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Sample records for induce store-operated calcium

  1. Store-Operated Calcium Channels.

    Science.gov (United States)

    Prakriya, Murali; Lewis, Richard S

    2015-10-01

    Store-operated calcium channels (SOCs) are a major pathway for calcium signaling in virtually all metozoan cells and serve a wide variety of functions ranging from gene expression, motility, and secretion to tissue and organ development and the immune response. SOCs are activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER), triggered physiologically through stimulation of a diverse set of surface receptors. Over 15 years after the first characterization of SOCs through electrophysiology, the identification of the STIM proteins as ER Ca(2+) sensors and the Orai proteins as store-operated channels has enabled rapid progress in understanding the unique mechanism of store-operate calcium entry (SOCE). Depletion of Ca(2+) from the ER causes STIM to accumulate at ER-plasma membrane (PM) junctions where it traps and activates Orai channels diffusing in the closely apposed PM. Mutagenesis studies combined with recent structural insights about STIM and Orai proteins are now beginning to reveal the molecular underpinnings of these choreographic events. This review describes the major experimental advances underlying our current understanding of how ER Ca(2+) depletion is coupled to the activation of SOCs. Particular emphasis is placed on the molecular mechanisms of STIM and Orai activation, Orai channel properties, modulation of STIM and Orai function, pharmacological inhibitors of SOCE, and the functions of STIM and Orai in physiology and disease.

  2. Atlastin regulates store-operated calcium entry for nerve growth factor-induced neurite outgrowth

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    Li, Jing; Yan, Bing; Si, Hongjiang; Peng, Xu; Zhang, Shenyuan L.; Hu, Junjie

    2017-01-01

    Homotypic membrane fusion of the endoplasmic reticulum (ER) is mediated by a class of dynamin-like GTPases known as atlastin (ATL). Depletion of or mutations in ATL cause an unbranched ER morphology and hereditary spastic paraplegia (HSP), a neurodegenerative disease characterized by axon shortening in corticospinal motor neurons and progressive spasticity of the lower limbs. How ER shaping is linked to neuronal defects is poorly understood. Here, we show that dominant-negative mutants of ATL1 in PC-12 cells inhibit nerve growth factor (NGF)-induced neurite outgrowth. Overexpression of wild-type or mutant ATL1 or depletion of ATLs alters ER morphology and affects store-operated calcium entry (SOCE) by decreasing STIM1 puncta formation near the plasma membrane upon calcium depletion of the ER. In addition, blockage of the STIM1-Orai pathway effectively abolishes neurite outgrowth of PC-12 cells stimulated by NGF. These results suggest that SOCE plays an important role in neuronal regeneration, and mutations in ATL1 may cause HSP, partly by undermining SOCE. PMID:28240257

  3. Mechanism of store-operated calcium entry

    Indian Academy of Sciences (India)

    Devkanya Dutta

    2000-12-01

    Activation of receptors coupled to the phospholipase C/IP3 signalling pathway results in a rapid release of calcium from its intracellular stores, eventually leading to depletion of these stores. Calcium store depletion triggers an influx of extracellular calcium across the plasma membrane, a mechanism known as the store-operated calcium entry or capacitative calcium entry. Capacitative calcium current plays a key role in replenishing calcium stores and activating various physiological processes. Despite considerable efforts, very little is known about the molecular nature of the capacitative channel and the signalling pathway that activates it. This review summarizes our current knowledge about store operated calcium entry and suggests possible hypotheses for its mode of activation.

  4. Blockade of store-operated calcium entry alleviates ethanol-induced hepatotoxicity via inhibiting apoptosis

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    Cui, Ruibing [Department of Hepatology and Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012 (China); Yan, Lihui [Shandong Normal University, Jinan, Shandong Province 250012 (China); Luo, Zheng; Guo, Xiaolan [Department of Hepatology and Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012 (China); Yan, Ming, E-mail: ymylh@163.com [Department of Hepatology and Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012 (China)

    2015-08-15

    Extracellular Ca{sup 2+} influx has been suggested to play a role in ethanol-induced hepatocyte apoptosis and necrosis. Previous studies indicated that store-operated Ca{sup 2+} entry (SOCE) was involved in liver injury induced by ethanol in HepG2 cells. However, the mechanisms underlying liver injury caused by SOCE remain unclear. We aimed to investigate the effects and mechanism of SOCE inhibition on liver injury induced by ethanol in BRL cells and Sprague–Dawley rats. Our data demonstrated that ethanol (0–400 mM) dose-dependently increased hepatocyte injury and 100 mM ethanol significantly upregulated the mRNA and protein expression of SOC for at least 72 h in BRL cells. Blockade of SOCE by pharmacological inhibitors and sh-RNA knockdown of STIM1 and Orai1 attenuated intracellular Ca{sup 2+} overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and inhibited ethanol-induced apoptosis. STIM1 and Orai1 expression was greater in ethanol-treated than control rats, and the SOCE inhibitor corosolic acid ameliorated the histopathological findings and alanine transaminase and aspartate transaminase activity as well as decreased cytochrome C release and inhibited alcohol-induced cell apoptosis. These findings suggest that SOCE blockade could alleviate alcohol-induced hepatotoxicity via inhibiting apoptosis. SOCE might be a useful therapeutic target in alcoholic liver diseases. - Highlights: • Blockade of SOCE alleviated overload of Ca{sup 2+} and hepatotoxicity after ethanol application. • Blockade of SOCE inhibited mitochondrial apoptosis after ethanol application. • SOCE might be a useful therapeutic target in alcoholic liver diseases.

  5. Elevation of extracellular Ca2+ induces store-operated calcium entry via calcium-sensing receptors: a pathway contributes to the proliferation of osteoblasts.

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    Fen Hu

    Full Text Available AIMS: The local concentration of extracellular Ca(2+ ([Ca(2+]o in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca(2+]o induced store-operated calcium entry (SOCE in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca(2+]o to osteoblastic proliferation. METHODS: Cytosolic Ca(2+ concentration ([Ca(2+]c of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR as well as inhibitors of phospholipase C (PLC, SOCE and voltage-gated calcium (Cav channels were applied to study the mechanism in detail. RESULTS: Our data showed that elevating [Ca(2+]o evoked a sustained increase of [Ca(2+]c in a dose-dependent manner. This [Ca(2+]c increase was blocked by TMB-8 (Ca(2+ release inhibitor, 2-APB and BTP-2 (both SOCE blockers, respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist or U73122 (a PLC inhibitor strongly reduced the [Ca(2+]o-induced [Ca(2+]c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca(2+]o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca(2+]o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator, 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by Cav channels antagonists. CONCLUSIONS: Elevating [Ca(2+]o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca(2+ concentration.

  6. Store-operated calcium signaling in neutrophils.

    Science.gov (United States)

    Clemens, Regina A; Lowell, Clifford A

    2015-10-01

    Calcium signals in neutrophils are initiated by a variety of cell-surface receptors, including formyl peptide and other GPCRs, FcRs, and integrins. The predominant pathway by which calcium enters immune cells is termed SOCE, whereby plasma membrane CRAC channels allow influx of extracellular calcium into the cytoplasm when intracellular ER stores are depleted. The identification of 2 key families of SOCE regulators, STIM calcium "sensors" and ORAI calcium channels, has allowed for genetic manipulation of SOCE pathways and provided valuable insight into the molecular mechanism of calcium signaling in immune cells, including neutrophils. This review focuses on our current knowledge of the molecules involved in neutrophil SOCE and how study of these molecules has further informed our understanding of the role of calcium signaling in neutrophil activation.

