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Ca2+ Flux (ca2+ + flux)
Selected AbstractsStatins inhibit NK-cell cytotoxicity by interfering with LFA-1-mediated conjugate formationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2009Patrick C. Raemer Abstract Inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, commonly referred to as statins, are inhibitors of cholesterol biosynthesis. They are broadly used for treating hypercholesterolemia and for prevention of cardio- and cerebrovascular diseases. Recent publications show that statins also act as immunomodulatory drugs. Here, we show that lipophilic statins inhibit NK-cell degranulation and cytotoxicity. This effect was reversible by addition of substrates of isoprenylation, but not by addition of cholesterol. In NK-target cell conjugates intracellular Ca2+ flux was unaffected by statin treatment. However, statins strongly reduced the amount of conjugate formation between NK and target cells. This inhibition was paralleled by a statin-dependent inhibition of LFA-1-mediated adhesion and a reduction of NK-cell polarization. This demonstrates that statins impair the formation of effector,target cell conjugates resulting in the disruption of early signaling and the loss of NK-cell cytotoxicity. [source] Chemokines integrate JAK/STAT and G-protein pathways during chemotaxis and calcium flux responsesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2003Silvia Abstract The JAK/STAT (Janus kinase / signaling transducer and activator of transcription) signaling pathway is implicated in converting stationary epithelial cells to migratory cells. In mammals, migratory responses are activated by chemoattractant proteins, including chemokines. We found that by binding to seven-transmembrane G-protein-coupled receptors, chemokines activate the JAK/STAT pathwayto trigger chemotactic responses. We show that chemokine-mediated JAK/STAT activation is critical for G-protein induction and for phospholipase C-, dependent Ca2+ flux; in addition, pharmacological inhibition of JAK or mutation of the JAK kinase domain causes defects in both responses. Furthermore, G,i association with the receptor is dependent on JAK activation, andthe chemokine-mediated Ca2+ flux that requires phospholipase C-, activity takes place downstream of JAK kinases. The chemokines thus employ a mechanism that links heterologous signaling pathways , G proteins and tyrosine kinases , in a network that may be essential for mediating their pleiotropic responses. [source] Decreased Triadin and Increased Calstabin2 Expression in Great Danes with Dilated CardiomyopathyJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 5 2009M.A. Oyama Background: Dilated cardiomyopathy (DCM) is a common cardiac disease of Great Dane dogs, yet very little is known about the underlying molecular abnormalities that contribute to disease. Objective: Discover a set of genes that are differentially expressed in Great Dane dogs with DCM as a way to identify candidate genes for further study as well as to better understand the molecular abnormalities that underlie the disease. Animals: Three Great Dane dogs with end-stage DCM and 3 large breed control dogs. Methods: Prospective study. Transcriptional activity of 42,869 canine DNA sequences was determined with a canine-specific oligonucleotide microarray. Genome expression patterns of left ventricular tissue samples from affected Great Dane dogs were evaluated by measuring the relative amount of complementary RNA hybridization to the microarray probes and comparing it with expression from large breed dogs with noncardiac disease. Results: Three hundred and twenty-three transcripts were differentially expressed (,2-fold change). The transcript with the greatest degree of upregulation (+61.3-fold) was calstabin2 (FKBP12.6), whereas the transcript with the greatest degree of downregulation (,9.07-fold) was triadin. Calstabin2 and triadin are both regulatory components of the cardiac ryanodine receptor (RyR2) and are critical to normal intracellular Ca2+ release and excitation-contraction coupling. Conclusion and clinical importance: Great Dane dogs with DCM demonstrate abnormal calstabin2 and triadin expression. These changes likely affect Ca2+ flux within cardiac cells and may contribute to the pathophysiology of disease. Microarray-based analysis identifies calstabin2, triadin, and RyR2 function as targets of future