SFK and calcium signaling in epithelia was important for late epimorphic regeneration of amputated fins. Wound-induced activation of SFKs in epithelia was dependent on injury-generated H(2)O(2). A SFK member, Fynb, was responsible for fin regeneration. This work provides a new link between early wound responses and late regeneration and suggests that redox, SFK, and calcium signaling are immediate "wound signals" that integrate early wound responses and late epimorphic regeneration.

  9. Calcium signaling in plant cell organelles delimited by a double membrane.

    PubMed

    Xiong, Tou-Cheu; Bourque, Stéphane; Lecourieux, David; Amelot, Nicolas; Grat, Sabine; Brière, Christian; Mazars, Christian; Pugin, Alain; Ranjeva, Raoul

    2006-11-01

    Increases in the concentration of free calcium in the cytosol are one of the general events that relay an external stimulus to the internal cellular machinery and allow eukaryotic organisms, including plants, to mount a specific biological response. Different lines of evidence have shown that other intracellular organelles contribute to the regulation of free calcium homeostasis in the cytosol. The vacuoles, the endoplasmic reticulum and the cell wall constitute storage compartments for mobilizable calcium. In contrast, the role of organelles surrounded by a double membrane (e.g. mitochondria, chloroplasts and nuclei) is more complex. Here, we review experimental data showing that these organelles harbor calcium-dependent biological processes. Mitochondria, chloroplasts as well as nuclei are equipped to generate calcium signal on their own. Changes in free calcium in a given organelle may also favor the relocalization of proteins and regulatory components and therefore have a profound influence on the integrated functioning of the cell. Studying, in time and space, the dynamics of different components of calcium signaling pathway will certainly give clues to understand the extraordinary flexibility of plants to respond to stimuli and mount adaptive responses. The availability of technical and biological resources should allow breaking new grounds by unveiling the contribution of signaling networks in integrative plant biology.

  10. Wnt/Calcium Signaling Mediates Axon Growth and Guidance in the Developing Corpus Callosum

    PubMed Central

    Hutchins, B Ian; Li, Li; Kalil, Katherine

    2011-01-01

    It has been shown in vivo that Wnt5a gradients surround the corpus callosum and guide callosal axons after the midline (postcrossing) by Wnt5a-induced repulsion via Ryk receptors. In dissociated cortical cultures we showed that Wnt5a simultaneously promotes axon outgrowth and repulsion by calcium signaling. Here to test the role of Wnt5a/calcium signaling in a complex in vivo environment we used sensorimotor cortical slices containing the developing corpus callosum. Plasmids encoding the cytoplasmic marker DsRed and the genetically encoded calcium indicator GCaMP2 were electroporated into one cortical hemisphere. Postcrossing callosal axons grew 50% faster than pre-crossing axons and higher frequencies of calcium transients in axons and growth cones correlated well with outgrowth. Application of pharmacological inhibitors to the slices showed that signaling pathways involving calcium release through IP3 receptors and calcium entry through TRP channels regulate post-crossing axon outgrowth and guidance. Co-electroporation of Ryk siRNA and DsRed revealed that knock down of the Ryk receptor reduced outgrowth rates of postcrossing but not precrossing axons by 50% and caused axon misrouting. Guidance errors in axons with Ryk knockdown resulted from reduced calcium activity. In the corpus callosum CaMKII inhibition reduced the outgrowth rate of postcrossing (but not precrossing) axons and caused severe guidance errors which resulted from reduced CaMKII-dependent repulsion downstream of Wnt/calcium. We show for the first time that Wnt/Ryk calcium signaling mechanisms regulating axon outgrowth and repulsion in cortical cultures are also essential for the proper growth and guidance of postcrossing callosal axons which involve axon repulsion through CaMKII. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 269–283, 2011. PMID:20936661

  11. 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

  12. Calcium signaling in lymphocytes and ELF fields. Evidence for an electric field metric and a site of interaction involving the calcium ion channel.

    PubMed

    Liburdy, R P

    1992-04-13

    Calcium influx increased during mitogen-activated signal transduction in thymic lymphocytes exposed to a 22 mT, 60 Hz magnetic field (E induced = 1.7 mV/cm, 37 degrees C, 60 min). To distinguish between an electric or a magnetic field dependence a special multi-ring annular cell culture plate based on Faraday's Law of Induction was employed. Studies show a dependence on the strength of the induced electric field at constant magnetic flux density. Moreover, exposure to a pure 60 Hz electric field or to a magnetically-induced electric field of identical strength resulted in similar changes in calcium transport. The first real-time monitoring of [Ca2+]i during application of a 60 Hz electric field revealed an increase in [Ca2+]i observed 100 s after mitogen stimulation; this suggests that the plateau phase rather than the early phase of calcium signaling was influenced. The hypothesis was tested by separating, in time, the early release of calcium from intracellular stores from the influx of extracellular calcium. In calcium-free buffer, 60 Hz field exerted little influence on the early release of calcium from intracellular stores. In contrast, addition of extracellular calcium during exposure enhanced calcium influx through the plasma membrane. Alteration of the plateau phase of calcium signaling implicates the calcium channel as a site of field interaction. In addition, an electric field exposure metric is mechanistically consistent with a cell-surface interaction site.

  13. Spontaneous and CRH-Induced Excitability and Calcium Signaling in Mice Corticotrophs Involves Sodium, Calcium, and Cation-Conducting Channels.

    PubMed

    Zemkova, Hana; Tomić, Melanija; Kucka, Marek; Aguilera, Greti; Stojilkovic, Stanko S

    2016-04-01

    Transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the proopiomelanocortin gene's regulatory elements are a useful model for studying corticotrophs. Using these mice, we studied the ion channels and mechanisms controlling corticotroph excitability. Corticotrophs were either quiescent or electrically active, with a 22-mV difference in the resting membrane potential (RMP) between the 2 groups. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in active cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory but not inhibitory action of CRH on electrical activity was mimicked by cAMP independently of the presence or absence of arachidonic acid. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling.

  14. Visualization of Plasticity in Fear-Evoked Calcium Signals in Midbrain Dopamine Neurons

    ERIC Educational Resources Information Center

    Gore, Bryan B.; Soden, Marta E.; Zweifel, Larry S.

    2014-01-01

    Dopamine is broadly implicated in fear-related processes, yet we know very little about signaling dynamics in these neurons during active fear conditioning. We describe the direct imaging of calcium signals of dopamine neurons during Pavlovian fear conditioning using fiber-optic confocal microscopy coupled with the genetically encoded calcium…

  15. Visualization of Plasticity in Fear-Evoked Calcium Signals in Midbrain Dopamine Neurons

    ERIC Educational Resources Information Center

    Gore, Bryan B.; Soden, Marta E.; Zweifel, Larry S.

    2014-01-01

    Dopamine is broadly implicated in fear-related processes, yet we know very little about signaling dynamics in these neurons during active fear conditioning. We describe the direct imaging of calcium signals of dopamine neurons during Pavlovian fear conditioning using fiber-optic confocal microscopy coupled with the genetically encoded calcium…

  16. Rapid, Long-Distance Electrical and Calcium Signaling in Plants.

    PubMed

    Choi, Won-Gyu; Hilleary, Richard; Swanson, Sarah J; Kim, Su-Hwa; Gilroy, Simon

    2016-04-29

    Plants integrate activities throughout their bodies using long-range signaling systems in which stimuli sensed by just a few cells are translated into mobile signals that can influence the activities in distant tissues. Such signaling can travel at speeds well in excess of millimeters per second and can trigger responses as diverse as changes in transcription and translation levels, posttranslational regulation, alterations in metabolite levels, and even wholesale reprogramming of development. In addition to the use of mobile small molecules and hormones, electrical signals have long been known to propagate throughout the plant. This electrical signaling network has now been linked to waves of Ca(2+) and reactive oxygen species that traverse the plant and trigger systemic responses. Analysis of cell type specificity in signal propagation has revealed the movement of systemic signals through specific cell types, suggesting that a rapid signaling network may be hardwired into the architecture of the plant.

  17. Spinning Disk Confocal Microscopy of Calcium Signalling in Blood Vessel Walls

    PubMed Central

    Nelson, Mark; Ledoux, Jonathan; Taylor, Mark; Bonev, Adrian; Hannah, Rachael; Solodushko, Viktoriya; Shui, Bo; Tallini, Yvonne; Kotlikoff, Michael

    2010-01-01

    Spinning disk confocal laser microscopy systems can be used for observing fast events occurring in a small volume when they include a sensitive electron-multiplying CCD camera. Such a confocal system was recently used to capture the first pictures of intracellular calcium signalling within the projections of endothelial cells to the adjacent smooth muscle cells in the blood vessel wall. Detection of these calcium signals required high spatial and temporal resolution. A newly developed calcium ion (Ca2+) biosensor was also used. This exclusively expressed in the endothelium and fluoresced when Ca2+ concentrations increased during signalling. This work gives insights into blood vessel disease because Ca2+ signalling is critical for blood flow and pressure regulation. PMID:22506097

  18. Calcium

    MedlinePlus

    ... in luck if you like sardines and canned salmon with bones. Almond milk. previous continue Working Calcium ... drinks, and cereals. Other Considerations for Building Bones Vitamin D is essential for calcium absorption, so it's ...

  19. Measurement and analysis of calcium signaling in heterogeneous cell cultures.

    PubMed

    Richards, Gillian R; Jack, Andrew D; Platts, Amy; Simpson, Peter B

    2006-01-01

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

  20. Bidirectional calcium signaling between satellite glial cells and neurons in cultured mouse trigeminal ganglia.

    PubMed

    Suadicani, Sylvia O; Cherkas, Pavel S; Zuckerman, Jonathan; Smith, David N; Spray, David C; Hanani, Menachem

    2010-02-01

    Astrocytes communicate with neurons, endothelial and other glial cells through transmission of intercellular calcium signals. Satellite glial cells (SGCs) in sensory ganglia share several properties with astrocytes, but whether this type of communication occurs between SGCs and sensory neurons has not been explored. In the present work we used cultured neurons and SGCs from mouse trigeminal ganglia to address this question. Focal electrical or mechanical stimulation of single neurons in trigeminal ganglion cultures increased intracellular calcium concentration in these cells and triggered calcium elevations in adjacent glial cells. Similar to neurons, SGCs responded to mechanical stimulation with increase in cytosolic calcium that spread to the adjacent neuron and neighboring glial cells. Calcium signaling from SGCs to neurons and among SGCs was diminished in the presence of the broad-spectrum P2 receptor antagonist suramin (50 muM) or in the presence of the gap junction blocker carbenoxolone (100 muM), whereas signaling from neurons to SGCs was reduced by suramin, but not by carbenoxolone. Following induction of submandibular inflammation by Complete Freund's Adjuvant injection, the amplitude of signaling among SGCs and from SGCs to neuron was increased, whereas the amplitude from neuron to SGCs was reduced. These results indicate for the first time the presence of bidirectional calcium signaling between neurons and SGCs in sensory ganglia cultures, which is mediated by the activation of purinergic P2 receptors, and to some extent by gap junctions. Furthermore, the results indicate that not only sensory neurons, but also SGCs release ATP. This form of intercellular calcium signaling likely plays key roles in the modulation of neuronal activity within sensory ganglia in normal and pathological states.

  1. Calcium

    MedlinePlus

    ... such as canned sardines and salmon Calcium-enriched foods such as breakfast cereals, fruit juices, soy and rice drinks, and tofu. Check the product labels. The exact amount of calcium you need depends on your age and other factors. Growing children and teenagers need more calcium than ...

  2. Controlling metabolism and cell death: at the heart of mitochondrial calcium signalling

    PubMed Central

    Murgia, Marta; Giorgi, Carlotta; Pinton, Paolo; Rizzuto, Rosario

    2009-01-01

    Transient increases in intracellular calcium concentration activate and coordinate a wide variety of cellular processes in virtually every cell type. This review describes the main homeostatic mechanisms that control Ca2+ transients, focusing on the mitochondrial checkpoint. We subsequently extend this paradigm to the cardiomyocyte and to the interplay between cytosol, endoplasmic reticulum and mitochondria that occurs beat-to-beat in excitation-contraction coupling. The mechanisms whereby mitochondria decode fast cytosolic calcium spikes are discussed in the light of the results obtained with recombinant photoproteins targeted to the mitochondrial matrix of contracting cardiomyocytes. Mitochondrial calcium homeostasis is then highlighted as a crucial point of convergence of the environmental signals that mediate cardiac cell death, both by necrosis and by apoptosis. Altogether we point to a role of the mitochondrion as an integrator of calcium signalling and fundamental decision maker in cardiomyocyte metabolism and survival. PMID:19285982

  3. Filamin and Phospholipase C-ε Are Required for Calcium Signaling in the Caenorhabditis elegans Spermatheca

    PubMed Central

    Kovacevic, Ismar; Orozco, Jose M.; Cram, Erin J.

    2013-01-01

    The Caenorhabditis elegans spermatheca is a myoepithelial tube that stores sperm and undergoes cycles of stretching and constriction as oocytes enter, are fertilized, and exit into the uterus. FLN-1/filamin, a stretch-sensitive structural and signaling scaffold, and PLC-1/phospholipase C-ε, an enzyme that generates the second messenger IP3, are required for embryos to exit normally after fertilization. Using GCaMP, a genetically encoded calcium indicator, we show that entry of an oocyte into the spermatheca initiates a distinctive series of IP3-dependent calcium oscillations that propagate across the tissue via gap junctions and lead to constriction of the spermatheca. PLC-1 is required for the calcium release mechanism triggered by oocyte entry, and FLN-1 is required for timely initiation of the calcium oscillations. INX-12, a gap junction subunit, coordinates propagation of the calcium transients across the spermatheca. Gain-of-function mutations in ITR-1/IP3R, an IP3-dependent calcium channel, and loss-of-function mutations in LFE-2, a negative regulator of IP3 signaling, increase calcium release and suppress the exit defect in filamin-deficient animals. We further demonstrate that a regulatory cassette consisting of MEL-11/myosin phosphatase and NMY-1/non-muscle myosin is required for coordinated contraction of the spermatheca. In summary, this study answers long-standing questions concerning calcium signaling dynamics in the C. elegans spermatheca and suggests FLN-1 is needed in response to oocyte entry to trigger calcium release and coordinated contraction of the spermathecal tissue. PMID:23671426

  4. The Acid Test: Calcium Signaling in the Skeletogenic Layer of Reef-Building Coral

    NASA Astrophysics Data System (ADS)

    Florn, A. M.

    2016-02-01

    Since the Industrial Revolution, carbon dioxide (CO2) emissions have increased more than 40%. This increased atmospheric CO2 drives ocean acidification and has potentially serious consequences for all marine life, especially calcifying organisms. The specific goal of this study was to examine calcium homeostasis and signaling dynamics within the skeletogenic tissue layers (calicodermal cells) of two coral species (Pavona maldivensis and Porites rus) at three pH treatments corresponding to present-future ocean acidification levels. Confocal microscopy techniques were used to analyze in vivo calcium dynamics of the calicodermal cells in Pavona maldivensis and Porites rus. The results show biological variation between the two reef-building coral species and their response to ocean acidification. Pavona maldivensis showed a significant difference (p < 0.01) in the ionomycin-induced calcium response among the pH treatments, but not among the microcolonies. Porites rus did not show a significant difference (p < 0.01) in the ionomycin-induced calcium response among the pH treatments or the microcolonies. Upon comparing the calcium response curves, the ionomycin-induced calcium response exhibited by Pavona maldivensis is phenomenologically similar to a calcium response that is commonly found in vertebrates. This well-studied phenomenon in vertebrate biology is known as store-operated calcium entry (SOCE) and is closely associated with the endoplasmic reticulum (ER) and mitochondria-associated endoplasmic reticulum (MAM) calcium stores. This study provides insight into the preliminary steps needed to understand in vivo calcium signaling in the calicodermis of reef-building coral and the associated consequences of ocean acidification.

  5. FRET imaging of calcium signaling in live cells in the microenvironment.

    PubMed

    Qian, Tongcheng; Lu, Shaoying; Ma, Hongwei; Fang, Jing; Zhong, Wenxuan; Wang, Yingxiao

    2013-02-01

    The microenvironment has been shown to regulate cellular functions including cell growth, differentiation, proliferation, migration, cancer development and metastasis. However, the underlying molecular mechanism remains largely unclear. We have integrated micro-pattern technology and molecular biosensors based on fluorescence resonance energy transfer (FRET) to visualize calcium responses in cells constrained to grow on a micro-patterned surface. Upon ATP stimulation, human umbilical vein endothelial cells (HUVECs) cultured on different surface micro-patterns had a shorter decay time and a reduced peak of a transient intracellular calcium rise compared to control cells without constraints. The decay time is regulated by the plasma membrane and the membrane calcium channels, while the peak by endoplasmic reticulum (ER) calcium release. Further results revealed that voltage operated channels (VOCs), coupling the plasma membrane and ER, can affect both the decay time and the peak of calcium response. The inhibition of VOCs can eliminate the effect of different micro-patterns on calcium signals. When two connected HUVECs were constrained to grow on a micro-pattern, drastically distinct calcium responses upon ATP stimulation can be observed, in contrast to the similar responses of two connected cells cultured without patterns. Interestingly, the inhibition of VOCs also blocked this difference of calcium responses between two connected cells on micro-patterns. These results indicate that a micro-patterned surface can have a profound effect on the calcium responses of HUVECs under ATP stimulation, largely mediated by VOCs. Therefore, our results shed new light on the molecular mechanism by which HUVECs perceive the microenvironment and regulate intracellular calcium signals.

  6. The impact of mitochondrial endosymbiosis on the evolution of calcium signaling.

    PubMed

    Blackstone, Neil W

    2015-03-01

    At high concentrations, calcium has detrimental effects on biological systems. Life likely arose in a low calcium environment, and the first cells evolved mechanisms to maintain this environment internally. Bursts of calcium influx followed by efflux or sequestration thus developed in a functional context. For example, in proto-cells with exterior energy-converting membranes, such bursts could be used to depolarize the membrane. In this way, proto-cells could maintain maximal phosphorylation (metabolic state 3) and moderate levels of reactive oxygen species (ROS), while avoiding the resting state (metabolic state 4) and high levels of ROS. This trait is likely a shared primitive characteristic of prokaryotes. When eukaryotes evolved, the α-proteobacteria that gave rise to proto-mitochondria inhabited a novel environment, the interior of the proto-eukaryote that had a low calcium concentration. In this environment, metabolic homeostasis was difficult to maintain, and there were inherent risks from ROS, yet depolarizing the proto-mitochondrial membrane by calcium influx was challenging. To maintain metabolic state 3, proto-mitochondria were required to congregate near calcium influx points in the proto-eukaryotic membrane. This behavior, resulting in embryonic forms of calcium signaling, may have occurred immediately after the initiation of the endosymbiosis. Along with ROS, calcium may have served as one of the key forms of crosstalk among the community of prokaryotes that led to the eukaryotic cell.

  7. Expressional Analysis and Role of Calcium Regulated Kinases in Abiotic Stress Signaling

    PubMed Central

    Das, Ritika; Pandey, Girdhar K

    2010-01-01

    Perception of stimuli and activation of a signaling cascade is an intrinsic characteristic feature of all living organisms. Till date, several signaling pathways have been elucidated that are involved in multiple facets of growth and development of an organism. Exposure to unfavorable stimuli or stress condition activates different signaling cascades in both plants and animal. Being sessile, plants cannot move away from an unfavorable condition, and hence activate the molecular machinery to cope up or adjust against that particular stress condition. In plants, role of calcium as second messenger has been studied in detail in both abiotic and biotic stress signaling. Several calcium sensor proteins such as calmodulin (CaM), calcium dependent protein kinases (CDPK) and calcinuerin B-like (CBL) were discovered to play a crucial role in abiotic stress signaling in plants. Unlike CDPK, CBL and CaM are calcium-binding proteins, which do not have any protein kinase enzyme activity and interact with a target protein kinase termed as CBL-interacting protein kinase (CIPK) and CaM kinases respectively. Genome sequence analysis of Arabidopsis and rice has led to the identification of multigene familes of these calcium signaling protein kinases. Individual and global gene expression analysis of these protein kinase family members has been analyzed under several developmental and different abiotic stress conditions. In this review, we are trying to overview and emphasize the expressional analysis of calcium signaling protein kinases under different abiotic stress and developmental stages, and linking the expression to possible function for these kinases. PMID:20808518

  8. Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization.

    PubMed

    Nikiforaki, D; Vanden Meerschaut, F; Qian, C; De Croo, I; Lu, Y; Deroo, T; Van den Abbeel, E; Heindryckx, B; De Sutter, P

    2014-01-01

    in vivo matured MII. In MI oocytes that reached the MII stage within 3 h the calcium oscillations additionally appear over a longer period of time (P < 0.05). In vivo MII oocytes show a different calcium oscillation pattern following V/W with calcium oscillations occurring over a longer period of time, with a higher amplitude and a lower frequency (P < 0.05). In vitro matured oocytes, either from the GV or the MI stage, also display an altered pattern of calcium oscillations after V/W and the parameters that were similarly affected in all these oocyte groups are the frequency and the amplitude of the calcium transients. Slow freezing/thawing differentially affects the calcium oscillation pattern of in vitro matured and in vitro aged oocytes. The relationship between a specific pattern of calcium oscillations and subsequent human embryonic development could not be evaluated since the calcium indicator used and the high-intensity excitation light impair development. Furthermore, all oocytes were derived from stimulated cycles and immature oocytes were denuded prior to in vitro maturation. Our data show for the first time how calcium signalling during human fertilization is affected by oocyte in vitro maturation, in vitro ageing as well as V/W and slow freezing/thawing. The analysis of calcium oscillations could be used as an oocyte quality indicator to evaluate in vitro culture and cryopreservation techniques of human oocytes. This work was supported by a clinical research mandate from the Flemish Foundation of Scientific Research (FWO-Vlaanderen, FWO09/ASP/063) to F.V.M, a fundamental clinical research mandate from the FWO-Vlaanderen (FWO05/FKM/001) to P.D.S and a Ghent University grant (KAN-BOF E/01321/01) to B.H. The authors have no conflict of interest to declare.

  9. Synergy of cAMP and calcium signaling pathways in CFTR regulation

    PubMed Central

    Bozoky, Zoltan; Ahmadi, Saumel; Milman, Tal; Kim, Tae Hun; Du, Kai; Di Paola, Michelle; Pasyk, Stan; Pekhletski, Roman; Keller, Jacob P.; Bear, Christine E.; Forman-Kay, Julie D.

    2017-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport. PMID:28242698

  10. Calcium signaling and cell volume regulation are altered in Sjögren's Syndrome.

    PubMed

    Enger, Tone Berge; Aure, Marit Høyberg; Jensen, Janicke Liaaen; Galtung, Hilde Kanli

    2014-10-01

    Sjögren's Syndrome (SS) is a chronic autoimmune disease, leading to deficient secretion from salivary and lacrimal glands. Saliva production is normally increased by cholinergic innervation, giving rise to intracellular calcium signaling and water transport through water channels (aquaporins, AQPs). The aim of this study was to investigate possible pathophysiological changes in cell volume regulation, AQP expression and localization, and intracellular calcium signaling in glandular cells from SS patients compared to controls. A total of 35 SS patients and 41 non-SS controls were included. Real time qPCR was combined with immunohistochemistry to analyze the mRNA expression and cellular distribution of AQP1, 3 and 5. Cell volume regulation and intracellular calcium signaling were examined in fresh acinar cells. We show for the first time a reduced mRNA expression of AQP1 and 5 in SS compared to controls, accompanied by a decrease in staining intensity of AQP1, 3 and 5 in areas adjacent to local lymphocytic infiltration. Furthermore, we observed that the SS cells' capacity for volume regulation was abnormal. Similarly, the calcium response after parasympathetic agonist (carbachol) stimulation was markedly decreased in SS cells. It is concluded that mRNA expression of AQP1 and 5, protein distribution of AQP1, 3 and 5, glandular cell volume regulation and intracellular calcium signaling are all altered in SS, pointing to possible pathophysiological mechanisms in SS.

  11. Targeted inhibition of calcineurin signaling blocks calcium-dependent reactivation of Kaposi sarcoma-associated herpesvirus.

    PubMed

    Zoeteweij, J P; Moses, A V; Rinderknecht, A S; Davis, D A; Overwijk, W W; Yarchoan, R; Orenstein, J M; Blauvelt, A

    2001-04-15

    Kaposi sarcoma-associated herpesvirus (KSHV) is associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman disease. Reactivation of KSHV in latently infected cells and subsequent plasma viremia occur before the development of KS. Intracellular signaling pathways involved in KSHV reactivation were studied. In latently infected PEL cells (BCBL-1), KSHV reactivation in single cells was determined by quantitative flow cytometry. Viral particle production was determined by electron microscope analyses and detection of minor capsid protein in culture supernatants. Agents that mobilized intracellular calcium (ionomycin, thapsigargin) induced expression of KSHV lytic cycle-associated proteins and led to increased virus production. Calcium-mediated virus reactivation was blocked by specific inhibitors of calcineurin-dependent signal transduction (cyclosporine, FK506). Similarly, calcium-mediated virus reactivation in KSHV-infected dermal microvascular endothelial cells was blocked by cyclosporine. Furthermore, retroviral transduction with plasmid DNA encoding VIVIT, a peptide specifically blocking calcineurin-NFAT interactions, inhibited calcium-dependent KSHV reactivation. By contrast, chemical induction of lytic-phase infection by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate was blocked by protein kinase C inhibitors, but not by calcineurin inhibitors. In summary, calcineurin-dependent signal transduction, an important signaling cascade in vivo, induces calcium-dependent KSHV replication, providing a possible target for the design of antiherpesvirus strategies in KSHV-infected patients.

  12. Synergy of cAMP and calcium signaling pathways in CFTR regulation.

    PubMed

    Bozoky, Zoltan; Ahmadi, Saumel; Milman, Tal; Kim, Tae Hun; Du, Kai; Di Paola, Michelle; Pasyk, Stan; Pekhletski, Roman; Keller, Jacob P; Bear, Christine E; Forman-Kay, Julie D

    2017-02-27

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport.

  13. Purinergic and Calcium Signaling in Macrophage Function and Plasticity

    PubMed Central

    Desai, Bimal N.; Leitinger, Norbert

    2014-01-01

    In addition to a fundamental role in cellular bioenergetics, the purine nucleotide adenosine triphosphate (ATP) plays a crucial role in the extracellular space as a signaling molecule. ATP and its metabolites serve as ligands for a family of receptors that are collectively referred to as purinergic receptors. These receptors were first described and characterized in the nervous system but it soon became evident that they are expressed ubiquitously. In the immune system, purinergic signals regulate the migration and activation of immune cells and they may also orchestrate the resolution of inflammation (1, 2). The intracellular signal transduction initiated by purinergic receptors is strongly coupled to Ca2+-signaling, and co-ordination of these pathways plays a critical role in innate immunity. In this review, we provide an overview of purinergic and Ca2+-signaling in the context of macrophage phenotypic polarization and discuss the implications on macrophage function in physiological and pathological conditions. PMID:25505897

  14. Noradrenaline effects on pyruvate decarboxylation: correlation with calcium signaling.

    PubMed

    Chen, Y; Hertz, L

    1999-11-15

    Noradrenaline effects on the rate of metabolism of pyruvate to acetyl coenzyme A, catalyzed by the pyruvate dehydrogenase complex, was measured in primary cultures of mouse astrocytes as rate of production of labeled CO(2) from 1-[(14) C]pyruvate in the absence of competing glucose in the medium. The subtype specificity of a noradrenaline-stimulated increase in rate of CO(2) formation was identical to that for noradrenaline-induced increase in free intracellular calcium ([Ca(2+)](i)), suggesting a causal relationship between these two phenomena. The noradrenaline-induced stimulation of pyruvate decarboxylation was abolished in the presence of 10 mM magnesium chloride in the medium, combined with the omission of calcium, a procedure known to prevent an increased [Ca(2+)] in the cytosol from raising intramitochondrial [Ca(2+)]. Thus, the stimulation of metabolic flux through the reaction catalyzed by the pyruvate dehydrogenase complex appears to result from an increase in intramitochondrial [Ca(2+)] ions in astrocytes. Such a mechanism for stimulation of the same enzyme has been convincingly demonstrated in other cell types, primarily heart muscle and hepatic cells, but it has not previously been demonstrated in any cell type from the central nervous system.

  15. Minireview: the intimate link between calcium sensing receptor trafficking and signaling: implications for disorders of calcium homeostasis.

    PubMed

    Breitwieser, Gerda E

    2012-09-01

    The calcium-sensing receptor (CaSR) regulates organismal Ca(2+) homeostasis. Dysregulation of CaSR expression or mutations in the CASR gene cause disorders of Ca(2+) homeostasis and contribute to the progression or severity of cancers and cardiovascular disease. This brief review highlights recent findings that define the CaSR life cycle, which controls the cellular abundance of CaSR and CaSR signaling. A novel mechanism, termed agonist-driven insertional signaling (ADIS), contributes to the unique hallmarks of CaSR signaling, including the high degree of cooperativity and the lack of functional desensitization. Agonist-mediated activation of plasma membrane-localized CaSR increases the rate of insertion of CaSR at the plasma membrane without altering the constitutive endocytosis rate, thereby acutely increasing the maximum signaling response. Prolonged CaSR signaling requires a large intracellular ADIS-mobilizable pool of CaSR, which is maintained by signaling-mediated increases in biosynthesis. This model provides a rational framework for characterizing the defects caused by CaSR mutations and the altered functional expression of wild-type CaSR in disease states. Mechanistic dissection of ADIS of CaSR should lead to optimized pharmacological approaches to normalize CaSR signaling in disorders of Ca(2+) homeostasis.

  16. Minireview: The Intimate Link Between Calcium Sensing Receptor Trafficking and Signaling: Implications for Disorders of Calcium Homeostasis

    PubMed Central

    2012-01-01

    The calcium-sensing receptor (CaSR) regulates organismal Ca2+ homeostasis. Dysregulation of CaSR expression or mutations in the CASR gene cause disorders of Ca2+ homeostasis and contribute to the progression or severity of cancers and cardiovascular disease. This brief review highlights recent findings that define the CaSR life cycle, which controls the cellular abundance of CaSR and CaSR signaling. A novel mechanism, termed agonist-driven insertional signaling (ADIS), contributes to the unique hallmarks of CaSR signaling, including the high degree of cooperativity and the lack of functional desensitization. Agonist-mediated activation of plasma membrane-localized CaSR increases the rate of insertion of CaSR at the plasma membrane without altering the constitutive endocytosis rate, thereby acutely increasing the maximum signaling response. Prolonged CaSR signaling requires a large intracellular ADIS-mobilizable pool of CaSR, which is maintained by signaling-mediated increases in biosynthesis. This model provides a rational framework for characterizing the defects caused by CaSR mutations and the altered functional expression of wild-type CaSR in disease states. Mechanistic dissection of ADIS of CaSR should lead to optimized pharmacological approaches to normalize CaSR signaling in disorders of Ca2+ homeostasis. PMID:22745192

  17. Disruption of Vitamin D and Calcium Signaling in Keratinocytes Predisposes to Skin Cancer

    PubMed Central

    Bikle, Daniel D.; Jiang, Yan; Nguyen, Thai; Oda, Yuko; Tu, Chia-ling

    2016-01-01

    1,25 dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D, and calcium regulate epidermal differentiation. 1,25(OH)2D exerts its effects through the vitamin D receptor (VDR), a transcription factor in the nuclear hormone receptor family, whereas calcium acts through the calcium sensing receptor (Casr), a membrane bound member of the G protein coupled receptor family. We have developed mouse models in which the Vdr and Casr have been deleted in the epidermis (epidVdr−∕− and epidCasr−∕−). Both genotypes show abnormalities in calcium induced epidermal differentiation in vivo and in vitro, associated with altered hedgehog (HH) and β–catenin signaling that when abnormally expressed lead to basal cell carcinomas (BCC) and trichofolliculomas, respectively. The Vdr−∕− mice are susceptible to tumor formation following UVB or chemical carcinogen exposure. More recently we found that the keratinocytes from these mice over express long non-coding RNA (lncRNA) oncogenes such as H19 and under express lncRNA tumor suppressors such as lincRNA-21. Spontaneous tumors have not been observed in either the epidVdr−∕− or epidCasr−∕−. But in mice with epidermal specific deletion of both Vdr and Casr (epidVdr−∕−/epidCasr−∕− [DKO]) tumor formation occurs spontaneously when the DKO mice are placed on a low calcium diet. These results demonstrate important interactions between vitamin D and calcium signaling through their respective receptors that lead to cancer when these signals are disrupted. The roles of the β–catenin, hedgehog, and lncRNA pathways in predisposing the epidermis to tumor formation when vitamin D and calcium signaling are disrupted will be discussed. PMID:27462278

  18. Calcium Signals: The Lead Currency of Plant Information Processing

    PubMed Central

    Kudla, Jörg; Batistič, Oliver; Hashimoto, Kenji

    2010-01-01

    Ca2+ signals are core transducers and regulators in many adaptation and developmental processes of plants. Ca2+ signals are represented by stimulus-specific signatures that result from the concerted action of channels, pumps, and carriers that shape temporally and spatially defined Ca2+ elevations. Cellular Ca2+ signals are decoded and transmitted by a toolkit of Ca2+ binding proteins that relay this information into downstream responses. Major transduction routes of Ca2+ signaling involve Ca2+-regulated kinases mediating phosphorylation events that orchestrate downstream responses or comprise regulation of gene expression via Ca2+-regulated transcription factors and Ca2+-responsive promoter elements. Here, we review some of the remarkable progress that has been made in recent years, especially in identifying critical components functioning in Ca2+ signal transduction, both at the single-cell and multicellular level. Despite impressive progress in our understanding of the processing of Ca2+ signals during the past years, the elucidation of the exact mechanistic principles that underlie the specific recognition and conversion of the cellular Ca2+ currency into defined changes in protein–protein interaction, protein phosphorylation, and gene expression and thereby establish the specificity in stimulus response coupling remain to be explored. PMID:20354197

  19. Interaction between Calcium and Actin in Guard Cell and Pollen Signaling Networks

    PubMed Central

    Chen, Dong-Hua; Acharya, Biswa R.; Liu, Wei; Zhang, Wei

    2013-01-01

    Calcium (Ca2+) plays important roles in plant growth, development, and signal transduction. It is a vital nutrient for plant physical design, such as cell wall and membrane, and also serves as a counter-cation for biochemical, inorganic, and organic anions, and more particularly, its concentration change in cytosol is a ubiquitous second messenger in plant physiological signaling in responses to developmental and environmental stimuli. Actin cytoskeleton is well known for its importance in cellular architecture maintenance and its significance in cytoplasmic streaming and cell division. In plant cell system, the actin dynamics is a process of polymerization and de-polymerization of globular actin and filamentous actin and that acts as an active regulator for calcium signaling by controlling calcium evoked physiological responses. The elucidation of the interaction between calcium and actin dynamics will be helpful for further investigation of plant cell signaling networks at molecular level. This review mainly focuses on the recent advances in understanding the interaction between the two aforementioned signaling components in two well-established model systems of plant, guard cell, and pollen. PMID:27137395

  20. Lipid rafts/caveolae as microdomains of calcium signaling

    PubMed Central

    Pani, Biswaranjan; Singh, Brij B

    2009-01-01

    Summary Ca2+ is a major signaling molecule in both excitable and non-excitable cells, where it serves critical functions ranging from cell growth to differentiation to cell death. The physiological functions of these cells are tightly regulated in response to changes in cytosolic Ca2+ that is achieved by the activation of several plasma membrane (PM) Ca2+ channels as well as release of Ca2+ from the internal stores. One such channel is referred to as store-operated Ca2+ channel that is activated by the release of endoplasmic reticulum (ER) Ca2+ which initiates store operated Ca2+ entry (SOCE). Recent advances in the field suggest that some members of TRPCs and Orai channels function as SOCE channels. However, the molecular mechanisms that regulate channel activity and the exact nature of where these channels are assembled and regulated remain elusive. Research from several laboratories has demonstrated that key proteins involved in Ca2+ signaling are localized in discrete PM lipid rafts/caveolar microdomains. Lipid rafts are cholesterol and sphingolipid enriched microdomains that function as unique signal transduction platforms. In addition lipid rafts are dynamic in nature which tends to scaffold certain signaling molecules while excluding others. By such spatial segregation, lipid rafts not only provide a favorable environment for intra-molecular cross talk but also aid to expedite the signal relay. Importantly, Ca2+ signaling is shown to initiate from these lipid raft microdomains. Clustering of Ca2+ channels and their regulators in such microdomains can provide an exquisite spatiotemporal regulation of Ca2+ mediated cellular function. Thus in this review we discuss PM lipid rafts and caveolae as Ca2+ signaling microdomains and highlight their importance in organizing and regulating SOCE channels. PMID:19324409

  1. Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer.

    PubMed

    Raynal, Noël J-M; Lee, Justin T; Wang, Youjun; Beaudry, Annie; Madireddi, Priyanka; Garriga, Judith; Malouf, Gabriel G; Dumont, Sarah; Dettman, Elisha J; Gharibyan, Vazganush; Ahmed, Saira; Chung, Woonbok; Childers, Wayne E; Abou-Gharbia, Magid; Henry, Ryan A; Andrews, Andrew J; Jelinek, Jaroslav; Cui, Ying; Baylin, Stephen B; Gill, Donald L; Issa, Jean-Pierre J

    2016-03-15

    Targeting epigenetic pathways is a promising approach for cancer therapy. Here, we report on the unexpected finding that targeting calcium signaling can reverse epigenetic silencing of tumor suppressor genes (TSG). In a screen for drugs that reactivate silenced gene expression in colon cancer cells, we found three classical epigenetic targeted drugs (DNA methylation and histone deacetylase inhibitors) and 11 other drugs that induced methylated and silenced CpG island promoters driving a reporter gene (GFP) as well as endogenous TSGs in multiple cancer cell lines. These newly identified drugs, most prominently cardiac glycosides, did not change DNA methylation locally or histone modifications globally. Instead, all 11 drugs altered calcium signaling and triggered calcium-calmodulin kinase (CamK) activity, leading to MeCP2 nuclear exclusion. Blocking CamK activity abolished gene reactivation and cancer cell killing by these drugs, showing that triggering calcium fluxes is an essential component of their epigenetic mechanism of action. Our data identify calcium signaling as a new pathway that can be targeted to reactivate TSGs in cancer.

  2. Reciprocal Interaction of Dendrite Geometry and Nuclear Calcium-VEGFD Signaling Gates Memory Consolidation and Extinction.

    PubMed

    Hemstedt, Thekla J; Bengtson, C Peter; Ramírez, Omar; Oliveira, Ana M M; Bading, Hilmar

    2017-07-19

    Nuclear calcium is an important signaling end point in synaptic excitation-transcription coupling that is critical for long-term neuroadaptations. Here, we show that nuclear calcium acting via a target gene, VEGFD, is required for hippocampus-dependent fear memory consolidation and extinction in mice. Nuclear calcium-VEGFD signaling upholds the structural integrity and complexity of the dendritic arbor of CA1 neurons that renders those cells permissive for the efficient generation of synaptic input-evoked nuclear calcium transients driving the expression of plasticity-related genes. Therefore, the gating of memory functions rests on the reciprocally reinforcing maintenance of an intact dendrite geometry and a functional synapse-to-nucleus communication axis. In psychiatric and neurodegenerative disorders, therapeutic application of VEGFD may help to stabilize dendritic structures and network connectivity, which may prevent cognitive decline and could boost the efficacy of extinction-based exposure therapies.SIGNIFICANCE STATEMENT This study uncovers a reciprocal relationship between dendrite geometry, the ability to generate nuclear calcium transients in response to synaptic inputs, and the subsequent induction of expression of plasticity-related and dendritic structure-preserving genes. Insufficient nuclear calcium signaling in CA1 hippocampal neurons and, consequently, reduced expression of the nuclear calcium target gene VEGFD, a dendrite maintenance factor, leads to reduced-complexity basal dendrites of CA1 neurons, which severely compromises the animals' consolidation of both memory and extinction memory. The structure-protective function of VEGFD may prove beneficial in psychiatric disorders as well as neurodegenerative and aging-related conditions that are associated with loss of neuronal structures, dysfunctional excitation-transcription coupling, and cognitive decline. Copyright © 2017 the authors 0270-6474/17/376946-10$15.00/0.

  3. Mitochondrial respiration links TOR Complex 2 signaling to calcium regulation and autophagy.

    PubMed

    Vlahakis, Ariadne; Lopez Muniozguren, Nerea; Powers, Ted

    2017-03-21

    The Target of Rapamycin (TOR) kinase is a conserved regulator of cell growth and functions within 2 different protein complexes, TORC1 and TORC2, where TORC2 positively controls macroautophagy/autophagy during amino acid starvation. Under these conditions, TORC2 signaling inhibits the activity of the calcium-regulated phosphatase calcineurin and promotes the general amino acid control (GAAC) response and autophagy. Here we demonstrate that TORC2 regulates calcineurin by controlling the respiratory activity of mitochondria. In particular, we find that mitochondrial oxidative stress affects the calcium channel regulatory protein Mid1, which we show is an essential upstream activator of calcineurin. Thus, these findings describe a novel regulation for autophagy that involves TORC2 signaling, mitochondrial respiration, and calcium homeostasis.

  4. 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

  5. Intracellular calcium signaling regulates autophagy via calcineurin-mediated TFEB dephosphorylation

    PubMed Central

    Tong, Yanju; Song, Fuyong

    2015-01-01

    The transcription-regulating activity of TFEB is dependent on its phosphorylation modification, but the phosphatase(s) involved in TFEB dephosphorylation have remained elusive. It has now become clear that lysosomal calcium signaling activates calcineurin, an endogenous serine/threonine phosphatase, which dephosphorylate TFEB leading to upregulation of autophagy. PMID:26043755

  6. Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans.

    PubMed

    Liu, Zhiyu; Wang, Bin; He, Ruijun; Zhao, Yanmei; Miao, Long

    2014-02-01

    In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

  7. Dissection of calcium signaling events in exocrine secretion.

    PubMed

    Ambudkar, Indu S

    2011-07-01

    The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple ion channel and transporter proteins, signaling components, and water transport. Importantly, neurotransmitter stimulated increase in the cytosolic free [Ca(2+)] ([Ca(2+)](i)) is critical for the regulation of salivary gland secretion as it regulates several major ion fluxes that together establish the sustained osmotic gradient to drive fluid secretion. The mechanisms that act to modulate these increases in [Ca(2+)](i) are therefore central to the process of salivary fluid secretion. Such modulation involves membrane receptors for neurotransmitters, as well as mechanisms that mediate intracellular Ca(2+) release, and Ca(2+) entry, as well as those that maintain cellular Ca(2+) homeostasis. Together, these mechanisms determine the spatial and temporal aspects of the [Ca(2+)](i) signals that regulate fluid secretion. Molecular cloning of these transporters and channels as well as development of mice lacking these proteins has established the physiological significance of key components that are involved in regulating [Ca(2+)](i) in salivary glands. This review will discuss these important studies and the findings which have led to resolution of the Ca(2+) signaling mechanisms that determine salivary gland fluid secretion.

  8. Calcium signaling and cell cycle: Progression or death.

    PubMed

    Humeau, Juliette; Bravo-San Pedro, José Manuel; Vitale, Ilio; Nuñez, Lucia; Villalobos, Carlos; Kroemer, Guido; Senovilla, Laura

    2017-07-25

    Cytosolic Ca(2+) concentration levels fluctuate in an ordered manner along the cell cycle, in line with the fact that Ca(2+) is involved in the regulation of cell proliferation. Cell proliferation should be an error-free process, yet is endangered by mistakes. In fact, a complex network of proteins ensures that cell cycle does not progress until the previous phase has been successfully completed. Occasionally, errors occur during the cell cycle leading to cell cycle arrest. If the error is severe, and the cell cycle checkpoints work perfectly, this results into cellular demise by activation of apoptotic or non-apoptotic cell death programs. Cancer is characterized by deregulated proliferation and resistance against cell death. Ca(2+) is a central key to these phenomena as it modulates signaling pathways that control oncogenesis and cancer progression. Here, we discuss how Ca(2+) participates in the exogenous and endogenous signals controlling cell proliferation, as well as in the mechanisms by which cells die if irreparable cell cycle damage occurs. Moreover, we summarize how Ca(2+) homeostasis remodeling observed in cancer cells contributes to deregulated cell proliferation and resistance to cell death. Finally, we discuss the possibility to target specific components of Ca(2+) signal pathways to obtain cytostatic or cytotoxic effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores.

    PubMed

    Gobin, V; De Bock, M; Broeckx, B J G; Kiselinova, M; De Spiegelaere, W; Vandekerckhove, L; Van Steendam, K; Leybaert, L; Deforce, D

    2015-09-01

    Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca(2+) signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca(2+) signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca(2+) signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca(2+) stores, thereby leaving less Ca(2+) available for release upon IP3- and ryanodine-receptor activation. The Ca(2+)-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca(2+) release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression.