  7. Homer1a attenuates glutamate-induced oxidative injury in HT-22 cells through regulation of store-operated calcium entry

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    Rao, Wei; Peng, Cheng; Zhang, Lei; Su, Ning; Wang, Kai; Hui, Hao; Dai, Shu-hui; Yang, Yue-fan; Luo, Peng; Fei, Zhou

    2016-01-01

    Calcium disequilibrium is extensively involved in oxidative stress-induced neuronal injury. Although Homer1a is known to regulate several neuronal calcium pathways, its effects on, or its exact relationship with, oxidative stress-induced neuronal injury has not yet been fully elucidated. We found that Homer1a protected HT-22 cells from glutamate-induced oxidative stress injury by inhibiting final-phase intracellular calcium overload and mitochondrial oxidative stress. In these cells, stromal interactive molecule 1 (STIM1) puncta, but not the protein level, was significantly increased after glutamate treatment. Store-operated calcium entry (SOCE) inhibitors and cells in which a key component of SOCE (STIM1) was knocked out were used as glutamate-induced oxidative stress injury models. Both models demonstrated significant improvement of HT-22 cell survival after glutamate treatment. Additionally, increased Homer1a protein levels significantly inhibited SOCE and decreased the association of STIM1-Orai1 triggered by glutamate. These results suggest that up-regulation of Homer1a can protect HT-22 cells from glutamate-induced oxidative injury by disrupting the STIM1-Oria1 association, and then by inhibiting the SOCE-mediated final-phrase calcium overload. Thus, regulation of Homer1a, either alone or in conjunction with SOCE inhibition, may serve as key therapeutic interventional targets for neurological diseases in which oxidative stress is involved in the etiology or progression of the disease. PMID:27681296

  8. Store-operated calcium channels and pro-inflammatory signals

    Institute of Scientific and Technical Information of China (English)

    Wei-chiao CHANG

    2006-01-01

    In non-excitable cells such as T lymphocytes,hepatocytes,mast cells,endothelia and epithelia,the major pathway for calcium(Ca2+)entry is through store-operated Ca2+ channels in the plasma membrane.These channels are activated by the emptying of intracellular Ca2+ stores,however,neither the gating mechanism nor the downstream targets of these channels has been clear established.Here,I review some of the proposed gating mechanisms of store-operated Ca2+ channels and the functional implications in regulating pro-inflammatory signals.

  9. The molecular choreography of a store-operated calcium channel.

    Science.gov (United States)

    Lewis, Richard S

    2007-03-15

    Store-operated calcium channels (SOCs) serve essential functions from secretion and motility to gene expression and cell growth. A fundamental mystery is how the depletion of Ca2+ from the endoplasmic reticulum (ER) activates Ca2+ entry through SOCs in the plasma membrane. Recent studies using genetic approaches have identified genes encoding the ER Ca2+ sensor and a prototypic SOC, the Ca2+-release-activated Ca2+ (CRAC) channel. New findings reveal a unique mechanism for channel activation, in which the CRAC channel and its sensor migrate independently to closely apposed sites of interaction in the ER and the plasma membrane.

  10. Phemindole, a synthetic di-indole derivative maneuvers the store operated calcium entry (SOCE to induce potent anti-carcinogenic activity in human triple negative breast cancer cells

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    Supriya eChakraborty

    2016-05-01

    Full Text Available Triple-negative breast cancer (TNBC, is a specific subtype of epithelial breast tumours that are immuno-histochemically negative for the protein expression of the estrogen receptor (ER, the progesterone receptor (PR and lack over expression/gene amplification of HER2. This subtype of breast cancers is highly metastatic, shows poor prognosis and hence represents an important clinical challenge to researchers worldwide. Thus alternative approaches of drug development for TNBC have gained utmost importance in the present times. Dietary indole and its derivatives have gained prominence as anti-cancer agents and new therapeutic approaches are being developed to target them against TNBC. But a major drawback with 3, 3’di Indolyl methane (DIM is their poor bioavailability and high effective concentration against TNBC. However the Aryl methyl ring substituted analogs of DIM display interesting anti-cancer activity in breast cancer cells. In the current study we report the synthesis of a novel synthetic aryl methyl ring substituted analog of DIM, named as Phemindole as an effective anti-tumor agent against TNBC cells. Furthermore, we enumerated that Phemindole caused ROS mediated mitochondrial-dependent apoptosis in MDAMB-231 cells. Furthermore, Phemindole mediated Store Operated Calcium Entry (SOCE retardation favored inactivation of STIM1 and henceforth activated ER stress to induce apoptosis in TNBC cells. Simultaneously, Phemindole was also found to restrict the in-vitro cell migration through its anti mitotic property and pFAK regulation. Studies extended to ex-ovo and in-vivo mice models further validated the efficacy of Phemindole. Thus our results cumulatively propose Phemindole as a new chemotherapeutic regime which might be effective to target the deadly aspects of the TNBC.

  11. Ginsenoside Rb1 Attenuates Agonist-Induced Contractile Response via Inhibition of Store-Operated Calcium Entry in Pulmonary Arteries of Normal and Pulmonary Hypertensive Rats

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    Rui-Xing Wang

    2015-03-01

    Full Text Available Background: Pulmonary hypertension (PH is characterized by sustained vasoconstriction, enhanced vasoreactivity and vascular remodeling, which leads to right heart failure and death. Despite several treatments are available, many forms of PH are still incurable. Ginsenoside Rb1, a principle active ingredient of Panax ginseng, exhibits multiple pharmacological effects on cardiovascular system, and suppresses monocrotaline (MCT-induced right heart hypertrophy. However, its effect on the pulmonary vascular functions related to PH is unknown. Methods: We examined the vasorelaxing effects of ginsenoside Rb1 on endothelin-1 (ET-1 induced contraction of pulmonary arteries (PAs and store-operated Ca2+ entry (SOCE in pulmonary arterial smooth muscle cells (PASMCs from chronic hypoxia (CH and MCT-induced PH. Results: Ginsenoside Rb1 elicited concentration-dependent relaxation of ET-1-induced PA contraction. The vasorelaxing effect was unaffected by nifedipine, but abolished by the SOCE blocker Gd3+. Ginsenoside Rb1 suppressed cyclopiazonic acid (CPA-induced PA contraction, and CPA-activated cation entry and Ca2+ transient in PASMCs. ET-1 and CPA-induced contraction, and CPA-activated cation entry and Ca2+ transients were enhanced in PA and PASMCs of CH and MCT-treated rats; the enhanced responses were abolished by ginsenoside Rb1. Conclusion: Ginsenoside Rb1 attenuates ET-1-induced contractile response via inhibition of SOCE, and it can effectively antagonize the enhanced pulmonary vasoreactivity in PH.

  12. Defective Store-Operated Calcium Entry Causes Partial Nephrogenic Diabetes Insipidus.

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    Mamenko, Mykola; Dhande, Isha; Tomilin, Viktor; Zaika, Oleg; Boukelmoune, Nabila; Zhu, Yaming; Gonzalez-Garay, Manuel L; Pochynyuk, Oleh; Doris, Peter A

    2016-07-01

    Store-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca(2+)) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca(2+) levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca(2+) mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca(2+) levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling.

  13. Involvement of phospholipase D in store-operated calcium influx in vascular smooth muscle cells.

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    Walter, M; Tepel, M; Nofer, J R; Neusser, M; Assmann, G; Zidek, W

    2000-08-11

    In non-excitable cells, sustained intracellular Ca2+ increase critically depends on influx of extracellular Ca2+. Such Ca2+ influx is thought to occur by a 'store-operated' mechanism, i.e. the signal for Ca2+ entry is believed to result from the initial release of Ca2+ from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Here we show that the depletion of cellular Ca2+ stores by thapsigargin or bradykinin is functionally linked to a phosphoinositide-specific phospholipase D (PLD) activity in cultured vascular smooth muscle cells (VSMC), and that phosphatidic acid formed via PLD enhances sustained calcium entry in this cell type. These results suggest a regulatory role for PLD in store-operated Ca2+ entry in VSMC.

