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Ca2+ Dynamics (ca2+ + dynamics)
Selected AbstractsVisualization of local Ca2+ dynamics with genetically encoded bioluminescent reportersEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2005Kelly L. Rogers Abstract Measurements of local Ca2+ signalling at different developmental stages and/or in specific cell types is important for understanding aspects of brain functioning. The use of light excitation in fluorescence imaging can cause phototoxicity, photobleaching and auto-fluorescence. In contrast, bioluminescence does not require the input of radiative energy and can therefore be measured over long periods, with very high temporal resolution. Aequorin is a genetically encoded Ca2+ -sensitive bioluminescent protein, however, its low quantum yield prevents dynamic measurements of Ca2+ responses in single cells. To overcome this limitation, we recently reported the bi-functional Ca2+ reporter gene, GFP-aequorin (GA), which was developed specifically to improve the light output and stability of aequorin chimeras [V. Baubet, et al., (2000) PNAS, 97, 7260,7265]. In the current study, we have genetically targeted GA to different microdomains important in synaptic transmission, including to the mitochondrial matrix, endoplasmic reticulum, synaptic vesicles and to the postsynaptic density. We demonstrate that these reporters enable ,real-time' measurements of subcellular Ca2+ changes in single mammalian neurons using bioluminescence. The high signal-to-noise ratio of these reporters is also important in that it affords the visualization of Ca2+ dynamics in cell,cell communication in neuronal cultures and tissue slices. Further, we demonstrate the utility of this approach in ex-vivo preparations of mammalian retina, a paradigm in which external light input should be controlled. This represents a novel molecular imaging approach for non-invasive monitoring of local Ca2+ dynamics and cellular communication in tissue or whole animal studies. [source] Calcium dynamics are altered in cortical neurons lacking the calmodulin-binding protein RC3EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2003Jacqueline J. W. Van Dalen Abstract RC3 is a neuronal calmodulin-binding protein and protein kinase C substrate that is thought to play an important regulatory role in synaptic transmission and neuronal plasticity. Two molecules known to regulate synaptic transmission and neuronal plasticity are Ca2+ and calmodulin, and proposed mechanisms of RC3 action involve both molecules. However, physiological evidence for a role of RC3 in neuronal Ca2+ dynamics is limited. In the current study we utilized cultured cortical neurons obtained from RC3 knockout (RC3,/,) and wildtype mice (RC3+/+) and fura-2-based microscopic Ca2+ imaging to investigate a role for RC3 in neuronal Ca2+ dynamics. Immunocytochemical characterization showed that the RC3,/, cultures lack RC3 immunoreactivity, whereas cultures prepared from wildtype mice showed RC3 immunoreactivity at all ages studied. RC3+/+ and RC3,/, cultures were indistinguishable with respect to neuron density, neuronal morphology, the formation of extensive neuritic networks and the presence of glial fibrillary acidic protein (GFAP)-positive astrocytes and ,-aminobutyric acid (GABA)ergic neurons. However, the absence of RC3 in the RC3,/, neurons was found to alter neuronal Ca2+ dynamics including baseline Ca2+ levels measured under normal physiological conditions or after blockade of synaptic transmission, spontaneous intracellular Ca2+ oscillations generated by network synaptic activity, and Ca2+ responses elicited by exogenous application of N-methyl- d -aspartate (NMDA) or class I metabotropic glutamate receptor agonists. Thus, significant changes in Ca2+ dynamics occur in cortical neurons when RC3 is absent and these changes do not involve changes in gross neuronal morphology or neuronal maturation. These data provide direct physiological evidence for a regulatory role of RC3 in neuronal Ca2+ dynamics. [source] Ca2+ entry through TRPC1 channels contributes to intracellular Ca2+ dynamics and consequent glutamate release from rat astrocytesGLIA, Issue 8 2008Erik B. Malarkey Abstract Astrocytes can respond to a variety of stimuli by elevating their cytoplasmic Ca2+ concentration and can in turn release glutamate to signal adjacent neurons. The majority of this Ca2+ is derived from internal stores while a portion also comes from outside of the cell. Astrocytes use Ca2+ entry through store-operated Ca2+ channels to refill their internal stores. Therefore, we