Evidence supporting a role for astrocytes in the regulation of cognitive flexibility and neuronal oscillations through the Ca2+ binding protein S100β.

Science.gov (United States)

Brockett, Adam T; Kane, Gary A; Monari, Patrick K; Briones, Brandy A; Vigneron, Pierre-Antoine; Barber, Gabriela A; Bermudez, Andres; Dieffenbach, Uma; Kloth, Alexander D; Buschman, Timothy J; Gould, Elizabeth

2018-01-01

The medial prefrontal cortex (mPFC) is important for cognitive flexibility, the ability to switch between two task-relevant dimensions. Changes in neuronal oscillations and alterations in the coupling across frequency ranges have been correlated with attention and cognitive flexibility. Here we show that astrocytes in the mPFC of adult male Sprague Dawley rats, participate in cognitive flexibility through the astrocyte-specific Ca2+ binding protein S100β, which improves cognitive flexibility and increases phase amplitude coupling between theta and gamma oscillations. We further show that reduction of astrocyte number in the mPFC impairs cognitive flexibility and diminishes delta, alpha and gamma power. Conversely, chemogenetic activation of astrocytic intracellular Ca2+ signaling in the mPFC enhances cognitive flexibility, while inactivation of endogenous S100β among chemogenetically activated astrocytes in the mPFC prevents this improvement. Collectively, our work suggests that astrocytes make important contributions to cognitive flexibility and that they do so by releasing a Ca2+ binding protein which in turn enhances coordinated neuronal oscillations.

Epigallocatechin-3-gallate increases intracellular [Ca2+] in U87 cells mainly by influx of extracellular Ca2+ and partly by release of intracellular stores.

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Kim, Hee Jung; Yum, Keun Sang; Sung, Jong-Ho; Rhie, Duck-Joo; Kim, Myung-Jun; Min, Do Sik; Hahn, Sang June; Kim, Myung-Suk; Jo, Yang-Hyeok; Yoon, Shin Hee

2004-02-01

Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [3H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca2+] ([Ca2+]i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca2+]i. The EGCG-induced [Ca2+]i increases were reduced to 20.9% of control by removal of extracellular Ca2+. The increases were also inhibited markedly by treatment with the non-specific Ca2+ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca2+ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca2+-ATPase thapsigargin (1 micro M) also significantly inhibited the EGCG-induced [Ca2+]i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 micro M) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 micro M) had no effect. EGCG increased [3H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 micro M) and flufenamic acid (100 micro M), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca2+]i increase in non-treated and thapsigargin-treated cells but indomethacin (100 micro M) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca2+]i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca2+ and partly by mobilizing intracellular Ca2+ stores by PLC activation. The EGCG-induced [Ca2+]i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.

TNF-α as a potential mediator of cardiac dysfunction due to intracellular Ca2+-overload

International Nuclear Information System (INIS)

Zhang Ming; Xu Yanjun; Saini, Harjot K.; Turan, Belma; Liu, Peter P.; Dhalla, Naranjan S.

2005-01-01

TNF-α has been shown to be involved in cardiac dysfunction during ischemia/reperfusion injury; however, no information regarding the status of TNF-α production in myocardial injury due to intracellular Ca 2+ -overload is available in the literature. The intracellular Ca 2+ -overload was induced in the isolated rat hearts subjected to 5 min Ca 2+ -depletion and 30 min Ca 2+ -repletion (Ca 2+ -paradox). The Ca 2+ -paradox hearts exhibited a dramatic depression in left ventricular developed pressure, a marked elevation in left ventricular end diastolic pressure, and more than a 4-fold increase in TNF-α content. The ratio of cytosolic to homogenate nuclear factor-κB (NFκB) was decreased whereas the ratio of phospho-NFκB to total NFκB was increased in the Ca 2+ -paradox hearts. All these changes due to Ca 2+ -paradox were significantly attenuated upon treating the hearts with 100 μM pentoxifylline. These results suggest that activation of NFκB and increased production of TNF-α may play an important role in cardiac injury due to intracellular Ca 2+ -overload

Involvement of intracellular Zn2+ signaling in LTP at perforant pathway-CA1 pyramidal cell synapse.

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Tamano, Haruna; Nishio, Ryusuke; Takeda, Atsushi

2017-07-01

Physiological significance of synaptic Zn 2+ signaling was examined at perforant pathway-CA1 pyramidal cell synapses. In vivo long-term potentiation (LTP) at perforant pathway-CA1 pyramidal cell synapses was induced using a recording electrode attached to a microdialysis probe and the recording region was locally perfused with artificial cerebrospinal fluid (ACSF) via the microdialysis probe. Perforant pathway LTP was not attenuated under perfusion with CaEDTA (10 mM), an extracellular Zn 2+ chelator, but attenuated under perfusion with ZnAF-2DA (50 μM), an intracellular Zn 2+ chelator, suggesting that intracellular Zn 2+ signaling is required for perforant pathway LTP. Even in rat brain slices bathed in CaEDTA in ACSF, intracellular Zn 2+ level, which was measured with intracellular ZnAF-2, was increased in the stratum lacunosum-moleculare where perforant pathway-CA1 pyramidal cell synapses were contained after tetanic stimulation. These results suggest that intracellular Zn 2+ signaling, which originates in internal stores/proteins, is involved in LTP at perforant pathway-CA1 pyramidal cell synapses. Because the influx of extracellular Zn 2+ , which originates in presynaptic Zn 2+ release, is involved in LTP at Schaffer collateral-CA1 pyramidal cell synapses, synapse-dependent Zn 2+ dynamics may be involved in plasticity of postsynaptic CA1 pyramidal cells. © 2017 Wiley Periodicals, Inc.

PLCζ Induced Ca2+ Oscillations in Mouse Eggs Involve a Positive Feedback Cycle of Ca2+ Induced InsP3 Formation From Cytoplasmic PIP2

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Sanders, Jessica R.; Ashley, Bethany; Moon, Anna; Woolley, Thomas E.; Swann, Karl

2018-01-01

Egg activation at fertilization in mammalian eggs is caused by a series of transient increases in the cytosolic free Ca2+ concentration, referred to as Ca2+ oscillations. It is widely accepted that these Ca2+ oscillations are initiated by a sperm derived phospholipase C isoform, PLCζ that hydrolyses its substrate PIP2 to produce the Ca2+ releasing messenger InsP3. However, it is not clear whether PLCζ induced InsP3 formation is periodic or monotonic, and whether the PIP2 source for generating InsP3 from PLCζ is in the plasma membrane or the cytoplasm. In this study we have uncaged InsP3 at different points of the Ca2+ oscillation cycle to show that PLCζ causes Ca2+ oscillations by a mechanism which requires Ca2+ induced InsP3 formation. In contrast, incubation in Sr2+ media, which also induces Ca2+ oscillations in mouse eggs, sensitizes InsP3-induced Ca2+ release. We also show that the cytosolic level Ca2+ is a key factor in setting the frequency of Ca2+ oscillations since low concentrations of the Ca2+ pump inhibitor, thapsigargin, accelerates the frequency of PLCζ induced Ca2+ oscillations in eggs, even in Ca2+ free media. Given that Ca2+ induced InsP3 formation causes a rapid wave during each Ca2+ rise, we use a mathematical model to show that InsP3 generation, and hence PLCζ's substate PIP2, has to be finely distributed throughout the egg cytoplasm. Evidence for PIP2 distribution in vesicles throughout the egg cytoplasm is provided with a rhodamine-peptide probe, PBP10. The apparent level of PIP2 in such vesicles could be reduced by incubating eggs in the drug propranolol which also reversibly inhibited PLCζ induced, but not Sr2+ induced, Ca2+ oscillations. These data suggest that the cytosolic Ca2+ level, rather than Ca2+ store content, is a key variable in setting the pace of PLCζ induced Ca2+ oscillations in eggs, and they imply that InsP3 oscillates in synchrony with Ca2+ oscillations. Furthermore, they support the hypothesis that PLCζ and sperm

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

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Zhenghang Zhao

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

Ca2+ controls slow NAD(P)H oscillations in glucose-stimulated mouse pancreatic islets

DEFF Research Database (Denmark)

Luciani, Dan Seriano; Misler, S.; Polonsky, K.S.

2006-01-01

Exposure of pancreatic islets of Langerhans to physiological concentrations of glucose leads to secretion of insulin in an oscillatory pattern. The oscillations in insulin secretion are associated with oscillations in cytosolic Ca2+ concentration ([Ca2+](c)). Evidence suggests that the oscillatio...

The mechanical environment modulates intracellular calcium oscillation activities of myofibroblasts.

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Charles Godbout

Full Text Available Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair.

Activity-dependent intracellular Ca2+ transients in unmyelinated nerve fibres of the isolated adult rat vagus nerve.

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Wächtler, J; Mayer, C; Grafe, P

1998-04-01

Confocal laser scanning microscopy was used to follow changes in the free intracellular calcium concentration ([Ca2+]i) in nerve fibres and adjacent Schwann cells in isolated rat vagus nerves. [Ca2+]i was monitored by the Ca2+-sensitive fluorescent dyes Calcium Green-1 and Fura Red. Intracellular Ca2+ transients were observed during repetitive (1-50 Hz) supramaximal electrical stimulation or by bath application of ATP. Trains of action potentials were more effective at elongated, fibre-like structures of the vagus nerves, whereas ATP-induced Ca2+ transients were found predominantly in regions of Schwann cell bodies. Activity-induced Ca2+ signals were unaffected by pharmacological manipulation of intracellular Ca2+ stores, during long-lasting application of purinergic receptor agonists, or by substitution of extracellular Na+ with Li+. However, they were abolished in the presence of Ca2+-free bathing solution or after the blocking of Ca2+ channels with Cd2+. Ca2+ transients were also observed during Ca2+ action potentials. Such "Ca2+ spikes" were elicited by electrical stimulation in the presence of a combination of tetrodotoxin and K+ channel blockers. These data suggest that voltage-dependent Ca2+ channels, activated during short trains of Na+ action potentials, produce an increase in intra-axonal [Ca2+] of rat vagus nerves. We did not find evidence for activity-dependent Ca2+ transients in the Schwann cells surrounding the unmyelinated axons.

IP3-dependent intracellular Ca2+ release is required for cAMP-induced c-fos expression in hippocampal neurons

International Nuclear Information System (INIS)

Zhang, Wenting; Tingare, Asmita; Ng, David Chi-Heng; Johnson, Hong W.; Schell, Michael J.; Lord, Rebecca L.; Chawla, Sangeeta

2012-01-01

Highlights: ► cAMP-induced c-fos expression in hippocampal neurons requires a submembraneous Ca 2+ pool. ► The submembraneous Ca 2+ pool derives from intracellular ER stores. ► Expression of IP 3 -metabolizing enzymes inhibits cAMP-induced c-fos expression. ► SRE-mediated and CRE-mediated gene expression is sensitive to IP 3 -metabolizing enzymes. ► Intracellular Ca 2+ release is required for cAMP-induced nuclear translocation of TORC1. -- Abstract: Ca 2+ and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca 2+ and cAMP signals, including some that are Ca 2+ -responsive, some that are cAMP-responsive and some that detect coincident Ca 2+ and cAMP signals. Because Ca 2+ and cAMP can influence each other’s amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca 2+ are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca 2+ buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP 3 levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements – the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP 3 metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited by depletion of intracellular Ca 2+ stores. Our data indicate that Ca 2+ release from IP 3 -sensitive pools is required for cAMP-induced transcription in hippocampal neurons.

Synaptically evoked Ca2+ release from intracellular stores is not influenced by vesicular zinc in CA3 hippocampal pyramidal neurones.

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Evstratova, Alesya; Tóth, Katalin

2011-12-01

The co-release of neuromodulatory substances in combination with classic neurotransmitters such as glutamate and GABA from individual presynaptic nerve terminals has the capacity to dramatically influence synaptic efficacy and plasticity. At hippocampal mossy fibre synapses vesicular zinc is suggested to serve as a cotransmitter capable of regulating calcium release from internal stores in postsynaptic CA3 pyramidal cells. Here we investigated this possibility using combined intracellular ratiometric calcium imaging and patch-clamp recording techniques. In acute hippocampal slices a brief train of mossy fibre stimulation produced a large, delayed postsynaptic Ca(2+) wave that was spatially restricted to the proximal apical dendrites of CA3 pyramidal cells within stratum lucidum. This calcium increase was sensitive to intracellularly applied heparin indicating reliance upon release from internal stores and was triggered by activation of both group I metabotropic glutamate and NMDA receptors. Importantly, treatment of slices with the membrane-impermeant zinc chelator CaEDTA did not influence the synaptically evoked postsynaptic Ca(2+) waves. Moreover, mossy fibre stimulus evoked postsynaptic Ca(2+) signals were not significantly different between wild-type and zinc transporter 3 (ZnT3) knock-out animals. Considered together our data do not support a role for vesicular zinc in regulating mossy fibre evoked Ca(2+) release from CA3 pyramidal cell internal stores.

Intracellular [Ca2+] and Vo2 after manipulation of the free-energy of the Na+/Ca(2+)-exchanger in isolated rat ventricular myocytes

NARCIS (Netherlands)

Fiolet, J. W.; Baartscheer, A.; Schumacher, C. A.

1995-01-01

We have investigated whether the Na+/Ca(2+)-exchanger has a functional regulatory role in the control of oxidative metabolism in suspensions of isolated rat ventricular myocytes. Therefore we simultaneously measured intracellular [Ca2+] ([Ca2+]i) with Indo-1 and respiratory rate (Vo2) after abrupt

Thymosin β4 promotes the migration of endothelial cells without intracellular Ca2+ elevation

International Nuclear Information System (INIS)

Selmi, Anna; Malinowski, Mariusz; Brutkowski, Wojciech; Bednarek, Radoslaw; Cierniewski, Czeslaw S.

2012-01-01

Numerous studies have demonstrated the effects of Tβ4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which Tβ4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with Tβ4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that Tβ4 interacts with Ku80, which may operate as a novel receptor for Tβ4 and mediates its intracellular activity. In this paper, we provide evidence that Tβ4 induces cellular processes without changes in the intracellular Ca 2+ concentration. External treatment of HUVECs with Tβ4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (Tβ4 AcSDKPT/4A ) or the actin-binding sequence KLKKTET (Tβ4 KLKKTET/7A ) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by Tβ4 was not associated with the intracellular Ca 2+ elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added Tβ4 induces HUVEC migration via the surface membrane receptors known to generate Ca 2+ influx. Our data confirm the concept that externally added Tβ4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.

Hydroxyhydroquinone, a by-product of coffee bean roasting, increases intracellular Ca2+ concentration in rat thymic lymphocytes.

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Kamae, Risa; Nojima, Shoko; Akiyoshi, Kenji; Setsu, Shoki; Honda, Sari; Masuda, Toshiya; Oyama, Yasuo

2017-04-01

Hydroxyhydroquinone (HHQ) is generated during coffee bean roasting. A cup of coffee contains 0.1-1.7 mg of HHQ. The actions of HHQ on mammalian DNA were examined because HHQ is a metabolite of benzene, which causes leukemia. Currently, information on the cellular actions of HHQ is limited. We examined the effects of sublethal levels of HHQ on the concentration of intracellular Ca 2+ in rat thymic lymphocytes by using a flow cytometric technique with fluorescent probes. HHQ at 10 μM or more significantly elevated intracellular Ca 2+ levels by increasing the membrane permeability of divalent cations, resulting in hyperpolarization via the activation of Ca 2+ -dependent K + channels. HHQ-induced changes in the intracellular Ca 2+ concentration and membrane potential may affect the cell functions of lymphocytes. HHQ-reduced coffee may be preferable in order to avoid the possible adverse effects of HHQ. Copyright © 2017 Elsevier Ltd. All rights reserved.

Lung Beractant Increases Free Cytosolic Levels of Ca2+ in Human Lung Fibroblasts.

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Alejandro Guzmán-Silva

Full Text Available Beractant, a natural surfactant, induces an antifibrogenic phenotype and apoptosis in normal human lung fibroblasts (NHLF. As intracellular Ca2+ signalling has been related to programmed cell death, we aimed to assess the effect of beractant on intracellular Ca2+ concentration ([Ca2+]i in NHLF in vitro. Cultured NHLF were loaded with Fura-2 AM (3 μM and Ca2+ signals were recorded by microfluorimetric techniques. Beractant causes a concentration-dependent increase in [Ca2+]i with a EC50 of 0.82 μg/ml. The application of beractant, at a concentration of 500 μg/ml, which has been shown to exert an apoptotic effect in human fibroblasts, elicited different patterns of Ca2+ signals in NHLF: a a single Ca2+ spike which could be followed by b Ca2+ oscillations, c a sustained Ca2+ plateau or d a sustained plateau overlapped by Ca2+ oscillations. The amplitude and pattern of Ca2+ transients evoked by beractant were dependent on the resting [Ca2+]i. Pharmacological manipulation revealed that beractant activates a Ca2+ signal through Ca2+ release from intracellular stores mediated by phospholipase Cβ (PLCβ, Ca2+ release from inositol 1,4,5-trisphosphate receptors (IP3Rs and Ca2+ influx via a store-operated pathway. Moreover, beractant-induced Ca2+ release was abolished by preventing membrane depolarization upon removal of extracellular Na+ and Ca2+. Finally, the inhibition of store-operated channels prevented beractant-induced NHLF apoptosis and downregulation of α1(I procollagen expression. Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF.

Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

DEFF Research Database (Denmark)

Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max

2012-01-01

of the physical and biochemical conditions in plant cells. As model system, we use a H(2)O(2) signal originating at the plasma membrane (PM) and spreading through the cytosol. We consider two maximally simple types of signals, isolated pulses and harmonic oscillations. First we consider the basic limits......Amplitude- and frequency-modulated waves of Ca(2+) ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...

Excitation contraction uncoupling by high intracellular [Ca2+] in frog skeletal muscle: a voltage clamp study.

Science.gov (United States)

Olivera, J Fernando; Pizarro, Gonzalo

2016-10-01

Raising the intracellular [Ca 2+ ] ([Ca 2+ ] i ) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca 2+ ] i in voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca 2+ release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca 2+ ] i was raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca 2+ ] i intracellular solution, permeabilization of the plasma membrane by a Ca 2+ ionophore) all of which produced impairment of excitation-contraction coupling. The charge movement was reduced from 20.2 ± 1.24 to 9.9 ± 0.94 nC/μF meanwhile the Ca 2+ release flux was reduced from 13.5 + 0.7 to 2.2 ± 0.3 μM/ms (n = 33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40 % of the uncoupled cells we observed that the [Ca 2+ ] i transient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca 2+ .

β adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca2+ release by tentacle extract from the jellyfish Cyanea capillata.

Science.gov (United States)

Wang, Qianqian; Zhang, Hui; Wang, Bo; Wang, Chao; Xiao, Liang; Zhang, Liming

2017-07-25

Intracellular Ca 2+ overload induced by extracellular Ca 2+ entry has previously been confirmed to be an important mechanism for the cardiotoxicity as well as the acute heart dysfunction induced by jellyfish venom, while the underlying mechanism remains to be elucidated. Under extracellular Ca 2+ -free or Ca 2+ -containing conditions, the Ca 2+ fluorescence in isolated adult mouse cardiomyocytes pre-incubated with tentacle extract (TE) from the jellyfish Cyanea capillata and β blockers was scanned by laser scanning confocal microscope. Then, the cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity in primary neonatal rat ventricular cardiomyocytes were determined by ELISA assay. Furthermore, the effect of propranolol against the cardiotoxicity of TE was evaluated in Langendorff-perfused rat hearts and intact rats. The increase of intracellular Ca 2+ fluorescence signal by TE was significantly attenuated and delayed when the extracellular Ca 2+ was removed. The β adrenergic blockers, including propranolol, atenolol and esmolol, partially inhibited the increase of intracellular Ca 2+ in the presence of 1.8 mM extracellular Ca 2+ and completely abolished the Ca 2+ increase under an extracellular Ca 2+ -free condition. Both cAMP concentration and PKA activity were stimulated by TE, and were inhibited by the β adrenergic blockers. Cardiomyocyte toxicity of TE was antagonized by β adrenergic blockers and the PKA inhibitor H89. Finally, the acute heart dysfuction by TE was antagonized by propranolol in Langendorff-perfused rat hearts and intact rats. Our findings indicate that β adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca 2+ overload through intracellular Ca 2+ release by TE from the jellyfish C. capillata.

Thymosin {beta}4 promotes the migration of endothelial cells without intracellular Ca{sup 2+} elevation

Energy Technology Data Exchange (ETDEWEB)

Selmi, Anna [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Malinowski, Mariusz [Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland); Brutkowski, Wojciech [Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw (Poland); Bednarek, Radoslaw [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Cierniewski, Czeslaw S., E-mail: czeslaw.cierniewski@umed.lodz.pl [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland)

2012-08-15

Numerous studies have demonstrated the effects of T{beta}4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which T{beta}4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with T{beta}4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that T{beta}4 interacts with Ku80, which may operate as a novel receptor for T{beta}4 and mediates its intracellular activity. In this paper, we provide evidence that T{beta}4 induces cellular processes without changes in the intracellular Ca{sup 2+} concentration. External treatment of HUVECs with T{beta}4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (T{beta}4{sub AcSDKPT/4A}) or the actin-binding sequence KLKKTET (T{beta}4{sub KLKKTET/7A}) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by T{beta}4 was not associated with the intracellular Ca{sup 2+} elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added T{beta}4 induces HUVEC migration via the surface membrane receptors known to generate Ca{sup 2+} influx. Our data confirm the concept that externally added T{beta}4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.

Dependence of cerebral arterial contractions on intracellularly stored Ca++.

Science.gov (United States)

Sasaki, T; Kassell, N F; Zuccarello, M

1986-01-01

The purpose of the present study was to evaluate the dependence of the arterial contractions induced by different vasoactive agents upon intracellularly stored calcium in canine versus monkey cerebral arteries. The potency for inducing contractions in Ca++-free media was in the order of 9,11-epithio-11,12-metano-thromboxane A2 (STXA2) greater than prostaglandin F2 alpha (PGF2 alpha) much greater than serotonin greater than K+ in canine basilar arteries, and STXA2 greater than PGF2 alpha much greater than serotonin = K+ in monkey basilar arteries.

Regulation of NF-κB oscillation by spatial parameters in true intracellular space (TiCS)

Science.gov (United States)

Ohshima, Daisuke; Sagara, Hiroshi; Ichikawa, Kazuhisa

2013-10-01

Transcription factor NF-κB is activated by cytokine stimulation, viral infection, or hypoxic environment leading to its translocation to the nucleus. The nuclear NF-κB is exported from the nucleus to the cytoplasm again, and by repetitive import and export, NF-κB shows damped oscillation with the period of 1.5-2.0 h. Oscillation pattern of NF-κB is thought to determine the gene expression profile. We published a report on a computational simulation for the oscillation of nuclear NF-κB in a 3D spherical cell, and showed the importance of spatial parameters such as diffusion coefficient and locus of translation for determining the oscillation pattern. Although the value of diffusion coefficient is inherent to protein species, its effective value can be modified by organelle crowding in intracellular space. Here we tested this possibility by computer simulation. The results indicate that the effective value of diffusion coefficient is significantly changed by the organelle crowding, and this alters the oscillation pattern of nuclear NF-κB.

Fast gamma oscillations are generated intrinsically in CA1 without the involvement of fast-spiking basket cells.

Science.gov (United States)

Craig, Michael T; McBain, Chris J

2015-02-25

Information processing in neuronal networks relies on the precise synchronization of ensembles of neurons, coordinated by the diverse family of inhibitory interneurons. Cortical interneurons can be usefully parsed by embryonic origin, with the vast majority arising from either the caudal or medial ganglionic eminences (CGE and MGE). Here, we examine the activity of hippocampal interneurons during gamma oscillations in mouse CA1, using an in vitro model where brief epochs of rhythmic activity were evoked by local application of kainate. We found that this CA1 KA-evoked gamma oscillation was faster than that in CA3 and, crucially, did not appear to require the involvement of fast-spiking basket cells. In contrast to CA3, we also found that optogenetic inhibition of pyramidal cells in CA1 did not significantly affect the power of the oscillation, suggesting that excitation may not be essential for gamma genesis in this region. We found that MGE-derived interneurons were generally more active than CGE interneurons during CA1 gamma, although a group of CGE-derived interneurons, putative trilaminar cells, were strongly phase-locked with gamma oscillations and, together with MGE-derived axo-axonic and bistratified cells, provide attractive candidates for being the driver of this locally generated, predominantly interneuron-driven model of gamma oscillations. Copyright © 2015 the authors 0270-6474/15/353616-09$15.00/0.

Involvement of intracellular free Ca2+ in enhanced release of herpes simplex virus by hydrogen peroxide

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Ogawa Yuzo

2006-08-01

Full Text Available Abstract Background It was reported that elevation of the intracellular concentration of free Ca2+ ([Ca2+]i by a calcium ionophore increased the release of herpes simplex virus type 1 (HSV-1. Freely diffusible hydrogen peroxide (H2O2 is implied to alter Ca2+ homeostasis, which further enhances abnormal cellular activity, causing changes in signal transduction, and cellular dysfunction. Whether H2O2 could affect [Ca2+]i in HSV-1-infected cells had not been investigated. Results H2O2 treatment increased the amount of cell-free virus and decreased the proportion of viable cells. After the treatment, an elevation in [Ca2+]i was observed and the increase in [Ca2+]i was suppressed when intracellular and cytosolic Ca2+ were buffered by Ca2+ chelators. In the presence of Ca2+ chelators, H2O2-mediated increases of cell-free virus and cell death were also diminished. Electron microscopic analysis revealed enlarged cell junctions and a focal disintegration of the plasma membrane in H2O2-treated cells. Conclusion These results indicate that H2O2 can elevate [Ca2+]i and induces non-apoptotic cell death with membrane lesions, which is responsible for the increased release of HSV-1 from epithelial cells.

Mastoparan-Induced Intracellular Ca2+ Fluxes May Regulate Cell-to-Cell Communication in Plants.

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Tucker, E. B.; Boss, W. F.

1996-06-01

The relationship of Ca2+ and plasmodesmatal closure was examined in staminal hairs of Setcreasea purpurea by microinjecting cells with active mastoparan (Mas-7), inactive mastoparan (Mas-17), active inositol-1,4,5-trisphosphate (IP3), or inactive IP3. Calcium green dextran 10,000 was used to study cellular free Ca2+, and carboxyfluorescein was used to monitor plasmodesmatal closure. When Mas-7 was microinjected into the cytoplasm of cell 1 (the tip cell of a chain of cells), a rapid increase in calcium green dextran-10,000 fluorescence was observed in the cytoplasmic areas on both sides of the plasmodesmata connecting cells 1 and 2 during the same time that the diffusion of carboxyfluorescein through them was blocked. The inhibition of cell-to-cell diffusion was transient, and the closed plasmodesmata reopened within 30 s. The elevated Ca2+ level near plasmodesmata was also transient and returned to base level in about 1.5 min. The transient increase in Ca2+, once initiated in cell 1, repeated with an oscillatory period of 3 min. Elevated Ca2+ and oscillations of Ca2+ were also observed near interconnecting cell walls throughout the chain of cells, indicating that the signal had been transmitted. Previously, we reported that IP3 closed plasmodesmata; now we report that it stimulated Ca2+ and oscillations similar to Mas-7. The effect was specific for similar concentrations of Mas-7 over Mas-17 and active IP3 over inactive IP3. It is important that the Ca2+ channel blocker La3+ eliminated the responses from Mas-7 and IP3, indicating that an influx of Ca2+ was required. These results support the contention that plasmodesmata functioning is regulated via Ca2+ and that IP3 may be an intermediary between the stimulus and Ca2+ elevations.

Hippocampal deletion of BDNF gene attenuates gamma oscillations in area CA1 by up-regulating 5-HT3 receptor.

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Ying Huang

2011-01-01

Full Text Available Pyramidal neurons in the hippocampal area CA3 express high levels of BDNF, but how this BDNF contributes to oscillatory properties of hippocampus is unknown.Here we examined carbachol-induced gamma oscillations in hippocampal slices lacking BDNF gene in the area CA3. The power of oscillations was reduced in the hippocampal area CA1, which coincided with increases in the expression and activity of 5-HT3 receptor. Pharmacological block of this receptor partially restored power of gamma oscillations in slices from KO mice, but had no effect in slices from WT mice.These data suggest that BDNF facilitates gamma oscillations in the hippocampus by attenuating signaling through 5-HT3 receptor. Thus, BDNF modulates hippocampal oscillations through serotonergic system.

The intercellular synchronization of Ca2+ oscillations evaluates Cx36-dependent coupling.

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Sabine Bavamian

Full Text Available Connexin36 (Cx36 plays an important role in insulin secretion by controlling the intercellular synchronization of Ca(2+ transients induced during stimulation. The lack of drugs acting on Cx36 channels is a major limitation in further unraveling the molecular mechanism underlying this effect. To screen for such drugs, we have developed an assay allowing for a semi-automatic, fluorimetric quantification of Ca(2+ transients in large populations of MIN6 cells. Here, we show that (1 compared to control cells, MIN6 cells with reduced Cx36 expression or function showed decreased synchrony of glucose-induced Ca(2+ oscillations; (2 glibenclamide, a sulphonylurea which promotes Cx36 junctions and coupling, increased the number of synchronous MIN6 cells, whereas quinine, an antimalarial drug which inhibits Cx36-dependent coupling, decreased this proportion; (3 several drugs were identified that altered the intercellular Ca(2+ synchronization, cell coupling and distribution of Cx36; (4 some of them also affected insulin content. The data indicate that the intercellular synchronization of Ca(2+ oscillations provides a reliable and non-invasive measurement of Cx36-dependent coupling, which is useful to identify novel drugs affecting the function of β-cells, neurons, and neuron-related cells that express Cx36.

Ca analysis: An Excel based program for the analysis of intracellular calcium transients including multiple, simultaneous regression analysis☆

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Greensmith, David J.

2014-01-01

Here I present an Excel based program for the analysis of intracellular Ca transients recorded using fluorescent indicators. The program can perform all the necessary steps which convert recorded raw voltage changes into meaningful physiological information. The program performs two fundamental processes. (1) It can prepare the raw signal by several methods. (2) It can then be used to analyze the prepared data to provide information such as absolute intracellular Ca levels. Also, the rates of change of Ca can be measured using multiple, simultaneous regression analysis. I demonstrate that this program performs equally well as commercially available software, but has numerous advantages, namely creating a simplified, self-contained analysis workflow. PMID:24125908

INTRACELLULAR Ca2+ HOMEOSTASIS

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Shahdevi Nandar Kurniawan

2015-01-01

Full Text Available Ca2+ signaling functions to regulate many cellular processes. Dynamics of Ca2+ signaling or homeostasis is regulated by the interaction between ON and OFF reactions that control Ca2+ flux in both the plasma membrane and internal organelles such as the endoplasmic reticulum (ER and mitochondria. External stimuli activate the ON reactions, which include Ca2+ into the cytoplasm either through channels in the plasma membrane or from internal storage like in ER. Most of the cells utilize both channels/sources, butthere area few cells using an external or internal source to control certain processes. Most of the Ca2+ entering the cytoplasm adsorbed to the buffer, while a smaller part activate effect or to stimulate cellular processes. Reaction OFF is pumping of cytoplasmic Ca2+ using a combination mechanism of mitochondrial and others. Changes in Ca2+ signal has been detected in various tissues isolated from animals induced into diabetes as well as patients with diabetes. Ca2+ signal interference is also found in sensory neurons of experimental animals with diabetes. Ca2+ signaling is one of the main signaling systems in the cell.

Single-cell resolution of intracellular T cell Ca2+ dynamics in response to frequency-based H2O2 stimulation.

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Kniss-James, Ariel S; Rivet, Catherine A; Chingozha, Loice; Lu, Hang; Kemp, Melissa L

2017-03-01

Adaptive immune cells, such as T cells, integrate information from their extracellular environment through complex signaling networks with exquisite sensitivity in order to direct decisions on proliferation, apoptosis, and cytokine production. These signaling networks are reliant on the interplay between finely tuned secondary messengers, such as Ca 2+ and H 2 O 2 . Frequency response analysis, originally developed in control engineering, is a tool used for discerning complex networks. This analytical technique has been shown to be useful for understanding biological systems and facilitates identification of the dominant behaviour of the system. We probed intracellular Ca 2+ dynamics in the frequency domain to investigate the complex relationship between two second messenger signaling molecules, H 2 O 2 and Ca 2+ , during T cell activation with single cell resolution. Single-cell analysis provides a unique platform for interrogating and monitoring cellular processes of interest. We utilized a previously developed microfluidic device to monitor individual T cells through time while applying a dynamic input to reveal a natural frequency of the system at approximately 2.78 mHz stimulation. Although our network was much larger with more unknown connections than previous applications, we are able to derive features from our data, observe forced oscillations associated with specific amplitudes and frequencies of stimuli, and arrive at conclusions about potential transfer function fits as well as the underlying population dynamics.

Decreased intracellular [Ca2+ ] coincides with reduced expression of Dhprα1s, RyR1, and diaphragmatic dysfunction in a rat model of sepsis.

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Wang, Meng-Meng; Hao, Li-Ying; Guo, Feng; Zhong, Bin; Zhong, Xiao-Mei; Yuan, Jing; Hao, Yi-Fei; Zhao, Shuang; Sun, Xue-Fei; Lei, Ming; Jiao, Guang-Yu

2017-12-01

Sepsis can cause decreased diaphragmatic contractility. Intracellular calcium as a second messenger is central to diaphragmatic contractility. However, changes in intracellular calcium concentration ([Ca 2+ ]) and the distribution and co-localization of relevant calcium channels [dihydropyridine receptors, (DHPRα1s) and ryanodine receptors (RyR1)] remain unclear during sepsis. In this study we investigated the effect of changed intracellular [Ca 2+ ] and expression and distribution of DHPRα1s and RyR1 on diaphragm function during sepsis. We measured diaphragm contractility and isolated diaphragm muscle cells in a rat model of sepsis. The distribution and co-localization of DHPRα1s and RyR1 were determined using immunohistochemistry and immunofluorescence, whereas intracellular [Ca 2+ ] was measured by confocal microscopy and fluorescence spectrophotometry. Septic rat diaphragm contractility, expression of DHPRα1s and RyR1, and intracellular [Ca 2+ ] were significantly decreased in the rat sepsis model compared with controls. Decreased intracellular [Ca 2+ ] coincides with diaphragmatic contractility and decreased expression of DHPRα1s and RyR1 in sepsis. Muscle Nerve 56: 1128-1136, 2017. © 2017 Wiley Periodicals, Inc.

Distribution of voltage-dependent and intracellular Ca2+ channels in submucosal neurons from rat distal colon.

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Rehn, Matthias; Bader, Sandra; Bell, Anna; Diener, Martin

2013-09-01

We recently observed a bradykinin-induced increase in the cytosolic Ca2+ concentration in submucosal neurons of rat colon, an increase inhibited by blockers of voltage-dependent Ca2+ (Ca(v)) channels. As the types of Ca(v) channels used by this part of the enteric nervous system are unknown, the expression of various Ca(v) subunits has been investigated in whole-mount submucosal preparations by immunohistochemistry. Submucosal neurons, identified by a neuronal marker (microtubule-associated protein 2), are immunoreactive for Ca(v)1.2, Ca(v)1.3 and Ca(v)2.2, expression being confirmed by reverse transcription plus the polymerase chain reaction. These data agree with previous observations that the inhibition of L- and N-type Ca2+ currents strongly inhibits the response to bradykinin. However, whole-cell patch-clamp experiments have revealed that bradykinin does not enhance Ca2+ inward currents under voltage-clamp conditions. Consequently, bradykinin does not directly interact with Ca(v) channels. Instead, the kinin-induced Ca2+ influx is caused indirectly by the membrane depolarization evoked by this peptide. As intracellular Ca2+ channels on Ca(2+)-storing organelles can also contribute to Ca2+ signaling, their expression has been investigated by imaging experiments and immunohistochemistry. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) have been functionally demonstrated in submucosal neurons loaded with the Ca(2+)-sensitive fluorescent dye, fura-2. Histamine, a typical agonist coupled to the phospholipase C pathway, induces an increase in the fura-2 signal ratio, which is suppressed by 2-aminophenylborate, a blocker of IP3 receptors. The expression of IP3R1 has been confirmed by immunohistochemistry. In contrast, ryanodine, tested over a wide concentration range, evokes no increase in the cytosolic Ca2+ concentration nor is there immunohistochemical evidence for the expression of ryanodine receptors in these neurons. Thus, rat submucosal neurons are equipped

Ca analysis: an Excel based program for the analysis of intracellular calcium transients including multiple, simultaneous regression analysis.

Science.gov (United States)

Greensmith, David J

2014-01-01

Here I present an Excel based program for the analysis of intracellular Ca transients recorded using fluorescent indicators. The program can perform all the necessary steps which convert recorded raw voltage changes into meaningful physiological information. The program performs two fundamental processes. (1) It can prepare the raw signal by several methods. (2) It can then be used to analyze the prepared data to provide information such as absolute intracellular Ca levels. Also, the rates of change of Ca can be measured using multiple, simultaneous regression analysis. I demonstrate that this program performs equally well as commercially available software, but has numerous advantages, namely creating a simplified, self-contained analysis workflow. Copyright © 2013 The Author. Published by Elsevier Ireland Ltd.. All rights reserved.

Induction of Intracellular Ca2+ and pH Changes in Sf9 Insect Cells by Rhodojaponin-III, A Natural Botanic Insecticide Isolated from Rhododendron molle

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Yan-Bo Zhang

2011-04-01

Full Text Available Many studies on intracellular calcium ([Ca2+]i and intracellular pH (pHi have been carried out due to their importance in regulation of different cellular functions. However, most of the previous studies are focused on human or mammalian cells. The purpose of the present study was to characterize the effect of Rhodojaponin-III (R-III on [Ca2+]i and pHi and the proliferation of Sf9 cells. R-III strongly inhibited Sf9 cells proliferation with a time- and dose-dependent manner. Flow cytometry established that R-III interfered with Sf9 cells division and arrested them in G2/M. By using confocal scanning technique, effects of R-III on intracellular free calcium ([Ca2+]i and intracellular pH (pHi in Sf9 cells were determined. R-III induced a significant dose-dependent (1, 10, 100, 200 μg/mL increase in [Ca2+]i and pHi of Sf9 cells in presence of Ca2+-containing solution (Hanks and an irreversible decrease in the absence of extra cellular Ca2+. We also found that both extra cellular Ca2+ and intracellular Ca2+ stores contributed to the increase of [Ca2+]i, because completely treating Sf9 cells with CdCl2 (5 mM, a Ca2+ channels blocker, R-III (100 μg/mL induced a transient elevation of [Ca2+]i in case of cells either in presence of Ca2+ containing or Ca2+ free solution. In these conditions, pHi showed similar changes with that of [Ca2+]i on the whole. Accordingly, we supposed that there was a certain linkage for change of [Ca2+]i, cell cycle arrest, proliferation inhibition in Sf9 cells induced by R-III.

Ca2+ Entry is Required for Mechanical Stimulation-induced ATP Release from Astrocyte

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Lee, Jaekwang; Chun, Ye-Eun; Han, Kyung-Seok; Lee, Jungmoo; Woo, Dong Ho

2015-01-01

Astrocytes and neurons are inseparable partners in the brain. Neurotransmitters released from neurons activate corresponding G protein-coupled receptors (GPCR) expressed in astrocytes, resulting in release of gliotransmitters such as glutamate, D-serine, and ATP. These gliotransmitters in turn influence neuronal excitability and synaptic activities. Among these gliotransmitters, ATP regulates the level of network excitability and is critically involved in sleep homeostasis and astrocytic Ca2+ oscillations. ATP is known to be released from astrocytes by Ca2+-dependent manner. However, the precise source of Ca2+, whether it is Ca2+ entry from outside of cell or from the intracellular store, is still not clear yet. Here, we performed sniffer patch to detect ATP release from astrocyte by using various stimulation. We found that ATP was not released from astrocyte when Ca2+ was released from intracellular stores by activation of Gαq-coupled GPCR including PAR1, P2YR, and B2R. More importantly, mechanical stimulation (MS)-induced ATP release from astrocyte was eliminated when external Ca2+ was omitted. Our results suggest that Ca2+ entry, but not release from intracellular Ca2+ store, is critical for MS-induced ATP release from astrocyte. PMID:25792866

Demand for Zn2+ in acid-secreting gastric mucosa and its requirement for intracellular Ca2+.

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JingJing Liu

Full Text Available Recent work has suggested that Zn(2+ plays a critical role in regulating acidity within the secretory compartments of isolated gastric glands. Here, we investigate the content, distribution and demand for Zn(2+ in gastric mucosa under baseline conditions and its regulation during secretory stimulation.Content and distribution of zinc were evaluated in sections of whole gastric mucosa using X-ray fluorescence microscopy. Significant stores of Zn(2+ were identified in neural elements of the muscularis, glandular areas enriched in parietal cells, and apical regions of the surface epithelium. In in vivo studies, extraction of the low abundance isotope, (70Zn(2+, from the circulation was demonstrated in samples of mucosal tissue 24 hours or 72 hours after infusion (250 µg/kg. In in vitro studies, uptake of (70Zn(2+ from media was demonstrated in isolated rabbit gastric glands following exposure to concentrations as low as 10 nM. In additional studies, demand of individual gastric parietal cells for Zn(2+ was monitored using the fluorescent zinc reporter, fluozin-3, by measuring increases in free intracellular concentrations of Zn(2+ {[Zn(2+](i} during exposure to standard extracellular concentrations of Zn(2+ (10 µM for standard intervals of time. Under resting conditions, demand for extracellular Zn(2+ increased with exposure to secretagogues (forskolin, carbachol/histamine and under conditions associated with increased intracellular Ca(2+ {[Ca(2+](i}. Uptake of Zn(2+ was abolished following removal of extracellular Ca(2+ or depletion of intracellular Ca(2+ stores, suggesting that demand for extracellular Zn(2+ increases and depends on influx of extracellular Ca(2+.This study is the first to characterize the content and distribution of Zn(2+ in an organ of the gastrointestinal tract. Our findings offer the novel interpretation, that Ca(2+ integrates basolateral demand for Zn(2+ with stimulation of secretion of HCl into the lumen of the gastric

Increased extracellular and intracellular Ca{sup 2+} lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms

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Hashimoto, Ryota, E-mail: hryota@juntendo.ac.jp [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Katoh, Youichi, E-mail: katoyo@juntendo-urayasu.jp [Juntendo University Faculty of International Liberal Arts, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Miyamoto, Yuki [Juntendo University Faculty of Health Care and Nursing, Takasu 2-5-1, Urayasu-shi, Chiba 279-0023 (Japan); Itoh, Seigo; Daida, Hiroyuki [Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Nakazato, Yuji [Center for Environmental Research, Department of Cardiology, Juntendo University Faculty of Medicine Urayasu Hospital, Tomioka 2-1-1, Urayasu-shi, Chiba 279-0022 (Japan); Okada, Takao [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

2015-02-20

Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca{sup 2+} ([Ca{sup 2+}]{sub o} and [Ca{sup 2+}]{sub i}) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} enhanced adipocyte accumulation, which suggested that increases in [Ca{sup 2+}]{sub o} caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca{sup 2+}]{sub i} (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca{sup 2+}]{sub o} (addition of CaCl{sub 2}) leads to increases in [Ca{sup 2+}]{sub i}. Flow cytometric methods revealed that high [Ca{sup 2+}]{sub o} suppressed the phosphorylation of ERK independently of intracellular Ca{sup 2+}. The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca{sup 2+} provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca{sup 2+}, which results in BMSC proliferation. - Highlights: �

Insulin-Like Growth Factor I (IGF-1) Deficiency Ameliorates Sex Difference in Cardiac Contractile Function and Intracellular Ca2+ Homeostasis

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Ceylan-Isik, Asli F.; Li, Qun; Ren, Jun

2011-01-01

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (± dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), fura-fluorescence intensity (FFI) and intracellular Ca2+ clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ± dL/dt, longer TPS, TR90 and intracellular Ca2+ clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na+-Ca2+ exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca2+ regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca2+ regulation. PMID:21763763

Insulin-like growth factor I (IGF-1) deficiency ameliorates sex difference in cardiac contractile function and intracellular Ca(2+) homeostasis.

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Ceylan-Isik, Asli F; Li, Qun; Ren, Jun

2011-10-10

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (±dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR(90)), fura-fluorescence intensity (FFI) and intracellular Ca(2+) clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ±dL/dt, longer TPS, TR(90) and intracellular Ca(2+) clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na(+)-Ca(2+) exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca(2+) regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca(2+) regulation. Copyright © 2011 Elsevier Ireland

[Establishment of oxygen and glucose deprive model of in vitro cultured hippocampal neuron and effect of ligustrazine on intracellular Ca+ level in model neurons].

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Wan, Hai-tong; Wang, Yu; Yang, Jie-hong

2007-03-01

To establish the oxygen and glucose deprive (OGD) model in cultured hippocampal neuron and study the effect of ligustrazine on intracellular Ca2+ level in the model neurons. The OGD model was established in cultured hippocampal neuron, and the intracellular Ca2+ level in it was detected by laser scanning confocal microscope (LSCM). The OGD model was successfully established in cultured hippocampal neurons; the intracellular Ca2+ level in the OGD model group was significantly higher than that in the blank control group (P neuron, which could be antagonized by ligustrazine, indicating that ligustrazine has a protective effect on hippocampal neuron from hypoxic-ischemic injury.

Ontogenic and sexual differences in pituitary GnRH receptors and intracellular Ca2+ mobilization induced by GnRH.

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Lacau-Mengido, I M; González Iglesias, A; Lux-Lantos, V; Libertun, C; Becú-Villalobos, D

1998-04-01

The present experiments were designed in order to elucidate the participation of the developing hypophysis in determining the changing sensitivity of gonadotrophins to gonadotropin-releasing hormone (GnRH) during ontogeny in the rat. To that end, we chose two well defined developmental ages that differ markedly in sexual and ontogenic characteristics of hypophyseal sensitivity to GnRH, 15 and 30 d. In order to study sex differences and the role of early sexual organization of the hypothalamus, experiments were carried out in males, females, and neonatally androgenized females (TP females). We evaluated (1) the characteristics of pituitary GnRH receptors, and (2) associated changes in GnRH-induced mobilization of intracellular Ca2+ (a second messenger involved in gonadotropins exocytosis). We measured binding characteristics of the GnRH analog D-Ser(TBu)6-des-Gly10-GnRH ethylamide in pituitary homogenates. We found that Kds did not vary among the different sex groups. Total number and concentration of receptors decreased in the female rat from 15-30 d of age, whereas in the male and TP female, receptors/pituitary increased, and the concentration/mg tissue did not change. Also, at 30 days of age, males presented higher content and concentration of receptors than females, and higher content than TP females. In order to evaluate if developmental and sexual differences in pituitary sensitivity to GnRH might be expressed through variations in the intracellular Ca2+ signal, we studied the mobilization of intracellular Ca2+ induced by GnRH (1 x 10(-8) to 1 x 10(-11) M) in a suspension of dispersed pituitary cells in the six groups. In cells from 15-d-old females, Ca2+ response was greater than in 30-d-old females at the doses of 10(-8) to 10(-10) M, indicating that in the infantile female rat activation of highly concentrated GnRH receptors is reflected in an increase in signal transduction mediated by Ca2+. In males and in female rats androgenized at birth, there was also

Anabolic Androgenic Steroids and Intracellular Calcium Signaling: A Mini Review on Mechanisms and Physiological Implications

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Vicencio, J.M.; Estrada, M.; Galvis, D.; Bravo, R.; Contreras, A.E.; Rotter, D.; Szabadkai, G.; Hill, J.A.; Rothermel, B.A.; Jaimovich, E.; Lavandero, S.

2015-01-01

Increasing evidence suggests that nongenomic effects of testosterone and anabolic androgenic steroids (AAS) operate concertedly with genomic effects. Classically, these responses have been viewed as separate and independent processes, primarily because nongenomic responses are faster and appear to be mediated by membrane androgen receptors, whereas long-term genomic effects are mediated through cytosolic androgen receptors regulating transcriptional activity. Numerous studies have demonstrated increases in intracellular Ca2+ in response to AAS. These Ca2+ mediated responses have been seen in a diversity of cell types, including osteoblasts, platelets, skeletal muscle cells, cardiac myocytes and neurons. The versatility of Ca2+ as a second messenger provides these responses with a vast number of pathophysiological implications. In cardiac cells, testosterone elicits voltage-dependent Ca2+ oscillations and IP3R-mediated Ca2+ release from internal stores, leading to activation of MAPK and mTOR signaling that promotes cardiac hypertrophy. In neurons, depending upon concentration, testosterone can provoke either physiological Ca2+ oscillations, essential for synaptic plasticity, or sustained, pathological Ca2+ transients that lead to neuronal apoptosis. We propose therefore, that Ca2+ acts as an important point of crosstalk between nongenomic and genomic AAS signaling, representing a central regulator that bridges these previously thought to be divergent responses. PMID:21443511

Suppression of the increasing level of acetylcholine-stimulated intracellular Ca2+ in guinea pig airway smooth muscle cells by mabuterol.

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Song, Xirui; Zhao, Chao; Dai, Cailing; Ren, Yanxin; An, Nan; Wen, Huimin; Pan, L I; Cheng, Maosheng; Zhang, Yuyang

2015-11-01

The present study aimed to establish an effective method for the in vitro culture of guinea pig airway smooth muscle (ASM) cells, and also investigate the suppressive effect of mabuterol hydrochloride (Mab) on the increased level of intracellular Ca 2+ in ASM cells induced with acetylcholine (Ach). Two different methods, i.e. with or without collagenase to pretreat tracheal tissues, were applied to the manufacture of ASM cells. Cell viability was determined with the 3-(4,5-dimethylthinazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Immunocytochemistry and immunofluorescence were used for the identification of ASM cells. Different concentration levels (10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 mmol/l) of Mab were administered 5 min before Ach (10 -4 M) treatment, respectively. The Ca 2+ fluorescent probe, Fura-2/AM or Fluo-3/AM were applied to the inspection of Ca 2+ fluorescent intensity with Varioskan Flash, immunocytometry systems and an inverted system microscope, respectively. The results showed that the fresh method, in which isolated tracheal tissues were previously treated with collagenase for 20 min, was more advantageous for the preparation of guinea pig ASM cells compared to when the enzyme was not used. The time for the ASM cells to initially migrate out of the 'tissue blocks' and the culture having to be generated due to the thick cell density was significantly less. On identification with immunocytochemistry or immunofluorescent staining, >95% of the cells were ASM cells. Mab (10 -3 -10 -7 mmol/l) significantly suppressed the elevation of intracellular Ca 2+ induced by Ach in a concentration-dependent manner. The inhibitory rates of intracellular Ca 2+ by different concentrations of Mab, from low to high, were 14.93, 24.73, 40.06, 48.54 and 57.13%, respectively, when Varioskan Flash was used for determination. In conclusion, this novel method has a shorter harvesting period for ASM cells. Mab can suppress the increasing level of intracellular Ca 2

Three types of ependymal cells with intracellular calcium oscillation are characterized by distinct cilia beating properties.