  14. Store-operated calcium entry modulates neuronal network activity in a model of chronic epilepsy.

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    Steinbeck, Julius A; Henke, Nadine; Opatz, Jessica; Gruszczynska-Biegala, Joanna; Schneider, Lars; Theiss, Stephan; Hamacher, Nadine; Steinfarz, Barbara; Golz, Stefan; Brüstle, Oliver; Kuznicki, Jacek; Methner, Axel

    2011-12-01

    Store-operated Ca(2+) entry (SOCE) over the plasma membrane is activated by depletion of intracellular Ca(2+) stores and has only recently been shown to play a role in CNS processes like synaptic plasticity. However, the direct effect of SOCE on the excitability of neuronal networks in vitro and in vivo has never been determined. We confirmed the presence of SOCE and the expression of the calcium sensors STIM1 and STIM2, which convey information about the calcium load of the stores to channel proteins at the plasma membrane, in neurons and astrocytes. Inhibition of SOCE by pharmacological agents 2-APB and ML-9 reduced the steady-state neuronal Ca(2+) concentration, reduced network activity, and increased synchrony of primary neuronal cultures grown on multi-electrode arrays, which prompted us to elucidate the relative expression of STIM proteins in conditions of pathologic excitability. Both proteins were increased in brains of chronic epileptic rodents and strongly expressed in hippocampal specimens from medial temporal lobe epilepsy patients. Pharmacologic inhibition of SOCE in chronic epileptic hippocampal slices suppressed interictal spikes and rhythmized epileptic burst activity. Our results indicate that SOCE modulates the activity of neuronal networks in vitro and in vivo and delineates SOCE as a potential drug target. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Increased store-operated and 1-oleoyl-2-acetyl-sn-glycerol-induced calcium influx in monocytes is mediated by transient receptor potential canonical channels in human essential hypertension

    DEFF Research Database (Denmark)

    Liu, Dao Yan; Thilo, Florian; Scholze, Alexandra;

    2007-01-01

    Activation of nonselective cation channels of the transient receptor potential canonical (TRPC) family has been associated with hypertension. Whether store-operated channels, which are activated after depletion of intracellular stores, or second-messenger-operated channels, which are activated by 1......-oleoyl-2-acetyl-sn-glycerol, are affected in essential hypertension is presently unknown....

  16. A key role for STIM1 in store operated calcium channel activation in airway smooth muscle

    Directory of Open Access Journals (Sweden)

    Peel Samantha E

    2006-09-01

    Full Text Available Abstract Background Control of cytosolic calcium plays a key role in airway myocyte function. Changes in intracellular Ca2+ stores can modulate contractile responses, modulate proliferation and regulate synthetic activity. Influx of Ca2+ in non excitable smooth muscle is believed to be predominantly through store operated channels (SOC or receptor operated channels (ROC. Whereas agonists can activate both SOC and ROC in a range of smooth muscle types, the specific trigger for SOC activation is depletion of the sarcoplasmic reticulum Ca2+ stores. The mechanism underlying SOC activation following depletion of intracellular Ca2+ stores in smooth muscle has not been identified. Methods To investigate the roles of the STIM homologues in SOC activation in airway myocytes, specific siRNA sequences were utilised to target and selectively suppress both STIM1 and STIM2. Quantitative real time PCR was employed to assess the efficiency and the specificity of the siRNA mediated knockdown of mRNA. Activation of SOC was investigated by both whole cell patch clamp electrophysiology and a fluorescence based calcium assay. Results Transfection of 20 nM siRNA specific for STIM1 or 2 resulted in robust decreases (>70% of the relevant mRNA. siRNA targeted at STIM1 resulted in a reduction of SOC associated Ca2+ influx in response to store depletion by cyclopiazonic acid (60% or histamine but not bradykinin. siRNA to STIM2 had no effect on these responses. In addition STIM1 suppression resulted in a more or less complete abrogation of SOC associated inward currents assessed by whole cell patch clamp. Conclusion Here we show that STIM1 acts as a key signal for SOC activation following intracellular Ca2+ store depletion or following agonist stimulation with histamine in human airway myocytes. These are the first data demonstrating a role for STIM1 in a physiologically relevant, non-transformed endogenous expression cell model.

  17. Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry

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    Irina V. Gorudko

    2013-07-01

    Myeloperoxidase (MPO is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist. In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca2+ through enhancement of store-operated Ca2+ entry (SOCE. Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations.

  18. Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes.

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    Casciano, Jessica C; Duchemin, Nicholas J; Lamontagne, R Jason; Steel, Laura F; Bouchard, Michael J

    2017-01-01

    Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases.

  19. Modeled microgravity suppressed invasion and migration of human glioblastoma U87 cells through downregulating store-operated calcium entry.

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    Shi, Zi-xuan; Rao, Wei; Wang, Huan; Wang, Nan-ding; Si, Jing-wen; Zhao, Jiao; Li, Jun-chang; Wang, Zong-ren

    2015-02-13

    Glioblastoma is the most common brain tumor and is characterized with robust invasion and migration potential resulting in poor prognosis. Previous investigations have demonstrated that modeled microgravity (MMG) could decline the cell proliferation and attenuate the metastasis potential in several cell lines. In this study, we studied the effects of MMG on the invasion and migration potentials of glioblastoma in human glioblastoma U87 cells. We found that MMG stimulation significantly attenuated the invasion and migration potentials, decreased thapsigargin (TG) induced store-operated calcium entry (SOCE) and downregulated the expression of Orai1 in U87 cells. Inhibition of SOCE by 2-APB or stromal interaction molecule 1 (STIM1) downregulation both mimicked the effects of MMG on the invasion and migration potentials in U87 cells. Furthermore, upregulation of Orai1 significantly weakened the effects of MMG on the invasion and migration potentials in U87 cells. Therefore, these findings indicated that MMG stimulation inhibited the invasion and migration potentials of U87 cells by downregulating the expression of Orai1 and sequentially decreasing the SOCE, suggesting that MMG might be a new potential therapeutic strategy in glioblastoma treatment in the future.

  20. Modeled microgravity suppressed invasion and migration of human glioblastoma U87 cells through downregulating store-operated calcium entry

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Zi-xuan [Department of Traditional Chinese Medicine, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 (China); Rao, Wei [Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 (China); Wang, Huan [Department of Dermatology, Tangdu Hospital, Fourth Military Medical University, Xi' an, 710032 (China); Wang, Nan-ding [Department of Cardiology, Xi' an Traditional Chinese Medicine Hospital, Xi' an, 710032 (China); Si, Jing-Wen; Zhao, Jiao; Li, Jun-chang [Department of Traditional Chinese Medicine, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 (China); Wang, Zong-ren, E-mail: zongren@fmmu.edu.cn [Department of Traditional Chinese Medicine, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 (China)

    2015-02-13

    Glioblastoma is the most common brain tumor and is characterized with robust invasion and migration potential resulting in poor prognosis. Previous investigations have demonstrated that modeled microgravity (MMG) could decline the cell proliferation and attenuate the metastasis potential in several cell lines. In this study, we studied the effects of MMG on the invasion and migration potentials of glioblastoma in human glioblastoma U87 cells. We found that MMG stimulation significantly attenuated the invasion and migration potentials, decreased thapsigargin (TG) induced store-operated calcium entry (SOCE) and downregulated the expression of Orai1 in U87 cells. Inhibition of SOCE by 2-APB or stromal interaction molecule 1 (STIM1) downregulation both mimicked the effects of MMG on the invasion and migration potentials in U87 cells. Furthermore, upregulation of Orai1 significantly weakened the effects of MMG on the invasion and migration potentials in U87 cells. Therefore, these findings indicated that MMG stimulation inhibited the invasion and migration potentials of U87 cells by downregulating the expression of Orai1 and sequentially decreasing the SOCE, suggesting that MMG might be a new potential therapeutic strategy in glioblastoma treatment in the future. - Highlights: • Modeled microgravity (MMG) suppressed migration and invasion in U87 cells. • MMG downregulated the SOCE and the expression of Orai1. • SOCE inhibition mimicked the effects of MMG on migration and invasion potentials. • Restoration of SOCE diminished the effects of MMG on migration and invasion.