  10. Role of nucleotide P2 receptors in calcium signaling and prolactin release in pituitary lactotrophs.

    PubMed

    He, Mu-Lan; Gonzalez-Iglesias, Arturo E; Stojilkovic, Stanko S

    2003-11-21

    Anterior pituitary cells express nucleotide-gated G protein-coupled P2 receptors (P2YRs) and cation-conducting channels (P2XRs). However, the identification of P2 receptors subtypes and their native ligands, and the distribution and function of these receptors within the secretory and non-secretory pituitary cells has been incompletely characterized. The focus in this study was on lactotroph subpopulation of cells. ATP and ADP, but not UTP and UDP, triggered calcium signaling in a majority (85%) of lactotrophs and prolactin release in mixed pituitary cells. Consistent with the role of P2 receptors in signaling and secretion, the actions of ATP and ADP were abolished in the presence of apyrase, an ectonucleotidase. Transcripts for Gq-coupled calcium-mobilizing P2Y1R, P2Y2R, P2Y4R, and P2Y6R, as well as Gi-coupled P2Y12R, were identified in mixed anterior pituitary cells. The ligand-selectivity profile of calcium mobilization-dependent signaling and prolactin secretion and the blockade of these responses by pyridoxal 5-phosphate 6-azophenyl-2',4'-disulphonic acid indicated that P2Y1R mediates the stimulatory action of ATP and ADP. Within the channels expressed in anterior pituitary (P2X2R, P2X3R, P2X4R, and P2X7R), the P2X4R subtype provides a major pathway for calcium influx-dependent signaling and prolactin secretion. This conclusion was based on comparison of native to recombinant channels with respect to their ligand preference, sensitivity to pyridoxal 5-phosphate 6-azophenyl-2',4'-disulphonic acid, and the rates of calcium signal desensitization.

  11. L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling

    PubMed Central

    Jacquemet, Guillaume; Baghirov, Habib; Georgiadou, Maria; Sihto, Harri; Peuhu, Emilia; Cettour-Janet, Pierre; He, Tao; Perälä, Merja; Kronqvist, Pauliina; Joensuu, Heikki; Ivaska, Johanna

    2016-01-01

    Mounting in vitro, in vivo and clinical evidence suggest an important role for filopodia in driving cancer cell invasion. Using a high-throughput microscopic-based drug screen, we identify FDA-approved calcium channel blockers (CCBs) as potent inhibitors of filopodia formation in cancer cells. Unexpectedly, we discover that L-type calcium channels are functional and frequently expressed in cancer cells suggesting a previously unappreciated role for these channels during tumorigenesis. We further demonstrate that, at filopodia, L-type calcium channels are activated by integrin inside-out signalling, integrin activation and Src. Moreover, L-type calcium channels promote filopodia stability and maturation into talin-rich adhesions through the spatially restricted regulation of calcium entry and subsequent activation of the protease calpain-1. Altogether we uncover a novel and clinically relevant signalling pathway that regulates filopodia formation in cancer cells and propose that cycles of filopodia stabilization, followed by maturation into focal adhesions, directs cancer cell migration and invasion. PMID:27910855

  12. Control of Insulin Secretion by Cytochrome c and Calcium Signaling in Islets with Impaired Metabolism*

    PubMed Central

    Rountree, Austin M.; Neal, Adam S.; Lisowski, Mark; Rizzo, Norma; Radtke, Jared; White, Sarah; Luciani, Dan S.; Kim, Francis; Hampe, Christiane S.; Sweet, Ian R.

    2014-01-01

    The aim of the study was to assess the relative control of insulin secretion rate (ISR) by calcium influx and signaling from cytochrome c in islets where, as in diabetes, the metabolic pathways are impaired. This was achieved either by culturing isolated islets at low (3 mm) glucose or by fasting rats prior to the isolation of the islets. Culture in low glucose greatly reduced the glucose response of cytochrome c reduction and translocation and ISR, but did not affect the response to the mitochondrial fuel α-ketoisocaproate. Unexpectedly, glucose-stimulated calcium influx was only slightly reduced in low glucose-cultured islets and was not responsible for the impairment in glucose-stimulated ISR. A glucokinase activator acutely restored cytochrome c reduction and translocation and ISR, independent of effects on calcium influx. Islets from fasted rats had reduced ISR and cytochrome c reduction in response to both glucose and α-ketoisocaproate despite normal responses of calcium. Our data are consistent with the scenario where cytochrome c reduction and translocation are essential signals in the stimulation of ISR, the loss of which can result in impaired ISR even when calcium response is normal. PMID:24841202

  13. 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

  14. Voltage-dependent calcium signaling in rat cerebellar unipolar brush cells.

    PubMed

    Birnstiel, S; Slater, N T; McCrimmon, D R; Mugnaini, E; Hartell, N A

    2009-09-01

    Unipolar brush cells (UBCs) are a class of excitatory interneuron found in the granule cell layer of the vestibulocerebellum. Mossy fibers form excitatory inputs on to the paint brush shaped dendrioles in the form of giant, glutamatergic synapses, activation of which results in prolonged bursts of action potentials in the postsynaptic UBC. The axons of UBCs themselves form mossy fiber contacts with other UBCs and granule cells, forming an excitatory, intrinsic cerebellar network that has the capacity to synchronize and amplify mossy fiber inputs to potentially large populations of granule cells. In this paper, we demonstrate that UBCs in rat cerebellar slices express low voltage activated (LVA) fast-inactivating and high voltage activated (HVA) slowly inactivating calcium channels. LVA calcium currents are mediated by T-type calcium channels and they are associated with calcium increases in the dendrites and to a lesser extent the cell soma. HVA currents, mediated by L-type calcium channels, are slowly inactivating and they produce larger overall increases in intracellular calcium but with a similar distribution pattern. We review these observations alongside several recent papers that examine how intrinsic membrane properties influence UBCs firing patterns and we discuss how UBC signaling may affect downstream cerebellar processing.

  15. Muscle mitochondrial metabolism and calcium signaling impairment in patients treated with statins

    SciTech Connect

    Sirvent, P.; Fabre, O.; Bordenave, S.; Hillaire-Buys, D.; Raynaud De Mauverger, E.; Lacampagne, A.; Mercier, J.

    2012-03-01

    The most common and problematic side effect of statins is myopathy. To date, the patho-physiological mechanisms of statin myotoxicity are still not clearly understood. In previous studies, we showed that acute application in vitro of simvastatin caused impairment of mitochondrial function and dysfunction of calcium homeostasis in human and rat healthy muscle samples. We thus evaluated in the present study, mitochondrial function and calcium signaling in muscles of patients treated with statins, who present or not muscle symptoms, by oxygraphy and recording of calcium sparks, respectively. Patients treated with statins showed impairment of mitochondrial respiration that involved mainly the complex I of the respiratory chain and altered frequency and amplitude of calcium sparks. The muscle problems observed in statin-treated patients appear thus to be related to impairment of mitochondrial function and muscle calcium homeostasis, confirming the results we previously reported in vitro. -- Highlights: ► The most common and problematic side effect of statins is myopathy. ► Patients treated with statins showed impairment of mitochondrial respiration. ► Statins-treated patients showed altered frequency and amplitude of calcium sparks.

  16. Nuclear proton dynamics and interactions with calcium signaling.

    PubMed

    Hulikova, Alzbeta; Swietach, Pawel

    2016-07-01

    Biochemical signals acting on the nucleus can regulate gene expression. Despite the inherent affinity of nucleic acids and nuclear proteins (e.g. transcription factors) for protons, little is known about the mechanisms that regulate nuclear pH (pHnuc), and how these could be exploited to control gene expression. Here, we show that pHnuc dynamics can be imaged using the DNA-binding dye Hoechst 33342. Nuclear pores allow the passage of medium-sized molecules (calcein), but protons must first bind to mobile buffers in order to gain access to the nucleoplasm. Fixed buffering residing in the nucleus of permeabilized cells was estimated to be very weak on the basis of the large amplitude of pHnuc transients evoked by photolytic H(+)-uncaging or exposure to weak acids/bases. Consequently, the majority of nuclear pH buffering is sourced from the cytoplasm in the form of mobile buffers. Effective proton diffusion was faster in nucleoplasm than in cytoplasm, in agreement with the higher mobile-to-fixed buffering ratio in the nucleus. Cardiac myocyte pHnuc changed in response to maneuvers that alter nuclear Ca(2+) signals. Blocking Ca(2+) release from inositol-1,4,5-trisphosphate receptors stably alkalinized the nucleus. This Ca(2+)-pH interaction may arise from competitive binding to common chemical moieties. Competitive binding to mobile buffers may couple the efflux of Ca(2+)via nuclear pores with a counterflux of protons. This would generate a stable pH gradient between cytoplasm and nucleus that is sensitive to the state of nuclear Ca(2+) signaling. The unusual behavior of protons in the nucleus provides new mechanisms for regulating cardiac nuclear biology. Copyright © 2015. Published by Elsevier Ltd.

  17. Force-dependent calcium signaling and its pathway of human neutrophils on P-selectin in flow.

    PubMed

    Huang, Bing; Ling, Yingchen; Lin, Jiangguo; Du, Xin; Fang, Ying; Wu, Jianhua

    2017-02-01

    P-selectin engagement of P-selectin glycoprotein ligand-1 (PSGL-1) causes circulating leukocytes to roll on and adhere to the vascular surface, and mediates intracellular calcium flux, a key but unclear event for subsequent arresting firmly at and migrating into the infection or injured tissue. Using a parallel plate flow chamber technique and intracellular calcium ion detector (Fluo-4 AM), the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin in the absence of chemokines at various wall shear stresses was investigated here in real time by fluorescence microscopy. The results demonstrated that P-selectin engagement of PSGL-1 induced the intracellular calcium flux of firmly adhered neutrophils in flow, increasing P-selectin concentration enhanced cellular calcium signaling, and, force triggered, enhanced and quickened the cytoplasmic calcium bursting of neutrophils on immobilized P-selectin. This P-selectin-induced calcium signaling should come from intracellular calcium release rather than extracellular calcium influx, and be along the mechano-chemical signal pathway involving the cytoskeleton, moesin and Spleen tyrosine kinase (Syk). These results provide a novel insight into the mechano-chemical regulation mechanism for P-selectin-induced calcium signaling of neutrophils in flow.

  18. Structural dynamics of the cell nucleus: basis for morphology modulation of nuclear calcium signaling and gene transcription.

    PubMed

    Queisser, Gillian; Wiegert, Simon; Bading, Hilmar

    2011-01-01

    Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons.

  19. Axial tubule junctions control rapid calcium signaling in atria

    PubMed Central

    Brandenburg, Sören; Kohl, Tobias; Williams, George S.B.; Rog-Zielinska, Eva A.; Hebisch, Elke; Dura, Miroslav; Didié, Michael; Nikolaev, Viacheslav O.; Hasenfuss, Gerd; Kohl, Peter; Ward, Christopher W.; Lehnart, Stephan E.

    2016-01-01

    The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface. Rapid Ca2+ release correlated with colocalization of highly phosphorylated RyR2 clusters at AT-SR junctions and earlier, more rapid shortening of central sarcomeres. In contrast, mice expressing phosphorylation-incompetent RyR2 displayed depressed AM sarcomere shortening and reduced in vivo atrial contractile function. Moreover, left atrial hypertrophy led to AT proliferation, with a marked increase in the highly phosphorylated RyR2-pS2808 cluster fraction, thereby maintaining cytosolic Ca2+ signaling despite decreases in RyR2 cluster density and RyR2 protein expression. AT couplon “super-hubs” thus underlie faster excitation-contraction coupling in health as well as hypertrophic compensatory adaptation and represent a structural and metabolic mechanism that may contribute to contractile dysfunction and arrhythmias. PMID:27643434

  20. Cell adhesion and intracellular calcium signaling in neurons

    PubMed Central

    2013-01-01

    Cell adhesion molecules (CAMs) play indispensable roles in the developing and mature brain by regulating neuronal migration and differentiation, neurite outgrowth, axonal fasciculation, synapse formation and synaptic plasticity. CAM-mediated changes in neuronal behavior depend on a number of intracellular signaling cascades including changes in various second messengers, among which CAM-dependent changes in intracellular Ca2+ levels play a prominent role. Ca2+ is an essential secondary intracellular signaling molecule that regulates fundamental cellular functions in various cell types, including neurons. We present a systematic review of the studies reporting changes in intracellular Ca2+ levels in response to activation of the immunoglobulin superfamily CAMs, cadherins and integrins in neurons. We also analyze current experimental evidence on the Ca2+ sources and channels involved in intracellular Ca2+ increases mediated by CAMs of these families, and systematically review the role of the voltage-dependent Ca2+ channels (VDCCs) in neurite outgrowth induced by activation of these CAMs. Molecular mechanisms linking CAMs to VDCCs and intracellular Ca2+ stores in neurons are discussed. PMID:24330678

  1. Vasopressin and interactive calcium, cyclic AMP and purinergic signaling in Polycystic Kidney Disease

    PubMed Central

    Chebib, Fouad T.; Sussman, Caroline R.; Wang, Xiaofang; Harris, Peter C.; Torres, Vicente E.

    2015-01-01

    Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common monogenic kidney disease and the fourth leading cause of end-stage renal disease, responsible for 5–10% of cases. The disease is characterized by relentless development and growth of cysts causing progressive kidney enlargement associated with hypertension, pain, reduced quality of life, and eventually kidney failure. It is caused by mutations to PKD1 or PKD2, encoding polycystin-1 and polycystin-2, respectively. Their function and the molecular mechanisms responsible for the development of polycystic kidney disease are not well understood. The objective of this review is to synthesize a large body of literature that examines how reduction of functional PC1 or PC2 at the primary cilia and/or the endoplasmic reticulum directly disrupts intracellular calcium signaling and indirectly disrupts calcium regulated cAMP and purinergic signaling. We propose a hypothetical model where dysregulated metabolism of cAMP and purinergic signaling increase the sensitivity of principal cells in collecting ducts and of tubular epithelial cells in the distal nephron to the constant tonic action of vasopressin. The resulting magnified response to vasopressin further enhances the disruption of calcium signaling initiated by mutations to PC1 or PC2 and activates downstream signaling pathways responsible for impaired tubulogenesis, cell proliferation, increased fluid secretion and interstitial inflammation. PMID:25870007

  2. Effect of sound on gap-junction-based intercellular signaling: Calcium waves under acoustic irradiation.

    PubMed

    Deymier, P A; Swinteck, N; Runge, K; Deymier-Black, A; Hoying, J B

    2015-01-01

    We present a previously unrecognized effect of sound waves on gap-junction-based intercellular signaling such as in biological tissues composed of endothelial cells. We suggest that sound irradiation may, through temporal and spatial modulation of cell-to-cell conductance, create intercellular calcium waves with unidirectional signal propagation associated with nonconventional topologies. Nonreciprocity in calcium wave propagation induced by sound wave irradiation is demonstrated in the case of a linear and a nonlinear reaction-diffusion model. This demonstration should be applicable to other types of gap-junction-based intercellular signals, and it is thought that it should be of help in interpreting a broad range of biological phenomena associated with the beneficial therapeutic effects of sound irradiation and possibly the harmful effects of sound waves on health.

  3. Effect of sound on gap-junction-based intercellular signaling: Calcium waves under acoustic irradiation

    NASA Astrophysics Data System (ADS)

    Deymier, P. A.; Swinteck, N.; Runge, K.; Deymier-Black, A.; Hoying, J. B.

    2015-11-01

    We present a previously unrecognized effect of sound waves on gap-junction-based intercellular signaling such as in biological tissues composed of endothelial cells. We suggest that sound irradiation may, through temporal and spatial modulation of cell-to-cell conductance, create intercellular calcium waves with unidirectional signal propagation associated with nonconventional topologies. Nonreciprocity in calcium wave propagation induced by sound wave irradiation is demonstrated in the case of a linear and a nonlinear reaction-diffusion model. This demonstration should be applicable to other types of gap-junction-based intercellular signals, and it is thought that it should be of help in interpreting a broad range of biological phenomena associated with the beneficial therapeutic effects of sound irradiation and possibly the harmful effects of sound waves on health.

  4. Label-Free Imaging of Dynamic and Transient Calcium Signaling in Single Cells.

    PubMed

    Lu, Jin; Li, Jinghong

    2015-11-09

    Cell signaling consists of diverse events that occur at various temporal and spatial scales, ranging from milliseconds to hours and from single biomolecules to cell populations. The pathway complexities require the development of new techniques that detect the overall signaling activities and are not limited to quantifying a single event. A plasmonic-based electrochemical impedance microscope (P-EIM) that can provide such data with excellent temporal and spatial resolution and does not require the addition of any labels for detection has now been developed. The highly dynamic and transient calcium signaling activities at the early stage of G-protein-coupled receptor (GPCR) stimulation were thus studied. It could be shown that a subpopulation of cells is more responsive towards agonist stimulation, and the heterogeneity of the local distributions and the transient activities of the ion channels during agonist-activated calcium flux in single HeLa cells were investigated.

  5. Role of Calcium in Signal Transduction of Commelina Guard Cells.

    PubMed Central

    Gilroy, S; Fricker, MD; Read, ND; Trewavas, AJ

    1991-01-01

    The role of cytosolic Ca2+ in signal transduction in stomatal guard cells of Commelina communis was investigated using fluorescence ratio imaging and photometry. By changing extracellular K+, extracellular Ca2+, or treatment with Br-A23187, substantive increases in cytosolic Ca2+ to over 1 micromolar accompanied stomatal closure. The increase in Ca2+ was highest in the cytoplasm around the vacuole and the nucleus. Similar increases were observed when the cells were pretreated with ethyleneglycol-bis-(o-aminoethyl)tetraacetic acid or the channel blocker La3+, together with the closing stimuli. This suggests that a second messenger system operates between the plasma membrane and Ca2+-sequestering organelle(s). The endogenous growth regulator abscisic acid elevated cytosolic Ca2+ levels in a minority of cells investigated, even though stomatal closure always occurred. Ca2+-dependent and Ca2+-independent transduction pathways linking abscisic acid perception to stomatal closure are thus indicated. PMID:12324599

  6. Plants, symbiosis and parasites: a calcium signalling connection.

    PubMed

    Harper, Jeffrey F; Harmon, Alice

    2005-07-01

    A unique family of protein kinases has evolved with regulatory domains containing sequences that are related to Ca(2+)-binding EF-hands. In this family, the archetypal Ca(2+)-dependent protein kinases (CDPKs) have been found in plants and some protists, including the malarial parasite, Plasmodium falciparum. Recent genetic evidence has revealed isoform-specific functions for a CDPK that is essential for Plasmodium berghei gametogenesis, and for a related chimeric Ca(2+) and calmodulin-dependent protein kinase (CCaMK) that is essential to the formation of symbiotic nitrogen-fixing nodules in plants. In Arabidopsis thaliana, the analysis of 42 isoforms of CDPK and related kinases is expected to delineate Ca(2+) signalling pathways in all aspects of plant biology.

  7. Signal sequence within FcγRIIA controls calcium wave propagation patterns: Apparent role in phagolysosome fusion

    PubMed Central

    Worth, Randall G.; Kim, Moo-Kyung; Kindzelskii, Andrei L.; Petty, Howard R.; Schreiber, Alan D.

    2003-01-01

    Calcium oscillations and traveling calcium waves have been observed in living cells, although amino acid sequences regulating wave directionality and downstream cell functions have not been reported. In this study we identify an amino acid sequence within the cytoplasmic domain of the leukocyte IgG receptor FcγRIIA that affects the amplitude of calcium spikes and the spatiotemporal dynamics of calcium waves in the vicinity of phagosomes. By using high-speed microscopy to map calcium-signaling routes within cells, we have discovered that bound IgG-coated targets trigger two calcium waves traveling in opposite directions about the perimeter of cells expressing FcγRIIA. After phagocytosis, one calcium wave propagates around the plasma membrane to the site of phagocytosis where it splits into two calcium signals: one traveling to and encircling the phagosome once, and the second continuing around the plasma membrane to the point of origin. However, in a genetically engineered form of FcγRIIA containing a mutation in the cytoplasmic L-T-L motif, the calcium signal travels around the plasma membrane, but is not properly routed to the phagosome. Furthermore, these calcium pattern-deficient mutants were unable to support phagolysosome fusion, although recruitment of phagolysosome-associated proteins lysosome-associated protein 1, Rab5, and Rab7 were normal. Our findings suggest that: (i) calcium signaling is a late step in phagolysosome fusion, (ii) a line of communication exists between the plasma membrane and phagosome, and (iii) the L-T-L motif is a signal sequence for calcium signal routing to the phagosome. PMID:12676989

  8. 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.

  9. 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

  10. Effect of TGFβ on calcium signaling in megakaryocytes

    SciTech Connect

    Yan, Jing; Schmid, Evi; Almilaji, Ahmad; Shumilina, Ekaterina; Borst, Oliver; Laufer, Stefan; Gawaz, Meinrad; Lang, Florian

    2015-05-22

    TGFβ is a powerful regulator of megakaryocyte maturation and platelet formation. As previously shown for other cell types, TGFβ may up-regulate the expression of the serum & glucocorticoid inducible kinase SGK1, an effect requiring p38 kinase. SGK1 has in turn recently been shown to participate in the regulation of cytosolic Ca{sup 2+} activity ([Ca{sup 2+}]{sub i}) in megakaryocytes and platelets. SGK1 phosphorylates the IκB kinase (IKKα/β), which in turn phosphorylates the inhibitor protein IκBα resulting in nuclear translocation of nuclear factor NFκB. Genes up-regulated by NFκB include Orai1, the pore forming ion channel subunit accomplishing store operated Ca{sup 2+} entry (SOCE). The present study explored whether TGFβ influences Ca{sup 2+} signaling in megakaryocytes. [Ca{sup 2+}]{sub i} was determined by Fura-2 fluorescence and SOCE from the increase of [Ca{sup 2+}]{sub i} following re-addition of extracellular Ca{sup 2+} after store depletion by removal of extracellular Ca{sup 2+} and inhibition of the sarcoendoplasmatic Ca{sup 2+} ATPase (SERCA) with thapsigargin (1 μM). As a result, TGFβ (60 ng, 24 h) increased SOCE, an effect significantly blunted by p38 kinase inhibitor Skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) and NFκB inhibitor wogonin (100 μM). In conclusion, TGFβ is a powerful regulator of store operated Ca{sup 2+} entry into megakaryocytes, an effect mediated by a signaling cascade involving p38 kinase, SGK1 and NFκB. - Highlights: • TGFβ up-regulates store operated Ca{sup 2+} entry (SOCE) in megakaryocytes. • The effect of TGFβ on SOCE is blunted by p38 kinase inhibitor Skepinone-L. • The effect of TGFβ on SOCE is virtually abrogated by SGK1 inhibitor EMD638683. • The effect of TGFβ on SOCE is almost abolished by NFκB inhibitor wogonin. • The effect of TGFβ is expected to enhance sensitivity of platelets to activation.

  11. Progesterone Inhibition of Neuronal Calcium Signaling Underlies Aspects of Progesterone-Mediated Neuroprotection

    PubMed Central

    Luoma, Jessie I; Stern, Christopher M; Mermelstein, Paul G.

    2011-01-01

    Progesterone is being utilized as a therapeutic means to ameliorate neuron loss and cognitive dysfunction following traumatic brain injury Although there have been numerous attempts to determine the means by which progesterone exerts neuroprotective effects, studies describing the underlying molecular mechanisms are lacking What has become clear, however, is the notion that progesterone can thwart several physiological processes that are detrimental to neuron function and survival, including inflammation, edema, demyelination and excitotoxicity One clue regarding the means by which progesterone has restorative value comes from the notion that these aforementioned biological processes all share the common theme of eliciting pronounced increases in intracellular calcium. Thus, we propose the hypothesis that progesterone regulation of calcium signaling underlies its ability to mitigate these cellular insults, ultimately leading to neuroprotection. Further, we describe recent findings that indicate neuroprotection is achieved via progesterone block of voltage-gated calcium channels, although additional outcomes may arise from blockade of various other ion channels and neurotransmitter receptors. PMID:22101209

  12. Microscopic spiral waves reveal positive feedback in subcellular calcium signaling.

    PubMed Central

    Lipp, P; Niggli, E

    1993-01-01

    The regenerative Ca(2+)-induced Ca2+ release mechanism is an important amplifier of signal transduction in diverse cells. In heart muscle cells, this mechanism contributes to the Ca2+ transient activating the mechanical contraction, but it is also believed to drive Ca2+ waves propagating within the cytosol. We investigated the subcellular Ca2+ distribution in heart muscle cells during spontaneous Ca2+ release using laser scanning confocal microscopy with a ratiometric fluorescent indicator technique. Besides planar Ca2+ waves with linear propagation, sequences of confocal optical sections also revealed spiral Ca2+ waves spinning around a subcellular core at approximately 1 Hz. Although the Ca2+ spirals were continuous processes they frequently exhibited an apparently oscillatory output function into the elongated cell body. These oscillatory waves emanating from the spiral at regular intervals were formally considered to be short outer segments of the spiral but could not be distinguished from planar Ca2+ waves propagating along the longitudinal cell axis. The complex spatiotemporal pattern of spiral Ca2+ waves implies the participation of an active process exhibiting a large degree of positive feedback, most likely the Ca(2+)-induced Ca2+ release mechanism. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:8312468

  13. D1-D2 Dopamine Receptor Synergy Promotes Calcium Signaling via Multiple Mechanisms

    PubMed Central

    Chun, Lani S.; Free, R. Benjamin; Doyle, Trevor B.; Huang, Xi-Ping; Rankin, Michele L.

    2013-01-01

    The D1 dopamine receptor (D1R) has been proposed to form a hetero-oligomer with the D2 dopamine receptor (D2R), which in turn results in a complex that couples to phospholipase C–mediated intracellular calcium release. We have sought to elucidate the pharmacology and mechanism of action of this putative signaling pathway. Dopamine dose-response curves assaying intracellular calcium mobilization in cells heterologously expressing the D1 and D2 subtypes, either alone or in combination, and using subtype selective ligands revealed that concurrent stimulation is required for coupling. Surprisingly, characterization of a putative D1-D2 heteromer-selective ligand, 6-chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3-benzazepine-7,8-diol (SKF83959), found no stimulation of calcium release, but it did find a broad range of cross-reactivity with other G protein–coupled receptors. In contrast, SKF83959 appeared to be an antagonist of calcium mobilization. Overexpression of Gqα with the D1 and D2 dopamine receptors enhanced the dopamine-stimulated calcium response. However, this was also observed in cells expressing Gqα with only the D1R. Inactivation of Gi or Gs with pertussis or cholera toxin, respectively, largely, but not entirely, reduced the calcium response in D1R and D2R cotransfected cells. Moreover, sequestration of Gβγ subunits through overexpression of G protein receptor kinase 2 mutants either completely or largely eliminated dopamine-stimulated calcium mobilization. Our data suggest that the mechanism of D1R/D2R–mediated calcium signaling involves more than receptor-mediated Gq protein activation, may largely involve downstream signaling pathways, and may not be completely heteromer-specific. In addition, SKF83959 may not exhibit selective activation of D1-D2 heteromers, and its significant cross-reactivity to other receptors warrants careful interpretation of its use in vivo. PMID:23680635

  14. Structural basis of Sorcin-mediated calcium-dependent signal transduction

    PubMed Central

    Ilari, Andrea; Fiorillo, Annarita; Poser, Elena; Lalioti, Vasiliki S.; Sundell, Gustav N.; Ivarsson, Ylva; Genovese, Ilaria; Colotti, Gianni

    2015-01-01

    Sorcin is an essential penta-EF hand calcium binding protein, able to confer the multi-drug resistance phenotype to drug-sensitive cancer cells and to reduce Endoplasmic Reticulum stress and cell death. Sorcin silencing blocks cell cycle progression in mitosis and induces cell death by triggering apoptosis. Sorcin participates in the modulation of calcium homeostasis and in calcium-dependent cell signalling in normal and cancer cells. The molecular basis of Sorcin action is yet unknown. The X-ray structures of Sorcin in the apo (apoSor) and in calcium bound form (CaSor) reveal the structural basis of Sorcin action: calcium binding to the EF1-3 hands promotes a large conformational change, involving a movement of the long D-helix joining the EF1-EF2 sub-domain to EF3 and the opening of EF1. This movement promotes the exposure of a hydrophobic pocket, which can accommodate in CaSor the portion of its N-terminal domain displaying the consensus binding motif identified by phage display experiments. This domain inhibits the interaction of sorcin with PDCD6, a protein that carries the Sorcin consensus motif, co-localizes with Sorcin in the perinuclear region of the cell and in the midbody and is involved in the onset of apoptosis. PMID:26577048

  15. Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death.

    PubMed

    Duchen, M R

    1999-04-01

    Over recent years, it has become clear that mitochondria play a central role in many key aspects of animal physiology and pathophysiology. Their central and ubiquitous task is clearly the production of ATP. Nevertheless, they also play subtle roles in glucose homeostasis, acting as the sensor for substrate supply in the transduction pathway that promotes insulin secretion by the pancreatic -cell and that modulates the excitability of the hypothalamic glucose-sensitive neurons involved in appetite control. Mitochondria may also act as sensors of availability of oxygen, the other major mitochondrial substrate, in the regulation of respiration. Mitochondria take up calcium, and the high opacity mitochondrial calcium uptake pathway provides a mechanism that couples energy demand to increased ATP production through the calcium-dependent upregulation of mitochondrial enzyme activity. Mitochondrial calcium accumulation may also have a substantial impact on the spatiotemporal dynamics of cellular calcium signals, with subtle differences of detail in different cell types. Recent work has also revealed the centrality of mitochondrial dysfunction as an irreversible step in the pathway to both necrotic and apoptotic cell death. This review looks at recent developments in these rapidly evolving areas of cell physiology in an attempt to draw together disparate areas of research into a common theme.

  16. Structural basis of Sorcin-mediated calcium-dependent signal transduction.

    PubMed

    Ilari, Andrea; Fiorillo, Annarita; Poser, Elena; Lalioti, Vasiliki S; Sundell, Gustav N; Ivarsson, Ylva; Genovese, Ilaria; Colotti, Gianni

    2015-11-18

    Sorcin is an essential penta-EF hand calcium binding protein, able to confer the multi-drug resistance phenotype to drug-sensitive cancer cells and to reduce Endoplasmic Reticulum stress and cell death. Sorcin silencing blocks cell cycle progression in mitosis and induces cell death by triggering apoptosis. Sorcin participates in the modulation of calcium homeostasis and in calcium-dependent cell signalling in normal and cancer cells. The molecular basis of Sorcin action is yet unknown. The X-ray structures of Sorcin in the apo (apoSor) and in calcium bound form (CaSor) reveal the structural basis of Sorcin action: calcium binding to the EF1-3 hands promotes a large conformational change, involving a movement of the long D-helix joining the EF1-EF2 sub-domain to EF3 and the opening of EF1. This movement promotes the exposure of a hydrophobic pocket, which can accommodate in CaSor the portion of its N-terminal domain displaying the consensus binding motif identified by phage display experiments. This domain inhibits the interaction of sorcin with PDCD6, a protein that carries the Sorcin consensus motif, co-localizes with Sorcin in the perinuclear region of the cell and in the midbody and is involved in the onset of apoptosis.

  17. Calcium.

    PubMed

    Williams, Robert J P

    2002-01-01

    This chapter describes the chemical and biological value of the calcium ion. In calcium chemistry, our main interest is in equilibria within static, nonflowing systems. Hence, we examined the way calcium formed precipitates and complex ions in solution. We observed thereafter its uses by humankind in a vast number of materials such as minerals, e.g., marble, concrete, mortars, which parallel the biological use in shells and bones. In complex formation, we noted that many combinations were of anion interaction with calcium for example in the uses of detergents and medicines. The rates of exchange of calcium from bound states were noted but they had little application. Calcium ions do not act as catalysts of organic reactions. In biological systems, interest is in the above chemistry, but extends to the fact that Ca2+ ions can carry information by flowing in one solution or from one solution to another through membranes. Hence, we became interested in the details of rates of calcium exchange. The fast exchange of this divalent ion from most organic binding sites has allowed it to develop as the dominant second messenger. Now the flow can be examined in vitro as calcium binds particular isolated proteins, which it activates as seen in physical mechanical changes or chemical changes and this piece-by-piece study of cells is common. Here, however, we have chosen to stress the whole circuit of Ca2+ action indicating that the cell is organized both at a basal and an activated state kinetic level by the steady state flow of the ion (see Fig. 11). Different time constants of exchange utilizing very similar binding constants lead to: 1) fast responses as in the muscle of an animal; or 2) slower change as in differentiation of an egg or seed. Many other changes of state may relate to Ca2+ steady-state levels of flow in the circuitry and here we point to two: 1) dormancy in reptiles and animals; and 2) sporulation in both bacteria and lower plants. In the other chapters of

  18. ATP releasing connexin 30 hemichannels mediate flow-induced calcium signaling in the collecting duct.

    PubMed

    Svenningsen, Per; Burford, James L; Peti-Peterdi, János

    2013-01-01

    ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC, and AQP2. Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30(-/-) mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca(2+)]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30(-/-) mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca(2+)]i in wild type CCDs. This response was blunted in Cx30(-/-) CCDs ([Ca(2+)]i increased only 1.2-fold, p < 0.0001 vs. WT, n = 6 each). To further test our hypothesis we performed CD [Ca(2+)]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca(2+)]i oscillations in free-flowing CDs of wild type but not Cx30(-/-) mice. The [Ca(2+)]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption.

  19. ATP Releasing Connexin 30 Hemichannels Mediate Flow-Induced Calcium Signaling in the Collecting Duct

    PubMed Central

    Svenningsen, Per; Burford, James L.; Peti-Peterdi, János

    2013-01-01

    ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC, and AQP2. Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30−/− mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca2+]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30−/− mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca2+]i in wild type CCDs. This response was blunted in Cx30−/− CCDs ([Ca2+]i increased only 1.2-fold, p < 0.0001 vs. WT, n = 6 each). To further test our hypothesis we performed CD [Ca2+]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca2+]i oscillations in free-flowing CDs of wild type but not Cx30−/− mice. The [Ca2+]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption. PMID:24137132

  20. The calcium signaling toolkit of the Apicomplexan parasites Toxoplasma gondii and Plasmodium spp.

    PubMed

    Lourido, Sebastian; Moreno, Silvia N J

    2015-03-01

    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 (Ca(2+)) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-ATPases are present in Apicomplexans and a putative mitochondrial Ca(2+)/H(+) exchanger has been identified. Apicomplexan genomes contain numerous genes encoding Ca(2+)-binding proteins, with the notable expansion of calcium-dependent protein kinases (CDPKs), whose study has revealed 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 Ca(2+) 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 Ca(2+) in these parasites.

  1. Distinct regulation of dopamine D2S and D2L autoreceptor signaling by calcium

    PubMed Central

    Gantz, Stephanie C; Robinson, Brooks G; Buck, David C; Bunzow, James R; Neve, Rachael L; Williams, John T; Neve, Kim A

    2015-01-01

    D2 autoreceptors regulate dopamine release throughout the brain. Two isoforms of the D2 receptor, D2S and D2L, are expressed in midbrain dopamine neurons. Differential roles of these isoforms as autoreceptors are poorly understood. By virally expressing the isoforms in dopamine neurons of D2 receptor knockout mice, this study assessed the calcium-dependence and drug-induced plasticity of D2S and D2L receptor-dependent G protein-coupled inwardly rectifying potassium (GIRK) currents. The results reveal that D2S, but not D2L receptors, exhibited calcium-dependent desensitization similar to that exhibited by endogenous autoreceptors. Two pathways of calcium signaling that regulated D2 autoreceptor-dependent GIRK signaling were identified, which distinctly affected desensitization and the magnitude of D2S and D2L receptor-dependent GIRK currents. Previous in vivo cocaine exposure removed calcium-dependent D2 autoreceptor desensitization in wild type, but not D2S-only mice. Thus, expression of D2S as the exclusive autoreceptor was insufficient for cocaine-induced plasticity, implying a functional role for the co-expression of D2S and D2L autoreceptors. DOI: http://dx.doi.org/10.7554/eLife.09358.001 PMID:26308580

  2. Intracellular calcium during signal transduction in the lymphocyte is altered by ELF magnetic and electric fields

    SciTech Connect

    Liburdy, R.P. )

    1992-02-26

    Research has shown that ELF magnetic and electric fields alter calcium transport in rat thymic T-lymphocytes during signal transduction initiated by mitogen. Interestingly activated T-lymphocytes display a nonlinear dose-response for this basic field interaction which scales with the induced electric field in contrast to the applied magnetic field. Specialized multiring annular well cell culture plates based on Faraday's Law of Current Induction were used to demonstrate that the electric field associated with the magnetic field is the exposure metric of biological interest. The first real-time measurements of (Ca{sup 2+}){sub i} were recently presented and (Ca{sup 2+}){sub i} was shown to be altered by sinusoidal 60 Hz electric fields; magnetic fields that induced comparable electric fields yielded similar alterations in (Ca{sup 2+}){sub i}. The author now presents evidence that both parameters, (Ca{sup 2+}){sub i} and calcium transport, are altered by ELF fields during calcium signaling in thymocytes and scale with the induced electric field. In addition, (Ca{sup 2+}){sub i} studies have been conducted that provide evidence supporting the hypothesis that the mitogen-gated calcium channel present in the plasma cell membrane represents a specific site of interaction for ELF fields.

  3. Ca2+ signalling in cardiovascular disease: the role of the plasma membrane calcium pumps.

    PubMed

    Cartwright, Elizabeth J; Oceandy, Delvac; Austin, Clare; Neyses, Ludwig

    2011-08-01

    The plasma membrane calcium ATPases (PMCA) are a family of genes which extrude Ca(2+) from the cell and are involved in the maintenance of intracellular free calcium levels and/or with Ca(2+) signalling, depending on the cell type. In the cardiovascular system, Ca(2+) is not only essential for contraction and relaxation but also has a vital role as a second messenger in signal transduction pathways. A complex array of mechanisms regulate intracellular free calcium levels in the heart and vasculature and a failure in these systems to maintain normal Ca(2+) homeostasis has been linked to both heart failure and hypertension. This article focuses on the functions of PMCA, in particular isoform 4 (PMCA4), in the heart and vasculature and the reported links between PMCAs and contractile function, cardiac hypertrophy, cardiac rhythm and sudden cardiac death, and blood pressure control and hypertension. It is becoming clear that this family of calcium extrusion pumps have essential roles in both cardiovascular health and disease.

  4. Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells.

    PubMed

    Chubinskiy-Nadezhdin, Vladislav I; Vasileva, Valeria Y; Pugovkina, Natalia A; Vassilieva, Irina O; Morachevskaya, Elena A; Nikolsky, Nikolay N; Negulyaev, Yuri A

    2017-01-22

    Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs). Functional expression of native mechanosensitive stretch-activated channels (SACs) and calcium-sensitive potassium channels of different conductances in hMESCs was shown. Single current analysis of stretch-induced channel activity revealed functional coupling of SACs and BK channels in plasma membrane. The combination of cell-attached and inside-out experiments have indicated that highly localized Ca(2+) entry via SACs triggers BK channel activity. At the same time, SK channels are not coupled with SACs despite of high calcium sensitivity as compared to BK. Our data demonstrate novel mechanism controlling BK channel activity in native cells. We conclude that SACs and BK channels are clusterized in functional mechanosensitive domains in the plasma membrane of hMESCs. Co-clustering of ion channels may significantly contribute to mechano-dependent calcium signalling in stem cells.

  5. Calcium signals drive cell shape changes during zebrafish midbrain-hindbrain boundary formation.

    PubMed

    Sahu, Srishti U; Visetsouk, Mike R; Garde, Ryan J; Hennes, Leah; Kwas, Constance; Gutzman, Jennifer H

    2017-02-01

    One of the first morphogenetic events in the vertebrate brain is the formation of the highly conserved midbrain-hindbrain boundary (MHB). Specific cell shape changes occur at the point of deepest constriction of the MHB, the midbrain-hindbrain boundary constriction (MHBC), and are critical for proper MHB formation. These cell shape changes are controlled by non-muscle myosin II (NMII) motor proteins which are tightly regulated via the phosphorylation of their associated myosin regulatory light chains (MRLC). However, the upstream signaling pathways that initiate the regulation of NMII to mediate cell shape changes during MHB morphogenesis are not known. We show that intracellular calcium signals are critical for the regulation of cell shortening during initial MHB formation. We demonstrate that the MHB region is poised to respond to calcium transients that occur in the MHB at the onset of MHB morphogenesis and that calcium mediates phosphorylation of MRLC specifically in MHB tissue. Our results indicate that calmodulin 1a (calm1a), expressed specifically in the MHB, and myosin light chain kinase (MLCK), together mediate MHBC cell length. Our data suggest that modulation of NMII activity by calcium is critical for proper regulation of cell length to determine embryonic brain shape during development.

  6. Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein.

    PubMed

    Apáti, Ágota; Pászty, Katalin; Hegedűs, Luca; Kolacsek, Orsolya; Orbán, Tamás I; Erdei, Zsuzsa; Szebényi, Kornélia; Péntek, Adrienn; Enyedi, Ágnes; Sarkadi, Balázs

    2013-04-01

    Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca(2+) indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca(2+) oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca(2+) signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging.

  7. Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem.

    PubMed

    Gazarini, Marcos L; Thomas, Andrew P; Pozzan, Tullio; Garcia, Célia R S

    2003-04-14

    Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

  8. Calcium signaling in mast cells: focusing on L-type calcium channels.

    PubMed

    Suzuki, Yoshihiro; Inoue, Toshio; Ra, Chisei

    2012-01-01

    Mast cells play central roles in adaptive and innate immunity. IgE-dependent stimulation of the high-affinity IgE receptor (FcεRI) results in rapid secretion of various proinflammatory chemical mediators and cytokines. All of the outputs depend to certain degrees on an increase in the intracellular Ca(2+) concentration, and influx of Ca(2+) from the extracellular space is often required for their full activation. There is strong evidence that FcεRI stimulation induces two different modes of Ca(2+) influx, store-operated Ca(2+) entry (SOCE) and non-SOCE, which are activated in response to endoplasmic reticulum Ca(2+) store depletion and independently of Ca(2+) store depletion, respectively, in mast cells. Although Ca(2+) release-activated Ca(2+) channels are the major route of SOCE, recent evidence indicates that they are not the only Ca(2+) channels activated by Ca(2+) store depletion. The recent data suggest that L-type Ca(2+) channels, which were thought to be a characteristic feature of excitable cells, exist in mast cells to mediate non-SOCE, which is critical for protecting mast cells against activation-induced mitochondrial cell death. In this chapter, we provide an overview of recent advances in our understanding of Ca(2+) signaling in mast cells with a special attention to the emerging role for the L-type Ca(2+) channels as a regulator of mast cell survival.

  9. Hormonal signaling and signal pathway crosstalk in the control of myometrial calcium dynamics

    PubMed Central

    Sanborn, Barbara M.

    2007-01-01

    Understanding the basis for the control of myometrial contractant and relaxant signaling pathways is important to understanding how to manage myometrial contractions. Signaling pathways are influenced by the level of expression of the signals and signal pathway components, the location of these components in the appropriate subcellular environment, and covalent modification. Crosstalk between these pathways regulates the effectiveness of signal transduction and represents an important way by which hormones can regulate phenotype. This review deals primarily with signaling pathways that control Ca2+ entry and intracellular release, as well as the interplay between these pathways. PMID:17627855

  10. Mutual independence of alkaline- and calcium-mediated signalling in Aspergillus fumigatus refutes the existence of a conserved druggable signalling nexus.

    PubMed

    Loss, Omar; Bertuzzi, Margherita; Yan, Yu; Fedorova, Natalie; McCann, Bethany L; Armstrong-James, Darius; Espeso, Eduardo A; Read, Nick D; Nierman, William C; Bignell, Elaine M

    2017-09-18

    Functional coupling of calcium- and alkaline responsive signalling occurs in multiple fungi to afford efficient cation homeostasis. Host microenvironments exert alkaline stress and potentially toxic concentrations of Ca(2+) , such that highly conserved regulators of both calcium- (Crz) and pH- (PacC/Rim) responsive signalling are crucial for fungal pathogenicity. Drugs targeting calcineurin are potent antifungal agents but also perturb human immunity thereby negating their use as anti-infectives, abrogation of alkaline signalling has therefore been postulated as an adjunctive antifungal strategy. We examined the interdependency of pH- and calcium-mediated signalling in Aspergillus fumigatus and found that calcium chelation severely impedes hyphal growth indicating a critical requirement for this ion independently of ambient pH. Transcriptomic responses to alkaline pH or calcium excess exhibited minimal similarity. Mutants lacking calcineurin, or its client CrzA, displayed normal alkaline tolerance, and nuclear translocation of CrzA was unaffected by ambient pH. Expression of a highly conserved, alkaline-regulated, sodium ATPase was tolerant of genetic or chemical perturbations of calcium-mediated signalling, but abolished in null mutants of the pH-responsive transcription factor PacC, and PacC proteolytic processing occurred normally during calcium excess. Taken together our data demonstrate that in A. fumigatus the regulatory hierarchy governing alkaline tolerance circumvents calcineurin signalling. This article is protected by copyright. All rights reserved. © 2017 John Wiley & Sons Ltd.

  11. Spontaneous calcium signals induced by gap junctions in a network model of astrocytes

    NASA Astrophysics Data System (ADS)

    Kazantsev, V. B.