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Liu, Tongyu; Jin, Xingjian; Prasad, Rahul M; Sari, Youssef; Nauli, Surya M

2014-09-01

Ependymal cells are multiciliated epithelial cells that line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia has been associated with various neurological deficits. For the first time, we report three distinct ependymal cell types, I, II, and III, based on their unique ciliary beating frequency and beating angle. These ependymal cells have specific localizations within the third ventricle of the mouse brain. Furthermore, neither ependymal cell types nor their localizations are altered by aging. Our high-speed fluorescence imaging analysis reveals that these ependymal cells have an intracellular pacing calcium oscillation property. Our study further shows that alcohol can significantly repress the amplitude of calcium oscillation and the frequency of ciliary beating, resulting in an overall decrease in volume replacement by the cilia. Furthermore, the pharmacological agent cilostazol could differentially increase cilia beating frequency in type II, but not in type I or type III, ependymal cells. In summary, we provide the first evidence of three distinct types of ependymal cells with calcium oscillation properties. © 2014 Wiley Periodicals, Inc.

Update on vascular endothelial Ca(2+) signalling: A tale of ion channels, pumps and transporters.

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Moccia, Francesco; Berra-Romani, Roberto; Tanzi, Franco

2012-07-26

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and forms a multifunctional transducing organ that mediates a plethora of cardiovascular processes. The activation of ECs from as state of quiescence is, therefore, regarded among the early events leading to the onset and progression of potentially lethal diseases, such as hypertension, myocardial infarction, brain stroke, and tumor. Intracellular Ca(2+) signals have long been know to play a central role in the complex network of signaling pathways regulating the endothelial functions. Notably, recent work has outlined how any change in the pattern of expression of endothelial channels, transporters and pumps involved in the modulation of intracellular Ca(2+) levels may dramatically affect whole body homeostasis. Vascular ECs may react to both mechanical and chemical stimuli by generating a variety of intracellular Ca(2+) signals, ranging from brief, localized Ca(2+) pulses to prolonged Ca(2+) oscillations engulfing the whole cytoplasm. The well-defined spatiotemporal profile of the subcellular Ca(2+) signals elicited in ECs by specific extracellular inputs depends on the interaction between Ca(2+) releasing channels, which are located both on the plasma membrane and in a number of intracellular organelles, and Ca(2+) removing systems. The present article aims to summarize both the past and recent literature in the field to provide a clear-cut picture of our current knowledge on the molecular nature and the role played by the components of the Ca(2+) machinery in vascular ECs under both physiological and pathological conditions.

Update on vascular endothelial Ca2+ signalling: A tale of ion channels, pumps and transporters

Science.gov (United States)

Moccia, Francesco; Berra-Romani, Roberto; Tanzi, Franco

2012-01-01

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and forms a multifunctional transducing organ that mediates a plethora of cardiovascular processes. The activation of ECs from as state of quiescence is, therefore, regarded among the early events leading to the onset and progression of potentially lethal diseases, such as hypertension, myocardial infarction, brain stroke, and tumor. Intracellular Ca2+ signals have long been know to play a central role in the complex network of signaling pathways regulating the endothelial functions. Notably, recent work has outlined how any change in the pattern of expression of endothelial channels, transporters and pumps involved in the modulation of intracellular Ca2+ levels may dramatically affect whole body homeostasis. Vascular ECs may react to both mechanical and chemical stimuli by generating a variety of intracellular Ca2+ signals, ranging from brief, localized Ca2+ pulses to prolonged Ca2+ oscillations engulfing the whole cytoplasm. The well-defined spatiotemporal profile of the subcellular Ca2+ signals elicited in ECs by specific extracellular inputs depends on the interaction between Ca2+ releasing channels, which are located both on the plasma membrane and in a number of intracellular organelles, and Ca2+ removing systems. The present article aims to summarize both the past and recent literature in the field to provide a clear-cut picture of our current knowledge on the molecular nature and the role played by the components of the Ca2+ machinery in vascular ECs under both physiological and pathological conditions. PMID:22905291

High-frequency sarcomeric auto-oscillations induced by heating in living neonatal cardiomyocytes of the rat

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Shintani, Seine A.; Oyama, Kotaro [Department of Pure and Applied Physics, School of Advanced Science and Engineering, Waseda University, Tokyo (Japan); Fukuda, Norio, E-mail: noriof@jikei.ac.jp [Department of Cell Physiology, The Jikei University School of Medicine, Tokyo (Japan); Ishiwata, Shin’ichi, E-mail: ishiwata@waseda.jp [Department of Pure and Applied Physics, School of Advanced Science and Engineering, Waseda University, Tokyo (Japan); WASEDA Bioscience Research Institute in Singapore (WABIOS) (Singapore)

2015-02-06

Highlights: • We tested the effects of infra-red laser irradiation on cardiac sarcomere dynamics. • A rise in temperature (>∼38 °C) induced high-frequency sarcomeric auto-oscillations. • These oscillations occurred with and without blockade of intracellular Ca{sup 2+} stores. • Cardiac sarcomeres can play a role as a temperature-dependent rhythm generator. - Abstract: In the present study, we investigated the effects of infra-red laser irradiation on sarcomere dynamics in living neonatal cardiomyocytes of the rat. A rapid increase in temperature to >∼38 °C induced [Ca{sup 2+}]{sub i}-independent high-frequency (∼5–10 Hz) sarcomeric auto-oscillations (Hyperthermal Sarcomeric Oscillations; HSOs). In myocytes with the intact sarcoplasmic reticular functions, HSOs coexisted with [Ca{sup 2+}]{sub i}-dependent spontaneous beating in the same sarcomeres, with markedly varying frequencies (∼10 and ∼1 Hz for the former and latter, respectively). HSOs likewise occurred following blockade of the sarcoplasmic reticular functions, with the amplitude becoming larger and the frequency lower in a time-dependent manner. The present findings suggest that in the mammalian heart, sarcomeres spontaneously oscillate at higher frequencies than the sinus rhythm at temperatures slightly above the physiologically relevant levels.

Hydrogen sulfide regulates intracellular Ca2+ concentration in endothelial cells from excised rat aorta.

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Moccia, Francesco; Bertoni, Giuseppe; Pla, Alessandra Florio; Dragoni, Silvia; Pupo, Emanuela; Merlino, Annalisa; Mancardi, Daniele; Munaron, Luca; Tanzi, Franco

2011-09-01

Hydrogen sulphide (H2S) is a recently discovered gasotransmitter that may regulate a growing number of endothelial functions, including nitric oxide (NO) release, proliferation, adhesion and migration, which are the key steps of angiogenesis. The mechanism whereby H2S impacts on endothelial physiology is still unclear: however, the aforementioned processes are driven by an increase in intracellular Ca2+ concentration ([Ca2+]i). In the present study, we exploited the excised rat aorta to gain insights into the regulation of [Ca2+]i by H2S within in situ endothelial cells (ECs). Sodium hydrosulphide (NaHS), a H2S donor, caused an elevation in [Ca2+]i, which disappeared in absence of extracellular Ca2+. NaHSinduced Ca2+ inflow was sensitive to high doses of Gd3+, but not BTP-2. Inhibition of the reverse-mode of the Na+-Ca2+ exchanger (NCX), with KB-R7943 or upon removal of extracellular Na+, abrogated the Ca2+ response to NaHS. Moreover, NaHS-elicited Ca2+ entry was significantly reduced by TEA and glybenclamide, which hinted at the involvement of ATP-dependent K+ (KATP) channels. Conversely, NaHS-evoked Ca2+ signal was not affected by the reducing agent, dithiothreitol. Acute addition of NaHS hindered both Ca2+ release and Ca2+ entry induced by ATP, a physiological agonist of ECs. Consistently, inhibition of endogenous H2S synthesis with DL-propargylglycine impaired ATP-induced Ca2+ inflow, whereas it did not affect Ca2+ mobilization. These data provide the first evidence that H2S may stimulate Ca2+ influx into ECs by recruiting the reverse-mode of NCX and KATP channels. In addition, they show that such gasotransmitter may modulate the Ca2+ signals elicited by physiological stimuli in intact endothelium.

Modulation of contraction by intracellular Na+ via Na(+)-Ca2+ exchange in single shark (Squalus acanthias) ventricular myocytes.

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Näbauer, M; Morad, M

1992-01-01

1. The effect of direct alteration of intracellular Na+ concentration on contractile properties of whole-cell clamped shark ventricular myocytes was studied using an array of 256 photodiodes to monitor the length of the isolated myocytes. 2. In myocytes dialysed with Na(+)-free solution, the voltage dependence of Ca2+ current (ICa) and contraction were similar and bell shaped. Contractions activated at all voltages were completely suppressed by nifedipine (5 microM), and failed to show significant tonic components, suggesting dependence of the contraction on Ca2+ influx through the L-type Ca2+ channel. 3. In myocytes dialysed with 60 mM Na+, a ICa-dependent and a ICa-independent component of contraction could be identified. The Ca2+ current-dependent component was prominent in voltages between -30 to +10 mV. The ICa-independent contractions were maintained for the duration of depolarization, increased with increasing depolarization between +10 to +100 mV, and were insensitive to nifedipine. 4. In such myocytes, repolarization produced slowly decaying inward tail currents closely related to the time course of relaxation and the degree of shortening prior to repolarization. 5. With 60 mM Na+ in the pipette solution, positive clamp potentials activated decaying outward currents which correlated to the size of contraction. These outward currents appeared to be generated by the Na(+)-Ca(2+)-exchanger since they depended on the presence of intracellular Na+, and were neither suppressed by nifedipine nor by K+ channel blockers. 6. The results suggest that in shark (Squalus acanthias) ventricular myocytes, which lack functionally relevant Ca2+ release pools, both Ca2+ channel and the Na(+)-Ca2+ exchanger deliver sufficient Ca2+ to activate contraction, though the effectiveness of the latter mechanism was highly dependent on the [Na+]i. PMID:1338467

Glucose and phosphate modulation of intracellular 45Ca incorporated into pancreatic islets during culture in the absence and presence of serum

International Nuclear Information System (INIS)

Bergsten, P.

1985-01-01

The effects of glucose and phosphate on the intracellular 45 Ca content were measured in β cell-rich pancreatic islets cultured in media containing or lacking serum. Irrespective of the glucose and serum concentrations there were no or very small increments of 45 Ca contents when phosphate was raised from 0.8 to 5.8 mM during culture for 1 day. However, after 7 days of culture in serum-free medium there was a massive accumulation of 45 Ca in the islets in response to the higher phosphate concentration. Glucose markedly reduced, and serum eliminated, the extensive accumulation probably due to increased cell viability. In the cells cultured in the presence of serum, raising the glucose concentration from 1.0 to 5.5 mM resulted in an increased incorporation of 45 Ca. This effect was particularly pronounced after culture for 7 days in 5.8 mM phosphate. A further increase of glucose to 20 mM reduced the 45 Ca content. The results are consistent with the concept that glucose both stimulates 45 Ca uptake into different β-cell pools and degranulates the cell with associated loss of intracellular calcium from the granular calcium pool. (author)

Irisin Controls Growth, Intracellular Ca2+ Signals, and Mitochondrial Thermogenesis in Cardiomyoblasts.

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Chao Xie

Full Text Available Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin's potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin and in HEK293 cells (hr-irisin for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca2+ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca2+ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work

Irisin Controls Growth, Intracellular Ca2+ Signals, and Mitochondrial Thermogenesis in Cardiomyoblasts.

Science.gov (United States)

Xie, Chao; Zhang, Yuan; Tran, Tran D N; Wang, Hai; Li, Shiwu; George, Eva Vertes; Zhuang, Haoyang; Zhang, Peilan; Kandel, Avi; Lai, Yimu; Tang, Dongqi; Reeves, Westley H; Cheng, Henrique; Ding, Yousong; Yang, Li-Jun

2015-01-01

Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin's potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin) and in HEK293 cells (hr-irisin) for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca2+ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca2+ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work supports the value

A low-dose β1-blocker in combination with milrinone improves intracellular Ca2+ handling in failing cardiomyocytes by inhibition of milrinone-induced diastolic Ca2+ leakage from the sarcoplasmic reticulum.

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Shigeki Kobayashi

Full Text Available OBJECTIVES: The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism. BACKGROUND: The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca(2+ handling in heart failure remains unclear. METHODS: We investigated the effect of milrinone plus landiolol on intracellular Ca(2+ transient (CaT, cell shortening (CS, the frequency of diastolic Ca(2+ sparks (CaSF, and sarcoplasmic reticulum Ca(2+ concentration ({Ca(2+}SR in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2 and phospholamban (PLB. RESULTS: In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca(2+}SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca(2+}SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808 in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17. Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz in failing cardiomyocytes. CONCLUSION: A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca(2+ leak.

A low-dose β1-blocker in combination with milrinone improves intracellular Ca2+ handling in failing cardiomyocytes by inhibition of milrinone-induced diastolic Ca2+ leakage from the sarcoplasmic reticulum.

Science.gov (United States)

Kobayashi, Shigeki; Susa, Takehisa; Ishiguchi, Hironori; Myoren, Takeki; Murakami, Wakako; Kato, Takayoshi; Fukuda, Masakazu; Hino, Akihiro; Suetomi, Takeshi; Ono, Makoto; Uchinoumi, Hitoshi; Tateishi, Hiroki; Mochizuki, Mamoru; Oda, Tetsuro; Okuda, Shinichi; Doi, Masahiro; Yamamoto, Takeshi; Yano, Masafumi

2015-01-01

The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism. The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca(2+) handling in heart failure remains unclear. We investigated the effect of milrinone plus landiolol on intracellular Ca(2+) transient (CaT), cell shortening (CS), the frequency of diastolic Ca(2+) sparks (CaSF), and sarcoplasmic reticulum Ca(2+) concentration ({Ca(2+)}SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB). In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca(2+)}SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca(2+)}SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes. A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca(2+) leak.

Bee venom induces apoptosis through intracellular Ca2+ -modulated intrinsic death pathway in human bladder cancer cells.

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Ip, Siu-Wan; Chu, Yung-Lin; Yu, Chun-Shu; Chen, Po-Yuan; Ho, Heng-Chien; Yang, Jai-Sing; Huang, Hui-Ying; Chueh, Fu-Shin; Lai, Tung-Yuan; Chung, Jing-Gung

2012-01-01

To focus on bee venom-induced apoptosis in human bladder cancer TSGH-8301 cells and to investigate its signaling pathway to ascertain whether intracellular calcium iron (Ca(2+)) is involved in this effect. Bee venom-induced cytotoxic effects, productions of reactive oxygen species and Ca(2+) and the level of mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry. Apoptosis-associated proteins were examined by Western blot analysis and confocal laser microscopy. Bee venom-induced cell morphological changes and decreased cell viability through the induction of apoptosis in TSGH-8301 cell were found. Bee venom promoted the protein levels of Bax, caspase-9, caspase-3 and endonuclease G. The enhancements of endoplasmic reticulum stress-related protein levels were shown in bee venom-provoked apoptosis of TSGH-8301 cells. Bee venom promoted the activities of caspase-3, caspase-8, and caspase-9, increased Ca(2+) release and decreased the level of ΔΨm. Co-localization of immunofluorescence analysis showed the releases of endonuclease G and apoptosis-inducing factor trafficking to nuclei for bee venom-mediated apoptosis. The images revealed evidence of nuclear condensation and formation of apoptotic bodies by 4',6-diamidino-2-phenylindole staining and DNA gel electrophoresis showed the DNA fragmentation in TSGH-8301 cells. Bee venom treatment induces both caspase-dependent and caspase-independent apoptotic death through intracellular Ca(2+) -modulated intrinsic death pathway in TSGH-8301 cells. © 2011 The Japanese Urological Association.

Tris-hydroxymethyl-aminomethane enhances capsaicin-induced intracellular Ca2+ influx through transient receptor potential V1 (TRPV1 channels

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Satoshi Murakami

2016-02-01

Full Text Available Non-selective transient receptor potential vanilloid (TRPV cation channels are activated by various insults, including exposure to heat, acidity, and the compound capsaicin, resulting in sensations of pain in the skin, visceral organs, and oral cavity. Recently, TRPV1 activation was also demonstrated in response to basic pH elicited by ammonia and intracellular alkalization. Tris-hydroxymethyl aminomethane (THAM is widely used as an alkalizing agent; however, the effects of THAM on TRPV1 channels have not been defined. In this study, we characterized the effects of THAM-induced TRPV1 channel activation in baby hamster kidney cells expressing human TRPV1 (hTRPV1 and the Ca2+-sensitive fluorescent sensor GCaMP2 by real-time confocal microscopy. Notably, both capsaicin (1 μM and pH 6.5 buffer elicited steep increases in the intracellular Ca2+ concentration ([Ca2+]i, while treatment with THAM (pH 8.5 alone had no effect. However, treatment with THAM (pH 8.5 following capsaicin application elicited a profound, long-lasting increase in [Ca2+]i that was completely inhibited by the TRPV1 antagonist capsazepine. Taken together, these results suggest that hTRPV1 pre-activation is required to provoke enhanced, THAM-induced [Ca2+]i increases, which could be a mechanism underlying pain induced by basic pH.

Glucose metabolism, islet architecture, and genetic homogeneity in imprinting of [Ca2+](i and insulin rhythms in mouse islets.

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Craig S Nunemaker

2009-12-01

Full Text Available We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca2+](i that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed "islet imprinting." We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J as well as outbred mouse strains (Swiss-Webster; CD1. Second, imprinting was observed in NAD(PH oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca2+](i oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a K(ATP-channel opener that blocks [Ca2+](i influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca2+](i oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca2+](i oscillations. Lastly, to test whether the imprinted [Ca2+](i patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca2+](i oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon.

Downstream effects of hippocampal sharp wave ripple oscillations on medial entorhinal cortex layer V neurons in vitro.

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Roth, Fabian C; Beyer, Katinka M; Both, Martin; Draguhn, Andreas; Egorov, Alexei V

2016-12-01

The entorhinal cortex (EC) is a critical component of the medial temporal lobe (MTL) memory system. Local networks within the MTL express a variety of state-dependent network oscillations that are believed to organize neuronal activity during memory formation. The peculiar pattern of sharp wave-ripple complexes (SPW-R) entrains neurons by a very fast oscillation at ∼200 Hz in the hippocampal areas CA3 and CA1 and then propagates through the "output loop" into the EC. The precise mechanisms of SPW-R propagation and the resulting cellular input patterns in the mEC are, however, largely unknown. We therefore investigated the activity of layer V (LV) principal neurons of the medial EC (mEC) during SPW-R oscillations in horizontal mouse brain slices. Intracellular recordings in the mEC were combined with extracellular monitoring of propagating network activity. SPW-R in CA1 were regularly followed by negative field potential deflections in the mEC. Propagation of SPW-R activity from CA1 to the mEC was mostly monosynaptic and excitatory, such that synaptic input to mEC LV neurons directly reflected unit activity in CA1. Comparison with propagating network activity from CA3 to CA1 revealed a similar role of excitatory long-range connections for both regions. However, SPW-R-induced activity in CA1 involved strong recruitment of rhythmic synaptic inhibition and corresponding fast field oscillations, in contrast to the mEC. These differences between features of propagating SPW-R emphasize the differential processing of network activity by each local network of the hippocampal output loop. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Nanomolar Bifenthrin Alters Synchronous Ca2+ Oscillations and Cortical Neuron Development Independent of Sodium Channel Activity

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Cao, Zhengyu; Cui, Yanjun; Nguyen, Hai M.; Jenkins, David Paul; Wulff, Heike; Pessah, Isaac N.

2014-01-01

Bifenthrin, a relatively stable type I pyrethroid that causes tremors and impairs motor activity in rodents, is broadly used. We investigated whether nanomolar bifenthrin alters synchronous Ca 2+ oscillations (SCOs) necessary for activity-dependent dendritic development. Primary mouse cortical neurons were cultured 8 or 9 days in vitro (DIV), loaded with the Ca2+ indicator Fluo-4, and imaged using a Fluorescence Imaging Plate Reader Tetra. Acute exposure to bifenthrin rapidly increased the fr...

Structure-activity relationships of dimethylsphingosine (DMS) derivatives and their effects on intracellular pH and Ca2+ in the U937 monocyte cell line.

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Chang, Young-Ja; Lee, Yun-Kyung; Lee, Eun-Hee; Park, Jeong-Ju; Chung, Sung-Kee; Im, Dong-Soon

2006-08-01

We recently reported that dimethylsphingosine (DMS), a metabolite of sphingolipids, increased intracellular pH and Ca2+ concentration in U937 human monocytes. In the present study, we found that dimethylphytosphingosine (DMPH) induced the above responses more robustly than DMS. However, phytosphingosine, monomethylphytosphingosine or trimethylsphingosine showed little or no activity. Synthetic C3 deoxy analogues of sphingosine did show similar activities, with the C16 analogue more so than C18. The following structure-activity relationships were observed between DMS derivatives and the intracellular pH and Ca2+ concentrations in U937 monocytes; 1) dimethyl modification is important for the DMS-induced increase of intracellular pH and Ca2+, 2) the addition of an OH group on C4 enhances both activities, 3) the deletion of the OH group on C3 has a negligible effect on the activities, and 4) C16 appears to be more effective than C18. We also found that W-7, a calmodulin inhibitor, blocked the DMS-induced pH increase, whereas, KN-62, ML9, and MMPX, specific inhibitors for calmodulin-dependent kinase II, myosin light chain kinase, and Ca(2+)-calmodulin-dependent phosphodiesterase, respectively, did not affect DMS-induced increases of pH in the U937 monocytes.

PLC-dependent intracellular Ca2+ release was associated with C6-ceramide-induced inhibition of Na+ current in rat granule cells.

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Liu, Zheng; Fei, Xiao-Wei; Fang, Yan-Jia; Shi, Wen-Jie; Zhang, Yu-Qiu; Mei, Yan-Ai

2008-09-01

In this report, the effects of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)), two types of main K(+) current [outward rectifier delayed K(+) current (I(K)) and outward transient K(+) current (I(A))], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) without alteration of the steady-state activation and inactivation properties. Treatment with C(2)-ceramide caused a similar inhibitory effect on I(Na). However, dihydro-C(6)-ceramide failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Furthermore, the C(6)-ceramide effect on I(Na) was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C(6)-ceramide decreased cell viability and blocking I(Na) by tetrodotoxin did not mimic the effect of C(6)-ceramide, and inhibiting intracellular Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) receptor may be responsible for the C(6)-ceramide-induced inhibition of I(Na), which does not seem to be associated with C(6)-ceramide-induced granule

Caffeine-Induced Ca2+ Oscillations in Type I Horizontal Cells of the Carp Retina and the Contribution of the Store-Operated Ca2+ Entry Pathway

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Lv, Ting; Gong, Hai-Qing; Liang, Pei-Ji

2014-01-01

The mechanisms of release, depletion, and refilling of endoplasmic reticulum (ER) Ca2+ were investigated in type I horizontal cells of the carp retina using a fluo-3-based Ca2+ imaging technique. Exogenous application of caffeine, a ryanodine receptor agonist, induced oscillatory intracellular free Ca2+ concentration ([Ca2+]i) responses in a duration- and concentration-dependent manner. In Ca2+-free Ringer’s solution, [Ca2+]i transients could also be induced by a brief caffeine application, whereas subsequent caffeine application induced no [Ca2+]i increase, which implied that extracellular Ca2+ was required for ER refilling, confirming the necessity of a Ca2+ influx pathway for ER refilling. Depletion of ER Ca2+ by thapsigargin triggered a Ca2+ influx which could be blocked by the store-operated channel inhibitor 2-APB, which proved the existence of the store-operated Ca2+ entry pathway. Taken together, these results suggested that after being depleted by caffeine, the ER was replenished by Ca2+ influx via store-operated channels. These results reveal the fine modulation of ER Ca2+ signaling, and the activation of the store-operated Ca2+ entry pathway guarantees the replenishment of the ER so that the cell can be ready for response to the subsequent stimulus. PMID:24918937

Attenuation of ischemia-reperfusion-induced alterations in intracellular Ca2+ in cardiomyocytes from hearts treated with N-acetylcysteine and N-mercaptopropionylglycine.

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Saini-Chohan, Harjot K; Dhalla, Naranjan S

2009-12-01

This study was undertaken to test whether Ca(2+)-handling abnormalities in cardiomyocytes after ischemia-reperfusion (I/R) are prevented by antioxidants such as N-acetyl L-cysteine (NAC), which is known to reduce oxidative stress by increasing the glutathione redox status, and N-(2-mercaptopropionyl)-glycine (MPG), which scavenges both peroxynitrite and hydroxyl radicals. For this purpose, isolated rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion, and cardiomyocytes were prepared to monitor changes in the intracellular concentration of free Ca(2+) ([Ca(2+)](i)). Marked depression in the left ventricular developed pressure and elevation in the left ventricular end-diastolic pressure in I/R hearts were attenuated by treatment with NAC or MPG. Cardiomyocytes obtained from I/R hearts showed an increase in the basal level of [Ca(2+)](i) as well as augmentation of the low Na(+)-induced increase in [Ca(2+)](i), with no change in the KCl-induced increase in [Ca(2+)](i). These I/R-induced alterations in Ca(2+) handling by cardiomyocytes were attenuated by treatment of hearts with NAC or MPG. Furthermore, reduction in the isoproterenol-, ATP-, ouabain-, and caffeine-induced increases in [Ca(2+)](i) in cardiomyocytes from I/R hearts were limited by treatment with NAC or MPG. The increases in the basal [Ca(2+)](i), unlike the KCl-induced increase in [Ca(2+)](i), were fully or partially prevented by both NAC and MPG upon exposing cardiomyocytes to hypoxia-reoxygenation, H(2)O(2), or a mixture of xanthine and xanthine oxidase. These results suggest that improvement in cardiac function of I/R hearts treated with NAC or MPG was associated with attenuation of changes in Ca(2+) handling by cardiomyocytes, and the results support the view that oxidative stress due to oxyradical generation and peroxynitrite formation plays an important role in the development of intracellular Ca(2+) overload in cardiomyocytes as a consequence of I/R injury.

Effects of hyperthermia on intracellular CA/sup 2+/ monitored by digitized video image fluorescence microscopy

International Nuclear Information System (INIS)

Asher, C.R.; Mikkelsen, R.B.

1987-01-01

With digitized video image fluorescence microscopy and the fluorescent Ca/sup 2+/ dye, fuca-2, the authors examined heat effects on intracellular free Ca/sup 2+/, [Ca/sup 2/]/sub f/. HT-29 human colon cancer cells grown on coverslip were equilibrated with 2.0 μM fura-2 in RPMI 1540 (20 0 , 15 min), washed three times and incubated at 20 0 for 1 h. Coverslips were mounted in a Dvorok perfusion chamber sitting within a temperature controlled microscope stage. Fluorescence was monitored at 500 nm by epi-illumination at 385 nm, excitation maximum for free dye, and 340 nm, maximum for Ca/sup 2+/ complexed dye, with a computer controlled filter wheel. The emission intensity ratio, I/sub 340//I/sub 385/, which corrects for dye leakage, photo-bleaching and cell thickness was used to calculate [Ca/sup 2+/]/sub f/. Measurements of 200 cells at 37 0 using a bit pad and mouse to select 0.6 x 0.6 μ cytoplasmi areas indicated 3 populations of cells in terms of [Ca/sup 2+/]/sub f/ (70%, 40-60nM; 15% 70-110nM; 15%, 120-200 nM). Heating to 43 0 for 1 h resulted in an overall decrease in [Ca/sup 2+/]/sub f/ with greater than 90% cells within 30-50 nM. Not all cells responded to heat. Post-incubation for 3 h at 37 0 showed the identical cell distribution; at 24 h, cell distribution was that of non-heated cells. The relationship of these results to cell killing and thermotolerance are not understood, but these results indicated the importance of cell heterogeneity in response to heat

β-Cell Ca(2+) dynamics and function are compromised in aging.

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Barker, Christopher J; Li, Luosheng; Köhler, Martin; Berggren, Per-Olof

2015-01-01

Defects in pancreatic β-cell function and survival are key components in type 2 diabetes (T2D). An age-dependent deterioration in β-cell function has also been observed, but little is known about the molecular mechanisms behind this phenomenon. Our previous studies indicate that the regulation of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) may be critical and that this is dependent on the proper function of the mitochondria. The [Ca(2+)]i dynamics of the pancreatic β-cell are driven by an interplay between glucose-induced influx of extracellular Ca(2+) via voltage-dependent Ca(2+) channels and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-mediated liberation of Ca(2+) from intracellular stores. Our previous work has indicated a direct relationship between disruption of Ins(1,4,5)P3-mediated Ca(2+) regulation and loss of β-cell function, including disturbed [Ca(2+)]i dynamics and compromised insulin secretion. To investigate these processes in aging we used three mouse models, a premature aging mitochondrial mutator mouse, a mature aging phenotype (C57BL/6) and an aging-resistant phenotype (129). Our data suggest that age-dependent impairment in mitochondrial function leads to modest changes in [Ca(2+)]i dynamics in mouse β-cells, particularly in the pattern of [Ca(2+)]i oscillations. These changes are driven by modifications in both PLC/Ins(1,4,5)P3-mediated Ca(2+) mobilization from intracellular stores and decreased β-cell Ca(2+) influx over the plasma membrane. Our findings underscore an important concept, namely that even relatively small, time-dependent changes in β-cell signal-transduction result in compromised insulin release and in a diabetic phenotype. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses

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Jinhua Li

2016-02-01

Full Text Available The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, ultramicrotomy, and scanning transmission X-ray microscopy (STXM. Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.

FREQUENCY CATASTROPHE AND CO-EXISTING ATTRACTORS IN A CELL Ca2+ NONLINEAR OSCILLATION MODEL WITH TIME DELAY*

Institute of Scientific and Technical Information of China (English)

应阳君; 黄祖洽

2001-01-01

Frequency catastrophe is found in a cell Ca2+ nonlinear oscillation model with time delay. The relation of the frequency transition to the time delay is studied by numerical simulations and theoretical analysis. There is a range of parameters in which two kinds of attractors with great frequency differences co-exist in the system. Along with parameter changes, a critical phenomenon occurs and the oscillation frequency changes greatly. This mechanism helps us to deepen the understanding of the complex dynamics of delay systems, and might be of some meaning in cell signalling.

Disruption in connexin-based communication is associated with intracellular Ca²⁺ signal alterations in astrocytes from Niemann-Pick type C mice.

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Pablo J Sáez

Full Text Available Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1⁻/⁻ showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1⁻/⁻ hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1⁻/⁻ astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1⁻/⁻ astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.

Disruption in connexin-based communication is associated with intracellular Ca²⁺ signal alterations in astrocytes from Niemann-Pick type C mice.

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Sáez, Pablo J; Orellana, Juan A; Vega-Riveros, Natalia; Figueroa, Vania A; Hernández, Diego E; Castro, Juan F; Klein, Andrés D; Jiang, Jean X; Zanlungo, Silvana; Sáez, Juan C

2013-01-01

Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1⁻/⁻) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1⁻/⁻ hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1⁻/⁻ astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1⁻/⁻ astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.

The hormesis effect of plasma-elevated intracellular ROS on HaCaT cells

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Szili, Endre J.; Harding, Frances J.; Hong, Sung-Ha; Herrmann, Franziska; Voelcker, Nicolas H.; Short, Robert D.

2015-12-01

We have examined the link between ionized-gas plasma delivery of reactive oxygen species (ROS) to immortalized keratinocyte (HaCaT) cells and cell fate, defined in terms of cell viability versus death. Phospholipid vesicles were used as cell mimics to measure the possible intracellular ROS concentration, [ROSi], delivered by various plasma treatments. Cells were exposed to a helium cold atmospheric plasma (CAP) jet for different plasma exposure times (5-60 s) and gas flow rates (50-1000 ml min-1). Based upon the [ROSi] data we argue that plasma-generated ROS in the cell culture medium can readily diffuse into real cells. Plasma exposure that equated to an [ROSi] in the range of 3.81  ×  10-10-9.47  ×  10-8 M, measured at 1 h after the plasma exposure, resulted in increased cell viability at 72 h; whereas a higher [ROSi] at 1 h decreased cell viability after 72 h of culture. This may be because of the manner in which the ROS are delivered by the plasma: HaCaT cells better tolerate a low ROS flux over an extended plasma exposure period of 1 min, compared to a high flux delivered in a few seconds, although the final [ROSi] may be the same. Our results suggest that plasma stimulation of HaCaT cells follows the principle of hormesis.

Ih tunes theta/gamma oscillations and cross-frequency coupling in an in silico CA3 model.

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Samuel A Neymotin

Full Text Available Ih channels are uniquely positioned to act as neuromodulatory control points for tuning hippocampal theta (4-12 Hz and gamma (25 Hz oscillations, oscillations which are thought to have importance for organization of information flow. contributes to neuronal membrane resonance and resting membrane potential, and is modulated by second messengers. We investigated oscillatory control using a multiscale computer model of hippocampal CA3, where each cell class (pyramidal, basket, and oriens-lacunosum moleculare cells, contained type-appropriate isoforms of . Our model demonstrated that modulation of pyramidal and basket allows tuning theta and gamma oscillation frequency and amplitude. Pyramidal also controlled cross-frequency coupling (CFC and allowed shifting gamma generation towards particular phases of the theta cycle, effected via 's ability to set pyramidal excitability. Our model predicts that in vivo neuromodulatory control of allows flexibly controlling CFC and the timing of gamma discharges at particular theta phases.

How Does the Ca2+-paradox Injury Induce Contracture in the Heart?—A Combined Study of the Intracellular Ca2+ Dynamics and Cell Structures in Perfused Rat Hearts—

International Nuclear Information System (INIS)

Mani, Hiroki; Tanaka, Hideo; Adachi, Tetsuya; Ikegawa, Masaya; Dai, Ping; Fujita, Naohisa; Takamatsu, Tetsuro

2015-01-01

The calcium (Ca 2+ )-paradox injury of the heart, induced by restoration of extracellular Ca 2+ after its short-term depletion, is known to provoke cardiomyocyte contracture. However, undetermined is how the Ca 2+ -paradox provokes such a distinctive presentation of myocytes in the heart. To address this, we imaged sequential intracellular Ca 2+ dynamics and concomitant structures of the subepicardial ventricular myocytes in fluo3-loaded, Langendorff-perfused rat hearts produced by the Ca 2+ paradox. Under rapid-scanning confocal microscopy, repletion of Ca 2+ following its depletion produced high-frequency Ca 2+ waves in individual myocytes with asynchronous localized contractions, resulting in contracture within 10 min. Such alterations of myocytes were attenuated by 5-mM NiCl 2 , but not by verapamil, SEA0400, or combination of ryanodine and thapsigargin, indicating a contribution of non-specific transmembrane Ca 2+ influx in the injury. However, saponin-induced membrane permeabilization of Ca 2+ showed no apparent contracture despite the emergence of high-frequency Ca 2+ waves, indicating an essential role of myocyte-myocyte and myocyte-extracellular matrix (ECM) mechanical connections in the Ca 2+ paradox. In immunohistochemistry Ca 2+ depletion produced separation of the intercalated disc that expresses cadherin and dissipation of β-dystroglycan located along the sarcolemma. Taken together, along with the trans-sarcolemmal Ca 2+ influx, disruption of cell-cell and cell-ECM connections is essential for contracture in the Ca 2+ -paradox injury

ß-Adrenergic stimulation increases RyR2 activity via intracellular Ca2+ and Mg2+ regulation.

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Jiao Li

Full Text Available Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs by intracellular Ca(2+ and Mg(2+ and the role of these changes in SR Ca(2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca(2+] 1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg(2+ and Ca(2+ inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca(2+ activation site were not affected by ß-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1 increasing the activating potency of Ca(2+ binding to the luminal Ca(2+ site and decreasing its affinity for luminal Mg(2+ and 2 decreasing affinity of the low-affinity Ca(2+/Mg(2+ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.

Ca2+ signals, cell membrane disintegration, and activation of TMEM16F during necroptosis.

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Ousingsawat, Jiraporn; Cabrita, Inês; Wanitchakool, Podchanart; Sirianant, Lalida; Krautwald, Stefan; Linkermann, Andreas; Schreiber, Rainer; Kunzelmann, Karl

2017-01-01

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca 2+ . We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT 29 , 16HBE, H441, and L929. Time-dependent changes in intracellular Ca 2+ , cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca 2+ , which was paralleled by the activation of outwardly rectifying Cl - currents, which were typical for TMEM16F/ANO6. Ca 2+ oscillations were due to Ca 2+ release from endoplasmic reticulum, and were independent of extracellular Ca 2+ . The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl - currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.

ß-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

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Li, Jiao; Imtiaz, Mohammad S.; Beard, Nicole A.; Dulhunty, Angela F.; Thorne, Rick; vanHelden, Dirk F.; Laver, Derek R.

2013-01-01

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca2+ and Mg2+ and the role of these changes in SR Ca2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca2+] adrenergic stimulation increased luminal Ca2+ activation of single RyR channels, decreased luminal Mg2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg2+. At cytoplasmic [Ca2+] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg2+ and Ca2+ inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca2+ activation site were not affected by ß-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca2+ binding to the luminal Ca2+ site and decreasing its affinity for luminal Mg2+ and 2) decreasing affinity of the low-affinity Ca2+/Mg2+ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter. PMID:23533585

Hepatic ischemia-reperfusion injury: roles of Ca2+ and other intracellular mediators of impaired bile flow and hepatocyte damage.

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Nieuwenhuijs, Vincent B; De Bruijn, Menno T; Padbury, Robert T A; Barritt, Gregory J

2006-06-01

Liver resection and liver transplantation have been successful in the treatment of liver tumors and end-stage liver disease. This success has led to an expansion in the pool of patients potentially treatable by liver surgery and, in the case of transplantation, to a shortage of liver donors. At present, there are significant numbers of potential candidates for liver resection and liver donation who have fatty livers, are aged, or have livers damaged by chemotherapy. All of these are at high risk for ischemic reperfusion (IR) injury. The aims of this review are to assess current knowledge of the clinical effectiveness of ischemic preconditioning and intermittent ischemia in reducing IR damage in liver surgery; to evaluate the use of bile flow as a sensitive indicator of IR liver damage; and to analyze the molecular mechanisms, especially intracellular Ca2+, involved in IR injury and ischemic preconditioning. It is concluded that bile flow is a sensitive indicator of IR injury. Together with reactive oxygen species (ROS) and other extracellular and intracellular signaling molecules, intracellular Ca2+ in hepatocytes plays a key role in the normal regulation of bile flow and in IR-induced injury and cell death. Ischemic preconditioning is an effective strategy to reduce IR injury but there is considerable scope for improvement, especially in patients with fatty and aged livers. The development of effective new strategies to reduce IR injury will depend on improved understanding of the molecular mechanisms involved, especially by gaining a better perspective of the relative importance of the various intrahepatocyte signaling pathways involved.

Chromospheric oscillations

NARCIS (Netherlands)

Lites, B.W.; Rutten, R.J.; Thomas, J.H.

1995-01-01

We show results from SO/Sacramento Peak data to discuss three issues: (i)--the spatial occurrence of chromospheric 3--min oscillations; (ii)--the validity of Ca II H&K line-center Doppler Shift measurements; (iii)--the signi ?cance of oscillation power and phase at frequencies above 10 mHz.

Activation of sodium channels by α-scorpion toxin, BmK NT1, produced neurotoxicity in cerebellar granule cells: an association with intracellular Ca2+ overloading.

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He, Yuwei; Zou, Xiaohan; Li, Xichun; Chen, Juan; Jin, Liang; Zhang, Fan; Yu, Boyang; Cao, Zhengyu

2017-02-01

Voltage-gated sodium channels (VGSCs) are responsible for the action potential generation in excitable cells including neurons and involved in many physiological and pathological processes. Scorpion toxins are invaluable tools to explore the structure and function of ion channels. BmK NT1, a scorpion toxin from Buthus martensii Karsch, stimulates sodium influx in cerebellar granule cells (CGCs). In this study, we characterized the mode of action of BmK NT1 on the VGSCs and explored the cellular response in CGC cultures. BmK NT1 delayed the fast inactivation of VGSCs, increased the Na + currents, and shifted the steady-state activation and inactivation to more hyperpolarized membrane potential, which was similar to the mode of action of α-scorpion toxins. BmK NT1 stimulated neuron death (EC 50  = 0.68 µM) and produced massive intracellular Ca 2+ overloading (EC 50  = 0.98 µM). TTX abrogated these responses, suggesting that both responses were subsequent to the activation of VGSCs. The Ca 2+ response of BmK NT1 was primary through extracellular Ca 2+ influx since reducing the extracellular Ca 2+ concentration suppressed the Ca 2+ response. Further pharmacological evaluation demonstrated that BmK NT1-induced Ca 2+ influx and neurotoxicity were partially blocked either by MK-801, an NMDA receptor blocker, or by KB-R7943, an inhibitor of Na + /Ca 2+ exchangers. Nifedipine, an L-type Ca 2+ channel inhibitor, slightly suppressed both Ca 2+ response and neurotoxicity. A combination of these three inhibitors abrogated both responses. Considered together, these data ambiguously demonstrated that activation of VGSCs by an α-scorpion toxin was sufficient to produce neurotoxicity which was associated with intracellular Ca 2+ overloading through both NMDA receptor- and Na + /Ca 2+ exchanger-mediated Ca 2+ influx.

CaV3.1 is a tremor rhythm pacemaker in the inferior olive

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Park, Young-Gyun; Park, Hye-Yeon; Lee, C. Justin; Choi, Soonwook; Jo, Seonmi; Choi, Hansol; Kim, Yang-Hann; Shin, Hee-Sup; Llinas, Rodolfo R.; Kim, Daesoo

2010-01-01

The rhythmic motor pathway activation by pacemaker neurons or circuits in the brain has been proposed as the mechanism for the timing of motor coordination, and the abnormal potentiation of this mechanism may lead to a pathological tremor. Here, we show that the potentiation of CaV3.1 T-type Ca2+ channels in the inferior olive contributes to the onset of the tremor in a pharmacological model of essential tremor. After administration of harmaline, 4- to 10-Hz synchronous neuronal activities arose from the IO and then propagated to cerebellar motor circuits in wild-type mice, but those rhythmic activities were absent in mice lacking CaV3.1 gene. Intracellular recordings in brain-stem slices revealed that the CaV3.1-deficient inferior olive neurons lacked the subthreshold oscillation of membrane potentials and failed to trigger 4- to 10-Hz rhythmic burst discharges in the presence of harmaline. In addition, the selective knockdown of CaV3.1 gene in the inferior olive by shRNA efficiently suppressed the harmaline-induced tremor in wild-type mice. A mathematical model constructed based on data obtained from patch-clamping experiments indicated that harmaline could efficiently potentiate CaV3.1 channels by changing voltage-dependent responsiveness in the hyperpolarizing direction. Thus, CaV3.1 is a molecular pacemaker substrate for intrinsic neuronal oscillations of inferior olive neurons, and the potentiation of this mechanism can be considered as a pathological cause of essential tremor. PMID:20498062

Local Optogenetic Induction of Fast (20-40 Hz Pyramidal-Interneuron Network Oscillations in the In Vitro and In Vivo CA1 Hippocampus: Modulation by CRF and Enforcement of Perirhinal Theta Activity

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Julien eDine

2016-04-01

Full Text Available The neurophysiological processes that can cause theta-to-gamma frequency range (4-80 Hz network oscillations in the rhinal cortical-hippocampal system and the potential connectivity-based interactions of such forebrain rhythms are a topic of intensive investigation. Here, using selective Channelrhodopsin-2 (ChR2 expression in mouse forebrain glutamatergic cells, we were able to locally, temporally precisely, and reliably induce fast (20-40 Hz field potential oscillations in hippocampal area CA1 in vitro (at 25°C and in vivo (i.e., slightly anaesthetized NEX-Cre-ChR2 mice. As revealed by pharmacological analyses and patch-clamp recordings from pyramidal cells and GABAergic interneurons in vitro, these light-triggered oscillations can exclusively arise from sustained suprathreshold depolarization (~200 ms or longer and feedback inhibition of CA1 pyramidal neurons, as being mandatory for prototypic pyramidal-interneuron network (P-I oscillations. Consistently, the oscillations comprised rhythmically occurring population spikes (generated by pyramidal cells and their frequency increased with increasing spectral power. We further demonstrate that the optogenetically driven CA1 oscillations, which remain stable over repeated evocations, are impaired by the stress hormone corticotropin-releasing factor (CRF, 125 nM in vitro and, even more remarkably, found that they are accompanied by concurrent states of enforced theta activity in the memory-associated perirhinal cortex (PrC in vivo. The latter phenomenon most likely derives from neurotransmission via a known, but poorly studied excitatory CA1PrC pathway. Collectively, our data provide evidence for the existence of a prototypic (CRF-sensitive P-I gamma rhythm generator in area CA1 and suggest that CA1 P-I oscillations can rapidly up-regulate theta activity strength in hippocampus-innervated rhinal networks, at least in the PrC.

Local Optogenetic Induction of Fast (20-40 Hz) Pyramidal-Interneuron Network Oscillations in the In Vitro and In Vivo CA1 Hippocampus: Modulation by CRF and Enforcement of Perirhinal Theta Activity.

Science.gov (United States)

Dine, Julien; Genewsky, Andreas; Hladky, Florian; Wotjak, Carsten T; Deussing, Jan M; Zieglgänsberger, Walter; Chen, Alon; Eder, Matthias

2016-01-01

The neurophysiological processes that can cause theta-to-gamma frequency range (4-80 Hz) network oscillations in the rhinal cortical-hippocampal system and the potential connectivity-based interactions of such forebrain rhythms are a topic of intensive investigation. Here, using selective Channelrhodopsin-2 (ChR2) expression in mouse forebrain glutamatergic cells, we were able to locally, temporally precisely, and reliably induce fast (20-40 Hz) field potential oscillations in hippocampal area CA1 in vitro (at 25°C) and in vivo (i.e., slightly anesthetized NEX-Cre-ChR2 mice). As revealed by pharmacological analyses and patch-clamp recordings from pyramidal cells and GABAergic interneurons in vitro, these light-triggered oscillations can exclusively arise from sustained suprathreshold depolarization (~200 ms or longer) and feedback inhibition of CA1 pyramidal neurons, as being mandatory for prototypic pyramidal-interneuron network (P-I) oscillations. Consistently, the oscillations comprised rhythmically occurring population spikes (generated by pyramidal cells) and their frequency increased with increasing spectral power. We further demonstrate that the optogenetically driven CA1 oscillations, which remain stable over repeated evocations, are impaired by the stress hormone corticotropin-releasing factor (CRF, 125 nM) in vitro and, even more remarkably, found that they are accompanied by concurrent states of enforced theta activity in the memory-associated perirhinal cortex (PrC) in vivo. The latter phenomenon most likely derives from neurotransmission via a known, but poorly studied excitatory CA1→PrC pathway. Collectively, our data provide evidence for the existence of a prototypic (CRF-sensitive) P-I gamma rhythm generator in area CA1 and suggest that CA1 P-I oscillations can rapidly up-regulate theta activity strength in hippocampus-innervated rhinal networks, at least in the PrC.

Subthreshold membrane potential oscillations in inferior olive neurons are dynamically regulated by P/Q- and T-type calcium channels: a study in mutant mice.

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Choi, Soonwook; Yu, Eunah; Kim, Daesoo; Urbano, Francisco J; Makarenko, Vladimir; Shin, Hee-Sup; Llinás, Rodolfo R

2010-08-15

The role of P/Q- and T-type calcium channels in the rhythmic oscillatory behaviour of inferior olive (IO) neurons was investigated in mutant mice. Mice lacking either the CaV2.1 gene of the pore-forming alpha1A subunit for P/Q-type calcium channel, or the CaV3.1 gene of the pore-forming alpha1G subunit for T-type calcium channel were used. In vitro intracellular recording from IO neurons reveals that the amplitude and frequency of sinusoidal subthreshold oscillations (SSTOs) were reduced in the CaV2.1-/- mice. In the CaV3.1-/- mice, IO neurons also showed altered patterns of SSTOs and the probability of SSTO generation was significantly lower (15%, 5 of 34 neurons) than that of wild-type (78%, 31 of 40 neurons) or CaV2.1-/- mice (73%, 22 of 30 neurons). In addition, the low-threshold calcium spike and the sustained endogenous oscillation following rebound potentials were absent in IO neurons from CaV3.1-/- mice. Moreover, the phase-reset dynamics of oscillatory properties of single neurons and neuronal clusters in IO were remarkably altered in both CaV2.1-/- and CaV3.1-/- mice. These results suggest that both alpha1A P/Q- and alpha1G T-type calcium channels are required for the dynamic control of neuronal oscillations in the IO. These findings were supported by results from a mathematical IO neuronal model that incorporated T and P/Q channel kinetics.

Quantum oscillations in antiferromagnetic CaFe2As2 on the brink of superconductivity

International Nuclear Information System (INIS)

Harrison, N; McDonald, R D; Mielke, C H; Bauer, E D; Ronning, F; Thompson, J D

2009-01-01

We report quantum oscillation measurements on CaFe 2 As 2 under strong magnetic fields-recently reported to become superconducting under pressures of as little as a kilobar. The largest observed carrier pocket occupies less than 0.05% of the paramagnetic Brillouin zone volume-consistent with Fermi surface reconstruction caused by antiferromagnetism. On comparing several alkaline earth AFe 2 As 2 antiferromagnets (with A = Ca, Sr and Ba), the dependences of the Fermi surface cross-sectional area F α and the effective mass m α * of the primary observed pocket on the antiferromagnetic/structural transition temperature T s are both found to be consistent with the case for quasiparticles in a conventional spin-density wave model. These findings suggest that the recently proposed strain-enhanced superconductivity in these materials occurs within a broadly conventional spin-density wave phase. (fast track communication)

Different types of bursting calcium oscillations in non-excitable cells

International Nuclear Information System (INIS)

Perc, Matjaz; Marhl, Marko

2003-01-01

In the paper different types of bursting Ca 2+ oscillations are presented. We analyse bursting behaviour in four recent mathematical models for Ca 2+ oscillations in non-excitable cells. Separately, regular, quasi-periodic, and chaotic bursting Ca 2+ oscillations are classified into several subtypes. The classification is based on the dynamics of separated fast and slow subsystems, the so-called fast-slow burster analysis. For regular bursting Ca 2+ oscillations two types of bursting are specified: Point-Point and Point-Cycle bursting. In particular, the slow passage effect, important for the Hopf-Hopf and SubHopf-SubHopf bursting subtypes, is explained by local divergence calculated for the fast subsystem. Quasi-periodic bursting Ca 2+ oscillations can be found in only one of the four studied mathematical models and appear via a homoclinic bifurcation with a homoclinic torus structure. For chaotic bursting Ca 2+ oscillations, we found that bursting patterns resulting from the period doubling root to chaos considerably differ from those appearing via intermittency and have to be treated separately. The analysis and classification of different types of bursting Ca 2+ oscillations provides better insight into mechanisms of complex intra- and intercellular Ca 2+ signalling. This improves our understanding of several important biological phenomena in cellular signalling like complex frequency-amplitude signal encoding and synchronisation of intercellular signal transduction between coupled cells in tissue

Chelation of intracellular calcium blocks insulin action in the adipocyte

International Nuclear Information System (INIS)

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

1987-01-01

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

Auranofin, an Anti-Rheumatic Gold Compound, Modulates Apoptosis by Elevating the Intracellular Calcium Concentration ([Ca{sup 2+}]{sub i}) in MCF-7 Breast Cancer Cells

Energy Technology Data Exchange (ETDEWEB)

Varghese, Elizabeth; Büsselberg, Dietrich, E-mail: dib2015@qatar-med.cornell.edu [Weil Cornell Medical College in Qatar, Qatar Foundation-Education City, P.O. Box 24144 Doha (Qatar)

2014-11-06

Auranofin, a transition metal complex is used for the treatment of rheumatoid arthritis but is also an effective anti-cancer drug. We investigate the effects of Auranofin in inducing cell death by apoptosis and whether these changes are correlated to changes of intracellular calcium concentration ([Ca{sup 2+}]{sub i}) in breast cancer cells (MCF-7). Cytotoxicity of Auranofin was evaluated using MTS assay and the Trypan blue dye exclusion method. With fluorescent dyes SR-FLICA and 7-AAD apoptotic death and necrotic death were differentiated by Flow cytometry. A concentration dependent decrease in the viability occurred and cells were shifted to the apoptotic phase. Intracellular calcium ([Ca{sup 2+}]{sub i}) was recorded using florescence microscopy and a calcium sensitive dye (Fluo-4 AM) with a strong negative correlation (r = −0.713) to viability. Pharmacological modulators 2-APB (50 μM), Nimodipine (10 μM), Caffeine (10 mM), SKF 96365(20 μM) were used to modify calcium entry and release. Auranofin induced a sustained increase of [Ca{sup 2+}]{sub i} in a concentration and time dependent manner. The use of different blockers of calcium channels did not reveal the source for the rise of [Ca{sup 2+}]{sub i}. Overall, elevation of [Ca{sup 2+}]{sub i} by Auranofin might be crucial for triggering Ca{sup 2+}-dependent apoptotic pathways. Therefore, in anti-cancer therapy, modulating [Ca{sup 2+}]{sub i} should be considered as a crucial factor for the induction of cell death in cancer cells.