  1. Mechanosensitive store-operated calcium entry regulates the formation of cell polarity.

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    Huang, Yi-Wei; Chang, Shu-Jing; I-Chen Harn, Hans; Huang, Hui-Ting; Lin, Hsi-Hui; Shen, Meng-Ru; Tang, Ming-Jer; Chiu, Wen-Tai

    2015-09-01

    Ca(2+) -mediated formation of cell polarity is essential for directional migration which plays an important role in physiological and pathological processes in organisms. To examine the critical role of store-operated Ca(2+) entry, which is the major form of extracellular Ca(2+) influx in non-excitable cells, in the formation of cell polarity, we employed human bone osteosarcoma U2OS cells, which exhibit distinct morphological polarity during directional migration. Our analyses showed that Ca(2+) was concentrated at the rear end of cells and that extracellular Ca(2+) influx was important for cell polarization. Inhibition of store-operated Ca(2+) entry using specific inhibitors disrupted the formation of cell polarity in a dose-dependent manner. Moreover, the channelosomal components caveolin-1, TRPC1, and Orai1 were concentrated at the rear end of polarized cells. Knockdown of TRPC1 or a TRPC inhibitor, but not knockdown of Orai1, reduced cell polarization. Furthermore, disruption of lipid rafts or overexpression of caveolin-1 contributed to the downregulation of cell polarity. On the other hand, we also found that cell polarity, store-operated Ca(2+) entry activity, and cell stiffness were markedly decreased by low substrate rigidity, which may be caused by the disorganization of actin filaments and microtubules that occurs while regulating the activity of the mechanosensitive TRPC1 channel.

  2. Dystrophin/α1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes.

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    Sabourin, Jessica; Harisseh, Rania; Harnois, Thomas; Magaud, Christophe; Bourmeyster, Nicolas; Déliot, Nadine; Constantin, Bruno

    2012-12-01

    In skeletal muscles from patient suffering of Duchenne Muscular Dystrophy and from mdx mice, the absence of the cytoskeleton protein dystrophin has been shown to be essential for maintaining a normal calcium influx. We showed that a TRPC store-dependent cation influx is increased by loss of dystrophin or a scaffolding protein α1-syntrophin, however the mechanisms of this calcium mishandling are incompletely understood. First of all, we confirmed that TRPC1 but also STIM1 and Orai1 are supporting the store-operated cation entry which is enhanced in dystrophin-deficient myotubes. Next, we demonstrated that inhibition of PLC or PKC in dystrophin-deficient myotubes restores elevated cation entry to normal levels similarly to enforced minidystrophin expression. In addition, silencing α1-syntrophin also increased cation influx in a PLC/PKC dependent pathway. We also showed that α1-syntrophin and PLCβ are part of a same protein complex reinforcing the idea of their inter-relation in calcium influx regulation. This elevated cation entry was decreased to normal levels by chelating intracellular free calcium with BAPTA-AM. Double treatments with BAPTA-AM and PLC or PKC inhibitors suggested that the elevation of cation influx by PLC/PKC pathway is dependent on cytosolic calcium. All these results demonstrate an involvement in dystrophin-deficient myotubes of a specific calcium/PKC/PLC pathway in elevation of store-operated cation influx supported by the STIM1/Orai1/TRPC1 proteins, which is normally regulated by the α1-syntrophin/dystrophin scaffold.

  3. Microtubule remodeling mediates the inhibition of store-operated calcium entry (SOCE) during mitosis in COS-7 cells.

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    Russa, Afadhali Denis; Ishikita, Naoyuki; Masu, Kazuki; Akutsu, Hitomi; Saino, Tomoyuki; Satoh, Yoh-ichi

    2008-12-01

    Regulation of the intracellular calcium ion concentration ([Ca(2+)](i)) is critical, because calcium signaling controls diverse and vital cellular processes such as secretion, proliferation, division, gene transcription, and apoptosis. Store-operated calcium entry (SOCE) is the main mechanism through which non-excitable cells replenish and thus maintain this delicate balance. There is limited evidence which indicates that SOCE may be inhibited during mitosis, and the mechanisms leading to the presumed inhibition has not been elucidated. In the present study, we examined and compared the [Ca(2+)](i) dynamics of COS-7 cells in mitotic and non-mitotic phases with special reference paid to SOCE. Laser scanning confocal microscopy to monitor [Ca(2+)](i) dynamics revealed that SOCE was progressively inhibited in mitosis and became virtually absent during the metaphase. We used various cytoskeletal modifying drugs and immunofluorescence to assess the contribution of microtubule and actin filaments in SOCE signaling. Nocodazole treatment caused microtubule reorganization and retraction from the cell periphery that mimicked the natural mitotic microtubule remodeling that was also accompanied by SOCE inhibition. Short exposure to paclitaxel, a microtubule-stabilizing drug, bolstered SOCE, whereas long exposure resulted in microtubule disruption and SOCE inhibition. Actin-modifying drugs did not affect SOCE. These findings indicate that mitotic microtubule remodeling plays a significant role in the inhibition of SOCE during mitosis.

  4. Astrocyte glycogenolysis is triggered by store-operated calcium entry and provides metabolic energy for cellular calcium homeostasis.

    Science.gov (United States)

    Müller, Margit S; Fox, Rebecca; Schousboe, Arne; Waagepetersen, Helle S; Bak, Lasse K

    2014-04-01

    Astrocytic glycogen, the only storage form of glucose in the brain, has been shown to play a fundamental role in supporting learning and memory, an effect achieved by providing metabolic support for neurons. We have examined the interplay between glycogenolysis and the bioenergetics of astrocytic Ca(2+) homeostasis, by analyzing interdependency of glycogen and store-operated Ca(2+) entry (SOCE), a mechanism in cellular signaling that maintains high endoplasmatic reticulum (ER) Ca(2+) concentration and thus provides the basis for store-dependent Ca(2+) signaling. We stimulated SOCE in primary cultures of murine cerebellar and cortical astrocytes, and determined glycogen content to investigate the effects of SOCE on glycogen metabolism. By blocking glycogenolysis, we tested energetic dependency of SOCE-related Ca(2+) dynamics on glycogenolytic ATP. Our results show that SOCE triggers astrocytic glycogenolysis. Upon inhibition of adenylate cyclase with 2',5'-dideoxyadenosine, glycogen content was no longer significantly different from that in unstimulated control cells, indicating that SOCE triggers astrocytic glycogenolysis in a cAMP-dependent manner. When glycogenolysis was inhibited in cortical astrocytes by 1,4-dideoxy-1,4-imino-D-arabinitol, the amount of Ca(2+) loaded into ER via sarco/endoplasmic reticulum Ca(2)-ATPase (SERCA) was reduced, which suggests that SERCA pumps preferentially metabolize glycogenolytic ATP. Our study demonstrates SOCE as a novel pathway in stimulating astrocytic glycogenolysis. We also provide first evidence for a new functional role of brain glycogen, in providing local ATP to SERCA, thus establishing the bioenergetic basis for astrocytic Ca(2+) signaling. This mechanism could offer a novel explanation for the impact of glycogen on learning and memory. Copyright © 2014 Wiley Periodicals, Inc.