    2009-01-01

    The dynamics of a network model of astrocytes coupled by gap junctions is investigated. Calcium dynamics of the single cell is described by the biophysical model comprising the set of three nonlinear differential equations. Intercellular dynamics is provided by the diffusion of inositol 1,4,5-trisphosphate (IP3) through gap junctions between neighboring astrocytes. It is found that the diffusion induces the appearance of spontaneous activity patterns in the network. Stability of the network steady state is analyzed. It is proved that the increase of the diffusion coefficient above a certain critical value yields the generation of low-amplitude subthreshold oscillatory signals in a certain frequency range. It is shown that such spontaneous oscillations can facilitate calcium pulse generation and provide a certain time scale in astrocyte signaling.

  12. NG2 glial cells integrate synaptic input in global and dendritic calcium signals

    PubMed Central

    Sun, Wenjing; Matthews, Elizabeth A; Nicolas, Vicky; Schoch, Susanne; Dietrich, Dirk

    2016-01-01

    Synaptic signaling to NG2-expressing oligodendrocyte precursor cells (NG2 cells) could be key to rendering myelination of axons dependent on neuronal activity, but it has remained unclear whether NG2 glial cells integrate and respond to synaptic input. Here we show that NG2 cells perform linear integration of glutamatergic synaptic inputs and respond with increasing dendritic calcium elevations. Synaptic activity induces rapid Ca2+ signals mediated by low-voltage activated Ca2+ channels under strict inhibitory control of voltage-gated A-type K+ channels. Ca2+ signals can be global and originate throughout the cell. However, voltage-gated channels are also found in thin dendrites which act as compartmentalized processing units and generate local calcium transients. Taken together, the activity-dependent control of Ca2+ signals by A-type channels and the global versus local signaling domains make intracellular Ca2+ in NG2 cells a prime signaling molecule to transform neurotransmitter release into activity-dependent myelination. DOI: http://dx.doi.org/10.7554/eLife.16262.001 PMID:27644104

  13. 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

  14. NG2 glial cells integrate synaptic input in global and dendritic calcium signals.

    PubMed

    Sun, Wenjing; Matthews, Elizabeth A; Nicolas, Vicky; Schoch, Susanne; Dietrich, Dirk

    2016-09-19

    Synaptic signaling to NG2-expressing oligodendrocyte precursor cells (NG2 cells) could be key to rendering myelination of axons dependent on neuronal activity, but it has remained unclear whether NG2 glial cells integrate and respond to synaptic input. Here we show that NG2 cells perform linear integration of glutamatergic synaptic inputs and respond with increasing dendritic calcium elevations. Synaptic activity induces rapid Ca(2+) signals mediated by low-voltage activated Ca(2+) channels under strict inhibitory control of voltage-gated A-type K(+) channels. Ca(2+) signals can be global and originate throughout the cell. However, voltage-gated channels are also found in thin dendrites which act as compartmentalized processing units and generate local calcium transients. Taken together, the activity-dependent control of Ca(2+) signals by A-type channels and the global versus local signaling domains make intracellular Ca(2+) in NG2 cells a prime signaling molecule to transform neurotransmitter release into activity-dependent myelination.

  15. 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

  16. Astrocytic Calcium Waves Signal Brain Injury to Neural Stem and Progenitor Cells.

    PubMed

    Kraft, Anna; Jubal, Eduardo Rosales; von Laer, Ruth; Döring, Claudia; Rocha, Adriana; Grebbin, Moyo; Zenke, Martin; Kettenmann, Helmut; Stroh, Albrecht; Momma, Stefan

    2017-03-14

    Brain injuries, such as stroke or trauma, induce neural stem cells in the subventricular zone (SVZ) to a neurogenic response. Very little is known about the molecular cues that signal tissue damage, even over large distances, to the SVZ. Based on our analysis of gene expression patterns in the SVZ, 48 hr after an ischemic lesion caused by middle cerebral artery occlusion, we hypothesized that the presence of an injury might be transmitted by an astrocytic traveling calcium wave rather than by diffusible factors or hypoxia. Using a newly established in vitro system we show that calcium waves induced in an astrocytic monolayer spread to neural stem and progenitor cells and increase their self-renewal as well as migratory behavior. These changes are due to an upregulation of the Notch signaling pathway. This introduces the concept of propagating astrocytic calcium waves transmitting brain injury signals over long distances. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-12-01

    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.

  18. From contraction to gene expression: nanojunctions of the sarco/endoplasmic reticulum deliver site- and function-specific calcium signals.

    PubMed

    Evans, A Mark; Fameli, Nicola; Ogunbayo, Oluseye A; Duan, Jingxian; Navarro-Dorado, Jorge

    2016-08-01

    Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum" hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2b and RyR1 are preferentially targeted to the sarcoplasmic reticulum (SR) proximal to the plasma membrane (PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. RyR3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of RyR2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear RyR3s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via RyR2 in order to elicit contraction. Data also suggest that unique SERCA and RyR are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.

  19. Influence of zinc on calcium-dependent signal transduction pathways during aluminium-induced neurodegeneration.

    PubMed

    Singla, Neha; Dhawan, D K

    2014-10-01

    Metals perform important functions in the normal physiological system, and alterations in their levels may lead to a number of diseases. Aluminium (Al) has been implicated as a major risk factor, which is linked to several neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. On the other hand, zinc (Zn) is considered as a neuromodulator and an essential dietary element that regulates a number of biological activities in our body. The aim of the present study was to investigate the effects of Zn supplementation, if any, in ameliorating the changes induced by Al on calcium signalling pathway. Male Sprague Dawley rats weighing 140-160 g were divided into four different groups viz.: normal control, aluminium treated (100 mg/kg b.wt./day via oral gavage), zinc treated (227 mg/l in drinking water) and combined aluminium and zinc treated. All the treatments were carried out for a total duration of 8 weeks. Al treatment decreased the Ca(2+) ATPase activity whereas increased the levels of 3', 5'-cyclic adenosine monophosphate, intracellular calcium and total calcium content in both the cerebrum and cerebellum, which, however, were modulated upon Zn supplementation. Al treatment exhibited a significant elevation in the protein expressions of phospholipase C, inositol triphosphate and protein kinase A but decreased the expression of protein kinase C, which, however, was reversed upon Zn co-treatment. Al treatment also revealed alterations in neurohistoarchitecture in the form of calcium deposits, which were improved upon zinc co-administration. The present study, therefore, suggests that zinc regulates the intracellular calcium signalling pathway during aluminium-induced neurodegeneration.

  20. Molecular Basis of the Extracellular Ligands Mediated Signaling by the Calcium Sensing Receptor

    PubMed Central

    Zhang, Chen; Miller, Cassandra L.; Gorkhali, Rakshya; Zou, Juan; Huang, Kenneth; Brown, Edward M.; Yang, Jenny J.

    2016-01-01

    Ca2+-sensing receptors (CaSRs) play a central role in regulating extracellular calcium concentration ([Ca2+]o) homeostasis and many (patho)physiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids, and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT) domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR's cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs) in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics. PMID:27746744

  1. Calcineurin-NFATc signaling pathway regulates AQP2 expression in response to calcium signals and osmotic stress.

    PubMed

    Li, Song-Zhe; McDill, Bradley W; Kovach, Paul A; Ding, Li; Go, William Y; Ho, Steffan N; Chen, Feng

    2007-05-01

    The aquaporin (AQP)2 channel mediates the reabsorption of water in renal collecting ducts in response to arginine vasopressin (AVP) and hypertonicity. Here we show that AQP2 expression is induced not only by the tonicity-responsive enhancer binding protein (TonEBP)/nuclear factor of activated T cells (NFAT)5-mediated hypertonic stress response but also by the calcium-dependent calcineurin-NFATc pathway. The induction of AQP2 expression by the calcineurin-NFATc pathway can occur in the absence of TonEBP/NFAT5. Mutational and chromatin immunoprecipitation analyses revealed the existence of functional NFAT binding sites within the proximal AQP2 promoter responsible for regulation of AQP2 by NFATc proteins and TonEBP/NFAT5. Contrary to the notion that TonEBP/NFAT5 is the only Rel/NFAT family member regulated by tonicity, we found that hypertonicity promotes the nuclear translocation of NFATc proteins for the subsequent induction of AQP2 expression. Calcineurin activity was also found to be involved in the induction of TonEBP/NFAT5 expression by hypertonicity, thus further defining the signaling mechanisms that underlie the TonEBP/NFAT5 osmotic stress response pathway. The coordinate regulation of AQP2 expression by both osmotic stress and calcium signaling appears to provide a means to integrate diverse extracellular signals into optimal cellular responses.

  2. The fragile X mental retardation protein developmentally regulates the strength and fidelity of calcium signaling in Drosophila mushroom body neurons.

    PubMed

    Tessier, Charles R; Broadie, Kendal

    2011-01-01

    Fragile X syndrome (FXS) is a broad-spectrum neurological disorder characterized by hypersensitivity to sensory stimuli, hyperactivity and severe cognitive impairment. FXS is caused by loss of the fragile X mental retardation 1 (FMR1) gene, whose FMRP product regulates mRNA translation downstream of synaptic activity to modulate changes in synaptic architecture, function and plasticity. Null Drosophila FMR1 (dfmr1) mutants exhibit reduced learning and loss of protein synthesis-dependent memory consolidation, which is dependent on the brain mushroom body (MB) learning and memory center. We targeted a transgenic GFP-based calcium reporter to the MB in order to analyze calcium dynamics downstream of neuronal activation. In the dfmr1 null MB, there was significant augmentation of the calcium transients induced by membrane depolarization, as well as elevated release of calcium from intracellular organelle stores. The severity of these calcium signaling defects increased with developmental age, although early stages were characterized by highly variable, low fidelity calcium regulation. At the single neuron level, both calcium transient and calcium store release defects were exhibited by dfmr1 null MB neurons in primary culture. Null dfmr1 mutants exhibit reduced brain mRNA expression of calcium-binding proteins, including calcium buffers calmodulin and calbindin, predicting that the inability to appropriately sequester cytosolic calcium may be the common mechanistic defect causing calcium accumulation following both influx and store release. Changes in the magnitude and fidelity of calcium signals in the absence of dFMRP likely contribute to defects in neuronal structure/function, leading to the hallmark learning and memory dysfunction of FXS.

  3. Shear stress-induced NO production is dependent on ATP autocrine signaling and capacitative calcium entry

    PubMed Central

    Andrews, Allison M.; Jaron, Dov; Buerk, Donald G.; Barbee, Kenneth A.

    2014-01-01

    Flow-induced production of nitric oxide (NO) by endothelial cells plays a fundamental role in vascular homeostasis. However, the mechanisms by which shear stress activates NO production remain unclear due in part to limitations in measuring NO, especially under flow conditions. Shear stress elicits the release of ATP, but the relative contribution of autocrine stimulation by ATP to flow-induced NO production has not been established. Furthermore, the importance of calcium in shear stress-induced NO production remains controversial, and in particular the role of capacitive calcium entry (CCE) has yet to be determined. We have utilized our unique NO measurement device to investigate the role of ATP autocrine signaling and CCE in shear stress-induced NO production. We found that endogenously released ATP and downstream activation of purinergic receptors and CCE plays a significant role in shear stress-induced NO production. ATP-induced eNOS phophorylation under static conditions is also dependent on CCE. Inhibition of protein kinase C significantly inhibited eNOS phosphorylation and the calcium response. To our knowledge, we are the first to report on the role of CCE in the mechanism of acute shear stress-induced NO response. In addition, our work highlights the importance of ATP autocrine signaling in shear stress-induced NO production. PMID:25386222

  4. Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo

    PubMed Central

    Boerman, Erika M.; Everhart, Jesse E.

    2016-01-01

    Aging is associated with vascular dysfunction that impairs tissue perfusion, physical activity, and the quality of life. Calcium signaling in endothelial cells (ECs) is integral to vasomotor control, exemplified by localized Ca2+ signals within EC projections through holes in the internal elastic lamina (IEL). Within these microdomains, endothelium-derived hyperpolarization is integral to smooth muscle cell (SMC) relaxation via coupling through myoendothelial gap junctions. However, the effects of aging on local EC Ca2+ signals (and thereby signaling between ECs and SMCs) remain unclear, and these events have not been investigated in vivo. Furthermore, it is unknown whether aging affects either the number or the size of IEL holes. In the present study, we tested the hypothesis that local EC Ca2+ signaling is impaired with advanced age along with a reduction in IEL holes. In anesthetized mice expressing a Ca2+-sensitive fluorescent protein (GCaMP2) selectively in ECs, our findings illustrate that for mesenteric arteries controlling splanchnic blood flow the frequency of spontaneous local Ca2+ signals in ECs was reduced by ∼85% in old (24–26 mo) vs. young (3–6 mo) animals. At the same time, the number (and total area) of holes per square millimeter of IEL was reduced by ∼40%. We suggest that diminished signaling between ECs and SMCs contributes to dysfunction of resistance arteries with advanced age. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/aging-impairs-endothelial-ca2-signaling/. PMID:26945073

  5. An ATP-dependent mechanism mediates intercellular calcium signaling in bone cell network under single cell nanoindentation.

    PubMed

    Huo, Bo; Lu, Xin L; Costa, Kevin D; Xu, Qiaobing; Guo, X Edward

    2010-03-01

    To investigate the roles of intercellular gap junctions and extracellular ATP diffusion in bone cell calcium signaling propagation in bone tissue, in vitro bone cell networks were constructed by using microcontact printing and self-assembled monolayer technologies. In the network, neighboring cells were interconnected through functional gap junctions. A single cell at the center of the network was mechanically stimulated by using an AFM nanoindenter. Intracellular calcium ([Ca2+](i)) responses of the bone cell network were recorded and analyzed. In the untreated groups, calcium propagation from the stimulated cell to neighboring cells was observed in 40% of the tests. No significant difference was observed in this percentage when the intercellular gap junctions were blocked. This number, however, decreased to 10% in the extracellular ATP-pathway-blocked group. When both the gap junction and ATP pathways were blocked, intercellular calcium waves were abolished. When the intracellular calcium store in ER was depleted, the indented cell can generate calcium transients, but no [Ca2+](i) signal can be propagated to the neighboring cells. No [Ca2+](i) response was detected in the cell network when the extracellular calcium source was removed. These findings identified the biochemical pathways involved in the calcium signaling propagation in bone cell networks. Published by Elsevier India Pvt Ltd.

  6. An ATP-Dependent Mechanism Mediates Intercellular Calcium Signaling in Bone Cell Network under Single Cell Nanoindentation

    PubMed Central

    Huo, Bo; Lu, Xin L.; Costa, Kevin D.; Xu, Qiaobing; Guo, X. Edward

    2010-01-01

    Summary To investigate the roles of intercellular gap junctions and extracellular ATP diffusion in bone cell calcium signaling propagation in bone tissue, in vitro bone cell networks were constructed by using microcontact printing and self-assembled monolayer technologies. In the network, neighboring cells were interconnected through functional gap junctions. A single cell at the center of the network was mechanically stimulated by using an AFM nanoindenter. Intracellular calcium ([Ca2+]i) responses of the bone cell network were recorded and analyzed. In the untreated groups, calcium propagation from the stimulated cell to neighboring cells was observed in 40% of the tests. No significant difference was observed in this percentage when the intercellular gap junctions were blocked. This number, however, decreased to 10% in the extracellular ATP-pathway-blocked group. When both the gap junction and ATP pathways were blocked, intercellular calcium waves were abolished. When the intracellular calcium store in ER was depleted, the indented cell can generate calcium transients, but no [Ca2+]i signal can be propagated to the neighboring cells. No [Ca2+]i response was detected in the cell network when the extracellular calcium source was removed. These findings identified the biochemical pathways involved in the calcium signaling propagation in bone cell networks. PMID:20060586

  7. Sleep deprivation impairs calcium signaling in mouse splenocytes and leads to a decreased immune response.

    PubMed

    Lungato, Lisandro; Gazarini, Marcos L; Paredes-Gamero, Edgar J; Tersariol, Ivarne L S; Tufik, Sergio; D'Almeida, Vânia

    2012-12-01

    Sleep is a physiological event that directly influences health by affecting the immune system, in which calcium (Ca(2+)) plays a critical signaling role. We performed live cell measurements of cytosolic Ca(2+) mobilization to understand the changes in Ca(2+) signaling that occur in splenic immune cells after various periods of sleep deprivation (SD). Adult male mice were subjected to sleep deprivation by platform technique for different periods (from 12 to 72h) and Ca(2+) intracellular fluctuations were evaluated in splenocytes by confocal microscopy. We also performed spleen cell evaluation by flow cytometry and analyzed intracellular Ca(2+) mobilization in endoplasmic reticulum and mitochondria. Additionally, Ca(2+) channel gene expression was evaluated Splenocytes showed a progressive loss of intracellular Ca(2+) maintenance from endoplasmic reticulum (ER) stores. Transient Ca(2+) buffering by the mitochondria was further compromised. These findings were confirmed by changes in mitochondrial integrity and in the performance of the store operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) Ca(2+) channels. These novel data suggest that SD impairs Ca(2+) signaling, most likely as a result of ER stress, leading to an insufficient Ca(2+) supply for signaling events. Our results support the previously described immunosuppressive effects of sleep loss and provide additional information on the cellular and molecular mechanisms involved in sleep function. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. 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

  9. Approaches and tools for modeling signaling pathways and calcium dynamics in neurons

    PubMed Central

    Blackwell, KT

    2013-01-01

    Signaling pathways are cascades of intracellular biochemical reactions that are activated by transmembrane receptors, and ultimately lead to transcription in the nucleus. In neurons, both calcium permeable synaptic and ionic channels as well as G protein coupled receptors initiate activation of signaling pathway molecules that interact with electrical activity at multiple spatial and time scales. At small temporal and spatial scales, calcium modifies the properties of ionic channels, whereas at larger temporal and spatial scales, various kinases and phosphatases modify the properties of ionic channels, producing phenomena such as synaptic plasticity and homeostatic plasticity. The elongated structure of neuronal dendrites and the organization of multi-protein complexes by anchoring proteins implies that the spatial dimension must be explicit. Therefore, modeling signaling pathways in neurons utilizes algorithms for both diffusion and reactions. The small size of spines coupled with small concentrations of some molecules implies that some reactions occur stochastically. The need for stochastic simulation of many reaction and diffusion events coupled with the multiple temporal and spatial scales makes modeling of signaling pathways a difficult problem. Several different software programs have achieved different aspects of these capabilities. This review explains some of the mathematical formulas used for modeling reactions and diffusion. In addition, it briefly presents the simulators used for modeling reaction-diffusion systems in neurons, together with scientific problems addressed. PMID:23743449

  10. Headpiece Domain of Dematin Regulates Calcium Mobilization and Signaling in Platelets*

    PubMed Central

    Wieschhaus, Adam J.; Le Breton, Guy C.; Chishti, Athar H.

    2012-01-01

    Dematin is a broadly expressed membrane cytoskeletal protein that has been well characterized in erythrocytes and to a lesser extent in non-erythroid cells. However, dematin's function in platelets is not known. Here, we show that dematin is abundantly expressed in both human and mouse platelets. Platelets harvested from the dematin headpiece knock-out (HPKO) mouse model exhibit a striking defect in the mobilization of calcium in response to multiple agonists of platelet activation. The reduced calcium mobilization in HPKO platelets is associated with concomitant inhibition of platelet aggregation and granule secretion. Integrin αIIbβ3 activation in response to agonists is attenuated in the HPKO platelets. The mutant platelets show nearly normal spreading on fibrinogen and an unaltered basal cAMP level; however, the clot retraction was compromised in the mutant mice. Immunofluorescence analysis indicated that dematin is present both at the dense tubular system and plasma membrane fractions of platelets. Proteomic analysis of dematin-associated proteins in human platelets identified inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) as a binding partner, which was confirmed by immunoprecipitation analysis. IP3KB, a dense tubular system protein, is a major regulator of calcium homeostasis. Loss of the dematin headpiece resulted in a decrease of IP3KB at the membrane and increased levels of IP3KB in the cytosol. Collectively, these findings unveil dematin as a novel regulator of internal calcium mobilization in platelets affecting multiple signaling and cytoskeletal functions. Implications of a conserved role of dematin in the regulation of calcium homeostasis in other cell types will be discussed. PMID:23060452

  11. Signal processing by T-type calcium channel interactions in the cerebellum

    PubMed Central

    Engbers, Jordan D. T.; Anderson, Dustin; Zamponi, Gerald W.; Turner, Ray W.

    2013-01-01

    T-type calcium channels of the Cav3 family are unique among voltage-gated calcium channels due to their low activation voltage, rapid inactivation, and small single channel conductance. These special properties allow Cav3 calcium channels to regulate neuronal processing in the subthreshold voltage range. Here, we review two different subthreshold ion channel interactions involving Cav3 channels and explore the ability of these interactions to expand the functional roles of Cav3 channels. In cerebellar Purkinje cells, Cav3 and intermediate conductance calcium-activated potassium (IKCa) channels form a novel complex which creates a low voltage-activated, transient outward current capable of suppressing temporal summation of excitatory postsynaptic potentials (EPSPs). In large diameter neurons of the deep cerebellar nuclei, Cav3-mediated calcium current (IT) and hyperpolarization-activated cation current (IH) are activated during trains of inhibitory postsynaptic potentials. These currents have distinct, and yet synergistic, roles in the subthreshold domain with IT generating a rebound burst and IH controlling first spike latency and rebound spike precision. However, by shortening the membrane time constant the membrane returns towards resting value at a faster rate, allowing IH to increase the efficacy of IT and increase the range of burst frequencies that can be generated. The net effect of Cav3 channels thus depends on the channels with which they are paired. When expressed in a complex with a KCa channel, Cav3 channels reduce excitability when processing excitatory inputs. If functionally coupled with an HCN channel, the depolarizing effect of Cav3 channels is accentuated, allowing for efficient inversion of inhibitory inputs to generate a rebound burst output. Therefore, signal processing relies not only on the activity of individual subtypes of channels but also on complex interactions between ion channels whether based on a physical complex or by indirect

  12. Calcium supplementation during sepsis exacerbates organ failure and mortality via calcium/calmodulin-dependent protein kinase kinase (CaMKK) signaling

    PubMed Central

    Collage, Richard D.; Howell, Gina M.; Zhang, Xianghong; Stripay, Jennifer L.; Lee, Janet S.; Angus, Derek C.; Rosengart, Matthew R.

    2013-01-01

    . This altered calcium signaling, transduced by the CaMKK cascade, mediates heightened inflammation and vascular leak that culminates in elevated organ dysfunction and mortality. In the clinical management of septic patients calcium supplementation provides no benefit and may impose harm. PMID:23887235

  13. Voltage-gated calcium channels function as Ca2+-activated signaling receptors.

    PubMed

    Atlas, Daphne

    2014-02-01

    Voltage-gated calcium channels (VGCCs) are transmembrane cell surface proteins responsible for multifunctional signals. In response to voltage, VGCCs trigger synaptic transmission, drive muscle contraction, and regulate gene expression. Voltage perturbations open VGCCs enabling Ca(2+) binding to the low affinity Ca(2+) binding site of the channel pore. Subsequent to permeation, Ca(2+) targets selective proteins to activate diverse signaling pathways. It is becoming apparent that the Ca(2+)-bound channel triggers secretion in excitable cells and drives contraction in cardiomyocytes prior to Ca(2+) permeation. Here, I highlight recent data implicating receptor-like function of the Ca(2+)-bound channel in converting external Ca(2+) into an intracellular signal. The two sequential mechanistic perspectives of VGCC function are discussed in the context of the prevailing and long-standing current models of depolarization-evoked secretion and cardiac contraction. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Expression of intracellular calcium signalling genes in cattle skin during tick infestation.

    PubMed

    Bagnall, N; Gough, J; Cadogan, L; Burns, B; Kongsuwan, K

    2009-04-01

    It is widely acknowledged that changes in intracellular calcium ion (Ca(2+)) concentration provide dynamic signals that control a plethora of cellular processes, including triggering and mediating host defence mechanisms. In this study, quantitative real-time PCR was used to analyse gene expression of 14 Ca(2+) signalling proteins in skin obtained from high tick-resistant (HR) and low tick-resistant (LR) cattle following artificial challenge with cattle tick (Rhipicephalus (Boophilus) microplus). Up-regulation of numerous genes was observed in both HR and LR skin following tick challenge, however substantially higher transcription activation was found in HR tissue. The elevated expression in HR skin of specific Ca(2+) signalling genes such as AHNAK, CASQ, IL2, NFAT2CIP and PLCG1 may be related to host resistance. Our data suggest that Ca(2+) and its associated proteins might play an important role in host response to ticks and that further investigation is warranted.

  15. Calcium signaling in mammalian egg activation and embryo development: Influence of subcellular localization

    PubMed Central

    Miao, Yi-Liang; Williams, Carmen J.

    2012-01-01

    Calcium (Ca2+) signals drive the fundamental events surrounding fertilization and the activation of development in all species examined to date. Initial studies of Ca2+ signaling at fertilization in marine animals were tightly linked to new discoveries of bioluminescent proteins and their use as fluorescent Ca2+ sensors. Since that time, there has been rapid progress in our understanding of the key functions for Ca2+ in many cell types and the impact of cellular localization on Ca2+ signaling pathways. In this review, which focuses on mammalian egg activation, we consider how Ca2+ is regulated and stored at different stages of oocyte development and examine the functions of molecules that serve as both regulators of Ca2+ release and effectors of Ca2+ signals. We then summarize studies exploring how Ca2+ directs downstream effectors mediating both egg activation and later signaling events required for successful preimplantation embryo development. Throughout this review, we focus attention on how localization of Ca2+ signals influences downstream signaling events, and attempt to highlight gaps in our knowledge that are ripe areas for future research. PMID:22888043

  16. Contractility and calcium signaling of human myometrium are profoundly affected by cholesterol manipulation: implications for labor?

    PubMed

    Jie Zhang; Kendrick, Annabelle; Quenby, Siobhan; Wray, Susan

    2007-07-01

    The authors elucidate cholesterol's effect on human uterine contractility and calcium signaling to test the hypotheses that elevation of cholesterol decreases uterine activity and that oxytocin cannot augment contraction when cholesterol is elevated. The effects of cholesterol extraction with methyl beta-cyclodextrin and enrichment with low-density lipoproteins and cholesterol on contractile activity and intracellular calcium signaling in spontaneous or oxytocin-stimulated myometrium are determined. Force occurring spontaneously and with oxytocin is significantly increased by cholesterol extraction. Cholesterol enrichment profoundly inhibits force production in a dose-dependent manner and could reverse the effects of cholesterol extraction. Qualitatively similar results are found for nonpregnant and pregnant laboring and non-laboring myometrium. These contractile changes are related to changes in intracellular Ca2+ . Thus, elevated cholesterol is deleterious to contractility and Ca2+ signaling in human myometrium. Cholesterol may contribute to uterine quiescence but could cause difficulties in labor in obese/dyslipidemic women, consistent with their increased cesarean delivery rates.

  17. Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.

    PubMed

    Tang, Zhurong; Wang, Zhe; Qing, Fangzhu; Ni, Yilu; Fan, Yujiang; Tan, Yanfei; Zhang, Xingdong

    2015-03-01

    Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway. © 2014 Wiley Periodicals, Inc.

  18. In Vivo Calcium Signaling during Synaptic Refinement at the Drosophila Neuromuscular Junction.

    PubMed

    Vonhoff, Fernando; Keshishian, Haig

    2017-05-31

    Neural activity plays a key role in pruning aberrant synapses in various neural systems, including the mammalian cortex, where low-frequency (0.01 Hz) calcium oscillations refine topographic maps. However, the activity-dependent molecular mechanisms remain incompletely understood. Activity-dependent pruning also occurs at embryonic Drosophila neuromuscular junctions (NMJs), where low-frequency Ca(2+) oscillations are required for synaptic refinement and the response to the muscle-derived chemorepellant Sema2a. We examined embryonic growth cone filopodia in vivo to directly observe their exploration and to analyze the episodic Ca(2+) oscillations involved in refinement. Motoneuron filopodia repeatedly contacted off-target muscle fibers over several hours during late embryogenesis, with episodic Ca(2+) signals present in both motile filopodia as well as in later-stabilized synaptic boutons. The Ca(2+) transients matured over several hours into regular low-frequency (0.03 Hz) oscillations. In vivo imaging of intact embryos of both sexes revealed that the formation of ectopic filopodia is increased in Sema2a heterozygotes. We provide genetic evidence suggesting a complex presynaptic Ca(2+)-dependent signaling network underlying refinement that involves the phosphatases calcineurin and protein phosphatase-1, as well the serine/threonine kinases CaMKII and PKA. Significantly, this network influenced the neuron's response to the muscle's Sema2a chemorepellant, critical for the removal of off-target contacts.SIGNIFICANCE STATEMENT To address the question of how synaptic connectivity is established during development, we examined the behavior of growth cone filopodia during the exploration of both correct and off-target muscle fibers in Drosophila embryos. We demonstrate that filopodia repeatedly contact off-target muscles over several hours, until they ultimately retract. We show that intracellular signals are observed in motile and stabilized "ectopic" contacts. Several

  19. 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

  20. Inhibition of calcium channels by neurokinin receptor and signal transduction in hamster submandibular ganglion cells.

    PubMed

    Yamada, T; Endoh, T; Suzuki, T

    1999-04-16

    Both substance P (SP) and neurokinin A (NKA) are known as neurotransmitters of the submandibular ganglion (SMG) neurons. SP released from collaterals of the sensory nerves also regulates the excitability of SMG neurons. It has recently been shown that neurokinins (NK) inhibit calcium channels in various neurons. In this study, the effects of NK on voltage-dependent calcium channel current (I(Ca)) in SMG cells were investigated using the whole-cell patch-clamp recording method. NK-1 receptor agonist and SP caused inhibition of I(Ca) in SMG cells in a dose-dependent manner. NK-1 receptor agonist inhibited L-, N- and P/Q-type I(Ca) components. GDP-beta-S included in the pipette solution reduced the NK-1 receptor agonist-induced inhibition of I(Ca). In addition, NK-1 receptor agonist-induced inhibition of I(Ca) was reduced by stimulation of protein kinase C (PKC) but not cyclic AMP-dependent protein kinase (PKA). The results provided evidence for a signal transduction pathway in which calcium channel inhibition by NK receptors required activation of G-protein and PKC-affected step phosphorylation in SMG neurons.

  1. Spatial separation of two different pathways accounting for the generation of calcium signals in astrocytes

    PubMed Central

    Oschmann, Franziska; Mergenthaler, Konstantin; Obermayer, Klaus

    2017-01-01

    Astrocytes integrate and process synaptic information and exhibit calcium (Ca2+) signals in response to incoming information from neighboring synapses. The generation of Ca2+ signals is mostly attributed to Ca2+ release from internal Ca2+ stores evoked by an elevated metabotropic glutamate receptor (mGluR) activity. Different experimental results associated the generation of Ca2+ signals to the activity of the glutamate transporter (GluT). The GluT itself does not influence the intracellular Ca2+ concentration, but it indirectly activates Ca2+ entry over the membrane. A closer look into Ca2+ signaling in different astrocytic compartments revealed a spatial separation of those two pathways. Ca2+ signals in the soma are mainly generated by Ca2+ release from internal Ca2+ stores (mGluR-dependent pathway). In astrocytic compartments close to the synapse most Ca2+ signals are evoked by Ca2+ entry over the plasma membrane (GluT-dependent pathway). This assumption is supported by the finding, that the volume ratio between the internal Ca2+ store and the intracellular space decreases from the soma towards the synapse. We extended a model for mGluR-dependent Ca2+ signals in astrocytes with the GluT-dependent pathway. Additionally, we included the volume ratio between the internal Ca2+ store and the intracellular compartment into the model in order to analyze Ca2+ signals either in the soma or close to the synapse. Our model results confirm the spatial separation of the mGluR- and GluT-dependent pathways along the astrocytic process. The model allows to study the binary Ca2+ response during a block of either of both pathways. Moreover, the model contributes to a better understanding of the impact of channel densities on the interaction of both pathways and on the Ca2+ signal. PMID:28192424

  2. Spatial separation of two different pathways accounting for the generation of calcium signals in astrocytes.

    PubMed

    Oschmann, Franziska; Mergenthaler, Konstantin; Jungnickel, Evelyn; Obermayer, Klaus

    2017-02-01

    Astrocytes integrate and process synaptic information and exhibit calcium (Ca2+) signals in response to incoming information from neighboring synapses. The generation of Ca2+ signals is mostly attributed to Ca2+ release from internal Ca2+ stores evoked by an elevated metabotropic glutamate receptor (mGluR) activity. Different experimental results associated the generation of Ca2+ signals to the activity of the glutamate transporter (GluT). The GluT itself does not influence the intracellular Ca2+ concentration, but it indirectly activates Ca2+ entry over the membrane. A closer look into Ca2+ signaling in different astrocytic compartments revealed a spatial separation of those two pathways. Ca2+ signals in the soma are mainly generated by Ca2+ release from internal Ca2+ stores (mGluR-dependent pathway). In astrocytic compartments close to the synapse most Ca2+ signals are evoked by Ca2+ entry over the plasma membrane (GluT-dependent pathway). This assumption is supported by the finding, that the volume ratio between the internal Ca2+ store and the intracellular space decreases from the soma towards the synapse. We extended a model for mGluR-dependent Ca2+ signals in astrocytes with the GluT-dependent pathway. Additionally, we included the volume ratio between the internal Ca2+ store and the intracellular compartment into the model in order to analyze Ca2+ signals either in the soma or close to the synapse. Our model results confirm the spatial separation of the mGluR- and GluT-dependent pathways along the astrocytic process. The model allows to study the binary Ca2+ response during a block of either of both pathways. Moreover, the model contributes to a better understanding of the impact of channel densities on the interaction of both pathways and on the Ca2+ signal.

  3. 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

  4. 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.

  5. Signaling domain of Sonic Hedgehog as cannibalistic calcium-regulated zinc-peptidase.

    PubMed

    Rebollido-Rios, Rocio; Bandari, Shyam; Wilms, Christoph; Jakuschev, Stanislav; Vortkamp, Andrea; Grobe, Kay; Hoffmann, Daniel

    2014-07-01

    Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined Zn2+ center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double-Ca2+ center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on pH.

  6. Orai and TRPC channel characterization in FcεRI-mediated calcium signaling and mediator secretion in human mast cells.

    PubMed

    Wajdner, Hannah E; Farrington, Jasmine; Barnard, Claire; Peachell, Peter T; Schnackenberg, Christine G; Marino, Joseph P; Xu, Xiaoping; Affleck, Karen; Begg, Malcolm; Seward, Elizabeth P

    2017-03-01

    Inappropriate activation of mast cells via the FcεRI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcεRI-initiated mediator release. However, until now the role of TRPC calcium channels in FcεRI-mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to FcεRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FcεRI-mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FcεRI-mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1-regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1-KK(684-685)EE - TRPC1 gating mutant) failed to alter FcεRI-mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that FcεRI-mediated calcium influx through Orai is necessary for histamine and TNFα release but is differentially involved in the generation of cytokines and eicosanoids.

  7. Bone Is a Major Target of PTH/PTHrP Receptor Signaling in Regulation of Fetal Blood Calcium Homeostasis.

    PubMed

    Hirai, Takao; Kobayashi, Tatsuya; Nishimori, Shigeki; Karaplis, Andrew C; Goltzman, David; Kronenberg, Henry M

    2015-08-01

    The blood calcium concentration during fetal life is tightly regulated within a narrow range by highly interactive homeostatic mechanisms that include transport of calcium across the placenta and fluxes in and out of bone; the mechanisms of this regulation are poorly understood. Our findings that endochondral bone-specific PTH/PTHrP receptor (PPR) knockout (KO) mice showed significant reduction of fetal blood calcium concentration compared with that of control littermates at embryonic day 18.5 led us to focus on bone as a possibly major determinant of fetal calcium homeostasis. We found that the fetal calcium concentration of Runx2 KO mice was significantly higher than that of control littermates, suggesting that calcium flux into bone had a considerable influence on the circulating calcium concentration. Moreover, Runx2:PTH double mutant fetuses showed calcium levels similar to those of Runx2 KO mice, suggesting that part of the fetal hypocalcemia in PTH KO mice was caused by the increment of the mineralized bone mass allowed by the formation of osteoblasts. Finally, Rank:PTH double mutant mice had a blood calcium concentration even lower than that of the either Rank KO or PTH KO mice alone at embryonic day 18.5. These observations in our genetic models suggest that PTH/PTHrP receptor signaling in bones has a significant role of the regulation of fetal blood calcium concentration and that both placental transport and osteoclast activation contribute to PTH's hypercalcemic action. They also show that PTH-independent deposition of calcium in bone is the major controller of fetal blood calcium level.

  8. Leptin regulated calcium channels of neuropeptide Y and proopiomelanocortin neurons by activation of different signal pathways.

    PubMed

    Wang, J-H; Wang, F; Yang, M-J; Yu, D-F; Wu, W-N; Liu, J; Ma, L-Q; Cai, F; Chen, J-G

    2008-09-22

    The fat-derived hormone leptin regulates food intake and body weight in part by modulating the activity of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC). To investigate the electrophysiological activity of these neurons and their responses to leptin, we recorded whole-cell calcium currents on NPY and POMC neurons in the ARC of rats, which we identified by morphologic features and immunocytochemical identification at the end of recording. Leptin decreased the peak amplitude of high voltage-activated calcium currents (I(HVA)) in the isolated neurons from ARC, which were subsequently shown to be immunoreactive for NPY. The inhibition was prevented by pretreatment with inhibitors of Janus kinase 2 (JAK2) and mitogen-activated protein kinases (MAPK). In contrast, leptin increased the amplitude of I(HVA) in POMC-containing neurons. The stimulations of I(HVA) were inhibited by blockers of JAK2 and phosphatidylino 3-kinase (PI3-k). Both of these effects were counteracted by the L-type calcium channel antagonist nifedipine, suggesting that L-type calcium channels were involved in the regulation induced by leptin. These data indicated that leptin exerted opposite effects on these two classes of neurons. Leptin directly inhibited I(HVA) in NPY neurons via leptin receptor (LEPR) -JAK2-MAPK pathways, whereas evoked I(HVA) in POMC neurons by LEPR-JAK2-PI3-k pathways. These neural pathways and intracellular signaling mechanisms may play key roles in regulating NPY and POMC neuron activity, anorectic action of leptin and, thereby, feeding.

  9. Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

    PubMed Central

    Sergé, Arnauld; Bernard, Anne-Marie; Phélipot, Marie-Claire; Bertaux, Nicolas; Fallet, Mathieu; Grenot, Pierre; Marguet, Didier; He, Hai-Tao; Hamon, Yannick

    2013-01-01

    We introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells. PMID:24086124

  10. 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

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

    PubMed

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

    2015-01-30

    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.

  12. Small caliber arterial endothelial cells calcium signals elicited by PAR2 are preserved from endothelial dysfunction

    PubMed Central

    Hennessey, John C; Stuyvers, Bruno D; McGuire, John J

    2015-01-01

    Endothelial cell (EC)-dependent vasodilation by proteinase-activated receptor 2 (PAR2) is preserved in small caliber arteries in disease states where vasodilation by muscarinic receptors is decreased. In this study, we identified and characterized the PAR2-mediated intracellular calcium (Ca2+)-release mechanisms in EC from small caliber arteries in healthy and diseased states. Mesenteric arterial EC were isolated from PAR2 wild-type (WT) and null mice, after saline (controls) or angiotensin II (AngII) infusion, for imaging intracellular calcium and characterizing the calcium-release system by immunofluorescence. EC Ca2+ signals comprised two forms of Ca2+-release events that had distinct spatial-temporal properties and occurred near either the plasmalemma (peripheral) or center of EC. In healthy EC, PAR2-dependent increases in the densities and firing rates of both forms of Ca2+-release were abolished by inositol 1,4,5- trisphosphate receptor (IP3R) inhibitor, but partially reduced by transient potential vanilloid channels inhibitor ruthenium red (RR). Acetylcholine (ACh)-induced less overall Ca2+-release than PAR2 activation, but enhanced selectively the incidence of central events. PAR2-dependent Ca2+-activity, inhibitors sensitivities, IP3R, small- and intermediate-conductance Ca2+-activated potassium channels expressions were unchanged in EC from AngII WT. However, the same cells exhibited decreases in ACh-induced Ca2+-release, RR sensitivity, and endothelial nitric oxide synthase expression, indicating AngII-induced dysfunction was differentiated by receptor, Ca2+-release, and downstream targets of EC activation. We conclude that PAR2 and muscarinic receptors selectively elicit two elementary Ca2+ signals in single EC. PAR2-selective IP3R-dependent peripheral Ca2+-release mechanisms are identical between healthy and diseased states. Further study of PAR2-selective Ca2+-release for eliciting pathological and/or normal EC functions is warranted. PMID:25729579

  13. Extracellular matrix stiffness modulates VEGF calcium signaling in endothelial cells: individual cell and population analysis.

    PubMed

    Derricks, Kelsey E; Trinkaus-Randall, Vickery; Nugent, Matthew A

    2015-09-01

    Vascular disease and its associated complications are the number one cause of death in the Western world. Both extracellular matrix stiffening and dysfunctional endothelial cells contribute to vascular disease. We examined endothelial cell calcium signaling in response to VEGF as a function of extracellular matrix stiffness. We developed a new analytical tool to analyze both population based and individual cell responses. Endothelial cells on soft substrates, 4 kPa, were the most responsive to VEGF, whereas cells on the 125 kPa substrates exhibited an attenuated response. Magnitude of activation, not the quantity of cells responding or the number of local maximums each cell experienced distinguished the responses. Individual cell analysis, across all treatments, identified two unique cell clusters. One cluster, containing most of the cells, exhibited minimal or slow calcium release. The remaining cell cluster had a rapid, high magnitude VEGF activation that ultimately defined the population based average calcium response. Interestingly, at low doses of VEGF, the high responding cell cluster contained smaller cells on average, suggesting that cell shape and size may be indicative of VEGF-sensitive endothelial cells. This study provides a new analytical tool to quantitatively analyze individual cell signaling response kinetics, that we have used to help uncover outcomes that are hidden within the average. The ability to selectively identify highly VEGF responsive cells within a population may lead to a better understanding of the specific phenotypic characteristics that define cell responsiveness, which could provide new insight for the development of targeted anti- and pro-angiogenic therapies.

  14. Hydrogen Peroxide Signaling in Plant Development and Abiotic Responses: Crosstalk with Nitric Oxide and Calcium.

    PubMed

    Niu, Lijuan; Liao, Weibiao

    2016-01-01

    Hydrogen peroxide (H2O2), as a reactive oxygen species, is widely generated in many biological systems. It has been considered as an important signaling molecule that mediates various physiological and biochemical processes in plants. Normal metabolism in plant cells results in H2O2 generation, from a variety of sources. Also, it is now clear that nitric oxide (NO) and calcium (Ca(2+)) function as signaling molecules in plants. Both H2O2 and NO are involved in plant development and abiotic responses. A wide range of evidences suggest that NO could be generated under similar stress conditions and with similar kinetics as H2O2. The interplay between H2O2 and NO has important functional implications to modulate transduction processes in plants. Moreover, close interaction also exists between H2O2 and Ca(2+) in response to development and abiotic stresses in plants. Cellular responses to H2O2 and Ca(2+) signaling systems are complex. There is quite a bit of interaction between H2O2 and Ca(2+) signaling in responses to several stimuli. This review aims to introduce these evidences in our understanding of the crosstalk among H2O2, NO, and Ca(2+) signaling which regulates plant growth and development, and other cellular and physiological responses to abiotic stresses.

  15. Hydrogen Peroxide Signaling in Plant Development and Abiotic Responses: Crosstalk with Nitric Oxide and Calcium

    PubMed Central

    Niu, Lijuan; Liao, Weibiao

    2016-01-01

    Hydrogen peroxide (H2O2), as a reactive oxygen species, is widely generated in many biological systems. It has been considered as an important signaling molecule that mediates various physiological and biochemical processes in plants. Normal metabolism in plant cells results in H2O2 generation, from a variety of sources. Also, it is now clear that nitric oxide (NO) and calcium (Ca2+) function as signaling molecules in plants. Both H2O2 and NO are involved in plant development and abiotic responses. A wide range of evidences suggest that NO could be generated under similar stress conditions and with similar kinetics as H2O2. The interplay between H2O2 and NO has important functional implications to modulate transduction processes in plants. Moreover, close interaction also exists between H2O2 and Ca2+ in response to development and abiotic stresses in plants. Cellular responses to H2O2 and Ca2+ signaling systems are complex. There is quite a bit of interaction between H2O2 and Ca2+ signaling in responses to several stimuli. This review aims to introduce these evidences in our understanding of the crosstalk among H2O2, NO, and Ca2+ signaling which regulates plant growth and development, and other cellular and physiological responses to abiotic stresses. PMID:26973673

  16. Graded boosting of synaptic signals by low-threshold voltage-activated calcium conductance

    PubMed Central

    Carbó Tano, Martín; Vilarchao, María Eugenia

    2015-01-01

    Low-threshold voltage-activated calcium conductances (LT-VACCs) play a substantial role in shaping the electrophysiological attributes of neurites. We have investigated how these conductances affect synaptic integration in a premotor nonspiking (NS) neuron of the leech nervous system. These cells exhibit an extensive neuritic tree, do not fire Na+-dependent spikes, but express an LT-VACC that was sensitive to 250 μM Ni2+ and 100 μM NNC 55-0396 (NNC). NS neurons responded to excitation of mechanosensory pressure neurons with depolarizing responses for which amplitude was a linear function of the presynaptic firing frequency. NNC decreased these synaptic responses and abolished the concomitant widespread Ca2+ signals. Coherent with the interpretation that the LT-VACC amplified signals at the postsynaptic level, this conductance also amplified the responses of NS neurons to direct injection of sinusoidal current. Synaptic amplification thus is achieved via a positive feedback in which depolarizing signals activate an LT-VACC that, in turn, boosts these signals. The wide distribution of LT-VACC could support the active propagation of depolarizing signals, turning the complex NS neuritic tree into a relatively compact electrical compartment. PMID:25972583

  17. Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo.