Neomysin inhibits Ca2+-stimulated phosphatidylinositol hydrolysis and protects cultured rat cardiomyocytes from Ca2+-dependent cell injury

International Nuclear Information System (INIS)

Babson, J.R.; Dougherty, J.M.

1991-01-01

Exposure of cultured rat cardiomyocytes to ionomycin and extracellular Ca 2+ leads to a rapid, sustained increase in intracellular free Ca 2+ as monitored by Ca 2+ -dependent phosphorylase a activation and to a subsequent loss of cardiomyocyte viability as determined by lactate dehydrogenase (LDH) leakage. The intracellular free Ca 2+ increase coincided with a rapid hydrolysis of phosphatidylinositol that preceded cell death. Phosphatidylinositol hydrolysis was monitored by the release of radiolabeled phosphoinositides from cardiomyocytes prelabeled with [2- 3 H]-myo-inositol. Neomycin, a known inhibitor of phospholipase C, inhibited the phosphatidylinositol hydrolysis and markedly reduced the extent of cell injury. Inhibitors of other Ca 2+ -activated processes, including intracellular proteases and phospholipase A 2 , had no effect on ionomycin-mediated cell injury. These data suggest that ionomycin-induced Ca 2+ -dependent cell injury in cultured cardiomyocytes may be due in part to the stimulation of phosphatidylinositol hydrolysis, presumably catalyzed by a Ca 2+ -dependent phospholipase C

Cyanohydrin reactions enhance glycolytic oscillations in yeast

DEFF Research Database (Denmark)

Hald, Bjørn Olav; Nielsen, Astrid Gram; Tortzen, Christian

2015-01-01

Synchronous metabolic oscillations can be induced in yeast by addition of glucose and removal of extracellular acetaldehyde (ACAx). Compared to other means of ACAx removal, cyanide robustly induces oscillations, indicating additional cyanide reactions besides ACA to lactonitrile conversion. Here......: a) by reducing [ACAx] relative to oscillation amplitude, b) by targeting multiple intracellular carbonyl compounds during fermentation, and c) by acting as a phase resetting stimulus....

Vasoinhibins Prevent Bradykinin-Stimulated Endothelial Cell Proliferation by Inactivating eNOS via Reduction of both Intracellular Ca2+ Levels and eNOS Phosphorylation at Ser1179

Directory of Open Access Journals (Sweden)

Carmen Clapp

2011-07-01

Full Text Available Vasoinhibins, a family of antiangiogenic peptides derived from prolactin proteolysis, inhibit the vascular effects of several proangiogenic factors, including bradykinin (BK. Here, we report that vasoinhibins block the BK-induced proliferation of bovine umbilical vein endothelial cells. This effect is mediated by the inactivation of endothelial nitric oxide synthase (eNOS, as the NO donor DETA-NONOate reverted vasoinhibin action. It is an experimentally proven fact that the elevation of intracellular Ca2+ levels ([Ca2+]i upon BK stimulation activates eNOS, and vasoinhibins blocked the BK-mediated activation of phospholipase C and the formation of inositol 1,4,5-triphosphate leading to a reduced release of Ca2+ from intracellular stores. The [Ca2+]i rise evoked by BK also involves the influx of extracellular Ca2+ via canonical transient receptor potential (TRPC channels. Vasoinhibins likely interfere with TRPC-mediated Ca2+ entry since La3+, which is an enhancer of TRPC4 and TRPC5 channel activity, prevented vasoinhibins from blocking the stimulation by BK of endothelial cell NO production and proliferation, and vasoinhibins reduced the BK-induced increase of TRPC5 mRNA expression. Finally, vasoinhibins prevented the BK-induced phosphorylation of eNOS at Ser1179, a post-translational modification that facilitates Ca2+-calmodulin activation of eNOS. Together, our data show that vasoinhibins, by lowering NO production through the inhibition of both [Ca2+]i mobilization and eNOS phosphorylation, prevent the BK-induced stimulation of endothelial cell proliferation. Thus, vasoinhibins help to regulate BK effects on angiogenesis and vascular homeostasis.

Ca2+ influx insensitive to organic Ca2+ entry blockers contributes to noradrenaline-induced contractions of the isolated guinea pig aorta

NARCIS (Netherlands)

Gouw, M. A.; Wilffert, B.; Wermelskirchen, D.; van Zwieten, P. A.

1990-01-01

We determined the contribution of intracellular Ca2+ to the noradrenaline (NA, 3 X 10(-5) mmol/l)-induced contraction of the isolated guinea pig aorta. Since only about 55% of the NA-induced contraction could be attributed to intracellular Ca2+ release, we assumed that a Ca2+ influx component

Transverse Oscillations in Slender Ca ii H Fibrils Observed with Sunrise/SuFI

Energy Technology Data Exchange (ETDEWEB)

Jafarzadeh, S. [Institute of Theoretical Astrophysics, University of Oslo, P.O. Box 1029 Blindern, NO-0315 Oslo (Norway); Solanki, S. K.; Gafeira, R.; Noort, M. van; Barthol, P.; Gandorfer, A.; Gizon, L.; Hirzberger, J.; Riethmüller, T. L. [Max Planck Institute for Solar System Research, Justus-von-Liebig-Weg 3, D-37077 Göttingen (Germany); Rodríguez, J. Blanco [Grupo de Astronomía y Ciencias del Espacio, Universidad de Valencia, E-46980 Paterna, Valencia (Spain); Iniesta, J. C. del Toro; Suárez, D. Orozco [Instituto de Astrofísica de Andalucía (CSIC), Apartado de Correos 3004, E-18080 Granada (Spain); Knölker, M. [High Altitude Observatory, National Center for Atmospheric Research, P.O. Box 3000, Boulder, CO 80307-3000 (United States); Schmidt, W., E-mail: shahin.jafarzadeh@astro.uio.no [Kiepenheuer-Institut für Sonnenphysik, Schöneckstr. 6, D-79104 Freiburg (Germany)

2017-03-01

We present observations of transverse oscillations in slender Ca ii H fibrils (SCFs) in the lower solar chromosphere. We use a 1 hr long time series of high- (spatial and temporal-) resolution seeing-free observations in a 1.1 Å wide passband covering the line core of Ca ii H 3969 Å from the second flight of the Sunrise balloon-borne solar observatory. The entire field of view, spanning the polarity inversion line of an active region close to the solar disk center, is covered with bright, thin, and very dynamic fine structures. Our analysis reveals the prevalence of transverse waves in SCFs with median amplitudes and periods on the order of 2.4 ± 0.8 km s{sup −1} and 83 ± 29 s, respectively (with standard deviations given as uncertainties). We find that the transverse waves often propagate along (parts of) the SCFs with median phase speeds of 9 ± 14 km s{sup −1}. While the propagation is only in one direction along the axis in some of the SCFs, propagating waves in both directions, as well as standing waves are also observed. The transverse oscillations are likely Alfvénic and are thought to be representative of magnetohydrodynamic kink waves. The wave propagation suggests that the rapid high-frequency transverse waves, often produced in the lower photosphere, can penetrate into the chromosphere with an estimated energy flux of ≈15 kW m{sup −2}. Characteristics of these waves differ from those reported for other fibrillar structures, which, however, were observed mainly in the upper solar chromosphere.

Mesurements of intracellular ATP provide new insight into the regulation of glycolysis in the yeast Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Ytting, Cecilie Karkov; Fuglsang, Anja Thoe; Hiltunen, J. Kalervo

2012-01-01

Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F0F1 ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating...... of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal...... activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose...

SEA0400 fails to alter the magnitude of intracellular Ca2+ transients and contractions in Langendorff-perfused guinea pig heart.

Science.gov (United States)

Szentandrássy, Norbert; Birinyi, Péter; Szigeti, Gyula; Farkas, Attila; Magyar, János; Tóth, András; Csernoch, László; Varró, András; Nánási, Péter P

2008-07-01

SEA0400 is a recently developed inhibitor of the Na+/Ca2+ exchanger (NCX) shown to suppress both forward and reverse mode operation of NCX. Present experiments were designed to study the effect of partial blockade of NCX on Ca handling and contractility in Langendorff-perfused guinea pig hearts loaded with the fluorescent Ca-sensitive dye fura-2. Left ventricular pressure and intracellular calcium concentration ([Ca2+]i) were synchronously recorded before and after cumulative superfusion with 0.3 and 1 muM SEA0400. SEA0400 caused no significant change in the systolic and diastolic values of left ventricular pressure and [Ca2+]i. Accordingly, pulse pressure and amplitude of the [Ca2+]i transient also remained unchanged in the presence of SEA0400. SEA0400 had no influence either on the time required to reach peak values of pressure and [Ca2+)]i or on half relaxation time. On the other hand, both 0.3 and 1 microM SEA0400 significantly increased the decay time constant of [Ca2+]i transients, obtained by fitting its descending limb between 30% and 90% of relaxation, from 127 +/- 7 to 165 +/- 7 and 177 +/- 14 ms, respectively (P hearts, rat hearts responded to SEA0400 treatment with increased [Ca2+]i transients and contractility. These interspecies differences observed in the effect of SEA0400 can be explained by the known differences in calcium handling between the two species.

Ca2+influx insensitive to organic Ca2+entry blockers contributes to noradrenaline-induced contractions of the isolated guinea pig aorta

NARCIS (Netherlands)

Gouw, M.A.M.; Wilffert, B.; Wermelskirchen, D.; Van Zwieten, P.A.

1990-01-01

We determined the contribution of intracellular Ca2+to the noradrenaline (NA, 3 x 10-5mmol/l)-induced contraction of the isolated guinea pig aorta. Since only about 55% of the NA-induced contraction could be attributed to intracellular Ca2+release, we assumed that a Ca2+influx component contributes

Physiology of spontaneous [Ca2+](i) oscillations in the isolated vasopressin and oxytocin neurones of the rat supraoptic nucleus

Czech Academy of Sciences Publication Activity Database

Kortus, Štěpán; Srinivasan, Ch.; Forostyak, O.; Ueta, Y.; Syková, E.; Chvátal, A.; Zápotocký, Martin; Verkhratsky, A.; Dayanithi, G.

2016-01-01

Roč. 59, č. 6 (2016), s. 280-288 ISSN 0143-4160 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : magnocellular neurosecretory cells * supraoptic nucleus * vasopressin * oxytocin * transgenic rats * Ca2+ oscillations Subject RIV: FH - Neurology Impact factor: 3.707, year: 2016

Bauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration.

Science.gov (United States)

Bilgin, Mehmet; Neuhof, Christiane; Doerr, Oliver; Benscheid, Utz; Andrade, Sheila S; Most, Astrid; Abdallah, Yaser; Parahuleva, Mariana; Guenduez, Dursun; Oliva, Maria L; Erdogan, Ali

2010-12-01

Proteinase inhibitors, isolated from different types of Bauhinia, have an effect on apoptosis, angiogenesis and inflammation. The Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from Trypanosoma cruzi. Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. The aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). For proliferation experiments, HUVEC were incubated with BbCI (10-100 μmol/L) for 48 h. The proliferation was detected by cell counting with a Neubauer chamber. The effect of BbCI (10-100 μM) on the membrane potential was measured with the fluorescence dye DiBAC4(3) and the effect on [Ca+2]i with the fluorescence probe Fluo-3 AM. The change of the fluorescence intensity was determined with a GENios plate reader (Tecan). The experiments showed that BbCI (10-100 μmol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 μmol/L (35.1±1.8% as compared to control (p≤0.05; n=45)). As compared to the control, the addition of BbCI (100 μmol/L) caused a significant increase of systolic Ca2+ of 28.4±5.0% after 30 min incubation. HUVEC treatment with BbCI (100 μmol/L) showed a weak but significant decrease of the membrane potential of 9.5±0.9% as compared to control (p≤0.05; n=80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca2+ concentration and the membrane potential.

Differential inhibitory response to telcagepant on αCGRP induced vasorelaxation and intracellular Ca(2+) levels in the perfused and non-perfused isolated rat middle cerebral artery

DEFF Research Database (Denmark)

Erdling, André; Sheykhzade, Majid; Edvinsson, Lars

2017-01-01

BACKGROUND: Calcitonin gene-related peptide (CGRP) is one of the most potent endogenous vasodilators identified to date. The present study elucidates the differential interaction of CGRP, its receptor and the effect of the CGRP-receptor antagonist telcagepant on intracellular Ca(2+) -levels...... and tension in rat middle cerebral arteries (MCA) by pressurized arteriography, FURA-2/wire myography and immunohistochemistry. METHODS: A pressurized arteriograph system was used to evaluate changes in MCA tension when subjected to CGRP and/or telcagepant. Intracellular calcium levels were evaluated using......, while abluminal telcagepant inhibited the relaxation (10(-6) M). Using the FURA-2 method in combination with wire myography we observed that αCGRP reduced intracellular calcium levels and in parallel the vascular tone. Telcagepant (10(-6) M) inhibited both vasorelaxation and drop in intracellular...

Brain-derived neurotrophic factor (BDNF) induces sustained intracellular Ca2+ elevation through the up-regulation of surface transient receptor potential 3 (TRPC3) channels in rodent microglia.

Science.gov (United States)

Mizoguchi, Yoshito; Kato, Takahiro A; Seki, Yoshihiro; Ohgidani, Masahiro; Sagata, Noriaki; Horikawa, Hideki; Yamauchi, Yusuke; Sato-Kasai, Mina; Hayakawa, Kohei; Inoue, Ryuji; Kanba, Shigenobu; Monji, Akira

2014-06-27

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca(2+) elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Hierarchic stochastic modelling applied to intracellular Ca(2+ signals.

Directory of Open Access Journals (Sweden)

Gregor Moenke

Full Text Available Important biological processes like cell signalling and gene expression have noisy components and are very complex at the same time. Mathematical analysis of such systems has often been limited to the study of isolated subsystems, or approximations are used that are difficult to justify. Here we extend a recently published method (Thurley and Falcke, PNAS 2011 which is formulated in observable system configurations instead of molecular transitions. This reduces the number of system states by several orders of magnitude and avoids fitting of kinetic parameters. The method is applied to Ca(2+ signalling. Ca(2+ is a ubiquitous second messenger transmitting information by stochastic sequences of concentration spikes, which arise by coupling of subcellular Ca(2+ release events (puffs. We derive analytical expressions for a mechanistic Ca(2+ model, based on recent data from live cell imaging, and calculate Ca(2+ spike statistics in dependence on cellular parameters like stimulus strength or number of Ca(2+ channels. The new approach substantiates a generic Ca(2+ model, which is a very convenient way to simulate Ca(2+ spike sequences with correct spiking statistics.

Primordial oscillations in life: Direct observation of glycolytic oscillations in individual HeLa cervical cancer cells

Science.gov (United States)

Amemiya, Takashi; Shibata, Kenichi; Itoh, Yoshihiro; Itoh, Kiminori; Watanabe, Masatoshi; Yamaguchi, Tomohiko

2017-10-01

We report the first direct observation of glycolytic oscillations in HeLa cervical cancer cells, which we regard as primordial oscillations preserved in living cells. HeLa cells starved of glucose or both glucose and serum exhibited glycolytic oscillations in nicotinamide adenine dinucleotide (NADH), exhibiting asynchronous intercellular behaviors. Also found were spatially homogeneous and inhomogeneous intracellular NADH oscillations in the individual cells. Our results demonstrate that starved HeLa cells may be induced to exhibit glycolytic oscillations by either high-uptake of glucose or the enhancement of a glycolytic pathway (Crabtree effect or the Warburg effect), or both. Their asynchronous collective behaviors in the oscillations were probably due to a weak intercellular coupling. Elucidation of the relationship between the mechanism of glycolytic dynamics in cancer cells and their pathophysiological characteristics remains a challenge in future.

A single amino acid residue controls Ca2+ signaling by an octopamine receptor from Drosophila melanogaster.

Science.gov (United States)

Hoff, Max; Balfanz, Sabine; Ehling, Petra; Gensch, Thomas; Baumann, Arnd

2011-07-01

Rhythmic activity of cells and cellular networks plays an important role in physiology. In the nervous system oscillations of electrical activity and/or second messenger concentrations are important to synchronize neuronal activity. At the molecular level, rhythmic activity can be initiated by different routes. We have recently shown that an octopamine-activated G-protein-coupled receptor (GPCR; DmOctα1Rb, CG3856) from Drosophila initiates Ca(2+) oscillations. Here, we have unraveled the molecular basis of cellular Ca(2+) signaling controlled by the DmOctα1Rb receptor using a combination of pharmacological intervention, site-directed mutagenesis, and functional cellular Ca(2+) imaging on heterologously expressed receptors. Phosphorylation of a single amino acid residue in the third intracellular loop of the GPCR by PKC is necessary and sufficient to desensitize the receptor. From its desensitized state, DmOctα1Rb is resensitized by dephosphorylation, and a new Ca(2+) signal occurs on octopamine stimulation. Our findings show that transient changes of the receptor's surface profile have a strong effect on its physiological signaling properties. We expect that the detailed knowledge of DmOctα1Rb-dependent signal transduction fosters the identification of specific drugs that can be used for GPCR-mediated pest control, since octopamine serves important physiological and behavioral functions in arthropods.

Intracellular Ca(2+) release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal.

Science.gov (United States)

Zhu, Mei Hong; Sung, Tae Sik; O'Driscoll, Kate; Koh, Sang Don; Sanders, Kenton M

2015-04-15

Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca(2+)-activated Cl(-) channels. We investigated the hypothesis that the Ca(2+) responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca(2+) stores. ICC, obtained from the small intestine of Kit(+/copGFP) mice, were studied under voltage and current clamp to determine the effects of blocking Ca(2+) uptake into stores and release of Ca(2+) via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca(2+) concentration, suggesting that pacemaker activity depends on Ca(2+) dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca(2+) from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC. Copyright © 2015 the American Physiological Society.

Intracellular Ca2+ release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal

Science.gov (United States)

Zhu, Mei Hong; Sung, Tae Sik; O'Driscoll, Kate; Koh, Sang Don

2015-01-01

Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca2+-activated Cl− channels. We investigated the hypothesis that the Ca2+ responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca2+ stores. ICC, obtained from the small intestine of Kit+/copGFP mice, were studied under voltage and current clamp to determine the effects of blocking Ca2+ uptake into stores and release of Ca2+ via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca2+ concentration, suggesting that pacemaker activity depends on Ca2+ dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca2+ from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC. PMID:25631870

Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation.

Science.gov (United States)

Hayashi, Kentaro; Yamamoto, Takamasa S; Ueno, Naoto

2018-02-05

During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca 2+ levels in the protrusion-forming leading cells are markedly higher than those of the following cells and the axial mesoderm cells. We also showed that inhibiting the intracellular Ca 2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca 2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca 2+ signals play an essential role in the active cell migration during gastrulation.

Physiology of spontaneous [Ca2+](i) oscillations in the isolated vasopressin and oxytocin neurones of the rat supraoptic nucleus

Czech Academy of Sciences Publication Activity Database

Kortus, Štěpán; Srinivasan, Chinnapaiyan; Forostyak, Oksana; Ueta, Y.; Syková, Eva; Chvátal, Alexandr; Zápotocký, M.; Verkhratsky, A.; Dayanithi, Govindan

2016-01-01

Roč. 6, č. 59 (2016), s. 280-288 ISSN 0143-4160 R&D Projects: GA ČR(CZ) GA14-34077S; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : Ca(2+) oscillations * electrical activity * enhanced green fluorescence protein * fluorescence spectrofluorimetry Subject RIV: FH - Neurology Impact factor: 3.707, year: 2016

Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Poulsen, Allan K.; Andersen, Ann Zahle; Brasen, Jens Christian

2008-01-01

, while mitochondrial membrane potential was measured using the fluorescent dye DiOC(2)(3). The results show that, as opposed to NADH and other intermediates in glycolysis, intracellular glucose is not oscillating. Furthermore, oscillations in NADH and membrane potential are inhibited by the ATP...

The other side of cardiac Ca2+ signaling: transcriptional control

Directory of Open Access Journals (Sweden)

Alejandro eDomínguez-Rodríquez

2012-11-01

Full Text Available Ca2+ is probably the most versatile signal transduction element used by all cell types. In the heart, it is essential to activate cellular contraction in each heartbeat. Nevertheless Ca2+ is not only a key element in excitation-contraction coupling (EC coupling, but it is also a pivotal second messenger in cardiac signal transduction, being able to control processes such as excitability, metabolism, and transcriptional regulation. Regarding the latter, Ca2+ activates Ca2+-dependent transcription factors by a process called excitation-transcription coupling (ET coupling. ET coupling is an integrated process by which the common signaling pathways that regulate EC coupling activate transcription factors. Although ET coupling has been extensively studied in neurons and other cell types, less is known in cardiac muscle. Some hints have been found in studies on the development of cardiac hypertrophy, where two Ca2+-dependent enzymes are key actors: Ca2+/Calmodulin kinase II (CaMKII and phosphatase calcineurin, both of which are activated by the complex Ca2+/ /Calmodulin. The question now is how ET coupling occurs in cardiomyocytes, where intracellular Ca2+ is continuously oscillating. In this focused review, we will draw attention to location of Ca2+ signaling: intranuclear ([Ca2+]n or cytoplasmic ([Ca2+]c, and the specific ionic channels involved in the activation of cardiac ET coupling. Specifically, we will highlight the role of the 1,4,5 inositol triphosphate receptors (IP3Rs in the elevation of [Ca2+]n levels, which are important to locally activate CaMKII, and the role of transient receptor potential channels canonical (TRPCs in [Ca2+]c, needed to activate calcineurin.

Effects of vitamin D metabolites on cellular Ca2+ and on Ca transport in primary cultures of bone cells.

Science.gov (United States)

Eilam, Y; Szydel, N; Harell, A

1980-09-01

Both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) exerted direct effects on Ca2+ transport and accumulation in primary cultures of bone cells. The following changes were recorded. (1) A significant decrease in the amount of intracellular exchangeable Ca2+. (2) A marked increase in the rate constants of efflux from the 'slow'-turnover intracellular Ca pool. (3) A marked increase in the 'initial rate' of Ca influx into the cells. Thus, vitamin D metabolites caused an increase in the turnover of Ca2+ in bone cells and altered the steady-stae level of intracellular exchangeable Ca2+. Whereas the changes in the rate of efflux were abolished in the presence of inhibitors of protein synthesis, the increase in the rate of influx was not sensitive to these inhibitors. It is suggested that the changes in the two fluxes were mediated by different mechanisms and that the changes in influx were due to a direct effect of vitamin D metabolites on the cellular membranes.

Mechanism of the negative force-frequency relationship in physiologically intact rat ventricular myocardium. Studies by intracellular Ca2+ monitor with iodo-1 and by 31P-nuclear magnetic resonance spectroscopy

International Nuclear Information System (INIS)

Morii, Isao; Kihara, Yasuki; Sasayama, Shigetake; Konishi, Takashi; Inubushi, Toshiro.

1996-01-01

We studied the subcellular mechanisms of the negative force-frequency relationship in rat myocardium by measuring intracellular Ca 2+ transients by indo-1 fluorometry and intracellular pH (pH i ) and phosphate compounds with 31 P-nuclear magnetic resonance (NMR). The data were compared with those from guinea pig hearts, which show a positive force-frequency relationship. By increasing the pacing rate from 3 Hz to 5 Hz, the peak positive first derivative of left ventricular pressure (LVdP/dt) in rat heart decreased by 10±1% (n=6). In contrast to this negative inotropic response, simultaneously measured peak Ca 2+ transients increased by 6±1%. Guinea pig heart (n=6) showed an increase in peak positive LVdP/dt (33±1%) which was associated with an increase in peak Ca 2+ transients (8±1%). Under equivalent experimental conditions in an NMR spectrometer, this increase in the pacing rate did not affect intracellular levels of phosphate compounds in either rat (n=6) or guinea pig heart (n=6). In contrast, pH i showed a decrease of 0.031±0.006 pH units in rat heart, while no changes were observed in guinea pig heart. These results suggest that in physiological rat myocardium, pH i is susceptible to changes in the stimulus frequency and may affect the Ca 2+ -responsiveness of contractile proteins, which results in the negative force-frequency relationship. (author)

The effect of theophylline on the labelling of nitracellular myocardial calcium with 45Ca

International Nuclear Information System (INIS)

Sausen, F.H.

1973-01-01

The effect of theophylline of varying concentration on the labelling of intracellular myocardial calcium was investigated by isotope tests with radioactively labelled Ca ( 45 Ca) in isolated, electrically stimulated left auricles of guinea pigs. The preparates were incubated in a 45 Ca solution for 60 minutes, and activity uptake and intracellular Ca concentration were determined. In contrast to earlier investigations, the extracellular Ca was removed after charge by rinsing the resting auricles with inactive, Ca ++ - and Na + -free choline chloride solution. Under controlled conditions, the intercellular Ca was found to be 45 Ca-labelled by about 36% theophylline in 'therapeutical' concentration (5 x 10 -4 g/ml) induced a significant increase of the labelled Ca fraction of 16.9% as compared to the controls. 'Toxic' theophylline concentrations, too, lead to a significantly higher absorption of 45 Ca. The interchangeable intracellular Ca fraction increased by 11.7% and 10.3% as compared to the untreated preparates. The findings are discussed with regard to earlier investigations on the influence of methylxanthines on the Ca metabolism of the heart. The author assumes that the positively inotropic effect of theophylline and the theophylline-induced contracture may be ascribed to a shift in the intracellular Ca distribution towards ionized Ca. This shift could be explained by a suppression of Ca rebinding in intracellular Ca storage places or by Ca release from these structures. (orig./AK) [de

A mathematical model of calcium dynamics in HSY cells.

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Jung Min Han

2017-02-01

Full Text Available Saliva is an essential part of activities such as speaking, masticating and swallowing. Enzymes in salivary fluid protect teeth and gums from infectious diseases, and also initiate the digestion process. Intracellular calcium (Ca2+ plays a critical role in saliva secretion and regulation. Experimental measurements of Ca2+ and inositol trisphosphate (IP3 concentrations in HSY cells, a human salivary duct cell line, show that when the cells are stimulated with adenosine triphosphate (ATP or carbachol (CCh, they exhibit coupled oscillations with Ca2+ spike peaks preceding IP3 spike peaks. Based on these data, we construct a mathematical model of coupled Ca2+ and IP3 oscillations in HSY cells and perform model simulations of three different experimental settings to forecast Ca2+ responses. The model predicts that when Ca2+ influx from the extracellular space is removed, oscillations gradually slow down until they stop. The model simulation of applying a pulse of IP3 predicts that photolysis of caged IP3 causes a transient increase in the frequency of the Ca2+ oscillations. Lastly, when Ca2+-dependent activation of PLC is inhibited, we see an increase in the oscillation frequency and a decrease in the amplitude. These model predictions are confirmed by experimental data. We conclude that, although concentrations of Ca2+ and IP3 oscillate, Ca2+ oscillations in HSY cells are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations.

Oxidative stress activates the TRPM2-Ca2+-CaMKII-ROS signaling loop to induce cell death in cancer cells.

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Wang, Qian; Huang, Lihong; Yue, Jianbo

2017-06-01

High intracellular levels of reactive oxygen species (ROS) cause oxidative stress that results in numerous pathologies, including cell death. Transient potential receptor melastatin-2 (TRPM2), a Ca 2+ -permeable cation channel, is mainly activated by intracellular adenosine diphosphate ribose (ADPR) in response to oxidative stress. Here we studied the role and mechanisms of TRPM2-mediated Ca 2+ influx on oxidative stress-induced cell death in cancer cells. We found that oxidative stress activated the TRPM2-Ca 2+ -CaMKII cascade to inhibit early autophagy induction, which ultimately led to cell death in TRPM2 expressing cancer cells. On the other hand, TRPM2 knockdown switched cells from cell death to autophagy for survival in response to oxidative stress. Moreover, we found that oxidative stress activated the TRPM2-CaMKII cascade to further induce intracellular ROS production, which led to mitochondria fragmentation and loss of mitochondrial membrane potential. In summary, our data demonstrated that oxidative stress activates the TRPM2-Ca 2+ -CaMKII-ROS signal loop to inhibit autophagy and induce cell death. Copyright © 2016 Elsevier B.V. All rights reserved.

Growth hormone receptor C-terminal domains required for growth hormone-induced intracellular free Ca2+ oscillations and gene transcription

DEFF Research Database (Denmark)

Billestrup, N; Bouchelouche, P; Allevato, G

1995-01-01

of varying frequency and amplitude. GH-induced transcription of the serine protease inhibitor 2.1 gene required the same C-terminal 52-amino acid domain of the receptor as for Ca2+ signaling. Mutation of the four proline residues in the conserved box 1 region of the GHR, which is responsible for binding...

Cardiac microvascular endothelial cells express a functional Ca+ -sensing receptor.

Science.gov (United States)

Berra Romani, Roberto; Raqeeb, Abdul; Laforenza, Umberto; Scaffino, Manuela Federica; Moccia, Francesco; Avelino-Cruz, Josè Everardo; Oldani, Amanda; Coltrini, Daniela; Milesi, Veronica; Taglietti, Vanni; Tanzi, Franco

2009-01-01

The mechanism whereby extracellular Ca(2+) exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca(2+)-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca(2+)-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd(3+), La(3+) and neomycin, elicited a heterogeneous intracellular Ca(2+) signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP(3)) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na(+)/Ca(2+) exchanger upon substitution of extracellular Na(+) unmasked the Ca(2+) signal triggered by an increase in extracellular Ca(2+) levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca(2+) response to the CaSR agonist La(3+). These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca(2+) from intracellular InsP(3)-sensitive stores. Copyright 2008 S. Karger AG, Basel.

Effects of hirsutine, an antihypertensive indole alkaloid from Uncaria rhynchophylla, on intracellular calcium in rat thoracic aorta.

Science.gov (United States)

Horie, S; Yano, S; Aimi, N; Sakai, S; Watanabe, K

1992-01-01

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla (MIQ.) Jackson, on cytosolic Ca2+ level ([Ca2+]cyt) were studied by using fura-2-Ca2+ fluorescence in smooth muscle of the isolated rat aorta. Noradrenaline and high K+ solution produced a sustained increase in [Ca2+]cyt. Application of hirsutine after the increases in [Ca2+]cyt induced by noradrenaline and high K+ notably decreased [Ca2+]cyt, suggesting that hirsutine inhibits Ca2+ influx mainly through a voltage-dependent Ca2+ channel. Furthermore, the effect of hirsutine on intracellular Ca2+ store was studied by using contractile responses to caffeine under the Ca(2+)-free nutrient condition in the rat aorta. When hirsutine was added at 30 microM before caffeine treatment, the agent slightly but significantly reduced the caffeine-induced contraction. When added during Ca2+ loading, hirsutine definitely augmented the contractile response to caffeine. These results suggest that hirsutine inhibits Ca2+ release from the Ca2+ store and increases Ca2+ uptake into the Ca2+ store, leading to a reduction of intracellular Ca2+ level. It is concluded that hirsutine reduces intracellular Ca2+ level through its effect on the Ca2+ store as well as through its effect on the voltage-dependent Ca2+ channel.

F NMR measurement of intracellular free calcium in human red blood cells

International Nuclear Information System (INIS)

Gupta, R.K.; Schanne, F.A.X.

1986-01-01

Optical techniques for the measurement of intracellular Ca are not readily applicable to the human red cell because of the intense absorption of hemoglobin. The authors have therefore examined the use of 19 F NMR of 5,5'-difluoro-1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid (5FBAPTA) introduced non-disruptively by intracellular hydrolysis of the membrane-permeant acetoxymethyl ester derivative. 19 F NMR spectra of 5FBAPTA-containing erythrocytes at 188 MHz displayed two well resolved resonances corresponding to the free and Ca-bound forms of the chelator, the resonance of the free form being ten-fold larger than that of the Ca-bound form. Addition of the ionophore A23187 resulted in the disappearance of the resonance of the free anion and a quantitative increase in the intensity of the resonance of the Ca-complex. From these data, and a K/sub D/ of 708 nM for the Ca-5FBAPTA complex, the authors estimate red cell free Ca to be 70 nM, which is in the range of values obtained for other cells, despite the fact that the human red cell, which lacks intracellular organelles for storing Ca, possesses only 1 μmol total Ca/1. cells in comparison to mmols of total Ca found in other cells. The authors ability to use 19 F NMR to measure free Ca in the red blood cell paves the way for future NMR studies of red cell free Ca concentrations in human essential hypertension as well as in other diseases states in which alterations in cellular Ca homeostasis may be involved

Dynamics of Ca2+i and pHi in Ehrlich ascites tumor cells after Ca2+-mobilizing agonists or exposure to hypertonic solution

DEFF Research Database (Denmark)

Pedersen, Stine F.; Jørgensen, Nanna K.; Hoffmann, Else Kay

1998-01-01

Intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi) were monitored in Ehrlich ascites tumor cells using Fura-2 or 2',7',-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), or both probes in combination. An increase in [Ca2+]i induced by thrombin or bradykinin, agonists...

Hippocampal network oscillations in APP/APLP2-deficient mice.

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Xiaomin Zhang

Full Text Available The physiological function of amyloid precursor protein (APP and its two homologues APP-like protein 1 (APLP1 and 2 (APLP2 is largely unknown. Previous work suggests that lack of APP or APLP2 impairs synaptic plasticity and spatial learning. There is, however, almost no data on the role of APP or APLP at the network level which forms a critical interface between cellular functions and behavior. We have therefore investigated memory-related synaptic and network functions in hippocampal slices from three lines of transgenic mice: APPsα-KI (mice expressing extracellular fragment of APP, corresponding to the secreted APPsα ectodomain, APLP2-KO, and combined APPsα-KI/APLP2-KO (APPsα-DM for "double mutants". We analyzed two prominent patterns of network activity, gamma oscillations and sharp-wave ripple complexes (SPW-R. Both patterns were generally preserved in all strains. We find, however, a significantly reduced frequency of gamma oscillations in CA3 of APLP2-KO mice in comparison to APPsα-KI and WT mice. Network activity, basic synaptic transmission and short-term plasticity were unaltered in the combined mutants (APPsα-DM which showed, however, reduced long-term potentiation (LTP. Together, our data indicate that APLP2 and the intracellular domain of APP are not essential for coherent activity patterns in the hippocampus, but have subtle effects on synaptic plasticity and fine-tuning of network oscillations.

In vivo optical microprobe imaging for intracellular Ca2+ dynamics in response to dopaminergic signaling in deep brain evoked by cocaine

Science.gov (United States)

Luo, Zhongchi; Pan, Yingtian; Du, Congwu

2012-02-01

Ca2+ plays a vital role as second messenger in signal transduction and the intracellular Ca2+ ([Ca2+]i) change is an important indicator of neuronal activity in the brain, including both cortical and subcortical brain regions. Due to the highly scattering and absorption of brain tissue, it is challenging to optically access the deep brain regions (e.g., striatum at >3mm under the brain surface) and image [Ca2+]i changes with cellular resolutions. Here, we present two micro-probe approaches (i.e., microlens, and micro-prism) integrated with a fluorescence microscope modified to permit imaging of neuronal [Ca2+]i signaling in the striatum using a calcium indicator Rhod2(AM). While a micro-prism probe provides a larger field of view to image neuronal network from cortex to striatum, a microlens probe enables us to track [Ca2+]i dynamic change in individual neurons within the brain. Both techniques are validated by imaging neuronal [Ca2+]i changes in transgenic mice with dopamine receptors (D1R, D2R) expressing EGFP. Our results show that micro-prism images can map the distribution of D1R- and D2R-expressing neurons in various brain regions and characterize their different mean [Ca2+]i changes induced by an intervention (e.g., cocaine administration, 8mg/kg., i.p). In addition, microlens images can characterize the different [Ca2+]i dynamics of D1 and D2 neurons in response to cocaine, including new mechanisms of these two types of neurons in striatum. These findings highlight the power of the optical micro-probe imaging for dissecting the complex cellular and molecular insights of cocaine in vivo.

Tributyltin-induced endoplasmic reticulum stress and its Ca2+-mediated mechanism

International Nuclear Information System (INIS)

Isomura, Midori; Kotake, Yaichiro; Masuda, Kyoichi; Miyara, Masatsugu; Okuda, Katsuhiro; Samizo, Shigeyoshi; Sanoh, Seigo; Hosoi, Toru; Ozawa, Koichiro; Ohta, Shigeru

2013-01-01

Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca 2+ signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca 2+ homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700 nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca 2+ depletion, and to test this idea, we examined the effect of TBT on intracellular Ca 2+ concentration using fura-2 AM, a Ca 2+ fluorescent probe. TBT increased intracellular Ca 2+ concentration in a TBT-concentration-dependent manner, and Ca 2+ increase in 700 nM TBT was mainly blocked by 50 μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca 2+ concentration by releasing Ca 2+ from ER, thereby causing ER stress. - Highlights: • We established that tributyltin induces endoplasmic reticulum (ER) stress. • Tributyltin induces ER stress markers in a concentration-dependent manner. • Tributyltin increases Ca 2+ release from ER, thereby causing ER stress. • Dibutyltin and monobutyltin did not increase GRP78 or intracellular Ca 2+

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

International Nuclear Information System (INIS)

Satheesh, Noothan Jyothi; Büsselberg, Dietrich

2015-01-01

Neuroblastoma is the second most common paediatric cancer. It develops from undifferentiated simpatico-adrenal lineage cells and is mostly sporadic; however, the aetiology behind the development of neuroblastoma is still not fully understood. Intracellular calcium ([Ca 2+ ] i ) is a secondary messenger which regulates numerous cellular processes and, therefore, its concentration is tightly regulated. This review focuses on the role of [Ca 2+ ] i in differentiation, apoptosis and proliferation in neuroblastoma. It describes the mechanisms by which [Ca 2+ ] i is regulated and how it modulates intracellular pathways. Furthermore, the importance of [Ca 2+ ] i for the function of anti-cancer drugs is illuminated in this review as [Ca 2+ ] i could be a target to improve the outcome of anti-cancer treatment in neuroblastoma. Overall, modulations of [Ca 2+ ] i could be a key target to induce apoptosis in cancer cells leading to a more efficient and effective treatment of neuroblastoma

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

Energy Technology Data Exchange (ETDEWEB)

Satheesh, Noothan Jyothi; Büsselberg, Dietrich, E-mail: dib2015@qatar-med.cornell.edu [Weill Cornell Medical College in Qatar, Qatar Foundation-Education City, POB 24144, Doha (Qatar)

2015-05-22

Neuroblastoma is the second most common paediatric cancer. It develops from undifferentiated simpatico-adrenal lineage cells and is mostly sporadic; however, the aetiology behind the development of neuroblastoma is still not fully understood. Intracellular calcium ([Ca{sup 2+}]{sub i}) is a secondary messenger which regulates numerous cellular processes and, therefore, its concentration is tightly regulated. This review focuses on the role of [Ca{sup 2+}]{sub i} in differentiation, apoptosis and proliferation in neuroblastoma. It describes the mechanisms by which [Ca{sup 2+}]{sub i} is regulated and how it modulates intracellular pathways. Furthermore, the importance of [Ca{sup 2+}]{sub i} for the function of anti-cancer drugs is illuminated in this review as [Ca{sup 2+}]{sub i} could be a target to improve the outcome of anti-cancer treatment in neuroblastoma. Overall, modulations of [Ca{sup 2+}]{sub i} could be a key target to induce apoptosis in cancer cells leading to a more efficient and effective treatment of neuroblastoma.

Intracellular calcium modulation of voltage-gated sodium channels in ventricular myocytes

NARCIS (Netherlands)

Casini, Simona; Verkerk, Arie O.; van Borren, Marcel M. G. J.; van Ginneken, Antoni C. G.; Veldkamp, Marieke W.; de Bakker, Jacques M. T.; Tan, Hanno L.

2009-01-01

AIMS: Cardiac voltage-gated sodium channels control action potential (AP) upstroke and cell excitability. Intracellular calcium (Ca(i)(2+)) regulates AP properties by modulating various ion channels. Whether Ca(i)(2+) modulates sodium channels in ventricular myocytes, is unresolved. We studied

GABAergic synapse properties may explain genetic variation in hippocampal network oscillations in mice

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Tim S Heistek

2010-06-01

Full Text Available Cognitive ability and the properties of brain oscillation are highly heritable in humans. Genetic variation underlying oscillatory activity might give rise to differences in cognition and behavior. How genetic diversity translates into altered properties of oscillations and synchronization of neuronal activity is unknown. To address this issue, we investigated cellular and synaptic mechanisms of hippocampal fast network oscillations in eight genetically distinct inbred mouse strains. The frequency of carbachol-induced oscillations differed substantially between mouse strains. Since GABAergic inhibition sets oscillation frequency, we studied the properties of inhibitory synaptic inputs (IPSCs received by CA3 and CA1 pyramidal cells of three mouse strains that showed the highest, lowest and intermediate frequencies of oscillations. In CA3 pyramidal cells, the frequency of rhythmic IPSC input showed the same strain differences as the frequency of field oscillations. Furthermore, IPSC decay times in both CA1 and CA3 pyramidal cells were faster in mouse strains with higher oscillation frequencies than in mouse strains with lower oscillation frequency, suggesting that differences in GABAA-receptor subunit composition exist between these strains. Indeed, gene expression of GABAA-receptor β2 (Gabrb2 and β3 (Gabrb2 subunits was higher in mouse strains with faster decay kinetics compared with mouse strains with slower decay kinetics. Hippocampal pyramidal neurons in mouse strains with higher oscillation frequencies and faster decay kinetics fired action potential at higher frequencies. These data indicate that differences in genetic background may result in different GABAA-receptor subunit expression, which affects the rhythm of pyramidal neuron firing and fast network activity through GABA synapse kinetics.

Tributyltin-induced endoplasmic reticulum stress and its Ca{sup 2+}-mediated mechanism

Energy Technology Data Exchange (ETDEWEB)

Isomura, Midori; Kotake, Yaichiro, E-mail: yaichiro@hiroshima-u.ac.jp; Masuda, Kyoichi; Miyara, Masatsugu; Okuda, Katsuhiro; Samizo, Shigeyoshi; Sanoh, Seigo; Hosoi, Toru; Ozawa, Koichiro; Ohta, Shigeru

2013-10-01

Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca{sup 2+} signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca{sup 2+} homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700 nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca{sup 2+} depletion, and to test this idea, we examined the effect of TBT on intracellular Ca{sup 2+} concentration using fura-2 AM, a Ca{sup 2+} fluorescent probe. TBT increased intracellular Ca{sup 2+} concentration in a TBT-concentration-dependent manner, and Ca{sup 2+} increase in 700 nM TBT was mainly blocked by 50 μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca{sup 2+} concentration by releasing Ca{sup 2+} from ER, thereby causing ER stress. - Highlights: • We established that tributyltin induces endoplasmic reticulum (ER) stress. • Tributyltin induces ER stress markers in a concentration-dependent manner. • Tributyltin increases Ca{sup 2+} release from ER, thereby causing ER stress. • Dibutyltin and monobutyltin did not increase GRP78 or intracellular Ca{sup 2+}.

Electrical control of calcium oscillations in mesenchymal stem cells using microsecond pulsed electric fields.

Science.gov (United States)

Hanna, Hanna; Andre, Franck M; Mir, Lluis M

2017-04-20

Human mesenchymal stem cells are promising tools for regenerative medicine due to their ability to differentiate into many cellular types such as osteocytes, chondrocytes and adipocytes amongst many other cell types. These cells present spontaneous calcium oscillations implicating calcium channels and pumps of the plasma membrane and the endoplasmic reticulum. These oscillations regulate many basic functions in the cell such as proliferation and differentiation. Therefore, the possibility to mimic or regulate these oscillations might be useful to regulate mesenchymal stem cells biological functions. One or several electric pulses of 100 μs were used to induce Ca 2+ spikes caused by the penetration of Ca 2+ from the extracellular medium, through the transiently electropermeabilized plasma membrane, in human adipose mesenchymal stem cells from several donors. Attached cells were preloaded with Fluo-4 AM and exposed to the electric pulse(s) under the fluorescence microscope. Viability was also checked. According to the pulse(s) electric field amplitude, it is possible to generate a supplementary calcium spike with properties close to those of calcium spontaneous oscillations, or, on the contrary, to inhibit the spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca 2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. None of the pulses used here, even those with the highest amplitude, caused a loss of cell viability. An easy way to control Ca 2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca 2+ spikes, is reported here. Indeed, the direct link between the microsecond electric pulse(s) delivery and the occurrence/cancellation of cytosolic Ca 2+ spikes allowed us to mimic and regulate the Ca 2+ oscillations in these cells. Since microsecond electric pulse delivery

Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte

Science.gov (United States)

Tombes, R. M.; Simerly, C.; Borisy, G. G.; Schatten, G.

1992-01-01

During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.

Estimating the biophysical properties of neurons with intracellular calcium dynamics.

Science.gov (United States)

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

2014-06-01

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

CaMKII in sinoatrial node physiology and dysfunction

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Yuejin eWu

2014-03-01

Full Text Available The calcium and calmodulin dependent protein kinase II (CaMKII is present in sinoatrial node (SAN pacemaker cells and is required for physiological fight or flight SAN beating rate responses. Inhibition of CaMKII in SAN does not affect baseline heart rate, but reduces heart rate increases in response to physiological stress. CaMKII senses intracellular calcium (Ca2+ changes, oxidation status and hyperglycemia to phosphorylate substrates that regulate Ca2+-sensitive proteins, such as L-type Ca2+ channels, phospholamban (PLN, and cardiac ryanodine receptors (RyR2. All of these substrates are involved in the SAN pacemaking mechanism. Excessive CaMKII activity, as occurs under pathological conditions such as heart failure, ischemia and diabetes, can promote intracellular Ca2+ overload and reactive oxygen species (ROS production. Oxidation of CaMKII (ox-CaMKII locks CaMKII into a constitutively active configuration that contributes to SAN cell apoptosis and fibrosis. This ox-CaMKII-mediated loss of functional SAN cells contributes to sinoatrial node dysfunction (SND and sudden death. Thus, CaMKII has emerged as a central regulator of physiological SAN responses and a key determinant of SND.

Spontaneous low-frequency oscillations in cerebral vessels

DEFF Research Database (Denmark)

Schytz, Henrik W; Hansson, Andreas; Phillip, Dorte

2010-01-01

). Analysis of CA by measurement of spontaneous oscillations in the low-frequency spectrum in cerebral vessels might be a useful tool for assessing risk and investigating different treatment strategies in carotid artery disease (CAD) and stroke. We reviewed studies exploring spontaneous oscillations...

Resolution of intracellular calcium metabolism in intact segments of rabbit aorta

International Nuclear Information System (INIS)

Phair, R.D.; Hai, C.M.

1986-01-01

A new method, based on computer-assisted kinetic analysis of 45 Ca efflux data, was used to measure calcium contents and fluxes for extracellular and intracellular compartments in intact segments of rabbit aorta. After a 1-hour loading period, efflux data were collected for 8 hours using a flow-through tissue chamber. These long-term effluxes were necessary because information on intracellular calcium metabolism was concentrated in the slow components of the efflux curves while earlier components appeared to be dominated by washout of extracellular calcium. Intracellular compartments were identified as those whose calcium contents were altered by 10 microM phenylephrine. This method complements previous approaches by providing simultaneous estimates of compartmental calcium contents and fluxes without requiring the assumption of isotopic equilibrium and without recourse to standard wash techniques for removal of extracellular calcium. In normal, calcium-containing, bicarbonate-buffered physiological salt solution these compartments contained a total of approximately 300 nmol Ca/g wet aorta. Of this total, 55 nmol/g were associated with the slowest resolvable compartment whose turnover time was 170 minutes and whose exchange flux was 0.32 nmol min-1g-1. Two other intracellular compartments had turnover times of 30 minutes. One of these was phenylephrine releasable and contained 145 nmol/g; it exchanged calcium at 4.9 nmol min-1g-1. In normal physiological salt solution the plasma membrane was, surprisingly, not rate limiting for Ca efflux; and in 10 microM phenylephrine the membrane Ca flux was even greater, increasing 3.5-fold compared to control

Oxidized CaMKII (Ca2+/Calmodulin-Dependent Protein Kinase II) Is Essential for Ventricular Arrhythmia in a Mouse Model of Duchenne Muscular Dystrophy.

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Wang, Qiongling; Quick, Ann P; Cao, Shuyi; Reynolds, Julia; Chiang, David Y; Beavers, David; Li, Na; Wang, Guoliang; Rodney, George G; Anderson, Mark E; Wehrens, Xander H T

2018-04-01

Duchenne muscular dystrophy patients are prone to ventricular arrhythmias, which may be caused by abnormal calcium (Ca 2+ ) homeostasis and elevated reactive oxygen species. CaMKII (Ca 2+ /calmodulin-dependent protein kinase II) is vital for normal Ca 2+ homeostasis, but excessive CaMKII activity contributes to abnormal Ca 2+ homeostasis and arrhythmias in cardiomyocytes. Reactive oxygen species induce CaMKII to become autonomously active. We hypothesized that genetic inhibition of CaMKII oxidation (ox-CaMKII) in a mouse model of Duchenne muscular dystrophy can alleviate abnormal Ca 2+ homeostasis, thus, preventing ventricular arrhythmia. The objective of this study was to test if selective loss of ox-CaMKII affects ventricular arrhythmias in the mdx mouse model of Duchenne muscular dystrophy. 5-(6)-Chloromethyl-2,7-dichlorodihydrofluorescein diacetate staining revealed increased reactive oxygen species production in ventricular myocytes isolated from mdx mice, which coincides with elevated ventricular ox-CaMKII demonstrated by Western blotting. Genetic inhibition of ox-CaMKII by knockin replacement of the regulatory domain methionines with valines (MM-VV [CaMKII M281/282V]) prevented ventricular tachycardia in mdx mice. Confocal calcium imaging of ventricular myocytes isolated from mdx :MM-VV mice revealed normalization of intracellular Ca 2+ release events compared with cardiomyocytes from mdx mice. Abnormal action potentials assessed by optical mapping in mdx mice were also alleviated by genetic inhibition of ox-CaMKII. Knockout of the NADPH oxidase regulatory subunit p47 phox normalized elevated ox-CaMKII, repaired intracellular Ca 2+ homeostasis, and rescued inducible ventricular arrhythmias in mdx mice. Inhibition of reactive oxygen species or ox-CaMKII protects against proarrhythmic intracellular Ca 2+ handling and prevents ventricular arrhythmia in a mouse model of Duchenne muscular dystrophy. © 2018 American Heart Association, Inc.