  5. Pathophysiological Significance of Store-Operated Calcium Entry in Megakaryocyte Function: Opening New Paths for Understanding the Role of Calcium in Thrombopoiesis

    Directory of Open Access Journals (Sweden)

    Christian A. Di Buduo

    2016-12-01

    Full Text Available Store-Operated Calcium Entry (SOCE is a universal calcium (Ca2+ influx mechanism expressed by several different cell types. It is now known that Stromal Interaction Molecule (STIM, the Ca2+ sensor of the intracellular compartments, together with Orai and Transient Receptor Potential Canonical (TRPC, the subunits of Ca2+ permeable channels on the plasma membrane, cooperate in regulating multiple cellular functions as diverse as proliferation, differentiation, migration, gene expression, and many others, depending on the cell type. In particular, a growing body of evidences suggests that a tight control of SOCE expression and function is achieved by megakaryocytes along their route from hematopoietic stem cells to platelet production. This review attempts to provide an overview about the SOCE dynamics in megakaryocyte development, with a focus on most recent findings related to its involvement in physiological and pathological thrombopoiesis.

  6. New insights into the activation mechanism of store-operated calcium channels:roles of STIM and Orai

    Institute of Scientific and Technical Information of China (English)

    Rui-wei GUO; Lan HUANG

    2008-01-01

    The activation of Ca2+ entry through store-operated channels by agonists that deplete Ca2+ from the endoplasmic reticulum (ER)is a ubiquitous signaling mechanism,the molecular basis of which has remained elusive for the past two decades.Store-operated Ca2+-release-activated Ca2+(CRAC)channels constitute the sole pathway for Ca2+ entry following antigen-receptor engagement.In a set of breakthrough studies over the past two years,stromal interaction molecule l(STIM1,tbe ER Ca2+ sensor) and Orail(a pore-forming subunit of the CRAC channel)have been identified.Here we review these recent studies and the insights they provide into the mechanism of store-operated Ca2+ channels(SOCCs).

  7. Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum.

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    Golovina, Vera A

    2005-05-01

    Unloading of endoplasmic reticulum (ER) Ca(2+) stores activates influx of extracellular Ca(2+) through 'store-operated' Ca(2+) channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca(2+) stores. Here, using high resolution imaging with the membrane-associated Ca(2+) indicator, FFP-18, it is shown that store-operated Ca(2+) entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane-ER junctions. In the absence of extracellular Ca(2+), depletion of ER Ca(2+) stores using cyclopiazonic acid, an ER Ca(2+)-ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca(2+) concentration ([Ca(2+)](SPM)) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca(2+) causes localized Ca(2+) influx that first increases [Ca(2+)](SPM) in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs, markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying 'junctional' ER. Thus, Ca(2+) entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca(2+) stores.

  8. Downregulation of ACE2/Ang-(1-7)/Mas axis promotes breast cancer metastasis by enhancing store-operated calcium entry.

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    Yu, Changhui; Tang, Wei; Wang, Yuhao; Shen, Qiang; Wang, Bin; Cai, Chunqing; Meng, Xiaojing; Zou, Fei

    2016-07-01

    The renin-angiotensin system (RAS) is an important component of the tumor microenvironment and plays a key role in promoting cancer cell proliferation, angiogenesis, metabolism, migration and invasion. Meanwhile, the arm of angiotensin-converting enzyme (ACE)2/angiotensin-(1-7) [Ang-(1-7)]/Mas axis in connection with RAS is associated with anti-proliferative, vasodilatory and anti-metastatic properties. Previous studies have shown that Ang-(1-7) reduces the proliferation of orthotopic human breast tumor growth by inhibiting cancer-associated fibroblasts. However, the role of ACE/Ang-(1-7)/Mas axis in the metastasis of breast cancer cells is still unknown. In the present study, we found that ACE2 protein level is negatively correlated with the metastatic ability of breast cancer cells and breast tumor grade. Upregulation of ACE2/Ang-(1-7)/Mas axis inhibits breast cancer cell migration and invasion in vivo and in vitro. Mechanistically, ACE2/Ang-(1-7)/Mas axis activation inhibits store-operated calcium entry (SOCE) and PAK1/NF-κB/Snail1 pathways, and induces E-cadherin expression. In summary, our results demonstrate that downregulation of ACE2/Ang-(1-7)/Mas axis stimulates breast cancer metastasis through the activation of SOCE and PAK1/NF-κB/Snail1 pathways. These results provide new mechanisms by which breast cancer develop metastasis and shed light on developing novel anti-metastasis therapeutics for metastatic breast cancer by modulating ACE2/Ang-(1-7)/Mas axis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. The polybasic lysine-rich domain of plasma membrane-resident STIM1 is essential for the modulation of store-operated divalent cation entry by extracellular calcium.

    Science.gov (United States)

    Jardin, Isaac; Dionisio, Natalia; Frischauf, Irene; Berna-Erro, Alejandro; Woodard, Geoffrey E; López, José J; Salido, Ginés M; Rosado, Juan A

    2013-05-01

    STIM1 acts as an endoplasmic reticulum Ca(2+) sensor that communicates the filling state of the intracellular stores to the store-operated channels. In addition, STIM1 is expressed in the plasma membrane, with the Ca(2+) binding EF-hand motif facing the extracellular medium; however, its role sensing extracellular Ca(2+) concentrations in store-operated Ca(2+) entry (SOCE), as well as the underlying mechanism remains unclear. Here we report that divalent cation entry stimulated by thapsigargin (TG) is attenuated by extracellular Ca(2+) in a concentration-dependent manner. Expression of the Ca(2+)-binding defective STIM1(D76A) mutant did not alter the surface expression of STIM1 but abolishes the regulation of divalent cation entry by extracellular Ca(2+). Orai1 and TRPC1 have been shown to play a major role in SOCE. Expression of the STIM1(D76A) mutant did not alter Orai1 phosphoserine content. TRPC1 silencing significantly attenuated TG-induced Mn(2+) entry. Expression of the STIM1(K684,685E) mutant impaired the association of plasma membrane STIM1 with TRPC1, as well as the regulation of TG-induced divalent cation entry by extracellular Ca(2+), which suggests that TRPC1 might be involved in the regulation of divalent cation entry by extracellular Ca(2+) mediated by plasma membrane-resident STIM1. Expression of the STIM1(D76A) or STIM1(K684,685E) mutants reduced store-operated divalent cation entry and resulted in loss of dependence on the extracellular Ca(2+) concentration, providing evidence for a functional role of plasma membrane-resident STIM1 in the regulation of store-operated divalent cation entry, which at least involves the EF-hand motif and the C-terminal polybasic lysine-rich domain. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Pharmacological characterization of the native store-operated calcium channels of cortical neuronal from embryonic mouse brain

    Directory of Open Access Journals (Sweden)

    Sylvain Chauvet

    2016-12-01

    Full Text Available In the murine brain, the first post-mitotic cortical neurons formed during embryogenesis express store-operated channels (SOCs sensitive to Pyr3, initially proposed as a blocker of transient receptor potential channel of C type 3 (TRPC3 channel. However Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to precise the molecular identity and the pharmacological profile of native SOCs from E13 cortical neurons. The mRNA expression of STIM1-2, Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR. E13 cortical neurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, GSK-7975A were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to the Orai blockers Pyr6, GSK-7975A, and also to EVP4593, zinc, copper and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ~10 nM. SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing the mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2 (iPLA2. Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex.