    PubMed

    Boerman, Erika M; Everhart, Jesse E; Segal, Steven S

    2016-05-01

    Aging is associated with vascular dysfunction that impairs tissue perfusion, physical activity, and the quality of life. Calcium signaling in endothelial cells (ECs) is integral to vasomotor control, exemplified by localized Ca(2+) signals within EC projections through holes in the internal elastic lamina (IEL). Within these microdomains, endothelium-derived hyperpolarization is integral to smooth muscle cell (SMC) relaxation via coupling through myoendothelial gap junctions. However, the effects of aging on local EC Ca(2+) signals (and thereby signaling between ECs and SMCs) remain unclear, and these events have not been investigated in vivo. Furthermore, it is unknown whether aging affects either the number or the size of IEL holes. In the present study, we tested the hypothesis that local EC Ca(2+) signaling is impaired with advanced age along with a reduction in IEL holes. In anesthetized mice expressing a Ca(2+)-sensitive fluorescent protein (GCaMP2) selectively in ECs, our findings illustrate that for mesenteric arteries controlling splanchnic blood flow the frequency of spontaneous local Ca(2+) signals in ECs was reduced by ∼85% in old (24-26 mo) vs. young (3-6 mo) animals. At the same time, the number (and total area) of holes per square millimeter of IEL was reduced by ∼40%. We suggest that diminished signaling between ECs and SMCs contributes to dysfunction of resistance arteries with advanced age.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/aging-impairs-endothelial-ca2-signaling/. Copyright © 2016 the American Physiological Society.

  18. CRH activation of different signaling pathways results in differential calcium signaling in human pregnant myometrium before and during labor.

    PubMed

    You, Xingji; Gao, Lu; Liu, Jie; Xu, Chen; Liu, Chunmin; Li, Yuan; Hui, Ning; Gu, Hang; Ni, Xin

    2012-10-01

    Our previous study has demonstrated that CRH has differential effects on human uterine contractility before and after onset of labor. Intracellular Ca2+ concentration ([Ca2+]i) mobilization plays an important role in the control of uterine contraction. Our objective was to investigate the effects of CRH on [Ca2+]i homeostasis in laboring and nonlaboring myometrial cells and determine subsequent signaling involved in [Ca2+]i regulation by CRH. The myometrial tissues were obtained from pregnant women who were undergoing or not undergoing labor at term. [Ca2+]i was determined by Ca2+ imaging system using the fluorescent dye fura-2-acetoxymethyl ester. Western blot analysis, ELISA, and RIA were used to determine the signaling pathways induced by CRH. CRH induced Ca2+ transient in laboring cells, which was blocked by CRH receptor type 1 (CRHR1) antagonist antalarmin. CRHR1 knockdown impaired this effect of CRH. CRH activated Gi protein, decreased cAMP production, and induced phosphorylated phospholipase C-β3 and inositol-1,4,5-triphosphate production. Phospholipase C and inositol-1,4,5-triphosphate receptor inhibitors blocked the CRH-induced Ca2+ transient in laboring cells. CRH did not induce whereas antalarmin induced the Ca2+ transient in nonlaboring cells. Knockdown of CRHR1 impaired the effect of antalarmin. CRH acted on CRHR1 to activate Gs in nonlaboring cells. Forskolin blocked antalarmin-induced Ca2+ transient. CRH acts on CRHR1 to activate different signaling pathways before and after onset of labor, thereby resulting in differential calcium signaling in response to CRH. The signaling pathways of CRHR1 might serve as a target for the development of new therapeutic strategies for preterm birth.

  19. A TRPC5-regulated calcium signaling pathway controls dendrite patterning in the mammalian brain.

    PubMed

    Puram, Sidharth V; Riccio, Antonio; Koirala, Samir; Ikeuchi, Yoshiho; Kim, Albert H; Corfas, Gabriel; Bonni, Azad

    2011-12-15

    Transient receptor potential (TRP) channels have been implicated as sensors of diverse stimuli in mature neurons. However, developmental roles for TRP channels in the establishment of neuronal connectivity remain largely unexplored. Here, we identify an essential function for TRPC5, a member of the canonical TRP subfamily, in the regulation of dendrite patterning in the mammalian brain. Strikingly, TRPC5 knockout mice harbor long, highly branched granule neuron dendrites with impaired dendritic claw differentiation in the cerebellar cortex. In vivo RNAi analyses suggest that TRPC5 regulates dendrite morphogenesis in the cerebellar cortex in a cell-autonomous manner. Correlating with impaired dendrite patterning in the cerebellar cortex, behavioral analyses reveal that TRPC5 knockout mice have deficits in gait and motor coordination. Finally, we uncover the molecular basis of TRPC5's function in dendrite patterning. We identify the major protein kinase calcium/calmodulin-dependent kinase II β (CaMKIIβ) as a critical effector of TRPC5 function in neurons. Remarkably, TRPC5 forms a complex specifically with CaMKIIβ, but not the closely related kinase CaMKIIα, and thereby induces the CaMKIIβ-dependent phosphorylation of the ubiquitin ligase Cdc20-APC at the centrosome. Accordingly, centrosomal CaMKIIβ signaling mediates the ability of TRPC5 to regulate dendrite morphogenesis in neurons. Our findings define a novel function for TRPC5 that couples calcium signaling to a ubiquitin ligase pathway at the centrosome and thereby orchestrates dendrite patterning and connectivity in the brain.

  20. The dynamic range and domain-specific signals of intracellular calcium in photoreceptors.

    PubMed

    Szikra, T; Krizaj, D

    2006-08-11

    Vertebrate photoreceptors consist of strictly delimited subcellular domains: the outer segment, ellipsoid, cell body and synaptic terminal, each hosting crucial cellular functions, including phototransduction, oxidative metabolism, gene expression and transmitter release. We used optical imaging to explore the spatiotemporal dynamics of Ca(2+) signaling in non-outer segment regions of rods and cones. Sustained depolarization, designed to emulate photoreceptor activation in the darkness, evoked a standing Ca(2+) gradient in tiger salamander photoreceptors with spatially-averaged intracellular Ca(2+) concentration within synaptic terminals of approximately 2 microM and lower (approximately 750 nM) intracellular calcium concentration in the ellipsoid. Measurements from axotomized cell bodies and isolated ellipsoids showed that Ca(2+) enters the two compartments via both local L-type Ca(2+) channels and diffusion. The results from optical imaging studies were supported by immunostaining analysis. L-type voltage-operated Ca(2+) channels and plasma membrane Ca(2+) ATPases were highly expressed in synaptic terminals with progressively lower expression levels in the cell body and ellipsoid. These results show photoreceptor Ca(2+) homeostasis is controlled in a region-specific manner by direct Ca(2+) entry and diffusion as well as Ca(2+) extrusion. Moreover, quantitative measurement of intracellular calcium concentration levels in different photoreceptor compartments indicates that the dynamic range of Ca(2+) signaling in photoreceptors is approximately 40-fold, from approximately 50 nM in the light to approximately 2 microM in darkness.

  1. THE DYNAMIC RANGE AND DOMAIN-SPECIFIC SIGNALS OF INTRACELLULAR CALCIUM IN PHOTORECEPTORS

    PubMed Central

    SZIKRA, T.; KRIŽAJ, D.

    2007-01-01

    Vertebrate photoreceptors consist of strictly delimited subcellular domains: the outer segment, ellipsoid, cell body and synaptic terminal, each hosting crucial cellular functions, including phototransduction, oxidative metabolism, gene expression and transmitter release. We used optical imaging to explore the spatiotemporal dynamics of Ca2+ signaling in non-outer segment regions of rods and cones. Sustained depolarization, designed to emulate photoreceptor activation in the darkness, evoked a standing Ca2+ gradient in tiger salamander photoreceptors with spatially-averaged intracellular Ca2+ concentration within synaptic terminals of ∼2 μM and lower (∼750 nM) intracellular calcium concentration in the ellipsoid. Measurements from axotomized cell bodies and isolated ellipsoids showed that Ca2+ enters the two compartments via both local L-type Ca2+ channels and diffusion. The results from optical imaging studies were supported by immunostaining analysis. L-type voltage-operated Ca2+ channels and plasma membrane Ca2+ ATPases were highly expressed in synaptic terminals with progressively lower expression levels in the cell body and ellipsoid. These results show photoreceptor Ca2+ homeostasis is controlled in a region-specific manner by direct Ca2+ entry and diffusion as well as Ca2+ extrusion. Moreover, quantitative measurement of intracellular calcium concentration levels in different photoreceptor compartments indicates that the dynamic range of Ca2+ signaling in photoreceptors is approximately 40-fold, from ∼50 nM in the light to ∼2 μM in darkness. PMID:16682126

  2. The involvement of calcium carriers and of the vacuole in the glucose-induced calcium signaling and activation of the plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells.

    PubMed

    Bouillet, L E M; Cardoso, A S; Perovano, E; Pereira, R R; Ribeiro, E M C; Trópia, M J M; Fietto, L G; Tisi, R; Martegani, E; Castro, I M; Brandão, R L

    2012-01-01

    Previous work from our laboratories demonstrated that the sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae is dependent on calcium metabolism with the contribution of calcium influx from external medium. Our results demonstrate that a glucose-induced calcium (GIC) transporter, a new and still unidentified calcium carrier, sensitive to nifedipine and gadolinium and activated by glucose addition, seems to be partially involved in the glucose-induced activation of the plasma membrane H(+)-ATPase. On the other hand, the importance of calcium carriers that can release calcium from internal stores was analyzed in glucose-induced calcium signaling and activation of plasma membrane H(+)-ATPase, in experimental conditions presenting very low external calcium concentrations. Therefore the aim was also to investigate how the vacuole, through the participation of both Ca(2+)-ATPase Pmc1 and the TRP homologue calcium channel Yvc1 (respectively, encoded by the genes PMC1 and YVC1) contributes to control the intracellular calcium availability and the plasma membrane H(+)-ATPase activation in response to glucose. In strains presenting a single deletion in YVC1 gene or a double deletion in YVC1 and PMC1 genes, both glucose-induced calcium signaling and activation of the H(+)-ATPase are nearly abolished. These results suggest that Yvc1 calcium channel is an important component of this signal transduction pathway activated in response to glucose addition. We also found that by a still undefined mechanism Yvc1 activation seems to correlate with the changes in the intracellular level of IP(3). Taken together, these data demonstrate that glucose addition to yeast cells exposed to low external calcium concentrations affects calcium uptake and the activity of the vacuolar calcium channel Yvc1, contributing to the occurrence of calcium signaling connected to plasma membrane H(+)-ATPase activation. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Science Signaling Podcast for 6 June 2017: Calcium signaling and dry mouth.

    PubMed

    Ambudkar, Indu; VanHook, Annalisa M

    2017-06-06

    This Podcast features a conversation with Indu Ambudkar, senior author of a Research Resource that appears in the 6 June 2017 issue of Science Signaling, about how activation of the cation channel TRPM2 is involved in radiation-induced dry mouth. Patients who receive radiation therapy for head and neck cancers often develop dry mouth as a side effect, and this condition is frequently permanent. Radiation does not kill cells in the salivary gland, yet it causes the acinar cells of the gland to reduce the amount of saliva they secrete. Liu et al found that radiation-induced activation of the cation channel TRPM2 triggered cleavage of the endoplasmic reticulum Ca(2+) sensor STIM1, thus inhibiting store-operated Ca(2+) entry and interfering with saliva production. These findings identify proteins that could potentially be targeted to prevent dry mouth in patients undergoing radiation therapy.Listen to Podcast. Copyright © 2017, American Association for the Advancement of Science.

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

    PubMed

    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 [Ca(2+)]i decreased (P < 0.001). The differences in [Ca(2+)]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 Ca(2+)-ATPases. Vitamin D3 supplementation had a beneficial effect on [Ca(2+)]i decreasing calcium entry via CRAC and P2X7 channels and reducing P2X7 receptors expression.

  5. Engendering biased signalling from the calcium-sensing receptor for the pharmacotherapy of diverse disorders

    PubMed Central

    Leach, K; Sexton, P M; Christopoulos, A; Conigrave, A D

    2014-01-01

    The human calcium-sensing receptor (CaSR) is widely expressed in the body, where its activity is regulated by multiple orthosteric and endogenous allosteric ligands. Each ligand stabilizes a unique subset of conformational states, which enables the CaSR to couple to distinct intracellular signalling pathways depending on the extracellular milieu in which it is bathed. Differential signalling arising from distinct receptor conformations favoured by each ligand is referred to as biased signalling. The outcome of CaSR activation also depends on the cell type in which it is expressed. Thus, the same ligand may activate diverse pathways in distinct cell types. Given that the CaSR is implicated in numerous physiological and pathophysiological processes, it is an ideal target for biased ligands that could be rationally designed to selectively regulate desired signalling pathways in preferred cell types. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5 PMID:24111791

  6. InsP3-mediated intracellular calcium signalling is altered by expression of synaptojanin-1

    PubMed Central

    2004-01-01

    Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] plays an important physiological role as a precursor for the InsP3-mediated intracellular calcium (Ca2+) signalling cascade. It also regulates membrane trafficking, actin function and transmembrane proteins. SJ-1 (synaptojanin-1), a phosphoinositide phosphatase, regulates the turnover of a PtdIns(4,5)P2 pool involved in clathrin and actin dynamics at the cell surface. We tested the interrelationship of this pool with PtdIns(4,5)P2 pools involved in Ca2+ signalling by expressing in Chinese-hamster ovary cells full-length SJ-1 or its 5-Pase (inositol 5-phosphatase) domain. SJ-1 significantly attenuated the generation of Ca2+ oscillations induced by ATP and the 5-Pase domain mimicked this effect. These changes correlated with increased PtdIns(4,5)P2 phosphatase activity of cellular extracts. Overexpression of the endoplasmic reticulum-anchored PtdIns(4)P phosphatase Sac1 did not affect Ca2+ oscillations, although it increased the Ca2+ efflux rate from intracellular stores. The ability of SJ-1 to alter intracellular Ca2+ signalling indicates a close functional interrelationship between plasma membrane PtdIns(4,5)P2 pools that control actin and endocytosis and those involved in the regulation of specific spatio-temporal Ca2+ signals. PMID:15080793

  7. Cdk9 T-loop Phosphorylation is Regulated by the Calcium Signaling Pathway

    PubMed Central

    Ramakrishnan, Rajesh; Rice, Andrew P.

    2011-01-01

    Eukaryotic RNA polymerase II transcriptional elongation is a tightly regulated process and is dependent upon positive transcription elongation factor-b (P-TEFb). The core P-TEFb complex is composed of Cdk9 and Cyclin T and is essential for the expression of most protein coding genes. Cdk9 kinase function is dependent upon phosphorylation of Thr186 in its T-loop. In this study, we examined kinases and signaling pathways that influence Cdk9 T-loop phosphorylation. Using an RNAi screen in HeLa cells, we found that Cdk9 T-loop phosphorylation is regulated by Calcium/Calmodulin- dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4+ T lymphocytes, we found that the Ca2+ signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca2+ signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca2+ signaling pathway repressed the PCNA promoter and HIV-1 Tat transactivation of the HIV-1 LTR, but not HTLV-1 Tax transactivation of the HTLV-1 LTR, suggesting that perturbation of the Ca2+ pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function. PMID:21448926

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

    PubMed

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

    2010-01-01

    Astrocytes have been identified to actively contribute to brain functions through Ca(2+) 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 (Ca(2+)) waves, providing a noninvasive and efficient approach with highly precise targeting. In this work, detailed characteristics of astrocytic Ca(2+) signaling induced by photostimulation are presented. In a purified astrocytic culture, after the illumination of a femtosecond laser onto one cell, a Ca(2+) wave throughout the network with reduced speed is induced, and intracellular Ca(2+) oscillations are observed. The intercellular propagation is pharmacologically confirmed to be mainly mediated by ATP through P(2)Y receptors. Different patterns of Ca(2+) 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 Ca(2+) signaling in astrocytes has a positive relationship with the stimulating laser power. Therefore, distinct Ca(2+) signaling is feasibly available for specific studies on astrocytes by employing precisely controlled photostimulation.

  9. 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.

  10. Serotonin Regulates Calcium Homeostasis in Lactation by Epigenetic Activation of Hedgehog Signaling

    PubMed Central

    Laporta, Jimena; Keil, Kimberly P.; Weaver, Samantha R.; Cronick, Callyssa M.; Prichard, Austin P.; Crenshaw, Thomas D.; Heyne, Galen W.; Vezina, Chad M.; Lipinski, Robert J.

    2014-01-01

    Calcium homeostasis during lactation is critical for maternal and neonatal health. We previously showed that nonneuronal/peripheral serotonin [5-hydroxytryptamine (5-HT)] causes the lactating mammary gland to synthesize and secrete PTHrP in an acute fashion. Here, using a mouse model, we found that genetic inactivation of tryptophan hydroxylase 1 (Tph1), which catalyzes the rate-limiting step in peripheral 5-HT synthesis, reduced circulating and mammary PTHrP expression, osteoclast activity, and maternal circulating calcium concentrations during the transition from pregnancy to lactation. Tph1 inactivation also reduced sonic hedgehog signaling in the mammary gland during lactation. Each of these deficiencies was rescued by daily injections of 5-hydroxy-L-tryptophan (an immediate precursor of 5-HT) to Tph1-deficient dams. We used immortalized mouse embryonic fibroblasts to demonstrate that 5-HT induces PTHrP through a sonic hedgehog-dependent signal transduction mechanism. We also found that 5-HT altered DNA methylation of the Shh gene locus, leading to transcriptional initiation at an alternate start site and formation of a variant transcript in mouse embryonic fibroblasts in vitro and in mammary tissue in vivo. These results support a new paradigm of 5-HT-mediated Shh regulation involving DNA methylation remodeling and promoter switching. In addition to having immediate implications for lactation biology, identification and characterization of a novel functional regulatory relationship between nonneuronal 5-HT, hedgehog signaling, and PTHrP offers new avenues for the study of these important factors in development and disease. PMID:25192038

  11. Cytosolic calcium and pH signaling in plants under salinity stress.

    PubMed

    Kader, Md Abdul; Lindberg, Sylvia

    2010-03-01

    Calcium is one of the essential nutrients for growth and development of plants. It is an important component of various structures in cell wall and membranes. Besides some fundamental roles under normal condition, calcium functions as a major secondary-messenger molecule in plants under different developmental cues and various stress conditions including salinity stress. Also changes in cytosolic pH, pH(cyt), either individually, or in coordination with changes in cytosolic Ca(2+) concentration, [Ca(2+)](cyt), evoke a wide range of cellular functions in plants including signal transduction in plant-defense responses against stresses. It is believed that salinity stress, like other stresses, is perceived at cell membrane, either extra cellular or intracellular, which then triggers an intracellular-signaling cascade including the generation of secondary messenger molecules like Ca(2+) and protons. The variety and complexity of Ca(2+) and pH signaling result from the nature of the stresses as well as the tolerance level of the plant species against that specific stress. The nature of changes in [Ca(2+)](cyt) concentration, in terms of amplitude, frequency and duration, is likely very important for decoding the specific downstream responses for salinity stress tolerance in planta. It has been observed that the signatures of [Ca(2+)](cyt) and pH differ in various studies reported so far depending on the techniques used to measure them, and also depending on the plant organs where they are measured, such as root, shoot tissues or cells. This review describes the recent advances about the changes in [Ca(2+)](cyt) and pH(cyt) at both cellular and whole-plant levels under salinity stress condition, and in various salinity-tolerant and -sensitive plant species.

  12. Modeling Local X-ROS and Calcium Signaling in the Heart.

    PubMed

    Limbu, Sarita; Hoang-Trong, Tuan M; Prosser, Benjamin L; Lederer, W Jonathan; Jafri, M Saleet

    2015-11-17

    Stretching single ventricular cardiac myocytes has been shown experimentally to activate transmembrane nicotinamide adenine dinucleotide phosphate oxidase type 2 to produce reactive oxygen species (ROS) and increase the Ca2+ spark rate in a process called X-ROS signaling. The increase in Ca2+ spark rate is thought to be due to an increase in ryanodine receptor type 2 (RyR2) open probability by direct oxidation of the RyR2 protein complex. In this article, a computational model is used to examine the regulation of ROS and calcium homeostasis by local, subcellular X-ROS signaling and its role in cardiac excitation-contraction coupling. To this end, a four-state RyR2 model was developed that includes an X-ROS-dependent RyR2 mode switch. When activated, [Ca2+]i-sensitive RyR2 open probability increases, and the Ca2+ spark rate changes in a manner consistent with experimental observations. This, to our knowledge, new model is used to study the transient effects of diastolic stretching and subsequent ROS production on RyR2 open probability, Ca2+ sparks, and the myoplasmic calcium concentration ([Ca2+]i) during excitation-contraction coupling. The model yields several predictions: 1) [ROS] is produced locally near the RyR2 complex during X-ROS signaling and increases by an order of magnitude more than the global ROS signal during myocyte stretching; 2) X-ROS activation just before the action potential, corresponding to ventricular filling during diastole, increases the magnitude of the Ca2+ transient; 3) during prolonged stretching, the X-ROS-induced increase in Ca2+ spark rate is transient, so that long-sustained stretching does not significantly increase sarcoplasmic reticulum Ca2+ leak; and 4) when the chemical reducing capacity of the cell is decreased, activation of X-ROS signaling increases sarcoplasmic reticulum Ca2+ leak and contributes to global oxidative stress, thereby increases the possibility of arrhythmia. The model provides quantitative information not

  13. Calcium signatures and signaling in cytosol and organelles of tobacco cells induced by plant defense elicitors.

    PubMed

    Manzoor, Hamid; Chiltz, Annick; Madani, Siham; Vatsa, Parul; Schoefs, Benoît; Pugin, Alain; Garcia-Brugger, Angela

    2012-06-01

    Calcium signatures induced by two elicitors of plant defense reactions, namely cryptogein and oligogalacturonides, were monitored at the subcellular level, using apoaequorin-transformed Nicotiana tabacum var Xanthi cells, in which the apoaequorin calcium sensor was targeted either to cytosol, mitochondria or chloroplasts. Our study showed that both elicitors induced specific Ca(2+) signatures in each compartment, with the most striking difference relying on duration. Common properties also emerged from the analysis of Ca(2+) signatures: both elicitors induced a biphasic cytosolic [Ca(2+)] elevation together with a single mitochondrial [Ca(2+)] elevation concomitant with the first cytosolic [Ca(2+)] peak. In addition, both elicitors induced a chloroplastic [Ca(2+)] elevation peaking later in comparison to cytosolic [Ca(2+)] elevation. In cryptogein-treated cells, pharmacological studies indicated that IP(3) should play an important role in Ca(2+) signaling contrarily to cADPR or nitric oxide, which have limited or no effect on [Ca(2+)] variations. Our data also showed that, depending on [Ca(2+)] fluxes at the plasma membrane, cryptogein triggered a mitochondrial respiration increase and affected excess energy dissipation mechanisms in chloroplasts. Altogether the results indicate that cryptogein profoundly impacted cell functions at many levels, including organelles.

  14. Moringa oleifera-rich diet and T-cell calcium signaling in hypertensive rats.

    PubMed

    Attakpa, E S; Chabi, N W; Bertin, G A; Ategbo, J-M; Seri, B; Khan, N A

    2017-04-12

    Moringa oleifera is a plant whose fruits, roots and leaves have been advocated for traditional medicinal uses. The physico-chemical analysis shows that, Moringa contains more dietary polyunsaturated fatty acids (PUFA) than saturated fatty acids (SFA). The consumption of an experimental diet enriched with Moringa oleifera extracts lowered blood pressure in spontaneously hypertensive rats (SHR), but not in normotensive Wistar-Kyoto (WKY) rats as compared to rats fed an unsupplemented control diet. Anti-CD3-stimulated T-cell proliferation was diminished in both strains of rats fed the Moringa oleifera. The experimental diet lowered secretion of interleukin-2 in SHR, but not in WKY rats compared with rats fed the control diet. Studies of platelets from patients with primary hypertension and from SHR support the notion that the concentration of intracellular free calcium [Ca(2+)]i is modified in both clinical and experimental hypertension. We observed that the basal, [Ca(2+)]i was lower in T cells of SHR than in those of WKY rats fed the control diet. Feeding the diet with Moringa oleifera extracts to WKY rats did not alter basal [Ca(2+)]i in T cells but increased basal [Ca(2+)]i in SHR. Our study clearly demonstrated that Moringa oleifera exerts antihypertensive effects by inhibiting the secretion of IL-2 and modulates T-cell calcium signaling in hypertensive rats.

  15. Astrocytes increase ATP exocytosis mediated calcium signaling in response to microgroove structures.

    PubMed

    Singh, Ajay V; Raymond, Michael; Pace, Fabiano; Certo, Anthony; Zuidema, Jonathan M; McKay, Christopher A; Gilbert, Ryan J; Lu, X Lucas; Wan, Leo Q

    2015-01-19

    Following central nervous system (CNS) injury, activated astrocytes form glial scars, which inhibit axonal regeneration, leading to long-term functional deficits. Engineered nanoscale scaffolds guide cell growth and enhance regeneration within models of spinal cord injury. However, the effects of micro-/nanosize scaffolds on astrocyte function are not well characterized. In this study, a high throughput (HTP) microscale platform was developed to study astrocyte cell behavior on micropatterned surfaces containing 1 μm spacing grooves with a depth of 250 or 500 nm. Significant changes in cell and nuclear elongation and alignment on patterned surfaces were observed, compared to on flat surfaces. The cytoskeleton components (particularly actin filaments and focal adhesions) and nucleus-centrosome axis were aligned along the grooved direction as well. More interestingly, astrocytes on micropatterned surfaces showed enhanced mitochondrial activity with lysosomes localized at the lamellipodia of the cells, accompanied by enhanced adenosine triphosphate (ATP) release and calcium activities. These data indicate that the lysosome-mediated ATP exocytosis and calcium signaling may play an important role in astrocytic responses to substrate topology. These new findings have furthered our understanding of the biomechanical regulation of astrocyte cell-substrate interactions, and may benefit the optimization of scaffold design for CNS healing.

  16. Use-dependent control of presynaptic calcium signalling at central synapses

    PubMed Central

    Scott, Ricardo

    2007-01-01

    Voltage-gated Ca2+ channels activated by action potentials evoke Ca2+ entry into presynaptic terminals thus briefly distorting the resting Ca2+ concentration. When this happens, a number of processes are initiated to re-establish the Ca2+ equilibrium. During the post-spike period, the increased Ca2+ concentration could enhance the presynaptic Ca2+ signalling. Some of the mechanisms contributing to presynaptic Ca2+ dynamics involve endogenous Ca2+ buffers, Ca2+ stores, mitochondria, the sodium–calcium exchanger, extraterminal Ca2+ depletion and presynaptic receptors. Additionally, subthreshold presynaptic depolarization has been proposed to have an effect on release of neurotransmitters through a mechanism involving changes in resting Ca2+. Direct evidence for the role of any of these participants in shaping the presynaptic Ca2+ dynamics comes from direct recordings of giant presynaptic terminals and from fluorescent Ca2+ imaging of axonal boutons. Here, some of this evidence is presented and discussed. PMID:17523936

  17. Phenotypic variability in unicellular organisms: from calcium signalling to social behaviour.

    PubMed

    Vogel, David; Nicolis, Stamatios C; Perez-Escudero, Alfonso; Nanjundiah, Vidyanand; Sumpter, David J T; Dussutour, Audrey

    2015-11-22

    Historically, research has focused on the mean and often neglected the variance. However, variability in nature is observable at all scales: among cells within an individual, among individuals within a population and among populations within a species. A fundamental quest in biology now is to find the mechanisms that underlie variability. Here, we investigated behavioural variability in a unique unicellular organism, Physarum polycephalum. We combined experiments and models to show that variability in cell signalling contributes to major differences in behaviour underpinning some aspects of social interactions. First, following thousands of cells under various contexts, we identified distinct behavioural phenotypes: 'slow-regular-social', 'fast-regular-social' and 'fast-irregular-asocial'. Second, coupling chemical analysis and behavioural assays we found that calcium signalling is responsible for these behavioural phenotypes. Finally, we show that differences in signalling and behaviour led to alternative social strategies. Our results have considerable implications for our understanding of the emergence of variability in living organisms. © 2015 The Author(s).

  18. Phenotypic variability in unicellular organisms: from calcium signalling to social behaviour

    PubMed Central

    Vogel, David; Nicolis, Stamatios C.; Perez-Escudero, Alfonso; Nanjundiah, Vidyanand; Sumpter, David J. T.; Dussutour, Audrey

    2015-01-01

    Historically, research has focused on the mean and often neglected the variance. However, variability in nature is observable at all scales: among cells within an individual, among individuals within a population and among populations within a species. A fundamental quest in biology now is to find the mechanisms that underlie variability. Here, we investigated behavioural variability in a unique unicellular organism, Physarum polycephalum. We combined experiments and models to show that variability in cell signalling contributes to major differences in behaviour underpinning some aspects of social interactions. First, following thousands of cells under various contexts, we identified distinct behavioural phenotypes: ‘slow–regular–social’, ‘fast–regular–social’ and ‘fast–irregular–asocial’. Second, coupling chemical analysis and behavioural assays we found that calcium signalling is responsible for these behavioural phenotypes. Finally, we show that differences in signalling and behaviour led to alternative social strategies. Our results have considerable implications for our understanding of the emergence of variability in living organisms. PMID:26609088

  19. The Calcium Ion Is a Second Messenger in the Nitrate Signaling Pathway of Arabidopsis1

    PubMed Central

    Riveras, Eleodoro; Alvarez, José M.; Vidal, Elena A.; Oses, Carolina; Vega, Andrea; Gutiérrez, Rodrigo A.

    2015-01-01

    Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca2+ concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca2+ levels in response to nitrate treatments were blocked by U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog U73343. In addition, increase in cytoplasmic Ca2+ levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca2+ channel blockers or phospholipase C inhibitors. These results indicate that Ca2+ acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca2+ in response to nitrate required for changes in expression of prototypical nitrate-responsive genes. PMID:26304850

  20. Proline induces calcium-mediated oxidative burst and salicylic acid signaling.

    PubMed

    Chen, Jiugeng; Zhang, Yueqin; Wang, Cuiping; Lü, Weitao; Jin, Jing Bo; Hua, Xuejun

    2011-05-01

    Although free proline accumulation is a well-documented phenomenon in many plants in response to a variety of environmental stresses, and is proposed to play protective roles, high intracellular proline content, by either exogenous application or endogenous over-production, in the absence of stresses, is found to be inhibitory to plant growth. We have shown here that exogenous application of proline significantly induced intracellular Ca(2+) accumulation in tobacco and calcium-dependent ROS production in Arabidopsis seedlings, which subsequently enhanced salicylic acid (SA) synthesis and PR genes expression. This suggested that proline can promote a reaction similar to hypersensitive response during pathogen infection. Other amino acids, such as glutamate, but not arginine and phenylalanine, were also found to be capable of inducing PR gene expression. In addition, proline at concentration as low as 0.5 mM could induce PR gene expression. However, proline could not induce the expression of PDF1.2 gene, the marker gene for jasmonic acid signaling pathway. Furthermore, proline-induced SA production is mediated by NDR1-dependent signaling pathway, but not that mediated by PAD4. Our data provide evidences that exogenous proline, and probably some other amino acids can specifically induce SA signaling and defense response.

  1. Local calcium signals induced by hyper-osmotic stress in mammalian skeletal muscle cells.

    PubMed

    Apostol, Simona; Ursu, Daniel; Lehmann-Horn, Frank; Melzer, Werner

    2009-01-01

    Strenuous activitiy of skeletal muscle leads to temporary osmotic dysbalance and isolated skeletal muscle fibers exposed to osmotic stress respond with characteristic micro-domain calcium signals. It has been suggested that osmotic stress targets transverse tubular (TT) dihydropyridine receptors (DHPRs) which normally serve as voltage-dependent activators of Ca release via ryanodine receptor (RyR1s) of the sarcoplasmic reticulum (SR). Here, we pursued this hypothesis by imaging the response to hyperosmotic solutions in both mouse skeletal muscle fibers and myotubes. Ca fluctuations in the cell periphery of fibers exposed to osmotic stress were accompanied by a substantial dilation of the peripheral TT. The Ca signals were completely inhibited by a conditioning depolarization that inactivates the DHPR. Dysgenic myotubes, lacking the DHP-receptor-alpha1-subunit, showed strongly reduced, yet not completely inhibited activity when stimulated with solutions of elevated tonicity. The results point to a modulatory, even though not essential, role of the DHP receptor for osmotic stress-induced Ca signals in skeletal muscle.

  2. Neuronal MHC Class I Expression Is Regulated by Activity Driven Calcium Signaling

    PubMed Central

    Peng, Yaqin; Liu, Jiane; Miao, Fengqin; Zhang, Jianqiong

    2015-01-01

    MHC class I (MHC-I) molecules are important components of the immune system. Recently MHC-I have been reported to also play important roles in brain development and synaptic plasticity. In this study, we examine the molecular mechanism(s) underlying activity-dependent MHC-I expression using hippocampal neurons. Here we report that neuronal expression level of MHC-I is dynamically regulated during hippocampal development after birth in vivo. Kainic acid (KA) treatment significantly increases the expression of MHC-I in cultured hippocampal neurons in vitro, suggesting that MHC-I expression is regulated by neuronal activity. In addition, KA stimulation decreased the expression of pre- and post-synaptic proteins. This down-regulation is prevented by addition of an MHC-I antibody to KA treated neurons. Further studies demonstrate that calcium-dependent protein kinase C (PKC) is important in relaying KA simulation activation signals to up-regulated MHC-I expression. This signaling cascade relies on activation of the MAPK pathway, which leads to increased phosphorylation of CREB and NF-κB p65 while also enhancing the expression of IRF-1. Together, these results suggest that expression of MHC-I in hippocampal neurons is driven by Ca2+ regulated activation of the MAPK signaling transduction cascade. PMID:26263390

  3. Comparative biology of sperm factors and fertilization-induced calcium signals across the animal kingdom.

    PubMed

    Kashir, Junaid; Deguchi, Ryusaku; Jones, Celine; Coward, Kevin; Stricker, Stephen A

    2013-10-01

    Fertilization causes mature oocytes or eggs to increase their concentrations of intracellular calcium ions (Ca²⁺) in all animals that have been examined, and such Ca²⁺ elevations, in turn, provide key activating signals that are required for non-parthenogenetic development. Several lines of evidence indicate that the Ca²⁺ transients produced during fertilization in mammals and other taxa are triggered by soluble factors that sperm deliver into oocytes after gamete fusion. Thus, for a broad-based analysis of Ca²⁺ dynamics during fertilization in animals, this article begins by summarizing data on soluble sperm factors in non-mammalian species, and subsequently reviews various topics related to a sperm-specific phospholipase C, called PLCζ, which is believed to be the predominant activator of mammalian oocytes. After characterizing initiation processes that involve sperm factors or alternative triggering mechanisms, the spatiotemporal patterns of Ca²⁺ signals in fertilized oocytes or eggs are compared in a taxon-by-taxon manner, and broadly classified as either a single major transient or a series of repetitive oscillations. Both solitary and oscillatory types of fertilization-induced Ca²⁺ signals are typically propagated as global waves that depend on Ca²⁺ release from the endoplasmic reticulum in response to increased concentrations of inositol 1,4,5-trisphosphate (IP₃). Thus, for taxa where relevant data are available, upstream pathways that elevate intraoocytic IP3 levels during fertilization are described, while other less-common modes of producing Ca²⁺ transients are also examined. In addition, the importance of fertilization-induced Ca²⁺ signals for activating development is underscored by noting some major downstream effects of these signals in various animals.

  4. Calcium Signaling via Phospholipase C Is Essential for Proline Accumulation upon Ionic But Not Nonionic Hyperosmotic Stresses in Arabidopsis1

    PubMed Central

    Parre, Elodie; Ghars, Mohamed Ali; Leprince, Anne-Sophie; Thiery, Laurent; Lefebvre, Delphine; Bordenave, Marianne; Richard, Luc; Mazars, Christian; Abdelly, Chedly; Savouré, Arnould

    2007-01-01

    Proline (Pro) accumulation occurs in various plant organisms in response to environmental stresses. To identify the signaling components involved in the regulation of Pro metabolism upon water stress in Arabidopsis (Arabidopsis thaliana), a pharmacological approach was developed. The role of phosphoinositide-specific phospholipases C (PLCs) in Pro accumulation was assessed by the use of the aminosteroid U73122, a commonly employed specific inhibitor of receptor-mediated PLCs. We found that U73122 reduced pyrroline-5-carboxylate synthetase transcript and protein as well as Pro levels in salt-treated seedlings. Inhibition of PLC activity by U73122 was quantified by measuring the decrease of inositol 1,4,5-trisphosphate (InsP3) levels. Moreover, the utilization of diacylglycerol kinase and InsP3-gated calcium release receptor inhibitors suggested that InsP3 or its derivatives are essential for Pro accumulation upon salt stress, involving calcium as a second messenger in ionic stress signaling. This observation was further supported by a partial restoration of Pro accumulation in salt- and U73122-treated seedlings after addition of extracellular calcium, or when calcium homeostasis was perturbed by cyclopiazonic acid, a blocker of plant type IIA calcium pumps. Taken together, our data indicate that PLC-based signaling is a committed step in Pro biosynthesis upon salinity but not in the case of mannitol stress. Calcium acts as a molecular switch to trigger downstream signaling events. These results also demonstrated the specific involvement of lipid signaling pathway to discriminate between ionic and nonionic stresses. PMID:17369432

  5. Tight mitochondrial control of calcium and exocytotic signals in chromaffin cells at embryonic life.

    PubMed

    Vestring, Stefan; Fernández-Morales, José C; Méndez-López, Iago; C Musial, Diego; G de Diego, Antonio-Miguel; Padín, J Fernando; G García, Antonio

    2015-12-01

    Calcium buffering by mitochondria plays a relevant physiological function in the regulation of Ca(2+) and exocytotic signals in mature chromaffin cells (CCs) from various adult mammals. Whether a similar or different role of mitochondrial Ca(2+) buffering is present in immature CCs at early life has not been explored. Here we present a comparative study in rat embryonic CCs and rat mother CCs, of various physiological parameters that are known to be affected by mitochondrial Ca(2+) buffering during cell activation. We found that the clearance of cytosolic Ca(2+) transients ([Ca(2+)]c) elicited by high K(+) was 7-fold faster in embryo CCs compared to mother CCs. This strongly suggests that at embryonic life, the mitochondria play a more significant role in the clearance of [Ca(2+)]c loads compared to adult life. Consistent with this view are the following results concerning the transient suppression of mitochondrial Ca(2+) buffering by protonophore FCCP, in embryonic CCs compared to mother CCs: (i) faster and greater inactivation of inward calcium currents, (ii) higher K(+)-elicited [Ca(2+)]c transients with 25-fold faster clearance, (iii) higher increase of basal catecholamine release and (iv) higher potentiation of K(+)-evoked secretion. These pronounced differences could be explained by two additional features (embryo versus mother CCs): (a) slower recovery of mitochondrial resting membrane potential after the application of a transient FCCP pulse and (b) greater relative density of the mitochondria in the cytosol. This tighter control by the mitochondria of Ca(2+) and exocytotic signals may be relevant to secure a healthy catecholamine secretory response at early life.

  6. Mitochondrial fission forms a positive feedback loop with cytosolic calcium signaling pathway to promote autophagy in hepatocellular carcinoma cells.

    PubMed

    Huang, Qichao; Cao, Haiyan; Zhan, Lei; Sun, Xiacheng; Wang, Gang; Li, Jibin; Guo, Xu; Ren, Tingting; Wang, Zhe; Lyu, Yinghua; Liu, Bingrong; An, Jiaze; Xing, Jinliang

    2017-09-10

    Both mitochondrial morphology and the level of cytosolic calcium [Ca(2+)]c are actively changed and play critical roles in a number of malignancies. However, whether communications existed between these two processes to ingeniously control the malignant phenotype are far from clear. We investigated the reciprocal regulation between mitochondrial fission and cytosolic calcium signaling in human hepatocellular carcinoma (HCC) cells. Furthermore, the underlying molecular mechanisms and the synergistic effect on autophagy were explored. Our results showed that mitochondrial fission increased the [Ca(2+)]c and calcium oscillation in HCC cells. We further found that mitochondrial fission-mediated calcium signaling was dependent on ROS-activated NF-κB pathways, which facilitated the expression of STIM1 and subsequent store-operated calciumentry. Additionally, we also demonstrated that increase in [Ca(2+)]c promoted mitochondrial fission by up-regulating expression of Drp1 and FIS1 via transcription factors NFATC2 and c-Myc, respectively. Moreover, the positive feedback loop significantly promoted HCC cell global autophagy by Ca(2+)/CAMKK/AMPK pathway. Our data demonstrate a positive feedback loop between mitochondrial fission and cytosolic calcium signaling and their promoting role in autophagy of HCC cells, which provides evidence for this loop as a potential drug target in tumor treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Spatiotemporal Properties of Intracellular Calcium Signaling in Osteocytic and Osteoblastic Cell Networks under Fluid Flow

    PubMed Central

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

    2013-01-01

    Mechanical stimuli can trigger intracellular calcium (Ca2+) responses in osteocytes and osteoblasts. Successful construction of bone cell networks necessitates more elaborate and systematic analysis for the spatiotemporal properties of Ca2+ signaling in the networks. In the present study, an unsupervised algorithm based on independent component analysis (ICA) was employed to extract the Ca2+ 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 Ca2+ 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 Ca2+ transients than MC3T3-E1 cells, evidenced by more Ca2+ 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 Ca2+ oscillating frequency. The spatial intercellular synchronous activities of Ca2+ 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 Ca2+ 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 Ca2+ signaling extraction in the cell networks. The present study also revealed a dramatic spatiotemporal difference in Ca2+ signaling for osteocytic and osteoblastic cell networks in processing the mechanical stimulus. The higher intracellular Ca2+ 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 processes. PMID:23328496

  8. Role of local vitamin D signaling and cellular calcium transport system in bone homeostasis.

    PubMed

    Masuyama, Ritsuko

    2014-01-01

    Mouse genetic studies have demonstrated that the 1,25-dihydroxyvitamin D [1,25(OH)2D] endocrine system is required for calcium (Ca(2+)) and bone homeostasis. These studies reported severe hypocalcemia and impaired bone mineralization associated with rickets in mutant mice. Specific phenotypes of these mice with an engineered deletion of 1,25(OH)2D cell signaling resemble the features observed in humans with the same congenital disease or severe 1,25(OH)2D deficiency. Decreased active intestinal Ca(2+) absorption because of reduced expression of epithelial Ca(2+) channels is a crucial mechanism that contributes to the major phenotypes observed in the mutant mice. The importance of intestinal Ca(2+) absorption supported by 1,25(OH)2D-mediated transport was further emphasized by the observation that Ca(2+) supplementation rescues hypocalcemia and restores bone mineralization in both patients and mice lacking 1,25(OH)2D signaling. This observation questions the direct role of 1,25(OH)2D signaling in bone tissue. Studies regarding tissue-specific manipulation of 1,25(OH)2D function have provided a consensus on this issue by demonstrating a direct action of 1,25(OH)2D on cells in bone tissue through bone metabolism and mineral homeostasis. In addition, movement of Ca(2+) from the bone as a result of osteoclastic bone resorption also provides a large Ca(2+) supply in Ca(2+) homeostasis; however, the system controlling Ca(2+) homeostasis in osteoclasts has not been fully identified. Transient receptor potential vanilloid (TRPV) 4 mediates Ca(2+) influx during the late stage of osteoclast differentiation, thereby regulating the Ca(2+) signaling essential for cellular events during osteoclast differentiation; however, the system-modifying effect of TRPV4 activity should be determined. Furthermore, it remains unknown how local Ca(2+) metabolism participates in systemic Ca(2+) homeostasis through bone remodeling. New insights are therefore required to understand this issue.