Cell Type-specific Intrinsic Perithreshold Oscillations in Hippocampal GABAergic Interneurons.

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Kang, Young-Jin; Lewis, Hannah Elisabeth Smashey; Young, Mason William; Govindaiah, Gubbi; Greenfield, Lazar John; Garcia-Rill, Edgar; Lee, Sang-Hun

2018-04-15

The hippocampus plays a critical role in learning, memory, and spatial processing through coordinated network activity including theta and gamma oscillations. Recent evidence suggests that hippocampal subregions (e.g., CA1) can generate these oscillations at the network level, at least in part, through GABAergic interneurons. However, it is unclear whether specific GABAergic interneurons generate intrinsic theta and/or gamma oscillations at the single-cell level. Since major types of CA1 interneurons (i.e., parvalbumin-positive basket cells (PVBCs), cannabinoid type 1 receptor-positive basket cells (CB 1 BCs), Schaffer collateral-associated cells (SCAs), neurogliaform cells and ivy cells) are thought to play key roles in network theta and gamma oscillations in the hippocampus, we tested the hypothesis that these cells generate intrinsic perithreshold oscillations at the single-cell level. We performed whole-cell patch-clamp recordings from GABAergic interneurons in the CA1 region of the mouse hippocampus in the presence of synaptic blockers to identify intrinsic perithreshold membrane potential oscillations. The majority of PVBCs (83%), but not the other interneuron subtypes, produced intrinsic perithreshold gamma oscillations if the membrane potential remained above -45 mV. In contrast, CB 1 BCs, SCAs, neurogliaform cells, ivy cells, and the remaining PVBCs (17%) produced intrinsic theta, but not gamma, oscillations. These oscillations were prevented by blockers of persistent sodium current. These data demonstrate that the major types of hippocampal interneurons produce distinct frequency bands of intrinsic perithreshold membrane oscillations. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Evidence for a Na+-Ca2+ exchanger in rat pancreatic ducts

DEFF Research Database (Denmark)

Hug, M; Pahl, C; Novak, I

1996-01-01

Only recently has it been recognized that intracellular Ca2+ is an important cellular mediator in pancreatic ducts. The aim of the present study was to characterize the Ca2+ efflux pathway in ducts freshly prepared from rat pancreas. Lowering of extracellular Na+ concentration resulted in a signi......Only recently has it been recognized that intracellular Ca2+ is an important cellular mediator in pancreatic ducts. The aim of the present study was to characterize the Ca2+ efflux pathway in ducts freshly prepared from rat pancreas. Lowering of extracellular Na+ concentration resulted...

Tributyltin-induced endoplasmic reticulum stress and its Ca(2+)-mediated mechanism.

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Isomura, Midori; Kotake, Yaichiro; Masuda, Kyoichi; Miyara, Masatsugu; Okuda, Katsuhiro; Samizo, Shigeyoshi; Sanoh, Seigo; Hosoi, Toru; Ozawa, Koichiro; Ohta, Shigeru

2013-10-01

Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca(2+) signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca(2+) homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca(2+) depletion, and to test this idea, we examined the effect of TBT on intracellular Ca(2+) concentration using fura-2 AM, a Ca(2+) fluorescent probe. TBT increased intracellular Ca(2+) concentration in a TBT-concentration-dependent manner, and Ca(2+) increase in 700nM TBT was mainly blocked by 50μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca(2+) concentration by releasing Ca(2+) from ER, thereby causing ER stress. Copyright © 2013 Elsevier Inc. All rights reserved.

Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats.

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Stern, Javier E; Potapenko, Evgeniy S

2013-08-15

An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca²⁺ imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca²⁺ levels (NMDA-ΔCa²⁺) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa²⁺ in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa²⁺ was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca²⁺ in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca²⁺ homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa²⁺ signaling during HF are discussed.

Reconstructing the history of the Atlantic Multidecadal Oscillation using high-resolution Mg/Ca paleothermometry from a Cariaco Basin core

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Wurtzel, J. B.; Black, D. E.; Rahman, S.; Thunell, R.; Peterson, L. C.; Tappa, E.

2010-12-01

Instrumental and proxy-reconstructions show the existence of an approximately 70-year periodicity in Atlantic sea surface temperature (SST), known as the Atlantic Multidecadal Oscillation (AMO). The AMO is correlated with circum-tropical Atlantic climate phenomena such as Sahel and Nordeste rainfall, and Atlantic hurricane patterns. Though it has been suggested that the AMO is controlled by thermohaline circulation, much debate exists as to whether the SST fluctuations are a result of anthropogenic forcing or a natural climate mode, or even if the AMO is a true oscillation at all. Our ability to address this issue has been limited by instrumental SST records that rarely extend back more than 50-100 years and proxy reconstructions that are mostly terrestrial-based. Additionally, the modern instrumental variability likely contains an anthropogenic component that is not easily distinguished from the natural background of the system. From a marine sediment core taken in the Cariaco Basin, we have developed a high-resolution SST reconstruction for the past ca. 1500 years using Mg/Ca paleothermometry on seasonally-representative foraminifera, with the most recent data calibrated to the instrumental record. Previous studies have shown Cariaco Basin Mg/Ca-SSTs to be well-correlated to the Caribbean Sea and much of the western tropical Atlantic, which allows us to create a record that can be used to determine pre-anthropogenic rates and ranges of SST variability and observe how they change over time. Averaging the seasonal temperatures derived from the two foraminiferal species over the instrumental period yields a strong correlation to the AMO index from A. D. 1880 through 1970 (r = 0.44, p<0.0001). Wavelet analysis of the proxy average annual SST data indicates that modern AMO variability is not a consistent feature through time, and may be a function of warm-period climate.

Automated analysis of calcium spiking profiles with CaSA software: two case studies from root-microbe symbioses.

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Russo, Giulia; Spinella, Salvatore; Sciacca, Eva; Bonfante, Paola; Genre, Andrea

2013-12-26

Repeated oscillations in intracellular calcium (Ca2+) concentration, known as Ca2+ spiking signals, have been described in plants for a limited number of cellular responses to biotic or abiotic stimuli and most notably the common symbiotic signaling pathway (CSSP) which mediates the recognition by their plant hosts of two endosymbiotic microbes, arbuscular mycorrhizal (AM) fungi and nitrogen fixing rhizobia. The detailed analysis of the complexity and variability of the Ca2+ spiking patterns which have been revealed in recent studies requires both extensive datasets and sophisticated statistical tools. As a contribution, we have developed automated Ca2+ spiking analysis (CaSA) software that performs i) automated peak detection, ii) statistical analyses based on the detected peaks, iii) autocorrelation analysis of peak-to-peak intervals to highlight major traits in the spiking pattern.We have evaluated CaSA in two experimental studies. In the first, CaSA highlighted unpredicted differences in the spiking patterns induced in Medicago truncatula root epidermal cells by exudates of the AM fungus Gigaspora margarita as a function of the phosphate concentration in the growth medium of both host and fungus. In the second study we compared the spiking patterns triggered by either AM fungal or rhizobial symbiotic signals. CaSA revealed the existence of different patterns in signal periodicity, which are thought to contribute to the so-called Ca2+ signature. We therefore propose CaSA as a useful tool for characterizing oscillatory biological phenomena such as Ca2+ spiking.

Control of local intracellular calcium concentration with dynamic-clamp controlled 2-photon uncaging.

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Erwin Idoux

Full Text Available The variations of the intracellular concentration of calcium ion ([Ca(2+](i are at the heart of intracellular signaling, and their imaging is therefore of enormous interest. However, passive [Ca(2+](i imaging provides no control over these variations, meaning that a full exploration of the functional consequences of [Ca(2+](i changes is difficult to attain. The tools designed so far to modify [Ca(2+](i, even qualitatively, suffer drawbacks that undermine their widespread use. Here, we describe an electro-optical technique to quantitatively set [Ca(2+](i, in real time and with sub-cellular resolution, using two-photon Ca(2+ uncaging and dynamic-clamp. We experimentally demonstrate, on neurons from acute olfactory bulb slices of Long Evans rats, various capabilities of this technique previously difficult to achieve, such as the independent control of the membrane potential and [Ca(2+](i variations, the functional knocking-in of user-defined virtual voltage-dependent Ca(2+ channels, and the standardization of [Ca(2+](i patterns across different cells. Our goal is to lay the groundwork for this technique and establish it as a new and versatile tool for the study of cell signaling.

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

International Nuclear Information System (INIS)

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

2011-01-01

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

Altered thalamocortical rhythmicity and connectivity in mice lacking CaV3.1 T-type Ca2+ channels in unconsciousness

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Choi, Soonwook; Yu, Eunah; Lee, Seongwon; Llinás, Rodolfo R.

2015-01-01

In unconscious status (e.g., deep sleep and anesthetic unconsciousness) where cognitive functions are not generated there is still a significant level of brain activity present. Indeed, the electrophysiology of the unconscious brain is characterized by well-defined thalamocortical rhythmicity. Here we address the ionic basis for such thalamocortical rhythms during unconsciousness. In particular, we address the role of CaV3.1 T-type Ca2+ channels, which are richly expressed in thalamic neurons. Toward this aim, we examined the electrophysiological and behavioral phenotypes of mice lacking CaV3.1 channels (CaV3.1 knockout) during unconsciousness induced by ketamine or ethanol administration. Our findings indicate that CaV3.1 KO mice displayed attenuated low-frequency oscillations in thalamocortical loops, especially in the 1- to 4-Hz delta band, compared with control mice (CaV3.1 WT). Intriguingly, we also found that CaV3.1 KO mice exhibited augmented high-frequency oscillations during unconsciousness. In a behavioral measure of unconsciousness dynamics, CaV3.1 KO mice took longer to fall into the unconscious state than controls. In addition, such unconscious events had a shorter duration than those of control mice. The thalamocortical interaction level between mediodorsal thalamus and frontal cortex in CaV3.1 KO mice was significantly lower, especially for delta band oscillations, compared with that of CaV3.1 WT mice, during unconsciousness. These results suggest that the CaV3.1 channel is required for the generation of a given set of thalamocortical rhythms during unconsciousness. Further, that thalamocortical resonant neuronal activity supported by this channel is important for the control of vigilance states. PMID:26056284

Obesity Resistance Promotes Mild Contractile Dysfunction Associated with Intracellular Ca2+ Handling

International Nuclear Information System (INIS)

Sá, Felipe Gonçalves dos Santos de; Lima-Leopoldo, Ana Paula; Jacobsen, Bruno Barcellos; Ferron, Artur Junio Togneri; Estevam, Wagner Muller; Campos, Dijon Henrique Salomé; Castardeli, Edson; Cunha, Márcia Regina Holanda da; Cicogna, Antonio Carlos; Leopoldo, André Soares

2015-01-01

Diet-induced obesity is frequently used to demonstrate cardiac dysfunction. However, some rats, like humans, are susceptible to developing an obesity phenotype, whereas others are resistant to that. To evaluate the association between obesity resistance and cardiac function, and the impact of obesity resistance on calcium handling. Thirty-day-old male Wistar rats were distributed into two groups, each with 54 animals: control (C; standard diet) and obese (four palatable high-fat diets) for 15 weeks. After the experimental protocol, rats consuming the high-fat diets were classified according to the adiposity index and subdivided into obesity-prone (OP) and obesity-resistant (OR). Nutritional profile, comorbidities, and cardiac remodeling were evaluated. Cardiac function was assessed by papillary muscle evaluation at baseline and after inotropic maneuvers. The high-fat diets promoted increase in body fat and adiposity index in OP rats compared with C and OR rats. Glucose, lipid, and blood pressure profiles remained unchanged in OR rats. In addition, the total heart weight and the weight of the left and right ventricles in OR rats were lower than those in OP rats, but similar to those in C rats. Baseline cardiac muscle data were similar in all rats, but myocardial responsiveness to a post-rest contraction stimulus was compromised in OP and OR rats compared with C rats. Obesity resistance promoted specific changes in the contraction phase without changes in the relaxation phase. This mild abnormality may be related to intracellular Ca2+ handling

Pacific ciguatoxin-1b effect over Na+ and K+ currents, inositol 1,4,5-triphosphate content and intracellular Ca2+ signals in cultured rat myotubes

Science.gov (United States)

Hidalgo, Jorge; Liberona, José Luis; Molgó, Jordi; Jaimovich, Enrique

2002-01-01

The action of the main ciguatoxin involved in ciguatera fish poisoning in the Pacific region (P-CTX-1b) was studied in myotubes originated from rat skeletal muscle cells kept in primary culture. The effect of P-CTX-1b on sodium currents at short times of exposure (up to 1 min) showed a moderate increase in peak Na+ current. During prolonged exposures, P-CTX-1b decreased the peak Na+ current. This action was always accompanied by an increase of leakage currents, tail currents and outward Na+ currents, resulting in an intracellular Na+ accumulation. This effect is blocked by prior exposure to tetrodotoxin (TTX) and becomes evident only after washout of TTX. Low to moderate concentrations of P-CTX-1b (2–5 nM) partially blocked potassium currents in a manner that was dependent on the membrane potential. P-CTX-1b (2–12 nM) caused a small membrane depolarization (3–5 mV) and an increase in the frequency of spontaneous action potential discharges that reached in general low frequencies (0.1–0.5 Hz). P-CTX-1b (10 nM) caused a transient increase of intracellular inositol 1,4,5-trisphosphate (IP3) mass levels, which was blocked by TTX. In the presence of P-CTX-1b (10 nM) and in the absence of external Ca2+, the intracellular Ca2+ levels show a transient increase in the cytoplasm as well as in the nuclei. The time course of this effect may reflect the action of IP3 over internal stores activated by P-CTX-1b-induced membrane depolarization. PMID:12429578

The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

Science.gov (United States)

Joseph, Noah; Reicher, Barak; Barda-Saad, Mira

2014-02-01

During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters

Ghrelin increases intracellular Ca²⁺ concentration in the various hormone-producing cell types of the rat pituitary gland.

Science.gov (United States)

Yamazaki, Mami; Aizawa, Sayaka; Tanaka, Toru; Sakai, Takafumi; Sakata, Ichiro

2012-09-20

Ghrelin, isolated from the stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has potent growth hormone release ability in vivo and in vitro. Although GHS-R is abundantly expressed in the pituitary gland, there is no direct evidence of a relationship between hormone-producing cells and functional GHS-R in the pituitary gland. The aim of this study was to determine which anterior pituitary cells respond to ghrelin stimulation in male rats. We performed Fura-2 Ca(2+) imaging analysis using isolated pituitary cells, and performed immunocytochemistry to identify the type of pituitary hormone-producing cells. In Fura-2 Ca(2+) imaging analysis, ghrelin administration increased the intracellular Ca(2+) concentration in approximately 50% of total isolated anterior pituitary cells, and 20% of these cells strongly responded to ghrelin. Immunocytochemical analysis revealed that 82.9 ± 1.3% of cells that responded to ghrelin stimulation were GH-immunopositive. On the other hand, PRL-, LH-, and ACTH-immunopositive cells constituted 2.0 ± 0.3%, 12.6 ± 0.3%, and 2.5 ± 0.8% of ghrelin-responding pituitary cells, respectively. TSH-immunopositive cells did not respond to ghrelin treatment. These results suggest that ghrelin directly acts not only on somatotrophs, but also on mammotrophs, gonadotrophs, and corticotrophs in the rat pituitary gland. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Depletion of intracellular calcium stores facilitates the influx of extracellular calcium in platelet derived growth factor stimulated A172 glioblastoma cells.

Science.gov (United States)

Vereb, G; Szöllösi, J; Mátyus, L; Balázs, M; Hyun, W C; Feuerstein, B G

1996-05-01

Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.

Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction.

Science.gov (United States)

Godin, Lindsay M; Suzuki, Sakiko; Jacobs, Christopher R; Donahue, Henry J; Donahue, Seth W

2007-11-01

An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca (2+)](i)). This study analyzed the [Ca (2+)](i) wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca (2+)](i) wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca (2+)](i) wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca (2+)](i) wave area in the first bout it was likely to have a large [Ca (2+)](i) wave area in the second bout (r (2) = 0.933). These findings support the idea that individual bone cells have "calcium fingerprints" (i.e., a unique [Ca (2+)](i) wave profile that is reproducible for repeated exposure to a given stimulus).

Intracellular calcium levels can regulate Importin-dependent nuclear import

International Nuclear Information System (INIS)

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

2014-01-01

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

Intracellular calcium levels can regulate Importin-dependent nuclear import

Energy Technology Data Exchange (ETDEWEB)

Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A., E-mail: David.Jans@monash.edu

2014-07-18

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

Nanomolar bifenthrin alters synchronous Ca2+ oscillations and cortical neuron development independent of sodium channel activity.

Science.gov (United States)

Cao, Zhengyu; Cui, Yanjun; Nguyen, Hai M; Jenkins, David Paul; Wulff, Heike; Pessah, Isaac N

2014-04-01

Bifenthrin, a relatively stable type I pyrethroid that causes tremors and impairs motor activity in rodents, is broadly used. We investigated whether nanomolar bifenthrin alters synchronous Ca(2+) oscillations (SCOs) necessary for activity-dependent dendritic development. Primary mouse cortical neurons were cultured 8 or 9 days in vitro (DIV), loaded with the Ca(2+) indicator Fluo-4, and imaged using a Fluorescence Imaging Plate Reader Tetra. Acute exposure to bifenthrin rapidly increased the frequency of SCOs by 2.7-fold (EC50 = 58 nM) and decreased SCO amplitude by 36%. Changes in SCO properties were independent of modifications in voltage-gated sodium channels since 100 nM bifenthrin had no effect on the whole-cell Na(+) current, nor did it influence neuronal resting membrane potential. The L-type Ca(2+) channel blocker nifedipine failed to ameliorate bifenthrin-triggered SCO activity. By contrast, the metabotropic glutamate receptor (mGluR)5 antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine] normalized bifenthrin-triggered increase in SCO frequency without altering baseline SCO activity, indicating that bifenthrin amplifies mGluR5 signaling independent of Na(+) channel modification. Competitive [AP-5; (-)-2-amino-5-phosphonopentanoic acid] and noncompetitive (dizocilpine, or MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate]) N-methyl-d-aspartate antagonists partially decreased both basal and bifenthrin-triggered SCO frequency increase. Bifenthrin-modified SCO rapidly enhanced the phosphorylation of cAMP response element-binding protein (CREB). Subacute (48 hours) exposure to bifenthrin commencing 2 DIV-enhanced neurite outgrowth and persistently increased SCO frequency and reduced SCO amplitude. Bifenthrin-stimulated neurite outgrowth and CREB phosphorylation were dependent on mGluR5 activity since MPEP normalized both responses. Collectively these data identify a new mechanism by which bifenthrin potently alters Ca(2

Intracellular calcium homeostasis and signaling.

Science.gov (United States)

Brini, Marisa; Calì, Tito; Ottolini, Denis; Carafoli, Ernesto

2013-01-01

Ca(2+) is a universal carrier of biological information: it controls cell life from its origin at fertilization to its end in the process of programmed cell death. Ca(2+) is a conventional diffusible second messenger released inside cells by the interaction of first messengers with plasma membrane receptors. However, it can also penetrate directly into cells to deliver information without the intermediation of first or second messengers. Even more distinctively, Ca(2+) can act as a first messenger, by interacting with a plasma membrane receptor to set in motion intracellular signaling pathways that involve Ca(2+) itself. Perhaps the most distinctive property of the Ca(2+) signal is its ambivalence: while essential to the correct functioning of cells, Ca(2+) becomes an agent that mediates cell distress, or even (toxic) cell death, if its concentration and movements inside cells are not carefully tuned. Ca(2+) is controlled by reversible complexation to specific proteins, which could be pure Ca(2+) buffers, or which, in addition to buffering Ca(2+), also decode its signal to pass it on to targets. The most important actors in the buffering of cell Ca(2+) are proteins that transport it across the plasma membrane and the membrane of the organelles: some have high Ca(2+) affinity and low transport capacity (e.g., Ca(2+) pumps), others have opposite properties (e.g., the Ca(2+) uptake system of mitochondria). Between the initial event of fertilization, and the terminal event of programmed cell death, the Ca(2+) signal regulates the most important activities of the cell, from the expression of genes, to heart and muscle contraction and other motility processes, to diverse metabolic pathways involved in the generation of cell fuels.

Cannabidiol induces intracellular calcium elevation and cytotoxicity in oligodendrocytes.

Science.gov (United States)

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

2010-11-01

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

Thalamocortical Oscillations in the Sleeping and Aroused Brain

Science.gov (United States)

Steriade, Mircea; McCormick, David A.; Sejnowski, Terrence J.

1993-10-01

Sleep is characterized by synchronized events in billions of synaptically coupled neurons in thalamocortical systems. The activation of a series of neuromodulatory transmitter systems during awakening blocks low-frequency oscillations, induces fast rhythms, and allows the brain to recover full responsiveness. Analysis of cortical and thalamic networks at many levels, from molecules to single neurons to large neuronal assemblies, with a variety of techniques, ranging from intracellular recordings in vivo and in vitro to computer simulations, is beginning to yield insights into the mechanisms of the generation, modulation, and function of brain oscillations.

Glucocorticoids can affect Pseudomonas aeruginosa (ATCC 27853) internalization and intracellular calcium concentration in cystic fibrosis bronchial epithelial cells.

Science.gov (United States)

Hussain, Rashida; Shahror, Rami; Karpati, Ferenc; Roomans, Godfried M

2015-01-01

Glucocorticoids (GCs) are anti-inflammatory agents, but their use in cystic fibrosis (CF) is controversial. In CF, the early colonization with Pseudomonas aeruginosa is mainly due to nonmucoid strains that can internalize, and induce apoptosis in the epithelial cells. Uptake of P. aeruginosa by the epithelial cells and subsequent apoptosis may prevent colonization of P. aeruginosa in CF airways. In the airway epithelia, several other biological effects, including an anti-secretory role by decreasing intracellular Ca(2+) concentration have been described for this anti-inflammatory drug. However, the effects of GCs on the nonmucoid P. aeruginosa internalization and intracellular Ca(2+) in CF bronchial epithelial cells have not been evaluated. We used cultured human CF bronchial airway epithelial cell (CFBE) monolayers to determine P. aeruginosa internalization, apoptosis, and intracellular Ca(2+)concentration in CF bronchial epithelial cells. Cells were treated with IL-6, IL-8, dexamethasone, betamethasone, or budesonide. GCs in co-treatments with IL-6 reversed the effect of IL-6 by decreasing the internalization of P. aeruginosa in the CFBE cells. GCs decreased the extent of apoptosis in CFBE cells infected with internalized P. aeruginosa, and increased the intracellular Ca(2+) concentration. These findings suggest that if internalization of P. aeruginosa reduces infection, GC therapy would increase the risk of pulmonary infection by decreasing the internalization of P. aeruginosa in CF cells, but GCs may improve airway hydration by increasing the intracellular Ca(2+) concentration. Whether the benefits of GC treatment outweigh the negative effects is questionable, and further clinical studies need to be carried out.

Fermi Surface with Dirac Fermions in CaFeAsF Determined via Quantum Oscillation Measurements

Science.gov (United States)

Terashima, Taichi; Hirose, Hishiro T.; Graf, David; Ma, Yonghui; Mu, Gang; Hu, Tao; Suzuki, Katsuhiro; Uji, Shinya; Ikeda, Hiroaki

2018-02-01

Despite the fact that 1111-type iron arsenides hold the record transition temperature of iron-based superconductors, their electronic structures have not been studied much because of the lack of high-quality single crystals. In this study, we comprehensively determine the Fermi surface in the antiferromagnetic state of CaFeAsF, a 1111 iron-arsenide parent compound, by performing quantum oscillation measurements and band-structure calculations. The determined Fermi surface consists of a symmetry-related pair of Dirac electron cylinders and a normal hole cylinder. From analyses of quantum-oscillation phases, we demonstrate that the electron cylinders carry a nontrivial Berry phase π . The carrier density is of the order of 10-3 per Fe. This unusual metallic state with the extremely small carrier density is a consequence of the previously discussed topological feature of the band structure which prevents the antiferromagnetic gap from being a full gap. We also report a nearly linear-in-B magnetoresistance and an anomalous resistivity increase above about 30 T for B ∥c , the latter of which is likely related to the quantum limit of the electron orbit. Intriguingly, the electrical resistivity exhibits a nonmetallic temperature dependence in the paramagnetic tetragonal phase (T >118 K ), which may suggest an incoherent state. Our study provides a detailed knowledge of the Fermi surface in the antiferromagnetic state of 1111 parent compounds and moreover opens up a new possibility to explore Dirac-fermion physics in those compounds.

Fermi Surface with Dirac Fermions in CaFeAsF Determined via Quantum Oscillation Measurements

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Taichi Terashima

2018-02-01

Full Text Available Despite the fact that 1111-type iron arsenides hold the record transition temperature of iron-based superconductors, their electronic structures have not been studied much because of the lack of high-quality single crystals. In this study, we comprehensively determine the Fermi surface in the antiferromagnetic state of CaFeAsF, a 1111 iron-arsenide parent compound, by performing quantum oscillation measurements and band-structure calculations. The determined Fermi surface consists of a symmetry-related pair of Dirac electron cylinders and a normal hole cylinder. From analyses of quantum-oscillation phases, we demonstrate that the electron cylinders carry a nontrivial Berry phase π. The carrier density is of the order of 10^{-3} per Fe. This unusual metallic state with the extremely small carrier density is a consequence of the previously discussed topological feature of the band structure which prevents the antiferromagnetic gap from being a full gap. We also report a nearly linear-in-B magnetoresistance and an anomalous resistivity increase above about 30 T for B∥c, the latter of which is likely related to the quantum limit of the electron orbit. Intriguingly, the electrical resistivity exhibits a nonmetallic temperature dependence in the paramagnetic tetragonal phase (T>118  K, which may suggest an incoherent state. Our study provides a detailed knowledge of the Fermi surface in the antiferromagnetic state of 1111 parent compounds and moreover opens up a new possibility to explore Dirac-fermion physics in those compounds.

Cell-permeable gomesin peptide promotes cell death by intracellular Ca(2+) overload.

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Paredes-Gamero, Edgar J; Casaes-Rodrigues, Rafael L; Moura, Gioconda E D D; Domingues, Tatiana M; Buri, Marcus V; Ferreira, Victor H C; Trindade, Edvaldo S; Moreno-Ortega, Ana J; Cano-Abad, María F; Nader, Helena B; Ferreira, Alice T; Miranda, Antonio; Justo, Giselle Z; Tersariol, Ivarne L S

2012-09-04

In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca(2+) signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 μM Gm increases the cytosolic Ca(2+) and induces membrane permeabilization after 30 min of treatment. Direct Ca(2+) measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca(2+) depletion, which in turn resulted in oscillatory mitochondrial Ca(2+) signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix (mit)Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca(2+)-dependent pathway involving Gm translocation into the cell, ER Ca(2+) depletion and disruption, mitochondrial Ca(2+) overload and oxidative stress.

Asymmetry in Signal Oscillations Contributes to Efficiency of Periodic Systems.

Science.gov (United States)

Bae, Seul-A; Acevedo, Alison; Androulakis, Ioannis P

2016-01-01

Oscillations are an important feature of cellular signaling that result from complex combinations of positive- and negative-feedback loops. The encoding and decoding mechanisms of oscillations based on amplitude and frequency have been extensively discussed in the literature in the context of intercellular and intracellular signaling. However, the fundamental questions of whether and how oscillatory signals offer any competitive advantages-and, if so, what-have not been fully answered. We investigated established oscillatory mechanisms and designed a study to analyze the oscillatory characteristics of signaling molecules and system output in an effort to answer these questions. Two classic oscillators, Goodwin and PER, were selected as the model systems, and corresponding no-feedback models were created for each oscillator to discover the advantage of oscillating signals. Through simulating the original oscillators and the matching no-feedback models, we show that oscillating systems have the capability to achieve better resource-to-output efficiency, and we identify oscillatory characteristics that lead to improved efficiency.

Effect of bauhinia bauhinioides kallikrein inhibitor on endothelial proliferation and intracellular calcium concentration.

Science.gov (United States)

Bilgin, M; Burgazli, K M; Rafiq, A; Mericliler, M; Neuhof, C; Oliva, M L; Parahuleva, M; Soydan, N; Doerr, O; Abdallah, Y; Erdogan, A

2014-01-01

Proteinase inhibitors act as a defensive system against predators e.g. insects, in plants. Bauhinia bauhinioides kallikrein inhibitor (BbKI) is a serine proteinase inhibitor, isolated from seeds of Bauhinia bauhinioides and is structurally similar to plant Kunitz-type inhibitors but lacks disulfide bridges. In this study we evaluated the antiproliferative effect of BbKI on endothelial cells and its impact on changes in membrane potential and intracellular calcium. HUVEC proliferation was significantly reduced by incubation with BbKI 50 and 100 µM 12% and 13%. Furthermore, BbKI (100 µM) exposure caused a significant increase in intracellular Ca2+ concentration by 35% as compared to untreated control. The intracellular rise in calcium was not affected by the absence of extracellular calcium. BBKI also caused a significant change in the cell membrane potential but the antiproliferative effect was independent of changes in membrane potential. BBKI has an antiproliferative effect on HUVEC, which is independent of the changes in membrane potential, and it causes an increase in intracellular Ca2+.

Obesity Resistance Promotes Mild Contractile Dysfunction Associated with Intracellular Ca{sup 2+} Handling

Energy Technology Data Exchange (ETDEWEB)

Sá, Felipe Gonçalves dos Santos de; Lima-Leopoldo, Ana Paula; Jacobsen, Bruno Barcellos; Ferron, Artur Junio Togneri; Estevam, Wagner Muller [Centro de Educação Física e Desportos - Departamento de Desportos - Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Campos, Dijon Henrique Salomé [Departamento de Clínica Médica - Faculdade de Medicina - Universidade Estadual Paulista, Botucatu, São Paulo (Brazil); Castardeli, Edson; Cunha, Márcia Regina Holanda da [Centro de Educação Física e Desportos - Departamento de Desportos - Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Cicogna, Antonio Carlos [Departamento de Clínica Médica - Faculdade de Medicina - Universidade Estadual Paulista, Botucatu, São Paulo (Brazil); Leopoldo, André Soares, E-mail: andresoaresleopoldo@gmail.com [Centro de Educação Física e Desportos - Departamento de Desportos - Universidade Federal do Espírito Santo, Vitória, ES (Brazil)

2015-12-15

Diet-induced obesity is frequently used to demonstrate cardiac dysfunction. However, some rats, like humans, are susceptible to developing an obesity phenotype, whereas others are resistant to that. To evaluate the association between obesity resistance and cardiac function, and the impact of obesity resistance on calcium handling. Thirty-day-old male Wistar rats were distributed into two groups, each with 54 animals: control (C; standard diet) and obese (four palatable high-fat diets) for 15 weeks. After the experimental protocol, rats consuming the high-fat diets were classified according to the adiposity index and subdivided into obesity-prone (OP) and obesity-resistant (OR). Nutritional profile, comorbidities, and cardiac remodeling were evaluated. Cardiac function was assessed by papillary muscle evaluation at baseline and after inotropic maneuvers. The high-fat diets promoted increase in body fat and adiposity index in OP rats compared with C and OR rats. Glucose, lipid, and blood pressure profiles remained unchanged in OR rats. In addition, the total heart weight and the weight of the left and right ventricles in OR rats were lower than those in OP rats, but similar to those in C rats. Baseline cardiac muscle data were similar in all rats, but myocardial responsiveness to a post-rest contraction stimulus was compromised in OP and OR rats compared with C rats. Obesity resistance promoted specific changes in the contraction phase without changes in the relaxation phase. This mild abnormality may be related to intracellular Ca2+ handling.

Influx of extracellular Zn(2+) into the hippocampal CA1 neurons is required for cognitive performance via long-term potentiation.

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Takeda, A; Suzuki, M; Tempaku, M; Ohashi, K; Tamano, H

2015-09-24

Physiological significance of synaptic Zn(2+) signaling was examined in the CA1 of young rats. In vivo CA1 long-term potentiation (LTP) was induced using a recording electrode attached to a microdialysis probe and the recording region was locally perfused with artificial cerebrospinal fluid (ACSF) via the microdialysis probe. In vivo CA1 LTP was inhibited under perfusion with CaEDTA and ZnAF-2DA, extracellular and intracellular Zn(2+) chelators, respectively, suggesting that the influx of extracellular Zn(2+) is required for in vivo CA1 LTP induction. The increase in intracellular Zn(2+) was chelated with intracellular ZnAF-2 in the CA1 1h after local injection of ZnAF-2DA into the CA1, suggesting that intracellular Zn(2+) signaling induced during learning is blocked with intracellular ZnAF-2 when the learning was performed 1h after ZnAF-2DA injection. Object recognition was affected when training of object recognition test was performed 1h after ZnAF-2DA injection. These data suggest that intracellular Zn(2+) signaling in the CA1 is required for object recognition memory via LTP. Surprisingly, in vivo CA1 LTP was affected under perfusion with 0.1-1μM ZnCl2, unlike the previous data that in vitro CA1 LTP was enhanced in the presence of 1-5μM ZnCl2. The influx of extracellular Zn(2+) into CA1 pyramidal cells has bidirectional action in CA1 LTP. The present study indicates that the degree of extracellular Zn(2+) influx into CA1 neurons is critical for LTP and cognitive performance. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

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Navarro, Gemma; Aguinaga, David; Moreno, Estefania; Hradsky, Johannes; Reddy, Pasham P; Cortés, Antoni; Mallol, Josefa; Casadó, Vicent; Mikhaylova, Marina; Kreutz, Michael R; Lluís, Carme; Canela, Enric I; McCormick, Peter J; Ferré, Sergi

2014-11-20

The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

Maintained LTP and Memory Are Lost by Zn2+ Influx into Dentate Granule Cells, but Not Ca2+ Influx.

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Takeda, Atsushi; Tamano, Haruna; Hisatsune, Marie; Murakami, Taku; Nakada, Hiroyuki; Fujii, Hiroaki

2018-02-01

The idea that maintained LTP and memory are lost by either increase in intracellular Zn 2+ in dentate granule cells or increase in intracellular Ca 2+ was examined to clarify significance of the increases induced by excess synapse excitation. Both maintained LTP and space memory were impaired by injection of high K + into the dentate gyrus, but rescued by co-injection of CaEDTA, which blocked high K + -induced increase in intracellular Zn 2+ but not high K + -induced increase in intracellular Ca 2+ . High K + -induced disturbances of LTP and intracellular Zn 2+ are rescued by co-injection of 6-cyano-7-nitroquinoxakine-2,3-dione, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, but not by co-injection of blockers of NMDA receptors, metabotropic glutamate receptors, and voltage-dependent calcium channels. Furthermore, AMPA impaired maintained LTP and the impairment was also rescued by co-injection of CaEDTA, which blocked increase in intracellular Zn 2+ , but not increase in intracellular Ca 2+ . NMDA and glucocorticoid, which induced Zn 2+ release from the internal stores, did not impair maintained LTP. The present study indicates that increase in Zn 2+ influx into dentate granule cells through AMPA receptors loses maintained LTP and memory. Regulation of Zn 2+ influx into dentate granule cells is more critical for not only memory acquisition but also memory retention than that of Ca 2+ influx.

The putative imidazoline receptor agonist, harmane, promotes intracellular calcium mobilisation in pancreatic beta-cells.

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Squires, Paul E; Hills, Claire E; Rogers, Gareth J; Garland, Patrick; Farley, Sophia R; Morgan, Noel G

2004-10-06

beta-Carbolines (including harmane and pinoline) stimulate insulin secretion by a mechanism that may involve interaction with imidazoline I(3)-receptors but which also appears to be mediated by actions that are additional to imidazoline receptor agonism. Using the MIN6 beta-cell line, we now show that both the imidazoline I(3)-receptor agonist, efaroxan, and the beta-carboline, harmane, directly elevate cytosolic Ca(2+) and increase insulin secretion but that these responses display different characteristics. In the case of efaroxan, the increase in cytosolic Ca(2+) was readily reversible, whereas, with harmane, the effect persisted beyond removal of the agonist and resulted in the development of a repetitive train of Ca(2+)-oscillations whose frequency, but not amplitude, was concentration-dependent. Initiation of the Ca(2+)-oscillations by harmane was independent of extracellular calcium but was sensitive to both dantrolene and high levels (20 mM) of caffeine, suggesting the involvement of ryanodine receptor-gated Ca(2+)-release. The expression of ryanodine receptor-1 and ryanodine receptor-2 mRNA in MIN6 cells was confirmed using reverse transcription-polymerase chain reaction (RT-PCR) and, since low concentrations of caffeine (1 mM) or thimerosal (10 microM) stimulated increases in [Ca(2+)](i), we conclude that ryanodine receptors are functional in these cells. Furthermore, the increase in insulin secretion induced by harmane was attenuated by dantrolene, consistent with the involvement of ryanodine receptors in mediating this response. By contrast, the smaller insulin secretory response to efaroxan was unaffected by dantrolene. Harmane-evoked changes in cytosolic Ca(2+) were maintained by nifedipine-sensitive Ca(2+)-influx, suggesting the involvement of L-type voltage-gated Ca(2+)-channels. Taken together, these data imply that harmane may interact with ryanodine receptors to generate sustained Ca(2+)-oscillations in pancreatic beta-cells and that this effect

Ozone (O{sub 3}) elicits neurotoxicity in spinal cord neurons (SCNs) by inducing ER Ca{sup 2+} release and activating the CaMKII/MAPK signaling pathway

Energy Technology Data Exchange (ETDEWEB)

Li, Yun; Lin, Xiaowen; Zhao, XueJun; Xie, Juntian; JunNan, Wang; Sun, Tao; Fu, Zhijian, E-mail: zhijian_fu@163.com

2014-11-01

Ozone (O{sub 3}) is widely used in the treatment of spinal cord related diseases. Excess or accumulation of this photochemical air can however be neurotoxic. In this study, in vitro cultured Wister rat spinal cord neurons (SCNs) were used to investigate the detrimental effects and underlying mechanisms of O{sub 3}. Ozone in a dose-dependent manner inhibited cell viability at a range of 20 to 500 μg/ml, with the dose at 40 μg/ml resulting in a decrease of cell viability to 75%. The cell death after O{sub 3} exposure was related to endoplasmic reticulum (ER) calcium (Ca{sup 2+}) release. Intracellular Ca{sup 2+} chelator, ER stabilizer (inositol 1,4,5-trisphosphate receptor (IP3R) antagonist and ryanodine receptor (RyR) antagonist) and calcium/calmodulin-dependent protein kinase II (CaMKII) antagonist could effectively block Ca{sup 2+} mobilization and inhibit cell death following 40 μg/ml O{sub 3} exposure. In addition, ER Ca{sup 2+} release due to O{sub 3} exposure enhanced phospho-p38 and phospho-JNK levels and apoptosis of SCNs through activating CaMKII. Based on these results, we confirm that ozone elicits neurotoxicity in SCNs via inducing ER Ca{sup 2+} release and activating CaMKII/MAPK signaling pathway. Therefore, physicians should get attention to the selection of treatment concentrations of oxygen/ozone. And, approaches, such as chelating intracellular Ca{sup 2+} and stabilizing neuronal Ca{sup 2+} homeostasis could effectively ameliorate the neurotoxicity of O{sub 3}. - Highlights: • Exposure to O{sub 3} can reduce the viability of SCNs and cause the cell death. • Exposure to O{sub 3} can trigger RyR and IP3R dependent intracellular Ca{sup 2+} release. • Exposure to O{sub 3} can enhance the phospho-CaMKII, phospho-JNK and phospho-p38 levels.

Decreasing extracellular Na+ concentration triggers inositol polyphosphate production and Ca2+ mobilization

International Nuclear Information System (INIS)

Smith, J.B.; Dwyer, S.D.; Smith, L.

1989-01-01

Removing extracellular Na+ (Na+o) evoked a large increase in cytosolic free Ca2+ concentration ([Ca2+]i in human skin fibroblasts. Decreasing [Na+]o from 120 to 14 mM caused the half-maximal peak increase in [Ca2+]i. Removing Na+o strongly stimulated 45 Ca2+ efflux and decreased total cell Ca2+ by about 40%. Bradykinin caused changes in [Ca2+]i, total Ca2+, and 45 Ca2+ fluxes similar to those evoked by removing Na+o. Prior stimulation of the cells with bradykinin prevented Na+o removal from increasing [Ca2+]i and vice versa. Na+o removal rapidly increased [ 3 H]inositol polyphosphate production. Loading the cells with Na+ had no effect on the increase in 45 Ca2+ efflux produced by Na+o removal. Therefore, decreasing [Na+]o probably stimulates a receptor(s) which is sensitive to extracellular, not intracellular, Na+. Removing Na+o also mobilized intracellular Ca2+ in smooth muscle and endothelial cells cultured from human umbilical and dog coronary arteries, respectively

Activation of PKA and Epac proteins by cyclic AMP depletes intracellular calcium stores and reduces calcium availability for vasoconstriction.

Science.gov (United States)

Cuíñas, Andrea; García-Morales, Verónica; Viña, Dolores; Gil-Longo, José; Campos-Toimil, Manuel

2016-06-15

We investigated the implication of PKA and Epac proteins in the endothelium-independent vasorelaxant effects of cyclic AMP (cAMP). Cytosolic Ca(2+) concentration ([Ca(2+)]c) was measured by fura-2 imaging in rat aortic smooth muscle cells (RASMC). Contraction-relaxation experiments were performed in rat aortic rings deprived of endothelium. In extracellular Ca(2+)-free solution, cAMP-elevating agents induced an increase in [Ca(2+)]c in RASMC that was reproduced by PKA and Epac activation and reduced after depletion of intracellular Ca(2+) reservoirs. Arginine-vasopressin (AVP)-evoked increase of [Ca(2+)]c and store-operated Ca(2+) entry (SOCE) were inhibited by cAMP-elevating agents, PKA or Epac activation in these cells. In aortic rings, the contractions induced by phenylephrine in absence of extracellular Ca(2+) were inhibited by cAMP-elevating agents, PKA or Epac activation. In these conditions, reintroduction of Ca(2+) induced a contraction that was inhibited by cAMP-elevating agents, an effect reduced by PKA inhibition and reproduced by PKA or Epac activators. Our results suggest that increased cAMP depletes intracellular, thapsigargin-sensitive Ca(2+) stores through activation of PKA and Epac in RASMC, thus reducing the amount of Ca(2+) released by IP3-generating agonists during the contraction of rat aorta. cAMP rise also inhibits the contraction induced by depletion of intracellular Ca(2+), an effect mediated by reduction of SOCE after PKA or Epac activation. Both effects participate in the cAMP-induced endothelium-independent vasorelaxation. Copyright © 2016 Elsevier Inc. All rights reserved.

Ca2+ transport in plant cells and mechanisms of transformation of phytochrome-induced photosignals

Science.gov (United States)

Volotovski, Igor D.

1995-01-01

The recent data on the influence of phytochrome on the efficiency of Ca2+ translocation across the membranes of oat protoplasts are given. Ca2+ uptake in the protoplasts was shown to be influenced by the red light (R) illumination. This effect was reverted by the following far-red light (FR) illumination. To elucidate the sensitivity to phytochrome-controlling action the screening between the mechanisms of Ca2+ transport across the plasma membranes of oat protoplasts, Na+/Ca2+ and Ca2+/H+ exchangers, Ca2+-pump and Ca2+-channel was done. It was established that phytochrome modulated the activity of Na+/Ca2+-exchanger and Ca2+-pump. The light-mediated oscillations of cytoplasmic Ca2+ concentration in the oat protoplasts were demonstrated using fluorescence probe quin2 loaded into the cells and laser monitoring of fluorescence signal. The evidences were obtained that the oscillations were not the result of the elevation of cytoplasmic Ca2+ concentration and had no connection with Ca2+ pool of mitochondria. The possibility of the relation between the Ca2+ oscillations and phosphoinositide metabolism in plant cell membranes is analyzed. The mechanisms of transformation of primary phytochrome signal into biological effects were discussed.

Interaction of Glycolysis and Mitochondrial Respiration in Metabolic Oscillations of Pancreatic Islets

DEFF Research Database (Denmark)

Bertram, Richard; Satin, Leslie S.; Pedersen, Morten Gram

2007-01-01

Insulin secretion from pancreatic ß-cells is oscillatory, with a typical period of 2–7 min, reflecting oscillations in membrane potential and the cytosolic Ca2+ concentration. Our central hypothesis is that the slow 2–7 min oscillations are due to glycolytic oscillations, whereas faster oscillati...

NMDA-dependent phase synchronization between septal and temporal CA3 hippocampal networks.

Science.gov (United States)

Gu, Ning; Jackson, Jesse; Goutagny, Romain; Lowe, Germaine; Manseau, Frédéric; Williams, Sylvain

2013-05-08

Increasing evidence suggests that synchronization between brain regions is essential for information exchange and memory processes. However, it remains incompletely known which synaptic mechanisms contribute to the process of synchronization. Here, we investigated whether NMDA receptor-mediated synaptic plasticity was an important player in synchronization between septal and temporal CA3 areas of the rat hippocampus. We found that both the septal and temporal CA3 regions intrinsically generate weakly synchronized δ frequency oscillations in the complete hippocampus in vitro. Septal and temporal oscillators differed in frequency, power, and rhythmicity, but both required GABAA and AMPA receptors. NMDA receptor activation, and most particularly the NR2B subunit, contributed considerably more to rhythm generation at the temporal than the septal region. Brief activation of NMDA receptors by application of extracellular calcium dramatically potentiated the septal-temporal coherence for long durations (>40 min), an effect blocked by the NMDA antagonist AP-5. This long-lasting NMDA-receptor-dependent increase in coherence was also associated with an elevated phase locking of spikes locally and across regions. Changes in coherence between oscillators were associated with increases in phase locking between oscillators independent of oscillator amplitude. Finally, although the septal CA3 rhythm preceded the oscillations in temporal regions in control conditions, this was reversed during the NMDA-dependent enhancement in coherence, suggesting that NMDA receptor activation can change the direction of information flow along the septotemporal CA3 axis. These data demonstrate that plastic changes in communication between septal and temporal hippocampal regions can arise from the NMDA-dependent phase locking of neural oscillators.

Closing in on the Mechanisms of Pulsatile Insulin Secretion.

Science.gov (United States)

Bertram, Richard; Satin, Leslie S; Sherman, Arthur S

2018-03-01

Insulin secretion from pancreatic islet β-cells occurs in a pulsatile fashion, with a typical period of ∼5 min. The basis of this pulsatility in mouse islets has been investigated for more than four decades, and the various theories have been described as either qualitative or mathematical models. In many cases the models differ in their mechanisms for rhythmogenesis, as well as other less important details. In this Perspective, we describe two main classes of models: those in which oscillations in the intracellular Ca 2+ concentration drive oscillations in metabolism, and those in which intrinsic metabolic oscillations drive oscillations in Ca 2+ concentration and electrical activity. We then discuss nine canonical experimental findings that provide key insights into the mechanism of islet oscillations and list the models that can account for each finding. Finally, we describe a new model that integrates features from multiple earlier models and is thus called the Integrated Oscillator Model. In this model, intracellular Ca 2+ acts on the glycolytic pathway in the generation of oscillations, and it is thus a hybrid of the two main classes of models. It alone among models proposed to date can explain all nine key experimental findings, and it serves as a good starting point for future studies of pulsatile insulin secretion from human islets. © 2018 by the American Diabetes Association.

F-actin-based Ca signaling-a critical comparison with the current concept of Ca signaling.

Science.gov (United States)

Lange, Klaus; Gartzke, Joachim

2006-11-01

A short comparative survey on the current idea of Ca signaling and the alternative concept of F-actin-based Ca signaling is given. The two hypotheses differ in one central aspect, the mechanism of Ca storage. The current theory rests on the assumption of Ca-accumulating endoplasmic/sarcoplasmic reticulum-derived vesicles equipped with an ATP-dependent Ca pump and IP3- or ryanodine-sensitive channel-receptors for Ca-release. The alternative hypothesis proceeds from the idea of Ca storage at the high-affinity binding sites of actin filaments. Cellular sites of F-actin-based Ca storage are microvilli and the submembrane cytoskeleton. Several specific features of Ca signaling such as store-channel coupling, quantal Ca release, spiking and oscillations, biphasic and "phasic" uptake kinetics, and Ca-induced Ca release (CICR), which are not adequately described by the current concept, are inherent properties of the F-actin system and its dynamic state of treadmilling. Copyright 2006 Wiley-Liss, Inc.

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. II. Changes in Na+ and Ca2+ fluxes, Na+/K+ pump activity, and intracellular pH

International Nuclear Information System (INIS)

Mendoza, S.A.; Schneider, J.A.; Lopez-Rivas, A.; Sinnett-Smith, J.W.; Rozengurt, E.