  11. Pharmacological Characterization of the Native Store-Operated Calcium Channels of Cortical Neurons from Embryonic Mouse Brain

    Science.gov (United States)

    Chauvet, Sylvain; Jarvis, Louis; Chevallet, Mireille; Shrestha, Niroj; Groschner, Klaus; Bouron, Alexandre

    2016-01-01

    In the murine brain, the first post-mitotic cortical neurons formed during embryogenesis express store-operated channels (SOCs) sensitive to Pyr3, initially proposed as a blocker of the transient receptor potential channel of C type 3 (TRPC3 channel). However, Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to clarify the molecular identity and the pharmacological profile of native SOCs from E13 cortical neurons. The mRNA expression of STIM1-2 and Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction. E13 cortical neurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, and GSK-7975A) were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to the Orai blockers Pyr6 and GSK-7975A, as well as to EVP4593, zinc, copper, and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ∼10 nM). SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2. Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex. PMID:28018223

  12. Orai1 and Orai3 in Combination with Stim1 Mediate the Majority of Store-operated Calcium Entry in Astrocytes

    Science.gov (United States)

    Kwon, Jea; An, Heeyoung; Sa, Moonsun; Won, Joungha; Shin, Jeong Im

    2017-01-01

    Astrocytes are non-excitable cells in the brain and their activity largely depends on the intracellular calcium (Ca2+) level. Therefore, maintaining the intracellular Ca2+ homeostasis is critical for proper functioning of astrocytes. One of the key regulatory mechanisms of Ca2+ homeostasis in astrocytes is the store-operated Ca2+ entry (SOCE). This process is mediated by a combination of the Ca2+-store-depletion-sensor, Stim, and the store-operated Ca2+-channels, Orai and TrpC families. Despite the existence of all those families in astrocytes, previous studies have provided conflicting results on the molecular identification of astrocytic SOCE. Here, using the shRNA-based gene-silencing approach and Ca2+-imaging from cultured mouse astrocytes, we report that Stim1 in combination with Orai1 and Orai3 contribute to the major portion of astrocytic SOCE. Gene-silencing of Stim1 showed a 79.2% reduction of SOCE, indicating that Stim1 is the major Ca2+-store-depletion-sensor. Further gene-silencing showed that Orai1, Orai2, Orai3, and TrpC1 contribute to SOCE by 35.7%, 20.3%, 26.8% and 12.2%, respectively. Simultaneous gene-silencing of all three Orai subtypes exhibited a 67.6% reduction of SOCE. Based on the detailed population analysis, we predict that Orai1 and Orai3 are expressed in astrocytes with a large SOCE, whereas TrpC1 is exclusively expressed in astrocytes with a small SOCE. This analytical approach allows us to identify the store operated channel (SOC) subtype in each cell by the degree of SOCE. Our results propose that Stim1 in combination with Orai1 and Orai3 are the major molecular components of astrocytic SOCE under various physiological and pathological conditions. PMID:28243166

  13. Activation of ERK1/2 by store-operated calcium entry in rat parotid acinar cells.

    Directory of Open Access Journals (Sweden)

    Stephen P Soltoff

    Full Text Available The regulation of intracellular Ca(2+ concentration ([Ca(2+]i plays a critical role in a variety of cellular processes, including transcription, protein activation, vesicle trafficking, and ion movement across epithelial cells. In many cells, the activation of phospholipase C-coupled receptors hydrolyzes membrane phosphoinositides and produces the depletion of endoplasmic reticulum Ca(2+ stores, followed by the sustained elevation of [Ca(2+]i from Ca(2+ entry across the plasma membrane via store-operated Ca(2+ entry (SOCE. Ca(2+ entry is also increased in a store-independent manner by arachidonate-regulated Ca(2+ (ARC channels. Using rat parotid salivary gland cells, we examined multiple pathways of Ca(2+ entry/elevation to determine if they activated cell signaling proteins and whether this occurred in a pathway-dependent manner. We observed that SOCE activates extracellular signal-related kinases 1 and 2 (ERK1/2 to ∼3-times basal levels via a receptor-independent mechanism when SOCE was initiated by depleting Ca(2+ stores using the endoplasmic reticulum Ca(2+-ATPase inhibitor thapsigargin (TG. TG-initiated ERK1/2 phosphorylation increased as rapidly as that initiated by the muscarinic receptor agonist carbachol, which promoted an increase to ∼5-times basal levels. Notably, ERK1/2 phosphorylation was not increased by the global elevation of [Ca(2+]i by Ca(2+ ionophore or by Ca(2+ entry via ARC channels in native cells, although ERK1/2 phosphorylation was increased by Ca(2+ ionophore in Par-C10 and HSY salivary cell lines. Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB, SKF96363, and removal of extracellular Ca(2+, also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation. TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol. These observations demonstrate

  14. Effects and mechanisms of store-operated calcium channel blockade on hepatic ischemia-reperfusion injury in rats

    Institute of Scientific and Technical Information of China (English)

    Li-Jie Pan; Zi-Chao Zhang; Zhen-Ya Zhang; Wen-Jun Wang; Yue Xu; Zong-Ming Zhang

    2012-01-01

    AIM: To further investigate the important role of storeoperated calcium channels (SOCs) in rat hepatocytes and to explore the effects of SOC blockers on hepatic ischemia-reperfusion injury (HIRI). METHODS: Using freshly isolated hepatocytes from a rat model of HIRI (and controls), we measured cytosolic free Ca2+ concentration (by calcium imaging), net Ca2+ fluxes (by a non-invasive micro-test technique), the SOC current (ISOC; by whole-cell patch-clamp recording), and taurocholate secretion [by high-performance liquid chromatography and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays]. RESULTS: Ca2+ oscillations and net Ca2+ fluxes mediated by Ca2+ entry via SOCs were observed in rat hepatocytes. ISOC was significantly higher in HIRI groups than in controls (57.0 ± 7.5 pA vs 31.6 ± 2.7 pA, P <0.05) and was inhibited by La3+. Taurocholate secretion by hepatocytes into culture supernatant was distinctly lower in HIRI hepatocytes than in controls, an effect reversed by SOC blockers. CONCLUSION: SOCs are pivotal in HIRI. SOC blockers protected against HIRI and assisted the recovery of secretory function in hepatocytes. Thus, they are likely to become a novel class of effective drugs for prevention or therapy of HIRI patients in the future.

  15. Differential involvement of L- and T-type Ca(2+) channels, store-operated calcium channel (TRPC) and Rho-kinase signaling pathway(s) in PGF2α-induced contractions in myometrium of non-pregnant and pregnant buffaloes (Bubalus bubalis).