  9. Role of Orai1 and store-operated calcium entry in mouse lacrimal gland signalling and function

    PubMed Central

    Xing, Juan; Petranka, John G; Davis, Felicity M; Desai, Pooja N; Putney, James W; Bird, Gary S

    2014-01-01

    Lacrimal glands function to produce an aqueous layer, or tear film, that helps to nourish and protect the ocular surface. Lacrimal glands secrete proteins, electrolytes and water, and loss of gland function can result in tear film disorders such as dry eye syndrome, a widely encountered and debilitating disease in ageing populations. To combat these disorders, understanding the underlying molecular signalling processes that control lacrimal gland function will give insight into corrective therapeutic approaches. Previously, in single lacrimal cells isolated from lacrimal glands, we demonstrated that muscarinic receptor activation stimulates a phospholipase C-coupled signalling cascade involving the inositol trisphosphate-dependent mobilization of intracellular calcium and the subsequent activation of store-operated calcium entry (SOCE). Since intracellular calcium stores are finite and readily exhausted, the SOCE pathway is a critical process for sustaining and maintaining receptor-activated signalling. Recent studies have identified the Orai family proteins as critical components of the SOCE channel activity in a wide variety of cell types. In this study we characterize the role of Orai1 in the function of lacrimal glands using a mouse model in which the gene for the calcium entry channel protein, Orai1, has been deleted. Our data demonstrate that lacrimal acinar cells lacking Orai1 do not exhibit SOCE following activation of the muscarinic receptor. In comparison with wild-type and heterozygous littermates, Orai1 knockout mice showed a significant reduction in the stimulated tear production following injection of pilocarpine, a muscarinic receptor agonist. In addition, calcium-dependent, but not calcium-independent exocytotic secretion of peroxidase was eliminated in glands from knockout mice. These studies indicate a critical role for Orai1-mediated SOCE in lacrimal gland signalling and function. PMID:24297846

  10. Role of Orai1 and store-operated calcium entry in mouse lacrimal gland signalling and function.

    PubMed

    Xing, Juan; Petranka, John G; Davis, Felicity M; Desai, Pooja N; Putney, James W; Bird, Gary S

    2014-03-01

    Lacrimal glands function to produce an aqueous layer, or tear film, that helps to nourish and protect the ocular surface. Lacrimal glands secrete proteins, electrolytes and water, and loss of gland function can result in tear film disorders such as dry eye syndrome, a widely encountered and debilitating disease in ageing populations. To combat these disorders, understanding the underlying molecular signalling processes that control lacrimal gland function will give insight into corrective therapeutic approaches. Previously, in single lacrimal cells isolated from lacrimal glands, we demonstrated that muscarinic receptor activation stimulates a phospholipase C-coupled signalling cascade involving the inositol trisphosphate-dependent mobilization of intracellular calcium and the subsequent activation of store-operated calcium entry (SOCE). Since intracellular calcium stores are finite and readily exhausted, the SOCE pathway is a critical process for sustaining and maintaining receptor-activated signalling. Recent studies have identified the Orai family proteins as critical components of the SOCE channel activity in a wide variety of cell types. In this study we characterize the role of Orai1 in the function of lacrimal glands using a mouse model in which the gene for the calcium entry channel protein, Orai1, has been deleted. Our data demonstrate that lacrimal acinar cells lacking Orai1 do not exhibit SOCE following activation of the muscarinic receptor. In comparison with wild-type and heterozygous littermates, Orai1 knockout mice showed a significant reduction in the stimulated tear production following injection of pilocarpine, a muscarinic receptor agonist. In addition, calcium-dependent, but not calcium-independent exocytotic secretion of peroxidase was eliminated in glands from knockout mice. These studies indicate a critical role for Orai1-mediated SOCE in lacrimal gland signalling and function.

  11. Calcium spike-mediated digital signaling increases glutamate output at the visual threshold of retinal bipolar cells.

    PubMed

    Lipin, Mikhail Y; Vigh, Jozsef

    2015-01-15

    Most retinal bipolar cells (BCs) transmit visual input from photoreceptors to ganglion cells using graded potentials, but some also generate calcium or sodium spikes. Sodium spikes are thought to increase temporal precision of light-evoked BC signaling; however, the role of calcium spikes in BCs is not fully understood. Here we studied how calcium spikes and graded responses mediate neurotransmitter release from Mb-type BCs, known to produce both. In dark-adapted goldfish retinal slices, light induced spikes in 40% of the axon terminals of intact Mbs; in the rest, light generated graded responses. These light-evoked membrane potentials were used to depolarize axotomized Mb terminals where depolarization-evoked calcium current (ICa) and consequent exocytosis-associated membrane capacitance increases (ΔCm) could be precisely measured. When evoked by identical dim light intensities, spiking responses transferred more calcium (Q(Ca)) and triggered larger exocytosis with higher efficiency (ΔCm/Q(Ca)) than graded potentials. Q(Ca) was translated into exocytosis linearly when transferred with spikes and supralinearly when transferred with graded responses. At the Mb output (ΔCm), spiking responses coded light intensity with numbers and amplitude whereas graded responses coded with amplitude, duration, and steepness. Importantly, spiking responses saturated exocytosis within scotopic range but graded potentials did not. We propose that calcium spikes in Mbs increase signal input-output ratio by boosting Mb glutamate release at threshold intensities. Therefore, spiking Mb responses are suitable to transfer low-light-intensity signals to ganglion cells with higher gain, whereas graded potentials signal for light over a wider range of intensities at the Mb output. Copyright © 2015 the American Physiological Society.

  12. Calcium spike-mediated digital signaling increases glutamate output at the visual threshold of retinal bipolar cells

    PubMed Central

    Lipin, Mikhail Y.

    2014-01-01

    Most retinal bipolar cells (BCs) transmit visual input from photoreceptors to ganglion cells using graded potentials, but some also generate calcium or sodium spikes. Sodium spikes are thought to increase temporal precision of light-evoked BC signaling; however, the role of calcium spikes in BCs is not fully understood. Here we studied how calcium spikes and graded responses mediate neurotransmitter release from Mb-type BCs, known to produce both. In dark-adapted goldfish retinal slices, light induced spikes in 40% of the axon terminals of intact Mbs; in the rest, light generated graded responses. These light-evoked membrane potentials were used to depolarize axotomized Mb terminals where depolarization-evoked calcium current (ICa) and consequent exocytosis-associated membrane capacitance increases (ΔCm) could be precisely measured. When evoked by identical dim light intensities, spiking responses transferred more calcium (QCa) and triggered larger exocytosis with higher efficiency (ΔCm/QCa) than graded potentials. QCa was translated into exocytosis linearly when transferred with spikes and supralinearly when transferred with graded responses. At the Mb output (ΔCm), spiking responses coded light intensity with numbers and amplitude whereas graded responses coded with amplitude, duration, and steepness. Importantly, spiking responses saturated exocytosis within scotopic range but graded potentials did not. We propose that calcium spikes in Mbs increase signal input-output ratio by boosting Mb glutamate release at threshold intensities. Therefore, spiking Mb responses are suitable to transfer low-light-intensity signals to ganglion cells with higher gain, whereas graded potentials signal for light over a wider range of intensities at the Mb output. PMID:25339710

  13. The clinical significance of calcium-signalling pathways mediating human sperm hyperactivation

    PubMed Central

    Alasmari, Wardah; Barratt, Christopher L.R.; Publicover, Stephen J.; Whalley, Katherine M.; Foster, Erica; Kay, Vanessa; Martins da Silva, Sarah; Oxenham, Senga K.

    2013-01-01

    STUDY QUESTION What is the prevalence of defects in the Ca2+-signalling pathways mediating hyperactivation (calcium influx and store mobilization) among donors and sub-fertile patients and are they functionally significant, i.e. related to fertilization success at IVF? SUMMARY ANSWER This study identifies, for the first time, the prevalence of Ca2+ store defects in sperm from research donors, IVF and ICSI patients. It highlights the biological role and importance of Ca2+ signalling (Ca2+ store mobilization) for fertilization at IVF. WHAT IS KNOWN ALREADY Sperm motility and hyperactivation (HA) are important for fertility, mice with sperm incapable of HA are sterile. Recently, there has been significant progress in our knowledge of the factors controlling these events, in particular the generation and regulation of calcium signals. Both pH-regulated membrane Ca2+ channels (CatSper) and Ca2+ stores (potentially activating store-operated Ca2+ channels) have been implicated in controlling HA. STUDY DESIGN, SIZE, AND DURATION This was a prospective study examining a panel of 68 donors and 181 sub-fertile patients attending the Assisted Conception Unit, Ninewells Hospital Dundee for IVF and ICSI. Twenty-five of the donors gave a second sample (∼4 weeks later) to confirm consistency/reliability of the recorded responses. Ca2+ signalling was manipulated using three agonists, NH4Cl (activates CatSper via pH), progesterone (direct activation of CatSper channels, potentially enhancing mobilization of stored Ca2+ by CICR) and 4-aminopyridine (4-AP) (effect on pH equivalent to NH4Cl and mobilizes stored Ca2+). The broad-spectrum phosphodiesterase inhibitor 3-isobutyl-1-methyxanthine (IBMX), a potent activator of HA was also used for comparison. For patient samples, an aliquot surplus to requirements for IVF/ICSI treatment was examined, allowing direct comparison of Ca2+ signalling and motility data with functional competence of the sperm. MATERIALS, SETTING, METHODS The

  14. 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

  15. Calcium signaling in live cells on elastic gels under mechanical vibration at subcellular levels.

    PubMed

    Nishitani, Wagner Shin; Saif, Taher A; Wang, Yingxiao

    2011-01-01

    A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels.

  16. Calcium Signaling in Live Cells on Elastic Gels under Mechanical Vibration at Subcellular Levels

    PubMed Central

    Nishitani, Wagner Shin; Saif, Taher A.; Wang, Yingxiao

    2011-01-01

    A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels. PMID:22053183

  17. Physiology and cell biology of acupuncture observed in calcium signaling activated by acoustic shear wave.

    PubMed

    Li, Geng; Liang, Jie-Ming; Li, Pei-Wen; Yao, Xiaoqiang; Pei, Peter Zhong; Li, Wei; He, Qi-Hua; Yang, Xifei; Chan, Queenie C C; Cheung, Paul Y S; Ma, Qi Yuan; Lam, Siu Kam; Cheng, Patrick Y C; Yang, Edward S

    2011-10-01

    This article presents a novel model of acupuncture physiology based on cellular calcium activation by an acoustic shear wave (ASW) generated by the mechanical movement of the needle. An acupuncture needle was driven by a piezoelectric transducer at 100 Hz or below, and the ASW in human calf was imaged by magnetic resonance elastography. At the cell level, the ASW activated intracellular Ca(2+) transients and oscillations in fibroblasts and endothelial, ventricular myocytes and neuronal PC-12 cells along with frequency-amplitude tuning and memory capabilities. Monitoring in vivo mammalian experiments with ASW, enhancement of endorphin in blood plasma and blocking by Gd(3+) were observed; and increased Ca(2+) fluorescence in mouse hind leg muscle was imaged by two-photon microscopy. In contrast with traditional acupuncture models, the signal source is derived from the total acoustic energy. ASW signaling makes use of the anisotropy of elasticity of tissues as its waveguides for transmission and that cell activation is not based on the nervous system.

  18. Neuronal calcium signaling pathways are associated with the development of epilepsy.

    PubMed

    Meng, Fanxin; You, Yu; Liu, Zhiliang; Liu, Jianming; Ding, Hu; Xu, Ruxiang

    2015-01-01

    Epilepsy is the most common serious neurological disorder worldwide, however, the specific causative factors and mechanisms underlying epilepsy remain unclear. The current study aimed to study the potential genes or pathways associated with epilepsy, based on rat miRNA expression profiles. The microarray dataset GSE49850 was downloaded and analyzed with the TimeCourse R software package, which was used to generate comparisons between the control and electrically-stimulated groups. The target genes of differentially expressed miRNAs were queried in the miRWalk database and functional enrichment was conducted using the Database for Annotation, Visualization and Integrated Discovery software tools. The interaction network of the target genes was constructed based on the Biomolecular Interaction Network Database and clustered using ClusterONE. In total, 152 differentially expressed miRNAs were identified, with rno-miR-21-5p being the most significantly differentially expressed. A total of 526 target genes of the differentially expressed miRNAs were obtained. Functional analysis indicated that these genes were predominantly involved in responses to stimuli. The interaction network showed that the GRIN and STX gene family, which are involved in synaptic signal transmission, were significant. In conclusion, the present study identified that the development of epilepsy was closely associated with neuronal calcium signaling pathways.

  19. Calcium Signaling of Lysophosphatidylethanolamine through LPA1 in Human SH-SY5Y Neuroblastoma Cells

    PubMed Central

    Lee, Jung-Min; Park, Soo-Jin; Im, Dong-Soon

    2017-01-01

    Lysophosphatidylethanolamine (LPE), a lyso-type metabolite of phosphatidylethanolamine, has been reported to be an intercellular signaling molecule. LPE mobilizes intracellular Ca2+ through G-protein-coupled receptor (GPCR) in some cells types. However, GPCRs for lysophosphatidic acid (LPA) were not implicated in the LPE-mediated activities in LPA GPCR overexpression systems or in SK-OV3 ovarian cancer cells. In the present study, in human SH-SY5Y neuroblastoma cells, experiments with LPA1 antagonists showed LPE induced intracellular Ca2+ increases in an LPA1 GPCR-dependent manner. Furthermore, LPE increased intracellular Ca2+ through pertussis-sensitive G proteins, edelfosine-sensitive-phospholipase C, 2-APB-sensitive IP3 receptors, Ca2+ release from intracellular Ca2+ stores, and subsequent Ca2+ influx across plasma membranes, and LPA acted on LPA1 and LPA2 receptors to induce Ca2+ response in a 2-APB-sensitive and insensitive manner. These findings suggest novel involvements for LPE and LPA in calcium signaling in human SH-SY5Y neuroblastoma cells. PMID:27302965

  20. Calcium Signaling of Lysophosphatidylethanolamine through LPA1 in Human SH-SY5Y Neuroblastoma Cells.

    PubMed

    Lee, Jung-Min; Park, Soo-Jin; Im, Dong-Soon

    2017-03-01

    Lysophosphatidylethanolamine (LPE), a lyso-type metabolite of phosphatidylethanolamine, has been reported to be an intercellular signaling molecule. LPE mobilizes intracellular Ca(2+) through G-protein-coupled receptor (GPCR) in some cells types. However, GPCRs for lysophosphatidic acid (LPA) were not implicated in the LPE-mediated activities in LPA GPCR overexpression systems or in SK-OV3 ovarian cancer cells. In the present study, in human SH-SY5Y neuroblastoma cells, experiments with LPA1 antagonists showed LPE induced intracellular Ca(2+) increases in an LPA1 GPCR-dependent manner. Furthermore, LPE increased intracellular Ca(2+) through pertussis-sensitive G proteins, edelfosine-sensitive-phospholipase C, 2-APB-sensitive IP3 receptors, Ca(2+) release from intracellular Ca(2+) stores, and subsequent Ca(2+) influx across plasma membranes, and LPA acted on LPA1 and LPA2 receptors to induce Ca(2+) response in a 2-APB-sensitive and insensitive manner. These findings suggest novel involvements for LPE and LPA in calcium signaling in human SH-SY5Y neuroblastoma cells.

  1. Computational modeling of calcium signaling from the nanoscale to multicellular systems

    NASA Astrophysics Data System (ADS)

    Ullah, Ghanim

    Calcium signaling is one of the most important signaling mechanisms controlling e.g. the contraction of muscle cells, the release of neurotransmitter from neurons and astrocytes, transcription inside the nucleus and metabolic processes in liver and pancreas [8, 44, 36]. Due to the general importance in cell biology, Ca2+ signals of a variety of forms have been the subject of much recent experimental research. A recent and particularly powerful approach towards the understanding of Ca2+ signaling is the combination of highly resolved fluorescent imaging methods and detailed mathematical modeling. Models for Ca2+ signaling are probably the most advanced and realistic modes in all areas of biological physics. Hence theoretical predictions are quantitative in nature and allow direct comparison with experiments. Ca2+ signaling patterns exhibit a hierarchical structure varying from single-channel release events (10's of nanometers) to Ca2+ waves sweeping over entire organs like the liver to globally orchestrate the efficient release of enzymes [48]. This multi-scale organization renders it an ideal tool for studying basic concepts of pattern formation, especially since access to the most important experimental parameters is given. The aim of this dissertation is to develop mathematical models that quantitatively describe the characteristics of elementary Ca2+ elements (called Ca2+ -puffs) on the nano-scale as well as the organization of global waves and oscillations on the cell and organ scale. We used oocytes, eggs and astrocytes as model cells for our theoretical studies. Particularly on the microscopic scale we report significant progress in modeling Ca 2+ release events that are accurate in time course and spatial shape. Experimental investigations have revealed recently that Ca 2+ signaling differentiates during the development of oocytes into mature eggs. The fertilization specific Ca2+ signal in mature eggs is characterized by a fast rise of intracellular Ca2+ and

  2. The endocytic receptor protein LRP also mediates neuronal calcium signaling via N-methyl-d-aspartate receptors

    PubMed Central

    Bacskai, B. J.; Xia, M. Q.; Strickland, D. K.; Rebeck, G. W.; Hyman, B. T.

    2000-01-01

    The low density lipoprotein receptor-related protein (LRP) is an endocytic receptor that is a member of the low density lipoprotein receptor family. We report that the LRP ligand, activated α2-macroglobulin (α2M*), induces robust calcium influx in cultured primary neurons, but not in nonneuronal LRP-containing cells in the same culture. The calcium influx is mediated through N-methyl-d-aspartate receptor channels, which explains the neuron specificity of the response. Microapplication of α2M* leads to a localized response at the site of application that dissipates rapidly, suggesting that the calcium signal is temporally and spatially discrete. Calcium influx to α2M* is blocked by the physiological LRP inhibitor, receptor-associated protein. Bivalent antibodies to the extracellular domain of LRP, but not Fab fragments of the same antibody, cause calcium influx, indicating that the response is specific to LRP and may require dimerization of the receptor. Thus, LRP is an endocytic receptor with a novel signaling role. PMID:11016955

  3. Internal calcium release and activation of sea urchin eggs by cGMP are independent of the phosphoinositide signaling pathway.

    PubMed Central

    Whalley, T; McDougall, A; Crossley, I; Swann, K; Whitaker, M

    1992-01-01

    We show that microinjecting cyclic GMP (cGMP) into unfertilized sea urchin eggs activates them by stimulating a rise in the intracellular free calcium ion concentration ([Ca2+]i). The increase in [Ca2+]i is similar in both magnitude and duration to the transient that activates the egg at fertilization. It is due to mobilization of calcium from intracellular stores but is not prevented by the inositol trisphosphate (InsP3) antagonist heparin. Furthermore, cGMP does not stimulate the eggs Na+/H+ antiport when the [Ca2+]i transient is blocked by the calcium chelator bis-(O-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), suggesting that cGMP does not activate eggs by interacting with the their phosphoinositide signaling pathway. However, the [Ca2+]i increase and activation are prevented in eggs in which the InsP3-sensitive calcium stores have been emptied by the prior microinjection of the InsP3 analogue inositol 1,4,5-trisphosphorothioate. These data indicate that cGMP activates eggs by stimulating the release of calcium from an InsP3-sensitive calcium store via a novel, though unidentified, route independent of the InsP3 receptor. PMID:1320962

  4. Impact of Acrylamide on Calcium Signaling and Cytoskeletal Filaments in Testes From F344 Rat.

    PubMed

    Recio, Leslie; Friedman, Marvin; Marroni, Dennis; Maynor, Timothy; Chepelev, Nikolai L

    Acrylamide (AA) at high exposure levels is neurotoxic, induces testicular toxicity, and increases dominant lethal mutations in rats. RNA-sequencing in testes was used to identify differentially expressed genes (DEG), explore AA-induced pathway perturbations that could contribute to AA-induced testicular toxicity and then used to derive a benchmark dose (BMD). Male F344/DuCrl rats were administered 0.0, 0.5, 1.5, 3.0, 6.0, or 12.0 mg AA/kg bw/d in drinking water for 5, 15, or 31 days. The experimental design used exposure levels that spanned and exceeded the exposure levels used in the rat dominant lethal, 2-generation reproductive toxicology, and cancer bioassays. The time of sample collection was based on previous studies that developed gene expression-based BMD. At 12.0 mg/kg, there were 38, 33, and 65 DEG ( P value <.005; fold change >1.5) in the testes after 5, 15, or 31 days of exposure, respectively. At 31 days, there was a dose-dependent increase in the number of DEG, and at 12.0 mg/kg/d the top three functional clusters affected by AA exposure were actin filament organization, response to calcium ion, and regulation of cell proliferation. The BMD lower 95% confidence limit using DEG ranged from 1.8 to 6.8 mg/kg compared to a no-observed-adverse-effect-level of 2.0 mg/kg/d for male reproductive toxicity. These results are consistent with the known effects of AA on calcium signaling and cytoskeletal actin filaments leading to neurotoxicity and suggest that AA can cause rat dominant lethal mutations by these same mechanisms leading to impaired chromosome segregation during cell division.

  5. Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions.

    PubMed

    Liu, Yi; Gianinazzi-Pearson, Vivienne; Arnould, Christine; Wipf, Daniel; Zhao, Bin; van Tuinen, Diederik

    2013-01-01

    Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca(2+) in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca(2+)-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca(2+)-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca(2+) ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca(2+)-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots. Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  6. The Calcium Ion Is a Second Messenger in the Nitrate Signaling Pathway of Arabidopsis.

    PubMed

    Riveras, Eleodoro; Alvarez, José M; Vidal, Elena A; Oses, Carolina; Vega, Andrea; Gutiérrez, Rodrigo A

    2015-10-01

    Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca(2+) concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca(2+) levels in response to nitrate treatments were blocked by U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog U73343. In addition, increase in cytoplasmic Ca(2+) levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca(2+) channel blockers or phospholipase C inhibitors. These results indicate that Ca(2+) acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca(2+) in response to nitrate required for changes in expression of prototypical nitrate-responsive genes. © 2015 American Society of Plant Biologists. All Rights Reserved.

  7. Calcium influx-mediated signaling is required for complete mouse egg activation.

    PubMed

    Miao, Yi-Liang; Stein, Paula; Jefferson, Wendy N; Padilla-Banks, Elizabeth; Williams, Carmen J

    2012-03-13

    Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca(2+)) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular stores. We tested the hypothesis that Ca(2+) influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca(2+) source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca(2+) influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca(2+) occurred, in the absence of Ca(2+) influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca(2+) chelator, BAPTA/AM, demonstrated that Ca(2+) influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca(2+) transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca(2+)-free medium remained arrested in metaphase II. Inhibition of store-operated Ca(2+) entry had no effect on ICSI-induced egg activation, so Ca(2+) influx through alternative channels must participate in egg activation signaling. Ca(2+) influx appears to be upstream of CaMKIIγ activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKIIγ in the absence of extracellular Ca(2+). These results suggest that Ca(2+) influx at fertilization not only maintains Ca(2+) oscillations by replenishing Ca(2+) stores, but also activates critical signaling pathways upstream of CaMKIIγ that are required for second polar body emission.

  8. Retinoic Acid Regulates Calcium Signaling to Promote Mouse Ovarian Granulosa Cell Proliferation.

    PubMed

    Demczuk, Michael; Huang, Huiya; White, Carl; Kipp, Jingjing L

    2016-09-01

    Normal development of ovarian follicles is critical for female reproduction and endocrine function. We have identified retinoic acid (RA) and the RA-degrading enzyme CYP26B1 as regulators of ovarian follicle development and showed that RA and a CYP26 inhibitor stimulated ovarian granulosa cell proliferation. The mechanism underpinning RA-dependent proliferation, however, is not known. The current study was designed to examine the role of intracellular calcium (Ca(2+)) signaling in mediating the effects of RA on primary mouse granulosa cell proliferation. In single-cell Ca(2+) imaging experiments, treatment of cultured granulosa cells with RA increased the steady-state Ca(2+) content of the endoplasmic reticulum (ER) stores. This correlated with increased store-operated Ca(2+) entry (SOCE) and enhanced inositol 1,4,5-trisphosphate receptor (IP3R)-dependent Ca(2+) release. In proliferation assays, RA treatment or Cyp26b1 knockdown stimulated proliferation, whereas Cyp26b1 overexpression inhibited proliferation. When RA was given together with 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3R-dependent ER Ca(2+) release and SOCE, with xestospongin C, a selective IP3R- receptor antagonist, or with 3,5-bis (trifluoromethyl)pyrazole (BTP-2), a specific SOCE blocker, the stimulatory effect of RA on cell proliferation was abolished. Further investigation showed that treatment with 2-APB or BTP-2 inhibited RA induction of RA response element (RARE) activation in granulosa cells, confirming an important role for Ca(2+) signaling in mediating RA actions. Overall, these data support a model in which RA regulates ovarian follicle development by stimulating granulosa cell proliferation and that this stimulatory effect is at least in part driven by the modulation of Ca(2+) signaling.

  9. Calcium influx-mediated signaling is required for complete mouse egg activation

    PubMed Central

    Miao, Yi-Liang; Stein, Paula; Jefferson, Wendy N.; Padilla-Banks, Elizabeth; Williams, Carmen J.

    2012-01-01

    Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca2+) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular stores. We tested the hypothesis that Ca2+ influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca2+ source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca2+ influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca2+ occurred, in the absence of Ca2+ influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca2+ chelator, BAPTA/AM, demonstrated that Ca2+ influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca2+ transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca2+-free medium remained arrested in metaphase II. Inhibition of store-operated Ca2+ entry had no effect on ICSI-induced egg activation, so Ca2+ influx through alternative channels must participate in egg activation signaling. Ca2+ influx appears to be upstream of CaMKIIγ activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKIIγ in the absence of extracellular Ca2+. These results suggest that Ca2+ influx at fertilization not only maintains Ca2+ oscillations by replenishing Ca2+ stores, but also activates critical signaling pathways upstream of CaMKIIγ that are required for second polar body emission. PMID:22371584

  10. Signal percolation through plants and the shape of the calcium signature.

    PubMed

    Plieth, Christoph

    2010-04-01

    Plants respond to almost any kind of external stimulus with transients in their cytoplasmic free calcium concentration ([Ca(2+)](c)). A huge variety of kinetics recorded by optical techniques has been reported in the past. This variety has been credited the specificity needed to explain how information about incoming stimuli is evaluated by the organism and turned into the right physiological responses which provide advantages for survival and reproduction. A physiological response often takes place away from the site of stimulation. This requires cell-to-cell communication. Hence, responding cells are not necessarily directly stimulated but rather receive an indirect stimulus via cell-to-cell communication. It appears unlikely that the '[Ca(2+)](c) signature' in the primarily stimulated cell is conveyed over long distances via cell-to-cell communication from the 'receptor cells' to the 'effector cells'. Here, a novel aspect is highlighted to explain the variety of [Ca(2+)] kinetics seen by integrating methods of [Ca(2+)](c) recording. Plants can generally be seen as cellular automata with specific morphology and capable for cell-to-cell communication. Just a few rules are needed to demonstrate how waves of [Ca(2+)](c)-increases percolate through the organism and thereby deliver a broad variety of 'signatures'. Modelling intercellular signalling may be a possible way to find explanations for different kinds of signal transmission, signal amplification, wave formation, oscillations and stimulus-response coupling. The basic examples presented here show that care has to be taken when interpreting cellular '[Ca(2+)](c) signatures' recorded by optical techniques which integrate over a big number of cells or even whole plants.

  11. PMCA2 VIA PSD-95 CONTROLS CALCIUM SIGNALING BY α7-CONTAINING NICOTINIC ACETYLCHOLINE RECEPTORS ON ASPINY INTERNEURONS

    PubMed Central

    Gomez-Varela, David; Schmidt, Manuela; Schoellerman, Jeff; Peters, Eric C.; Berg, Darwin K.

    2012-01-01

    Local control of calcium concentration within neurons is critical for signaling and regulation of synaptic communication in neural circuits. How local control can be achieved in the absence of physical compartmentalization is poorly understood. Challenging examples are provided by nicotinic acetylcholine receptors that contain α7 nicotinic receptor subunits (α7-nAChRs). These receptors are highly permeable to calcium and are concentrated on aspiny dendrites of interneurons which lack obvious physical compartments for constraining calcium diffusion. Using functional proteomics on rat brain, we show that α7-nAChRs are associated with the membrane calcium pump PMCA2. Analysis of α7-nAChR function in hippocampal interneurons in culture shows that PMCA2 activity limits the duration of calcium elevations produced by the receptors. Unexpectedly, PMCA2 inhibition triggers rapid calcium-dependent loss of α7-nAChR clusters. This extreme regulatory response is mediated by CaMKII, involves proteasome activity, depends on the second intracellular loop of α7-nAChR subunits, and is specific in that it does not alter two other classes of calcium-permeable ionotropic receptors on the same neurons. A critical link is provided by the scaffold protein PSD-95, which is associated with α7-nAChRs and constrains their mobility as revealed by single-particle tracking on neurons. The PSD-95 link is required for PMCA2-mediated removal of α7-nAChR clusters. This three-component combination of PMCA2/PSD-95/α7-nAChR offers a novel mechanism for tight control of calcium dynamics in neurons. PMID:22593058

  12. The APP670/671 mutation alters calcium signaling and response to hyperosmotic stress in rat primary hippocampal neurons.

    PubMed

    Kloskowska, Ewa; Bruton, Joseph D; Winblad, Bengt; Benedikz, Eirikur

    2008-10-31

    Altered calcium homeostasis is implicated in the pathogenesis of Alzheimer's disease and much effort has been put into understanding the association between the autosomal dominant gene mutations causative of this devastating disease and perturbed calcium signaling. We have focused our attention on the effect of the APP670/671 mutation on spontaneous calcium oscillations in embryonic hippocampal neurons derived from the tg6590 transgenic rat. Intracellular free calcium levels were imaged by confocal microscopy using the fluorescent dye fluo-3AM. Hyperosmotic shrinkage, which can occur in a variety of pathophysiological conditions, has been shown to induce multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. When exposed to hyperosmotic stress (addition of 50mM sucrose) the frequency of calcium oscillations was suppressed to an equal extent in both wild-type and transgenic cultures, but the transgenic neurons, in contrast to the wild-type neurons, responded with a significantly higher increase in the amplitude of oscillations. A decrease in cell viability was observed by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay in neurons exposed to the hyperosmotic medium for 30h. Whereas this loss in cell viability was comparable in both sets of cultures, the amplitude of calcium oscillations in transgenic neurons exhibited a significantly greater decrease in the presence of the L-type calcium channel antagonist, nimodipine. These results suggest that APP670/671 transgenic neurons have impaired calcium homeostasis.

  13. Effects of Sclerocarya birrea (A. rich) hochst (anacardiaceae) leaf extracts on calcium signalling in cultured rat skeletal muscle cells.

    PubMed

    Belemtougri, R G; Constantin, B; Cognard, C; Raymond, G; Sawadogo, L

    2001-08-01

    Sclerocarya birrea is a plant used widely to treat many diseases in Burkina Faso, although no scientific data has been reported about its mechanism of action. In the present study the effects of its leaf extracts were investigated on calcium signalling in rat cultured skeletal muscle cells. The results show that the different extracts (crude decoction, aqueous, ethanolic and chloroformic extracts) have significant antagonistic effect on caffeine-induced calcium release from sarcoplasmic reticulum. Crude decoction is the most active followed by ethanolic, aqueous and chloroformic extracts in dose-dependent manner and can partly justify the use of the plant in traditional medicine.

  14. Wnt11-R signaling regulates a calcium sensitive EMT event essential for dorsal fin development of Xenopus

    PubMed Central

    Garriock, Robert J.; Krieg, Paul A.

    2007-01-01

    In the frog embryo, a sub-population of trunk neural crest (NC) cells undergoes a dorsal route of migration to contribute to the mesenchyme in the core of the dorsal fin. Here we show that a second population of cells, originally located in the dorsomedial region of the somite, also contributes to the fin mesenchyme. We find that the frog orthologue of Wnt11 (Wnt11-R) is expressed in both the NC and somite cell populations that migrate into the fin matrix. Wnt11-R is expressed prior to migration and persists in the mesenchymal cells after they have distributed throughout the fin. Loss of function studies demonstrate that Wnt11-R activity is required for an epithelial to mesenchymal transformation (EMT) event that precedes migration of cells into the fin matrix. In Wnt11-R depleted embryos, the absence of fin core cells leads to defective dorsal fin development and to collapse of the fin structure. Experiments using small molecule inhibitors indicate that dorsal migration of fin core cells depends on calcium signaling through calcium/calmodulin-dependent kinase II (CaMKII). In Wnt11-R depleted embryos, normal migration of NC cells and dorsal somite cells into the fin and normal fin development can be rescued by stimulation of calcium release. These studies are consistent with a model in which Wnt11-R signaling, via a downstream calcium pathway, regulates fin cell migration and, more generally, indicates a role for non-canonical Wnt signaling in regulation of EMT. PMID:17240368

  15. Chondrocyte calcium signaling in response to fluid flow is regulated by matrix adhesion in 3-D alginate scaffolds.

    PubMed

    Degala, Satish; Zipfel, Warren R; Bonassar, Lawrence J

    2011-01-01

    The interaction between chondrocytes and their surrounding extracellular matrix plays an important role in regulating cartilage metabolism in response to environmental cues. This study characterized the role of cell adhesion on the calcium signaling response of chondrocytes to fluid flow. Bovine chondrocytes were suspended in alginate hydrogels functionalized with RGD at concentrations of 0-400μM. The hydrogels were perfused and the calcium signaling response of the cells was measured over a range of fluid velocities from 0 to 68μm/s. Attachment to RGD-alginate doubled the sensitivity of chondrocytes to flows in the range of 8-13μm/s, but at higher fluid velocities, the contribution of cell adhesion to the observed calcium signaling response was no longer apparent. The enhanced sensitivity to flow was dependent on the density of RGD-ligand present in the scaffolds. The RGD-enhanced sensitivity to flow was completely inhibited by the addition of soluble RGD which acted as a competitive inhibitor. The results of this study indicate a role for matrix adhesion in regulating chondrocyte response to fluid flow through a calcium dependent mechanism.

  16. Modeling of [Formula: see text]-mediated calcium signaling in vascular endothelial cells induced by fluid shear stress and ATP.

    PubMed

    Li, Long-Fei; Xiang, Cheng; Qin, Kai-Rong

    2015-10-01

    The calcium signaling plays a vital role in flow-dependent vascular endothelial cell (VEC) physiology. Variations in fluid shear stress and ATP concentration in blood vessels can activate dynamic responses of cytosolic-free [Formula: see text] through various calcium channels on the plasma membrane. In this paper, a novel dynamic model has been proposed for transient receptor potential vanilloid 4 [Formula: see text]-mediated intracellular calcium dynamics in VECs induced by fluid shear stress and ATP. Our model includes [Formula: see text] signaling pathways through P2Y receptors and [Formula: see text] channels (indirect mechanism) and captures the roles of the [Formula: see text] compound channels in VEC [Formula: see text] signaling in response to fluid shear stress (direct mechanism). In particular, it takes into account that the [Formula: see text] compound channels are regulated by intracellular [Formula: see text] and [Formula: see text] concentrations. The simulation studies have demonstrated that the dynamic responses of calcium concentration produced by the proposed model correlate well with the existing experimental observations. We also conclude from the simulation studies that endogenously released ATP may play an insignificant role in the process of intracellular [Formula: see text] response to shear stress.

  17. Dexamethasone-induced cardiac deterioration is associated with both calcium handling abnormalities and calcineurin signaling pathway activation.

    PubMed

    de Salvi Guimarães, Fabiana; de Moraes, Wilson Max Almeida Monteiro; Bozi, Luis Henrique Marchesi; Souza, Pâmela R; Antonio, Ednei Luiz; Bocalini, Danilo Sales; Tucci, Paulo José Ferreira; Ribeiro, Daniel Araki; Brum, Patricia Chakur; Medeiros, Alessandra

    2017-01-01

    Dexamethasone is a potent and widely used anti-inflammatory and immunosuppressive drug. However, recent evidences suggest that dexamethasone cause pathologic cardiac remodeling, which later impairs cardiac function. The mechanism behind the cardiotoxic effect of dexamethasone is elusive. The present study aimed to verify if dexamethasone-induced cardiotoxicity would be associated with changes in the cardiac net balance of calcium handling protein and calcineurin signaling pathway activation. Wistar rats (~400 g) were treated with dexamethasone (35 µg/g) in drinking water for 15 days. After dexamethasone treatment, we analyzed cardiac function, cardiomyocyte diameter, cardiac fibrosis, and the expression of proteins involved in calcium handling and calcineurin signaling pathway. Dexamethasone-treated rats showed several cardiovascular abnormalities, including elevated blood pressure, diastolic dysfunction, cardiac fibrosis, and cardiomyocyte apoptosis. Regarding the expression of proteins involved in calcium handling, dexamethasone increased phosphorylation of phospholamban at threonine 17, reduced protein levels of Na(+)/Ca(2+) exchanger, and had no effect on protein expression of Serca2a. Protein levels of NFAT and GATA-4 were increased in both cytoplasmic and nuclear faction. In addition, dexamethasone increased nuclear protein levels of calcineurin. Altogether our findings suggest that dexamethasone causes pathologic cardiac remodeling and diastolic dysfunction, which is associated with impaired calcium handling and calcineurin signaling pathway activation.

  18. The GAR-3 muscarinic receptor cooperates with calcium signals to regulate muscle contraction in the Caenorhabditis elegans pharynx.

    PubMed Central

    Steger, Katherine A; Avery, Leon

    2004-01-01

    Muscarinic acetylcholine receptors regulate the activity of neurons and muscle cells through G-protein-coupled cascades. Here, we identify a pathway through which the GAR-3 muscarinic receptor regulates both membrane potential and excitation-contraction coupling in the Caenorhabditis elegans pharyngeal muscle. GAR-3 signaling is enhanced in worms overexpressing gar-3 or lacking GPB-2, a G-protein beta-subunit involved in RGS-mediated inhibition of G(o)alpha- and G(q)alpha-linked pathways. High levels of signaling through GAR-3 inhibit pharyngeal muscle relaxation and impair feeding--but do not block muscle repolarization--when worms are exposed to arecoline, a muscarinic agonist. Loss of gar-3 function results in shortened action potentials and brief muscle contractions in the pharyngeal terminal bulb. High levels of calcium entry through voltage-gated channels also impair terminal bulb relaxation and sensitize worms to the toxic effects of arecoline. Mutation of gar-3 reverses this sensitivity, suggesting that GAR-3 regulates calcium influx or calcium-dependent processes. Because the effects of GAR-3 signaling on membrane depolarization and muscle contraction can be separated, we conclude that GAR-3 regulates multiple calcium-dependent processes in the C. elegans pharyngeal muscle. PMID:15238517

  19. Intra-articular basic calcium phosphate and monosodium urate crystals inhibit anti-osteoclastogenic cytokine signalling.

    PubMed

    Cunningham, C C; Corr, E M; McCarthy, G M; Dunne, A

    2016-12-01

    Basic calcium phosphate (BCP) and monosodium urate (MSU) crystals are particulates with potent pro-inflammatory effects, associated with osteoarthritis (OA) and gout, respectively. Bone erosion, due to increased osteoclastogenesis, is a hallmark of both arthropathies and results in severe joint destruction. The aim of this study was to investigate the effect of these endogenous particulates on anti-osteoclastogenic cytokine signalling. Human osteoclast precursors (OcP) were treated with BCP and MSU crystals prior to stimulation with Interleukin (IL-6) or Interferon (IFN-γ) and the effect on Signal Transducer and Activator of Transcription (STAT)-3 and STAT-1 activation in addition to Mitogen Activated Protein Kinase (MAPK) activation was examined by immunoblotting. Crystal-induced suppressor of cytokine signalling (SOCS) protein and SH-2 containing tyrosine phosphatase (SHP) expression was assessed by real-time polymerase chain reaction (PCR) in the presence and absence of MAPK inhibitors. Pre-treatment with BCP or MSU crystals for 1 h inhibited IL-6-induced STAT-3 activation in human OcP, while pre-treatment for 3 h inhibited IFN-γ-induced STAT-1 activation. Both crystals activated p38 and extracellular signal-regulated (ERK) MAPKs with BCP crystals also activating c-Jun N-terminal kinase (JNK). Inhibition of p38 counteracted the inhibitory effect of BCP and MSU crystals and restored STAT-3 phosphorylation. In contrast, STAT-1 phosphorylation was not restored by MAPK inhibition. Finally, both crystals potently induced the expression of SOCS-3 in a MAPK dependent manner, while BCP crystals also induced expression of SHP-1 and SHP-2. This study provides further insight into the pathogenic effects of endogenous particulates in joint arthropathies and demonstrates how they may contribute to bone erosion via the inhibition of anti-osteoclastogenic cytokine signalling. Potential targets to overcome these effects include p38 MAPK, SOCS-3 and SHP phosphatases

  20. Amyloid β Peptide Enhances RANKL-Induced Osteoclast Activation through NF-κB, ERK, and Calcium Oscillation Signaling

    PubMed Central

    Li, Shangfu; Yang, Bu; Teguh, Dian; Zhou, Lin; Xu, Jiake; Rong, Limin

    2016-01-01

    Osteoporosis and Alzheimer’s disease (AD) are common chronic degenerative disorders which are strongly associated with advanced age. We have previously demonstrated that amyloid beta peptide (Aβ), one of the pathological hallmarks of AD, accumulated abnormally in osteoporotic bone specimens in addition to having an activation effect on osteoclast (Bone 2014,61:164-75). However, the underlying molecular mechanisms remain unclear. Activation of NF-κB, extracellular signal-regulated kinase (ERK) phosphorylates, and calcium oscillation signaling pathways by receptor activator NF-κB ligand (RANKL) plays a pivotal role in osteoclast activation. Targeting this signaling to modulate osteoclast function has been a promising strategy for osteoclast-related diseases. In this study, we investigated the effects of Aβ on RANKL-induced osteoclast signaling pathways in vitro. In mouse bone marrow monocytes (BMMs), Aβ exerted no effect on RANKL-induced osteoclastogenesis but promoted osteoclastic bone resorption. In molecular levels, Aβ enhanced NF-κB activity and IκB-α degradation, activated ERK phosphorylation and stimulated calcium oscillation, thus leading to upregulation of NFAT-c1 expression during osteoclast activation. Taken together, our data demonstrate that Aβ enhances RANKL-induced osteoclast activation through IκB-α degradation, ERK phosphorylation, and calcium oscillation signaling pathways and that Aβ may be a promising agent in the treatment of osteoclast-related disease such as osteoporosis. PMID:27735865

  1. Linoleic acid induces calcium signaling, Src kinase phosphorylation, and neurotransmitter release in mouse CD36-positive gustatory cells.

    PubMed

    El-Yassimi, Abdelghani; Hichami, Aziz; Besnard, Philippe; Khan, Naim Akhtar

    2008-05-09

    We have recently demonstrated that the cells expressing CD36, localized apically on the taste buds of mouse lingual circumvallate papillae, act as gustatory cells. In the present study we isolated these CD36-positive cells from mouse circumvallate papillae and investigated intracellular signaling events, triggered by a long-chain polyunsaturated fatty acid, i.e. linoleic acid (LA). LA induced increases in free intracellular calcium concentrations, [Ca(2+)](i), by recruiting calcium from endoplasmic reticulum pool via inositol 1,4,5-triphosphate production followed by calcium influx via opening of store-operated calcium (SOC) channels. LA also induced phosphorylation of Src-protein-tyrosine kinases (Src-PTKs), particularly of Fyn(59) and Yes(62). LA-evoked phosphorylation of Fyn(59) and Yes(62) was implicated in the activation of SOC channels. Reverse transcription-quantitative PCR revealed that the CD36-positive gustatory cells possessed mRNA of enzymes like tryptophan hydroxylase-1, l-aromatic amino acid decarboxylase, tyrosine hydroxylase, and dopamine beta-hydroxylase, involved in the synthesis of monoamine neurotransmitters. Interestingly, the addition of LA to these cells induced the release of 5-hydroxytryptamine and noradrenalin to the extracellular environment. The LA-induced release of these neurotransmitters was curtailed by SOC channel blockers and Src-PTK inhibitors. These results altogether demonstrate that LA binds to mouse CD36-positive gustatory cells, induces Src-PTKs phosphorylation, triggers calcium signaling, and evokes the release of 5-hydroxytryptamine and noradrenalin, which in turn may be implicated in the downstream signaling to the afferent nerve fibers, thus transmitting the output signal from taste buds to the central nervous system.