study. [source] Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammalsJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 4 2002Erich W. Zinser Oxindole alkaloids in the paraherquamide/marcfortine family exhibit broad-spectrum anthelmintic activity that includes drug-resistant strains of nematodes. Paraherquamide (PHQ), 2-deoxoparaherquamide (2DPHQ), and close structural analogs of these compounds rapidly induce flaccid paralysis in parasitic nematodes in vitro, without affecting adenosine triphosphate (ATP) levels. The mechanism of action of this anthelmintic class was investigated using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum. None of the compounds altered A. suum muscle tension or membrane potential. However, PHQ blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by acetylcholine (ACh) and the nicotinic agonists levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic activity of PHQ and marcfortines is due to blockade of cholinergic neuromuscular transmission. The effects of these compounds were also examined on subtypes of human nicotinic ACh receptors expressed in mammalian cells with a Ca2+ flux assay. 2DPHQ blocked nicotinic stimulation of cells expressing ,3 ganglionic (IC50 , 9 µm) and muscle-type (IC50 , 3 µm) nicotinic cholinergic receptors, but was inactive at 100 µm vs. the ,7 CNS subtype. PHQ anthelmintics are nicotinic cholinergic antagonists in both nematodes and mammals, and this mechanism appears to underlie both their efficacy and toxicity. [source] Rapid Ca2+ flux through the transverse tubular membrane, activated by individual action potentials in mammalian skeletal muscleTHE JOURNAL OF PHYSIOLOGY, Issue 10 2009Bradley S. Launikonis Periods of low frequency stimulation are known to increase the net Ca2+ uptake in skeletal muscle but the mechanism responsible for this Ca2+ entry is not known. In this study a novel high-resolution fluorescence microscopy approach allowed the detection of an action potential-induced Ca2+ flux across the tubular (t-) system of rat extensor digitorum longus muscle fibres that appears to be responsible for the net uptake of Ca2+ in working muscle. Action potentials were triggered in the t-system of mechanically skinned fibres from rat by brief field stimulation and t-system [Ca2+] ([Ca2+]t-sys) and cytoplasmic [Ca2+] ([Ca2+]cyto) were simultaneously resolved on a confocal microscope. When initial [Ca2+]t-sys was , 0.2 mm a Ca2+ flux from t-system to the cytoplasm was observed following a single action potential. The action potential-induced Ca2+ flux and associated t-system Ca2+ permeability decayed exponentially and displayed inactivation characteristics such that further Ca2+ entry across the t-system could not be observed after 2,3 action potentials at 10 Hz stimulation rate. When [Ca2+]t-sys was closer to 0.1 mm, a transient rise in [Ca2+]t-sys was observed almost concurrently with the increase in [Ca2+]cyto following the action potential. The change in direction of Ca2+ flux was consistent with changes in the direction of the driving force for Ca2+. This is the first demonstration of a rapid t-system Ca2+ flux associated with a single action potential in mammalian skeletal muscle. The properties of this channel are inconsistent with a flux through the L-type Ca2+ channel suggesting that an as yet unidentified t-system protein is conducting this current. This action potential-activated Ca2+ flux provides an explanation for the previously described Ca2+ entry and accumulation observed with prolonged, intermittent muscle activity. [source] Simultaneous flux and current measurement from single plant protoplasts reveals a strong link between K+ fluxes and current, but no link between Ca2+ fluxes and currentTHE PLANT JOURNAL, Issue 1 2006Matthew Gilliham Summary We present a thorough calibration and verification of a combined non-invasive self-referencing microelectrode-based ion-flux measurement and whole-cell patch clamp system as a novel and powerful tool for the study of ion transport. The system is shown to be capable of revealing the movement of multiple ions across the plasma membrane of a single protoplast at multiple voltages and in complex physiologically relevant solutions. Wheat root protoplasts are patch clamped in the whole-cell configuration and current,voltage relations obtained whilst monitoring net K+ and Ca2+ flux adjacent to the membrane with ion-selective electrodes. At