    Science.gov (United States)

    Sharma, Abhishek; Nakade, Udayraj P; Jaitley, Pooja; Sharma, Vipin; Choudhury, Soumen; Garg, Satish Kumar

    2017-09-12

    This study unravels the differential involvement of calcium signaling pathway(s) in PGF2α-induced contractions in myometrium of non-pregnant and pregnant buffaloes. Compared to the myometrium of pregnant animals, myometrium of non-pregnant buffaloes was more sensitive to PGF2α-induced contractile effect as manifested by the changes in mean integral tension (MIT) and tonicity. However, phasic contraction was significantly more in myometrium of pregnant animals. The uterotonic effect of PGF2α was dependent on extracellular Ca(2+) and its influx through nifedipine-sensitive L-type Ca(2+) channels both in non-pregnant and pregnant animals, but T-type Ca(2+) channels play an additional role during pregnancy. Entry of extracellular Ca(2+) is triggered by enhanced functional involvement of Pyr3-sensitive TRPC3 channels and Rho-kinase pathways to regulate uterotonic action of PGF2α in myometrium of non-pregnant buffaloes while these are down-regulated during pregnancy as there was significantly reduced expression of Rho-A proteins in myometrium of pregnant buffaloes and down-regulation of these pathways facilitate uterine quiescence. Intracellular Ca(2+) plays minor role in myometrium of both the non-pregnant and pregnant buffaloes. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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

  17. Store-operated calcium entry and the localisation of STIM1 and Orai1 proteins in isolated mouse sinoatrial node cells

    Directory of Open Access Journals (Sweden)

    Jie eLiu

    2015-03-01

    Full Text Available In many nonexcitable and excitable cells, store-operated calcium entry (SOCE represents an additional pathway for calcium entry upon Ca2+ store depletion. In a previous study, we demonstrated SOCE activity in intact mouse cardiac pacemaker tissue, specifically from sinoatrial node (SAN tissue. However, store content as a key determinant of SOCE activity is difficult to measure in intact SAN tissue. Therefore, to investigate the interaction between SOCE and store content and its role in cardiac pacemaking, it is necessary to investigate SOCE activity in single cardiac pacemaker cells. Furthermore, recent studies in other tissues have identified two new proteins involved in SOCE, stromal interacting molecule (STIM, which is an ER Ca2+ sensor, and the surface membrane channel Orai, a prototypic gene encoding for SOCE. However, whether STIM and Orai are expressed in native pacemaker cells is still unknown.In this current study, we examined SOCE activity in single firing pacemaker cells isolated from mouse sinoatrial node tissue. We found a significant rise in Ca2+ entry in response to Ca2+ store depletion. SOCE blockers reduced the amplitude and frequency of spontaneous Ca2+ transients and reduced Ca2+ store content. We demonstrated for the first time that STIM and Orai are expressed in pacemaker cells. After store depletion, STIM1 redistributed to the cell periphery and showed increased co-localization with surface membrane located Orai1, indicating a possible involvement of these proteins in SOCE activity in native cardiac pacemaker cells. These results suggest the novel concept that SOCE plays a functional role in regulating intracellular Ca2+ of cardiac pacemaker cells.

  18. Store-operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells.

    Science.gov (United States)

    Shi, Jian; Miralles, Francesc; Birnbaumer, Lutz; Large, William A; Albert, Anthony P

    2017-02-15

    Depletion of Ca(2+) stores activates store-operated channels (SOCs), which mediate Ca(2+) entry pathways that regulate cellular processes such as contraction, proliferation and gene expression. In vascular smooth muscle cells (VSMCs), stimulation of SOCs composed of canonical transient receptor potential channel 1 (TRPC1) proteins requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1/protein kinase C (PKC) activity. We studied the role of stromal interaction molecule 1 (STIM1) in coupling store depletion to this activation pathway using patch clamp recording, GFP-PLCδ1-PH imaging and co-localization techniques. Store-operated TRPC1 channel and PLCβ1 activities were inhibited by STIM1 short hairpin RNA (shRNA) and absent in TRPC1(-/-) cells, and store-operated PKC phosphorylation of TRPC1 was inhibited by STIM1 shRNA. Store depletion induced interactions between STIM1 and TRPC1, Gαq and PLCβ1, which required STIM1 and TRPC1. Similar effects were produced with noradrenaline. These findings identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, where store-operated STIM1-TRPC1 interactions stimulate Gαq/PLCβ1/PKC activity to induce channel gating. In vascular smooth muscle cells (VSMCs), stimulation of canonical transient receptor potential channel 1 (TRPC1) protein-based store-operated channels (SOCs) mediates Ca(2+) entry pathways that regulate contractility, proliferation and migration. It is therefore important to understand how these channels are activated. Studies have shown that stimulation of TRPC1-based SOCs requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1 activities and protein kinase C (PKC) phosphorylation, although it is unclear how store depletion stimulates this gating pathway. The present study examines this issue by focusing on the role of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum Ca(2+) sensor. Store-operated TRPC1 channel activity was inhibited by TRPC

  19. Dissociation of the store-operated calcium current ICRAC and the Mg-nucleotide-regulated metal ion current MagNuM

    Science.gov (United States)

    Hermosura, Meredith C; Monteilh-Zoller, Mahealani K; Scharenberg, Andrew M; Penner, Reinhold; Fleig, Andrea

    2002-01-01

    Rat basophilic leukaemia cells (RBL-2H3-M1) were used to study the characteristics of the store-operated Ca2+ release-activated Ca2+ current (ICRAC) and the magnesium-nucleotide-regulated metal cation current (MagNuM) (which is conducted by the LTRPC7 channel). Pipette solutions containing 10 mm BAPTA and no added ATP induced both currents in the same cell, but the time to half-maximal activation for MagNuM was about two to three times slower than that of ICRAC. Differential suppression of ICRAC was achieved by buffering free [Ca2+]i to 90 nm and selective inhibition of MagNuM was accomplished by intracellular solutions containing 6 mm Mg.ATP, 1.2 mm free [Mg2+]i or 100 μm GTP-γ-S, allowing investigations on these currents in relative isolation. Removal of extracellular Ca2+ and Mg2+ caused both currents to be carried significantly by monovalent ions. In the absence or presence of free [Mg2+]i, ICRAC carried by monovalent ions inactivated more rapidly and more completely than MagNuM carried by monovalent ions. Since several studies have used divalent-free solutions on either side of the membrane to study selectivity and single-channel behaviour of ICRAC, these experimental conditions would have favoured the contribution of MagNuM to monovalent conductance and call for caution in interpreting results where both ICRAC and MagNuM are activated. PMID:11882677

  20. Gypenosides Induce Apoptosis by Ca2+ Overload Mediated by Endoplasmic-Reticulum and Store-Operated Ca2+ Channels in Human Hepatoma Cells

    Science.gov (United States)

    Sun, Da-Peng; Li, Xiao-Xi; Liu, Xin-Li; Zhao, Dan; Qiu, Feng-Qi; Li, Yan

    2013-01-01

    Abstract Gypenosides (Gyps) are triterpenoid saponins contained in an extract from Gynostemma pentaphyllum Makino and reported to induce apoptosis in human hepatoma cells through Ca2+-implicated endoplasmic reticulum (ER) stress and mitochondria-dependent pathways. The mechanism underlying the Gyp-increased intracellular Ca2+ concentration ([Ca2+]i) is unclear. Here, we examined Gyp-induced necrosis and apoptosis in human hepatoma HepG2 cells. Gyp-induced apoptotic cell death was accompanied by a sustained increase in [Ca2+]i level. Gyp-increased [Ca2+]i level was partly inhibited by removal of extracellular Ca2+ by Ca2+ chelator EGTA, store-operated Ca2+ channel (SOC) inhibitor 2- aminoethoxydiphenyl borate (2-APB), and ER Ca2+-release-antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8). The strongest inhibitory effect was observed with TMB-8. EGTA, 2-APB, and TMB-8 also protected against Gyp-induced apoptosis in HepG2 cells. The combination of 2-APB and TMB-8 almost completely abolished the Gyp-induced Ca2+ response and apoptosis. In contrast, the sarco/endoplasmic-reticulum-Ca2+-ATPase (SERCA) inhibitor thapsigargin slightly elevated Gyp-induced [Ca2+]i increase and apoptosis in HepG2 cells. Exposure to 300 μg/mL Gyp for 24 hours upregulated protein levels of inositol 1,4,5-trisphosphate receptor and SOC and downregulated that of SERCA for at least 72 hours. Thus, Gyp-induced increase in [Ca2+]i level and consequent apoptosis in HepG2 cells may be mainly due to enhanced Ca2+ release from ER stores and increased store-operated Ca2+ entry. PMID:25310348