  2. Agonist-Biased Signaling via Proteinase Activated Receptor-2: Differential Activation of Calcium and Mitogen-Activated Protein Kinase Pathways

    PubMed Central

    Ramachandran, Rithwik; Mihara, Koichiro; Mathur, Maneesh; Rochdi, Moulay Driss; Bouvier, Michel; DeFea, Kathryn

    2009-01-01

    We evaluated the ability of different trypsin-revealed tethered ligand (TL) sequences of rat proteinase-activated receptor 2 (rPAR2) and the corresponding soluble TL-derived agonist peptides to trigger agonist-biased signaling. To do so, we mutated the proteolytically revealed TL sequence of rPAR2 and examined the impact on stimulating intracellular calcium transients and mitogen-activated protein (MAP) kinase. The TL receptor mutants, rPAR2-Leu37Ser38, rPAR2-Ala37–38, and rPAR2-Ala39–42 were compared with the trypsin-revealed wild-type rPAR2 TL sequence, S37LIGRL42—. Upon trypsin activation, all constructs stimulated MAP kinase signaling, but only the wt-rPAR2 and rPAR2-Ala39–42 triggered calcium signaling. Furthermore, the TL-derived synthetic peptide SLAAAA-NH2 failed to cause PAR2-mediated calcium signaling but did activate MAP kinase, whereas SLIGRL-NH2 triggered both calcium and MAP kinase signaling by all receptors. The peptides AAIGRL-NH2 and LSIGRL-NH2 triggered neither calcium nor MAP kinase signals. Neither rPAR2-Ala37–38 nor rPAR2-Leu37Ser38 constructs recruited β-arrestins-1 or -2 in response to trypsin stimulation, whereas both β-arrestins were recruited to these mutants by SLIGRL-NH2. The lack of trypsin-triggered β-arrestin interactions correlated with impaired trypsin-activated TL-mutant receptor internalization. Trypsin-stimulated MAP kinase activation by the TL-mutated receptors was not blocked by inhibitors of Gαi (pertussis toxin), Gαq [N-cyclohexyl-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methylindeno[1,2-c]pyrazole-3-carboxamide (GP2A)], Src kinase [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], or the epidermal growth factor (EGF) receptor [4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478)], but was inhibited by the Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27362). The data indicate that the proteolytically revealed TL sequence(s) and the mode

  3. Calcium and bone disease

    PubMed Central

    Blair, Harry C.; Robinson, Lisa J.; Huang, Christopher L.-H.; Sun, Li; Friedman, Peter A.; Schlesinger, Paul H.; Zaidi, Mone

    2013-01-01

    Calcium transport and calcium signaling are of basic importance in bone cells. Bone is the major store of calcium and a key regulatory organ for calcium homeostasis. Bone, in major part, responds to calcium-dependent signals from the parathyroids and via vitamin D metabolites, although bone retains direct response to extracellular calcium if parathyroid regulation is lost. Improved understanding of calcium transporters and calcium-regulated cellular processes has resulted from analysis of genetic defects, including several defects with low or high bone mass. Osteoblasts deposit calcium by mechanisms including phosphate and calcium transport with alkalinization to absorb acid created by mineral deposition; cartilage calcium mineralization occurs by passive diffusion and phosphate production. Calcium mobilization by osteoclasts is mediated by acid secretion. Both bone forming and bone resorbing cells use calcium signals as regulators of differentiation and activity. This has been studied in more detail in osteoclasts, where both osteoclast differentiation and motility are regulated by calcium. PMID:21674636

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

    PubMed

    Liu, Pei-Shan; Chiung, Yin-Mei; Kao, Yi-Yun; Chen, Han-Ting

    2006-02-15

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

  5. Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling.

    PubMed

    Shih, Yu-Ru V; Hwang, YongSung; Phadke, Ameya; Kang, Heemin; Hwang, Nathaniel S; Caro, Eduardo J; Nguyen, Steven; Siu, Michael; Theodorakis, Emmanuel A; Gianneschi, Nathan C; Vecchio, Kenneth S; Chien, Shu; Lee, Oscar K; Varghese, Shyni

    2014-01-21

    Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.

  6. Effect of HeNe laser on calcium signals in sperm cells

    NASA Astrophysics Data System (ADS)

    Lubart, Rachel; Friedmann, Harry; Cohen, Natalie; Brietbart, Haim

    1998-12-01

    Irradiation of mouse spermatozoa by 630 nm HeNe laser was found to enhance calcium transport in these cells. The change in Ca transport was investigated through two approaches, the first employing the fluorescent Ca indicator, Fluo-3 AM and a fluorescence microscopic system, and the second the radiolabeled Ca uptake. In both approaches the effect of light on Ca transport was abrogated in the absence of Ca during the irradiation time, indicating that the effect of light is Ca-dependent. The stimulatory effect of light on Ca uptake was inhibited by treatment with catalase, suggesting H2O2 to be involved in light stimulated Ca2+ uptake. The stimulatory effect of light on Ca uptake was abolished in the presence of a voltage-dependent Ca-channel inhibitor, nifedipine, indicating the involvement of a plasma membrane, voltage- dependent Ca-channel. In contrast, addition of nifedipine prior to the HeNe laser irradiation did not affect the light-induced rise in intracellular Ca levels, as measured with Fluo-3 loaded sperm cells. Therefore, it can be concluded that this Ca influx occurs via a voltage- insensitive Ca-channel. The stimulatory effect of light on Ca uptake was almost completely abolished by the mitochondrial uncoupler FCCP. These data imply that light affects the mitochondrial Ca transport mechanisms. It is well known that Ca influx from an extracellular environment is an essential component of a signaling cascade leading to fertilization.

  7. Analysing calcium signalling of cells under high shear flows using discontinuous dielectrophoresis

    NASA Astrophysics Data System (ADS)

    Soffe, Rebecca; Baratchi, Sara; Tang, Shi-Yang; Nasabi, Mahyar; McIntyre, Peter; Mitchell, Arnan; Khoshmanesh, Khashayar

    2015-07-01

    Immobilisation of cells is an important feature of many cellular assays, as it enables the physical/chemical stimulation of cells; whilst, monitoring cellular processes using microscopic techniques. Current approaches for immobilising cells, however, are hampered by time-consuming processes, the need for specific antibodies or coatings, and adverse effects on cell integrity. Here, we present a dielectrophoresis-based approach for the robust immobilisation of cells, and analysis of their responses under high shear flows. This approach is quick and label-free, and more importantly, minimises the adverse effects of electric field on the cell integrity, by activating the field for a short duration of 120 s, just long enough to immobilise the cells, after which cell culture media (such as HEPES) is flushed through the platform. In optimal conditions, at least 90% of the cells remained stably immobilised, when exposed to a shear stress of 63 dyn/cm2. This approach was used to examine the shear-induced calcium signalling of HEK-293 cells expressing a mechanosensitive ion channel, transient receptor potential vaniloid type 4 (TRPV4), when exposed to the full physiological range of shear stress.

  8. Nuclear BK Channels Regulate Gene Expression via the Control of Nuclear Calcium Signaling

    PubMed Central

    Li, Boxing; Jie, Wei; Huang, Lianyan; Wei, Peng; Li, Shuji; Luo, Zhengyi; Friedman, Allyson K.; Meredith, Andrea L.; Han, Ming-Hu; Zhu, Xin-Hong; Gao, Tian-Ming

    2014-01-01

    Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal ion channels in the regulation of Ca2+ is well established. In contrast, surprisingly less is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally blockade of nuclear BK channels (nBK channels) induces NE-derived Ca2+ release, nucleoplasmic Ca2+ elevation, and cAMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca2+-sensitive gene expression and promotes dendritic arborization in a nuclear Ca2+-dependent manner. These results suggest that nBK channel functions as a molecular linker between neuronal activity and nuclear Ca2+ to convey the signals from synapse to nucleus and is a new modulator for synaptic activity-dependent neuronal functions at the NE level. PMID:24952642

  9. Calcium Signaling During Meiotic Cell Cycle Regulation and Apoptosis in Mammalian Oocytes.

    PubMed

    Tiwari, Meenakshi; Prasad, Shilpa; Shrivastav, Tulsidas G; Chaube, Shail K

    2017-05-01

    Calcium (Ca(++) ) is one of the major signal molecules that regulate various aspects of cell functions including cell cycle progression, arrest, and apoptosis in wide variety of cells. This review summarizes current knowledge on the differential roles of Ca(++) in meiotic cell cycle resumption, arrest, and apoptosis in mammalian oocytes. Release of Ca(++) from internal stores and/or Ca(++) influx from extracellular medium causes moderate increase of intracellular Ca(++) ([Ca(++) ]i) level and reactive oxygen species (ROS). Increase of Ca(++) as well as ROS levels under physiological range trigger maturation promoting factor (MPF) destabilization, thereby meiotic resumption from diplotene as well as metaphase-II (M-II) arrest in oocytes. A sustained increase of [Ca(++) ]i level beyond physiological range induces generation of ROS sufficient enough to cause oxidative stress (OS) in aging oocytes. The increased [Ca(++) ]i triggers Fas ligand-mediated oocyte apoptosis. Further, OS triggers mitochondria-mediated oocyte apoptosis in several mammalian species. Thus, Ca(++) exerts differential roles on oocyte physiology depending upon its intracellular concentration. A moderate increase of [Ca(++) ]i as well as ROS mediate spontaneous resumption of meiosis from diplotene as well as M-II arrest, while their high levels cause meiotic cell cycle arrest and apoptosis by operating both mitochondria- as well as Fas ligand-mediated apoptotic pathways. Indeed, Ca(++) regulates cellular physiology by modulating meiotic cell cycle and apoptosis in mammalian oocytes. J. Cell. Physiol. 232: 976-981, 2017. © 2016 Wiley Periodicals, Inc.

  10. A clinically relevant model of osteoinduction: a process requiring calcium phosphate and BMP/Wnt signalling

    PubMed Central

    Eyckmans, J; Roberts, S J; Schrooten, J; Luyten, F P

    2010-01-01

    Abstract In this study, we investigated a clinically relevant model of in vivo ectopic bone formation utilizing human periosteum derived cells (HPDCs) seeded in a Collagraft™ carrier and explored the mechanisms by which this process is driven. Bone formation occurred after eight weeks when a minimum of one million HPDCs was loaded on Collagraft™ carriers and implanted subcutaneously in NMRI nu/nu mice. De novo bone matrix, mainly secreted by the HPDCs, was found juxta-proximal of the calcium phosphate (CaP) granules suggesting that CaP may have triggered the ‘osteoinductive program’. Indeed, removal of the CaP granules by ethylenediaminetetraacetic acid decalcification prior to cell seeding and implantation resulted in loss of bone formation. In addition, inhibition of endogenous bone morphogenetic protein and Wnt signalling by overexpression of the secreted antagonists Noggin and Frzb, respectively, also abrogated osteoinduction. Proliferation of the engrafted HPDCs was strongly reduced in the decalcified scaffolds or when seeded with adenovirus-Noggin/Frzb transduced HPDCs indicating that cell division of the engrafted HPDCs is required for the direct bone formation cascade. These data suggest that this model of bone formation is similar to that observed during physiological intramembranous bone development and may be of importance when investigating tissue engineering strategies. PMID:19538476

  11. Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR{sub 1} activation

    SciTech Connect

    Blanc-Brude, Olivier P. . E-mail: olivier.blanc-brude@larib.inserm.fr; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.

    2005-03-10

    Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR{sub 1}). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR{sub 1}-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR{sub 1}-specific agonists and inhibitors were used to demonstrate that PAR{sub 1} mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR{sub 1} and not PAR{sub 2}. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.

  12. A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death

    PubMed Central

    Wang, Z; Liu, D; Varin, A; Nicolas, V; Courilleau, D; Mateo, P; Caubere, C; Rouet, P; Gomez, A-M; Vandecasteele, G; Fischmeister, R; Brenner, C

    2016-01-01

    Although cardiac cytosolic cyclic 3′,5′-adenosine monophosphate (cAMP) regulates multiple processes, such as beating, contractility, metabolism and apoptosis, little is known yet on the role of this second messenger within cardiac mitochondria. Using cellular and subcellular approaches, we demonstrate here the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1), and show a protective role for sACt against cell death, apoptosis as well as necrosis in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3−) and Ca2+, sACt produces cAMP, which in turn stimulates oxygen consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. cAMP is rate limiting for matrix Ca2+ entry via Epac1 and the mitochondrial calcium uniporter and, as a consequence, prevents mitochondrial permeability transition (MPT). The mitochondrial cAMP effects involve neither protein kinase A, Epac2 nor the mitochondrial Na+/Ca2+ exchanger. In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3− rescued the sensitization of mitochondria to Ca2+-induced MPT. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism and cell death in the heart, which is independent of cytosolic cAMP signaling. Our results might have implications for therapeutic prevention of cell death in cardiac pathologies. PMID:27100892

  13. Possible Signaling Pathways Mediating Neuronal Calcium Sensor-1-Dependent Spatial Learning and Memory in Mice

    PubMed Central

    Nakamura, Tomoe Y.; Nakao, Shu; Nakajo, Yukako; Takahashi, Jun C.; Wakabayashi, Shigeo; Yanamoto, Hiroji

    2017-01-01

    Intracellular Ca2+ signaling regulates diverse functions of the nervous system. Many of these neuronal functions, including learning and memory, are regulated by neuronal calcium sensor-1 (NCS-1). However, the pathways by which NCS-1 regulates these functions remain poorly understood. Consistent with the findings of previous reports, we revealed that NCS-1 deficient (Ncs1-/-) mice exhibit impaired spatial learning and memory function in the Morris water maze test, although there was little change in their exercise activity, as determined via treadmill-analysis. Expression of brain-derived neurotrophic factor (BDNF; a key regulator of memory function) and dopamine was significantly reduced in the Ncs1-/- mouse brain, without changes in the levels of glial cell-line derived neurotrophic factor or nerve growth factor. Although there were no gross structural abnormalities in the hippocampi of Ncs1-/- mice, electron microscopy analysis revealed that the density of large dense core vesicles in CA1 presynaptic neurons, which release BDNF and dopamine, was decreased. Phosphorylation of Ca2+/calmodulin-dependent protein kinase II-α (CaMKII-α, which is known to trigger long-term potentiation and increase BDNF levels, was significantly reduced in the Ncs1-/- mouse brain. Furthermore, high voltage electric potential stimulation, which increases the levels of BDNF and promotes spatial learning, significantly increased the levels of NCS-1 concomitant with phosphorylated CaMKII-α in the hippocampus; suggesting a close relationship between NCS-1 and CaMKII-α. Our findings indicate that NCS-1 may regulate spatial learning and memory function at least in part through activation of CaMKII-α signaling, which may directly or indirectly increase BDNF production. PMID:28122057

  14. Atorvastatin calcium inhibits phenotypic modulation of PDGF-BB-induced VSMCs via down-regulation the Akt signaling pathway.

    PubMed

    Chen, Shuang; Liu, Baoqin; Kong, Dehui; Li, Si; Li, Chao; Wang, Huaqin; Sun, Yingxian

    2015-01-01

    Plasticity of vascular smooth muscle cells (VSMCs) plays a central role in the onset and progression of proliferative vascular diseases. In adult tissue, VSMCs exist in a physiological contractile-quiescent phenotype, which is defined by lack of the ability of proliferation and migration, while high expression of contractile marker proteins. After injury to the vessel, VSMC shifts from a contractile phenotype to a pathological synthetic phenotype, associated with increased proliferation, migration and matrix secretion. It has been demonstrated that PDGF-BB is a critical mediator of VSMCs phenotypic switch. Atorvastatin calcium, a selective inhibitor of 3-hydroxy-3-methyl-glutaryl l coenzyme A (HMG-CoA) reductase, exhibits various protective effects against VSMCs. In this study, we investigated the effects of atorvastatin calcium on phenotype modulation of PDGF-BB-induced VSMCs and the related intracellular signal transduction pathways. Treatment of VSMCs with atorvastatin calcium showed dose-dependent inhibition of PDGF-BB-induced proliferation. Atorvastatin calcium co-treatment inhibited the phenotype modulation and cytoskeleton rearrangements and improved the expression of contractile phenotype marker proteins such as α-SM actin, SM22α and calponin in comparison with PDGF-BB alone stimulated VSMCs. Although Akt phosphorylation was strongly elicited by PDGF-BB, Akt activation was attenuated when PDGF-BB was co-administrated with atorvastatin calcium. In conclusion, atorvastatin calcium inhibits phenotype modulation of PDGF-BB-induced VSMCs and activation of the Akt signaling pathway, indicating that Akt might play a vital role in the modulation of phenotype.

  15. Calcium signaling in pathogenic and beneficial plant microbe interactions: what can we learn from the interaction between Piriformospora indica and Arabidopsis thaliana.

    PubMed

    Vadassery, Jyothilakshmi; Oelmüller, Ralf

    2009-11-01

    Elevation of intracellular calcium levels in a plant cell is an early signaling event in many mutualistic and pathogenic plant/microbe interactions. In pathogenic plant/fungus interactions, receptor-mediated cytoplasmic calcium elevations induce defense genes via the activation of ion fluxes at the plasma membrane, an oxidative burst and MAPK activation. Mycorrhizal and beneficial endophytic plant/fungus interactions result in a better plant performance through sequencial cytoplasmic and nuclear calcium elevations. The specificity of the calcium responses depends on the calcium signature, its amplitude, duration, frequency and location, a selective activation of calcium channels in the diverse cellular membranes and the stimulation of calcium-dependent signaling components. Arabidopsis contains more than 100 genes for calcium-binding proteins and channels and the response to pathogens and beneficial fungi relies on a highly specific activation of individual members of these protein families. Genetic tools are required to understand this complex response patterns and the cross talks between the individual calcium-dependent signaling pathways. The beneficial interaction of Arabidopsis with the growth-promoting endophyte Piriformospora indica provides a nice model system to unravel signaling events leading to mutualistic or pathogenic plant/fungus interactions.

  16. B cell receptor-mediated calcium signaling is impaired in B lymphocytes of type Ia patients with common variable immunodeficiency.

    PubMed

    Foerster, Christian; Voelxen, Nadine; Rakhmanov, Mirzokhid; Keller, Baerbel; Gutenberger, Sylvia; Goldacker, Sigune; Thiel, Jens; Feske, Stefan; Peter, Hans-Hartmut; Warnatz, Klaus

    2010-06-15

    Several lines of evidence have demonstrated B cell intrinsic activation defects in patients with common variable immunodeficiency (CVID). The rapid increase of intracellular free calcium concentrations after engagement of the BCR represents one crucial element in this activation process. The analysis of 53 patients with CVID for BCR-induced calcium flux identified a subgroup of patients with significantly reduced Ca2+ signals in primary B cells. This subgroup strongly corresponded to the class Ia of the Freiburg classification. Comparison at the level of defined B cell subpopulations revealed reduced Ca2+ signals in all mature B cell populations of patients with CVID class Ia when compared with healthy individuals and other groups of patients with CVID but not in circulating transitional B cells. BCR-induced Ca2+ responses were the lowest in CD21low B cells in patients as well as healthy donors, indicating an additional cell-specific mechanism inhibiting the Ca2+ flux. Although proximal BCR signaling events are unperturbed in patients' B cells, including normal phospholipase Cgamma2 phosphorylation and Ca2+ release from intracellular stores, Ca2+ influx from the extracellular space is significantly impaired. CD22, a negative regulator of calcium signals in B cells, is highly expressed on CD21low B cells from patients with CVID Ia and might be involved in the attenuated Ca2+ response of this B cell subpopulation. These data from patients with CVID suggest that a defect leading to impaired BCR-induced calcium signaling is associated with the expansion of CD21low B cells, hypogammaglobulinemia, autoimmune dysregulation, and lymphadenopathy.

  17. Honokiol blocks store operated calcium entry in CHO cells expressing the M3 muscarinic receptor: honokiol and muscarinic signaling

    PubMed Central

    2013-01-01

    Background Honokiol, a cell-permeable phenolic compound derived from the bark of magnolia trees and present in Asian herbal teas, has a unique array of pharmacological actions, including the inhibition of multiple autonomic responses. We determined the effects of honokiol on calcium signaling underlying transmission mediated by human M3 muscarinic receptors expressed in Chinese hamster ovary (CHO) cells. Receptor binding was determined in radiolabelled ligand binding assays; changes in intracellular calcium concentrations were determined using a fura-2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay. Results Honokiol had a potent (EC50 ≈ 5 μmol/l) inhibitory effect on store operated calcium entry (SOCE) that was induced by activation of the M3 receptors. This effect was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor-mediated calcium release, depletion of ER calcium stores, or disruption of M3 receptor binding. Conclusions It is likely that an inhibition of SOCE contributes to honokiol disruption of parasympathetic motor functions, as well as many of its beneficial pharmacological properties. PMID:23432810

  18. Transient Receptor Potential-Like Channels Are Essential for Calcium Signaling and Fluid Transport in a Drosophila Epithelium

    PubMed Central

    MacPherson, Matthew R.; Pollock, Valerie P.; Kean, Laura; Southall, Tony D.; Giannakou, Maria E.; Broderick, Kate E.; Dow, Julian A. T.; Hardie, Roger C.; Davies, Shireen A.

    2005-01-01

    Calcium signaling is an important mediator of neuropeptide-stimulated fluid transport by Drosophila Malpighian (renal) tubules. We demonstrate the first epithelial role, in vivo, for members of the TRP family of calcium channels. RT-PCR revealed expression of trp, trpl, and trpγ in tubules. Use of antipeptide polyclonal antibodies for TRP, TRPL, and TRPγ showed expression of all three channels in type 1 (principal) cells in the tubule main segment. Neuropeptide (CAP2b)-stimulated fluid transport rates were significantly reduced in tubules from the trpl302 mutant and the trpl;trp double mutant, trpl302;trp343. However, a trp null, trp343, had no impact on stimulated fluid transport. Measurement of cytosolic calcium concentrations ([Ca2+]i) in tubule principal cells using an aequorin transgene in trp and trpl mutants showed a reduction in calcium responses in trpl302. Western blotting of tubule preparations from trp and trpl mutants revealed a correlation between TRPL levels and CAP2b-stimulated fluid transport and calcium signaling. Rescue of trpl302 with a trpl transgene under heat-shock control resulted in a stimulated fluid transport phenotype that was indistinguishable from wild-type tubules. Furthermore, restoration of normal stimulated rates of fluid transport by rescue of trpl302 was not compromised by introduction of the trp null, trp343. Thus, in an epithelial context, TRPL is sufficient for wild-type responses. Finally, a scaffolding component of the TRPL/TRP-signaling complex, INAD, is not expressed in tubules, suggesting that inaD is not essential for TRPL/TRP function in Drosophila tubules. PMID:15695363

  19. MYB30 transcription factor regulates oxidative and heat stress responses through ANNEXIN-mediated cytosolic calcium signaling in Arabidopsis.

    PubMed

    Liao, Chancan; Zheng, Yuan; Guo, Yan

    2017-10-01

    Cytosolic calcium signaling is critical for regulating downstream responses in plants encountering unfavorable environmental conditions. In a genetic screen for Arabidopsis thaliana mutants defective in stress-induced cytosolic free Ca(2+) ([Ca(2+) ]cyt ) elevations, we identified the R2R3-MYB transcription factor MYB30 as a regulator of [Ca(2+) ]cyt in response to H2 O2 and heat stresses. Plants lacking MYB30 protein exhibited greater elevation of [Ca(2+) ]cyt in response to oxidative and heat stimuli. Real-time reverse transcription-polymerase chain reaction (RT-PCR) results indicated that the expression of a number of ANNEXIN (ANN) genes, which encode Ca(2+) -regulated membrane-binding proteins modulating cytosolic calcium signatures, were upregulated in myb30 mutants. Further analysis showed that MYB30 bound to the promoters of ANN1 and ANN4 and repressed their expression. myb30 mutants were sensitive to methyl viologen (MV) and heat stresses. The H2 O2 - and heat-induced abnormal [Ca(2+) ]cyt in myb30 was dependent on the function of ANN proteins. Moreover, the MV and heat sensitivity of myb30 was suppressed in mutants lacking ANN function or by application of LaCl3 , a calcium channel blocker. These results indicate that MYB30 regulates oxidative and heat stress responses through calcium signaling, which is at least partially mediated by ANN1 and ANN4. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  20. 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.

  1. 1alpha,25(OH)2-vitamin D3 membrane-initiated calcium signaling modulates exocytosis and cell survival.

    PubMed

    Xiaoyu, Zhang; Payal, Biswas; Melissa, Owraghi; Zanello, Laura P

    2007-03-01

    1alpha,25(OH)(2)-vitamin D(3) (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca(2+) channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca(2+) concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca(2+)-dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca(2+)-dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.

  2. The role of extracellular free-calcium gradients in gravitropic signalling in maize roots

    NASA Technical Reports Server (NTRS)

    Bjorkman, T.; Cleland, R. E.

    1991-01-01

    Gravitropism in roots has been proposed to depend on a downward redistribution of calcium across the root cap. However, because of the many calcium-binding sites in the apoplast, redistribution might not result in a physiologically effective change in the apoplasmic calcium activity. To test whether there is such a change, we measured the effect of gravistimulation on the calcium activity of statocyte cell walls with calcium-specific microelectrodes. Such a measurement must be made on a tissue with gravity sensing cells at the surface. To obtain such a tissue, decapped maize roots (Zea mays L. cv. Golden Cross Bantam) were grown for 31 h to regenerate gravitropic sensitivity, but not root caps. The calcium activity in the apoplasm surrounding the gravity-sensing cells could then be measured. The initial pCa was 2.60 +/- 0.28 (approx 2.5 mM). The calcium activity on the upper side of the root tip remained constant for 10 min after gravistimulation, then decreased 1.7-fold. On the lower side, after a similar lag the calcium activity increased 1.6-fold. Control roots, which were decapped but measured before recovering gravisensitivity (19 h), showed no change in calcium activity. To test whether this gradient is necessary for gravitropic curvature, we eliminated the calcium activity gradient during gravitropism by applying a mobile calcium-binding site (dinitro-BAPTA; 1,2-bis(2-amino-5-nitro-phenoxy)ethane-N,N,N',N'-tetraacetic acid) to the root cap; this treatment eliminated gravicurvature. A calcium gradient may be formed by proton-induced calcium desorption if there is a proton gradient. Preventing the formation of apoplastic pH gradients, using 10 and 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer or 10 mM fusicoccin to stimulate proton excretion maximally, did not inhibit curvature; therefore the calcium gradient is not a secondary effect of a proton gradient. We have found a distinct and rapid differential in the apoplasmic calcium activity between the

  3. Decoding of calcium signal through calmodulin: calmodulin-binding proteins in plants

    USDA-ARS?s Scientific Manuscript database

    Many abiotic and biotic stimuli such as heat, cold, drought, salt, light, wind, touch, wounding, symbionts and pathogens as well as growth, developmental and hormonal cues can quickly induce cytosolic calcium increases. Calmodulin, the most thoroughly studied calcium sensor, mediates interpretation...

  4. Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

    PubMed Central

    Jiang, Ruotian; Haustein, Martin D.; Sofroniew, Michael V.; Khakh, Baljit S.

    2014-01-01

    Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry. PMID:25490346

  5. Imaging intracellular Ca²⁺ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators.

    PubMed

    Jiang, Ruotian; Haustein, Martin D; Sofroniew, Michael V; Khakh, Baljit S

    2014-11-19

    Astrocytes display spontaneous intracellular Ca(2+) concentration fluctuations ([Ca(2+)]i) and in several settings respond to neuronal excitation with enhanced [Ca(2+)]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca(2+)]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca(2+)]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca(2+)]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca(2+)]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca(2+)]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca(2+)]i signals in the striatal microcircuitry.

  6. FM dyes enter via a store-operated calcium channel and modify calcium signaling of cultured astrocytes

    PubMed Central

    Li, Dongdong; Hérault, Karine; Oheim, Martin; Ropert, Nicole

    2009-01-01

    The amphiphilic fluorescent styryl pyridinium dyes FM1-43 and FM4-64 are used to probe activity-dependent synaptic vesicle cycling in neurons. Cultured astrocytes can internalize FM1-43 and FM4-64 inside vesicles but their uptake is insensitive to the elevation of cytosolic calcium (Ca2+) concentration and the underlying mechanism remains unclear. Here we used total internal reflection fluorescence microscopy and pharmacological tools to study the mechanisms of FM4-64 uptake into cultured astrocytes from mouse neocortex. Our data show that: (i) endocytosis is not a major route for FM4-64 uptake into astrocytes; (ii) FM4-64 enters astrocytes through an aqueous pore and strongly affects Ca2+ homeostasis; (iii) partitioning of FM4-64 into the outer leaflet of the plasma membrane results in a facilitation of store-operated Ca2+ entry (SOCE) channel gating; (iv) FM4-64 permeates and competes with Ca2+ for entry through a SOCE channel; (v) intracellular FM4-64 mobilizes Ca2+ from the endoplasmic reticulum stores, conveying a positive feedback to activate SOCE and to sustain dye uptake into astrocytes. Our study demonstrates that FM dyes are not markers of cycling vesicles in astrocytes and calls for a careful interpretation of FM fluorescence. PMID:20007370

  7. Neuron class-specific requirements for Fragile X Mental Retardation Protein in critical period development of calcium signaling in learning and memory circuitry.

    PubMed

    Doll, Caleb A; Broadie, Kendal

    2016-05-01

    Neural circuit optimization occurs through sensory activity-dependent mechanisms that refine synaptic connectivity and information processing during early-use developmental critical periods. Fragile X Mental Retardation Protein (FMRP), the gene product lost in Fragile X syndrome (FXS), acts as an activity sensor during critical period development, both as an RNA-binding translation regulator and channel-binding excitability regulator. Here, we employ a Drosophila FXS disease model to assay calcium signaling dynamics with a targeted transgenic GCaMP reporter during critical period development of the mushroom body (MB) learning/memory circuit. We find FMRP regulates depolarization-induced calcium signaling in a neuron-specific manner within this circuit, suppressing activity-dependent calcium transients in excitatory cholinergic MB input projection neurons and enhancing calcium signals in inhibitory GABAergic MB output neurons. Both changes are restricted to the developmental critical period and rectified at maturity. Importantly, conditional genetic (dfmr1) rescue of null mutants during the critical period corrects calcium signaling defects in both neuron classes, indicating a temporally restricted FMRP requirement. Likewise, conditional dfmr1 knockdown (RNAi) during the critical period replicates constitutive null mutant defects in both neuron classes, confirming cell-autonomous requirements for FMRP in developmental regulation of calcium signaling dynamics. Optogenetic stimulation during the critical period enhances depolarization-induced calcium signaling in both neuron classes, but this developmental change is eliminated in dfmr1 null mutants, indicating the activity-dependent regulation requires FMRP. These results show FMRP shapes neuron class-specific calcium signaling in excitatory vs. inhibitory neurons in developing learning/memory circuitry, and that FMRP mediates activity-dependent regulation of calcium signaling specifically during the early

  8. In vivo photoacoustic neuronal imaging of odor-evoked calcium signals in the drosophila brain (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Zhang, Ruiying; Rao, Bin; Rong, Haoyang; Raman, Baranidharan; Wang, Lihong V.

    2016-03-01

    Neural scientists can benefit greatly from imaging tools that can penetrate thick brain tissue. Compared with traditional optical microscopy methods, photoacoustic imaging can beat the optical diffusion limit and achieve such deep tissue imaging with high spatial resolution. In this study, we used an optical-resolution photoacoustic microscope to image the odor-evoked neuronal activities in a drosophila model. Drosophila brain neurons stably express GCaMP5G, a calcium-sensitive fluorescent protein whose optical absorption coefficient changes with calcium influx during action potentials. We recorded an ~20% odor-evoked fractional photoacoustic signal increase at all depths of the drosophila brain in vivo, with and without removal of the brain cuticle, at a recording rate of 1 kHz. Our results were confirmed by concurrent fluorescent recordings. Furthermore, by performing fast 2D scanning, we imaged the antenna lobe region, which is of particular interest in neuroscience, at a volumetric rate of ~1 Hz with a sub-neuron resolution of 3 μm. Unlike optical imaging, which requires surgical removal of the scattering brain cuticle, our photoacoustic system can image through the cuticle and measure neuronal signals of the whole drosophila brain without invasive surgery, enabling minimal disturbance to the animal's behaviors. In conclusion, we have demonstrated photoacoustic imaging of calcium signals in drosophila brains for the first time. Utilizing the deep imaging capability of photoacoustic tomography, our methods could potentially be extended to in vivo imaging of neuronal activities from deep brains in other animal models.

  9. Heteromerization of dopamine D2 receptors with dopamine D1 or D5 receptors generates intracellular calcium signaling by different mechanisms

    PubMed Central

    Hasbi, Ahmed; O’Dowd, Brian F.; George, Susan R.

    2009-01-01

    The repertoire of signal transduction pathways activated by dopamine in brain includes the increase of intracellular calcium. However the mechanism(s) by which dopamine activated this important second messenger system was unknown. Although we showed that activation of the D5 dopamine receptor increased calcium concentrations, the restricted anatomic distribution of this receptor made this unlikely to be the major mechanism in brain. We have identified novel heteromeric dopamine receptor complexes that are linked to calcium signaling. The calcium pathway activated through the D1–D2 receptor heteromer involved coupling to Gq, through phospholipase C and IP3 receptors to result in a rise in intracellular calcium. The calcium rise activated through the D2–D5 receptor heteromer involved a small rise in intracellular calcium through the Gq pathway that triggered a store operated channel mediated influx of extracellular calcium. These novel receptor heteromeric complexes, for the first time, establish the link between dopamine action and rapid calcium signaling. PMID:19897420

  10. Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons.

    PubMed

    Bickler, P E; Fahlman, C S

    2004-01-01

    Although large increases in neuronal intracellular calcium concentrations ([Ca(2+)](i)) are lethal, moderate increases in [Ca(2+)](i) of 50-200 nM may induce immediate or long-term tolerance of ischemia or other stresses. In neurons in rat hippocampal slice cultures, we determined the relationship between [Ca(2+)](i), cell death, and Ca(2+)-dependent neuroprotective signals before and after a 45 min period of oxygen and glucose deprivation (OGD). Thirty minutes before OGD, [Ca(2+)](i) was increased in CA1 neurons by 40-200 nM with 1 nM-1 microM of a Ca(2+)-selective ionophore (calcimycin or ionomycin-"Ca(2+) preconditioning"). Ca(2+) preconditioning greatly reduced cell death in CA1, CA3 and dentate during the following 7 days, even though [Ca(2+)](i) was similar (approximately 2 microM) in preconditioned and control neurons 1 h after the OGD. When pre-OGD [Ca(2+)](i) was lowered to 25 nM (10 nM ionophore in Ca(2+)-free medium) or increased to 8 microM (10 microM ionophore), more than 90% of neurons died. Increased levels of the anti-apoptotic protein protein kinase B (Akt) and the MAP kinase ERK (p42/44) were present in preconditioned slices after OGD. Reducing Ca(2+) influx, inhibiting calmodulin, and preventing Akt or MAP kinase p42/44 upregulation prevented Ca(2+) preconditioning, supporting a specific role for Ca(2+) in the neuroprotective process. Further, in continuously oxygenated cultured hippocampal/cortical neurons, preconditioning for 30 min with 10 nM ionomycin reduced cell death following a 4 microM increase in [Ca(2+)](i) elicited by 1 microM ionomycin. Thus, a zone of moderately increased [Ca(2+)](i) before a potentially lethal insult promotes cell survival, uncoupling subsequent large increases in [Ca(2+)](i) from initiating cell death processes.

  11. Calcium Influx through Cav1.2 Is a Proximal Signal for Pathological Cardiomyocyte Hypertrophy

    PubMed Central

    Chen, Xiongwen; Nakayama, Hiroyuki; Zhang, Xiaoying; Ai, Xiaojie; Harris, David M.; Tang, Mingxin; Zhang, Hongyu; Szeto, Christopher; Stockbower, Kathryn; Berretta, Remus M.; Eckhart, Andrea D.; Koch, Walter J.; Molkentin, Jeffery D.; Houser, Steven R.

    2010-01-01

    Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca2+) is implicated in hypertrophic signaling pathways, the specific role of Ca2+ influx through the L-type Ca2+ channel (ICa-L) has been controversial and is the topic of this study. To determine if and how sustained increases in ICa-L induces PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the β2a subunit of Ca2+ channels (β2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a β2a-GFP. Results In vivo, β2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume and cross sectional area and the expression of PCH markers in a time- and dose- dependent manner. PCH was associated with a hypercontractile phenotype including enhanced ICa-L, fractional shortening, peak Ca2+ transient, at the myocyte level, greater ejection fraction and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of β2a in cultured AFVMs increased ICa-L, cell volume, protein synthesis, NFAT and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of ICa-L, intracellular Ca2+, calcineurin, CaMK II and SERCA. Conclusion Increasing ICa-L is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca2+ pools were shown to be involved. PMID:21111744

  12. Modulation of action potential and calcium signaling by levetiracetam in rat sensory neurons.

    PubMed

    Ozcan, Mete; Ayar, Ahmet

    2012-06-01

    Levetiracetam (LEV), a new anticonvulsant agent primarily used to treat epilepsy, has been used in pain treatment but the cellular mechanism of this action remains unclear. This study aimed to investigate effects of LEV on the excitability and membrane depolarization-induced calcium signaling in isolated rat sensory neurons using the whole-cell patch clamp and fura 2-based ratiometric Ca(2+)-imaging techniques. Dorsal root ganglia (DRG) were excised from neonatal rats, and cultured following enzymatic and mechanical dissociation. Under current clamp conditions, acute application of LEV (30 µM, 100 µM and 300 µM) significantly increased input resistance and caused the membrane to hyperpolarize from resting membrane potential in a dose-dependent manner. Reversal potentials of action potential (AP) after hyperpolarising amplitudes were shifted to more negative, toward to potassium equilibrium potentials, after application of LEV. It also caused a decrease in number of APs in neurons fired multiple APs in response to prolonged depolarization. Fura-2 fluorescence Ca(2+) imaging protocols revealed that HiK(+) (30 mM)-induced intracellular free Ca(2+) ([Ca(2+)](i)) was inhibited to 97.8 ± 4.6% (n = 17), 92.6 ± 4.8% (n = 17, p < 0.01) and 89.1 ± 5.1% (n = 18, p < 0.01) after application of 30 µM, 100 µM and 300 µM LEV (respectively), without any significant effect on basal levels of [Ca(2+)](i). This is the first evidence for the effect of LEV on the excitability of rat sensory neurons through an effect which might involve activation of potassium channels and inhibition of entry of Ca(2+), providing new insights for cellular mechanism(s) of LEV in pain treatment modalities.

  13. Calcium signalling indicates bilateral power balancing in the Drosophila flight muscle during manoeuvring flight

    PubMed Central

    Lehmann, Fritz-Olaf; Skandalis, Dimitri A.; Berthé, Ruben

    2013-01-01

    Manoeuvring flight in animals requires precise adjustments of mechanical power output produced by the flight musculature. In many insects such as fruit flies, power generation is most likely varied by altering stretch-activated tension, that is set by sarcoplasmic calcium levels. The muscles reside in a thoracic shell that simultaneously drives both wings during wing flapping. Using a genetically expressed muscle calcium indicator, we here demonstrate in vivo the ability of this animal to bilaterally adjust its calcium activation to the mechanical power output required to sustain aerodynamic costs during flight. Motoneuron-specific comparisons of calcium activation during lift modulation and yaw turning behaviour suggest slightly higher calcium activation for dorso-longitudinal than for dorsoventral muscle fibres, which corroborates the elevated need for muscle mechanical power during the wings’ downstroke. During turning flight, calcium activation explains only up to 54 per cent of the required changes in mechanical power, suggesting substantial power transmission between both sides of the thoracic shell. The bilateral control of muscle calcium runs counter to the hypothesis that the thorax of flies acts as a single, equally proportional source for mechanical power production for both flapping wings. Collectively, power balancing highlights the precision with which insects adjust their flight motor to changing energetic requirements during aerial steering. This potentially enhances flight efficiency and is thus of interest for the development of technical vehicles that employ bioinspired strategies of power delivery to flapping wings. PMID:23486171

  14. Calcium signalling indicates bilateral power balancing in the Drosophila flight muscle during manoeuvring flight.

    PubMed

    Lehmann, Fritz-Olaf; Skandalis, Dimitri A; Berthé, Ruben

    2013-05-06

    Manoeuvring flight in animals requires precise adjustments of mechanical power output produced by the flight musculature. In many insects such as fruit flies, power generation is most likely varied by altering stretch-activated tension, that is set by sarcoplasmic calcium levels. The muscles reside in a thoracic shell that simultaneously drives both wings during wing flapping. Using a genetically expressed muscle calcium indicator, we here demonstrate in vivo the ability of this animal to bilaterally adjust its calcium activation to the mechanical power output required to sustain aerodynamic costs during flight. Motoneuron-specific comparisons of calcium activation during lift modulation and yaw turning behaviour suggest slightly higher calcium activation for dorso-longitudinal than for dorsoventral muscle fibres, which corroborates the elevated need for muscle mechanical power during the wings' downstroke. During turning flight, calcium activation explains only up to 54 per cent of the required changes in mechanical power, suggesting substantial power transmission between both sides of the thoracic shell. The bilateral control of muscle calcium runs counter to the hypothesis that the thorax of flies acts as a single, equally proportional source for mechanical power production for both flapping wings. Collectively, power balancing highlights the precision with which insects adjust their flight motor to changing energetic requirements during aerial steering. This potentially enhances flight efficiency and is thus of interest for the development of technical vehicles that employ bioinspired strategies of power delivery to flapping wings.

  15. Involvement of oxidative stress and calcium signaling in airborne particulate matter - induced damages in human pulmonary artery endothelial cells.

    PubMed

    Deweirdt, J; Quignard, J F; Crobeddu, B; Baeza-Squiban, A; Sciare, J; Courtois, A; Lacomme, S; Gontier, E; Muller, B; Savineau, J P; Marthan, R; Guibert, C; Baudrimont, I

    2017-07-05

    Recent studies have revealed that particulate matter (PM) exert deleterious effects on vascular function. Pulmonary artery endothelial cells (HPAEC), which are involved in the vasomotricity regulation, can be a direct target of inhaled particles. Modifications in calcium homeostasis and oxidative stress are critical events involved in the physiopathology of vascular diseases. The objectives of this study were to assess the effects of PM2.5 on oxidative stress and calcium signaling in HPAEC. Different endpoints were studied, (i) intrinsic and intracellular production of reactive oxygen species (ROS) by the H2DCF-DA probe, (ii) intrinsic, intracellular and mitochondrial production of superoxide anion (O2(-)) by electronic paramagnetic resonance spectroscopy and MitoSOX probe, (iii) reactive nitrosative species (RNS) production by Griess reaction, and (vi) calcium signaling by the Fluo-4 probe. In acellular conditions, PM2.5 leads to an intrinsic free radical production (ROS, O2(-)) and a 4h-exposure to PM2.5 (5-15μg/cm(2)), induced, in HPAEC, an increase of RNS, of global ROS and of cytoplasmic and mitochondrial O2(-) levels. The basal intracellular calcium ion level [Ca(2+)]i was also increased after 4h-exposure to PM2.5 and a pre-treatment with superoxide dismutase and catalase significantly reduced this response. This study provides evidence that the alteration of intracellular calcium homeostasis induced by PM2.5 is closely correlated to an increase of oxidative stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. 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.

  17. 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

  18. Visualizing context-dependent calcium signaling in encephalitogenic T cells in vivo by two-photon microscopy

    PubMed Central

    Kyratsous, Nikolaos I.; Bauer, Isabel J.; Zhang, Guokun; Pesic, Marija; Bartholomäus, Ingo; Mues, Marsilius; Fang, Ping; Wörner, Miriam; Everts, Stephanie; Ellwart, Joachim W.; Watt, Joanna M.; Potter, Barry V. L.; Hohlfeld, Reinhard; Wekerle, Hartmut; Kawakami, Naoto

    2017-01-01

    In experimental autoimmune encephalitis (EAE), autoimmune T cells are activated in the periphery before they home to the CNS. On their way, the T cells pass through a series of different cellular milieus where they receive signals that instruct them to invade their target tissues. These signals involve interaction with the surrounding stroma cells, in the presence or absence of autoantigens. To portray the serial signaling events, we studied a T-cell–mediated model of EAE combining in vivo two-photon microscopy with two different activation reporters, the FRET-based calcium biosensor Twitch1 and fluorescent NFAT. In vitro activated T cells first settle in secondary (2°) lymphatic tissues (e.g., the spleen) where, in the absence of autoantigen, they establish transient contacts with stroma cells as indicated by sporadic short-lived calcium spikes. The T cells then exit the spleen for the CNS where they first roll and crawl along the luminal surface of leptomeningeal vessels without showing calcium activity. Having crossed the blood–brain barrier, the T cells scan the leptomeningeal space for autoantigen-presenting cells (APCs). Sustained contacts result in long-lasting calcium activity and NFAT translocation, a measure of full T-cell activation. This process is sensitive to anti-MHC class II antibodies. Importantly, the capacity to activate T cells is not a general property of all leptomeningeal phagocytes, but varies between individual APCs. Our results identify distinct checkpoints of T-cell activation, controlling the capacity of myelin-specific T cells to invade and attack the CNS. These processes may be valuable therapeutic targets. PMID:28716943

  19. 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.

  20. A secreted chitinase-like protein (OsCLP) supports root growth through calcium signaling in Oryza sativa.

    PubMed

    Wu, Jingni; Wang, Yiming; Kim, Sang Gon; Jung, Ki-Hong; Gupta, Ravi; Kim, Joonyup; Park, Younghoon; Kang, Kyu Young; Kim, Sun Tae

    2017-04-12

    Chitinases belong to a conserved protein family and play multiple roles in defense, development, and growth regulation in plants. Here, we identified a secreted chitinase-like protein, OsCLP, which functions in rice growth. A T-DNA insertion mutant of OsCLP (osclp) showed significant retardation of root and shoot growth. A comparative proteomic analysis was carried out using root tissue of wild-type and the osclp mutant to understand the OsCLP-mediated rice growth retardation. Results obtained revealed that proteins related to glycolysis (phosphoglycerate kinase), stress adaption (chaperonin) and calcium signaling (calreticulin and CDPK1) were differentially regulated in osclp roots. Fura-2 molecular probe staining, which is an intracellular calcium indicator, and ICP-MS analysis suggested that the intracellular calcium content was significantly lower in roots of osclp as compared to the wild-type. Exogenous application of Ca(2+) resulted in successful recovery of both primary and lateral root growth in osclp. Moreover, overexpression of OsCLP resulted in improved growth with modified seed shape and starch structure; however, the overall yield remained unaffected. Taken together, our results highlight the involvement of OsCLP in rice growth by regulating the intracellular calcium concentrations.