each voltage, net ion flux (nmol m,2 sec,1) is converted to an equivalent current density (mA m,2) taking into account geometry and electrode efficiency, and compared with the net current density measured with the patch clamp system. Using this technique, it is demonstrated that the K+ -permeable outwardly rectifying conductance (KORC) is responsible for net outward K+ movement across the plasma membrane [1:1 flux-to-current ratio (1.21 ± 0.14 SEM, n = 15)]. Variation in the K+ flux-to-current ratio among single protoplasts suggests a heterogeneous distribution of KORC channels on the membrane surface. As a demonstration of the power of the technique we show that despite a significant Ca2+ permeability being associated with KORC (analysis of tail current reversal potentials), there is no correlation between Ca2+ flux and KORC activity. A very significant observation is that large Ca2+ fluxes are electrically silent and probably tightly coupled to compensatory charge movements. This analysis demonstrates that it is mandatory to measure flux and currents simultaneously to investigate properly Ca2+ transport mechanisms and selectivity of ion channels in general. [source] Stroma-derived factor 1, induces a selective inhibition of human erythroid development via the functional upregulation of Fas/CD95 ligandBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2000Davide Gibellini CXC chemokine receptor 4 (CXCR4), the high-affinity receptor for stroma-derived factor 1, (SDF-1,), shows distinct patterns of expression in human CD34+ haematopoietic progenitor cells induced to differentiate in vitro along the granulocytic and erythroid lineages. In serum-free liquid cultures supplemented with stem cell factor (SCF), interleukin 3 (IL-3) and granulocyte colony-stimulating factor, the expression of surface CXCR4 progressively increased in cells differentiating along the granulocytic lineage. The addition in culture of 200 ng/ml of SDF-1,, a concentration which maximally activated intracellular Ca2+ flux, only modestly affected the expression levels of CD15 and CD11b granulocytic antigens, as well as the total number of viable cells. On the other hand, in liquid cultures supplemented with SCF, IL-3 and erythropoietin, SDF-1, induced the downregulation of glycophorin A erythroid antigen, accompanied by a progressive decline in the number of viable erythroblasts. Moreover, in semisolid assays, SDF-1, significantly reduced the number of plurifocal erythroid colonies (erythroid blast-forming units; BFU-E), whereas it did not affect granulocyte,macrophage colony-forming units (CFU-GM). We also demonstrated that the inhibitory effect of SDF-1, on glycophorin A+ erythroid cell development was mediated by the functional upregulation of CD95L in erythroid cultures. These data indicate that SDF-1, plays a role as a negative regulator of erythropoiesis. [source] Developmental characteristics of AMPA receptors in chick lumbar motoneuronsDEVELOPMENTAL NEUROBIOLOGY, Issue 11 2007Xianglian Ni Abstract Ca2+ fluxes through ionotropic glutamate receptors regulate a variety of developmental processes, including neurite outgrowth and naturally occurring cell death. In the CNS, NMDA receptors were originally thought to be the sole source of Ca2+ influx through glutamate receptors; however, AMPA receptors also allow a significant influx of Ca2+ ions. The Ca2+ permeability of AMPA receptors is regulated by the insertion of one or more edited GluR2 subunits. In this study, we tested the possibility that changes in GluR2 expression regulate the Ca2+ permeability of AMPA receptors during a critical period of neuronal development in chick lumbar motoneurons. GluR2 expression is absent between embryonic day (E) 5 and E7, but increases significantly by E8 in the chick ventral spinal cord. Increased GluR2 protein expression is correlated with parallel changes in GluR2 mRNA in the motoneuron pool. Electrophysiological recordings of kainate-evoked currents indicate a significant reduction in the Ca2+ -permeability of AMPA receptors between E6 and E11. Kainate-evoked currents were sensitive to the AMPA receptor blocker GYKI 52466. Application of AMPA or kainate generates a significant increase in the intracellular Ca2+ concentration in E6 spinal motoneurons, but generates a small response in older neurons. Changes in the Ca2+ -permeability of AMPA receptors are not mediated by age-dependent changes in the editing pattern of GluR2 subunits. These findings raise the possibility that Ca2+ influx through Ca2+ -permeable AMPA receptors plays an important role during early embryonic development in chick spinal motoneurons. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Econazole-induced Ca2+ fluxes and apoptosis in human oral cancer cellsDRUG DEVELOPMENT RESEARCH, Issue 4 2010Daih-Huang Kuo Abstract The effect of econazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability was explored in human oral cancer cells (OC2), using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations of >1,µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The econazole-induced Ca2+ influx was sensitive to blockade of aristolochic acid (phospholipase A2 inhibitor) and GF109203X (PKC inhibitor). In Ca2+ -free medium, after treatment with 1,µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 30,µM econazole failed to induce a [Ca2+]i rise. Inhibition of phospholipase C with 2,µM U73122 substantially suppressed econazole-induced [Ca2+]i rise. At concentrations of 5,70,µM econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50,µM econazole was enhanced by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,,N,-tetraacetic acid (BAPTA). The ERK MAPK inhibitor, PD98059 (10,µM), also enhanced 20,µM econazole-induced cell death. Propidium iodide staining data suggest that econazole induced apoptosis between concentrations of 10,70,µM. Collectively, in OC2 cells, econazole induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from phospholipase A2/PKC-regulated Ca2+ channels. Furthermore, econazole caused cell death appeared to be regulated by ERK MAPK. Drug Dev Res 71: 240,248, 2010. © 2010 Wiley-Liss, Inc. [source] Effect of capsaicin on Ca2+ fluxes in Madin-Darby canine renal tubular cellsDRUG DEVELOPMENT RESEARCH, Issue 2 2010Jeng-Hsien Yeh Abstract The effect of capsaicin, a transient receptor potential vanniloid-1 (TRPV1) receptor agonist, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether capsaicin changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+ -sensitive fluorescent dye. Capsaicin at concentrations between 10,100,µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 80% by removing extracellular Ca2+. Capsacin induced Mn2+ influx, leading to quench of fura-2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid and the non-selective Ca2+ entry blocker La3+, but not by store-operated Ca2+ channel blockers nifedipine, econazole, and SK&F96365, and protein kinase C/A modulators. In Ca2+ -free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished capsaicin-induced Ca2+ release. Conversely, pretreatment with capsaicin partly reduced thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter capsaicin-induced [Ca2+]i rise. The TRPV1 receptor antagonist capsazepine also induced significant Ca2+ entry and Ca2+ release. Collectively, in MDCK cells, capsaicin induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-regulated, La3+ -sensitive Ca2+ channels in a manner dissociated from stimulation of TRPV1 receptors. Drug Dev Res, 2009. © 2009 Wiley-Liss, Inc. [source] Nuclear pore disassembly from endoplasmic reticulum membranes promotes Ca2+ signalling competencyTHE JOURNAL OF PHYSIOLOGY, Issue 12 2008Michael J. Boulware The functionality of the endoplasmic reticulum (ER) as a Ca2+ storage organelle is supported by families of Ca2+ pumps, buffers and channels that regulate Ca2+ fluxes between the ER lumen and cytosol. Although many studies have identified heterogeneities in Ca2+ fluxes throughout the ER, the question of how differential functionality of Ca2+ channels is regulated within proximal regions of the same organelle is unresolved. Here, we studied the in vivo dynamics of an ER subdomain known as annulate lamellae (AL), a cytoplasmic nucleoporin-containing organelle widely used in vitro to study the mechanics of nuclear envelope breakdown. We show that nuclear pore complexes (NPCs) within AL suppress local Ca2+ signalling activity, an inhibitory influence relieved by heterogeneous dissociation of nucleoporins to yield NPC-denuded ER domains competent at Ca2+ signalling. Consequently, we propose a novel generalized role for AL , reversible attenuation of resident protein activity , such that regulated AL (dis)assembly via a kinase/phosphatase cycle allows cells to support rapid gain/loss-of-function transitions in cellular