1986-01-01

The amphibian tetradecapeptide, bombesin, and structurally related peptides caused a marked increase in ouabain-sensitive 86 Rb + uptake (a measure of Na + /K + pump activity) in quiescent Swiss 3T3 cells. This effect occurred within seconds after the addition of the peptide and appeared to be mediated by an increase in Na + entry into the cells. The effect of bombesin on Na + entry and Na + /K + pump activity was concentration dependent with half-maximal stimulation occurring at 0.3-0.4 nM. The structurally related peptides litorin, gastrin-releasing peptide, and neuromedin B also stimulated ouabain-sensitive 86 Rb + uptake; the relative potencies of these peptides in stimulating the Na + /K + pump were comparable to their potencies in increasing DNA synthesis. Bombesin increased Na + influx, at least in part, through an Na + /H + antiport. The peptide augmented intracellular pH and this effect was abolished in the absence of extracellular Na + . In addition to monovalent ion transport, bombesin and the structurally related peptides rapidly increased the efflux of 45 Ca 2+ from quiescent Swiss 3T3 cells. This Ca 2+ came from an intracellular pool and the efflux was associated with a 50% decrease in total intracellular Ca 2+ . The peptides also caused a rapid increase in cytosolic free calcium concentration. Prolonged pretreatment of Swiss 3T3 cells with phorbol dibutyrate, which causes a loss of protein kinase C activity, greatly decreased the stimulation of 86 Rb + uptake and Na + entry by bombesin implicating this phosphotransferase system in the mediation of part of these responses to bombesin. Since some activation of monovalent ion transport by bombesin was seen in phorbol dibutyrate-pretreated cells, it is likely that the peptide also stimulates monovalent ion transport by a second mechanism

Dynamic force microscopy with quartz tuning forks at high oscillation amplitudes

International Nuclear Information System (INIS)

Labardi, M

2007-01-01

Dynamic force microscopy (DFM) with the self-oscillator (SO) method allows reasonably high scanning rates even with high Q-factors of the resonant force sensor, typical of cantilevers in ultra-high vacuum and of quartz tuning forks. However, due to simpler interpretation of force spectroscopy measurements, small oscillation amplitudes (sub-nm level) are generally preferred. In applications like 'apertureless' scanning near-field optical microscopy (SNOM), oscillation amplitudes of the order of 5-10 nm are needed to increase optical sensitivity and to apply standard optical artefact suppression methods. This motivates the study of the behaviour of tuning forks driven at such high amplitudes, as compared to usual air-operated cantilevers. Both constant-excitation-amplitude (CE) and constant-oscillation-amplitude (CA) modes of SO-DFM are analysed, since the CA mode is more convenient for SNOM applications, denoting remarkable differences. In particular, possible instability effects, previously found in CE mode, are not anticipated for CA mode. It is shown how resonance and approach ('isophase') curves in both modes can be conveniently described in terms of the usual 'normalized frequency shift' γ and of a 'normalized gain' η, defined as a measurement of surface dissipation

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

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

2016-02-01

Industrial solvents pose a significant threat to the humankind. The mechanisms of their toxicity still remain in debate. Trichloroethylene (TCE) is a widespread industrial solvent responsible for severe liver dysfunction, cutaneous toxicity in occupationally exposed humans. We utilized an in vitro system of human epidermal keratinocyte (HaCaT) cells in this study to avoid complex cell and extracellular interactions. We report the cytotoxicity of organic solvent TCE in HaCaT and its reversal by a natural flavanone, naringenin (Nar). The cytotoxicity was attributed to the rapid intracellular free calcium (Ca(2+)) release, which might lead to the elevation of protein kinase C along with robust free radical generation, instability due to energy depletion, and sensitization of intracellular stress signal transducer nuclear factor κB. These effects were actually seen to induce significant amount of genomic DNA fragmentation. Furthermore, all these effects of TCE were effectively reversed by the treatment of Nar, a natural flavanone. Our studies identify intracellular Ca as a unique target used by organic solvents in the cytotoxicity and highlight the Ca(2+) ion stabilizer properties of Nar. © The Author(s) 2015.

An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis.

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Wei Xiong

Full Text Available AIM: Neurotransmitter release is elicited by an elevation of intracellular Ca(2+ concentration ([Ca(2+](i. The action potential triggers Ca(2+ influx through Ca(2+ channels which causes local changes of [Ca(2+](i for vesicle release. However, any direct role of extracellular Ca(2+ (besides Ca(2+ influx on Ca(2+-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG neurons and chromaffin cells, widely used models for studying vesicle exocytosis. RESULTS: Using photolysis of caged Ca(2+ and caffeine-induced release of stored Ca(2+, we found that extracellular Ca(2+ inhibited exocytosis following moderate [Ca(2+](i rises (2-3 µM. The IC(50 for extracellular Ca(2+ inhibition of exocytosis (ECIE was 1.38 mM and a physiological reduction (∼30% of extracellular Ca(2+ concentration ([Ca(2+](o significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca(2+](o. The calcimimetics Mg(2+, Cd(2+, G418, and neomycin all inhibited exocytosis. The extracellular Ca(2+-sensing receptor (CaSR was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE. CONCLUSION/SIGNIFICANCE: As an extension of the classic Ca(2+ hypothesis of synaptic release, physiological levels of extracellular Ca(2+ play dual roles in evoked exocytosis by providing a source of Ca(2+ influx, and by directly regulating quantal size and release probability in neuronal cells.

Advanced glycation end-products (AGEs acutely impair Ca2+ signalling in bovine aortic endothelial cells

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Nadim eNaser

2013-03-01

Full Text Available Post-translational modification of proteins in diabetes, including formation of advanced glycation end products (AGEs are believed to contribute to vascular dysfunction and disease. Impaired function of the endothelium is an early indicator of vascular dysfunction in diabetes and as many endothelial cell processes are dependent upon intracellular [Ca2+] and Ca2+ signalling, the aim of this study was to examine the acute effects of AGEs on Ca2+ signalling in bovine aortic endothelial cells (BAEC. Ca2+ signalling was studied using the fluorescent indicator dye Fura2-AM. AGEs were generated by incubating bovine serum albumin with 0 - 250 mM glucose or glucose-6-phosphate for 0 to 120 days at 37ºC. Under all conditions, the main AGE species generated was carboxymethyl lysine (CML as assayed using both GC-MS and HPLC. In Ca2+-replete solution, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca2+] and attenuated the increase in intracellular [Ca2+] caused by ATP (100 µM. In the absence of extracellular Ca2+, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca2+] and attenuated subsequent intracellular Ca2+ release caused by ATP, thapsigargin (0.1 µM and ionomycin (3 µM, but AGEs did not affect extracellular Ca2+ entry induced by the re-addition of Ca2+ to the bathing solution in the presence of any of these agents. The anti-oxidant α-lipoic acid (2 µM and NAD(PH oxidase inhibitors apocynin (500 µM and diphenyleneiodonium (DPI, 1 µM abolished these effects of AGEs on BAECs, as did the IP3 receptor antagonist xestospongin C (1 µM. In summary, AGEs caused an acute depletion of Ca2+ from the intracellular store in BAECs, such that the Ca2+ signal stimulated by the subsequent application other agents acting upon this store is reduced. The mechanism may involve generation of ROS from NAD(PH oxidase and possible activation of the IP3 receptor.

Endothelial remodelling and intracellular calcium machinery.

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Moccia, F; Tanzi, F; Munaron, L

2014-05-01

Rather being an inert barrier between vessel lumen and surrounding tissues, vascular endothelium plays a key role in the maintenance of cardiovascular homeostasis. The de-endothelialization of blood vessels is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm. Restoration of the endothelial lining may be accomplished by the activation of neighbouring endothelial cells (ECs) freed by contact inhibition and by circulating endothelial progenitor cells (EPCs). Intracellular Ca(2+) signalling is essential to promote wound healing: however, the molecular underpinnings of the Ca(2+) response to injury are yet to be fully elucidated. Similarly, the components of the Ca(2+) toolkit that drive EPC incorporation into denuded vessels are far from being fully elucidated. The present review will survey the current knowledge on the role of Ca(2+) signalling in endothelial repair and in EPC activation. We propose that endothelial regeneration might be boosted by intraluminal release of specific Ca(2+) channel agonists or by gene transfer strategies aiming to enhance the expression of the most suitable Ca(2+) channels at the wound site. In this view, connexin (Cx) channels/hemichannels and store-operated Ca(2+) entry (SOCE) stand amid the most proper routes to therapeutically induce the regrowth of denuded vessels. Cx stimulation might trigger the proliferative and migratory behaviour of ECs facing the lesion site, whereas activation of SOCE is likely to favour EPC homing to the wounded vessel.

Transition in complex calcium bursting induced by IP3 degradation

International Nuclear Information System (INIS)

Zhang Feng; Lu Qishao; Su Jianzhong

2009-01-01

Complex intracellular Ca 2+ oscillations are systematically investigated in a mathematical model based on the mechanism of Ca 2+ -induced Ca 2+ release (CICR), taking account of the Ca 2+ -stimulated degradation of inositol 1,4,5-trisphosphate (IP 3 ) by a 3-kinase. Periodic, quasi-periodic and chaotic bursting oscillations exist in a wide range of parameter values and occur alternatively as the parameters change slightly. The transition among them can be observed by the evidence in their interspike interval and the Lyapunov exponent. These results reveal the role of agonist-stimulated of IP 3 degradation as a possible source for complex patterns in Ca 2+ signaling.

Emergence of noise-induced oscillations in the central circadian pacemaker.

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Caroline H Ko

2010-10-01

Full Text Available Bmal1 is an essential transcriptional activator within the mammalian circadian clock. We report here that the suprachiasmatic nucleus (SCN of Bmal1-null mutant mice, unexpectedly, generates stochastic oscillations with periods that overlap the circadian range. Dissociated SCN neurons expressed fluctuating levels of PER2 detected by bioluminescence imaging but could not generate circadian oscillations intrinsically. Inhibition of intercellular communication or cyclic-AMP signaling in SCN slices, which provide a positive feed-forward signal to drive the intracellular negative feedback loop, abolished the stochastic oscillations. Propagation of this feed-forward signal between SCN neurons then promotes quasi-circadian oscillations that arise as an emergent property of the SCN network. Experimental analysis and mathematical modeling argue that both intercellular coupling and molecular noise are required for the stochastic rhythms, providing a novel biological example of noise-induced oscillations. The emergence of stochastic circadian oscillations from the SCN network in the absence of cell-autonomous circadian oscillatory function highlights a previously unrecognized level of circadian organization.

Intracellular Zn(2+) signaling in the dentate gyrus is required for object recognition memory.

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Takeda, Atsushi; Tamano, Haruna; Ogawa, Taisuke; Takada, Shunsuke; Nakamura, Masatoshi; Fujii, Hiroaki; Ando, Masaki

2014-11-01

The role of perforant pathway-dentate granule cell synapses in cognitive behavior was examined focusing on synaptic Zn(2+) signaling in the dentate gyrus. Object recognition memory was transiently impaired when extracellular Zn(2+) levels were decreased by injection of clioquinol and N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylendediamine. To pursue the effect of the loss and/or blockade of Zn(2+) signaling in dentate granule cells, ZnAF-2DA (100 pmol, 0.1 mM/1 µl), an intracellular Zn(2+) chelator, was locally injected into the dentate molecular layer of rats. ZnAF-2DA injection, which was estimated to chelate intracellular Zn(2+) signaling only in the dentate gyrus, affected object recognition memory 1 h after training without affecting intracellular Ca(2+) signaling in the dentate molecular layer. In vivo dentate gyrus long-term potentiation (LTP) was affected under the local perfusion of the recording region (the dentate granule cell layer) with 0.1 mM ZnAF-2DA, but not with 1-10 mM CaEDTA, an extracellular Zn(2+) chelator, suggesting that the blockade of intracellular Zn(2+) signaling in dentate granule cells affects dentate gyrus LTP. The present study demonstrates that intracellular Zn(2+) signaling in the dentate gyrus is required for object recognition memory, probably via dentate gyrus LTP expression. Copyright © 2014 Wiley Periodicals, Inc.

Mobilisation of store Ca2+ activates tyrosine hydroxylase in bovine adrenal chromaffin cells

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McKenzie, S.; Marley, P.D.

2001-01-01

Full text: Many receptor agonists are able to activate tyrosine hydroxylase (TOH) in bovine adrenal chromaffin cells. The majority of these are dependent on extracellular Ca 2+ for this action. Entry of extracellular Ca 2+ through voltage-operated Ca 2+ channels is very effective at activating TOH. The contribution of the intracellular Ca 2+ stores to TOH activation however is not known. Previous studies have shown that mobilisation of intracellular Ca 2+ stores is effective at increasing phosphorylation of TOH, but its effect on TOH activity has not been studied. Therefore, in the present study, the effect of mobilisation of store Ca 2+ on TOH activity was investigated using primary cultures of bovine adrenal chromaffin cells. Cells were prepared from abattoir tissue and cultured for 3-6 days. TOH activity was determined over 10 minutes, measuring the 14 CO 2 produced following the hydroxylation and rapid decarboxylation of 14 C-tyrosine offered to intact cells. Caffeine increased TOH activity in a concentration-dependent manner with a maximum response of 100% increase at 20mM. This effect was not due to osmolarity since 20mM sucrose had no effect.Nor was it due to inhibition of phosphodiesterases, since the effect of caffeine was still seen in the presence of 1mM IBMX. However,caffeine-induced TOH activation was substantially reduced in the absence of extracellular Ca 2+ . The results suggest that TOH activity can be increased by mobilising intracellular Ca 2+ stores, but that this effect involves extracellular Ca 2+ influx, possibly through store-operated channels. Copyright (2001) Australian Neuroscience Society

Measurement of Intracellular Ionized Calcium in a Free-living Soil Nematode, Caenorhabditis elegans.

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Kawaii, S; Yoshizawa, Y; Mizutani, J

1993-01-01

A calcium chelating fluorescence indicator, fura-2, was used to measure intracellular ionized calcium in Caenorhabditis elegans. The indicator loading process was harmless to the nematode, and completed within 2-3 h. Fura-2 was loaded mainly at its intestinal tract. The effects of DOPA on locomotion and the level of intracellular calcium were investigated and measured by using a microfluorometer. The addition of DOPA temporarily increased [Ca(2+)]i for several minutes.

Influence of extracellular oscillations on neural communication: a computational perspective

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Zoran eTiganj

2014-02-01

Full Text Available Neural communication generates oscillations of electric potential in the extracellular medium. In feedback, these oscillations affect the electrochemical processes within the neurons, influencing the timing and the number of action potentials. It is unclear whether this influence should be considered only as noise or it has some functional role in neural communication. Through computer simulations we investigated the effect of various sinusoidal extracellular oscillations on the timing and number of action potentials. Each simulation is based on a multicompartment model of a single neuron, which is stimulated through spatially distributed synaptic activations. A thorough analysis is conducted on a large number of simulations with different models of CA3 and CA1 pyramidal neurons which are modeled using realistic morphologies and active ion conductances. We demonstrated that the influence of the weak extracellular oscillations, which are commonly present in the brain, is rather stochastic and modest. We found that the stronger fields, which are spontaneously present in the brain only in some particular cases (e.g. during seizures or that can be induced externally, could significantly modulate spike timings.

To Break or to Brake Neuronal Network Accelerated by Ammonium Ions?

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Vladimir V Dynnik

Full Text Available The aim of present study was to investigate the effects of ammonium ions on in vitro neuronal network activity and to search alternative methods of acute ammonia neurotoxicity prevention.Rat hippocampal neuronal and astrocytes co-cultures in vitro, fluorescent microscopy and perforated patch clamp were used to monitor the changes in intracellular Ca2+- and membrane potential produced by ammonium ions and various modulators in the cells implicated in neural networks.Low concentrations of NH4Cl (0.1-4 mM produce short temporal effects on network activity. Application of 5-8 mM NH4Cl: invariably transforms diverse network firing regimen to identical burst patterns, characterized by substantial neuronal membrane depolarization at plateau phase of potential and high-amplitude Ca2+-oscillations; raises frequency and average for period of oscillations Ca2+-level in all cells implicated in network; results in the appearance of group of «run out» cells with high intracellular Ca2+ and steadily diminished amplitudes of oscillations; increases astrocyte Ca2+-signalling, characterized by the appearance of groups of cells with increased intracellular Ca2+-level and/or chaotic Ca2+-oscillations. Accelerated network activity may be suppressed by the blockade of NMDA or AMPA/kainate-receptors or by overactivation of AMPA/kainite-receptors. Ammonia still activate neuronal firing in the presence of GABA(A receptors antagonist bicuculline, indicating that «disinhibition phenomenon» is not implicated in the mechanisms of networks acceleration. Network activity may also be slowed down by glycine, agonists of metabotropic inhibitory receptors, betaine, L-carnitine, L-arginine, etc.Obtained results demonstrate that ammonium ions accelerate neuronal networks firing, implicating ionotropic glutamate receptors, having preserved the activities of group of inhibitory ionotropic and metabotropic receptors. This may mean, that ammonia neurotoxicity might be prevented by

Tau-Induced Ca2+/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation.

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Wei, Yu-Ping; Ye, Jin-Wang; Wang, Xiong; Zhu, Li-Ping; Hu, Qing-Hua; Wang, Qun; Ke, Dan; Tian, Qing; Wang, Jian-Zhi

2018-04-01

Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca 2+ concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca 2+ concentration with a simultaneous increase in the phosphorylation of Ca 2+ /calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca 2+ /CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca 2+ /calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca 2+ /CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca 2+ concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.

Modeling caspian sea water level oscilLations Under Diffrent Scenarioes of Increasing Atmospheric Carbon Dioxide Concentrations

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GholamReza Roshan

2012-12-01

Full Text Available The rapid rise of Caspian Sea water level (about 2.25 meters since 1978 has caused much concern to all five surrounding countries, primarily because flooding has destroyed or damaged buildings and other engineering structures, roads, beaches and farm lands in thecoastal zone. Given that climate, and more specifically climate change, is a primary factor influencing oscillations in Caspian Sea water levels, the effect of different climate change scenarios on future Caspian Sea levels was stimulated. Variations in environmentalparameters such as temperature, precipitation, evaporation, tmospheric carbon dioxide and water level oscillations of the Caspian sea and surrounding regions, are considered for bothpast (1951-2006 and future (2025-2100 time frames. The output of the UKHADGEM general circulation model and five alternative scenarios including A1CAI, BIASF, BIMES WRE450 and WRE750 were extracted using the MAGICC SCENGEN Model software(version 5.3. The results suggest that the mean temperature of the Caspian Sea region (Bandar-E-Anzali monitoring site has increased by ca. 0.17ºC per decade under the impacts of atmospheric carbon dioxide changes (r=0.21. The Caspian Sea water level has increasedby ca. +36 mm per decade (r=0.82 between the years 1951-2006. Mean results from all modeled scenarios indicate that the temperature will increase by ca. 3.64ºC and precipitation will decrease by ca. 10% (182 mm over the Caspian Sea, whilst in the Volga river basin,temperatures are projected to increase by ca. 4.78ºC and precipitation increase by ca. 12% (58 mm by the year 2100. Finally, statistical modeling of the Caspian Sea water levels projectfuture water level increases of between 86 cm and 163 cm by the years 2075 and 2100, respectively.

Protein Kinase Cα and P-Type Ca2+ Channel CaV2.1 in Red Blood Cell Calcium Signalling

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Lisa Wagner-Britz

2013-06-01

Full Text Available Background/Aims: Protein kinase Cα (PKCα is activated by an increase in cytosolic Ca2+ in red blood cells (RBCs. Previous work has suggested that PKCα directly stimulates the CaV2.1 channel, whereas other studies revealed that CaV2.1 is insensitive to activation by PKC. The aim of this study was to resolve this discrepancy. Methods: We performed experiments based on a single cell read-out of the intracellular Ca2+ concentration in terms of Fluo-4 fluorescence intensity and phosphatidylserine exposure to the external membrane leaflet. Measurement modalities included flow cytometry and live cell imaging. Results: Treatment of RBCs with phorbol 12-myristate 13-acetate (PMA led to two distinct populations of cells with an increase in intracellular Ca2+: a weak-responding and a strong-responding population. The EC50 of PMA for the number of cells with Ca2+ elevation was 2.7±1.2 µM; for phosphatidylserine exposure to the external membrane surface, it was 2.8±0.5 µM; and for RBC haemolysis, it was 2.9±0.5 µM. Using pharmacological manipulation with the CaV2.1 inhibitor ω-agatoxin TK and the broad protein kinase C inhibitor Gö6983, we are able to show that there are two independent PMA-activated Ca2+ entry processes: the first is independent of CaV2.1 and directly PKCα-activated, while the second is associated with a likely indirect activation of CaV2.1. Further studies using lysophosphatidic acid (LPA as a stimulation agent have provided additional evidence that PKCα and CaV2.1 are not directly interconnected in a signalling chain. Conclusion: Although we provide evidence for a lack of interaction between PKCα and CaV2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKCα and CaV2.1.

Cyclic ADP ribose-dependent Ca2+ release by group I metabotropic glutamate receptors in acutely dissociated rat hippocampal neurons.

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Jong-Woo Sohn

Full Text Available Group I metabotropic glutamate receptors (group I mGluRs; mGluR1 and mGluR5 exert diverse effects on neuronal and synaptic functions, many of which are regulated by intracellular Ca(2+. In this study, we characterized the cellular mechanisms underlying Ca(2+ mobilization induced by (RS-3,5-dihydroxyphenylglycine (DHPG; a specific group I mGluR agonist in the somata of acutely dissociated rat hippocampal neurons using microfluorometry. We found that DHPG activates mGluR5 to mobilize intracellular Ca(2+ from ryanodine-sensitive stores via cyclic adenosine diphosphate ribose (cADPR, while the PLC/IP(3 signaling pathway was not involved in Ca(2+ mobilization. The application of glutamate, which depolarized the membrane potential by 28.5±4.9 mV (n = 4, led to transient Ca(2+ mobilization by mGluR5 and Ca(2+ influx through L-type Ca(2+ channels. We found no evidence that mGluR5-mediated Ca(2+ release and Ca(2+ influx through L-type Ca(2+ channels interact to generate supralinear Ca(2+ transients. Our study provides novel insights into the mechanisms of intracellular Ca(2+ mobilization by mGluR5 in the somata of hippocampal neurons.

Apoptosis of leukemia K562 and Molt-4 cells induced by emamectin benzoate involving mitochondrial membrane potential loss and intracellular Ca2+ modulation.

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Yun, Xinming; Rao, Wenbing; Xiao, Ciying; Huang, Qingchun

2017-06-01

Leukemia threatens millions of people's health and lives, and the pesticide-induced leukemia has been increasingly concerned because of the etiologic exposure. In this paper, cytotoxic effect of emamectin benzoate (EMB), an excellent natural-product insecticide, was evaluated through monitoring cell viability, cell apoptosis, mitochondrial membrane potential and intracellular Ca 2+ concentration ([Ca 2+ ] i ) in leukemia K562 and Molt-4 cells. Following the exposure to EMB, cell viability was decreased and positive apoptosis of K562 and Molt-4 cells was increased in a concentration- and time- dependent fashion. In the treatment of 10μM EMB, apoptotic cells accounted for 93.0% to K562 cells and 98.9% to Molt-4 cells based on the control, meanwhile, 63.47% of K562 cells and 81.15% of Molt-4 cells exhibited late apoptotic and necrotic features with damaged cytoplasmic membrane. 48h exposure to 10μM EMB increased significantly the great number of cells with mitochondrial membrane potential (MMP) loss, and the elevation of [Ca 2+ ] i level was peaked and persisted within 70s in K562 cells whilst 50s in Molt-4 cells. Moreover, a stronger cytotoxicity of EMB was further observed than that of imatinib. The results authenticate the efficacious effect of EMB as a potential anti-leukemia agent and an inconsistency with regard to insecticide-induced leukemia. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes

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Glushakova Svetlana

2013-01-01

Full Text Available Abstract Background Egress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites. Methods Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ homeostasis on the egress programme. Results A steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. Conclusions The parasite egress

Kinetic identification of an intracellular calcium compartment sensitive to phosphate and dinitrophenol in intact isolated rabbit aorta

International Nuclear Information System (INIS)

Hai, C.M.; Phair, R.D.

1986-01-01

Previous work from this laboratory revealed the presence of at least three distinct intracellular calcium compartments in intact segments of rabbit aorta. In this study one of these intracellular compartments is shown to be sensitive to dinitrophenol and to increased extracellular phosphate. Intact aortic segments were loaded with 45 Ca in bicarbonate-buffered physiologic salt solution for 1 hour, and then transferred to a flow-through chamber perfused with physiologic salt solution. Effluent from the chamber was collected for 8 hours, and 45 Ca efflux curves were analyzed using compartmental analysis. When aortic segments were loaded and washed out in dinitrophenol, the slowest component of the efflux curve was less prominent; in high phosphate it was more prominent. The rate constant changes required to account for these data were primarily in the exchange between the cytosolic and slowest intracellular calcium compartment, suggesting that the slowest calcium compartment resolved during the 8-hour washout was mitochondrial. This compartment contained 5.4 +/- 3.2 nmol calcium/g wet wt. tissue. The calcium flux across its membranes was 0.32 +/- 0.04 nmol min-1g-1. Because this flux is much smaller than the plasma-membrane calcium flux, we suggest that, in normal physiological circumstances, plasma-membrane extrusion is more important for the removal of Ca from the smooth muscle cytosol than is uptake into this slow intracellular compartment

Extracellular ATP elevates cytoplasmatic free Ca2+ in HeLa cells by the interaction with a 5'-nucleotide receptor

NARCIS (Netherlands)

Smit, M J; Leurs, R; Bloemers, S M; Tertoolen, L G; Bast, A; De Laat, S W; Timmerman, H

1993-01-01

In the present study we have characterized the effects of ATP and several other nucleotides on the intracellular Ca2+ levels of HeLa cells. Using fura-2 microscopy fluorescence measurements, the ATP-mediated increase in intracellular Ca2+ was shown to consist of a rapid rise which decreased after a

Glu¹⁰⁶ in the Orai1 pore contributes to fast Ca²⁺-dependent inactivation and pH dependence of Ca²⁺ release-activated Ca²⁺ (CRAC) current.

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Scrimgeour, Nathan R; Wilson, David P; Rychkov, Grigori Y

2012-01-15

FCDI (fast Ca²⁺-dependent inactivation) is a mechanism that limits Ca²⁺ entry through Ca²⁺ channels, including CRAC (Ca²⁺ release-activated Ca²⁺) channels. This phenomenon occurs when the Ca²⁺ concentration rises beyond a certain level in the vicinity of the intracellular mouth of the channel pore. In CRAC channels, several regions of the pore-forming protein Orai1, and STIM1 (stromal interaction molecule 1), the sarcoplasmic/endoplasmic reticulum Ca²⁺ sensor that communicates the Ca²⁺ load of the intracellular stores to Orai1, have been shown to regulate fast Ca²⁺-dependent inactivation. Although significant advances in unravelling the mechanisms of CRAC channel gating have occurred, the mechanisms regulating fast Ca²⁺-dependent inactivation in this channel are not well understood. We have identified that a pore mutation, E106D Orai1, changes the kinetics and voltage dependence of the ICRAC (CRAC current), and the selectivity of the Ca²⁺-binding site that regulates fast Ca²⁺-dependent inactivation, whereas the V102I and E190Q mutants when expressed at appropriate ratios with STIM1 have fast Ca²⁺-dependent inactivation similar to that of WT (wild-type) Orai1. Unexpectedly, the E106D mutation also changes the pH dependence of ICRAC. Unlike WT ICRAC, E106D-mediated current is not inhibited at low pH, but instead the block of Na⁺ permeation through the E106D Orai1 pore by Ca²⁺ is diminished. These results suggest that Glu¹⁰⁶ inside the CRAC channel pore is involved in co-ordinating the Ca²⁺-binding site that mediates fast Ca²⁺-dependent inactivation.

Regulation of intracellular calcium in resting and stimulated rat basophilic leukemia cells

International Nuclear Information System (INIS)

Mohr, F.C.

1988-01-01

Intracellular calcium regulation was studied in a cell line of mast cells, the rat basophilic leukemia (RBL) cells with the purpose of determining (1) The properties of the plasma membrane calcium permeability pathway and (2) The role of intracellular calcium stores. The first set of experiments showed that depolarization did not induce calcium entry or secretion in resting cells and did inhibit antigen-stimulated calcium uptake and secretion. In the second set of experiments the ionic basis of antigen-induced depolarization was studied using the fluorescent potential-sensitive probe bis-oxonol. The properties of the calcium entry pathway were more consistent with a calcium channel than a calcium transport mechanism such as Na:Ca exchange. The third set of experiments examined the effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) on RBL cells. CCCP inhibited antigen-stimulated 45 Ca uptake and secretion by depolarizing the plasma membrane

Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase

DEFF Research Database (Denmark)

Thastrup, Ole; Cullen, P J; Drøbak, B K

1990-01-01

. This hypothesis is strongly supported by the demonstration that thapsigargin causes a rapid inhibition of the Ca2(+)-activated ATPase activity of rat liver microsomes, with an identical dose dependence to that seen in whole cell or isolated microsome Ca2+ discharge. The inhibition of the endoplasmic reticulum...

Regulation of Endoplasmic Reticulum–Mitochondria Ca2+ Transfer and Its Importance for Anti-Cancer Therapies

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Gaia Pedriali

2017-08-01

Full Text Available Inter-organelle membrane contact sites are emerging as major sites for the regulation of intracellular Ca2+ concentration and distribution. Here, extracellular stimuli operate on a wide array of channels, pumps, and ion exchangers to redistribute intracellular Ca2+ among several compartments. The resulting highly defined spatial and temporal patterns of Ca2+ movement can be used to elicit specific cellular responses, including cell proliferation, migration, or death. Plasma membrane (PM also can directly contact mitochondria and endoplasmic reticulum (ER through caveolae, small invaginations of the PM that ensure inter-organelle contacts, and can contribute to the regulation of numerous cellular functions through scaffolding proteins such as caveolins. PM and ER organize specialized junctions. Here, many components of the receptor-dependent Ca2+ signals are clustered, including the ORAI1-stromal interaction molecule 1 complex. This complex constitutes a primary mechanism for Ca2+ entry into non-excitable cells, modulated by intracellular Ca2+. Several contact sites between the ER and mitochondria, termed mitochondria-associated membranes, show a very complex and specialized structure and host a wide number of proteins that regulate Ca2+ transfer. In this review, we summarize current knowledge of the particular action of several oncogenes and tumor suppressors at these specialized check points and analyze anti-cancer therapies that specifically target Ca2+ flow at the inter-organelle contacts to alter the metabolism and fate of the cancer cell.

[Alpha but not beta-adrenergic stimulation has a positive inotropic effect associated with alkalinization of intracellular pH].

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Gambassi, G; Lakatta, E G; Capogrossi, M C

1991-01-01

There is increasing evidence that alpha-adrenoceptors also exist in the myocardium and that an increase in force of contraction may be produced by stimulation of these sites. This positive inotropism seems to be dependent either on an increased amount of Ca++ released into the cytosol with each action potential or on increased myofilament responsiveness. In contrast, beta-adrenergic stimulation reduces the sensitivity of the contractile proteins and the positive inotropic effect is due to the activation of L-type calcium channels on the sarcolemma. We used single, isolated, enzymatically dissociated, adult rat ventricular myocytes. Cells were loaded either with the ester derivative of the Ca++ probe Indo-1 or with the intracellular pH probe Snarf-1 and at the same time we measured the contractile parameters and monitored the fluorescence as an index of intracellular calcium concentration or pH value. The single cells (bicarbonate buffer continuously gassed with O2 95%, CO2 5%, Ca++ 1.5 mM, field stimulation 0.5 Hz) were exposed to phenylephrine (50 microM) and nadolol (1 microM). Alpha-adrenergic stimulation increased twitch amplitude (delta ES = 1.93 +/- 0.77, n = 8; p less than 0.05) and showed only a slight increase in Ca++ transient. On the other end, the positive inotropic effect (delta ES = 2.84 +/- 0.86, n = 4; p less than 0.02) obtained with beta-adrenergic stimulation (isoproterenol 50 nM, bicarbonate buffer, Ca++ 0.5 mM, field stimulation 0.2 Hz) was always associated with a large increase in intracellular Ca++ concentration. Isoproterenol did not change intracellular pH (delta pH = 0.006 +/- 0.006, n = 4; NS) while phenylephrine increased it significantly (delta pH = 0.055 +/- 0.011, n = 8; p less than 0.002). Moreover, there was a statistically significant correlation between delta ES and delta pH (R2 = 0.532; p less than 0.05) when phenylephrine was present. This alkalinization as well as the increased contractility was antagonized by treatment with

Amyloid beta-peptide(25-35) changes [Ca2+] in hippocampal neurons

DEFF Research Database (Denmark)

Mogensen, Helle Smidt; Beatty, D M; Morris, S J

1998-01-01

of A beta(25-35) on [Ca2+]i and intracellular H+ concentration ([H+]i) in single hippocampal neurons by real time fluorescence imaging using the Ca(2+)- and H(+)-specific ratio dyes, indo-1 and SNARF-1. Incubation of these cultures with A beta(25-35) for 3-12 days in vitro increased [Ca2+]i and [H......+]i in large, NMDA-responsive neurons....

DA-6034-induced mucin secretion via Ca2+-dependent pathways through P2Y receptor stimulation.

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Lee, Hun; Kim, Eung Kweon; Kim, Ji Yeon; Yang, Yu-Mi; Shin, Dong Min; Kang, Kyung Koo; Kim, Tae-im

2014-09-11

We evaluated whether DA-6034 is involved in mucin secretion via P2Y receptor activation and/or intracellular Ca2+ concentration ([Ca2+]i) change. Also, we investigated the effect of P2Y receptor inhibitors or Ca2+ chelators on the DA-6034-induced mucin secretion and [Ca2+]i increases. Effects of DA-6034 on mucin expression in primary, cultured, conjunctival epithelial cells was studied using RT-PCR, Western blot analysis, and periodic acid-schiff (PAS) staining. To evaluate thin film layer thickness generated by mucin and fluid secretion, cells were incubated in DA-6034 with/without P2Y antagonists or extracellular/intracellular Ca2+ chelators, and were imaged with confocal microscope using Texas Red-dextran dye. In addition, DA-6034-induced Ca2+-dependent Cl- channels opening was evaluated using perforated patch clamp. Fluo-4/AM was used to measure changes in [Ca2+]i induced by DA-6034 in Ca2+-free or Ca2+-containing buffered condition, as well as P2Y antagonists. DA-6034 induced the expression of mucin genes, production of mucin protein, and increase of number of mucin-secreting cells. P2Y antagonists inhibited DA-6034-induced mucin and fluid secretion, which was also affected by extracellular/intracellular Ca2+ chelators. DA-6034 stimulated Cl- channel opening and [Ca2+]i elevation. Further, [Ca2+]i increases induced by DA-6034 were lacking in either P2Y antagonists or Ca2+-free buffered condition, and diminished when endoplasmic reticulum Ca2+ was depleted by cyclopiazonic acid in Ca2+-free buffered condition. This study demonstrated that DA-6034 has a potential to induce mucin secretion via Ca2+-dependent pathways through P2Y receptors in multilayer, cultured, human conjunctival epithelial cells. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Up-regulation of Ca2+/CaMKII/CREB signaling in salicylate-induced tinnitus in rats.

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Zhao, Jiuhan; Wang, Biao; Wang, Xiaohong; Shang, Xiuli

2018-02-09

The purpose of the study was to investigate the changes of Ca 2+ /calmodulin-dependent protein kinases II (CaMKII)/cAMP response element-binding protein (CREB) signaling pathway in a rat tinnitus model. Eighteen Wistar rats were randomly divided into three groups: normal control (NC), normal saline (NS), and tinnitus model (TM) groups. Tinnitus model was induced by intraperitoneal injection of salicylate. The concentration of intracellular calcium level in auditory cortex cells was determined using Fura-2 acetoxymethyl ester (Fura-2 AM) method with fluorospectrophotometer. Expressions of calmodulin (CaM), N-methyl-D-aspartate receptor 2B subunit (NR2B), calcium-calmodulin kinase II (CaMKII), and cAMP response element-binding protein (CREB) were detected with Western blot. Tinnitus model was successfully established by the intraperitoneal administration of salicylate in rats. Compared with rats in NC and NS groups, salicylate administration significantly elevated CaM, NR2B, phospho-CaMKII and phospho-CREB expression in auditory cortex from tinnitus model group (p salicylate administration causes tinnitus symptoms and elevates Ca 2+ /CaMKII/CREB signaling pathway in auditory cortex cells. Our study likely provides a new understanding of the development of tinnitus.

A shell-formation related carbonic anhydrase in Crassostrea gigas modulates intracellular calcium against CO2 exposure: Implication for impacts of ocean acidification on mollusk calcification.

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Wang, Xiudan; Wang, Mengqiang; Jia, Zhihao; Song, Xiaorui; Wang, Lingling; Song, Linsheng

2017-08-01

Ocean acidification (OA) could decrease the shells and skeletons formation of mollusk by reducing the availability of carbonate ions at calcification sites. Carbonic anhydrases (CAs) convert CO 2 to HCO 3 - and play important roles in biomineralization process from invertebrate to vertebrate. In the present study, a CA (designated as CgCA) was identified and characterized in Pacific oyster C. gigas. The cDNA of CgCA was of 927bp encoding a predicted polypeptide of 308 amino acids with a signal peptide and a CA catalytic function domain. The mRNA transcripts of CgCA were constitutively expressed in all tested tissues with the highest levels in mantle and hemocytes. During the early development period, the mRNA transcripts of CgCA could be detected in all the stages with the highest level in D-veliger larvae. Elevated CO 2 increased the mRNA transcripts of CgCA in muscle, mantle, hepatopancreas, gill and hemocytes significantly (p<0.05) and induced the translocation of CgCA in hemocytes and mantle. Moreover, elevated CO 2 also caused the decrease of intracellular Ca 2+ in hemocytes (p<0.05). The inhibition of CA by acetazolamide and suppression of CgCA gene via RNA interference could increase the intracellular Ca 2+ in hemocytes (p<0.05). Besides, the decrease of intracellular Ca 2+ content caused by Ca 2+ reagent ionomycin could affect localization of CgCA in mantle tissue. The results indicated CgCA played essential roles in calcification and elevated CO 2 accelerated the mutual modulation between calcium and CgCA, implying reduced calcification rate and dissolved shells under OA. Copyright © 2017 Elsevier B.V. All rights reserved.

a-Band Oscillations in Intracellular Membrane Potentials of Dentate Gyrus Neurons in Awake Rodents

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Anderson, Ross W.; Strowbridge, Ben W.

2014-01-01

The hippocampus and dentate gyrus play critical roles in processing declarative memories and spatial information. Dentate granule cells, the first relay in the trisynaptic circuit through the hippocampus, exhibit low spontaneous firing rates even during locomotion. Using intracellular recordings from dentate neurons in awake mice operating a…

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

International Nuclear Information System (INIS)

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

2013-01-01

A non-thermal plasma is known to induce apoptosis of various cells but the mechanism is not yet clear. A eukaryotic model organism Saccharomyces cerevisiaewas used to investigate the cellular and biochemical regulations of cell apoptosis and cell cycle after an atmospheric-pressure cold plasma treatment. More importantly, intracellular calcium (Ca 2+ ) was first involved in monitoring the process of plasma-induced apoptosis in this study. We analysed the cell apoptosis and cell cycle by flow cytometry and observed the changes in intracellular reactive oxygen species (ROS) and Ca 2+ concentration, cell mitochondrial membrane potential (Δψ m ) as well as nuclear DNA morphology via fluorescence staining assay. All experimental results indicated that plasma-generated ROS leads to the accumulation of intracellular ROS and Ca 2+ that ultimately contribute to apoptosis associated with cell cycle arrest at G1 phase through depolarization of Δψ m and fragmenting nuclear DNA. This work provides a novel insight into the physical and biological mechanism of apoptosis induced by a plasma which could benefit for promoting the development of plasmas applied to cancer therapy. (paper)

DISC1 Modulates Neuronal Stress Responses by Gate-Keeping ER-Mitochondria Ca2+ Transfer through the MAM

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Sung Jin Park

2017-12-01

Full Text Available Summary: A wide range of Ca2+-mediated functions are enabled by the dynamic properties of Ca2+, all of which are dependent on the endoplasmic reticulum (ER and mitochondria. Disrupted-in-schizophrenia 1 (DISC1 is a scaffold protein that is involved in the function of intracellular organelles and is linked to cognitive and emotional deficits. Here, we demonstrate that DISC1 localizes to the mitochondria-associated ER membrane (MAM. At the MAM, DISC1 interacts with IP3R1 and downregulates its ligand binding, modulating ER-mitochondria Ca2+ transfer through the MAM. The disrupted regulation of Ca2+ transfer caused by DISC1 dysfunction leads to abnormal Ca2+ accumulation in mitochondria following oxidative stress, which impairs mitochondrial functions. DISC1 dysfunction alters corticosterone-induced mitochondrial Ca2+ accumulation in an oxidative stress-dependent manner. Together, these findings link stress-associated neural stimuli with intracellular ER-mitochondria Ca2+ crosstalk via DISC1, providing mechanistic insight into how environmental risk factors can be interpreted by intracellular pathways under the control of genetic components in neurons. : Park et al. show that DISC1 regulates ER-mitochondria Ca2+ transfer through mitochondria-associated ER membrane (MAM. DISC1 dysfunction at MAM increases ER-mitochondria Ca2+ transfer during oxidative stress and excessive amounts of corticosterone, which impairs mitochondrial function. Keywords: DISC1, MAM, mitochondria, Ca2+, IP3R1, oxidative stress

[Cognitive Function and Calcium. Structures and functions of Ca2+-permeable channels].

Science.gov (United States)

Kaneko, Shuji

2015-02-01

Calcium is essential for living organisms where the increase in intracellular Ca2+ concentration functions as a second messenger for many cellular processes including synaptic transmission and neural plasticity. The cytosolic concentration of Ca2+ is finely controlled by many Ca2+-permeable ion channels and transporters. The comprehensive view of their expression, function, and regulation will advance our understanding of neural and cognitive functions of Ca2+, which leads to the future drug discovery.

Ca2+-induced uncoupling of Aplysia bag cell neurons.

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Dargaei, Zahra; Standage, Dominic; Groten, Christopher J; Blohm, Gunnar; Magoski, Neil S

2015-02-01

Electrical transmission is a dynamically regulated form of communication and key to synchronizing neuronal activity. The bag cell neurons of Aplysia are a group of electrically coupled neuroendocrine cells that initiate ovulation by secreting egg-laying hormone during a prolonged period of synchronous firing called the afterdischarge. Accompanying the afterdischarge is an increase in intracellular Ca2+ and the activation of protein kinase C (PKC). We used whole cell recording from paired cultured bag cell neurons to demonstrate that electrical coupling is regulated by both Ca2+ and PKC. Elevating Ca2+ with a train of voltage steps, mimicking the onset of the afterdischarge, decreased junctional current for up to 30 min. Inhibition was most effective when Ca2+ entry occurred in both neurons. Depletion of Ca2+ from the mitochondria, but not the endoplasmic reticulum, also attenuated the electrical synapse. Buffering Ca2+ with high intracellular EGTA or inhibiting calmodulin kinase prevented uncoupling. Furthermore, activating PKC produced a small but clear decrease in junctional current, while triggering both Ca2+ influx and PKC inhibited the electrical synapse to a greater extent than Ca2+ alone. Finally, the amplitude and time course of the postsynaptic electrotonic response were attenuated after Ca2+ influx. A mathematical model of electrically connected neurons showed that excessive coupling reduced recruitment of the cells to fire, whereas less coupling led to spiking of essentially all neurons. Thus a decrease in electrical synapses could promote the afterdischarge by ensuring prompt recovery of electrotonic potentials or making the neurons more responsive to current spreading through the network. Copyright © 2015 the American Physiological Society.

Resveratrol and polydatin as modulators of Ca2+ mobilization in the cardiovascular system.

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Liu, Wenjuan; Chen, Peiya; Deng, Jianxin; Lv, Jingzhang; Liu, Jie

2017-09-01

In the cardiovascular system, Ca 2+ controls cardiac excitation-contraction coupling and vascular contraction and dilation. Disturbances in intracellular Ca 2+ homeostasis induce malfunctions of the cardiovascular system, including cardiac pump dysfunction, arrhythmia, remodeling, and apoptosis, as well as hypertension and impairment of vascular reactivity. Therefore, developing drugs and strategies manipulating Ca 2+ handling are highly valued in the treatment of cardiovascular disease. Resveratrol (Res) and polydatin (PD), a Res glucoside, have been well established to have beneficial effects on improving cardiovascular function. Studies from our laboratory and others have demonstrated that they exhibit inotropic effects on normal heart and therapeutic effects on hypertension, cardiac ischemia/reperfusion injury, hypertrophy, and heart failure by manipulating Ca 2+ mobilization. The actions of Res and PD on Ca 2+ signals delicately manipulated by multiple Ca 2+ -handling proteins are pleiotropic and somewhat controversial, depending on cellular species and intracellular oxidative status. Here, we focus on the effects of Res and PD on controlling Ca 2+ homeostasis in the heart and vasculature under normal and diseased conditions and highlight the key direct and indirect molecules mediating these effects. © 2017 New York Academy of Sciences.

Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors.

Science.gov (United States)

Bkaily, Ghassan; El-Bizri, Nesrine; Bui, Michel; Sukarieh, Rami; Jacques, Danielle; Fu, Michael L X

2003-03-01

The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.

Altered Ca fluxes and contractile state during pH changes in cultured heart cells

International Nuclear Information System (INIS)

Kim, D.; Smith, T.W.

1987-01-01

The authors studied mechanisms underlying changes in myocardial contractile state produced by intracellular (pH/sub i/) or extracellular (pH 0 ) changes in pH using cultured chick embryo ventricular cells. A change in pH 0 of HEPES-buffered medium from 7.4 to 6.0 or to 8.8 changed the amplitude of cell motion by -85 or +60%, and 45 Ca uptake at 10 s by -29 or +22%, respectively. The pH 0 induced change in Ca uptake was not sensitive to nifedipine but was Na gradient dependent. Changes in pH/sub i/ produced by NH 4 Cl or preincubation in media at pH values ranging from 6.0 to 8.8 failed to alter significantly 45 Ca uptake or efflux. However, larger changes in pH/sub i/ were associated with altered Ca uptake. Changes in pH 0 from 7.5 to 6.0 or to 8.8 were associated with initial changes in 45 Ca efflux by +17 or -18%, respectively, and these effects were not Na dependent. Exposure of cells to 20 mM NH 4 Cl produced intracellular alkalinization and a positive inotropic effect, whereas subsequent removal of NH 4 Cl caused intracellular acidification and a negative inotropic effect. There was, however, a lack of close temporal relationships between pH/sub i/ and contractile state. These results indicated that pH 0 -induced changes in contractile state in cultured heart cells are closely correlated with altered transarcolemmal Ca movements and presumably are due to these Ca flux changes

MicroRNA-145 suppresses ROS-induced Ca2+ overload of cardiomyocytes by targeting CaMKIIδ

International Nuclear Information System (INIS)

Cha, Min-Ji; Jang, Jin-Kyung; Ham, Onju; Song, Byeong-Wook; Lee, Se-Yeon; Lee, Chang Yeon; Park, Jun-Hee; Lee, Jiyun; Seo, Hyang-Hee; Choi, Eunhyun; Jeon, Woo-min; Hwang, Hye Jin; Shin, Hyun-Taek

2013-01-01

Highlights: •CaMKIIδ mediates H 2 O 2 -induced Ca 2+ overload in cardiomyocytes. •miR-145 can inhibit Ca 2+ overload. •A luciferase assay confirms that miR-145 functions as a CaMKIIδ-targeting miRNA. •Overexpression of miR-145 regulates CaMKIIδ-related genes and ameliorates apoptosis. -- Abstract: A change in intracellular free calcium (Ca 2+ ) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca 2+ signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca 2+ -mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H 2 O 2 -mediated Ca 2+ overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca 2+ overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca 2+ -related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca 2+ overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses

Depletion of intracellular zinc from neurons by use of an extracellular chelator in vivo and in vitro.

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Frederickson, Christopher J; Suh, Sang W; Koh, Jae-Young; Cha, Yoo K; Thompson, Richard B; LaBuda, Christopher J; Balaji, Rengarajan V; Cuajungco, Math P

2002-12-01

The membrane-impermeable chelator CaEDTA was introduced extracellularly among neurons in vivo and in vitro for the purpose of chelating extracellular Zn(2+). Unexpectedly, this treatment caused histochemically reactive Zn(2+) in intracellular compartments to drop rapidly. The same general result was seen with intravesicular Zn(2+), which fell after CaEDTA infusion into the lateral ventricle of the brain, with perikaryal Zn(2+) in Purkinje neurons (in vivo) and with cortical neurons (in vitro). These findings suggest either that the volume of zinc ion efflux and reuptake is higher than previously suspected or that EDTA can enter cells and vesicles. Caution is therefore warranted in attempting to manipulate extracellular or intracellular Zn(2+) selectively.

The Relationship between Respiration-Related Membrane Potential Slow Oscillations and Discharge Patterns in Mitral/Tufted Cells: What Are the Rules?

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Briffaud, Virginie; Fourcaud-Trocmé, Nicolas; Messaoudi, Belkacem; Buonviso, Nathalie; Amat, Corine

2012-01-01

Background A slow respiration-related rhythm strongly shapes the activity of the olfactory bulb. This rhythm appears as a slow oscillation that is detectable in the membrane potential, the respiration-related spike discharge of the mitral/tufted cells and the bulbar local field potential. Here, we investigated the rules that govern the manifestation of membrane potential slow oscillations (MPSOs) and respiration-related discharge activities under various afferent input conditions and cellular excitability states. Methodology and Principal Findings We recorded the intracellular membrane potential signals in the mitral/tufted cells of freely breathing anesthetized rats. We first demonstrated the existence of multiple types of MPSOs, which were influenced by odor stimulation and discharge activity patterns. Complementary studies using changes in the intracellular excitability state and a computational model of the mitral cell demonstrated that slow oscillations in the mitral/tufted cell membrane potential were also modulated by the intracellular excitability state, whereas the respiration-related spike activity primarily reflected the afferent input. Based on our data regarding MPSOs and spike patterns, we found that cells exhibiting an unsynchronized discharge pattern never exhibited an MPSO. In contrast, cells with a respiration-synchronized discharge pattern always exhibited an MPSO. In addition, we demonstrated that the association between spike patterns and MPSO types appeared complex. Conclusion We propose that both the intracellular excitability state and input strength underlie specific MPSOs, which, in turn, constrain the types of spike patterns exhibited. PMID:22952828

Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma.

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Chen, Jun; Miller, Marina; Unno, Hirotoshi; Rosenthal, Peter; Sanderson, Michael J; Broide, David H

2017-09-07

Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3 Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3 Zp3-Cre mice, which do not have a blood supply. Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3 Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes.

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Zhang, Xiao-Dong; Coulibaly, Zana A; Chen, Wei Chun; Ledford, Hannah A; Lee, Jeong Han; Sirish, Padmini; Dai, Gu; Jian, Zhong; Chuang, Frank; Brust-Mascher, Ingrid; Yamoah, Ebenezer N; Chen-Izu, Ye; Izu, Leighton T; Chiamvimonvat, Nipavan

2018-03-16

Small-conductance Ca 2+ -activated K + (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca 2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca 2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca 2+ microdomains necessary for SK channel activation. SK currents coupled with Ca 2+ influx via L-type Ca 2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca 2+ store, suggesting that LTCCs provide the immediate Ca 2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Ca v 1.2 Ca 2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Ca v 1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca 2+ signaling.

Alpha-2 adrenoceptors and imidazoline receptors in cardiomyocytes mediate counterbalancing effect of agmatine on NO synthesis and intracellular calcium handling.

Science.gov (United States)

Maltsev, Alexander V; Kokoz, Yuri M; Evdokimovskii, Edward V; Pimenov, Oleg Y; Reyes, Santiago; Alekseev, Alexey E

2014-03-01

Evidence suggests that intracellular Ca(2+) levels and contractility of cardiomyocytes can be modulated by targeting receptors other than already identified adrenergic or non-adrenergic sarcolemmal receptors. This study uncovers the presence in myocardial cells of adrenergic α2 (α2-AR) and imidazoline I1 (I1R) receptors. In isolated left ventricular myocytes generating stationary spontaneous Ca(2+) transients in the absence of triggered action potentials, the prototypic agonist of both receptors agmatine can activate corresponding signaling cascades with opposing outcomes on nitric oxide (NO) synthesis and intracellular Ca(2+) handling. Specifically, activation of α2-AR signaling through PI3 kinase and Akt/protein kinase B stimulates NO production and abolishes Ca(2+) transients, while targeting of I1R signaling via phosphatidylcholine-specific phospholipase C (PC-PLC) and protein kinase C (PKC) suppresses NO synthesis and elevates averaged intracellular Ca(2+). We identified that endothelial NO synthase (eNOS) is a major effector for both signaling cascades. According to the established eNOS transitions between active (Akt-dependent) and inactive (PKC-dependent) conformations, we suggest that balance between α2-AR and I1R signaling pathways sets eNOS activity, which by defining operational states of myocellular sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) can adjust Ca(2+) re-uptake and thereby cardiac inotropy. These results indicate that the conventional catalog of cardiomyocyte sarcolemmal receptors should be expanded by the α2-AR and I1R populations, unveiling previously unrecognized targets for endogenous ligands as well as for existing and potential pharmacological agents in cardiovascular medicine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Eukaryotic translation initiation factor 3 subunit e controls intracellular calcium homeostasis by regulation of cav1.2 surface expression.