  1. Osteogenic Differentiation of MSC through Calcium Signaling Activation: Transcriptomics and Functional Analysis

    PubMed Central

    Viti, Federica; Landini, Martina; Mezzelani, Alessandra; Petecchia, Loredana; Milanesi, Luciano; Scaglione, Silvia

    2016-01-01

    The culture of progenitor mesenchymal stem cells (MSC) onto osteoconductive materials to induce a proper osteogenic differentiation and mineralized matrix regeneration represents a promising and widely diffused experimental approach for tissue-engineering (TE) applications in orthopaedics. Among modern biomaterials, calcium phosphates represent the best bone substitutes, due to their chemical features emulating the mineral phase of bone tissue. Although many studies on stem cells differentiation mechanisms have been performed involving calcium-based scaffolds, results often focus on highlighting production of in vitro bone matrix markers and in vivo tissue ingrowth, while information related to the biomolecular mechanisms involved in the early cellular calcium-mediated differentiation is not well elucidated yet. Genetic programs for osteogenesis have been just partially deciphered, and the description of the different molecules and pathways operative in these differentiations is far from complete, as well as the activity of calcium in this process. The present work aims to shed light on the involvement of extracellular calcium in MSC differentiation: a better understanding of the early stage osteogenic differentiation program of MSC seeded on calcium-based biomaterials is required in order to develop optimal strategies to promote osteogenesis through the use of new generation osteoconductive scaffolds. A wide spectrum of analysis has been performed on time-dependent series: gene expression profiles are obtained from samples (MSC seeded on calcium-based scaffolds), together with related microRNAs expression and in vivo functional validation. On this basis, and relying on literature knowledge, hypotheses are made on the biomolecular players activated by the biomaterial calcium-phosphate component. Interestingly, a key role of miR-138 was highlighted, whose inhibition markedly increases osteogenic differentiation in vitro and enhance ectopic bone formation in vivo

  2. Involvement of three annexin genes in the ripening of strawberry fruit regulated by phytohormone and calcium signal transduction.

    PubMed

    Chen, Jingxin; Mao, Linchun; Mi, Hongbo; Lu, Wenjing; Ying, Tiejin; Luo, Zisheng

    2016-04-01

    Three annexin genes may be involved in the ripening progress of strawberry fruit. Phytohormones and calcium regulate the expressions of three annexin genes during strawberry fruit ripening. Plant annexins are multi-functional membrane- and Ca(2+)-binding proteins that are involved in various developmental progresses and stress responses. Three annexins FaAnn5a, FaAnn5b and FaAnn8 cDNA obtained from strawberry fruit encode amino acid sequences of approximately 35 kDa containing four annexin repeats, Ca(2+)-binding site, GTP-binding motif, peroxidase residue, and conserved amino acid residues of tryptophan, arginine and cysteine. During fruit development, the transcript levels of FaAnn5a and FaAnn5b increased while FaAnn5b declined after 3/4R stage. The expression patterns of annexins suggested their potential roles in strawberry fruit development and ripening. Expressions of annexin genes were also highly correlated with hormone levels. In addition, exogenous abscisic acid (ABA) enhanced the expressions of FaAnn5a and FaAnn8 while exogenous auxin (IAA) retarded it. However, both ABA and IAA promoted the transcript levels of FaAnn5b, indicating the independent regulation of annexins in fruit likely due to multi-functions of their large family. The responses of annexin genes to exogenous ABA and IAA inhibitors verified the involvement of annexins in plant hormone signaling. Besides, calcium restrained the expressions of FaAnn5s (FaAnn5a and FaAnn5b) but promoted the expression of FaAnn8. Effects of calcium and ethylene glycol tetraacetic acid (EGTA) on the transcript levels of annexins confirmed that calcium likely mediated hormone signal transduction pathways, which helped to elucidate the mechanism of calcium in fruit ripening. Therefore, FaAnn5s and FaAnn8 might be involved in plant hormones' regulation in the development and ripening of strawberry fruit through calcium signaling in the downstream.

  3. Differential regulation of calcium signalling pathways by components of Piper methysticum (‘Awa)

    PubMed Central

    Shimoda, L.M.N; Showman, A.; Baker, J.D.; Lange, I.; Koomoa, D.L.; Stokes, A.J.; Borris, R.P.; Turner, H.

    2015-01-01

    Kava is a soporific, anxiolytic and relaxant in widespread ritual and recreational use throughout the Pacific. Traditional uses of kava by indigenous Pacific Island peoples reflect a complex pharmacopeia, centered on GABA-ergic effects of the well-characterized kavalactones. However, peripheral effects of kava suggest active components other than the CNS-targeted kavalactones. We have previously shown that immunocytes exhibit calcium mobilization in response to traditionally-prepared kava extracts, and that the kavalactones do not induce these calcium responses. Here, we characterize the complex calcium-mobilizing activity of traditionally-prepared and partially HPLC-purified kava extracts, noting induction of both calcium entry and store release pathways. Kava components activate intracellular store depletion of thapsigargin-sensitive and –insensitive stores that are coupled to the calcium release activated (CRAC) current, and cause calcium entry through non-store-operated pathways. Together with the pepper-like potency reported by kava users, these studies lead us to hypothesize that kava extracts contain one or more ligands for the transient receptor potential (TRP) family of ion channels. Indeed, TRP-like conductances are observed in kava-treated cells under patch clamp. Thus TRP-mediated cellular effects may be responsible for some of the reported pharmacology of kava. PMID:25640812

  4. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

    PubMed

    Supper, Verena; Schiller, Herbert B; Paster, Wolfgang; Forster, Florian; Boulègue, Cyril; Mitulovic, Goran; Leksa, Vladimir; Ohradanova-Repic, Anna; Machacek, Christian; Schatzlmaier, Philipp; Zlabinger, Gerhard J; Stockinger, Hannes

    2016-02-01

    The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression.

  6. Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants

    PubMed Central

    Sinha, Deepak K.; Chandran, Predeesh; Timm, Alicia E.; Aguirre-Rojas, Lina; Smith, C. Michael

    2016-01-01

    The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion. PMID:26815857

  7. Up-regulation of ryanodine receptor expression increases the calcium-induced calcium release and spontaneous calcium signals in cerebral arteries from hindlimb unloaded rats.

    PubMed

    Morel, Jean-Luc; Dabertrand, Fabrice; Porte, Yves; Prevot, Anne; Macrez, Nathalie

    2014-08-01

    Microgravity induces a redistribution of blood volume. Consequently, astronauts' body pressure is modified so that the upright blood pressure gradient is abolished, thereby inducing a modification in cerebral blood pressure. This effect is mimicked in the hindlimb unloaded rat model. After a duration of 8 days of unloading, Ca2+ signals activated by depolarization and inositol-1,4,5-trisphosphate intracellular release were increased in cerebral arteries. In the presence of ryanodine and thapsigargin, the depolarization-induced Ca2+ signals remained increased in hindlimb suspended animals, indicating that Ca2+ influx and Ca2+-induced Ca2+ release mechanism were both increased. Spontaneous Ca2+ waves and localized Ca2+ events were also investigated. Increases in both amplitude and frequency of spontaneous Ca2+ waves were measured in hindlimb suspension conditions. After pharmacological segregation of Ca2+ sparks and Ca2+ sparklets, their kinetic parameters were characterized. Hindlimb suspension induced an increase in the frequencies of both Ca2+ localized events, suggesting an increase of excitability. Labeling with bodipy compounds suggested that voltage-dependent Ca2+ channels and ryanodine receptor expressions were increased. Finally, the expression of the ryanodine receptor subtype 1 (RyR1) was increased in hindlimb unloading conditions. Taken together, these results suggest that RyR1 expression and voltage-dependent Ca2+ channels activity are the focal points of the regulation of Ca2+ signals activated by vasoconstriction in rat cerebral arteries with an increase of the voltage-dependent Ca2+ influx.

  8. Role of calcium signaling in the activation of mitochondrial nitric oxide synthase and citric acid cycle.

    PubMed

    Traaseth, Nathaniel; Elfering, Sarah; Solien, Joseph; Haynes, Virginia; Giulivi, Cecilia

    2004-07-23

    An apparent discrepancy arises about the role of calcium on the rates of oxygen consumption by mitochondria: mitochondrial calcium increases the rate of oxygen consumption because of the activation of calcium-activated dehydrogenases, and by activating mitochondrial nitric oxide synthase (mtNOS), decreases the rates of oxygen consumption because nitric oxide is a competitive inhibitor of cytochrome oxidase. To this end, the rates of oxygen consumption and nitric oxide production were followed in isolated rat liver mitochondria in the presence of either L-Arg (to sustain a mtNOS activity) or N(G)-monomethyl-L-Arg (NMMA, a competitive inhibitor of mtNOS) under State 3 conditions. In the presence of NMMA, the rates of State 3 oxygen consumption exhibited a K(0.5) of 0.16 microM intramitochondrial free calcium, agreeing with those required for the activation of the Krebs cycle. By plotting the difference between the rates of oxygen consumption in State 3 with L-Arg and with NMMA at various calcium concentrations, a K(0.5) of 1.2 microM intramitochondrial free calcium was obtained, similar to the K(0.5) (0.9 microM) of the dependence of the rate of nitric oxide production on calcium concentrations. The activation of dehydrogenases, followed by the activation of mtNOS, would lead to the modulation of the Krebs cycle activity by the modulation of nitric oxide on the respiratory rates. This would ensue in changes in the NADH/NAD and ATP/ADP ratios, which would influence the rate of the cycle and the oxygen diffusion.

  9. The Effect of Chemically Defined Medium on Spontaneous Calcium Signaling of In Situ Chondrocytes during Long-term Culture

    PubMed Central

    Zhou, Yilu; Park, Miri; Cheung, Enoch; Wang, Liyun; Lu, X. Lucas

    2015-01-01

    Chemically defined serum-free medium has been shown to maintain the mechanical integrity of articular cartilage explants better than serum-supplemented medium during long-term in vitro culture, but little is known about its effect on cellular mechanisms. We hypothesized that the chemically defined culture medium can regulate the spontaneous calcium signaling of in situ chondrocytes, which may modulate the cellular metabolic activities. Bovine cartilage explants were cultured in chemically defined serum-free or serum-supplemented medium for four weeks. The spontaneous intracellular calcium ([Ca2+]i) signaling of in situ chondrocytes was longitudinally measured together along with the biomechanical properties of the explants. The spontaneous [Ca2+]i oscillations in chondrocytes were enhanced at the initial exposure of serum-supplemented medium, but were significantly dampened afterwards. In contrast, cartilage explants in chemically defined medium preserved the level of calcium signaling, and showed more responsive cells with higher and more frequent [Ca2+]i peaks after one to four week culture in comparison to those in serum medium. Regardless of the culture medium that the explants were exposed, a positive correlation was detected between the [Ca2+]i responsive rate and the stiffness of cartilage (Spearman's rank correlation coefficient = 0.762). A stable pattern of [Ca2+]i peaks was revealed for each chondrocyte, i.e., the spatiotemporal features of [Ca2+]i peaks from a cell were highly consistent during the observation period (15 minutes). This study showed that the beneficial effect of chemically defined culture of cartilage explants is associated with the spontaneous [Ca2+]i signaling of chondrocytes in cartilage. PMID:25700610

  10. Effects of nonylphenol on the calcium signal and catecholamine secretion coupled with nicotinic acetylcholine receptors in bovine adrenal chromaffin cells.

    PubMed

    Liu, Pei-Shan; Liu, Ging-Hui; Chao, Wei-Liang

    2008-02-03

    Nonylphenol (NP) is the most critical metabolite of alkylphenol polyethoxylate detergents. NP is known as an endocrine disruptor with estrogenic activities and as an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. Estrogen has modulatory roles on ligand-gated ion channels, such as nicotinic acetylcholine receptors (nAChRs). Ca(2+)-ATPase inhibitors can modulate the cytosolic calcium concentration ([Ca(2+)](c)]) and thus can affect the calcium signaling coupled with nAChRs. Therefore, NP is predicted to have complex effects on the Ca(2+) signaling and secretion coupled with nAChRs. This study investigated these effects using bovine adrenal chromaffin cells. The results show that NP suppressed the Ca(2+) signaling coupled with nAChRs and voltage-operated Ca(2+) channels in a dose-dependent manner, with IC(50)s of 1 and 5.9 microM, respectively. Estradiol exhibits similar suppression but much lower inhibitory potencies. NP alone induced a transient rise in [Ca(2+)](c) in the presence or absence of extracellular calcium. Thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, partially suppressed the [Ca(2+)](c) rise induced by NP, but NP totally blocked the [Ca(2+)](c) rise induced by thapsigargin. This illustrates that NP can cause Ca(2+) release from thapsigargin-insensitive pools. Thapsigargin suppressed the Ca(2+) signaling coupled with nAChRs but increased that coupled with voltage-operated Ca(2+) channels. We propose that three routes are responsible for the effects of NP on nAChRs: named receptor channels, voltage-gated Ca(2+) channels, and Ca(2+)-induced Ca(2+) release. Three routes are related to the characteristics of NP as steroid-like compounds and Ca(2+)-ATPase inhibitor.

  11. The effect of chemically defined medium on spontaneous calcium signaling of in situ chondrocytes during long-term culture.

    PubMed

    Zhou, Yilu; Park, Miri; Cheung, Enoch; Wang, Liyun; Lu, X Lucas

    2015-04-13

    Chemically defined serum-free medium has been shown to better maintain the mechanical integrity of articular cartilage explants than serum-supplemented medium during long-term in vitro culture, but little is known about its effect on cellular mechanisms. We hypothesized that the chemically defined culture medium could regulate the spontaneous calcium signaling of in situ chondrocytes, which may modulate the cellular metabolic activities. Bovine cartilage explants were cultured in chemically defined serum-free or serum-supplemented medium for four weeks. The spontaneous intracellular calcium ([Ca(2+)]i) signaling of in situ chondrocytes was longitudinally measured together along with the biomechanical properties of the explants. The spontaneous [Ca(2+)]i oscillations in chondrocytes were enhanced at the initial exposure of serum-supplemented medium, but were significantly dampened afterwards. In contrast, cartilage explants in chemically defined medium preserved the level of calcium signaling, and showed more responsive cells with higher and more frequent [Ca(2+)]i peaks throughout the four week culture in comparison to those in serum medium. Regardless of the culture medium that the explants were exposed, a positive correlation was detected between the [Ca(2+)]i responsive rate and the stiffness of cartilage (Spearman's rank correlation coefficient=0.762). A stable pattern of [Ca(2+)]i peaks was revealed for each chondrocyte, i.e., the spatiotemporal features of [Ca(2+)]i peaks from a cell were highly consistent during the observation period (15 min). This study showed that the beneficial effect of chemically defined culture of cartilage explants is associated with the spontaneous [Ca(2+)]i signaling of chondrocytes in cartilage.

  12. Regulation of anaphase chromosome motion in Tradescantia stamen hair cells by calcium and related signaling agents

    PubMed Central

    1990-01-01

    Several lines of evidence support the idea that increases in the intracellular free calcium concentration [( Ca2+]i) regulate chromosome motion. To directly test this we have iontophoretically injected Ca2+ or related signaling agents into Tradescantia stamen hair cells during anaphase and measured their effect on chromosome motion and on the Ca2+ levels. Ca2+ at (+)1 nA for 10 s (approximately 1 microM) causes a transient (20 s) twofold increase in the rate of chromosome motion, while at higher levels it slows or completely stops motion. Ca2+ buffers, EGTA, and 5,5'-dibromo-1,2- bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid, which transiently suppress the ion level, also momentarily stop motion. Injection of K+, Cl-, or Mg2+, as controls, have no effect on motion. The injection of GTP gamma S, and to a lesser extent GTP, enhances motion similarly to a low level of Ca2+. However, inositol 1,4,5-trisphosphate, ATP gamma S, ATP, and GDP beta S have no effect. Measurement of the [Ca2+]i with indo-1 reveals that the direct injections of Ca2+ produce the expected increases. GTP gamma S, on the other hand, causes only a small [Ca2+]i rise, which by itself is insufficient to increase the rate of chromosome motion. Further studies reveal that any negative ion injection, presumably through hyperpolarization of the membrane potential, generates a similar small pulse of Ca2+, yet these agents have no effect on motion. Two major conclusions from these studies are as follows. (a) Increased [Ca2+]i can enhance the rate of motion, if administered in a narrow physiological window around 1 microM; concentrations above 1 microM or below the physiological resting level will slow or stop chromosomes. (b) GTP gamma S enhances motion by a mechanism that does not cause a sustained uniform rise of [Ca2+]i in the spindle; this effect may be mediated through very localized [Ca2+]i changes or Ca2(+)-independent effectors. PMID:2114409

  13. Measurements of intracellular calcium signals in polarized primary cultures of normal and cystic fibrosis human airway epithelia.

    PubMed

    Ribeiro, Carla M P

    2011-01-01

    The airways are continuously challenged by a variety of stimuli including bacteria, viruses, allergens, and inflammatory factors that act as agonists for G protein-coupled receptors (GPCR). Intracellular calcium (Ca(2+) (i)) mobilization in airway epithelia in response to extracellular stimuli regulates key airway innate defense functions, e.g., Ca(2+)-activated Cl(-) secretion, ciliary beating, mucin secretion, and inflammatory responses. Because Ca(2+) (i) mobilization in response to luminal stimuli is larger in CF vs. normal human airway epithelia, alterations in Ca(2+) (i) signals have been associated with the pathogenesis of CF airway disease. Hence, assessment of Ca(2+) (i) signaling has become an important area of CF research. This chapter will focus on measurements of cytoplasmic and mitochondrial Ca(2+) signals resulting from GPCR activation in polarized primary cultures of normal and CF human bronchial epithelia (HBE).

  14. 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

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

    PubMed Central

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

    2015-01-01

    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 Ca2+. Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca2+-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 Ca2+ in astrocytic processes. Thus, the regulation of intracellular Ca2+ signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca2+ wave propagation, gliotransmission, and ultimately neuronal function. SIGNIFICANCE STATEMENT Mitochondria are key cellular organelles that play important roles in providing cellular energy and buffering intracellular calcium ions. The mechanisms that control mitochondrial distribution within the processes of glial cells called astrocytes and the impact this may have on calcium signaling remains unclear. We show that activation of glutamate receptors or increased neuronal

  16. A calcium sensor – protein kinase signaling module diversified in plants and is retained in all lineages of Bikonta species

    PubMed Central

    Beckmann, Linda; Edel, Kai H.; Batistič, Oliver; Kudla, Jörg

    2016-01-01

    Calcium (Ca2+) signaling is a universal mechanism of signal transduction and involves Ca2+ signal formation and decoding of information by Ca2+ binding proteins. Calcineurin B-like proteins (CBLs), which upon Ca2+ binding activate CBL-interacting protein kinases (CIPKs) regulate a multitude of physiological processes in plants. Here, we combine phylogenomics and functional analyses to investigate the occurrence and structural conservation of CBL and CIPK proteins in 26 species representing all major clades of eukaryotes. We demonstrate the presence of at least singular CBL-CIPK pairs in representatives of Archaeplastida, Chromalveolates and Excavates and their general absence in Opisthokonta and Amoebozoa. This denotes CBL-CIPK complexes as evolutionary ancient Ca2+ signaling modules that likely evolved in the ancestor of all Bikonta. Furthermore, we functionally characterize the CBLs and CIPK from the parabasalid human pathogen Trichomonas vaginalis. Our results reveal strict evolutionary conservation of functionally important structural features, preservation of biochemical properties and a remarkable cross-kingdom protein-protein interaction potential between CBLs and CIPKs from Arabidopsis thaliana and T. vaginalis. Together our findings suggest an ancient evolutionary origin of a functional CBL-CIPK signaling module close to the root of eukaryotic evolution and provide insights into the initial evolution of signaling networks and Ca2+ signaling specificity. PMID:27538881

  17. hsBAFF promotes proliferation and survival in cultured B lymphocytes via calcium signaling activation of mTOR pathway.

    PubMed

    Ke, Zhen; Liang, Dingfang; Zeng, Qingyu; Ren, Qian; Ma, Hongwei; Gui, Lin; Chen, Sujuan; Guo, Min; Xu, Yijiao; Gao, Wei; Zhang, Shuangquan; Chen, Long

    2013-05-01

    B-cell activating factor of the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) has revealed its critical function in B lymphocyte proliferation and survival, as well as the pathogenesis of autoimmune disease. However, the molecular mechanisms of excess BAFF-extended aggressive B lymphocytes have not been completely defined. Here we show that excessive hsBAFF-elevated [Ca(2+)]i activated mammalian target of rapamycin (mTOR) signaling pathway, leading to proliferation and survival in B lymphocytes. This is supported by the findings that intracellular Ca(2+) chelator (BAPTA/AM) or mTOR inhibitor (rapamycin) abolished the events. Sequentially, we observed that preventing [Ca(2+)]i elevation using EGTA or 2-APB dramatically inhibited hsBAFF activation of mTOR signaling, as well as cell growth and survival, suggesting that hsBAFF-induced extracellular Ca(2+) influx and ER Ca(2+) release elevates [Ca(2+)]i contributing to B lymphocyte proliferation and survival via activation of mTOR signaling. Further, we noticed that pretreatment with BAPTA/AM, EGTA or 2-APB blocked hsBAFF-increased phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII), and inhibiting CaMKII with KN93 attenuated hsBAFF-activated mTOR signaling, as well as cell growth and survival, revealing that the effects of hsBAFF-elevated [Ca(2+)]i on mTOR signaling as well as proliferation and survival in B lymphocytes is through stimulating phosphorylation of CaMKII. The results indicate that hsBAFF activates mTOR pathway triggering B lymphocyte proliferation and survival by calcium signaling. Our findings suggest that manipulation of intracellular Ca(2+) level or CaMKII and mTOR activity may be exploited for the prevention of excessive BAFF-induced aggressive B lymphocyte disorders and autoimmune diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. The colon-selective spasmolytic otilonium bromide inhibits muscarinic M3 receptor-coupled calcium signals in isolated human colonic crypts

    PubMed Central

    Lindqvist, Susanne; Hernon, James; Sharp, Paul; Johns, Neil; Addison, Sarah; Watson, Mark; Tighe, Richard; Greer, Shaun; Mackay, Jean; Rhodes, Michael; Lewis, Michael; Stebbings, William; Speakman, Chris; Evangelista, Stefano; Johnson, Ian; Williams, Mark

    2002-01-01

    Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M3 receptor subtype (CHO-M3). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC50 of 14 μM. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC50 (pKb) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M3 receptor subtype expression at the crypt-base. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC50=880 nM). In CHO-M3 cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. The present studies have demonstrated that OB inhibited M3 receptor-coupled calcium signals in human colonic crypts and CHO-M3 cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M3 receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action in IBS patients suffering with diarrhoea. PMID

  19. The colon-selective spasmolytic otilonium bromide inhibits muscarinic M(3) receptor-coupled calcium signals in isolated human colonic crypts.

    PubMed

    Lindqvist, Susanne; Hernon, James; Sharp, Paul; Johns, Neil; Addison, Sarah; Watson, Mark; Tighe, Richard; Greer, Shaun; Mackay, Jean; Rhodes, Michael; Lewis, Michael; Stebbings, William; Speakman, Chris; Evangelista, Stefano; Johnson, Ian; Williams, Mark

    2002-12-01

    1. Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. 2. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M(3) receptor subtype (CHO-M(3)). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. 3. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC(50) of 14 micro M. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC(50) (pK(b)) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). 4. Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M(3) receptor subtype expression at the crypt-base. 5. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC(50)=880 nM). 6. In CHO-M(3) cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. 7. The present studies have demonstrated that OB inhibited M(3) receptor-coupled calcium signals in human colonic crypts and CHO-M(3) cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M(3) receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action

  20. Calcium/calmodulin-dependent protein kinase type IV is a target gene of the Wnt/beta-catenin signaling pathway.

    PubMed

    Arrázola, Macarena S; Varela-Nallar, Lorena; Colombres, Marcela; Toledo, Enrique M; Cruzat, Fernando; Pavez, Leonardo; Assar, Rodrigo; Aravena, Andrés; González, Mauricio; Montecino, Martín; Maass, Alejandro; Martínez, Servet; Inestrosa, Nibaldo C

    2009-12-01

    Calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. The expression of CaMKIV and the activation of CREB regulated genes are involved in memory and neuronal survival. We report here that: (a) a bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes, being CaMKIV a very interesting candidate; (b) CaMKIV promoter contains TCF/LEF transcription motifs similar to those present in Wnt target genes; (c) biochemical studies indicate that lithium and the canonical ligand Wnt-3a induce CaMKIV mRNA and protein expression levels in rat hippocampal neurons as well as CaMKIV promoter activity; (d) treatment of hippocampal neurons with Wnt-3a increases the binding of beta-catenin to the CaMKIV promoter: (e) In vivo activation of the Wnt signaling improve spatial memory impairment and restores the expression of CaMKIV in a mice double transgenic model for Alzheimer's disease which shows decreased levels of the kinase. We conclude that CaMKIV is regulated by the Wnt signaling pathway and that its expression could play a role in the neuroprotective function of the Wnt signaling against the Alzheimer's amyloid peptide.

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

    PubMed

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

    2011-09-09

    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.

  2. Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors.

    PubMed

    Hamada, Kozo; Terauchi, Akiko; Nakamura, Kyoko; Higo, Takayasu; Nukina, Nobuyuki; Matsumoto, Nagisa; Hisatsune, Chihiro; Nakamura, Takeshi; Mikoshiba, Katsuhiko

    2014-09-23

    The inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP3Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP3 at a distance. Defective allostery in IP3R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP3R type 1 (IP3R1) and negatively regulates IP3R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP3R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.

  3. Tespa1 regulates T cell receptor-induced calcium signals by recruiting inositol 1,4,5-trisphosphate receptors

    PubMed Central

    Liang, Jingjing; Lyu, Jun; Zhao, Meng; Li, Dan; Zheng, Mingzhu; Fang, Yan; Zhao, Fangzhu; Lou, Jun; Guo, Chuansheng; Wang, Lie; Wang, Di; Liu, Wanli; Lu, Linrong

    2017-01-01

    Thymocyte-expressed, positive selection-associated 1 (Tespa1) is important in T cell receptor (TCR)-driven thymocyte development. Downstream of the TCR, Tespa1 is a crucial component of the linker for activation of T cells (LAT) signalosome, facilitating calcium signalling and subsequent MAPK activation. However, it is unknown how Tespa1 elicits calcium signalling. Here, we show that inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is crucial for Tespa1-optimized, TCR-induced Ca2+ flux and thymocyte development. Upon TCR stimulation, Tespa1 directly interacts with IP3R1 and recruits it to the TCR complex, where IP3R1 is phosphorylated at Y353 by Fyn. This Tespa1-IP3R1 interaction is mediated by the F187 and F188 residues of Tespa1 and the amino-terminus of IP3R1. Tespa1-F187A/F188A mutant mice phenocopy Tespa1-deficient mice with impaired late thymocyte development due to reduced IP3R1 translocation to the TCR-proximal region. Our work elucidates the function of Tespa1 in T cell development and the regulation of TCR-induced Ca2+ signalling through IP3R1. PMID:28598420

  4. A new system for profiling drug-induced calcium signal perturbation in human embryonic stem cell-derived cardiomyocytes.

    PubMed

    Lewis, Kimberley J; Silvester, Nicole C; Barberini-Jammaers, Steven; Mason, Sammy A; Marsh, Sarah A; Lipka, Magdalena; George, Christopher H

    2015-03-01

    The emergence of human stem cell-derived cardiomyocyte (hSCCM)-based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling "fingerprint" in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ≈ control. This rank order of drug-induced Ca(2+) signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework.

  5. Pathway Network Analyses for Autism Reveal Multisystem Involvement, Major Overlaps with Other Diseases and Convergence upon MAPK and Calcium Signaling

    PubMed Central

    Wen, Ya; Alshikho, Mohamad J.; Herbert, Martha R.

    2016-01-01

    We used established databases in standard ways to systematically characterize gene ontologies, pathways and functional linkages in the large set of genes now associated with autism spectrum disorders (ASDs). These conditions are particularly challenging—they lack clear pathognomonic biological markers, they involve great heterogeneity across multiple levels (genes, systemic biological and brain characteristics, and nuances of behavioral manifestations)—and yet everyone with this diagnosis meets the same defining behavioral criteria. Using the human gene list from Simons Foundation Autism Research Initiative (SFARI) we performed gene set enrichment analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database, and then derived a pathway network from pathway-pathway functional interactions again in reference to KEGG. Through identifying the GO (Gene Ontology) groups in which SFARI genes were enriched, mapping the coherence between pathways and GO groups, and ranking the relative strengths of representation of pathway network components, we 1) identified 10 disease-associated and 30 function-associated pathways 2) revealed calcium signaling pathway and neuroactive ligand-receptor interaction as the most enriched, statistically significant pathways from the enrichment analysis, 3) showed calcium signaling pathways and MAPK signaling pathway to be interactive hubs with other pathways and also to be involved with pervasively present biological processes, 4) found convergent indications that the process “calcium-PRC (protein kinase C)-Ras-Raf-MAPK/ERK” is likely a major contributor to ASD pathophysiology, and 5) noted that perturbations associated with KEGG’s category of environmental information processing were common. These findings support the idea that ASD-associated genes may contribute not only to core features of ASD themselves but also to vulnerability to other chronic and systemic problems potentially including cancer, metabolic

  6. Unilateral vestibular deafferentation-induced changes in calcium signaling-related molecules in the rat vestibular nuclear complex.

    PubMed

    Masumura, Chisako; Horii, Arata; Mitani, Kenji; Kitahara, Tadashi; Uno, Atsuhiko; Kubo, Takeshi

    2007-03-23

    Inquiries into the neurochemical mechanisms of vestibular compensation, a model of lesion-induced neuronal plasticity, reveal the involvement of both voltage-gated Ca(2+) channels (VGCC) and intracellular Ca(2+) signaling. Indeed, our previous microarray analysis showed an up-regulation of some calcium signaling-related genes such as the alpha2 subunit of L-type calcium channels, calcineurin, and plasma membrane Ca(2+) ATPase 1 (PMCA1) in the ipsilateral vestibular nuclear complex (VNC) following unilateral vestibular deafferentation (UVD). To further elucidate the role of calcium signaling-related molecules in vestibular compensation, we used a quantitative real-time polymerase chain reaction (PCR) method to confirm the microarray results and investigated changes in expression of these molecules at various stages of compensation (6 h to 2 weeks after UVD). We also investigated the changes in gene expression during Bechterew's phenomenon and the effects of a calcineurin inhibitor on vestibular compensation. Real-time PCR showed that genes for the alpha2 subunit of VGCC, PMCA2, and calcineurin were transiently up-regulated 6 h after UVD in ipsilateral VNC. A subsequent UVD, which induced Bechterew's phenomenon, reproduced a complete mirror image of the changes in gene expressions of PMCA2 and calcineurin seen in the initial UVD, while the alpha2 subunit of VGCC gene had a trend to increase in VNC ipsilateral to the second lesion. Pre-treatment by FK506, a calcineurin inhibitor, decelerated the vestibular compensation in a dose-dependent manner. Although it is still uncertain whether these changes in gene expression are causally related to the molecular mechanisms of vestibular compensation, this observation suggests that after increasing the Ca(2+) influx into the ipsilateral VNC neurons via up-regulated VGCC, calcineurin may be involved in their synaptic plasticity. Conversely, an up-regulation of PMCA2, a brain-specific Ca(2+) pump, would increase an efflux of Ca

  7. Integrin binding and MAPK signal pathways in primary cell responses to surface chemistry of calcium silicate cements.

    PubMed

    Shie, Ming-You; Ding, Shinn-Jyh

    2013-09-01

    Cell attachment, proliferation and differentiation on different materials depend largely on the surface properties of the materials. This study sheds light on the mechanism by which the modulation of the chemical composition of calcium silicate cements with different Si/Ca molar ratios could produce different cell responses. Two primary cell types (human mesenchymal stem cells (hMSCs) and human dental pulp cells (hDPCs)) were used to elicit the changes in total DNA content, integrin subunit levels, phosphor-focal adhesion kinase (pFAK) levels, and mitogen-activated protein kinase (MAPK) signaling pathway activity at the cell attachment stage. The effect of small interfering RNA (siRNA) transfection targeting collagen type I (COL I) and fibronectin (FN) was also evaluated. The results indicated that increased total DNA content, pFAK and total integrin levels were observed upon an increase in cement Si content. Cements with different Si/Ca ratios did not cause the variations of interleukin 1β (IL-1β), epidermal growth factor (EGF) and tumor necrosis factor-α (TNF-α) ligands. The Si-rich cement facilitated COL I and α2β1 subintegrin expression, while Ca-rich cement promoted FN and αvβ3 subintegrin expression. Si component of the calcium silicates stimulated cell adhesion via activation of MAPK/extracellular signal-regulated kinase (ERK) and p38 signaling pathways more effectively than did by Ca component, but it did not affect c-Jun NH2-terminal kinase (JNK) activity. Inhibition of MAPK/ERK and MAPK/p38 signaling pathways in hMSCs and hDPCs significantly attenuated adhesion, proliferation and differentiation as assessed according to total DNA content and alkaline phosphatase activity. hMSCs and hDPCs from the three different donors exhibited a similar preference for cell behaviors. The results of the current study suggest that calcium silicate cements with a higher Si content have the potential to serve as excellent supports for primary cells. Unraveling the

  8. Imaging calcium in neurons.

    PubMed

    Grienberger, Christine; Konnerth, Arthur

    2012-03-08

    Calcium ions generate versatile intracellular signals that control key functions in all types of neurons. Imaging calcium in neurons is particularly important because calcium signals exert their highly specific functions in well-defined cellular subcompartments. In this Primer, we briefly review the general mechanisms of neuronal calcium signaling. We then introduce the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices that are used in freely moving animals. We provide an overview of the classical chemical fluorescent calcium indicators and of the protein-based genetically encoded calcium indicators. Using application examples, we introduce new developments in the field, such as calcium imaging in awake, behaving animals and the use of calcium imaging for mapping single spine sensory inputs in cortical neurons in vivo. We conclude by providing an outlook on the prospects of calcium imaging for the analysis of neuronal signaling and plasticity in various animal models.

  9. Aluminum Chloride Induces Osteoblasts Apoptosis via Disrupting Calcium Homeostasis and Activating Ca(2+)/CaMKII Signal Pathway.

    PubMed

    Cao, Zheng; Liu, Dawei; Zhang, Qiuyue; Sun, Xudong; Li, Yanfei

    2016-02-01

    Aluminum promotes osteoblast (OB) apoptosis. Apoptosis is induced by the disordered calcium homeostasis. Therefore, to investigate the relationship between Al-induced OB apoptosis and calcium homeostasis, calvarium OBs from neonatal rats (3-4 days) were cultured and exposed to 0.048-mg/mL Al(3+) or 0.048-mg/mL Al(3+) combined with 5 μM BAPTA-AM (OBs were pretreated with 5 μM BAPTA-AM for 1 h, then added 0.048 mg/mL Al(3+)), respectively. Then OB apoptosis rate, intracellular calcium ions concentration ([Ca(2+)]i), mRNA expression level of calmodulin (CaM), and protein expression levels of CaM and p-CaMKII in OBs were examined. The result showed that AlCl3 increased OB apoptosis rate, and [Ca(2+)]i and p-CaMKII expression levels and decreased CaM expression levels, whereas BAPTA-AM relieved the effects. These results proved that AlCl3 induced OB apoptosis by disrupting the intracellular Ca(2+) homeostasis and activating the Ca(2+)/CaMKII signal pathway. Our findings can provide new insights for revealing the apoptosis mechanism of OBs exposed to AlCl3.

  10. Calcium-sensing receptors signal constitutive macropinocytosis and facilitate the uptake of NOD2 ligands in macrophages

    PubMed Central

    Canton, Johnathan; Schlam, Daniel; Breuer, Christian; Gütschow, Michael; Glogauer, Michael; Grinstein, Sergio

    2016-01-01

    Macropinocytosis can be induced in several cell types by stimulation with growth factors. In selected cell types, notably macrophages and dendritic cells, macropinocytosis occurs constitutively, supporting the uptake of antigens for subsequent presentation. Despite their different mode of initiation and contrasting physiological roles, it is tacitly assumed that both types of macropinocytosis are mechanistically identical. We report that constitutive macropinocytosis is stringently calcium dependent, while stimulus-induced macropinocytosis is not. Extracellular calcium is sensed by G-protein-coupled calcium-sensing receptors (CaSR) that signal macropinocytosis through Gα-, phosphatidylinositol 3-kinase and phospholipase C. These pathways promote the recruitment of exchange factors that stimulate Rac and/or Cdc42, driving actin-dependent formation of ruffles and macropinosomes. In addition, the heterologous expression of CaSR in HEK293 cells confers on them the ability to perform constitutive macropinocytosis. Finally, we show that CaSR-induced constitutive macropinocytosis facilitates the sentinel function of macrophages, promoting the efficient delivery of ligands to cytosolic pattern-recognition receptors. PMID:27050483

  11. Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion

    PubMed Central

    Nishitani, Wagner Shin; Alencar, Adriano Mesquita; Wang, Yingxiao

    2015-01-01

    A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs) in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7) expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount) and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment) and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion. PMID:25946314

  12. Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion.

    PubMed

    Nishitani, Wagner Shin; Alencar, Adriano Mesquita; Wang, Yingxiao

    2015-01-01

    A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs) in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7) expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount) and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment) and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion.

  13. Calcium Signaling Involvement in Cadmium-Induced Astrocyte Cytotoxicity and Cell Death Through Activation of MAPK and PI3K/Akt Signaling Pathways.

    PubMed

    Jiang, Jiao Hua; Ge, Guo; Gao, Kai; Pang, Ying; Chai, Rui Chao; Jia, Xi Hua; Kong, Jin Ge; Yu, Albert Cheung-Hoi

    2015-09-01

    Cadmium (Cd), a highly ubiquitous toxic heavy metal, can contaminate the environment, including agricultural soil, water and air, via industrial runoff and other sources of pollution. Cd accumulated in the body via direct exposure or through the food chain results in neurodegeneration and many other diseases. Previous studies on its toxicity in the central nervous system (CNS) focused mainly on neurons. To obtain a more comprehensive understanding of Cd toxicity for the CNS, we investigated how astrocytes respond to acute and chronic Cd exposure and its toxic molecular mechanisms. When primary cultures of cerebral cortical astrocytes incubated with 1-300 μM CdCl2, morphological changes, LDH release and cell death were observed in a time and dose-dependent manner. Further studies demonstrated that acute and chronic Cd treatment phosphorylated JNK, p38 and Akt to different degrees, while ERK1/2 was only phosphorylated under low doses of Cd (10 μM) exposure. Inhibition of JNK and PI3K/Akt, but not of p38, could partially protect astrocyte from cytotoxicity in chronic and acute Cd exposure. Moreover, Cd also induced a strong calcium signal, while BAPTA, a specific intracellular calcium (Ca(2+)) chelator, prevented Cd-induced intracellular increase of calcium levels in astrocytes; inhibited the Cd-induced activation of ERK1/2, JNK, p38 and Akt; and also significantly reduced astrocyte cell death. All of these results suggested that the Cd-Ca(2+)-MAPK and PI3K/Akt signaling pathways were involved in Cd-induced toxicity in astrocytes. This toxicity involvement indicates that these pathways may be exploited as a target for the prevention of Cd-induced neurodegenerative diseases.

  14. Redox Modulation of Cellular Signaling and Metabolism Through Reversible Oxidation of Methionine Sensors in Calcium Regulatory Proteins

    SciTech Connect

    Bigelow, Diana J.; Squier, Thomas C.

    2005-01-17

    Adaptive responses associated with environmental stressors are critical to cell survival. These involve the modulation of central signaling protein functions through site-specific and enzymatically reversible oxidative modifications of methionines to coordinate cellular metabolism, energy utilization, and calcium signaling. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. Thus, depending on either the ecological niche of the organism or the cellular milieu of different organ systems, cellular metabolism can be fine-tuned to maintain optimal function in the face of variable amounts of collateral oxidative damage. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met{sup 144} or Met{sup 145} results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylationdependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in

  15. Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system

    PubMed Central

    Cordiglieri, Chiara; Odoardi, Francesca; Zhang, Bo; Nebel, Merle; Kawakami, Naoto; Klinkert, Wolfgang E. F.; Lodygin, Dimtri; Lühder, Fred; Breunig, Esther; Schild, Detlev; Ulaganathan, Vijay Kumar; Dornmair, Klaus; Dammermann, Werner; Potter, Barry V. L.; Guse, Andreas H.

    2010-01-01

    Nicotinic acid adenine dinucleotide phosphate represents a newly identified second messenger in T cells involved in antigen receptor-mediated calcium signalling. Its function in vivo is, however, unknown due to the lack of biocompatible inhibitors. Using a recently developed inhibitor, we explored the role of nicotinic acid adenine dinucleotide phosphate in autoreactive effector T cells during experimental autoimmune encephalomyelitis, the animal model for multiple sclerosis. We provide in vitro and in vivo evidence that calcium signalling controlled by nicotinic acid adenine dinucleotide phosphate is relevant for the pathogenic potential of autoimmune effector T cells. Live two photon imaging and molecular analyses revealed that nicotinic acid adenine dinucleotide phosphate signalling regulates T cell motility and re-activation upon arrival in the nervous tissues. Treatment with the nicotinic acid adenine dinucleotide phosphate inhibitor significantly reduced both the number of stable arrests of effector T cells and their invasive capacity. The levels of pro-inflammatory cytokines interferon-gamma and interleukin-17 were strongly diminished. Consecutively, the clinical symptoms of experimental autoimmune encephalomyelitis were ameliorated. In vitro, antigen-triggered T cell proliferation and cytokine production were evenly suppressed. These inhibitory effects were reversible: after wash-out of the nicotinic acid adenine dinucleotide phosphate antagonist, the effector T cells fully regained their functions. The nicotinic acid derivative BZ194 induced this transient state of non-responsiveness specifically in post-activated effector T cells. Naïve and long-lived memory T cells, which express lower levels of the putative nicotinic acid adenine dinucleotide phosphate receptor, type 1 ryanodine receptor, were not targeted. T cell priming and recall responses in vivo were not reduced. These data indicate that the nicotinic acid adenine dinucleotide phosphate/calcium

  16. Calcium Signaling Pathway Genes RUNX2 and CACNA1C Are Associated With Calcific Aortic Valve Disease

    PubMed Central

    Guauque-Olarte, Sandra; Messika-Zeitoun, David; Droit, Arnaud; Lamontagne, Maxime; Tremblay-Marchand, Joël; Lavoie-Charland, Emilie; Gaudreault, Nathalie; Arsenault, Benoit J.; Dubé, Marie-Pierre; Tardif, Jean-Claude; Body, Simon C.; Seidman, Jonathan G.; Boileau, Catherine; Mathieu, Patrick; Pibarot, Philippe; Bossé, Yohan

    2016-01-01

    Background Calcific aortic valve stenosis (AS) is a life-threatening disease with no medical therapy. The genetic architecture of AS remains elusive. This study combines genome-wide association studies, gene expression, and expression quantitative trait loci mapping in human valve tissues to identify susceptibility genes of AS. Methods and Results A meta-analysis was performed combining the results of 2 genome-wide association studies in 474 and 486 cases from Quebec City (Canada) and Paris (France), respectively. Corresponding controls consisted of 2988 and 1864 individuals with European ancestry from the database of genotypes and phenotypes. mRNA expression levels were evaluated in 9 calcified and 8 normal aortic valves by RNA sequencing. The results were integrated with valve expression quantitative trait loci data obtained from 22 AS patients. Twenty-five single-nucleotide polymorphisms had P<5×10−6 in the genome-wide association studies meta-analysis. The calcium signaling pathway was the top gene set enriched for genes mapped to moderately AS-associated single-nucleotide polymorphisms. Genes in this pathway were found differentially expressed in valves with and without AS. Two single-nucleotide polymorphisms located in RUNX2 (runt-related transcription factor 2), encoding an osteogenic transcription factor, demonstrated some association with AS (genome-wide association studies P=5.33×10−5). The mRNA expression levels of RUNX2 were upregulated in calcified valves and associated with eQTL-SNPs. CACNA1C encoding a subunit of a voltage-dependent calcium channel was upregulated in calcified valves. The eQTL-SNP with the most significant association with AS located in CACNA1C was associated with higher expression of the gene. Conclusions This integrative genomic study confirmed the role of RUNX2 as a potential driver of AS and identified a new AS susceptibility gene, CACNA1C, belonging to the calcium signaling pathway. PMID:26553695

  17. Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes.

    PubMed

    Devi, Sulochana; Markandeya, Yogananda; Maddodi, Nityanand; Dhingra, Anuradha; Vardi, Noga; Balijepalli, Ravi C; Setaluri, Vijayasaradhi

    2013-05-01

    Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gαo and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L-AP4, a type III mGluR-selective agonist, enhances Ca(2+) uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L-AP4-induced Ca(2+) influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gαo protein is absent in melanocytes. However, forced expression of Gαo restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of Gi /Go proteins, did not affect basal or mGluR6-induced Ca(2+) uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.