physiology. [source] Modulation of Ca2+ signalling in rat atrial myocytes: possible role of the ,1c carboxyl terminalTHE JOURNAL OF PHYSIOLOGY, Issue 2 2003Sun-Hee Woo Ca2+ influx through L-type Cav1.2 (,1c) Ca2+ channels is a critical step in the activation of cardiac ryanodine receptors (RyRs) and release of Ca2+ via Ca2+ -induced Ca2+ release(CICR). The released Ca2+, in turn, is the dominant determinant of inactivation of the Ca2+ current (ICa) and termination of release. Although Ca2+ cross-signalling is mediated by high Ca2+ fluxes in the microdomains of ,1c -RyR complexes, ICa -gated Ca2+ cross-signalling is surprisingly resistant to intracellular Ca2+ buffering and has steeply voltage-dependent gain, inconsistent with a strict CICR mechanism, suggesting the existence of additional regulatory step(s). To explore the possible regulatory role of the carboxyl (C)-terminal tail of ,1c in modulating Ca2+ signalling, we tested the effects of introducing two ,1c C-terminal peptides, LA (1571,1599) and K (1617,1636) on the central ,1c -unassociated Ca2+ -release sites of atrial myocytes, using rapid (240 Hz) two-dimensional confocal Ca2+ imaging. The frequency of spontaneously activating central sparks increased by approximately fourfold on dialysing LA- but not K-peptide into myocytes voltage-clamped at -80 mV. The rate but not the magnitude of caffeine (10 mM)-triggered central Ca2+ release was significantly accelerated by LA- but not K-peptide. Individual Ca2+ spark size and flux were larger in LA- but not in K-peptide-dialysed myocytes. Although LA-peptide did not change the amplitude or inactivation kinetics of ICa, LA-peptide did strongly enhance the central Ca2+ transients triggered by ICa at -30 mV (small ICa) but not at +20 mV (large ICa). In contrast, K-peptide had no effect on either ICa or the local Ca2+ transients. LA-peptide with a deleted calmodulin-binding region (LM1-peptide) had no significant effects on the central spark frequency but suppressed spontaneous spark frequency in the periphery. Our results indicate that the calmodulin-binding LA motif of the ,1c C-terminal tail may sensitize the RyRs, thereby increasing their open probability and providing for both the voltage-dependence of CICR and the higher frequency of spark occurrence in the periphery of atrial myocytes where the native ,1c -RyR complexes are intact. [source] Simultaneous flux and current measurement from single plant protoplasts reveals a strong link between K+ fluxes and current, but no link between Ca2+ fluxes and currentTHE PLANT JOURNAL, Issue 1 2006Matthew Gilliham Summary We present a thorough calibration and verification of a combined non-invasive self-referencing microelectrode-based ion-flux measurement and whole-cell patch clamp system as a novel and powerful tool for the study of ion transport. The system is shown to be capable of revealing the movement of multiple ions across the plasma membrane of a single protoplast at multiple voltages and in complex physiologically relevant solutions. Wheat root protoplasts are patch clamped in the whole-cell configuration and current,voltage relations obtained whilst monitoring net K+ and Ca2+ flux adjacent to the membrane with ion-selective electrodes. At each voltage, net ion flux (nmol m,2 sec,1) is converted to an equivalent current density (mA m,2) taking into account geometry and electrode efficiency, and compared with the net current density measured with the patch clamp system. Using this technique, it is demonstrated that the K+ -permeable outwardly rectifying conductance (KORC) is responsible for net outward K+ movement across the plasma membrane [1:1 flux-to-current ratio (1.21 ± 0.14 SEM, n = 15)]. Variation in the K+ flux-to-current ratio among single protoplasts suggests a heterogeneous distribution of KORC channels on the membrane surface. As a demonstration of the power of the technique we show that despite a significant Ca2+ permeability being associated with KORC (analysis of tail current reversal potentials), there is no correlation between Ca2+ flux and KORC activity. A very significant observation is that large Ca2+ fluxes are electrically silent and probably tightly coupled to compensatory charge movements. This analysis demonstrates that it is mandatory to measure flux and currents simultaneously to investigate properly Ca2+ transport mechanisms and selectivity of ion channels in general. [source] | |||||||||||||