investigated what role this store-operated Ca2+ entry plays in astrocytic Ca2+ responses and subsequent glutamate release. Astrocytes express canonical transient receptor potential (TRPC) channels that have been implicated in mediating store-operated Ca2+ entry. Here, we show that astrocytes in culture and freshly isolated astrocytes from visual cortex express TRPC1, TRPC4, and TRPC5. Indirect immunocytochemistry reveals that these proteins are present throughout the cell; the predominant expression of functionally tested TRPC1, however, is on the plasma membrane. Labeling in freshly isolated astrocytes reveals changes in TRPC expression throughout development. Using an antibody against TRPC1 we were able to block the function of TRPC1 channels and determine their involvement in mechanically and agonist-evoked Ca2+ entry in cultured astrocytes. Blocking TRPC1 was also found to reduce mechanically induced Ca2+ -dependent glutamate release. These data indicate that Ca2+ entry through TRPC1 channels contributes to Ca2+ signaling in astrocytes and the consequent glutamate release from these cells. © 2008 Wiley-Liss, Inc. [source] Vesicular release of glutamate mediates bidirectional signaling between astrocytes and neuronsJOURNAL OF NEUROCHEMISTRY, Issue 4 2007Yingchun Ni Abstract The major excitatory neurotransmitter in the CNS, glutamate, can be released exocytotically by neurons and astrocytes. Glutamate released from neurons can affect adjacent astrocytes by changing their intracellular Ca2+ dynamics and, vice versa, glutamate released from astrocytes can cause a variety of responses in neurons such as: an elevation of [Ca2+]i, a slow inward current, an increase of excitability, modulation of synaptic transmission, synchronization of synaptic events, or some combination of these. This astrocyte-neuron signaling pathway might be a widespread phenomenon throughout the brain with astrocytes possessing the means to be active participants in many functions of the CNS. Thus, it appears that the vesicular release of glutamate can serve as a common denominator for two of the major cellular components of the CNS, astrocytes and neurons, in brain function. [source] Melanotrope Cells of Xenopus laevis Express Multiple Types of High-Voltage-Activated Ca2+ ChannelsJOURNAL OF NEUROENDOCRINOLOGY, Issue 1 2005H.-Y. Zhang Abstract Pituitary melanotrope cells are neuroendocrine signal transducing cells that translate physiological stimuli into adaptive hormonal responses. In this translation process, Ca2+ channels play essential roles. We have characterised which types of Ca2+ current are present in melanotropes of the amphibian Xenopus laevis, using whole-cell, voltage-clamp, patch-clamp experiments and specific blockers of the various current types. Running an activation current,voltage relationship protocol from a holding potential (HP) of ,80 mV/or ,110 mV, shows that Xenopus melanotropes possess only high-voltage activated (HVA) Ca2+ currents. Steady-state inactivation protocols reveal that no inactivation occurs at ,80 mV, whereas 30% of the current is inactivated at ,30 mV. We determined the contribution of individual channel types to the total HVA Ca2+ current, examining the effect of each channel blocker at an HP of ,80 mV and ,30 mV. At ,80 mV, ,-conotoxin GVIA, ,-agatoxin IVA, nifedipine and SNX-482 inhibit Ca2+ currents by 21.8 ± 4.1%, 26.1 ± 3.1%, 24.2 ± 2.4% and 17.9 ± 4.7%, respectively. At ,30 mV, ,-conotoxin GVIA, nifedipine and ,-agatoxin IVA inhibit Ca2+ currents by 33.8 ± 3.0, 24.2 ± 2.6 and 16.0 ± 2.8%, respectively, demonstrating that these blockers substantially inhibit part of the Ca2+ current, independently from the HP. We have previously demonstrated that ,-conotoxin GVIA can block Ca2+ oscillations and steps. We now show that nifedipine and ,-agatoxin IVA do not affect the intracellular Ca2+ dynamics, whereas SNX-482 reduces the Ca2+ step amplitude. We conclude that Xenopus melanotrope cells express all four major types of HVA Ca2+ channel, as well as the resulting currents, but no low-voltage activated channels. The results provide the basis for future studies on the complex regulation of channel-mediated Ca2+ influxes into this neuroendocrine cell type as a function of its role in the animal's adaptation to external challenges. [source] Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin-induced morphological changes in 1321N1 human astrocytoma cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2008Kenji Nakao Abstract Activated astrocytes show various patterns of Ca2+ mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca2+ dynamics through investigation of thrombin-induced unique Ca2+ entry. Using 1321N1 human astrocytoma cells, which have been shown to be a good model for detecting morphological dynamics, we observed rapid retraction of bipolar protrusions that were reversibly evoked by 0.03,3 U/mL thrombin. Morphological changes were predominantly dependent on a specific thrombin receptor subtype, proteinase-activated receptor 1 (PAR-1). In parallel, Fura-2 imaging of intracellular Ca2+ concentration ([Ca2+]i) showed that thrombin induced heterogeneous Ca2+ responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca2+ release from intracellular stores, followed by refilling of Ca2+ from the extracellular region without a direct [Ca2+]i increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca2+ mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca2+ entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca2+ responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca2+ influx. Moreover, TRPC3-mediated Ca2+ dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca2+ dynamics, including Ca2+ entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca2+ channel and might modulate astrocytic activation in response to brain hemorrhaging. © 2008 Wiley-Liss, Inc. [source] Modulatory effects of static magnetic fields on blood pressure in rabbitsBIOELECTROMAGNETICS, Issue 6 2001Hideyuki Okano Abstract Acute effects of locally applied static magnetic fields (SMF) on pharmacologically altered blood pressure (BP) in a central artery of the ear lobe of a conscious rabbit were evaluated. Hypotensive and vasodilator actions were induced by a Ca2+ channel blocker, nicardipine (NIC). Hypertensive and vasoconstrictive actions were induced by a nitric oxide synthase (NOS) inhibitor, N, -nitro- L -arginine methyl ester (L-NAME). The hemodynamic changes in the artery exposed to SMF were measured continuously and analyzed by penetrating microphotoelectric plethysmography (MPPG). Concurrently, BP changes in a central artery contralateral to that of the exposed ear lobe were monitored. SMF intensity was 1,mT and the duration of exposure was 30,min. A total of 180 experimental trials were carried out in 34 healthy adult male rabbits weighing 2.6,3.8,kg. Six experimental procedures were chosen at random: (1) sham exposure without pharmacological treatment; (2) SMF exposure alone; (3) decreased BP induced by a single intravenous (iv) bolus injection of NIC (100,,M/kg) without SMF exposure; (4) decreased BP induced by injection of NIC with SMF exposure; (5) increased BP induced by a constant iv infusion of L-NAME (10,mM/kg/h) without SMF exposure; (6) increased BP induced by infusion of L-NAME with SMF exposure. The results demonstrated that SMF significantly reduced the vasodilatation with enhanced vasomotion and antagonized the reduction of BP via NIC-blocked Ca2+ channels in vascular smooth muscle cells. In addition, SMF significantly attenuated the vasoconstriction and suppressed the elevation of BP via NOS inhibition in vascular endothelial cells and/or central nervous system neurons. These results suggest that these modulatory effects of SMF on BP might, in part, involve a feedback control system for alteration in NOS activity in conjunction with modulation of Ca2+ dynamics. Bioelectromagnetics 22:408,418, 2001. © 2001 Wiley-Liss, Inc. [source] Dynamical analysis of the calcium signaling pathway in cardiac myocytes based on logarithmic sensitivity analysisBIOTECHNOLOGY JOURNAL, Issue 5 2008Tae-Hwan Kim Abstract Many cellular functions are regulated by the Ca2+ signal which contains specific information in the form of frequency, amplitude, and duration of the oscillatory dynamics. Any alterations or dysfunctions of components in the calcium signaling pathway of cardiac myocytes may lead to a diverse range of cardiac diseases including hypertrophy and heart failure. In this study, we have investigated the hidden dynamics of the intracellular Ca2+ signaling and the functional roles of its regulatory mechanism through in silico simulations and parameter sensitivity analysis based on an experimentally verified mathematical model. It was revealed that the Ca2+ dynamics of cardiac myocytes are determined by the balance among various system parameters. Moreover, it was found through the parameter sensitivity analysis that the self-oscillatory Ca2+ dynamics are most sensitive to the Ca2+ leakage rate of the sarcolemmal membrane and the maximum rate of NCX, suggesting that these two components have dominant effects on circulating the cytosolic Ca2+. [source] | ||||||||||