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Pawel Buda

Full Text Available Inappropriate surface expression of voltage-gated Ca(2+channels (CaV in pancreatic ß-cells may contribute to the development of type 2 diabetes. First, failure to increase intracellular Ca(2+ concentrations at the sites of exocytosis impedes insulin release. Furthermore, excessive Ca(2+ influx may trigger cytotoxic effects. The regulation of surface expression of CaV channels in the pancreatic β-cells remains unknown. Here, we used real-time 3D confocal and TIRFM imaging, immunocytochemistry, cellular fractionation, immunoprecipitation and electrophysiology to study trafficking of L-type CaV1.2 channels upon β-cell stimulation. We found decreased surface expression of CaV1.2 and a corresponding reduction in L-type whole-cell Ca(2+ currents in insulin-secreting INS-1 832/13 cells upon protracted (15-30 min stimulation. This internalization occurs by clathrin-dependent endocytosis and could be prevented by microtubule or dynamin inhibitors. eIF3e (Eukaryotic translation initiation factor 3 subunit E is part of the protein translation initiation complex, but its effect on translation are modest and effects in ion channel trafficking have been suggested. The factor interacted with CaV1.2 and regulated CaV1.2 traffic bidirectionally. eIF3e silencing impaired CaV1.2 internalization, which resulted in an increased intracellular Ca(2+ load upon stimulation. These findings provide a mechanism for regulation of L-type CaV channel surface expression with consequences for β-cell calcium homeostasis, which will affect pancreatic β-cell function and insulin production.

3-Methylcholanthrene inhibits lymphocyte proliferation and increases intracellular calcium levels in common carp (Cyprinus carpio L)

International Nuclear Information System (INIS)

Reynaud, S.; Duchiron, C.; Deschaux, P.

2003-01-01

Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. Many authors have focused on macrophage activities in fish exposed to PAHs. However, fewer studies have reported decrease in specific immunity in such fish. We investigated the intracellular mechanisms by which the 3-methylcholanthrene (3-MC) decreased lymphocyte proliferation in carp. T- and B-lymphocyte proliferation induced by Concanavalin A (Con A) and lipopolysaccharide (LPS) were inhibited by 3-MC (0.5-50 μM). 3-MC also produced a rapid and a sustained increase in intracellular calcium concentration ([Ca 2+ ] i ) (2 h minimum). However, the cytochrome P450 1A and Ah receptor inhibitor, α-naphtoflavone (a-NF), also inhibited lymphocyte proliferation and did not reverse the effects of 3-MC. Moreover, since a-NF and 3-MC increased [Ca 2+ ] i and inhibited lymphocyte proliferation it was possible that calcium release played a role in 3-MC-inhibited lymphocyte proliferation. The rise in [Ca 2+ ] i induced by 3-MC was potentiated by the inhibitor of the endoplasmic reticulum calcium ATPases, thapsigargin. Treating cells with 3-MC decreased calcium mobilization caused by thapsigargin. These results suggest that 3-MC acts on the endoplasmic reticulum, perhaps directly on calcium ATPases, to increase intracellular calcium levels in carp leucocytes

Gliadin peptides induce tissue transglutaminase activation and ER-stress through Ca2+ mobilization in Caco-2 cells.

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Ivana Caputo

Full Text Available BACKGROUND: Celiac disease (CD is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca(2+ homeostasis and tTG activity. METHODS/PRINCIPAL FINDINGS: We studied Ca(2+ homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31-43 and 57-68 rapidly mobilized Ca(2+ from intracellular stores. Specifically, peptide 31-43 mobilized Ca(2+ from the endoplasmic reticulum (ER and mitochondria, whereas peptide 57-68 mobilized Ca(2+ only from mitochondria. We also found that gliadin peptide-induced Ca(2+ mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31-43, but not peptide 57-68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31-43, but not of peptide 57-68, induces the expression of both genes. CONCLUSIONS: By inducing Ca(2+ mobilization from the ER, peptide 31-43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31-43 and 57-68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin-tTG crosslinking in enterocytes and specialized antigen-presenting cells.

The activity of spontaneous action potentials in developing hair cells is regulated by Ca(2+-dependence of a transient K+ current.

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Snezana Levic

Full Text Available Spontaneous action potentials have been described in developing sensory systems. These rhythmic activities may have instructional roles for the functional development of synaptic connections. The importance of spontaneous action potentials in the developing auditory system is underpinned by the stark correlation between the time of auditory system functional maturity, and the cessation of spontaneous action potentials. A prominent K(+ current that regulates patterning of action potentials is I(A. This current undergoes marked changes in expression during chicken hair cell development. Although the properties of I(A are not normally classified as Ca(2+-dependent, we demonstrate that throughout the development of chicken hair cells, I(A is greatly reduced by acute alterations of intracellular Ca(2+. As determinants of spike timing and firing frequency, intracellular Ca(2+ buffers shift the activation and inactivation properties of the current to more positive potentials. Our findings provide evidence to demonstrate that the kinetics and functional expression of I(A are tightly regulated by intracellular Ca(2+. Such feedback mechanism between the functional expression of I(A and intracellular Ca(2+ may shape the activity of spontaneous action potentials, thus potentially sculpting synaptic connections in an activity-dependent manner in the developing cochlea.

Ned-19 inhibition of parasite growth and multiplication suggests a role for NAADP mediated signalling in the asexual development of Plasmodium falciparum.

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Suárez-Cortés, Pablo; Gambara, Guido; Favia, Annarita; Palombi, Fioretta; Alano, Pietro; Filippini, Antonio

2017-09-12

Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca 2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca 2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca 2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca 2+ levels. This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca 2+ oscillations suggests a potential role of NAADP in regulating Ca 2+ signalling of P. falciparum.

Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-ATPase 4 in Ca2+-transporting epithelia

DEFF Research Database (Denmark)

Alexander, R Todd; Beggs, Megan R; Zamani, Reza

2015-01-01

role in transcellular Ca(2+) flux and investigated the localization and regulation of Pmca4 in Ca(2+)-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestine, while pan-specific Pmca antibodies...... the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In human kidney, a similar pattern of distribution was observed, with highest PMCA4 expression in NCC positive tubules. Electron microscopy demonstrated Pmca4 localization...... in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments, but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca(2+) balance, pointing...

Modeling Intracellular Oscillations and Polarity Transition in Fission Yeast

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Drake, Tyler; Das, Maitreyi; Verde, Fulvia; Vavylonis, Dimitrios

2011-03-01

Fission yeast, a pill-shaped model organism, restricts growth to its tips. These cells maintain an asymmetric growth state, growing at only one tip, until they meet length and cell-cycle requirements. With these met, they grow at both. The mechanism of this transition, new-end take-off (NETO), remains unclear. We find that NETO occurs due to long-range competition for fast-diffusing signaling protein Cdc42 between the old and new tips. From experimental results, we suppose that symmetric tips compete for Cdc42, which triggers growth. We describe a symmetric growth model based on competition between tips. This model restricts short cells to monopolar states while allowing longer cells to be bipolar. Autocatalytic Cdc42 recruiting at both cells tips leads to broken symmetry, and the recruiting cuts off as tip Cdc42 levels saturate. Non-linear differential equations describe the model, with stable attractors indicating valid distributions. Linear stability analysis and numerical methods identify stable fixed points over a twofold increase in cell length. The model reproduces qualitative behavior of the organism. We show that observed pole-to-pole Cdc42 oscillations may facilitate the polarity transition and discuss their relationship to the Min system in E. Coli.

Mechanisms underlying odorant-induced and spontaneous calcium signals in olfactory receptor neurons of spiny lobsters, Panulirus argus.

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Tadesse, Tizeta; Derby, Charles D; Schmidt, Manfred

2014-01-01

We determined if a newly developed antennule slice preparation allows studying chemosensory properties of spiny lobster olfactory receptor neurons under in situ conditions with Ca(2+) imaging. We show that chemical stimuli reach the dendrites of olfactory receptor neurons but not their somata, and that odorant-induced Ca(2+) signals in the somata are sufficiently stable over time to allow stimulation with a substantial number of odorants. Pharmacological manipulations served to elucidate the source of odorant-induced Ca(2+) transients and spontaneous Ca(2+) oscillations in the somata of olfactory receptor neurons. Both Ca(2+) signals are primarily mediated by an influx of extracellular Ca(2+) through voltage-activated Ca(2+) channels that can be blocked by CoCl2 and the L-type Ca(2+) channel blocker verapamil. Intracellular Ca(2+) stores contribute little to odorant-induced Ca(2+) transients and spontaneous Ca(2+) oscillations. The odorant-induced Ca(2+) transients as well as the spontaneous Ca(2+) oscillations depend on action potentials mediated by Na(+) channels that are largely TTX-insensitive but blocked by the local anesthetics tetracaine and lidocaine. Collectively, these results corroborate the conclusion that odorant-induced Ca(2+) transients and spontaneous Ca(2+) oscillations in the somata of olfactory receptor neurons closely reflect action potential activity associated with odorant-induced phasic-tonic responses and spontaneous bursting, respectively. Therefore, both types of Ca(2+) signals represent experimentally accessible proxies of spiking.

The effect of NO-donors on chloride efflux, intracellular Ca(2+) concentration and mRNA expression of CFTR and ENaC in cystic fibrosis airway epithelial cells.

Science.gov (United States)

Oliynyk, Igor; Hussain, Rashida; Amin, Ahmad; Johannesson, Marie; Roomans, Godfried M

2013-06-01

Since previous studies showed that the endogenous bronchodilator, S-nitrosglutathione (GSNO), caused a marked increase in CFTR-mediated chloride (Cl(-)) efflux and improved the trafficking of CFTR to the plasma membrane, and that also the nitric oxide (NO)-donor GEA3162 had a similar, but smaller, effect on Cl(-) efflux, it was investigated whether the NO-donor properties of GSNO were relevant for its effect on Cl(-) efflux from airway epithelial cells. Hence, the effect of a number of other NO-donors, sodium nitroprusside (SNP), S-nitroso-N-acetyl-DL-penicillamine (SNAP), diethylenetriamine/nitric oxide adduct (DETA-NO), and diethylenetriamine/nitric oxide adduct (DEA-NONOate) on Cl(-) efflux from CFBE (∆F508/∆F508-CFTR) airway epithelial cells was tested. Cl(-) efflux was determined using the fluorescent N-(ethoxycarbonylmethyl)-6-methoxyquinoliniu bromide (MQAE)-technique. Possible changes in the intracellular Ca(2+) concentration were tested by the fluorescent fluo-4 method in a confocal microscope system. Like previously with GSNO, after 4 h incubation with the NO-donor, an increased Cl(-) efflux was found (in the order SNAP>DETA-NO>SNP). The effect of DEA-NONOate on Cl(-) efflux was not significant, and the compound may have (unspecific) deleterious effects on the cells. Again, as with GSNO, after a short (5 min) incubation, SNP had no significant effect on Cl(-) efflux. None of the NO-donors that had a significant effect on Cl(-) efflux caused significant changes in the intracellular Ca(2+) concentration. After 4 h preincubation, SNP caused a significant increase in the mRNA expression of CFTR. SNAP and DEA-NONOate decreased the mRNA expression of all ENaC subunits significantly. DETA-NO caused a significant decrease only in α-ENaC expression. After a short preincubation, none of the NO-donors had a significant effect, neither on the expression of CFTR, nor on that of the ENaC subunits in the presence and absence of L-cysteine. It can be concluded that

Physiology of Ca2+ signalling in stem cells of different origins and differentiation stages

Czech Academy of Sciences Publication Activity Database

Forostyak, O.; Forostyak, S.; Kortus, Štěpán; Syková, E.; Verkhratsky, A.; Dayanithi, G.

2016-01-01

Roč. 59, 2-3 (2016), s. 57-66 ISSN 0143-4160 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : stem cells * Ca2+ channels * Ca2+ signalling * spontaneous Ca2+ oscillations Subject RIV: FH - Neurology Impact factor: 3.707, year: 2016

Fs-laser-induced Ca2+ concentration change during membrane perforation for cell transfection.

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Baumgart, J; Bintig, W; Ngezahayo, A; Lubatschowski, H; Heisterkamp, A

2010-02-01

Fs-laser based opto-perforation is a gentle method for gene transfer into sensitive cells such as stem cells or primary cells. The high selectivity and the low damage to the cell lead to a high efficiency of transfection. However, there are side effects which induce stress to the cell due to the exchange of intra- and extracellular media as well as the disintegration of the structure of biomolecules resulting from the laser exposure. Moreover, the mechanisms of the optical transfection are still unclear. In this paper, we present our study on calcium (Ca(2+)) homeostasis during cell surgery, especially during laser induced membrane perforation. We show that the manipulation of cells can induce an increase in the cytosolic Ca(2+) concentration. This increase was not observed if the manipulation of the cells was performed in absence of the extracellular calcium indicating the importance of the Ca(2+) uptake. We found, that the uptake of extracellular Ca(2+) strongly depends on the repetition rate and the irradiation time of the laser pulses. The exposure for several seconds to kHz pulses even induces Ca(2+) induced Ca(2+) release. Dependent on the location of perforation, probably in the vicinity of an intracellular Ca(2+) stock, an instantaneous intracellular Ca(2+) release can be induced. Since Ca(2+) could be involved in negative side effect by cell surgery, we propose an application of the optoperforation technique in nominal Ca(2+)-free external solution.

Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER

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Elisa Greotti

2016-09-01

Full Text Available Calcium ion (Ca2+ is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+] within its lumen ([Ca2+]ER can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET-based, Cameleon probe, named D4ER, characterized by suitable Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2. The lower Ca2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca2+ content in cells expressing mutated PS2, compared to controls.

TPC2 is a novel NAADP-sensitive Ca2+ release channel, operating as a dual sensor of luminal pH and Ca2+.

Science.gov (United States)

Pitt, Samantha J; Funnell, Tim M; Sitsapesan, Mano; Venturi, Elisa; Rietdorf, Katja; Ruas, Margarida; Ganesan, A; Gosain, Rajendra; Churchill, Grant C; Zhu, Michael X; Parrington, John; Galione, Antony; Sitsapesan, Rebecca

2010-11-05

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca(2+) required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca(2+) from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca(2+) release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca(2+) that will enable it to act as a Ca(2+) release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca(2+)] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca(2+) release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca(2+) release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μM but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

Growth-rate-dependent dynamics of a bacterial genetic oscillator

Science.gov (United States)

Osella, Matteo; Lagomarsino, Marco Cosentino

2013-01-01

Gene networks exhibiting oscillatory dynamics are widespread in biology. The minimal regulatory designs giving rise to oscillations have been implemented synthetically and studied by mathematical modeling. However, most of the available analyses generally neglect the coupling of regulatory circuits with the cellular “chassis” in which the circuits are embedded. For example, the intracellular macromolecular composition of fast-growing bacteria changes with growth rate. As a consequence, important parameters of gene expression, such as ribosome concentration or cell volume, are growth-rate dependent, ultimately coupling the dynamics of genetic circuits with cell physiology. This work addresses the effects of growth rate on the dynamics of a paradigmatic example of genetic oscillator, the repressilator. Making use of empirical growth-rate dependencies of parameters in bacteria, we show that the repressilator dynamics can switch between oscillations and convergence to a fixed point depending on the cellular state of growth, and thus on the nutrients it is fed. The physical support of the circuit (type of plasmid or gene positions on the chromosome) also plays an important role in determining the oscillation stability and the growth-rate dependence of period and amplitude. This analysis has potential application in the field of synthetic biology, and suggests that the coupling between endogenous genetic oscillators and cell physiology can have substantial consequences for their functionality.

Atrial remodelling in atrial fibrillation: CaMKII as a nodal proarrhythmic signal

Science.gov (United States)

Mesubi, Olurotimi O.; Anderson, Mark E.

2016-01-01

CaMKII is a serine–threonine protein kinase that is abundant in myocardium. Emergent evidence suggests that CaMKII may play an important role in promoting atrial fibrillation (AF) by targeting a diverse array of proteins involved in membrane excitability, cell survival, calcium homeostasis, matrix remodelling, inflammation, and metabolism. Furthermore, CaMKII inhibition appears to protect against AF in animal models and correct proarrhythmic, defective intracellular Ca2+ homeostasis in fibrillating human atrial cells. This review considers current concepts and evidence from animal and human studies on the role of CaMKII in AF. PMID:26762270

Neural cell adhesion molecule induces intracellular signaling via multiple mechanisms of Ca2+ homeostasis

DEFF Research Database (Denmark)

Kiryushko, Darya; Korshunova, Irina; Berezin, Vladimir

2006-01-01

. The first pathway was associated with activation of FGFR, phospholipase Cgamma, and production of diacylglycerol, and the second pathway involved Src-family kinases. Moreover, NCAM-mediated Ca2+ entry required activation of nonselective cation and T-type voltage-gated Ca2+ channels. These channels, together...

MicroRNA-145 suppresses ROS-induced Ca{sup 2+} overload of cardiomyocytes by targeting CaMKIIδ

Energy Technology Data Exchange (ETDEWEB)

Cha, Min-Ji [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Jang, Jin-Kyung [College of Pharmacy, Sookmyung Women’s University, 52 HyoChangWon-Gil, Yongsan-ku, Seoul 140-742 (Korea, Republic of); Ham, Onju; Song, Byeong-Wook; Lee, Se-Yeon [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Lee, Chang Yeon; Park, Jun-Hee [Department of Integrated Omics for Biomedical Sciences, Graduate School, Yonsei University, 50 Yonsei-ro, Seodamun-gu, Seoul 120-759 (Korea, Republic of); Lee, Jiyun; Seo, Hyang-Hee [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Choi, Eunhyun [Severance Integrative Research Institute for Cerebral and Cardiovascular Disease, Yonsei University Health System, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Jeon, Woo-min [Department of Animal Resource, Sahmyook University, Seoul 139-742 (Korea, Republic of); Hwang, Hye Jin [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Shin, Hyun-Taek [College of Pharmacy, Sookmyung Women’s University, 52 HyoChangWon-Gil, Yongsan-ku, Seoul 140-742 (Korea, Republic of); and others

2013-06-14

Highlights: •CaMKIIδ mediates H{sub 2}O{sub 2}-induced Ca{sup 2+} overload in cardiomyocytes. •miR-145 can inhibit Ca{sup 2+} overload. •A luciferase assay confirms that miR-145 functions as a CaMKIIδ-targeting miRNA. •Overexpression of miR-145 regulates CaMKIIδ-related genes and ameliorates apoptosis. -- Abstract: A change in intracellular free calcium (Ca{sup 2+}) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca{sup 2+} signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca{sup 2+}-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H{sub 2}O{sub 2}-mediated Ca{sup 2+} overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca{sup 2+} overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca{sup 2+}-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca{sup 2+} overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

DMS triggers apoptosis associated with the inhibition of SPHK1/NF-κB activation and increase in intracellular Ca2+ concentration in human cancer cells.

Science.gov (United States)

Chen, Kan; Pan, Qiuwei; Gao, Ying; Yang, Xinyan; Wang, Shibing; Peppelenbosch, Maikel P; Kong, Xiangdong

2014-01-01

N,N-Dimethyl-D-erythro-sphingosine (DMS) is known to induce cell apoptosis by specifically inhibiting sphingosine kinase 1 (SPHK1) and modulating the activity of cellular ceramide levels. The present study investigated the effects and the mechanism(s) of action of DMS in human lung cancer cells. We found that DMS dose-dependently suppressed cell proliferation and induced cell apoptosis in the human lung cancer cell line, A549. Mechanistically, treatment with DMS suppressed the activation of SPHK1 and nuclear factor-κB (NF-κB) p65, but increased intracellular [Ca2+]i in A549 cells. This study demonstrates that DMS triggers the apoptosis of human lung cancer cells through the modulation of SPHK1, NF-κB and calcium signaling. These molecules may represent targets for anticancer drug design.

Sphingosine inhibits the sarco(endo)plasmic reticulum Ca"2"+-ATPase (SERCA) activity

International Nuclear Information System (INIS)

Benaim, Gustavo; Pimentel, Adriana A.; Felibertt, Pimali; Mayora, Adriana; Colman, Laura; Sojo, Felipe; Rojas, Héctor; De Sanctis, Juan B.

2016-01-01

The increase in the intracellular Ca"2"+ concentration ([Ca"2"+]_i) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca"2"+]_i by inhibiting the sarco(endo)plasmic reticulum Ca"2"+-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca"2"+ pump. The results showed that addition of sphingosine produced a release of Ca"2"+ from the endoplasmic reticulum followed by a Ca"2"+ entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca"2"+]_I in mammalian cells.

Trigeminal ganglion neurons of mice show intracellular chloride accumulation and chloride-dependent amplification of capsaicin-induced responses.

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Nicole Schöbel

Full Text Available Intracellular Cl(- concentrations ([Cl(-](i of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG and olfactory sensory neurons (OSNs, Cl(- is accumulated by the Na(+-K(+-2Cl(- cotransporter 1 (NKCC1, resulting in a [Cl(-](i above electrochemical equilibrium and a depolarizing Cl(- efflux upon Cl(- channel opening. Here, we investigate the [Cl(-](i and function of Cl(- in primary sensory neurons of trigeminal ganglia (TG of wild type (WT and NKCC1(-/- mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl(-](i of WT TG neurons indicated active NKCC1-dependent Cl(- accumulation. Gamma-aminobutyric acid (GABA(A receptor activation induced a reduction of [Cl(-](i as well as Ca(2+ transients in a corresponding fraction of TG neurons. Ca(2+ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca(2+ channels (VGCCs. Ca(2+ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1 were diminished in NKCC1(-/- TG neurons, but elevated under conditions of a lowered [Cl(-](o suggesting a Cl(--dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS, we found expression of different Ca(2+-activated Cl(- channels (CaCCs in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca(2+ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1(-/- mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca(2+-activated Cl(--dependent signal amplification mechanism in TG neurons that requires intracellular Cl(- accumulation by NKCC1 and the activation of CaCCs.

Cerebellar Kainate Receptor-Mediated Facilitation of Glutamate Release Requires Ca2+-Calmodulin and PKA

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Rafael Falcón-Moya

2018-06-01

Full Text Available We elucidated the mechanisms underlying the kainate receptor (KAR-mediated facilitatory modulation of synaptic transmission in the cerebellum. In cerebellar slices, KA (3 μM increased the amplitude of evoked excitatory postsynaptic currents (eEPSCs at synapses between axon terminals of parallel fibers (PF and Purkinje neurons. KA-mediated facilitation was antagonized by NBQX under condition where AMPA receptors were previously antagonized. Inhibition of protein kinase A (PKA suppressed the effect of KA on glutamate release, which was also obviated by the prior stimulation of adenylyl cyclase (AC. KAR-mediated facilitation of synaptic transmission was prevented by blocking Ca2+ permeant KARs using philanthotoxin. Furthermore, depletion of intracellular Ca2+ stores by thapsigargin, or inhibition of Ca2+-induced Ca2+-release by ryanodine, abrogated the synaptic facilitation by KA. Thus, the KA-mediated modulation was conditional on extracellular Ca2+ entry through Ca2+-permeable KARs, as well as and mobilization of Ca2+ from intracellular stores. Finally, KAR-mediated facilitation was sensitive to calmodulin inhibitors, W-7 and calmidazolium, indicating that the increased cytosolic [Ca2+] sustaining KAR-mediated facilitation of synaptic transmission operates through a downstream Ca2+/calmodulin coupling. We conclude that, at cerebellar parallel fiber-Purkinje cell synapses, presynaptic KARs mediate glutamate release facilitation, and thereby enhance synaptic transmission through Ca2+-calmodulin dependent activation of adenylyl cyclase/cAMP/protein kinase A signaling.

Rapid efflux of Ca2+ from heart mitochondria in the presence of inorganic pyrophosphate.

Science.gov (United States)

Vercesi, A; Lehninger, A L

1984-01-13

Inorganic pyrophosphate (PPi) in the intracellular concentration range causes rapid efflux of Ca2+ from rat heart mitochondria oxidizing pyruvate + malate in a low Na+ medium. Half-maximal rates of Ca2+ efflux were given by 20 microM PPi. During and after PPi-stimulated Ca2+ efflux the mitochondria retain their structural integrity and complete respiratory control. Carboxyatractyloside inhibits PPi-stimulated Ca2+ efflux, indicating PPi must enter the matrix in order to promote Ca2+ efflux. Heart mitochondria have a much higher affinity for PPi uptake and PPi-induced Ca2+ efflux than liver mitochondria.

Interaction between Na+/K+-pump and Na+/Ca2+-exchanger modulates intercellular communication.

Science.gov (United States)

Matchkov, Vladimir V; Gustafsson, Helena; Rahman, Awahan; Briggs Boedtkjer, Donna M; Gorintin, Sarah; Hansen, Anne Kirstine; Bouzinova, Elena V; Praetorius, Helle A; Aalkjaer, Christian; Nilsson, Holger

2007-04-13

Ouabain, a specific inhibitor of the Na(+)/K(+)-pump, has previously been shown to interfere with intercellular communication. Here we test the hypothesis that the communication between vascular smooth muscle cells is regulated through an interaction between the Na(+)/K(+)-pump and the Na(+)/Ca(2+)-exchanger leading to an increase in the intracellular calcium concentration ([Ca(2+)](i)) in discrete areas near the plasma membrane. [Ca(2+)](i) in smooth muscle cells was imaged in cultured rat aortic smooth muscle cell pairs (A7r5) and in rat mesenteric small artery segments simultaneously with force. In A7r5 coupling between cells was estimated by measuring membrane capacitance. Smooth muscle cells were uncoupled when the Na(+)/K(+)-pump was inhibited either by a low concentration of ouabain, which also caused a localized increase of [Ca(2+)](i) near the membrane, or by ATP depletion. Reduction of Na(+)/K(+)-pump activity by removal of extracellular potassium ([K(+)](o)) also uncoupled cells, but only after inhibition of K(ATP) channels. Inhibition of the Na(+)/Ca(2+)-exchange activity by SEA0400 or by a reduction of the equilibrium potential (making it more negative) also uncoupled the cells. Depletion of intracellular Na(+) and clamping of [Ca(2+)](i) at low concentrations prevented the uncoupling. The experiments suggest that the Na(+)/K(+)-pump may affect gap junction conductivity via localized changes in [Ca(2+)](i) through modulation of Na(+)/Ca(2+)-exchanger activity.

Development of the Wintertime Sr/Ca-SST Record from Red Sea Corals as a Proxy for the North Atlantic Oscillation

Science.gov (United States)

Bernstein, W. N.; Hughen, K. A.

2009-12-01

The North Atlantic Oscillation (NAO) is one of the most pronounced and influential patterns in winter atmospheric circulation variability. This meridional redistribution of atmospheric mass across the Atlantic Ocean produces large changes in the intensity, number and direction of storms generated within the basin, and the regional climate of surrounding continents. The NAO exerts a significant impact on society, through influences on agriculture, fisheries, water management, energy generation and coastal development. NAO effects on climate extend from eastern North America across Europe to the eastern Mediterranean and Middle East. Changes in NAO behavior during the late 20th century have been linked to global warming; yet despite its importance, the causes and long-term patterns of NAO variability in the past remain poorly understood. In order to better predict the influence of the NAO on climate in the future, it is critical to examine multi-century NAO variability. The Red Sea is an excellent location from which to generate long NAO records for two reasons. First, patterns of wintertime sea surface temperature (SST) and salinity (SSS) in the Red Sea are highly correlated with NAO variability (Visbeck et al. 2001; Hurrell et al. 2003). Second, the tropical/subtropical Red Sea region contains fast growing long-lived massive Porites spp. corals with annually banded skeletons. These corals are ideal for generating well-dated high-resolution paleoclimatic records that extend well beyond the instrumental period. Here we present a study of winter SST and NAO variability in the Red sea region based on coral Sr/Ca data. In 2008, we collected multiple drill cores ranging in length from 1 to 4.1 meters from Porites corals at six sites spanning a large SST gradient. Sr/Ca measurements from multiple corals will be regressed against 23 years of satellite SST data, expanding the SST range over which we calibrate. A sampling resolution of 0.5mm will yield greater than bi

Regulation of capacitative and non-capacitative Ca2+ entry in A7r5 vascular smooth muscle cells

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COLIN W TAYLOR

2004-01-01

Full Text Available A capacitative Ca2+ entry (CCE pathway, activated by depletion of intracellular Ca2+ stores, is thought to mediate much of the Ca2+ entry evoked by receptors that stimulate phospholipase C (PLC. However, in A7r5 vascular smooth muscle cells, vasopressin, which stimulates PLC, empties intracellular Ca2+ stores but simultaneously inhibits their ability to activate CCE. The diacylglycerol produced with the IP3 that empties the stores is metabolized to arachidonic and this leads to activation of nitric oxide (NO synthase, production of NO and cyclic GMP, and consequent activation of protein kinase G. The latter inhibits CCE. In parallel, NO directly activates a non-capacitative Ca2+ entry (NCCE pathway, which is entirely responsible for the Ca2+ entry that occurs in the presence of vasopressin. This reciprocal regulation of two Ca2+ entry pathways ensures that there is sequential activation of first NCCE in the presence of vasopressin, and then a transient activation of CCE when vasopressin is removed. We suggest that the two routes for Ca2+ entry may selectively direct Ca2+ to processes that mediate activation and then recovery of the cell.

Intracellular pH in sperm physiology.

Science.gov (United States)

Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

2014-08-01

Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. Copyright © 2014 Elsevier Inc. All rights reserved.

Coupled Ca2+/H+ transport by cytoplasmic buffers regulates local Ca2+ and H+ ion signaling.

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Swietach, Pawel; Youm, Jae-Boum; Saegusa, Noriko; Leem, Chae-Hun; Spitzer, Kenneth W; Vaughan-Jones, Richard D

2013-05-28

Ca(2+) signaling regulates cell function. This is subject to modulation by H(+) ions that are universal end-products of metabolism. Due to slow diffusion and common buffers, changes in cytoplasmic [Ca(2+)] ([Ca(2+)]i) or [H(+)] ([H(+)]i) can become compartmentalized, leading potentially to complex spatial Ca(2+)/H(+) coupling. This was studied by fluorescence imaging of cardiac myocytes. An increase in [H(+)]i, produced by superfusion of acetate (salt of membrane-permeant weak acid), evoked a [Ca(2+)]i rise, independent of sarcolemmal Ca(2+) influx or release from mitochondria, sarcoplasmic reticulum, or acidic stores. Photolytic H(+) uncaging from 2-nitrobenzaldehyde also raised [Ca(2+)]i, and the yield was reduced following inhibition of glycolysis or mitochondrial respiration. H(+) uncaging into buffer mixtures in vitro demonstrated that Ca(2+) unloading from proteins, histidyl dipeptides (HDPs; e.g., carnosine), and ATP can underlie the H(+)-evoked [Ca(2+)]i rise. Raising [H(+)]i tonically at one end of a myocyte evoked a local [Ca(2+)]i rise in the acidic microdomain, which did not dissipate. The result is consistent with uphill Ca(2+) transport into the acidic zone via Ca(2+)/H(+) exchange on diffusible HDPs and ATP molecules, energized by the [H(+)]i gradient. Ca(2+) recruitment to a localized acid microdomain was greatly reduced during intracellular Mg(2+) overload or by ATP depletion, maneuvers that reduce the Ca(2+)-carrying capacity of HDPs. Cytoplasmic HDPs and ATP underlie spatial Ca(2+)/H(+) coupling in the cardiac myocyte by providing ion exchange and transport on common buffer sites. Given the abundance of cellular HDPs and ATP, spatial Ca(2+)/H(+) coupling is likely to be of general importance in cell signaling.

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum

Science.gov (United States)

López-Sanjurjo, Cristina I.; Tovey, Stephen C.; Prole, David L.; Taylor, Colin W.

2013-01-01

Summary Most intracellular Ca2+ signals result from opening of Ca2+ channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca2+ into mitochondria. Ca2+ channels in lysosomes contribute to endo-lysosomal trafficking and Ca2+ signalling, but the role of lysosomal Ca2+ uptake in Ca2+ signalling is unexplored. Inhibition of lysosomal Ca2+ uptake by dissipating the H+ gradient (using bafilomycin A1), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca2+ signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation. Bafilomycin A1 amplified the Ca2+ signals evoked by photolysis of caged Ins(1,4,5)P3 or by inhibition of ER Ca2+ pumps, and it slowed recovery from them. Ca2+ signals evoked by store-operated Ca2+ entry were unaffected by bafilomycin A1. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca2+ released by Ins(1,4,5)P3 receptors. PMID:23097044

Cannabinoids disrupt memory encoding by functionally isolating hippocampal CA1 from CA3.

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Roman A Sandler

2017-07-01

Full Text Available Much of the research on cannabinoids (CBs has focused on their effects at the molecular and synaptic level. However, the effects of CBs on the dynamics of neural circuits remains poorly understood. This study aims to disentangle the effects of CBs on the functional dynamics of the hippocampal Schaffer collateral synapse by using data-driven nonparametric modeling. Multi-unit activity was recorded from rats doing an working memory task in control sessions and under the influence of exogenously administered tetrahydrocannabinol (THC, the primary CB found in marijuana. It was found that THC left firing rate unaltered and only slightly reduced theta oscillations. Multivariate autoregressive models, estimated from spontaneous spiking activity, were then used to describe the dynamical transformation from CA3 to CA1. They revealed that THC served to functionally isolate CA1 from CA3 by reducing feedforward excitation and theta information flow. The functional isolation was compensated by increased feedback excitation within CA1, thus leading to unaltered firing rates. Finally, both of these effects were shown to be correlated with memory impairments in the working memory task. By elucidating the circuit mechanisms of CBs, these results help close the gap in knowledge between the cellular and behavioral effects of CBs.

Ca2+ influx and ATP release mediated by mechanical stretch in human lung fibroblasts

International Nuclear Information System (INIS)

Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

2014-01-01

Highlights: • Uniaxial stretching activates Ca 2+ signaling in human lung fibroblasts. • Stretch-induced intracellular Ca 2+ elevation is mainly via Ca 2+ influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca 2+ influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca 2+ concentration ([Ca 2+ ] i ) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca 2+ ] i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca 2+ ] i . The stretch-induced [Ca 2+ ] i elevation was attenuated in Ca 2+ -free solution. In contrast, the increase of [Ca 2+ ] i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd 3+ , ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca 2+ ] i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca 2+ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP

Hypoxia activates a Ca2+-permeable cation conductance sensitive to carbon monoxide and to GsMTx-4 in human and mouse sickle erythrocytes.

OpenAIRE

David H Vandorpe; Chang Xu; Boris E Shmukler; Leo E Otterbein; Marie Trudel; Frederick Sachs; Philip A Gottlieb; Carlo Brugnara; Seth L Alper

2010-01-01

Background: Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca2+] ([Ca2+]i) and subsequent activation of KCa 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded...

Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation

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Fox Jane

2010-12-01

Full Text Available Abstract Background Agonist stimulation of airway smooth muscle (ASM results in IP3 mediated Ca2+ release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na+ influx. This localised increase in Na+ levels can potentially switch the Na+/Ca2+ exchanger into reverse mode and so result in a further influx of Ca2+. The aim of this study was to characterise the expression and physiological function of the Na+/Ca2+ exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca2+ influx into these cells. Methods The expression profile of NCX (which encodes the Na+/Ca2+ exchanger homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca2+ measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX and target specific siRNA were utilised as tools to inhibit NCX function. Results NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na+ loading and addition of extracellular Ca2+ induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1 and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca2+ and induced relaxation of porcine

Ca2+ influx and efflux in animal cells in the presence of panax notoginseng extracts: investigated by using 45Ca as a radioactive tracer

International Nuclear Information System (INIS)

Yang Yuanyou; Liu Ning; Mo Shangwu; Liao Jiali; Xu Falun

2010-01-01

In this paper, the influence of extracts of Panax notoginseng on Ca 2+ influx and efflux in isolated rat visceral organs was investigated by using 45 Ca as a radioactive tracer. The results indicated that both extracts, the total flavonoids and total saponins of Panax notoginseng had significant influence on Ca 2+ influx and efflux in the isolated rat aorta, heart, and kidney, in those organs it could markedly block 45 Ca entering into cell and could facilitate efflux of intracellular Ca 2+ . Compared with the total flavonoids, total saponins had stronger role in the regulation of Ca 2+ influx and efflux. Also, regulation effects of Ca 2+ influx and efflux of the total saponins were compared with positive drug Verapamil, or even better. This implies that the total flavonoids and total saponins of Panax notoginseng have calcium antagonistic effect, and both may be the active ingredients in Panax notoginseng for coronary heart disease treatment. (authors)

DDPH ameliorated oxygen and glucose deprivation-induced injury in rat hippocampal neurons via interrupting Ca2+ overload and glutamate release.

Science.gov (United States)

He, Zhi; Lu, Qing; Xu, Xulin; Huang, Lin; Chen, Jianguo; Guo, Lianjun

2009-01-28

Our previous work has demonstrated that DDPH (1-(2, 6-dimethylphenoxy)-2-(3, 4-dimethoxyphenylethylamino) propane hydrochloride), a competitive alpha(1)-adrenoceptor antagonist, could improve cognitive deficits, reduce histopathological damage and facilitate synaptic plasticity in vivo possibly via increasing NR2B (NMDA receptor 2B) expression and antioxidation of DDPH itself. The present study further evaluated effects of DDPH on OGD (Oxygen and glucose deprivation)-induced neuronal damage in rat primary hippocampal cells. The addition of DDPH to the cultured cells 12 h before OGD for 4 h significantly reduced neuronal damage as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and LDH (lactate dehydrogenase) release experiments. The effects of DDPH on intracellular calcium concentration were explored by Fura-2 based calcium imaging techniques and results showed that DDPH at the dosages of 5 microM and 10 microM suppressed the increase of intracellular calcium ([Ca(2+)](i)) stimulated by 50 mM KCl in Ca(2+)-containing extracellular solutions. However, DDPH couldn't suppress the increase of [Ca(2+)](i) induced by both 50 microM glutamate in Ca(2+)-containing extracellular solutions and 20 microM ATP (Adenosine Triphosphate) in Ca(2+)-free solution. These results indicated that DDPH prevented [Ca(2+)](i) overload in hippocampal neurons by blocking Ca(2+) influx (voltage-dependent calcium channel) but not Ca(2+) mobilization from the intracellular Ca(2+) store in endoplasm reticulum (ER). We also demonstrated that DDPH could decrease glutamate release when hippocampal cells were subjected to OGD. These observations demonstrated that DDPH protected hippocampal neurons against OGD-induced damage by preventing the Ca(2+) influx and decreasing glutamate release.

Bax regulates neuronal Ca2+ homeostasis.

Science.gov (United States)

D'Orsi, Beatrice; Kilbride, Seán M; Chen, Gang; Perez Alvarez, Sergio; Bonner, Helena P; Pfeiffer, Shona; Plesnila, Nikolaus; Engel, Tobias; Henshall, David C; Düssmann, Heiko; Prehn, Jochen H M

2015-01-28

Excessive Ca(2+) entry during glutamate receptor overactivation ("excitotoxicity") induces acute or delayed neuronal death. We report here that deficiency in bax exerted broad neuroprotection against excitotoxic injury and oxygen/glucose deprivation in mouse neocortical neuron cultures and reduced infarct size, necrotic injury, and cerebral edema formation after middle cerebral artery occlusion in mice. Neuronal Ca(2+) and mitochondrial membrane potential (Δψm) analysis during excitotoxic injury revealed that bax-deficient neurons showed significantly reduced Ca(2+) transients during the NMDA excitation period and did not exhibit the deregulation of Δψm that was observed in their wild-type (WT) counterparts. Reintroduction of bax or a bax mutant incapable of proapoptotic oligomerization equally restored neuronal Ca(2+) dynamics during NMDA excitation, suggesting that Bax controlled Ca(2+) signaling independently of its role in apoptosis execution. Quantitative confocal imaging of intracellular ATP or mitochondrial Ca(2+) levels using FRET-based sensors indicated that the effects of bax deficiency on Ca(2+) handling were not due to enhanced cellular bioenergetics or increased Ca(2+) uptake into mitochondria. We also observed that mitochondria isolated from WT or bax-deficient cells similarly underwent Ca(2+)-induced permeability transition. However, when Ca(2+) uptake into the sarco/endoplasmic reticulum was blocked with the Ca(2+)-ATPase inhibitor thapsigargin, bax-deficient neurons showed strongly elevated cytosolic Ca(2+) levels during NMDA excitation, suggesting that the ability of Bax to support dynamic ER Ca(2+) handling is critical for cell death signaling during periods of neuronal overexcitation. Copyright © 2015 the authors 0270-6474/15/351706-17$15.00/0.

Silver ions increase plasma membrane permeability through modulation of intracellular calcium levels in tobacco BY-2 cells.

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Klíma, Petr; Laňková, Martina; Vandenbussche, Filip; Van Der Straeten, Dominique; Petrášek, Jan

2018-05-01

Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides a dramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO 3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO 3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca 2+ ) as shown by comparison of transport assays in Ca 2+ -rich and Ca 2+ -free buffers and upon treatment with inhibitors of plasma membrane Ca 2+ -permeable channels Al 3+ and ruthenium red, both abolishing the effect of AgNO 3 . Confocal microscopy of Ca 2+ -sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca 2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca 2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca 2+ -permeable channels at the plasma membrane.

Involvement of glucocorticoid-mediated Zn2+ signaling in attenuation of hippocampal CA1 LTP by acute stress.

Science.gov (United States)

Takeda, Atsushi; Suzuki, Miki; Tamano, Haruna; Takada, Shunsuke; Ide, Kazuki; Oku, Naoto

2012-03-01

Glucocorticoid-glutamatergic interactions have been proposed as a potential model to explain stress-mediated impairment of cognition. However, it is unknown whether glucocorticoid-zincergic interactions are involved in this impairment. Histochemically reactive zinc (Zn(2+)) is co-released with glutamate from zincergic neurons. In the present study, involvement of synaptic Zn(2+) in stress-induced attenuation of CA1 LTP was examined in hippocampal slices from young rats after exposure to tail suspension stress for 30s, which significantly increased serum corticosterone. Stress-induced attenuation of CA1 LTP was ameliorated by administration of clioquinol, a membrane permeable zinc chelator, to rats prior to exposure to stress, implying that the reduction of synaptic Zn(2+) by clioquinol participates in this amelioration. To pursue the involvement of corticosterone-mediated Zn(2+) signal in the attenuated CA1 LTP by stress, dynamics of synaptic Zn(2+) was checked in hippocampal slices exposed to corticosterone. Corticosterone increased extracellular Zn(2+) levels measured with ZnAF-2 dose-dependently, as well as the intracellular Ca(2+) levels measured with calcium orange AM, suggesting that corticosterone excites zincergic neurons in the hippocampus and increases Zn(2+) release from the neuron terminals. Intracellular Zn(2+) levels measured with ZnAF-2DA were also increased dose-dependently, but not in the coexistence of CaEDTA, a membrane-impermeable zinc chelator, suggesting that intracellular Zn(2+) levels is increased by the influx of extracellular Zn(2+). Furthermore, corticosterone-induced attenuation of CA1 LTP was abolished in the coexistence of CaEDTA. The present study suggests that corticosterone-mediated increase in postsynaptic Zn(2+) signal in the cytosolic compartment is involved in the attenuation of CA1 LTP after exposure to acute stress. Copyright © 2012 Elsevier Ltd. All rights reserved.

Physiology of Ca2+ signalling in stem cells of different origins and differentiation stages.

Czech Academy of Sciences Publication Activity Database

Forostyak, Oksana; Forostyak, Serhiy; Kortus, Štěpán; Syková, Eva; Verkhratsky, A.; Dayanithi, Govindan

2016-01-01

Roč. 59, 2-3 (2016), s. 57-66 ISSN 0143-4160 R&D Projects: GA ČR(CZ) GAP304/11/2373; GA ČR(CZ) GA14-34077S; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : Stem cells * Ca2+ channels * Ca2+ signalling * Spontaneous Ca2+ oscillations Subject RIV: FH - Neurology Impact factor: 3.707, year: 2016

Effect of metabolic and respiratory acidosis on intracellular calcium in osteoblasts.

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Frick, Kevin K; Bushinsky, David A

2010-08-01

In vivo, metabolic acidosis {decreased pH from decreased bicarbonate concentration ([HCO(3)(-)])} increases urine calcium (Ca) without increased intestinal Ca absorption, resulting in a loss of bone Ca. Conversely, respiratory acidosis [decreased pH from increased partial pressure of carbon dioxide (Pco(2))] does not appreciably alter Ca homeostasis. In cultured bone, chronic metabolic acidosis (Met) significantly increases cell-mediated net Ca efflux while isohydric respiratory acidosis (Resp) does not. The proton receptor, OGR1, appears critical for cell-mediated, metabolic acid-induced bone resorption. Perfusion of primary bone cells or OGR1-transfected Chinese hamster ovary (CHO) cells with Met induces transient peaks of intracellular Ca (Ca(i)). To determine whether Resp increases Ca(i), as does Met, we imaged Ca(i) in primary cultures of bone cells. pH for Met = 7.07 ([HCO(3)(-)] = 11.8 mM) and for Resp = 7.13 (Pco(2) = 88.4 mmHg) were similar and lower than neutral (7.41). Both Met and Resp induced a marked, transient increase in Ca(i) in individual bone cells; however, Met stimulated Ca(i) to a greater extent than Resp. We used OGR1-transfected CHO cells to determine whether OGR1 was responsible for the greater increase in Ca(i) in Met than Resp. Both Met and Resp induced a marked, transient increase in Ca(i) in OGR1-transfected CHO cells; however, in these cells Met was not different than Resp. Thus, the greater induction of Ca(i) by Met in primary bone cells is not a function of OGR1 alone, but must involve H(+) receptors other than OGR1, or pathways sensitive to Pco(2), HCO(3)(-), or total CO(2) that modify the effect of H(+) in primary bone cells.

A single and rapid calcium wave at egg activation in Drosophila

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Anna H. York-Andersen

2015-03-01

Full Text Available Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca2+ concentration in mammals, ascidians and polychaete worms and a single Ca2+ peak in fish and frogs, in insects, such as Drosophila, to date, it has not been shown what changes in intracellular Ca2+ levels occur. Here, we utilise ratiometric imaging of Ca2+ indicator dyes and genetically encoded Ca2+ indicator proteins to identify and characterise a single, rapid, transient wave of Ca2+ in the Drosophila egg at activation. Using genetic tools, physical manipulation and pharmacological treatments we demonstrate that the propagation of the Ca2+ wave requires an intact actin cytoskeleton and an increase in intracellular Ca2+ can be uncoupled from egg swelling, but not from progression of the cell cycle. We further show that mechanical pressure alone is not sufficient to initiate a Ca2+ wave. We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca2+ transient. Based on this data we propose the following model for egg activation in Drosophila: exposure to lateral oviduct fluid initiates an increase in intracellular Ca2+ at the egg posterior via osmotic swelling, possibly through mechano-sensitive Ca2+ channels; a single Ca2+ wave then propagates in an actin dependent manner; this Ca2+ wave co-ordinates key developmental events including resumption of the cell cycle and initiation of translation of mRNAs such as bicoid.

Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons.

Science.gov (United States)

Bak, Lasse K; Obel, Linea F; Walls, Anne B; Schousboe, Arne; Faek, Sevan A A; Jajo, Farah S; Waagepetersen, Helle S

2012-04-05

We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca2+ during activation cause influx of Ca2+ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate-aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca2+ (i.e. independent of synaptic activity) on neuronal energy metabolism employing 13C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca2+ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca2+ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca2+-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca2+ signalling.

Regulation of the Na+/Ca2+ exchanger in rat pancreatic ducts

DEFF Research Database (Denmark)

Ankorina-Stark, I; Amstrup, J; Novak, I

2002-01-01

by hormones/agonists affecting pancreatic secretion. Whole pancreas, pure pancreatic acini and ducts were obtained from rats and used for RT-PCR and Western blot analysis, immunohistochemistry and intracellular Ca2+ measurements using Fura-2. RT-PCR analysis indicated Na+/Ca2+-exchanger isoforms NCX1.......3 and NCX1.7 in acini and pancreas. Western blot with NCX1 antibody identified bands of 70, 120 and 150 kDa in isolated ducts, acini and pancreas. Immunofluorescence experiments showed the Na+/Ca2+ exchanger on the basolateral membrane of acini and small intercalated/intralobular ducts, but in larger...

Back to the future with the AGP–Ca2+ flux capacitor

Science.gov (United States)

Lamport, Derek T. A.; Varnai, Peter; Seal, Charlotte E.

2014-01-01

Background Arabinogalactan proteins (AGPs) are ubiquitous in green plants. AGPs comprise a widely varied group of hydroxyproline (Hyp)-rich cell surface glycoproteins (HRGPs). However, the more narrowly defined classical AGPs massively predominate and cover the plasma membrane. Extensive glycosylation by pendant polysaccharides O-linked to numerous Hyp residues like beads of a necklace creates a unique ionic compartment essential to a wide range of physiological processes including germination, cell extension and fertilization. The vital clue to a precise molecular function remained elusive until the recent isolation of small Hyp–arabinogalactan polysaccharide subunits; their structural elucidation by nuclear magentic resonance imaging, molecular simulations and direct experiment identified a 15-residue consensus subunit as a β-1,3-linked galactose trisaccharide with two short branched sidechains each with a single glucuronic acid residue that binds Ca2+ when paired with its adjacent sidechain. Scope AGPs bind Ca2+ (Kd ∼ 6 μm) at the plasma membrane (PM) at pH ∼5·5 but release it when auxin-dependent PM H+-ATPase generates a low periplasmic pH that dissociates AGP–Ca2+ carboxylates (pka ∼3); the consequential large increase in free Ca2+ drives entry into the cytosol via Ca2+ channels that may be voltage gated. AGPs are thus arguably the primary source of cytosolic oscillatory Ca2+ waves. This differs markedly from animals, in which cytosolic Ca2+ originates mostly from internal stores such as the sarcoplasmic reticulum. In contrast, we propose that external dynamic Ca2+ storage by a periplasmic AGP capacitor co-ordinates plant growth, typically involving exocytosis of AGPs and recycled Ca2+, hence an AGP–Ca2+ oscillator. Conclusions The novel concept of dynamic Ca2+ recycling by an AGP–Ca2+ oscillator solves the long-standing problem of a molecular-level function for classical AGPs and thus integrates three fields: AGPs, Ca2+ signalling and auxin

Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

International Nuclear Information System (INIS)

Ryu, S.H.; Lee, S.Y.; Lee, K.Y.; Rhee, S.G.