  18. Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes

    PubMed Central

    Devi, Sulochana; Markandeya, Yogananda; Maddodi, Nityanand; Dhingra, Anuradha; Vardi, Noga; Balijepalli, Ravi C; Setaluri, Vijayasaradhi

    2013-01-01

    SUMMARY Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gαo and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6 and treatment of melanocytes with L-AP4, a type III mGluR-selective agonist, enhances Ca2+ uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L-AP4-induced Ca2+ influx and TRPM1 currents showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gαo protein is absent in melanocytes. However, forced expression of Gαo restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with and pertussis toxin, an inhibitor of Gi/Go proteins, did not affect basal or mGluR6-induced Ca2+ uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin. PMID:23452348

  19. Science Signaling Podcast for 24 January 2017: Tissue-specific regulation of L-type calcium channels

    PubMed Central

    Hell, Johannes W.; Navedo, Manuel F.; VanHook, Annalisa M.

    2017-01-01

    This Podcast features an interview with Johannes Hell and Manuel Navedo, senior authors of two Research Articles that appear in the 24 January 2017 issue of Science Signaling, about tissue-specific regulation of the L-type calcium channel CaV1.2. This channel is present in many tissues, including the heart, vasculature, and brain, and allows calcium to flow into cells when it is activated. Signaling through the β-adrenergic receptor (βAR) stimulates CaV1.2 activity in heart cells and neurons to accelerate heart rate and increase neuronal excitability, respectively. Using mouse models, Qian et al. found that βAR-mediated enhancement of CaV1.2 activity in the brain required phosphorylation of Ser1928, whereas βAR-mediated enhancement of CaV1.2 activity in the heart did not require phosphorylation of this residue. In a related study, Nystoriak et al. demonstrated that phosphorylation of Ser1928 in arterial myocytes was required for vasoconstriction during acute hyperglycemia and in diabetic mice. These findings demonstrate tissue-specific differences in CaV1.2 regulation and suggest that it may be possible to design therapies to target this channel in specific tissues. PMID:28119457

  20. Science Signaling Podcast for 24 January 2017: Tissue-specific regulation of L-type calcium channels.

    PubMed

    Hell, Johannes W; Navedo, Manuel F; VanHook, Annalisa M

    2017-01-24

    This Podcast features an interview with Johannes Hell and Manuel Navedo, senior authors of two Research Articles that appear in the 24 January 2017 issue of Science Signaling, about tissue-specific regulation of the L-type calcium channel CaV1.2. This channel is present in many tissues, including the heart, vasculature, and brain, and allows calcium to flow into cells when it is activated. Signaling through the β-adrenergic receptor (βAR) stimulates CaV1.2 activity in heart cells and neurons to accelerate heart rate and increase neuronal excitability, respectively. Using mouse models, Qian et al found that βAR-mediated enhancement of CaV1.2 activity in the brain required phosphorylation of Ser(1928), whereas βAR-mediated enhancement of CaV1.2 activity in the heart did not require phosphorylation of this residue. In a related study, Nystoriak et al demonstrated that phosphorylation of Ser(1928) in arterial myocytes was required for vasoconstriction during acute hyperglycemia and in diabetic mice. These findings demonstrate tissue-specific differences in CaV1.2 regulation and suggest that it may be possible to design therapies to target this channel in specific tissues.Listen to Podcast. Copyright © 2017, American Association for the Advancement of Science.

  1. Nitric oxide and cGMP signaling in calcium-dependent development of cell polarity in Ceratopteris richardii.

    PubMed

    Salmi, Mari L; Morris, Kacey E; Roux, Stanley J; Porterfield, D Marshall

    2007-05-01

    Single-celled spores of the fern Ceratopteris richardii undergo gravity-directed cell polarity development that is driven by polar calcium currents. Here we present results that establish a role for nitric oxide (NO)/cGMP signaling in transducing the stimulus of gravity to directed polarization of the spores. Application of specific NO donors and scavengers inhibited the calcium-dependent gravity response in a dose-dependent manner. The effects of NO donor exposure were antagonized by application of NO scavenger compounds. Similarly, the guanylate cyclase inhibitors 6-anilino-5,8-quinolinedione and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin, and the phosphodiesterase inhibitor Viagra, which modulate NO-dependent cGMP levels in the cells, disrupted gravity-directed cell polarity in a dose-dependent manner. Viagra effects were antagonized by application of NO scavengers, consistent with the postulate that NO and cGMP are linked in the signaling pathway. To identify other components of the signaling system we analyzed gene expression changes induced by Viagra treatment using microarrays and quantitative real-time reverse transcription-polymerase chain reaction. Preliminary microarray analysis revealed several genes whose expression was significantly altered by Viagra treatment. Three of these genes had strong sequence similarity to key signal transduction or stress response genes and quantitative real-time reverse transcription-polymerase chain reaction was used to more rigorously quantify the effects of Viagra on their expression in spores and to test how closely these effects could be mimicked by treatment with dibutyryl cGMP. Taken together our results implicate NO and cGMP as downstream effectors that help link the gravity stimulus to polarized growth in C. richardii spores. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers BE 640669 to BE 643506, BQ 086920 to BQ 087668, and CV 734654 to CV 736151.

  2. Endogenous lysophosphatidic acid (LPA1 ) receptor agonists demonstrate ligand bias between calcium and ERK signalling pathways in human lung fibroblasts.

    PubMed

    Sattikar, Afrah; Dowling, Mark R; Rosethorne, Elizabeth M

    2017-02-01

    Human lung fibroblasts (HLF) express high levels of the LPA1 receptor, a GPCR that responds to the endogenous lipid mediator, lysophosphatidic acid (LPA). Several molecular species or analogues of LPA exist and have been detected in biological fluids such as serum and plasma. The most widely expressed of the LPA receptor family is the LPA1 receptor, which predominantly couples to Gq/11 , Gi/o and G12/13 proteins. This promiscuity of coupling raises the possibility that some of the LPA analogues may bias the LPA1 receptor towards one signalling pathway over another. Here, we have explored the signalling profiles of a range of LPA analogues in HLF that endogenously express the LPA1 receptor. HLF were treated with LPA analogues and receptor activation monitored via calcium mobilization and ERK phosphorylation. These analyses demonstrated that the 16:0, 17:0, 18:2 and C18:1 LPA analogues appear to exhibit ligand bias between ERK phosphorylation and calcium mobilization when compared with 18:1 LPA, one of the most abundant forms of LPA that has been found in human plasma. The importance of LPA as a key signalling molecule is shown by its widespread occurrence in biological fluids and its association with disease conditions such as fibrosis and cancer. These findings have important, as yet unexplored, implications for the (patho-) physiological signalling of the LPA1 receptor, as it may be influenced not only by the concentration of endogenous ligand but the isoform as well. © 2016 The British Pharmacological Society.

  3. Ryanodine receptors, calcium signaling and regulation of vascular tone in the cerebral parenchymal microcirculation

    PubMed Central

    Dabertrand, Fabrice; Nelson, Mark T.; Brayden, Joseph E.

    2012-01-01

    The cerebral blood supply is delivered by a surface network of pial arteries and arterioles from which arise (parenchymal) arterioles that penetrate into the cortex and terminate in a rich capillary bed. The critical regulation of cerebral blood flow, locally and globally, requires precise vasomotor regulation of the intracerebral microvasculature. This vascular region is anatomically unique as illustrated by the presence of astrocytic processes that envelope almost the entire basolateral surface of parenchymal arterioles. There are, moreover, notable functional differences between pial arteries and parenchymal arterioles. For example, in pial vascular smooth muscle cells (VSMCs), local calcium release events (“calcium sparks”) through ryanodine receptor (RyR) channels in sarcoplasmic reticulum membrane activate large conductance, calcium-sensitive potassium (BK) channels to modulate vascular diameter. In contrast, VSMCs in parenchymal arterioles express functional RyR and BK channels, but under physiological conditions these channels do not oppose pressure-induced vasoconstriction. Here we summarize the roles of ryanodine receptors in the parenchymal microvasculature under physiologic and pathologic conditions, and discuss their importance in the control of cerebral blood flow. PMID:23216877

  4. Nitric oxide signals are interlinked with calcium signals in normal pancreatic stellate cells upon oxidative stress and inflammation

    PubMed Central

    2016-01-01

    The mammalian diffuse stellate cell system comprises retinoid-storing cells capable of remarkable transformations from a quiescent to an activated myofibroblast-like phenotype. Activated pancreatic stellate cells (PSCs) attract attention owing to the pivotal role they play in development of tissue fibrosis in chronic pancreatitis and pancreatic cancer. However, little is known about the actual role of PSCs in the normal pancreas. These enigmatic cells have recently been shown to respond to physiological stimuli in a manner that is markedly different from their neighbouring pancreatic acinar cells (PACs). Here, we demonstrate the capacity of PSCs to generate nitric oxide (NO), a free radical messenger mediating, for example, inflammation and vasodilatation. We show that production of cytosolic NO in PSCs is unambiguously related to cytosolic Ca2+ signals. Only stimuli that evoke Ca2+ signals in the PSCs elicit consequent NO generation. We provide fresh evidence for the striking difference between signalling pathways in PSCs and adjacent PACs, because PSCs, in contrast to PACs, generate substantial Ca2+-mediated and NOS-dependent NO signals. We also show that inhibition of NO generation protects both PSCs and PACs from necrosis. Our results highlight the interplay between Ca2+ and NO signalling pathways in cell–cell communication, and also identify a potential therapeutic target for anti-inflammatory therapies. PMID:27488376

  5. The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins.

    PubMed

    Mola, Maria Grazia; Sparaneo, Angelo; Gargano, Concetta Domenica; Spray, David C; Svelto, Maria; Frigeri, Antonio; Scemes, Eliana; Nicchia, Grazia Paola

    2016-01-01

    Regulatory volume decrease (RVD) is a process by which cells restore their original volume in response to swelling. In this study, we have focused on the role played by two different Aquaporins (AQPs), Aquaporin-4 (AQP4), and Aquaporin-1 (AQP1), in triggering RVD and in mediating calcium signaling in astrocytes under hypotonic stimulus. Using biophysical techniques to measure water flux through the plasma membrane of wild-type (WT) and AQP4 knockout (KO) astrocytes and of an astrocyte cell line (DI TNC1) transfected with AQP4 or AQP1, we here show that AQP-mediated fast swelling kinetics play a key role in triggering and accelerating RVD. Using calcium imaging, we show that AQP-mediated fast swelling kinetics also significantly increases the amplitude of calcium transients inhibited by Gadolinium and Ruthenium Red, two inhibitors of the transient receptor potential vanilloid 4 (TRPV4) channels, and prevented by removing extracellular calcium. Finally, inhibition of TRPV4 or removal of extracellular calcium does not affect RVD. All together our study provides evidence that (1) AQP influenced swelling kinetics is the main trigger for RVD and in mediating calcium signaling after hypotonic stimulus together with TRPV4, and (2) calcium influx from the extracellular space and/or TRPV4 are not essential for RVD to occur in astrocytes.

  6. Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter.

    PubMed

    Xiong, Tou Cheu; Ronzier, Elsa; Sanchez, Frédéric; Corratgé-Faillie, Claire; Mazars, Christian; Thibaud, Jean-Baptiste

    2014-01-01

    Calcium (Ca(2+)) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca(2+) signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca(2+) signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca(2+) signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca(2+) imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca(2+) sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca(2+)-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca(2+) signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca(2+) dynamics in intact plants. We describe a simple method to image Ca(2+) signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca(2+) signals. It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

  7. Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter

    PubMed Central

    Xiong, Tou Cheu; Ronzier, Elsa; Sanchez, Frédéric; Corratgé-Faillie, Claire; Mazars, Christian; Thibaud, Jean-Baptiste

    2014-01-01

    Calcium (Ca2+) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca2+ signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca2+ signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca2+ signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca2+ imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca2+ sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca2+-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca2+ signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca2+ dynamics in intact plants. We describe a simple method to image Ca2+ signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca2+ signals. It is shown that Ca2+ signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins. PMID:24600459

  8. Magnolol and honokiol regulate the calcium-activated potassium channels signaling pathway in Enterotoxigenic Escherichia coli-induced diarrhea mice.

    PubMed

    Deng, Yanli; Han, Xuefeng; Tang, Shaoxun; Xiao, Wenjun; Tan, Zhiliang; Zhou, Chuanshe; Wang, Min; Kang, Jinghe

    2015-05-15

    To explore the regulatory mechanisms of magnolol and honokiol on calcium-activated potassium channels signaling pathway in Enterotoxigenic Escherichia coli (ETEC)-induced diarrhea mice, the concentrations of serum chloride ion (Cl(-)), sodium ion (Na(+)), potassium ion (K(+)) and calcium ion (Ca(2+)) were measured. Additionally, the mRNA expressions of calmodulin 1 (CaM), calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) and beta subunit (CaMKIIβ), ryanodine receptor 1, inositol 1,4,5-trisphosphate receptors (IP3 receptors), protein kinases C (PKC), potassium intermediate/small conductance calcium-activated channels (SK) and potassium large conductance calcium-activated channels(BK)were determined. A diarrhea mouse model was established using ETEC suspensions (3.29×10(9)CFU/ml) at a dosage of 0.02ml/g live body weight (BW). Magnolol or honokiol was intragastrically administered at dosages of 100 (M100 or H100), 300 (M300 or H300) and 500 (M500 or H500) mg/kg BW according to a 3×3 factorial arrangement. Magnolol and honokiol increased the Cl(-) and K(+) concentrations, further, upregulated the CaM, BKα1 and BKβ3 mRNA levels but downregulated the IP3 receptors 1, PKC, SK1, SK2, SK3, SK4 and BKβ4 mRNA expressions. Magnolol and honokiol did not alter the CaMKIIα, CaMKIIβ, ryanodine receptor 1, IP3 receptor 2, IP3 receptor 3, BKβ1 and BKβ2 mRNA expressions. These results clarify that magnolol and honokiol, acting through Ca(2+) channel blockade, inhibit the activation of IP3 receptor 1 to regulate the IP3-Ca(2+) store release, activate CaM to inhibit SK channels, and effectively suppress PKC kinases to promote BKα1 and BKβ3 channels opening and BKβ4 channel closing, which modulates the intestinal ion secretion. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Effect of sildenafil on cyclic nucleotide phosphodiesterase activity, vascular tone and calcium signaling in rat pulmonary artery

    PubMed Central

    Pauvert, O; Lugnier, C; Keravis, T; Marthan, R; Rousseau, E; Savineau, J P

    2003-01-01

    Sildenafil (viagra) is a potent PDE5 inhibitor and thus a relaxant drug in corpus carvernosum smooth muscle. In the present work, we evidenced the presence of PDE5 isozyme and investigated the effect of sildenafil on the specific cyclic nucleotide phosphodiesterase (PDE) activity, smooth muscle tone and calcium signaling in the rat main pulmonary artery (MPA). The PDE activity was measured in cytosolic and microsomal fractions. Total cAMP and cGMP-PDE activities were mainly present in the cytosolic fraction. Sildenafil (0.1 μM) reduced by 72% cGMP-PDE activity, whereas zaprinast (10 μM), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Sildenafil (0.1 μM) also inhibited significantly (22%) the cAMP-PDE activity. Western blot analysis revealed the expression of PDE5 mainly in the cytosolic fraction of MPA. Sildenafil concentration-dependently inhibited (IC50=3.4 nM) the activity of MPA PDE5 partially purified by HPLC. Sildenafil (0.1 nM–50 μM) concentration-dependently relaxed MPA rings precontracted with phenylephrine (0.5 μM). The potency of sildenafil (IC50=11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC50=600 nM). The vasorelaxant effect of sildenafil was not altered by endothelium removal or in the presence of KT 5823 (1 μM) and H89 (1 μM), potent inhibitors of PKG and PKA, respectively. In isolated MPA myocytes, which had been loaded with the calcium fluorophore indo-1, sildenafil (10–100 nM) antagonized ATP- and endothelin-1-induced calcium oscillations but had no effect on the transient caffeine-induced [Ca2+]i response. This study demonstrates the presence of a functional and highly sildenafil-sensitive PDE5 isozyme in rat MPA. Inhibition of this isozyme mainly accounts for the potent pulmonary vasodilator action of sildenafil, which involves alteration in the inositol triphosphate-mediated calcium signaling pathway. PMID:12788811

  10. Effect of sildenafil on cyclic nucleotide phosphodiesterase activity, vascular tone and calcium signaling in rat pulmonary artery.

    PubMed

    Pauvert, O; Lugnier, C; Keravis, T; Marthan, R; Rousseau, E; Savineau, J P

    2003-06-01

    (1) Sildenafil (viagra) is a potent PDE5 inhibitor and thus a relaxant drug in corpus carvernosum smooth muscle. In the present work, we evidenced the presence of PDE5 isozyme and investigated the effect of sildenafil on the specific cyclic nucleotide phosphodiesterase (PDE) activity, smooth muscle tone and calcium signaling in the rat main pulmonary artery (MPA). (2) The PDE activity was measured in cytosolic and microsomal fractions. Total cAMP and cGMP-PDE activities were mainly present in the cytosolic fraction. Sildenafil (0.1 micro M) reduced by 72% cGMP-PDE activity, whereas zaprinast (10 micro M), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Sildenafil (0.1 micro M) also inhibited significantly (22%) the cAMP-PDE activity. (3) Western blot analysis revealed the expression of PDE5 mainly in the cytosolic fraction of MPA. Sildenafil concentration-dependently inhibited (IC(50)=3.4 nM) the activity of MPA PDE5 partially purified by HPLC. (4) Sildenafil (0.1 nM-50 micro M) concentration-dependently relaxed MPA rings precontracted with phenylephrine (0.5 micro M). The potency of sildenafil (IC(50)=11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC(50)=600 nM). The vasorelaxant effect of sildenafil was not altered by endothelium removal or in the presence of KT 5823 (1 micro M) and H89 (1 micro M), potent inhibitors of PKG and PKA, respectively. (5) In isolated MPA myocytes, which had been loaded with the calcium fluorophore indo-1, sildenafil (10-100 nM) antagonized ATP- and endothelin-1-induced calcium oscillations but had no effect on the transient caffeine-induced [Ca(2+)](i) response. (6) This study demonstrates the presence of a functional and highly sildenafil-sensitive PDE5 isozyme in rat MPA. Inhibition of this isozyme mainly accounts for the potent pulmonary vasodilator action of sildenafil, which involves alteration in the inositol triphosphate-mediated calcium

  11. Differential volume regulation and calcium signaling in two ciliary body cell types is subserved by TRPV4 channels

    PubMed Central

    Jo, Andrew O.; Lakk, Monika; Frye, Amber M.; Phuong, Tam T. T.; Redmon, Sarah N.; Roberts, Robin; Berkowitz, Bruce A.; Yarishkin, Oleg; Križaj, David

    2016-01-01

    Fluid secretion by the ciliary body plays a critical and irreplaceable function in vertebrate vision by providing nutritive support to the cornea and lens, and by maintaining intraocular pressure. Here, we identify TRPV4 (transient receptor potential vanilloid isoform 4) channels as key osmosensors in nonpigmented epithelial (NPE) cells of the mouse ciliary body. Hypotonic swelling and the selective agonist GSK1016790A (EC50 ∼33 nM) induced sustained transmembrane cation currents and cytosolic [Ca2+]i elevations in dissociated and intact NPE cells. Swelling had no effect on [Ca2+]i levels in pigment epithelial (PE) cells, whereas depolarization evoked [Ca2+]i elevations in both NPE and PE cells. Swelling-evoked [Ca2+]i signals were inhibited by the TRPV4 antagonist HC067047 (IC50 ∼0.9 μM) and were absent in Trpv4−/− NPE. In NPE, but not PE, swelling-induced [Ca2+]i signals required phospholipase A2 activation. TRPV4 localization to NPE was confirmed with immunolocalization and excitation mapping approaches, whereas in vivo MRI analysis confirmed TRPV4-mediated signals in the intact mouse ciliary body. Trpv2 and Trpv4 were the most abundant vanilloid transcripts in CB. Overall, our results support a model whereby TRPV4 differentially regulates cell volume, lipid, and calcium signals in NPE and PE cell types and therefore represents a potential target for antiglaucoma medications. PMID:27006502

  12. Differential volume regulation and calcium signaling in two ciliary body cell types is subserved by TRPV4 channels.

    PubMed

    Jo, Andrew O; Lakk, Monika; Frye, Amber M; Phuong, Tam T T; Redmon, Sarah N; Roberts, Robin; Berkowitz, Bruce A; Yarishkin, Oleg; Križaj, David

    2016-04-05

    Fluid secretion by the ciliary body plays a critical and irreplaceable function in vertebrate vision by providing nutritive support to the cornea and lens, and by maintaining intraocular pressure. Here, we identify TRPV4 (transient receptor potential vanilloid isoform 4) channels as key osmosensors in nonpigmented epithelial (NPE) cells of the mouse ciliary body. Hypotonic swelling and the selective agonist GSK1016790A (EC50 ∼33 nM) induced sustained transmembrane cation currents and cytosolic [Formula: see text] elevations in dissociated and intact NPE cells. Swelling had no effect on [Formula: see text] levels in pigment epithelial (PE) cells, whereas depolarization evoked [Formula: see text] elevations in both NPE and PE cells. Swelling-evoked [Formula: see text] signals were inhibited by the TRPV4 antagonist HC067047 (IC50 ∼0.9 μM) and were absent in Trpv4(-/-) NPE. In NPE, but not PE, swelling-induced [Formula: see text] signals required phospholipase A2 activation. TRPV4 localization to NPE was confirmed with immunolocalization and excitation mapping approaches, whereas in vivo MRI analysis confirmed TRPV4-mediated signals in the intact mouse ciliary body. Trpv2 and Trpv4 were the most abundant vanilloid transcripts in CB. Overall, our results support a model whereby TRPV4 differentially regulates cell volume, lipid, and calcium signals in NPE and PE cell types and therefore represents a potential target for antiglaucoma medications.

  13. Voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic development.

    PubMed

    Yamashita, Naoya; Aoki, Reina; Chen, Sandy; Jitsuki-Takahashi, Aoi; Ohura, Shunsuke; Kamiya, Haruyuki; Goshima, Yoshio

    2016-01-15

    Growing axons rely on local signaling at the growth cone for guidance cues. Semaphorin3A (Sema3A), a secreted repulsive axon guidance molecule, regulates synapse maturation and dendritic branching. We previously showed that local Sema3A signaling in the growth cones elicits retrograde retrograde signaling via PlexinA4 (PlexA4), one component of the Sema3A receptor, thereby regulating dendritic localization of AMPA receptor GluA2 and proper dendritic development. In present study, we found that nimodipine (voltage-gated L-type Ca(2+) channel blocker) and tetrodotoxin (TTX; voltage-gated Na(+) channel blocker) suppress Sema3A-induced dendritic localization of GluA2 and dendritic branch formation in cultured hippocampal neurons. The local application of nimodipine or TTX to distal axons suppresses retrograde transport of Venus-Sema3A that has been exogenously applied to the distal axons. Sema3A facilitates axonal transport of PlexA4, which is also suppressed in neurons treated with either TTX or nimodipine. These data suggest that voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic GluA2 localization and branch formation.

  14. Calcium alterations signal either to senescence or to autophagy induction in stem cells upon oxidative stress.

    PubMed

    Borodkina, Aleksandra V; Shatrova, Alla N; Deryabin, Pavel I; Griukova, Anastasiia A; Abushik, Polina A; Antonov, Sergei M; Nikolsky, Nikolay N; Burova, Elena B

    2016-12-08

    Intracellular calcium ([Ca(2+)]i) has been reported to play an important role in autophagy, apoptosis and necrosis, however, a little is known about its impact in senescence. Here we investigated [Ca(2+)]i contribution to oxidative stress-induced senescence of human endometrium-derived stem cells (hMESCs). In hMESCs sublethal H2O2-treatment resulted in a rapid calcium release from intracellular stores mediated by the activation of PLC/IP3/IP3R pathway. Notably, further senescence development was accompanied by persistently elevated [Ca(2+)]i levels. In H2O2-treated hMESCs, [Ca(2+)]i chelation by BAPTA-AM (BAPTA) was sufficient to prevent the expansion of the senescence phenotype, to decrease endogenous reactive oxygen species levels, to avoid G0/G1 cell cycle arrest, and finally to retain proliferation. Particularly, loading with BAPTA attenuated phosphorylation of the main DNA damage response members, including ATM, 53BP1 and H2A.X and reduced activation of the p53/p21/Rb pathway in H2O2-stimulated cells. Next, we revealed that BAPTA induced an early onset of AMPK-dependent autophagy in H2O2-treated cells as confirmed by both the phosphorylation status of AMPK/mTORC1 pathway and the dynamics of the LC3 lipidization. Summarizing the obtained data we can assume that calcium chelation is able to trigger short-term autophagy and to prevent the premature senescence of hMESCs under oxidative stress.

  15. Calcium alterations signal either to senescence or to autophagy induction in stem cells upon oxidative stress

    PubMed Central

    Borodkina, Aleksandra V.; Shatrova, Alla N.; Deryabin, Pavel I.; Griukova, Anastasiia A.; Abushik, Polina A.; Antonov, Sergei M.; Nikolsky, Nikolay N.; Burova, Elena B.

    2016-01-01

    Intracellular calcium ([Ca2+]i) has been reported to play an important role in autophagy, apoptosis and necrosis, however, a little is known about its impact in senescence. Here we investigated [Ca2+]i contribution to oxidative stress-induced senescence of human endometrium-derived stem cells (hMESCs). In hMESCs sublethal H2O2-treatment resulted in a rapid calcium release from intracellular stores mediated by the activation of PLC/IP3/IP3R pathway. Notably, further senescence development was accompanied by persistently elevated [Ca2+]i levels. In H2O2-treated hMESCs, [Ca2+]i chelation by BAPTA-AM (BAPTA) was sufficient to prevent the expansion of the senescence phenotype, to decrease endogenous reactive oxygen species levels, to avoid G0/G1 cell cycle arrest, and finally to retain proliferation. Particularly, loading with BAPTA attenuated phosphorylation of the main DNA damage response members, including ATM, 53BP1 and H2A.X and reduced activation of the p53/p21/Rb pathway in H2O2-stimulated cells. Next, we revealed that BAPTA induced an early onset of AMPK-dependent autophagy in H2O2-treated cells as confirmed by both the phosphorylation status of AMPK/mTORC1 pathway and the dynamics of the LC3 lipidization. Summarizing the obtained data we can assume that calcium chelation is able to trigger short-term autophagy and to prevent the premature senescence of hMESCs under oxidative stress. PMID:27941214

  16. Intracellular calcium promotes radioresistance of non-small cell lung cancer A549 cells through activating Akt signaling.

    PubMed

    Wang, Yiling; He, Jiantao; Zhang, Shenghui; Yang, Qingbo

    2017-03-01

    Radiotherapy is a major therapeutic approach in non-small cell lung cancer but is restricted by radioresistance. Although Akt signaling promotes radioresistance in non-small cell lung cancer, it is not well understood how Akt signaling is activated. Since intracellular calcium (Ca(2+)) could activate Akt in A549 cells, we investigated the relationship between intracellular calcium (Ca(2+)) and Akt signaling in radioresistant A549 cells by establishing radioresistant non-small cell lung cancer A549 cells. The radioresistant cell line A549 was generated by dose-gradient irradiation of the parental A549 cells. The cell viability, proliferation, and apoptosis were, respectively, assessed using the cell counting kit-8, EdU labeling, and flow cytometry analysis. The phosphorylation of Akt was evaluated by Western blotting, and the intracellular Ca(2+) concentration was assessed by Fluo 4-AM. The radioresistant A549 cells displayed mesenchymal morphology. After additional irradiation, the radioresistant A549 cells showed decreased cell viability and proliferation but increased apoptosis. Moreover, the intracellular Ca(2+) concentration and the phosphorylation level on the Akt473 site in radioresistant A549 cells were higher than those in original cells, whereas the percentage of apoptosis in radioresistant A549 cells was less. All these results could be reversed by verapamil. In conclusion, our study found that intracellular Ca(2+) could promote radioresistance of non-small cell lung cancer cells through phosphorylating of Akt on the 473 site, which contributes to a better understanding on the non-small cell lung cancer radioresistance, and may provide a new target for radioresistance management.

  17. The Glycosyltransferase QUA1 Regulates Chloroplast-Associated Calcium Signaling During Salt and Drought Stress in Arabidopsis.

    PubMed

    Zheng, Yuan; Liao, Chancan; Zhao, Shuangshuang; Wang, Chongwu; Guo, Yan

    2017-02-01

    Cytoplasmic Ca2+ ([Ca2+]cyt) elevation induced by various signals is responsible for appropriate downstream responses. Through a genetic screen of Arabidopsis thaliana mutants defective in stress-induced [Ca2+]cyt elevation, the glycosyltransferase QUASIMODO1 (QUA1) was identified as a regulator of [Ca2+]cyt in response to salt stress. Compared with the wild type, the qua1-4 mutant exhibited a dramatically greater increase in [Ca2+]cyt under NaCl treatment. Functional analysis showed that QUA1 is a novel chloroplast protein that regulates cytoplasmic Ca2+ signaling. QUA1 was detected in chloroplast thylakoids, and the qua1-4 mutant exhibited irregularly stacked grana. The observed greater increase in [Ca2+]cyt was inhibited upon recovery of chloroplast function in the qua1-4 mutant. Further analysis showed that CAS, a thylakoid-localized calcium sensor, also displayed irregularly stacked grana, and the chloroplasts of the qua1-4 cas-1 double mutant were similar to those of cas-1 plants. In QUA1-overexpressing plants, the protein level of CAS was decreased, and CAS was readily degraded under osmotic stress. When CAS was silenced in the qua1-4 mutant, the large [Ca2+]cyt increase was blocked, and the higher expression of PLC3 and PLC4 was suppressed. Under osmotic stress, the qua1-4 mutant showed an even greater elevation in [Ca2+]cyt and was hypersensitive to drought stress. However, this sensitivity was inhibited when the increase in [Ca2+]cyt was repressed in the qua1-4 mutant. Collectively, our data indicate that QUA1 may function in chloroplast-dependent calcium signaling under salt and drought stresses. Additionally, CAS may function downstream of QUA1 to mediate these processes. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Transient Oxygen/Glucose Deprivation Causes a Delayed Loss of Mitochondria and Increases Spontaneous Calcium Signaling in Astrocytic Processes

    PubMed Central

    O'Donnell, John C.; Jackson, Joshua G.

    2016-01-01

    brain, are vital integrators of signaling and metabolism. Each astrocyte consists of many long, thin branches, called processes, which ensheathe vasculature and thousands of synapses. Mitochondria occupy the majority of each process. This occupancy is decreased by ∼50% 24 h after an in vitro model of ischemia/reperfusion injury, due to delayed fragmentation and mitophagy. The mechanism appears to be independent of neuropathology, instead involving an extended period of high glutamate uptake into astrocytes. Our data suggest that mitochondria serve as spatial buffers, and possibly even as a source of calcium signals in astrocytic processes. Loss of mitochondria resulted in drastically altered calcium signaling that could disrupt neurovascular coupling and gliotransmission. PMID:27383588

  19. Transient Oxygen/Glucose Deprivation Causes a Delayed Loss of Mitochondria and Increases Spontaneous Calcium Signaling in Astrocytic Processes.

    PubMed

    O'Donnell, John C; Jackson, Joshua G; Robinson, Michael B

    2016-07-06

    , are vital integrators of signaling and metabolism. Each astrocyte consists of many long, thin branches, called processes, which ensheathe vasculature and thousands of synapses. Mitochondria occupy the majority of each process. This occupancy is decreased by ∼50% 24 h after an in vitro model of ischemia/reperfusion injury, due to delayed fragmentation and mitophagy. The mechanism appears to be independent of neuropathology, instead involving an extended period of high glutamate uptake into astrocytes. Our data suggest that mitochondria serve as spatial buffers, and possibly even as a source of calcium signals in astrocytic processes. Loss of mitochondria resulted in drastically altered calcium signaling that could disrupt neurovascular coupling and gliotransmission. Copyright © 2016 the authors 0270-6474/16/367110-19$15.00/0.

  20. The Speed of Swelling Kinetics Modulates Cell Volume Regulation and Calcium Signaling in Astrocytes: A Different Point of View on the Role of Aquaporins

    PubMed Central

    Mola, Maria Grazia; Sparaneo, Angelo; Gargano, Concetta Domenica; Spray, David C.; Svelto, Maria; Frigeri, Antonio; Scemes, Eliana; Nicchia, Grazia Paola

    2016-01-01

    Regulatory volume decrease (RVD) is a process by which cells restore their original volume in response to swelling. In this study, we have focused on the role played by two different Aquaporins (AQPs), Aquaporin-4 (AQP4), and Aquaporin-1 (AQP1), in triggering RVD and in mediating calcium signaling in astrocytes under hypotonic stimulus. Using biophysical techniques to measure water flux through the plasma membrane of wild-type (WT) and AQP4 knockout (KO) astrocytes and of an astrocyte cell line (DI TNC1) transfected with AQP4 or AQP1, we here show that AQP-mediated fast swelling kinetics play a key role in triggering and accelerating RVD. Using calcium imaging, we show that AQP-mediated fast swelling kinetics also significantly increases the amplitude of calcium transients inhibited by Gadolinium and Ruthenium Red, two inhibitors of the transient receptor potential vanilloid 4 (TRPV4) channels, and prevented by removing extracellular calcium. Finally, inhibition of TRPV4 or removal of extracellular calcium does not affect RVD. All together our study provides evidence that (1) AQP influenced swelling kinetics is the main trigger for RVD and in mediating calcium signaling after hypotonic stimulus together with TRPV4, and (2) calcium influx from the extracellular space and/or TRPV4 are not essential for RVD to occur in astrocytes. Main Points: (1) The speed of swelling kinetics is the main trigger for Regulatory Volume Decrease (RVD) and for calcium response in astrocytes; (2) Calcium influx from the extracellular space and TRPV4 are not essential for RVD. PMID:26413835

  1. Chromaffin cell calcium signal and morphology study based on multispectral images

    NASA Astrophysics Data System (ADS)

    Wu, Hongxiu; Wei, Shunhui; Qu, Anlian; Zhou, Zhuan

    1998-09-01

    Increasing or decreasing the internal calcium concentration can promote or prevent programmed cell death (PCD). We therefore performed a Ca2+ imaging study using Ca2+ indicator dye fura-2 and a sensitive cooled-CCD camera with a 12 bit resolution. Monochromatic beams of light with a wavelength of 345,380 nm were isolated from light emitted by a xenon lamp using a monochromator. The concentration of free calcium can be directly calculated from the ratio of two fluorescence values taken at two appropriately selected wavelength. Fluorescent light emitted from the cells was capture using a camera system. The cell morphology study is based on multispectral scanning, with smear images provided as three monochromatic images by illumination with light of 610,535 and 470 nm wavelengths. The nuclear characteristic parameters extracted from individual nuclei by system are nuclear area, nuclear diameter, nuclear density vector. The results of the restoration of images and the performance of a primitive logic for the detection of nuclei with PCD proved the usefulness of the system and the advantages of using multispectral images in the restoration and detection procedures.

  2. Intracellular calcium signals regulate growth of hepatic stellate cells via specific effects on cell cycle progression.

    PubMed

    Soliman, Elwy M; Rodrigues, Michele Angela; Gomes, Dawidson Assis; Sheung, Nina; Yu, Jin; Amaya, Maria Jimina; Nathanson, Michael H; Dranoff, Jonathan A

    2009-03-01

    Hepatic stellate cells (HSC) are important mediators of liver fibrosis. Hormones linked to downstream intracellular Ca(2+) signals upregulate HSC proliferation, but the mechanisms by which this occurs are unknown. Nuclear and cytosolic Ca(2+) signals may have distinct effects on cell proliferation, so we expressed plasmid and adenoviral constructs containing the Ca(2+) chelator parvalbumin (PV) linked to either a nuclear localization sequence (NLS) or a nuclear export sequence (NES) to block Ca(2+) signals in distinct compartments within LX-2 immortalized human HSC and primary rat HSC. PV-NLS and PV-NES constructs each targeted to the appropriate intracellular compartment and blocked Ca(2+) signals only within that compartment. PV-NLS and PV-NES constructs inhibited HSC growth. Furthermore, blockade of nuclear or cytosolic Ca(2+) signals arrested growth at the G2/mitosis (G2/M) cell-cycle interface and prevented the onset of mitosis. Blockade of nuclear or cytosolic Ca(2+) signals downregulated phosphorylation of the G2/M checkpoint phosphatase Cdc25C. Inhibition of calmodulin kinase II (CaMK II) had identical effects on LX-2 growth and Cdc25C phosphorylation. We propose that nuclear and cytosolic Ca(2+) are critical signals that regulate HSC growth at the G2/M checkpoint via CaMK II-mediated regulation of Cdc25C phosphorylation. These data provide a new logical target for pharmacological therapy directed against progression of liver fibrosis.

  3. Subcellular propagation of calcium waves in Müller glia does not require autocrine/paracrine purinergic signaling.

    PubMed

    Phuong, Tam T T; Yarishkin, Oleg; Križaj, David

    2016-09-02

    The polarized morphology of radial glia allows them to functionally interconnect different layers of CNS tissues including the retina, cerebellum, and cortex. A likely mechanism involves propagation of transcellular Ca(2+) waves which were proposed to involve purinergic signaling. Because it is not known whether ATP release is required for astroglial Ca(2+) wave propagation we investigated this in mouse Müller cells, radial astroglia-like retinal cells in which in which waves can be induced and supported by Orai/TRPC1 (transient receptor potential isoform 1) channels. We found that depletion of endoplasmic reticulum (ER) stores triggers regenerative propagation of transcellular Ca(2+) waves that is independent of ATP release and activation of P2X and P2Y receptors. Both the amplitude and kinetics of transcellular, depletion-induced waves were resistant to non-selective purinergic P2 antagonists such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Thus, store-operated calcium entry (SOCE) is itself sufficient for the initiation and subcellular propagation of calcium waves in radial glia.

  4. Acrolein induces Hsp72 via both PKCdelta/JNK and calcium signaling pathways in human umbilical vein endothelial cells.

    PubMed

    Misonou, Yoshiko; Takahashi, Motoko; Park, Yong Seek; Asahi, Michio; Miyamoto, Yasuhide; Sakiyama, Haruhiko; Cheng, Xinyao; Taniguchi, Naoyuki

    2005-05-01

    Acrolein is a highly electrophilic alpha,beta-unsaturated aldehydes to which humans are exposed in a variety of environment situations and is also a product of lipid peroxidation. Increased levels of unsaturated aldehydes play an important role in the pathogenesis of a number of human diseases such as Alzheimer's disease, atherosclerosis and diabetes. A number of studies have reported that acrolein evokes downstream signaling via an elevation in cellular oxidative stress. Here, we report that low concentrations of acrolein induce Hsp72 in human umbilical vein endothelial cells (HUVEC) and that both the PKCdelta/JNK pathway and calcium pathway were involved in the induction. The findings confirm that the production of reactive oxygen species (ROS) is not directly involved in the pathway. The induction of Hsp72 was not observed in other cells such as smooth muscle cells (SMC) or COS-1 cells. The results suggest that HUVEC have a unique defense system against cell damage by acrolein in which Hsp72 is induced via activation of both the PKCd/JNK and the calcium pathway.

  5. Odontogenic differentiation of human dental pulp cells by calcium silicate materials stimulating via FGFR/ERK signaling pathway.

    PubMed

    Liu, Chao-Hsin; Hung, Chi-Jr; Huang, Tsui-Hsien; Lin, Chi-Chang; Kao, Chia-Tze; Shie, Ming-You

    2014-10-01

    Bone healing needs a complex interaction of growth factors that establishes an environment for efficient bone formation. We examine how calcium silicate (CS) and tricalcium phosphate (β-TCP) cements influence the behavior of human dental pulp cells (hDPCs) through fibroblast growth factor receptor (FGFR) and active MAPK pathways, in particular ERK. The hDPCs are cultured with β-TCP and CS, after which the cells' viability and odontogenic differentiation markers are determined by using PrestoBlue® assay and western blot, respectively. The effect of small interfering RNA (siRNA) transfection targeting FGFR was also evaluated. The results showed that CS promoted cell proliferation and enhances FGFR expression. It was also found that CS increases ERK and p38 activity in hDPCs, and furthermore, raises the expression and secretion of DSP, and DMP-1. Additionally, statistically significant differences (p<0.05) have been found in the calcium deposition in si-FGFR transfection and ERK inhibitor between CS and β-TCP; these variations indicated that ERK/MAPK signaling is involved in the silicon-induced odontogenic differentiation of hDPCs. The current study shows that CS substrates play a key role in odontoblastic differentiation of hDPCs through FGFR and modulate ERK/MAPK activation. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. KChIP2 Attenuates Cardiac Hypertrophy Through Regulation of Ito and Intracellular Calcium Signaling

    PubMed Central

    Jin, Hongwei; Hadri, Lahouaria; Palomeque, Julieta; Morel, Charlotte; Karakikes, Ioannis; Kaprielian, Roger; Hajjar, Roger; Lebeche, Djamel

    2010-01-01

    Recent evidence shows that the auxiliary subunit KChIP2, which assembles with pore-forming Kv4-subunits, represents a new potential regulator of the cardiac calcium-independent transient outward potassium current (Ito) density. In hypertrophy and heart failure, KChIP2 expression has been found to be significantly decreased. Our aim was to examine the role of KChIP2 in cardiac hypertrophy and the effect of restoring its expression on electrical remodeling and cardiac mechanical function using a combination of molecular, biochemical and gene targeting approaches. KChIP2 overexpression through gene transfer of Ad.KChIP2 in neonatal cardiomyocytes resulted in a significant increase in Ito-channel forming Kv4.2 and Kv4.3 protein levels. In vivo gene transfer of KChIP2 in aortic banded adult rats showed that, compared to sham-operated or Ad.β-gal-transduced hearts, KChIP2 significantly attenuated the developed left ventricular hypertrophy, robustly increased Ito densities, shortened action potential duration, and significantly altered myocyte mechanics by shortening contraction amplitudes and maximal rates of contraction and relaxation velocities and decreasing Ca2+ transients. Interestingly, blocking Ito with 4-aminopyridine in KChIP2-overexpressing adult cardiomyocytes significantly increased the Ca2+ transients to control levels. One-day old rat pups intracardially transduced with KChIP2 for two months then subjected to aortic banding for 6–8 weeks (to induce hypertrophy) showed similar echocardiographic, electrical and mechanical remodeling parameters. In addition, in cultured adult cardiomyocytes, KChIP2 overexpression increased the expression of Ca2+-ATPase (SERCA2a) and sodium calcium exchanger but had no effect on ryanodine receptor 2 or phospholamban expression. In neonatal myocytes, KChIP2 notably reversed Ang II-induced hypertrophic changes in protein synthesis and MAP-kinase activation. It also significantly decreased calcineurin expression, NFATc1

  7. Insights into the early evolution of animal calcium signaling machinery: A unicellular point of view

    PubMed Central

    Cai, Xinjiang; Wang, Xiangbing; Patel, Sandip; Clapham, David E.

    2014-01-01

    The basic principles of Ca2+ regulation emerged early in prokaryotes. Ca2+ signaling acquired more extensive and varied functions when life evolved into multicellular eukaryotes with intracellular organelles. Animals, fungi and plants display differences in the mechanisms that control cytosolic Ca2+ concentrations. The aim of this review is to examine recent findings from comparative genomics of Ca2+ signaling molecules in close unicellular relatives of animals and in common unicellular ancestors of animals and fungi. Also discussed are the evolution and origins of the sperm-specific CatSper channel complex, cation/Ca2+ exchangers and four-domain voltage-gated Ca2+ channels. Newly identified evolutionary evidence suggests that the distinct Ca2+ signaling machineries in animals, plants and fungi likely originated from an ancient Ca2+ signaling machinery prior to early eukaryotic radiation. PMID:25498309

  8. Insights into the early evolution of animal calcium signaling machinery: a unicellular point of view.

    PubMed

    Cai, Xinjiang; Wang, Xiangbing; Patel, Sandip; Clapham, David E

    2015-03-01

    The basic principles of Ca(2+) regulation emerged early in prokaryotes. Ca(2+) signaling acquired more extensive and varied functions when life evolved into multicellular eukaryotes with intracellular organelles. Animals, fungi and plants display differences in the mechanisms that control cytosolic Ca(2+) concentrations. The aim of this review is to examine recent findings from comparative genomics of Ca(2+) signaling molecules in close unicellular relatives of animals and in common unicellular ancestors of animals and fungi. Also discussed are the evolution and origins of the sperm-specific CatSper channel complex, cation/Ca(2+) exchangers and four-domain voltage-gated Ca(2+) channels. Newly identified evolutionary evidence suggests that the distinct Ca(2+) signaling machineries in animals, plants and fungi likely originated from an ancient Ca(2+) signaling machinery prior to early eukaryotic radiation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Glial Cell Calcium Signaling Mediates Capillary Regulation of Blood Flow in the Retina

    PubMed Central

    Biesecker, Kyle R.; Srienc, Anja I.; Shimoda, Angela M.; Agarwal, Amit; Bergles, Dwight E.; Kofuji, Paulo

    2016-01-01