1987-01-01

Inositol 1,4,5-triphosphate (Ins-1,4,5-P 3 ) is an important second-messenger molecule that mobilizes Ca 2+ from intracellular stores in response to the occupancy of receptor by various Ca 2+ -mobilizing agonists. The fate of Ins-1,4,5-P 3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P 3 to Ins-1,3,4,5-P 4 , whereas the latter forms Ins-1,4-P 2 . Recent studies suggest that Ins-1,3,4,5-P 4 might modulate the entry of Ca 2+ from an extracellular source. In the current report, the authors describe the partial purification of the 3-kinase from the cytosolic fraction of bovine brain and studies of its catalytic properties. They found that the 3-kinase activity is significantly activated by the Ca 2+ /calmodulin complex. Therefore, they propose that Ca 2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P 3 forms a complex with calmodulin, and that the Ca 2+ /calmodulin complex stimulates the conversion of Ins-1,4,5-P 3 , and intracellular Ca 2+ mobilizer, to Ins-1,3,4,5-P 4 , an extracellular Ca 2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3- 32 P]Ins-1,3,4,5-P 4 and [γ- 32 P]ATP by thin-layer chromatography. Using this new assay method, they evaluated kinetic parameters (K/sub m/ for ATP = 40 μM, K/sub m/ for Ins-1,4,5-P 3 = 0.7 μM, K/sub i/ for ADP = 12 μM) and divalent cation specificity (Mg 2+ > > Mn 2+ > Ca 2+ ) for the 3-kinase

Lead perturbs epidermal growth factor (EGF) modulation of intracellular calcium metabolism in clonal rat osteoblastic (ROS 17/2.8) cells

International Nuclear Information System (INIS)

Long, G.J.; Rosen, J.F.

1991-01-01

EGF, a single chain polypeptide growth factor important for many cellular functions including glycolysis and protein phophorylation, is known to modulate calcium metabolism in several cell systems. It has been shown that EGF causes an increase in Ca 2+ influx and accumulation of inositol triphosphate, and probably exhibits many, if not all, of its effects via the calcium messenger system. Lead is known to interact with and perturb normal calcium signaling pathways; hence, the purpose of this work was to determine if lead perturbs EGF modulation of calcium metabolism in ROS 17/2.8 cells and if cell functions controlled by EGF were impaired. Cells were labelled with 45 Ca (1.87 mM Ca) for 20 hr in the presence of 5 μM Pb, 50 ng/ml EGF or μM Pb and 50 ng/ml EGF. Following an EGTA rinse, kinetic parameters were determined from 45 Ca efflux curves. Three kinetic compartments described the intracellular metabolism of 45 Ca. 5 μM Pb significantly altered the effect of EGF on intracellular calcium metabolism. Calcium distribution was shifted from the fast exchanging, quantitatively small calcium pools, S 1 and S 2 to the slow exchanging, quantitatively large S 2 . There was also a 50% increase in total cell calcium in cells treated with 5 μM Pb and 50 ng/ml EGF over cells treated with 50 ng/ml EGF alone. There was also a 25% decrease in the half-time for calcium exchange from S 3 to S 1 was also decreased. These data show that Pb impairs the normal modulation of intracellular calcium homeostasis by EGF and may therefore perturb functions that are modulated by EGF via the calcium messenger system

Association of serine protease with the rise of intracellular calcium in cytotoxic T lymphocytes.

Science.gov (United States)

Koo, G C; Luk, Y; Talento, A; Wu, J; Sirotina, A; Fischer, P A; Blake, J T; Nguyen, M P; Parsons, W; Poe, M

1996-12-15

The precise role of the granular enzyme A (granzyme A), a serine protease, in the lytic process of cytotoxic T lymphocytes (CTL) is not clear. We have recently constructed a CTL line transfected with the antisense gene of granzyme A (a-GrA). These a-GrA CTL had lower GrA activity as well as decreased lytic activities, as measured by 51Cr and by DNA degradation assays. Furthermore, at low effector:target ratio (1:8) in prolonged lytic assays, they could not lyse targets as rapidly as the control CTL. When we examined their ability to exocytose BLT (CBZ-L-lys-thiobenzyl)-esterase in the presence of anti-CD3 antibody, the a-GrA CTL exocytosed poorly compared to the parental CTL or control transfectant with a CAT gene. Most strikingly, a-GrA cells could not release intracellular stores of Ca2+ in response to anti-CD3 induction, although the Ca2+ flux was normal when they were stimulated with ionomycin. When the parental CTL was treated with a specific benzyllactam inhibitor of BLT-esterase or N-tosyl-L-phenylalanylchloromethyl ketone, the Ca2+ flux induced by anti-CD3 was also suppressed. We propose that granzyme A is involved in the signal transduction pathway that causes the rise of the intracellular calcium.

Intensely oscillating cavitation bubble in microfluidics

International Nuclear Information System (INIS)

Siew-Wan, Ohl; Tandiono; Klaseboer, Evert; Dave, Ow; Choo, Andre; Claus-Dieter, Ohl

2015-01-01

This study reports the technical breakthrough in generating intense ultrasonic cavitation in the confinement of a microfluidics channel [1], and applications that has been developed on this platform for the past few years [2,3,4,5]. Our system consists of circular disc transducers (10-20 mm in diameter), the microfluidics channels on PDMS (polydimethylsiloxane), and a driving circuitry. The cavitation bubbles are created at the gas- water interface due to strong capillary waves which are generated when the system is driven at its natural frequency (around 100 kHz) [1]. These bubbles oscillate and collapse within the channel. The bubbles are useful for sonochemistry and the generation of sonoluminescence [2]. When we add bacteria (Escherichia coli), and yeast cells (Pichia pastoris) into the microfluidics channels, the oscillating and collapsing bubbles stretch and lyse these cells [3]. Furthermore, the system is effective (DNA of the harvested intracellular content remains largely intact), and efficient (yield reaches saturation in less than 1 second). In another application, human red blood cells are added to a microchamber. Cell stretching and rapture are observed when a laser generated cavitation bubble expands and collapses next to the cell [4]. A numerical model of a liquid pocket surrounded by a membrane with surface tension which was placed next to an oscillating bubble was developed using the Boundary Element Method. The simulation results showed that the stretching of the liquid pocket occurs only when the surface tension is within a certain range. (paper)

Network models provide insights into how oriens–lacunosum-moleculare and bistratified cell interactions influence the power of local hippocampal CA1 theta oscillations

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Katie A Ferguson

2015-08-01

Full Text Available Hippocampal theta is a 4-12 Hz rhythm associated with episodic memory, and although it has been studied extensively, the cellular mechanisms underlying its generation are unclear. The complex interactions between different interneuron types, such as those between oriens--lacunosum-moleculare (OLM interneurons and bistratified cells (BiCs, make their contribution to network rhythms difficult to determine experimentally. We created network models that are tied to experimental work at both cellular and network levels to explore how these interneuron interactions affect the power of local oscillations. Our cellular models were constrained with properties from patch clamp recordings in the CA1 region of an intact hippocampus preparation in vitro. Our network models are composed of three different types of interneurons: parvalbumin-positive (PV+ basket and axo-axonic cells (BC/AACs, PV+ BiCs, and somatostatin-positive OLM cells. Also included is a spatially extended pyramidal cell model to allow for a simplified local field potential representation, as well as experimentally-constrained, theta frequency synaptic inputs to the interneurons. The network size, connectivity, and synaptic properties were constrained with experimental data. To determine how the interactions between OLM cells and BiCs could affect local theta power, we explored a number of OLM-BiC connections and connection strengths.We found that our models operate in regimes in which OLM cells minimally or strongly affected the power of network theta oscillations due to balances that, respectively, allow compensatory effects or not. Inactivation of OLM cells could result in no change or even an increase in theta power. We predict that the dis-inhibitory effect of OLM cells to BiCs to pyramidal cell interactions plays a critical role in the power of network theta oscillations. Our network models reveal a dynamic interplay between different classes of interneurons in influencing local theta

Ca2+ uptake and cellular integrity in rat EDL muscle exposed to electrostimulation, electroporation, or A23187

DEFF Research Database (Denmark)

Gissel, Hanne; Clausen, Torben

2003-01-01

We tested the hypothesis that increased Ca2+ uptake in rat extensor digitorum longus (EDL) muscle elicits cell membrane damage as assessed from release of the intracellular enzyme lactate dehydrogenase (LDH). This was done by using 1) electrostimulation, 2) electroporation, and 3) the Ca2+ ionoph...

Chronic Loss of CA2 Transmission Leads to Hippocampal Hyperexcitability.

Science.gov (United States)

Boehringer, Roman; Polygalov, Denis; Huang, Arthur J Y; Middleton, Steven J; Robert, Vincent; Wintzer, Marie E; Piskorowski, Rebecca A; Chevaleyre, Vivien; McHugh, Thomas J

2017-05-03

Hippocampal CA2 pyramidal cells project into both the neighboring CA1 and CA3 subfields, leaving them well positioned to influence network physiology and information processing for memory and space. While recent work has suggested unique roles for CA2, including encoding position during immobility and generating ripple oscillations, an interventional examination of the integrative functions of these connections has yet to be reported. Here we demonstrate that CA2 recruits feedforward inhibition in CA3 and that chronic genetically engineered shutdown of CA2-pyramidal-cell synaptic transmission consequently results in increased excitability of the recurrent CA3 network. In behaving mice, this led to spatially triggered episodes of network-wide hyperexcitability during exploration accompanied by the emergence of high-frequency discharges during rest. These findings reveal CA2 as a regulator of network processing in hippocampus and suggest that CA2-mediated inhibition in CA3 plays a key role in establishing the dynamic excitatory and inhibitory balance required for proper network function. Copyright © 2017 Elsevier Inc. All rights reserved.

Pyramidal cell-interneuron interactions underlie hippocampal ripple oscillations.

Science.gov (United States)

Stark, Eran; Roux, Lisa; Eichler, Ronny; Senzai, Yuta; Royer, Sebastien; Buzsáki, György

2014-07-16

High-frequency ripple oscillations, observed most prominently in the hippocampal CA1 pyramidal layer, are associated with memory consolidation. The cellular and network mechanisms underlying the generation, frequency control, and spatial coherence of the rhythm are poorly understood. Using multisite optogenetic manipulations in freely behaving rodents, we found that depolarization of a small group of nearby pyramidal cells was sufficient to induce high-frequency oscillations, whereas closed-loop silencing of pyramidal cells or activation of parvalbumin- (PV) or somatostatin-immunoreactive interneurons aborted spontaneously occurring ripples. Focal pharmacological blockade of GABAA receptors abolished ripples. Localized PV interneuron activation paced ensemble spiking, and simultaneous induction of high-frequency oscillations at multiple locations resulted in a temporally coherent pattern mediated by phase-locked interneuron spiking. These results constrain competing models of ripple generation and indicate that temporally precise local interactions between excitatory and inhibitory neurons support ripple generation in the intact hippocampus. Copyright © 2014 Elsevier Inc. All rights reserved.

Importance of the assessment of intracellular Ca2+ level as diagnostic tool of dysfunctional sperm

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Wardah Alasmari

2017-09-01

Full Text Available Sperm functions are an important factor for fertility/pregnancy to be achieved. Sperm dysfunction is the most common cause of male infertility. The best option to help couples with such male factor to achieve a pregnancy, is using Assisted Reproductive Technology (ART, without defining the underlying cause of sperm dysfunction or male-factor infertility in general at the cellular and molecular levels. Thus, the limited success of ART in a proportion of male infertility cases is unsurprising. Ca2+ signalling plays a fundamental role in the regulation of sperm function. Interestingly, it appears that the potential exists to diagnose abnormalities in the Ca2+ channels that underlie sperm dysfunction. This raises the potential for future drug discovery to try to correct this defect by augmenting Ca2+ signalling such as Ca2+ store mobilisation or activating CatSper, as possible rational treatments for sperm dysfunction that may temporarily increase the capacity to interact with the egg. Such a pharmacological agent may provide a useful way of increasing the effectiveness of IUI or IVF over conventional IUI and IVF procedures.

Disruption of Intracellular ATP Generation and Tight Junction Protein Expression during the Course of Brain Edema Induced by Subacute Poisoning of 1,2-Dichloroethane

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Gaoyang Wang

2018-01-01

Full Text Available The aim of this study was to explore changes in intracellular ATP generation and tight junction protein expression during the course of brain edema induced by subacute poisoning of 1,2-dichloroethane (1,2-DCE. Mice were exposed to 1.2 g/m3 1,2-DCE for 3.5 h per day for 1, 2, or 3 days, namely group A, B, and C. Na+-K+-ATPase and Ca2+-ATPase activity, ATP and lactic acid content, intracellular free Ca2+ concentration and ZO-1 and occludin expression in the brain were measured. Results of present study disclosed that Ca2+-ATPase activities in group B and C, and Na+/K+-ATPase activity in group C decreased, whereas intracellular free Ca2+ concentrations in group B and C increased significantly compared with control. Moreover, ATP content decreased, whereas lactic acid content increased significantly in group C compared with control. On the other hand, expressions of ZO-1 and occludin at both the protein and gene levels in group B and C decreased significantly compared with control. In conclusion, findings from this study suggest that calcium overload and depressed expression of tight junction associated proteins, such as ZO-1 and occludin might play an important role in the early phase of brain edema formation induced by subacute poisoning of 1,2-DCE.

Sphingosine inhibits the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA) activity

Energy Technology Data Exchange (ETDEWEB)

Benaim, Gustavo, E-mail: gbenaim@idea.gob.ve [Instituto de Estudios Avanzados (IDEA), Caracas (Venezuela, Bolivarian Republic of); Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Pimentel, Adriana A., E-mail: adriana.pimentel@ucv.ve [Facultad de Farmacia, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Felibertt, Pimali [Facultad de Ciencias, Universidad de Carabobo, Valencia (Venezuela, Bolivarian Republic of); Mayora, Adriana [Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Colman, Laura [Instituto Pasteur de Montevideo, Montevideo (Uruguay); Sojo, Felipe [Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Rojas, Héctor [Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas (Venezuela, Bolivarian Republic of); De Sanctis, Juan B. [Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of)

2016-04-29

The increase in the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca{sup 2+}]{sub i} by inhibiting the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca{sup 2+} pump. The results showed that addition of sphingosine produced a release of Ca{sup 2+} from the endoplasmic reticulum followed by a Ca{sup 2+} entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca{sup 2+}]{sub I} in mammalian cells.

Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Murata, Naohiko [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Furuya, Kishio [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Takahara, Norihiro [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Naruse, Keiji [Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama 700-8558 (Japan); Aso, Hiromichi; Kondo, Masashi [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Sokabe, Masahiro [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Hasegawa, Yoshinori [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan)

2014-10-10

Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology.

Science.gov (United States)

Abbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, Hongkun

2018-03-20

, clarifying how the nanoelectrode attains intracellular access. This understanding will be translated into a circuit model for the nanobio interface, which we will then use to lay out the strategies for improving the interface. The intracellular interface of the nanoelectrode is currently inferior to that of the patch clamp electrode; reaching this benchmark will be an exciting challenge that involves optimization of electrode geometries, materials, chemical modifications, electroporation protocols, and recording/stimulation electronics, as we describe in the Account. Another important theme of this Account, beyond the optimization of the individual nanoelectrode-cell interface, is the scalability of the nanoscale electrodes. We will discuss this theme using a recent development from our groups as an example, where an array of ca. 1000 nanoelectrode pixels fabricated on a CMOS integrated circuit chip performs parallel intracellular recording from a few hundreds of cardiomyocytes, which marks a new milestone in electrophysiology.

Inhibition of PaCaMKII-E isoform in the dorsal unpaired median neurosecretory cells of cockroach reduces nicotine- and clothianidin-induced currents.

Science.gov (United States)

List, Olivier; Calas-List, Delphine; Taillebois, Emiliane; Juchaux, Marjorie; Heuland, Emilie; Thany, Steeve H

2014-08-01

Cellular responses to Ca(2+) require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII), which transduces the signal into downstream effects. We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms, and only PaCaMKII-E isoform is specifically expressed in the dorsal unpaired median neurosecretory cells. In the present study, using antisense oligonucleotides, we demonstrated that PaCaMKII-E isoform inhibition reduced nicotine-induced currents through α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptor subtypes. Specifically, PaCaMKII-E isoform is sufficient to repress nicotinic current amplitudes as a result of its depression by antisense oligonucleotides. Similar results were found using the neonicotinoid insecticide clothianidin, which acted as a full agonist of dorsal unpaired median neuron nicotinic acetylcholine receptors. Clothianidin current amplitudes are strongly reduced under bath application of PaCaMKII-E antisense oligonucleotides but no significant results are found with α-bungarotoxin co-applied, demonstrating that CaMKII-E isoform affects nicotine currents through α-bungarotoxin-sensitive and -insensitive receptor subtypes whereas clothianidin currents are reduced via α-bungarotoxin-insensitive receptors. In addition, we found that intracellular calcium increase induced by nicotine and clothianidin were reduced by PaCaMKII-E antisense oligonucleotides, demonstrating that intracellular calcium increase induced by nicotine and clothianidin are affected by PaCaMKII-E inhibition. Cellular responses to Ca(2+) require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII). We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms and only PaCaMKII-E isoform was specifically expressed in the dorsal unpaired median neurosecretory cells. Here we show that specific inhibition of PaCaMKII-E isoform is

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

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Michael P Grant

Full Text Available 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.

Heterogeneous Cytoskeletal Force Distribution Delineates the Onset Ca2+ Influx Under Fluid Shear Stress in Astrocytes

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Mohammad M. Maneshi

2018-03-01

Full Text Available Mechanical perturbations increase intracellular Ca2+ in cells, but the coupling of mechanical forces to the Ca2+ influx is not well understood. We used a microfluidic chamber driven with a high-speed pressure servo to generate defined fluid shear stress to cultured astrocytes, and simultaneously measured cytoskeletal forces using a force sensitive actinin optical sensor and intracellular Ca2+. Fluid shear generated non-uniform forces in actinin that critically depended on the stimulus rise time emphasizing the presence of viscoelasticity in the activating sequence. A short (ms shear pulse with fast rise time (2 ms produced an immediate increase in actinin tension at the upstream end of the cell with minimal changes at the downstream end. The onset of Ca2+ rise began at highly strained areas. In contrast to stimulus steps, slow ramp stimuli produced uniform forces throughout the cells and only a small Ca2+ response. The heterogeneity of force distribution is exaggerated in cells having fewer stress fibers and lower pre-tension in actinin. Disruption of cytoskeleton with cytochalasin-D (Cyt-D eliminated force gradients, and in those cells Ca2+ elevation started from the soma. Thus, Ca2+ influx with a mechanical stimulus depends on local stress within the cell and that is time dependent due to viscoelastic mechanics.

Histone deacetylase 6 inhibition reduces cysts by decreasing cAMP and Ca2+ in knock-out mouse models of polycystic kidney disease.

Science.gov (United States)

Yanda, Murali K; Liu, Qiangni; Cebotaru, Valeriu; Guggino, William B; Cebotaru, Liudmila

2017-10-27

Autosomal dominant polycystic kidney disease (ADPKD) is associated with progressive enlargement of multiple renal cysts, often leading to renal failure that cannot be prevented by a current treatment. Two proteins encoded by two genes are associated with ADPKD: PC1 ( pkd1 ), primarily a signaling molecule, and PC2 ( pkd2 ), a Ca 2+ channel. Dysregulation of cAMP signaling is central to ADPKD, but the molecular mechanism is unresolved. Here, we studied the role of histone deacetylase 6 (HDAC6) in regulating cyst growth to test the possibility that inhibiting HDAC6 might help manage ADPKD. Chemical inhibition of HDAC6 reduced cyst growth in PC1-knock-out mice. In proximal tubule-derived, PC1-knock-out cells, adenylyl cyclase 6 and 3 (AC6 and -3) are both expressed. AC6 protein expression was higher in cells lacking PC1, compared with control cells containing PC1. Intracellular Ca 2+ was higher in PC1-knock-out cells than in control cells. HDAC inhibition caused a drop in intracellular Ca 2+ and increased ATP-simulated Ca 2+ release. HDAC6 inhibition reduced the release of Ca 2+ from the endoplasmic reticulum induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+ -ATPase. HDAC6 inhibition and treatment of cells with the intracellular Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane- N , N , N ', N '-tetraacetic acid tetrakis(acetoxymethyl ester) reduced cAMP levels in PC1-knock-out cells. Finally, the calmodulin inhibitors W-7 and W-13 reduced cAMP levels, and W-7 reduced cyst growth, suggesting that AC3 is involved in cyst growth regulated by HDAC6. We conclude that HDAC6 inhibition reduces cell growth primarily by reducing intracellular cAMP and Ca 2+ levels. Our results provide potential therapeutic targets that may be useful as treatments for ADPKD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Chronic ethanol exposure induces SK-N-SH cell apoptosis by increasing N-methyl-D-aspartic acid receptor expression and intracellular calcium.

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Wang, Hongbo; Wang, Xiaolong; Li, Yan; Yu, Hao; Wang, Changliang; Feng, Chunmei; Xu, Guohui; Chen, Jiajun; You, Jiabin; Wang, Pengfei; Wu, Xu; Zhao, Rui; Zhang, Guohua

2018-04-01

It has been identified that chronic ethanol exposure damages the nervous system, particularly neurons. There is scientific evidence suggesting that neuronal loss caused by chronic ethanol exposure has an association with neuron apoptosis and intracellular calcium oscillation is one of the primary inducers of apoptosis. Therefore, the present study aimed to investigate the inductive effects of intracellular calcium oscillation on apoptosis in SK-N-SH human neuroblastoma cells and the protective effects of the N-methyl-D-aspartic acid receptor (NMDAR) antagonist, memantine, on SK-N-SH cell apoptosis caused by chronic ethanol exposure. SK-N-SH cells were treated with 100 mM ethanol and memantine (4 µM) for 2 days. Protein expression of NR1 was downregulated by RNA interference (RNAi). Apoptosis was detected by Annexin V/propidium iodide (PI) double-staining and flow cytometry and cell viability was detected using an MTS kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration and the levels of NR1 and caspase-3 were detected using western blotting. NR1 mRNA levels were also detected using qPCR. It was found that chronic ethanol exposure reduced neuronal cell viability and caused apoptosis of SK-N-SH cells, and the extent of damage in SK-N-SH cells was associated with ethanol exposure concentration and time. In addition, chronic ethanol exposure increased the concentration of intracellular calcium in SK-N-SH cells by inducing the expression of NMDAR, resulting in apoptosis, and memantine treatment reduced ethanol-induced cell apoptosis. The results of the present study indicate that the application of memantine may provide a novel strategy for the treatment of alcoholic dementia.

Behavior-Dependent Activity and Synaptic Organization of Septo-hippocampal GABAergic Neurons Selectively Targeting the Hippocampal CA3 Area.

Science.gov (United States)

Joshi, Abhilasha; Salib, Minas; Viney, Tim James; Dupret, David; Somogyi, Peter

2017-12-20

Rhythmic medial septal (MS) GABAergic input coordinates cortical theta oscillations. However, the rules of innervation of cortical cells and regions by diverse septal neurons are unknown. We report a specialized population of septal GABAergic neurons, the Teevra cells, selectively innervating the hippocampal CA3 area bypassing CA1, CA2, and the dentate gyrus. Parvalbumin-immunopositive Teevra cells show the highest rhythmicity among MS neurons and fire with short burst duration (median, 38 ms) preferentially at the trough of both CA1 theta and slow irregular oscillations, coincident with highest hippocampal excitability. Teevra cells synaptically target GABAergic axo-axonic and some CCK interneurons in restricted septo-temporal CA3 segments. The rhythmicity of their firing decreases from septal to temporal termination of individual axons. We hypothesize that Teevra neurons coordinate oscillatory activity across the septo-temporal axis, phasing the firing of specific CA3 interneurons, thereby contributing to the selection of pyramidal cell assemblies at the theta trough via disinhibition. VIDEO ABSTRACT. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

Science.gov (United States)

Yang, Yu-Mi; Park, Soonhong; Ji, Hyewon; Kim, Tae-Im; Kim, Eung Kweon; Kang, Kyung Koo; Shin, Dong Min

2014-04-01

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca(2+) signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca(2+) signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca(2+)-activated Cl(-) channels (CaCCs) and increased intracellular calcium concentrations ([Ca(2+)]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca(2+)]i in mouse salivary gland cells and human corneal epithelial cells. [Ca(2+)]i increase of DA-6034 was dependent on the Ca(2+) entry from extracellular and Ca(2+) release from internal Ca(2+) stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca(2+) stores. These results suggest that DA-6034 induces Ca(2+) signaling via extracellular Ca(2+) entry and RyRs-sensitive Ca(2+) release from internal Ca(2+) stores in epithelial cells.

Ghrelin inhibits proliferation and increases T-type Ca2+ channel expression in PC-3 human prostate carcinoma cells

International Nuclear Information System (INIS)

Diaz-Lezama, Nundehui; Hernandez-Elvira, Mariana; Sandoval, Alejandro; Monroy, Alma; Felix, Ricardo; Monjaraz, Eduardo

2010-01-01

Research highlights: → Ghrelin decreases prostate carcinoma PC-3 cells proliferation. → Ghrelin favors apoptosis in PC-3 cells. → Ghrelin increase in intracellular free Ca 2+ levels in PC-3 cells. → Grelin up-regulates expression of T-type Ca 2+ channels in PC-3 cells. → PC-3 cells express T-channels of the Ca V 3.1 and Ca V 3.2 subtype. -- Abstract: Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating the cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca 2+ levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca 2+ channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca 2+ channel expression.

Cholinergic induction of input-specific late-phase LTP via localized Ca2+ release in the visual cortex.

Science.gov (United States)

Cho, Kwang-Hyun; Jang, Hyun-Jong; Jo, Yang-Hyeok; Singer, Wolf; Rhie, Duck-Joo

2012-03-28

Acetylcholine facilitates long-term potentiation (LTP) and long-term depression (LTD), substrates of learning, memory, and sensory processing, in which acetylcholine also plays a crucial role. Ca(2+) ions serve as a canonical regulator of LTP/LTD but little is known about the effect of acetylcholine on intracellular Ca(2+) dynamics. Here, we investigated dendritic Ca(2+) dynamics evoked by synaptic stimulation and the resulting LTP/LTD in layer 2/3 pyramidal neurons of the rat visual cortex. Under muscarinic stimulation, single-shock electrical stimulation (SES) inducing ∼20 mV EPSP, applied via a glass electrode located ∼10 μm from the basal dendrite, evoked NMDA receptor-dependent fast Ca(2+) transients and the subsequent Ca(2+) release from the inositol 1,4,5-trisphosphate (IP(3))-sensitive stores. These secondary dendritic Ca(2+) transients were highly localized within 10 μm from the center (SD = 5.0 μm). The dendritic release of Ca(2+) was a prerequisite for input-specific muscarinic LTP (LTPm). Without the secondary Ca(2+) release, only muscarinic LTD (LTDm) was induced. D(-)-2-amino-5-phosphopentanoic acid and intracellular heparin blocked LTPm as well as dendritic Ca(2+) release. A single burst consisting of 3 EPSPs with weak stimulus intensities instead of the SES also induced secondary Ca(2+) release and LTPm. LTPm and LTDm were protein synthesis-dependent. Furthermore, LTPm was confined to specific dendritic compartments and not inducible in distal apical dendrites. Thus, cholinergic activation facilitated selectively compartment-specific induction of late-phase LTP through IP(3)-dependent Ca(2+) release.

Intermittent Hypoxia Inhibits Na+-H+ Exchange-Mediated Acid Extrusion Via Intracellular Na+ Accumulation in Cardiomyocytes

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Huai-Ren Chang

2018-04-01

Full Text Available Background/Aims: Intermittent hypoxia (IH has been shown to exert preconditioning-like cardioprotective effects. It also has been reported that IH preserves intracellular pH (pHi during ischemia and protects cardiomyocytes against ischemic reperfusion injury. However, the exact mechanism is still unclear. Methods: In this study, we used proton indicator BCECF-AM to analyze the rate of pHi recovery from acidosis in the IH model of rat neonatal cardiomyocytes. Neonatal cardiomyocytes were first treated with repetitive hypoxia-normoxia cycles for 1-4 days. Cells were then acid loaded with NH4Cl, and the rate of pHi recovery from acidosis was measured. Results: We found that the pHi recovery rate from acidosis was much slower in the IH group than in the room air (RA group. When we treated cardiomyocytes with Na+-H+ exchange (NHE inhibitors (Amiloride and HOE642 or Na+-free Tyrode solution during the recovery, there was no difference between RA and IH groups. We also found intracellular Na+ concentration ([Na+]i significantly increased after IH exposure for 4 days. However, the phenomenon could be abolished by pretreatment with ROS inhibitors (SOD and phenanathroline, intracellular calcium chelator or Na+-Ca2+ exchange (NCX inhibitor. Furthermore, the pHi recovery rate from acidosis became faster in the IH group than in the RA group when inhibition of NCX activity. Conclusions: These results suggest that IH would induce the elevation of ROS production. ROS then activates Ca2+-efflux mode of NCX and results in intracellular Na+ accumulation. The rise of [Na+]i further inhibits the activity of NHE-mediated acid extrusion and retards the rate of pHi recovery from acidosis during IH.

Back to the future with the AGP-Ca2+ flux capacitor.

Science.gov (United States)

Lamport, Derek T A; Varnai, Peter; Seal, Charlotte E

2014-10-01

Arabinogalactan proteins (AGPs) are ubiquitous in green plants. AGPs comprise a widely varied group of hydroxyproline (Hyp)-rich cell surface glycoproteins (HRGPs). However, the more narrowly defined classical AGPs massively predominate and cover the plasma membrane. Extensive glycosylation by pendant polysaccharides O-linked to numerous Hyp residues like beads of a necklace creates a unique ionic compartment essential to a wide range of physiological processes including germination, cell extension and fertilization. The vital clue to a precise molecular function remained elusive until the recent isolation of small Hyp-arabinogalactan polysaccharide subunits; their structural elucidation by nuclear magentic resonance imaging, molecular simulations and direct experiment identified a 15-residue consensus subunit as a β-1,3-linked galactose trisaccharide with two short branched sidechains each with a single glucuronic acid residue that binds Ca(2+) when paired with its adjacent sidechain. AGPs bind Ca(2+) (Kd ∼ 6 μm) at the plasma membrane (PM) at pH ∼5·5 but release it when auxin-dependent PM H(+)-ATPase generates a low periplasmic pH that dissociates AGP-Ca(2+) carboxylates (pka ∼3); the consequential large increase in free Ca(2+) drives entry into the cytosol via Ca(2+) channels that may be voltage gated. AGPs are thus arguably the primary source of cytosolic oscillatory Ca(2+) waves. This differs markedly from animals, in which cytosolic Ca(2+) originates mostly from internal stores such as the sarcoplasmic reticulum. In contrast, we propose that external dynamic Ca(2+) storage by a periplasmic AGP capacitor co-ordinates plant growth, typically involving exocytosis of AGPs and recycled Ca(2+), hence an AGP-Ca(2+) oscillator. The novel concept of dynamic Ca(2+) recycling by an AGP-Ca(2+) oscillator solves the long-standing problem of a molecular-level function for classical AGPs and thus integrates three fields: AGPs, Ca(2+) signalling and auxin. This

Hypoxia activates a Ca2+-permeable cation conductance sensitive to carbon monoxide and to GsMTx-4 in human and mouse sickle erythrocytes.

Science.gov (United States)

Vandorpe, David H; Xu, Chang; Shmukler, Boris E; Otterbein, Leo E; Trudel, Marie; Sachs, Frederick; Gottlieb, Philip A; Brugnara, Carlo; Alper, Seth L

2010-01-15

Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca(2+)] ([Ca(2+)](i)) and subsequent activation of K(Ca) 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration. We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca(2+)- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 microM), dipyridamole (100 microM), DIDS (100 microM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca(2+)](i), in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca(2+)](i) in mouse sickle erythrocytes did not require KCa3.1 activity. The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca(2+)-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca(2+) for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

Hypoxia activates a Ca2+-permeable cation conductance sensitive to carbon monoxide and to GsMTx-4 in human and mouse sickle erythrocytes.

Directory of Open Access Journals (Sweden)

David H Vandorpe

2010-01-01

Full Text Available Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle, leading to elevated intracellular [Ca(2+] ([Ca(2+](i and subsequent activation of K(Ca 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration.We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca(2+- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 microM, dipyridamole (100 microM, DIDS (100 microM, and carbon monoxide (25 ppm pretreatment. Deoxygenation also elevates sickle erythrocyte [Ca(2+](i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca(2+](i in mouse sickle erythrocytes did not require KCa3.1 activity.The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca(2+-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca(2+ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

A memristor-based third-order oscillator: beyond oscillation

KAUST Repository

Talukdar, Abdul Hafiz Ibne

2012-10-06

This paper demonstrates the first third-order autonomous linear time variant circuit realization that enhances parametric oscillation through the usage of memristor in conventional oscillators. Although the output has sustained oscillation, the linear features of the conventional oscillators become time dependent. The poles oscillate in nonlinear behavior due to the oscillation of memristor resistance. The mathematical formulas as well as SPICE simulations are introduced for the memristor-based phase shift oscillator showing a great matching.

A memristor-based third-order oscillator: beyond oscillation

KAUST Repository

Talukdar, Abdul Hafiz Ibne; Radwan, Ahmed G.; Salama, Khaled N.

2012-01-01

This paper demonstrates the first third-order autonomous linear time variant circuit realization that enhances parametric oscillation through the usage of memristor in conventional oscillators. Although the output has sustained oscillation, the linear features of the conventional oscillators become time dependent. The poles oscillate in nonlinear behavior due to the oscillation of memristor resistance. The mathematical formulas as well as SPICE simulations are introduced for the memristor-based phase shift oscillator showing a great matching.

Oscillations in the wake of a flare blast wave

Science.gov (United States)

Tothova, D.; Innes, D. E.; Stenborg, G.

2011-04-01

Context. Oscillations of coronal loops in the Sun have been reported in both imaging and spectral observations at the onset of flares. Images reveal transverse oscillations, whereas spectra detect line-of-sight velocity or Doppler-shift oscillations. The Doppler-shift oscillations are commonly interpreted as longitudinal modes. Aims: Our aim is to investigate the relationship between loop dynamics and flows seen in TRACE 195 Å images and Doppler shifts observed by SUMER in Si iii 1113.2 Å and FeXIX 1118.1 Å at the time of a C.8-class limb flare and an associated CME. Methods: We carefully co-aligned the sequence of TRACE 195 Å images to structures seen in the SUMER Si iii, CaX, and FeXIX emission lines. Additionally, Hα observations of a lifting prominence associated with the flare and the coronal mass ejection (CME) are available in three bands around 6563.3 Å. They give constraints on the timing and geometry. Results: Large-scale Doppler-shift oscillations in FeXIX and transverse oscillations in intensity images were observed over a large region of the corona after the passage of a wide bright extreme-ultraviolet (EUV) disturbance, which suggests ionization, heating, and acceleration of hot plasma in the wake of a blast wave. The online movie associated to Fig. 2 is available at http://www.aanda.org and at http://www.mps.mpg.de/data/outgoing/tothova/movie.gif

The driving force of the Na+/Ca2+-exchanger during metabolic inhibition

NARCIS (Netherlands)

Baartscheer, Antonius; Schumacher, Cees A.; Coronel, Ruben; Fiolet, Jan W. T.

2011-01-01

Objective: Metabolic inhibition causes a decline in mechanical performance and, if prolonged, myocardial contracture and cell death. The decline in mechanical performance is mainly due to altered intracellular calcium handling, which is under control of the Na+/Ca2+-exchanger (NCX) The driving force

Effects of calcium (Ca(2+)) extrusion mechanisms on electrophysiological properties in a hypoglossal motoneuron: insight from a mathematical model.

Science.gov (United States)

Horn, Kyle G; Solomon, Irene C

2014-01-01

Spike-frequency dynamics and spike shape can provide insight into the types of ion channels present in any given neuron and give a sense for the precise response any neuron may have to a given input stimulus. Motoneuron firing frequency over time is especially important due to its direct effect on motor output. Of particular interest is intracellular Ca(2+), which exerts a powerful influence on both firing properties over time and spike shape. In order to better understand the cellular mechanisms for the regulation of intracellular Ca(2+) and their effect on spiking behavior, we have modified a computational model of an HM to include a variety of Ca(2+) handling processes. For the current study, a series of HM models that include Ca(2+) pumps, Na(+)/Ca(2+) exchangers, and a generic exponential decay of excess Ca(2+) were generated. Simulations from these models indicate that although each extrusion mechanism exerts a similar effect on voltage, the firing properties change distinctly with the inclusion of additional Ca(2+)-related mechanisms: BK channels, Ca(2+) buffering, and diffusion of [Ca(2+)]i modeled via a linear diffusion partial differential equation. While an exponential decay of Ca(2+) seems to adequately capture short-term changes in firing frequency seen in biological data, internal diffusion of Ca(2+) appears to be necessary for capturing longer term frequency changes. © 2014 Elsevier B.V. All rights reserved.

Dysregulation of Neuronal Ca2+ Channel Linked to Heightened Sympathetic Phenotype in Prohypertensive States

OpenAIRE

Larsen, Hege E.; Bardsley, Emma N.; Lefkimmiatis, Konstantinos; Paterson, David J.

2016-01-01

Hypertension is associated with impaired nitric oxide (NO)–cyclic nucleotide (CN)-coupled intracellular calcium (Ca2+) homeostasis that enhances cardiac sympathetic neurotransmission. Because neuronal membrane Ca2+ currents are reduced by NO-activated S-nitrosylation, we tested whether CNs affect membrane channel conductance directly in neurons isolated from the stellate ganglia of spontaneously hypertensive rats (SHRs) and their normotensive controls. Using voltage-clamp and cAMP–protein kin...

A critical comparison of the current view of Ca signaling with the novel concept of F-actin-based Ca signaling.

Science.gov (United States)

Lange, Klaus; Gartzke, Joachim

2006-01-01

A detailed comparative survey on the current idea of Ca signaling and the alternative concept of F-actin-based Ca signaling is given. The two hypotheses differ in one central aspect - the mechanism of Ca storage. The current theory rests on the assumption of Ca-accumulating vesicles derived from the endoplasmic/ sarcoplasmic reticulum, which are equipped with an ATP-dependent Ca pump and IP3- or ryanodine-sensitive Ca-release channels/receptors. The alternative hypothesis proceeds from the idea of Ca storage at the high-affinity binding sites of F-actin subunits. Several prominent features of Ca signaling, which are not adequately described by the current concept, are inherent properties of the F-actin system and its dynamic state of treadmilling. F-actin is the only known biological Ca-binding system that has been proven by in vitro experiments to work within the physiological range of Ca concentrations and the only system that meets all necessary conditions to function as receptor-operated Ca store and as a coupling device between the Ca store and the store-operated Ca influx pathway. The most important properties of Ca signaling, such as store-channel coupling, quantal Ca release, spiking and oscillations, biphasic and "phasic" uptake kinetics, and Ca-induced Ca release, turn out to be systematic features of the new concept but remain unexplained by the classical vesicle storage hypothesis. A number of novel findings, specifically recent reports about direct effects of actin-specific toxins on Ca stores, have strengthened the new concept. The concept of F-actin-based Ca signaling combined with the notion of microvillar regulation of ion and substrate fluxes opens new aspects and far-reaching consequences, not only for cellular Ca signaling but also for various other cell functions, and represents an opportunity to connect several fields of cell physiology on the basis of a common mechanism.

Influence of a chinese crude drug on Ca2+ influx and efflux in rat visceral organs:Investigation and evaluation by 45Ca

International Nuclear Information System (INIS)

Yang Yuanyou; Liu Ning; Mo Zhengji; Xie Jianping; Liao Jiali; Mo Shangwu

2006-01-01

The influences of a Chinese crude drug, Herba Epimedii (HE), on Ca 2+ influx and efflux in the isolated rat aorta and some visceral organs were evaluated by using 45 Ca as a radioactive tracer. Additionally, its protective effect on myocardial ischemia was investigated in live animals. The results indicated that HE has significant influence on Ca 2+ influx and efflux in the isolated rat aorta, heart, and kidney, in that it can markedly block 45 Ca entering into cell and can facilitate efflux of intracellular Ca 2+ . However, among the three kinds of extracts from HE, the alkali extracts have the most obvious effect on calcium channels in visceral organs. Even if the alkali extracts are diluted by water for 10 times, the material still has a rather strong inhibition effect on calcium channels. Fortunately, the three kinds of extracts have favorable protective effect on myocardial ischemia induced by drugs or by the ligation of the coronary artery. This is consistent with the results about the Ca 2+ influx and efflux obtained by isotope tracer technique, and implies that the Chinese crude drug has attractive potential for the treatment of heart, cerebrovascular and other diseases

Altered myoplasmic Ca(2+) handling in rat fast-twitch skeletal muscle fibres during disuse atrophy.

Science.gov (United States)

Weiss, Norbert; Andrianjafiniony, Tina; Dupré-Aucouturier, Sylvie; Pouvreau, Sandrine; Desplanches, Dominique; Jacquemond, Vincent

2010-03-01

Calcium-dependent signalling pathways are believed to play an important role in skeletal muscle atrophy, but whether intracellular Ca(2+) homeostasis is affected in that situation remains obscure. We show here that there is a 20% atrophy of the fast-type flexor digitorum brevis (FDB) muscle in rats hind limb unloaded (HU) for 2 weeks, with no change in fibre type distribution. In voltage-clamp experiments, the amplitude of the slow Ca(2+) current was found similar in fibres from control and HU animals. In fibres loaded with the Ca(2+) dye indo-1, the value for the rate of [Ca(2+)] decay after the end of 5-100-ms-long voltage-clamp depolarisations from -80 to +10 mV was found to be 30-50% lower in fibres from HU animals. This effect was consistent with a reduced contribution of both saturable and non-saturable components of myoplasmic Ca(2+) removal. However, there was no change in the relative amount of parvalbumin, and type 1 sarco-endoplasmic reticulum Ca(2+)-ATPase was increased by a factor of three in the atrophied muscles. Confocal imaging of mitochondrial membrane potential showed that atrophied FDB fibres had significantly depolarized mitochondria as compared to control fibres. Depolarization of mitochondria in control fibres with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone induced a slowing of the decay of [Ca(2+)] transients accompanied by an increase in resting [Ca(2+)] and a reduction of the peak amplitude of the transients. Overall results provide the first functional evidence for severely altered intracellular Ca(2+) removal capabilities in atrophied fast-type muscle fibres and highlight the possible contribution of reduced mitochondrial polarisation.

Na+/Ca2+ exchange and Na+/K+-ATPase in the heart

Science.gov (United States)

Shattock, Michael J; Ottolia, Michela; Bers, Donald M; Blaustein, Mordecai P; Boguslavskyi, Andrii; Bossuyt, Julie; Bridge, John H B; Chen-Izu, Ye; Clancy, Colleen E; Edwards, Andrew; Goldhaber, Joshua; Kaplan, Jack; Lingrel, Jerry B; Pavlovic, Davor; Philipson, Kenneth; Sipido, Karin R; Xie, Zi-Jian

2015-01-01

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation–contraction coupling and arrhythmias: Na+ channel and Na+ transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na+/Ca2+ exchange (NCX) and Na+/K+-ATPase (NKA). While the relevance of Ca2+ homeostasis in cardiac function has been extensively investigated, the role of Na+ regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na+ content have multiple effects on the heart by influencing intracellular Ca2+ and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na+ homeostasis. Among the proteins that accomplish this task are the Na+/Ca2+ exchanger (NCX) and the Na+/K+ pump (NKA). By transporting three Na+ ions into the cytoplasm in exchange for one Ca2+ moved out, NCX is one of the main Na+ influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na+ ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na+ and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na+ homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na+/Ca2+ exchanger (NCX1) and Na+/K+ pump and the controversies that still persist in the field. PMID:25772291

Sodium-calcium exchanger and R-type Ca2+ channels mediate spontaneous [Ca2+](i) oscillations in magnocellular neurones of the rat supraoptic nucleus

Czech Academy of Sciences Publication Activity Database

Kortus, Štěpán; Srinivasan, Chinnapaiyan; Forostyak, Oksana; Zápotocký, M.; Ueta, Y.; Syková, Eva; Chvátal, Alexandr; Verkhratsky, A.; Dayanithi, Govindan

2016-01-01

Roč. 59, č. 6 (2016), s. 289-298 ISSN 0143-4160 R&D Projects: GA ČR(CZ) GA14-34077S; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : 1,4,5-Trisphosphate * Ca(2+) channel toxins * Ca(2+) clearance * Ca(2+) homeostasis Subject RIV: FH - Neurology Impact factor: 3.707, year: 2016

Intercellular Ca2+ Waves: Mechanisms and Function

Science.gov (United States)

Sanderson, Michael J.

2012-01-01

Intercellular calcium (Ca2+) waves (ICWs) represent the propagation of increases in intracellular Ca2+ through a syncytium of cells and appear to be a fundamental mechanism for coordinating multicellular responses. ICWs occur in a wide diversity of cells and have been extensively studied in vitro. More recent studies focus on ICWs in vivo. ICWs are triggered by a variety of stimuli and involve the release of Ca2+ from internal stores. The propagation of ICWs predominately involves cell communication with internal messengers moving via gap junctions or extracellular messengers mediating paracrine signaling. ICWs appear to be important in both normal physiology as well as pathophysiological processes in a variety of organs and tissues including brain, liver, retina, cochlea, and vascular tissue. We review here the mechanisms of initiation and propagation of ICWs, the key intra- and extracellular messengers (inositol 1,4,5-trisphosphate and ATP) mediating ICWs, and the proposed physiological functions of ICWs. PMID:22811430

Cell enlargement of plant tissue explants oscillates with a temperature-compensated period of ca. 24 min

Science.gov (United States)

Morre, D. James; Ternes, Philipp; Morre, Dorothy M.

2002-01-01

Rate of plant cell enlargement, measured at intervals of 3 min using a sensitive linear transducer, oscillates with a minimum period of about 24 min that parallels the 24-min periodicity observed with the oxidation of NADH by the external plasma membrane NADH oxidase and of single cells measured previously by video-enhanced light microscopy. Also exhibiting 24-min oscillations is the steady-state rate of cell enlargement induced by the addition of the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges. The length of the 24-min period is temperature compensated and remains constant at 24 min when measured at 15, 25 or 35 degrees C, despite the fact that the rate of cell enlargement approximately doubles for each 10 degree C rise over this same range of temperatures.

Ca2+ signaling in pancreatic acinar cells: physiology and pathophysiology

Directory of Open Access Journals (Sweden)

O.H. Petersen

2009-01-01

Full Text Available The pancreatic acinar cell is a classical model for studies of secretion and signal transduction mechanisms. Because of the extensive endoplasmic reticulum and the large granular compartment, it has been possible - by direct measurements - to obtain considerable insights into intracellular Ca2+ handling under both normal and pathological conditions. Recent studies have also revealed important characteristics of stimulus-secretion coupling mechanisms in isolated human pancreatic acinar cells. The acinar cells are potentially dangerous because of the high intra-granular concentration of proteases, which become inappropriately activated in the human disease acute pancreatitis. This disease is due to toxic Ca2+ signals generated by excessive liberation of Ca2+ from both the endoplasmic reticulum and the secretory granules.

Ghrelin inhibits proliferation and increases T-type Ca{sup 2+} channel expression in PC-3 human prostate carcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Diaz-Lezama, Nundehui; Hernandez-Elvira, Mariana [Laboratory of Neuroendocrinology, Institute of Physiology, Autonomous University of Puebla (BUAP), Puebla (Mexico); Sandoval, Alejandro [School of Medicine FES Iztacala, National Autonomous University of Mexico (UNAM), Tlalnepantla (Mexico); Monroy, Alma; Felix, Ricardo [Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico City (Mexico); Monjaraz, Eduardo, E-mail: emguzman@siu.buap.mx [Laboratory of Neuroendocrinology, Institute of Physiology, Autonomous University of Puebla (BUAP), Puebla (Mexico)

2010-12-03

Research highlights: {yields} Ghrelin decreases prostate carcinoma PC-3 cells proliferation. {yields} Ghrelin favors apoptosis in PC-3 cells. {yields} Ghrelin increase in intracellular free Ca{sup 2+} levels in PC-3 cells. {yields} Grelin up-regulates expression of T-type Ca{sup 2+} channels in PC-3 cells. {yields} PC-3 cells express T-channels of the Ca{sub V}3.1 and Ca{sub V}3.2 subtype. -- Abstract: Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating the cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca{sup 2+} levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca{sup 2+} channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca{sup 2+} channel expression.

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

KAUST Repository

Ren, Jian

2012-03-06

Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E 2) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E 2 elevated [Ca 2+] i and increased Ca 2+ oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E 2 mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E 2 activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E 2 induces the non-genomic responses Ca 2+ release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E 2 responses. © 2012 Springer Science+Business Media, LLC.

Teaching the Modes of Ca[superscript 2+] Transport between the Plasma Membrane and Endoplasmic Reticulum Using a Classic Paper by Kwan et al.

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Liang, Willmann

2009-01-01

This teaching article uses the report by Kwan et al., "Effects of methacholine, thapsigargin, and La[superscript 3+] on plasmalemmal and intracellular Ca[superscript 2+] transport in lacrimal acinar cells," where the effects of Ca[superscript 2+]-mobilizing agents in regulating Ca[superscript 2+] fluxes were examined under various conditions.…

Phosphorylation of CaMKII in the rat dorsal raphe nucleus plays an important role in sleep-wake regulation.

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Cui, Su-Ying; Li, Sheng-Jie; Cui, Xiang-Yu; Zhang, Xue-Qiong; Yu, Bin; Sheng, Zhao-Fu; Huang, Yuan-Li; Cao, Qing; Xu, Ya-Ping; Lin, Zhi-Ge; Yang, Guang; Song, Jin-Zhi; Ding, Hui; Wang, Zi-Jun; Zhang, Yong-He

2016-02-01

The Ca(2+) modulation in the dorsal raphe nucleus (DRN) plays an important role in sleep-wake regulation. Calmodulin-dependent kinase II (CaMKII) is an important signal-transducing molecule that is activated by Ca(2+) . This study investigated the effects of intracellular Ca(2+) /CaMKII signaling in the DRN on sleep-wake states in rats. Maximum and minimum CaMKII phosphorylation was detected at Zeitgeber time 21 (ZT 21; wakefulness state) and ZT 3 (sleep state), respectively, across the light-dark rhythm in the DRN in rats. Six-hour sleep deprivation significantly reduced CaMKII phosphorylation in the DRN. Microinjection of the CAMKII activation inhibitor KN-93 (5 or 10 nmol) into the DRN suppressed wakefulness and enhanced rapid-eye-movement sleep (REMS) and non-REM sleep (NREMS). Application of a high dose of KN-93 (10 nmol) increased slow-wave sleep (SWS) time, SWS bouts, the mean duration of SWS, the percentage of SWS relative to total sleep, and delta power density during NREMS. Microinjection of CaCl2 (50 nmol) in the DRN increased CaMKII phosphorylation and decreased NREMS, SWS, and REMS. KN-93 abolished the inhibitory effects of CaCl2 on NREMS, SWS, and REMS. These data indicate a novel wake-promoting and sleep-suppressing role for the Ca(2+) /CaMKII signaling pathway in DRN neurons. We propose that the intracellular Ca(2+) /CaMKII signaling in the dorsal raphe nucleus (DRN) plays wake-promoting and sleep-suppressing role in rats. Intra-DRN application of KN-93 (CaMKII activation inhibitor) suppressed wakefulness and enhanced rapid-eye-movement sleep (REMS) and non-REMS (NREMS). Intra-DRN application of CaCl2 attenuated REMS and NREMS. We think these findings should provide a novel cellular and molecular mechanism of sleep-wake regulation. © 2015 International Society for Neurochemistry.

Detection and Measurement of the Intracellular Calcium Variation in Follicular Cells

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Ana M. Herrera-Navarro

2014-01-01

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

Pb2+ Modulates Ca2+ Membrane Permeability In Paramecium

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Bernal-Martínez, Juan; Ortega Soto, Arturo

2004-09-01

Intracellular recording experiments in current clamp configuration were done to evaluate whether Pb2+ modulates ionic membrane permeability in the fresh water Paramecium tetraurelia. It was found that Pb2+ triggers in a dose-dependent manner, a burst of spontaneous action potentials followed by a robust and sustained after hyper-polarization. In addition, Pb2+ increased the frequency of firing the spontaneous Ca2+-Action Potential and also, the duration of Ca2+-Action Potential, in a dose and reversibly-dependent manner. These results suggest that Pb2+ increases calcium membrane permeability of Paramecium and probably activates a calcium-dependent-potassium conductance in the ciliate.

Line list for the ground state of CaF

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Hou, Shilin; Bernath, Peter F.

2018-05-01

The molecular potential energy function and electronic dipole moment function for the ground state of CaF were studied with MRCI, ACPF, and RCCSD(T) ab initio calculations. The RCCSD(T) potential function reproduces the experimental vibrational intervals to within ∼2 cm-1. The RCCSD(T) dipole moment at the equilibrium internuclear separation agrees well with the experimental value. Over a wide range of internuclear separations, far beyond the range associated with the observed spectra, the ab initio dipole moment functions are similar and highly linear. An extended Morse oscillator (EMO) potential function was also obtained by fitting the observed lines of the laboratory vibration-rotation and pure rotation spectra of the 40CaF X2Σ+ ground state. The fitted potential reproduces the observed transitions (v ≤ 8, N ≤ 121, Δv = 0, 1) within their experimental uncertainties. With this EMO potential and the RCCSD(T) dipole moment function, line lists for 40CaF, 42CaF, 43CaF, 44CaF, 46CaF, and 48CaF were computed for v ≤ 10, N ≤ 121, Δv = 0-10. The calculated emission spectra are in good agreement with an observed laboratory spectrum of CaF at a sample temperature of 1873 K.

A mitochondrial-targeted antioxidant improves myofilament Ca2+ sensitivity during prolonged low frequency force depression at low PO2.

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Gandra, Paulo G; Shiah, Amy A; Nogueira, Leonardo; Hogan, Michael C

2018-03-15

Skeletal muscle contractile activity is associated with an enhanced reactive oxygen species (ROS) generation. At very low PO2, ROS generation by mitochondria can be elevated in intact cells. An elevated intracellular oxidant activity may affect muscle force development and recovery from fatigue. We treated intact single muscle fibres with a mitochondrial antioxidant and stimulated the fibres to contract at a low extracellular PO2 that is similar to the intracellular PO2 that is observed during moderate to intense exercise in vivo. The mitochondrial antioxidant prevented a sustained decrease in the myofibrillar Ca 2+ sensitivity and improved muscle submaximal force development after fatigue at low extracellular PO2. Skeletal muscle can develop a prolonged low frequency-stimulation force depression (PLFFD) following fatigue-inducing contractions. Increased levels of reactive oxygen species (ROS) have been implicated in the development of PLFFD. During exercise the skeletal muscle intracellular PO2 decreases to relatively low levels, and can be further decreased when there is an impairment in O 2 diffusion or availability, such as in certain chronic diseases and during exercise at high altitude. Since ROS generation by mitochondria is elevated at very low PO2 in cells, we tested the hypothesis that treatment of muscle fibres with a mitochondrial-targeted antioxidant at a very low, near hypoxic, PO2 can attenuate PLFFD. We treated intact single fibres from mice with the mitochondrial-specific antioxidant SS31, and measured force development and intracellular [Ca 2+ ] 30 min after fatigue at an extracellular PO2 of ∼5 Torr. After 30 min following the end of the fatiguing contractions, fibres treated with SS31 showed significantly less impairment in force development compared to untreated fibres at submaximal frequencies of stimulation. The cytosolic peak [Ca 2+ ] transients (peak [Ca 2+ ] c ) were equally decreased in both groups compared to pre-fatigue values. The

Ca2+ transport and signalling in enamel cells

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Nurbaeva, Meerim K.; Eckstein, Miriam; Feske, Stefan

2016-01-01

Abstract Dental enamel is one of the most remarkable examples of matrix‐mediated biomineralization. Enamel crystals form de novo in a rich extracellular environment in a stage‐dependent manner producing complex microstructural patterns that are visually stunning. This process is orchestrated by specialized epithelial cells known as ameloblasts which themselves undergo striking morphological changes, switching function from a secretory role to a cell primarily engaged in ionic transport. Ameloblasts are supported by a host of cell types which combined represent the enamel organ. Fully mineralized enamel is the hardest tissue found in vertebrates owing its properties partly to the unique mixture of ionic species represented and their highly organized assembly in the crystal lattice. Among the main elements found in enamel, Ca2+ is the most abundant ion, yet how ameloblasts modulate Ca2+ dynamics remains poorly known. This review describes previously proposed models for passive and active Ca2+ transport, the intracellular Ca2+ buffering systems expressed in ameloblasts and provides an up‐dated view of current models concerning Ca2+ influx and extrusion mechanisms, where most of the recent advances have been made. We also advance a new model for Ca2+ transport by the enamel organ. PMID:27510811

Dynamic analysis of the conditional oscillator underlying slow waves in thalamocortical neurons

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Francois eDavid

2016-02-01

Full Text Available During non-REM sleep the EEG shows characteristics waves that are generated by the dynamic interactions between cortical and thalamic oscillators. In thalamic neurons, low-threshold T-type Ca2+ channels play a pivotal role in almost every type of neuronal oscillations, including slow (<1 Hz waves, sleep spindles and delta waves. The transient opening of T channels gives rise to the low threshold spikes (LTSs, and associated high frequency bursts of action potentials, that are characteristically present during sleep spindles and delta waves, whereas the persistent opening of a small fraction of T channels, (i.e. ITwindow is responsible for the membrane potential bistability underlying sleep slow oscillations. Surprisingly thalamocortical (TC neurons express a very high density of T channels that largely exceed the amount required to generate LTSs and therefore, to support certain, if not all, sleep oscillations. Here, to clarify the relationship between T current density and sleep oscillations, we systematically investigated the impact of the T conductance level on the intrinsic rhythmic activities generated in TC neurons, combining in vitro experiments and TC neuron simulation. Using bifurcation analysis, we provide insights into the dynamical processes taking place at the transition between slow and delta oscillations. Our results show that although stable delta oscillations can be evoked with minimal T conductance, the full range of slow oscillation patterns, including groups of delta oscillations separated by Up states (grouped-delta slow waves requires a high density of T channels. Moreover, high levels of T conductance ensure the robustness of different types of slow oscillations.

Differential contribution of Ca2+ sources to day and night BK current activation in the circadian clock.

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Whitt, Joshua P; McNally, Beth A; Meredith, Andrea L

2018-02-05

Large conductance K + (BK) channels are expressed widely in neurons, where their activation is regulated by membrane depolarization and intracellular Ca 2+ (Ca 2+ i ). To enable this regulation, BK channels functionally couple to both voltage-gated Ca 2+ channels (VGCCs) and channels mediating Ca 2+ release from intracellular stores. However, the relationship between BK channels and their specific Ca 2+ source for particular patterns of excitability is not well understood. In neurons within the suprachiasmatic nucleus (SCN)-the brain's circadian clock-BK current, VGCC current, and Ca 2+ i are diurnally regulated, but paradoxically, BK current is greatest at night when VGCC current and Ca 2+ i are reduced. Here, to determine whether diurnal regulation of Ca 2+ is relevant for BK channel activation, we combine pharmacology with day and night patch-clamp recordings in acute slices of SCN. We find that activation of BK current depends primarily on three types of channels but that the relative contribution changes between day and night. BK current can be abrogated with nimodipine during the day but not at night, establishing that L-type Ca 2+ channels (LTCCs) are the primary daytime Ca 2+ source for BK activation. In contrast, dantrolene causes a significant decrease in BK current at night, suggesting that nighttime BK activation is driven by ryanodine receptor (RyR)-mediated Ca 2+ i release. The N- and P/Q-type Ca 2+ channel blocker ω-conotoxin MVIIC causes a smaller reduction of BK current that does not differ between day and night. Finally, inhibition of LTCCs, but not RyRs, eliminates BK inactivation, but the BK β2 subunit was not required for activation of BK current by LTCCs. These data reveal a dynamic coupling strategy between BK channels and their Ca 2+ sources in the SCN, contributing to diurnal regulation of SCN excitability. © 2018 Whitt et al.

Transient increase in Zn2+ in hippocampal CA1 pyramidal neurons causes reversible memory deficit.

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Atsushi Takeda

Full Text Available The translocation of synaptic Zn(2+ to the cytosolic compartment has been studied to understand Zn(2+ neurotoxicity in neurological diseases. However, it is unknown whether the moderate increase in Zn(2+ in the cytosolic compartment affects memory processing in the hippocampus. In the present study, the moderate increase in cytosolic Zn(2+ in the hippocampus was induced with clioquinol (CQ, a zinc ionophore. Zn(2+ delivery by Zn-CQ transiently attenuated CA1 long-term potentiation (LTP in hippocampal slices prepared 2 h after i.p. injection of Zn-CQ into rats, when intracellular Zn(2+ levels was transiently increased in the CA1 pyramidal cell layer, followed by object recognition memory deficit. Object recognition memory was transiently impaired 30 min after injection of ZnCl(2 into the CA1, but not after injection into the dentate gyrus that did not significantly increase intracellular Zn(2+ in the granule cell layer of the dentate gyrus. Object recognition memory deficit may be linked to the preferential increase in Zn(2+ and/or the preferential vulnerability to Zn(2+ in CA1 pyramidal neurons. In the case of the cytosolic increase in endogenous Zn(2+ in the CA1 induced by 100 mM KCl, furthermore, object recognition memory was also transiently impaired, while ameliorated by co-injection of CaEDTA to block the increase in cytosolic Zn(2+. The present study indicates that the transient increase in cytosolic Zn(2+ in CA1 pyramidal neurons reversibly impairs object recognition memory.

Transient increase in Zn2+ in hippocampal CA1 pyramidal neurons causes reversible memory deficit.

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Takeda, Atsushi; Takada, Shunsuke; Nakamura, Masatoshi; Suzuki, Miki; Tamano, Haruna; Ando, Masaki; Oku, Naoto

2011-01-01

The translocation of synaptic Zn(2+) to the cytosolic compartment has been studied to understand Zn(2+) neurotoxicity in neurological diseases. However, it is unknown whether the moderate increase in Zn(2+) in the cytosolic compartment affects memory processing in the hippocampus. In the present study, the moderate increase in cytosolic Zn(2+) in the hippocampus was induced with clioquinol (CQ), a zinc ionophore. Zn(2+) delivery by Zn-CQ transiently attenuated CA1 long-term potentiation (LTP) in hippocampal slices prepared 2 h after i.p. injection of Zn-CQ into rats, when intracellular Zn(2+) levels was transiently increased in the CA1 pyramidal cell layer, followed by object recognition memory deficit. Object recognition memory was transiently impaired 30 min after injection of ZnCl(2) into the CA1, but not after injection into the dentate gyrus that did not significantly increase intracellular Zn(2+) in the granule cell layer of the dentate gyrus. Object recognition memory deficit may be linked to the preferential increase in Zn(2+) and/or the preferential vulnerability to Zn(2+) in CA1 pyramidal neurons. In the case of the cytosolic increase in endogenous Zn(2+) in the CA1 induced by 100 mM KCl, furthermore, object recognition memory was also transiently impaired, while ameliorated by co-injection of CaEDTA to block the increase in cytosolic Zn(2+). The present study indicates that the transient increase in cytosolic Zn(2+) in CA1 pyramidal neurons reversibly impairs object recognition memory.

Temporal and spatial differences in intracellular Ca++ changes elicited by K+ and glutamate in single cultured neocortical neurons

DEFF Research Database (Denmark)

Belhage, B; Frandsen, A; Schousboe, A

1996-01-01

characteristics of voltage gated Ca++ channels are dramatically different in cell bodies and neurites. Moreover, the distribution of L-type channels activated by glutamate differs in cell bodies and neurites. Such differences in the spatial distribution of Ca++ channels are likely to be of major importance...... after exposure to K+. The Ca++ channel blockers verapamil and nifedipine affecting N- and L-type channels, respectively had differential effects on K+ stimulated increases in [Ca++]i. Nifedipine only affected the increase marginally whereas verapamil inhibited the response by 50-60% both in cell bodies...

Anticancer ruthenium(III) complex KP1019 interferes with ATP-dependent Ca2+ translocation by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA).

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Sadafi, Fabrizio-Zagros; Massai, Lara; Bartolommei, Gianluca; Moncelli, Maria Rosa; Messori, Luigi; Tadini-Buoninsegni, Francesco

2014-08-01

Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), a P-type ATPase that sustains Ca2+ transport and plays a major role in intracellular Ca2+ homeostasis, represents a therapeutic target for cancer therapy. Here, we investigated whether ruthenium-based anticancer drugs, namely KP1019 (indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)]), NAMI-A (imidazolium [trans-tetrachloro(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)]) and RAPTA-C ([Ru(η6-p-cymene)dichloro(1,3,5-triaza-7-phosphaadamantane)]), and cisplatin (cis-diammineplatinum(II) dichloride) might act as inhibitors of SERCA. Charge displacement by SERCA adsorbed on a solid-supported membrane was measured after ATP or Ca2+ concentration jumps. Our results show that KP1019, in contrast to the other metal compounds, is able to interfere with ATP-dependent translocation of Ca2+ ions. An IC50 value of 1 μM was determined for inhibition of calcium translocation by KP1019. Conversely, it appears that KP1019 does not significantly affect Ca2+ binding to the ATPase from the cytoplasmic side. Inhibition of SERCA at pharmacologically relevant concentrations may represent a crucial aspect in the overall pharmacological and toxicological profile of KP1019. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biphasic regulation of intracellular calcium by gemfibrozil contributes to inhibiting L6 myoblast differentiation: implications for clinical myotoxicity.

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Liu, Aiming; Yang, Julin; Gonzalez, Frank J; Cheng, Gary Q; Dai, Renke

2011-02-18

Gemfibrozil is the most myotoxic fibrate drug commonly used for dyslipidemia, but the mechanism is poorly understood. The current study revealed that gemfibrozil inhibits myoblast differentiation through the regulation of intracellular calcium ([Ca(2+)]i) as revealed in L6 myoblasts by use of laser scan confocal microscopy and flow cytometry using Fluo-4 AM as a probe. Gemfibrozil at 20-400 μM, could regulate [Ca(2+)]i in L6 cells in a biphasic manner, and sustained reduction was observed when the concentration reached 200 μM. Inhibition of L6 differentiation by gemfibrozil was concentration-dependent with maximal effect noted between 200 and 400 μM, as indicated by creatine kinase activities and the differentiation index, respectively. In differentiating L6 myoblasts, gemfibrozil at concentrations below 400 μM led to no significant signs of apoptosis or cytotoxicity, whereas differentiation, inhibited by 200 μM gemfibrozil, was only partially recovered. A good correlation was noted between gemfibrozil concentrations that regulate [Ca(2+)]i and inhibit L6 myoblasts differentiation, and both are within the range of total serum concentrations found in the clinic. These data suggest a potential pharmacodynamic effect of gemfibrozil on myogenesis as a warning sign, in addition to the complex pharmacokinetic interactions. It is also noteworthy that mobilization of [Ca(2+)]i by gemfibrozil may trigger complex biological responses besides myocyte differentiation. Information revealed in this study explores the mechanism of gemfibrozil-induced myotoxicity through the regulation of intracellular calcium.

Ketamine Protects Gamma Oscillations by Inhibiting Hippocampal LTD

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Huang, Lanting; Yang, Xiu-Juan; Huang, Ying; Sun, Eve Y.

2016-01-01

NMDA receptors have been widely reported to be involved in the regulation of synaptic plasticity through effects on long-term potentiation (LTP) and long-term depression (LTD). LTP and LTD have been implicated in learning and memory processes. Besides synaptic plasticity, it is known that the phenomenon of gamma oscillations is critical in cognitive functions. Synaptic plasticity has been widely studied, however it is still not clear, to what degree synaptic plasticity regulates the oscillations of neuronal networks. Two NMDA receptor antagonists, ketamine and memantine, have been shown to regulate LTP and LTD, to promote cognitive functions, and have even been reported to bring therapeutic effects in major depression and Alzheimer’s disease respectively. These compounds allow us to investigate the putative interrelationship between network oscillations and synaptic plasticity and to learn more about the mechanisms of their therapeutic effects. In the present study, we have identified that ketamine and memantine could inhibit LTD, without impairing LTP in the CA1 region of mouse hippocampus, which may underlie the mechanism of these drugs’ therapeutic effects. Our results suggest that NMDA-induced LTD caused a marked loss in the gamma power, and pretreatment with 10 μM ketamine prevented the oscillatory loss via its inhibitory effect on LTD. Our study provides a new understanding of the role of NMDA receptors on hippocampal plasticity and oscillations. PMID:27467732

Aequorin chimeras as valuable tool in the measurement of Ca2+ concentration during cadmium injury

International Nuclear Information System (INIS)

Biagioli, M.; Pinton, P.; Scudiero, R.; Ragghianti, M.; Bucci, S.; Rizzuto, R.

2005-01-01

The ability of cadmium to disrupt calcium homeostasis has been known since a long time, but the precise cellular targets of its toxic action are still debated. A great problem in the interpretation of data has been associated with the ability of cadmium to strongly bind traditional calcium probes. Aequorin, the well-characterized calcium-sensitive photoprotein, was used as intracellular calcium indicator during cadmium injury in NIH 3T3 murine fibroblasts. NIH 3T3 cells were transfected with a cDNA construct containing aequorin fused to a truncated glutamate receptor, which directs the probe to the outer surface of intracellular membranes. At first, we tested if different cadmium concentrations were able to modify the rate of light emission by aequorin showing that cadmium concentrations 2+ /Ca 2+ interference. To directly investigate the role of Cd 2+ in Ca 2+ homeostasis, we have started to selectively measure the free Ca 2+ concentration in different cell compartments. Here, we report that cadmium reduces the transient free calcium signal after stimulation of cells with bradykinin. Further studies are in progress to clarify the role of mitochondria and endoplasmic reticulum in cadmium-induced alterations of Ca 2+ homeostasis in order to link signal transduction modifications with the onset of apoptosis induced by cadmium exposure

Fluorescence methods for analysis of interactions between Ca(2+) signaling, lysosomes, and endoplasmic reticulum.

Science.gov (United States)

Prole, David L; López-Sanjurjo, Cristina I; Tovey, Stephen C; Taylor, Colin W

2015-01-01

The endoplasmic reticulum (ER) is both the major source of intracellular Ca(2+) for cell signaling and the organelle that forms the most extensive contacts with the plasma membrane and other organelles. Lysosomes fulfill important roles in degrading cellular materials and in cholesterol handling, but they also contribute to Ca(2+) signaling by both releasing and sequestering Ca(2+). Interactions between ER and other Ca(2+)-transporting membranes, notably mitochondria and the plasma membrane, often occur at sites where the two membranes are closely apposed, allowing local Ca(2+) signaling between them. These interactions are often facilitated by scaffold proteins. Recent evidence suggests similar local interactions between ER and lysosomes. We describe simple fluorescence-based methods that allow the interplay between Ca(2+) signals, the ER, and lysosomes to be examined. Copyright © 2015 Elsevier Inc. All rights reserved.

A common behaviour of thermoelectric layered cobaltites: incommensurate spin density wave states in [Ca2Co4/3Cu2/3O4]0.62[CoO2] and [Ca2CoO3]0.62[CoO2

International Nuclear Information System (INIS)

Sugiyama, J; Brewer, J H; Ansaldo, E J; Itahara, H; Dohmae, K; Xia, C; Seno, Y; Hitti, B; Tani, T

2003-01-01

Magnetism of a misfit layered cobaltite [Ca 2 Co 4/3 Cu 2/3 O 4 ] x RS [CoO 2 ] (x ∼ 0.62, RS denotes a rocksalt-type block) was investigated by a positive muon spin rotation and relaxation (μ + SR) experiment. A transition to an incommensurate (IC) spin density wave (SDW) state was found below 180 K (= T C on ); and a clear oscillation due to a static internal magnetic field was observed below 140 K(= T C ). Furthermore, an anisotropic behaviour of the zero-field μ + SR experiment indicated that the IC-SDW lies in the a-b plane, with oscillating moments directed along the c axis. These results were quite similar to those for the related compound [Ca 2 CoO 3 ] 0.62 RS [CoO 2 ], i.e., Ca 3 Co 4 O 9 . Since the IC-SDW field in [Ca 2 Co 4/3 Cu 2/3 O 4 ] 0.62 RS [CoO 2 ] was approximately the same as those in pure and doped [Ca 2 CoO 3 ] 0.62 RS [CoO 2 ], it was concluded that the IC-SDW exists in the [CoO 2 ] planes

The influence of statins on the free intracellular calcium concentration in human umbilical vein endothelial cells

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Figulla Hans R

2004-05-01

Full Text Available Abstract Background Statins are cholesterol-lowering drugs that are widely used to reduce the risk of cardiac infarction. Their beneficial clinical effects, however, are not restricted to their influence on cholesterol production. As several studies have shown that they have a potency of relaxing blood vessels. Methods We measured the effects of statins on the intracellular free calcium concentration ([Ca2+]i in human umbilical vein endothelial cells (HUVEC after acute application and 24-h-preincubation of statins. Results Incubation of the cells for 24 h with cerivastatin or fluvastatin significantly increased the resting [Ca2+]i. For cerivastatin this effect manifested at a concentration of 1 μM. Increase of resting [Ca2+]i in the presence of cerivastatin also occurred when the nitric oxide synthase was inhibited. Transient Ca2+ release induced by histamine was not affected. Conclusions The increase of resting [Ca2+]i after incubation with cerivastatin or fluvastatin may provide an explanation for the direct effects of statins on the endothelial-dependent vasodilatation and restoration of endothelial activity in vivo.

Photoelectric observations of propagating sunspot oscillations

International Nuclear Information System (INIS)

Lites, B.W.; White, O.R.; Packman, D.

1982-01-01

The Sacramento Park Observatory Vacuum Tower Telescope and diode array were used to make repeated intensity and velocity images of a large, isolated sunspot in both a chromospheric (lambda8542 Ca II) and a photospheric (lambda5576 Fe I) line. The movie of the digital data for the chromospheric line shows clearly a relationship between the propagating umbral disturbances and the running penumbral waves. The velocities for transverse propagating of the umbral and penumbral disturbances are 60--70 km s -1 and 20--35 km s -1 , respectively. Power spectra of the oscillations show a sharp peak at a period of about 170 s in both the velocity and intensity signals. The rms velocity fluctuation of this power peak is 0.26 km s -1 . The oscillations at any given point in the sunspot are very regular, and the phase relationship between the velocity and intensity of the chromospheric oscillations is radically different than that for the quiet Sun. Our preliminary interpretation of the phase relationship involves acoustic waves with wave vector directed downwards along the magnetic field lines; however, this interpretation relies on assumptions involved in the data reduction scheme. The mechanical energy flux carried by the observed umbral disturbances does not appear to be a significant contributor to the overall energy budget of the sunspot or the surrounding active region

Effects of cell entrapment in Ca-alginate on the metabolism of yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Galazzo, J.L.

1989-01-01

Saccharomyces cerevisiae cells grown in suspension have been immobilized in calcium-alginate beads. Fermentation rates and intracellular composition have been determined under nongrowing conditions in these Ca-alginate entrapped cells and for identical cells in suspension. Glucose uptake and ethanol and glycerol production are approximately two times faster in immobilized cells than in suspended cells. Intermediate metabolite levels such as fructose-1,6-diphosphate, glucose-6-phosphate and 3-phosphoglycerate have been determined by phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy under glucose fermenting conditions. Carbon-13 NMR shows an increase in polysaccharide production in immobilized cells. S. cerevisiae cells grown within a Ca-alginate matrix have a specific growth rate 40% lower than the growth rate of similar cells cultivated in suspension. Alginate-grown cells have been used to compare glucose fermentation under nongrowing conditions in suspended and Ca-entrapped cells. Fermentation rate is higher in immobilized cells than in suspended cells. The observed differences in intracellular components between suspended and immobilized cells are qualitatively similar to the differences observed for cells grown in suspension. Ethanol production rate is 2.7 times faster in immobilized alginate-grown cells than in suspended suspension-grown cells

Intracellular calcium and the mechanism of anodal supernormal excitability in langendorff perfused rabbit ventricles.

Science.gov (United States)

Joung, Boyoung; Park, Hyung-Wook; Maruyama, Mitsunori; Tang, Liang; Song, Juan; Han, Seongwook; Piccirillo, Gianfranco; Weiss, James N; Lin, Shien-Fong; Chen, Peng-Sheng

2011-01-01

Anodal stimulation hyperpolarizes the cell membrane and increases the intracellular Ca(2+) (Ca(i)) transient. This study tested the hypothesis that the maximum slope of the Ca(i) decline (-(dCa(i)/dt)(max)) corresponds to the timing of anodal dip on the strength-interval curve and the initiation of repetitive responses and ventricular fibrillation (VF) after a premature stimulus (S(2)). We simultaneously mapped the membrane potential (V(m)) and Ca(i) in 23 rabbit ventricles. A dip in the anodal strength-interval curve was observed. During the anodal dip, ventricles were captured by anodal break excitation directly under the S(2) electrode. The Ca(i) following anodal stimuli is larger than that following cathodal stimuli. The S(1)-S(2) intervals of the anodal dip (203±10 ms) coincided with the -(dCa(i)/dt)(max) (199±10 ms, P=NS). BAPTA-AM (n=3), inhibition of the electrogenic Na(+)-Ca(2+) exchanger current (I(NCX)) by low extracellular Na(+) (n=3), and combined ryanodine and thapsigargin infusion (n=2) eliminated the anodal supernormality. Strong S(2) during the relative refractory period (n=5) induced 29 repetitive responses and 10 VF episodes. The interval between S(2) and the first non-driven beat was coincidental with the time of -(dCa(i)/dt)(max). Larger Ca(i) transient and I(NCX) activation induced by anodal stimulation produces anodal supernormality. The time of maximum I(NCX) activation is coincidental to the induction of non-driven beats from the Ca(i) sinkhole after a strong premature stimulation. All rights are reserved to the Japanese Circulation Society.

The potential roles of T-type Ca2+ channels in motor coordination

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Young-Gyun ePark

2013-10-01

Full Text Available Specific behavioral patterns are expressed by complex combinations of muscle coordination. Tremors are simple behavioral patterns and are the focus of studies investigating motor coordination mechanisms in the brain. T-type Ca2+ channels mediate intrinsic neuronal oscillations and rhythmic burst spiking, and facilitate the generation of tremor rhythms in motor circuits. Despite substantial evidence that T-type Ca2+ channels mediate pathological tremors, their roles in physiological motor coordination and behavior remain unknown. Here, we review recent progress in understanding the roles that T-type Ca2+ channels play under pathological conditions, and discuss the potential relevance of these channels in mediating physiological motor coordination.

A model of cardiac ryanodine receptor gating predicts experimental Ca2+-dynamics and Ca2+-triggered arrhythmia in the long QT syndrome

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Wilson, Dan; Ermentrout, Bard; Němec, Jan; Salama, Guy

2017-09-01

Abnormal Ca2+ handling is well-established as the trigger of cardiac arrhythmia in catecholaminergic polymorphic ventricular tachycardia and digoxin toxicity, but its role remains controversial in Torsade de Pointes (TdP), the arrhythmia associated with the long QT syndrome (LQTS). Recent experimental results show that early afterdepolarizations (EADs) that initiate TdP are caused by spontaneous (non-voltage-triggered) Ca2+ release from Ca2+-overloaded sarcoplasmic reticulum (SR) rather than the activation of the L-type Ca2+-channel window current. In bradycardia and long QT type 2 (LQT2), a second, non-voltage triggered cytosolic Ca2+ elevation increases gradually in amplitude, occurs before overt voltage instability, and then precedes the rise of EADs. Here, we used a modified Shannon-Puglisi-Bers model of rabbit ventricular myocytes to reproduce experimental Ca2+ dynamics in bradycardia and LQT2. Abnormal systolic Ca2+-oscillations and EADs caused by SR Ca2+-release are reproduced in a modified 0-dimensional model, where 3 gates in series control the ryanodine receptor (RyR2) conductance. Two gates control RyR2 activation and inactivation and sense cytosolic Ca2+ while a third gate senses luminal junctional SR Ca2+. The model predicts EADs in bradycardia and low extracellular [K+] and cessation of SR Ca2+-release terminate salvos of EADs. Ca2+-waves, systolic cell-synchronous Ca2+-release, and multifocal diastolic Ca2+ release seen in subcellular Ca2+-mapping experiments are observed in the 2-dimensional version of the model. These results support the role of SR Ca2+-overload, abnormal SR Ca2+-release, and the subsequent activation of the electrogenic Na+/Ca2+-exchanger as the mechanism of TdP. The model offers new insights into the genesis of cardiac arrhythmia and new therapeutic strategies.

Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.

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Francesco Lodola

Full Text Available Endothelial progenitor cells (EPCs may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca(2+ entry (SOCE, which is activated by a depletion of the intracellular Ca(2+ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca(2+-sensor, Stim1, and the plasmalemmal Ca(2+ channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca(2+ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.The present study employed Ca(2+ imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs as compared to control EPCs (N-EPCs. SOCE, activated by either pharmacological (i.e. cyclopiazonic acid or physiological (i.e. ATP stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La(3+ and Gd(3+. Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI, blocked both SOCE and the intracellular Ca(2+ release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1 at both mRNA and protein level The intracellular Ca(2+ buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.SOCE is remodelled in EPCs

Transcription factor CREB is involved in CaSR-mediated cytoskeleton gene expression.

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Huang, Shuaishuai; Ren, Yu; Wang, Ping; Li, Yanyuan; Wang, Xue; Zhuang, Haihui; Fang, Rong; Wang, Yuduo; Liu, Ningsheng; Hehir, Michael; Zhou, Jeff X

2015-03-01

Our previous studies illustrated that a steady increase of intracellular calcium concentration ([Ca2+]i) was important for maintaining microtubules (MTs) rearrangement in apoptotic cells. However, little is known about the effect of calcium sensing receptor (CaSR)-mediated increase in [Ca2+]i on cytoskeleton gene expression. We examined the impact of taxol or CaSR agonist/antagonist on the regulation of [Ca2+]i concentration, cytoskeleton arrangement, phosphorylated CREB and cytoskeleton gene expressions in HeLa cells with dominant negative plasmid of CREB (PM). This study demonstrated that Gdcl3 (a specific CaSR agonist) evoked a rapid increase of [Ca2+]i, formed a rigid bundle of MTs which surrounded the nucleus and decreased the cytoskeleton gene expressions in HeLa cells. These effects were rescued by addition of NPS2390 (a specific CaSR antagonist). Moreover, CaSR activity affected cytoskeleton gene expression through transcription factor CREB. Histoscores of pCREB immunoreactivity in tissues of cervical adenocarcinoma, renal clear cell carcinoma, and diffuse large B-cell lymphoma were markedly increased compared with non malignant tissue. These data demonstrate, for the first time, that CaSR-mediated increase in [Ca2+]i probably modulate cytoskeleton organization and gene expression via transcription factor. © 2014 Wiley Periodicals, Inc.

Sonic hedgehog stimulates the proliferation of rat gastric mucosal cells through ERK activation by elevating intracellular calcium concentration

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Osawa, Hiroyuki; Ohnishi, Hirohide; Takano, Koji; Noguti, Takasi; Mashima, Hirosato; Hoshino, Hiroko; Kita, Hiroto; Sato, Kiichi; Matsui, Hirofumi; Sugano, Kentaro

2006-01-01

Sonic Hedgehog (Shh), a member of hedgehog peptides family, is expressed in gastric gland epithelium. To elucidate Shh function to gastric mucosal cells, we examined the effect of Shh on the proliferation of a rat normal gastric mucosal cell line, RGM-1. RGM-1 cells express essential components of Shh receptor system, patched-1, and smoothened. Shh enhanced DNA synthesis in RGM-1 cells and elevated intracellular calcium concentration ([Ca 2+ ] i ). In addition, Shh as well as calcium ionophore A32187 rapidly activated ERK. However, Shh failed to activate ERK under calcium-free culture condition. Pretreatment of cells with PD98059 attenuated the DNA synthesis promoted by Shh. Moreover, when cells were pretreated with cyclopamine, Shh could not elevate [Ca 2+ ] i , activate ERK or promote DNA synthesis. On the other hand, although Shh induced Gli-1 nuclear accumulation in RGM-1 cells, Shh activated ERK even in cells pretreated with actinomycin D. These results indicate that Shh promotes the proliferation of RGM-1 cells through an intracellular calcium- and ERK-dependent but transcription-independent pathway via Patched/Smoothened receptor system

Evidence for Long-Timescale Patterns of Synaptic Inputs in CA1 of Awake Behaving Mice.

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Kolb, Ilya; Talei Franzesi, Giovanni; Wang, Michael; Kodandaramaiah, Suhasa B; Forest, Craig R; Boyden, Edward S; Singer, Annabelle C

2018-02-14

Repeated sequences of neural activity are a pervasive feature of neural networks in vivo and in vitro In the hippocampus, sequential firing of many neurons over periods of 100-300 ms reoccurs during behavior and during periods of quiescence. However, it is not known whether the hippocampus produces longer sequences of activity or whether such sequences are restricted to specific network states. Furthermore, whether long repeated patterns of activity are transmitted to single cells downstream is unclear. To answer these questions, we recorded intracellularly from hippocampal CA1 of awake, behaving male mice to examine both subthreshold activity and spiking output in single neurons. In eight of nine recordings, we discovered long (900 ms) reoccurring subthreshold fluctuations or "repeats." Repeats generally were high-amplitude, nonoscillatory events reoccurring with 10 ms precision. Using statistical controls, we determined that repeats occurred more often than would be expected from unstructured network activity (e.g., by chance). Most spikes occurred during a repeat, and when a repeat contained a spike, the spike reoccurred with precision on the order of ≤20 ms, showing that long repeated patterns of subthreshold activity are strongly connected to spike output. Unexpectedly, we found that repeats occurred independently of classic hippocampal network states like theta oscillations or sharp-wave ripples. Together, these results reveal surprisingly long patterns of repeated activity in the hippocampal network that occur nonstochastically, are transmitted to single downstream neurons, and strongly shape their output. This suggests that the timescale of information transmission in the hippocampal network is much longer than previously thought. SIGNIFICANCE STATEMENT We found long (≥900 ms), repeated, subthreshold patterns of activity in CA1 of awake, behaving mice. These repeated patterns ("repeats") occurred more often than expected by chance and with 10 ms

The Ca2+ pump inhibitor, thapsigargin, inhibits root gravitropism in Arabidopsis thaliana

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DANIELA C URBINA

2006-01-01

Full Text Available Thapsigargin, a specific inhibitor of most animal intracellular SERCA-type Ca2+ pumps present in the sarcoplasmic/endoplasmic reticulum, was originally isolated from the roots of the Mediterranean plant Thapsia gargancia L. Here, we demonstrate that this root-derived compound is capable of altering root gravitropism in Arabidopsis thaliana. Thapsigargin concentrations as low as 0.1 µM alter root gravitropism whereas under similar conditions cyclopiazonic acid does not. Furthermore, a fluorescently conjugated thapsigargin (BODIPY FL thapsigargin suggests that target sites for thapsigargin are located in intracellular organelles in the root distal elongation zone and the root cap, regions known to regulate root gravitropism

AMP Affects Intracellular Ca2+ Signaling, Migration, Cytokine Secretion and T Cell Priming Capacity of Dendritic Cells

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Panther, Elisabeth; Dürk, Thorsten; Ferrari, Davide; Di Virgilio, Francesco; Grimm, Melanie; Sorichter, Stephan; Cicko, Sanja; Herouy, Yared; Norgauer, Johannes; Idzko, Marco; Müller, Tobias

2012-01-01

The nucleotide adenosine-5′-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A1 and A2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4+CD45RA+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5′-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders. PMID:22624049

AMP affects intracellular Ca2+ signaling, migration, cytokine secretion and T cell priming capacity of dendritic cells.

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Elisabeth Panther

Full Text Available The nucleotide adenosine-5'-monophosphate (AMP can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A(1 and A(2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC function. AMP increased intracellular Ca(2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A(1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A(2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4(+CD45RA(+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5'-(alpha,beta-methylene diphosphate (APCP. Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.

Ca2+-Dependent Regulations and Signaling in Skeletal Muscle: From Electro-Mechanical Coupling to Adaptation

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Gehlert, Sebastian; Bloch, Wilhelm; Suhr, Frank

2015-01-01

Calcium (Ca2+) plays a pivotal role in almost all cellular processes and ensures the functionality of an organism. In skeletal muscle fibers, Ca2+ is critically involved in the innervation of skeletal muscle fibers that results in the exertion of an action potential along the muscle fiber membrane, the prerequisite for skeletal muscle contraction. Furthermore and among others, Ca2+ regulates also intracellular processes, such as myosin-actin cross bridging, protein synthesis, protein degradation and fiber type shifting by the control of Ca2+-sensitive proteases and transcription factors, as well as mitochondrial adaptations, plasticity and respiration. These data highlight the overwhelming significance of Ca2+ ions for the integrity of skeletal muscle tissue. In this review, we address the major functions of Ca2+ ions in adult muscle but also highlight recent findings of critical Ca2+-dependent mechanisms essential for skeletal muscle-regulation and maintenance. PMID:25569087

Cortisol rapidly reduces prolactin release and cAMP and 45Ca2+ accumulation in the cichlid fish pituitary in vitro

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Borski, R.J.; Helms, L.M.H.; Richman, N.H. III; Grau, E.G.

1991-01-01

During in vitro incubation, prolactin release is inhibited in a dose-related manner by cortisol. This action is mimicked by the synthetic glucocorticoid agonist dexamethasone but not by other steroids tested. Perifusion studies indicate that the inhibition of [ 3 H]prolactin release by cortisol occurs within 20 min. Cortisol (50 nM) also inhibits cAMP accumulation and reduces 45 Ca 2+ accumulation in the tilapia rostral pars distalis within 15 min. Cortisol's action on prolactin release is blocked in the presence of either the Ca 2+ ionophore A23187 or a combination of dibutyryl cAMP and 3-isobutyl-1-methylxanthine, which increase intracellular Ca 2+ and cAMP, respectively. Taken together, these findings suggest that cortisol may play a physiologically relevant role in the rapid modulation of prolactin secretion in vivo. These studies also suggest that the inhibition of prolactin release by cortisol is a specific glucocorticoid action that may be mediated, in part, through cortisol's ability to inhibit intracellular cAMP and Ca 2+ metabolism

Cross-talk between signaling pathways can generate robust oscillations in calcium and cAMP.

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Fernando Siso-Nadal

Full Text Available BACKGROUND: To control and manipulate cellular signaling, we need to understand cellular strategies for information transfer, integration, and decision-making. A key feature of signal transduction is the generation of only a few intracellular messengers by many extracellular stimuli. METHODOLOGY/PRINCIPAL FINDINGS: Here we model molecular cross-talk between two classic second messengers, cyclic AMP (cAMP and calcium, and show that the dynamical complexity of the response of both messengers increases substantially through their interaction. In our model of a non-excitable cell, both cAMP and calcium concentrations can oscillate. If mutually inhibitory, cross-talk between the two second messengers can increase the range of agonist concentrations for which oscillations occur. If mutually activating, cross-talk decreases the oscillation range, but can generate 'bursting' oscillations of calcium and may enable better filtering of noise. CONCLUSION: We postulate that this increased dynamical complexity allows the cell to encode more information, particularly if both second messengers encode signals. In their native environments, it is unlikely that cells are exposed to one stimulus at a time, and cross-talk may help generate sufficiently complex responses to allow the cell to discriminate between different combinations and concentrations of extracellular agonists.

Altered gamma oscillations during pregnancy through loss of δ subunit-containing GABAA receptors on parvalbumin interneurons

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Isabella eFerando

2013-09-01

Full Text Available Gamma (γ oscillations (30-120 Hz, an emergent property of neuronal networks, correlate with memory, cognition and encoding. In the hippocampal CA3 region, locally generated γ oscillations emerge through feedback between inhibitory parvalbumin-positive basket cells (PV+BCs and the principal (pyramidal cells. PV+BCs express δ-subunit-containing GABAARs (-GABAARs and NMDA receptors (NMDA-Rs that balance the frequency of γ oscillations. Neuroactive steroids (NS, such as the progesterone-derived (3α,5α-3-hydroxy-pregnan-20-one (allopregnanolone; ALLO, modulate the expression of δ-GABAARs and the tonic conductance they mediate. Pregnancy produces large increases in ALLO and brain-region-specific homeostatic changes in δ-GABAARs expression. Here we show that in CA3, where most PV+ interneurons (INs express δ-GABAARs, expression of δ-GABAARs on INs diminishes during pregnancy, but reverts to control levels within 48 hours postpartum. These anatomical findings were corroborated by a pregnancy-related increase in the frequency of kainate-induced CA3 γ oscillations in vitro that could be countered by the NMDA-R antagonists D-AP5 and PPDA. Mimicking the typical hormonal conditions during pregnancy by supplementing 100 nM ALLO lowered the γ frequencies to levels found in virgin or postpartum mice. Our findings show that states of altered NS levels (e.g., pregnancy may provoke perturbations in γ oscillatory activity through direct effects on the GABAergic system, and underscore the importance of δ-GABAARs homeostatic plasticity in maintaining constant network output despite large hormonal changes. Inaccurate coupling of NS levels to δ-GABAAR expression may facilitate abnormal neurological and psychiatric conditions such as epilepsy, post-partum depression, and post-partum psychosis, thus providing insights into potential new treatments.

BMP-2 Overexpression Augments Vascular Smooth Muscle Cell Motility by Upregulating Myosin Va via Erk Signaling

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Ming Zhang

2014-01-01

Full Text Available Background. The disruption of physiologic vascular smooth muscle cell (VSMC migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2. Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy.

Comparison of the effects of caffeine and other methylxanthines on [Ca2+]i in rat ventricular myocytes.

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Donoso, P.; O'Neill, S. C.; Dilly, K. W.; Negretti, N.; Eisner, D. A.

1994-01-01

1. The effects of caffeine and other methylxanthines were investigated on intracellular calcium concentration ([Ca2+]i) and contraction in rat isolated ventricular myocytes. The use of the fluorescent indicator, Indo-1, allowed simultaneous measurement of [Ca2+]i and the intracellular concentration of the methylxanthines. 2. Rapid application of caffeine (10 mM) produced a transient rise of [Ca2+]i which decayed to resting levels. This was accompanied by a transient contraction which decayed to a level above baseline. The addition of theophylline also produced a transient increase of [Ca2+]i. However, following the initial transient, contraction decayed before redeveloping to a maintained level. 3. Direct measurements showed that [caffeine]i rose more quickly than did [theophylline]i. The slower rise of [theophylline]i was associated with a delay in the increase of [Ca2+]i. At lower concentrations of the methylxanthines, theophylline was less effective than caffeine at initiating Ca release. The rate of entry of theobromine was similar to that of theophylline. 4. Isocaffeine did not produce a rise of [Ca2+]i. The rate of rise of [isocaffeine]i was much slower than that of either caffeine or theophylline. 5. Measurements of the oil:water partition coefficient showed that the order of relative partitioning into oil was: caffeine > theophylline > theobromine > isocaffeine. This is similar to the order of rate of entry into the cell. 6. We conclude that many of the differences in the effects of these methylxanthines can be attributed to differences in membrane permeability due to differences in oil:water partition. PMID:8004389

Is the Langevin phase equation an efficient model for oscillating neurons?

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Ota, Keisuke; Tsunoda, Takamasa; Omori, Toshiaki; Watanabe, Shigeo; Miyakawa, Hiroyoshi; Okada, Masato; Aonishi, Toru

2009-12-01

The Langevin phase model is an important canonical model for capturing coherent oscillations of neural populations. However, little attention has been given to verifying its applicability. In this paper, we demonstrate that the Langevin phase equation is an efficient model for neural oscillators by using the machine learning method in two steps: (a) Learning of the Langevin phase model. We estimated the parameters of the Langevin phase equation, i.e., a phase response curve and the intensity of white noise from physiological data measured in the hippocampal CA1 pyramidal neurons. (b) Test of the estimated model. We verified whether a Fokker-Planck equation derived from the Langevin phase equation with the estimated parameters could capture the stochastic oscillatory behavior of the same neurons disturbed by periodic perturbations. The estimated model could predict the neural behavior, so we can say that the Langevin phase equation is an efficient model for oscillating neurons.

Is the Langevin phase equation an efficient model for oscillating neurons?

International Nuclear Information System (INIS)

Ota, Keisuke; Tsunoda, Takamasa; Aonishi, Toru; Omori, Toshiaki; Okada, Masato; Watanabe, Shigeo; Miyakawa, Hiroyoshi

2009-01-01

The Langevin phase model is an important canonical model for capturing coherent oscillations of neural populations. However, little attention has been given to verifying its applicability. In this paper, we demonstrate that the Langevin phase equation is an efficient model for neural oscillators by using the machine learning method in two steps: (a) Learning of the Langevin phase model. We estimated the parameters of the Langevin phase equation, i.e., a phase response curve and the intensity of white noise from physiological data measured in the hippocampal CA1 pyramidal neurons. (b) Test of the estimated model. We verified whether a Fokker-Planck equation derived from the Langevin phase equation with the estimated parameters could capture the stochastic oscillatory behavior of the same neurons disturbed by periodic perturbations. The estimated model could predict the neural behavior, so we can say that the Langevin phase equation is an efficient model for oscillating neurons.

Intracellular mediators of Na+-K+ pump activity in guinea pig pancreatic acinar cells

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Hootman, S.R.; Ochs, D.L.; Williams, J.A.

1985-01-01

The involvement of Ca 2+ and cyclic nucleotides in neurohormonal regulation of Na + -K + -ATPase (Na + -K + pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by [3H]ouabain binding and by ouabain-sensitive 86 Rb + uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of [ 3 H]ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in Ca 2+ -free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular Ca 2+ , while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free Ca 2+ levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent Ca 2+ chelator. Basal intracellular Ca 2+ concentration ([Ca 2+ ]i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively

A quantitative description of tubular system Ca2+ handling in fast‐ and slow‐twitch muscle fibres

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Cully, Tanya R.; Edwards, Joshua N.; Murphy, Robyn M.

2016-01-01

Key points Current methods do not allow a quantitative description of Ca2+ movements across the tubular (t‐) system membrane without isolating the membranes from their native skeletal muscle fibre.Here we present a fluorescence‐based method that allows determination of the t‐system [Ca2+] transients and derivation of t‐system Ca2+ fluxes in mechanically skinned skeletal muscle fibres. Differences in t‐system Ca2+‐handling properties between fast‐ and slow‐twitch fibres from rat muscle are resolved for the first time using this new technique.The method can be used to study Ca2+ handling of the t‐system and allows direct comparisons of t‐system Ca2+ transients and Ca2+ fluxes between groups of fibres and fibres from different strains of animals. Abstract The tubular (t‐) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca2+ gradient and exchanges Ca2+ between the extracellular and intracellular environments. Little is known of the Ca2+‐handling properties of the t‐system as the small Ca2+ fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t‐system‐trapped rhod‐5N inside skinned fibres from rat and [Ca2+]t‐sys, allowing confocal measurements of Ca2+‐dependent changes in rhod‐5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca2+] transients in the t‐system ([Ca2+]t‐sys (t)). Furthermore, t‐system Ca2+‐buffering power was determined so that t‐system Ca2+ fluxes could be derived from [Ca2+]t‐sys (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca2+ induced a robust store‐operated Ca2+ entry (SOCE) in fast‐ and slow‐twitch fibres, reducing [Ca2+]t‐sys to fibre types. Abruptly introducing internal solutions with 1 mm Mg2+ and [Ca2+]cyto (28 nm–1.3 μm) to Ca2+‐depleted fibres generated t‐system Ca2+ uptake rates

A quantitative description of tubular system Ca(2+) handling in fast- and slow-twitch muscle fibres.

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Cully, Tanya R; Edwards, Joshua N; Murphy, Robyn M; Launikonis, Bradley S

2016-06-01

Current methods do not allow a quantitative description of Ca(2+) movements across the tubular (t-) system membrane without isolating the membranes from their native skeletal muscle fibre. Here we present a fluorescence-based method that allows determination of the t-system [Ca(2+) ] transients and derivation of t-system Ca(2+) fluxes in mechanically skinned skeletal muscle fibres. Differences in t-system Ca(2+) -handling properties between fast- and slow-twitch fibres from rat muscle are resolved for the first time using this new technique. The method can be used to study Ca(2+) handling of the t-system and allows direct comparisons of t-system Ca(2+) transients and Ca(2+) fluxes between groups of fibres and fibres from different strains of animals. The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca(2+) gradient and exchanges Ca(2+) between the extracellular and intracellular environments. Little is known of the Ca(2+) -handling properties of the t-system as the small Ca(2+) fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system-trapped rhod-5N inside skinned fibres from rat and [Ca(2+) ]t-sys , allowing confocal measurements of Ca(2+) -dependent changes in rhod-5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca(2+) ] transients in the t-system ([Ca(2+) ]t-sys (t)). Furthermore, t-system Ca(2+) -buffering power was determined so that t-system Ca(2+) fluxes could be derived from [Ca(2+) ]t-sys (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca(2+) induced a robust store-operated Ca(2+) entry (SOCE) in fast- and slow-twitch fibres, reducing [Ca(2+) ]t-sys to fibre types. Abruptly introducing internal solutions with 1 mm Mg(2+) and [Ca(2+) ]cyto (28 nm-1.3 μm) to Ca(2+) -depleted fibres generated t-system Ca(2+) uptake rates dependent on [Ca(2

Age-dependent changes in diastolic Ca2+ and Na+ concentrations in dystrophic cardiomyopathy: Role of Ca2+ entry and IP3

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Mijares, Alfredo; Altamirano, Francisco; Kolster, Juan; Adams, José A.; López, José R.

2014-01-01

Highlights: • Age-dependent increase in [Ca 2+ ] d and [Na + ] d in mdx cardiomyocytes. • Gadolinium significantly reduced both [Ca 2+ ] d and [Na + ] d at all ages. • IP 3 -pathway inhibition reduced cations concentrations in dystrophic cardiomyocytes. - Abstract: Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca 2+ concentration ([Ca 2+ ] d ) and diastolic Na + concentration ([Na + ] d ) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd 3+ )-sensitive Ca 2+ entry and inositol triphosphate (IP 3 ) signaling pathways in abnormal [Ca 2+ ] d and [Na + ] d were investigated. Our results showed an age-dependent increase in both [Ca 2+ ] d and [Na + ] d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd 3+ treatment significantly reduced both [Ca 2+ ] d and [Na + ] d at all ages. In addition, blockade of the IP 3 -pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd 3+ normalized both [Ca 2+ ] d and [Na + ] d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca 2+ and Na + overload mediated at least in part by enhanced Ca 2+ entry through Gd 3+ sensitive transient receptor potential channels (TRPC), and by IP 3 receptors