  1. STIM1 and STIM2 proteins differently regulate endogenous store-operated channels in HEK293 cells.

    Science.gov (United States)

    Shalygin, Alexey; Skopin, Anton; Kalinina, Vera; Zimina, Olga; Glushankova, Lyuba; Mozhayeva, Galina N; Kaznacheyeva, Elena

    2015-02-20

    The endoplasmic reticulum calcium sensors stromal interaction molecules 1 and 2 (STIM1 and STIM2) are key modulators of store-operated calcium entry. Both these sensors play a major role in physiological functions in normal tissue and in pathology, but available data on native STIM2-regulated plasma membrane channels are scarce. Only a few studies have recorded STIM2-induced CRAC (calcium release-activated calcium) currents. On the other hand, many cell types display store-operated currents different from CRAC. The STIM1 protein regulates not only CRAC but also transient receptor potential canonical (TRPC) channels, but it has remained unclear whether STIM2 is capable of regulating store-operated non-CRAC channels. Here we present for the first time experimental evidence for the existence of endogenous non-CRAC STIM2-regulated channels. As shown in single-channel patch clamp experiments on HEK293 cells, selective activation of native STIM2 proteins or STIM2 overexpression results in store-operated activation of Imin channels, whereas STIM1 activation blocks this process. Changes in the ratio between active STIM2 and STIM1 proteins can switch the regulation of Imin channels between store-operated and store-independent modes. We have previously characterized electrophysiological properties of different Ca(2+) influx channels coexisting in HEK293 cells. The results of this study show that STIM1 and STIM2 differ in the ability to activate these store-operated channels; Imin channels are regulated by STIM2, TRPC3-containing INS channels are induced by STIM1, and TRPC1-composed Imax channels are activated by both STIM1 and STIM2. These new data about cross-talk between STIM1 and STIM2 and their different roles in store-operated channel activation are indicative of an additional level in the regulation of store-operated calcium entry pathways.

  2. Mitochondrial calcium uniporter MCU supports cytoplasmic Ca2+ oscillations, store-operated Ca2+ entry and Ca2+-dependent gene expression in response to receptor stimulation.

    Directory of Open Access Journals (Sweden)

    Krishna Samanta

    Full Text Available Ca2+ flux into mitochondria is an important regulator of cytoplasmic Ca2+ signals, energy production and cell death pathways. Ca2+ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU but whether the MCU is involved in shaping Ca2+ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC4 evokes a series of cytoplasmic Ca2+ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca2+ entry into the matrix, prevents the mitochondrial Ca2+ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca2+ oscillations appeared to be a consequence of enhanced Ca2+-dependent inactivation of InsP3 receptors, which arose from the loss of mitochondrial Ca2+ buffering. Ca2+ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca2+ release, mitochondria also sequestrated Ca2+ entry through store-operated Ca2+ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca2+.

  3. Mitochondrial calcium uniporter MCU supports cytoplasmic Ca2+ oscillations, store-operated Ca2+ entry and Ca2+-dependent gene expression in response to receptor stimulation.

    Science.gov (United States)

    Samanta, Krishna; Douglas, Sophie; Parekh, Anant B

    2014-01-01

    Ca2+ flux into mitochondria is an important regulator of cytoplasmic Ca2+ signals, energy production and cell death pathways. Ca2+ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU) but whether the MCU is involved in shaping Ca2+ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC4 evokes a series of cytoplasmic Ca2+ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca2+ entry into the matrix, prevents the mitochondrial Ca2+ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca2+ oscillations appeared to be a consequence of enhanced Ca2+-dependent inactivation of InsP3 receptors, which arose from the loss of mitochondrial Ca2+ buffering. Ca2+ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca2+ release, mitochondria also sequestrated Ca2+ entry through store-operated Ca2+ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca2+.

  4. Niflumic acid affects store-operated Ca(2+)-permeable (SOC) and Ca (2+)-dependent K (+) and Cl (-) ion channels and induces apoptosis in K562 cells.

    Science.gov (United States)

    Kucherenko, Yuliya V; Lang, Florian

    2014-07-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl(-) and Ca(2+)-activated Cl(-) (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8-10 %) apoptotic effect of endoplasmic reticulum Ca(2+) mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca(2+)-activated K(+) (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC50 for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.

  5. Expression and function of store-operated calcium channels in human circulating fibrocytes%钙库操纵性钙通道在人循环纤维细胞中的表达及功能

    Institute of Scientific and Technical Information of China (English)

    钟金男; 兰兰; 何光珍; 黄革; 杨炯; 高亚东

    2016-01-01

    目的:研究钙库操纵性钙通道(store-operated calcium channels,SOCC)相关功能蛋白ORAI1-3和STIM1-2在人循环纤维细胞(circulating fibrocytes)中的表达及SOCC对人循环纤维细胞分化的影响.方法:采集健康人外周静脉血,分离出单个核细胞,体外培养分化为循环纤维细胞.采用RT-PCR和real-time PCR检测循环纤维细胞中ORAI1-3及STIM1-2的mRNA表达情况,并检测SOCC抑制剂对循环纤维细胞分化的影响.结果:Real-time PCR检测结果显示ORAI1-3和STIM1-2 mRNA在循环纤维细胞中有较高的表达水平,并且SOCC抑制剂SKF-96365对循环纤维细胞分化具有明显的抑制作用.结论:SOCC表达于循环纤维细胞中,并且影响循环纤维细胞的分化.%AIM:To investigate the expression and function of store-operated calcium channels ( SOCC) in human circulating fibrocytes.METHODS:Peripheral blood mononuclear cells ( PBMCs) were isolated and cultured in ser-um-free media.After 7 d, the PBMCs differentiated into fibrocytes.RT-PCR and real-time PCR were performed to deter-mine the mRNA expression of ORAI1-3 and STIM1-2 in the fibrocytes.SOCC inhibitor SKF-96365 was used to elucidate the role of SOCC in the differentiation of fibrocytes.RESULTS:The results of real-time PCR showed that the mRNA ex-pression of ORAI1-3 and STIM1-2 was positive in cultured fibrocytes.SKF-96365 (10μmol/L) significantly inhibited the differentiation of fibrocytes.CONCLUSION:SOCC-related proteins ORAI1-3 and STIM1-2 are abundantly expressed in the fibrocytes, and may play an important role in the differentiation of these cells.

  6. Store operations to maintain cache coherence

    Energy Technology Data Exchange (ETDEWEB)

    Evangelinos, Constantinos; Nair, Ravi; Ohmacht, Martin

    2017-09-12

    In one embodiment, a computer-implemented method includes encountering a store operation during a compile-time of a program, where the store operation is applicable to a memory line. It is determined, by a computer processor, that no cache coherence action is necessary for the store operation. A store-without-coherence-action instruction is generated for the store operation, responsive to determining that no cache coherence action is necessary. The store-without-coherence-action instruction specifies that the store operation is to be performed without a cache coherence action, and cache coherence is maintained upon execution of the store-without-coherence-action instruction.

  7. Store operations to maintain cache coherence

    Energy Technology Data Exchange (ETDEWEB)

    Evangelinos, Constantinos; Nair, Ravi; Ohmacht, Martin

    2017-08-01

    In one embodiment, a computer-implemented method includes encountering a store operation during a compile-time of a program, where the store operation is applicable to a memory line. It is determined, by a computer processor, that no cache coherence action is necessary for the store operation. A store-without-coherence-action instruction is generated for the store operation, responsive to determining that no cache coherence action is necessary. The store-without-coherence-action instruction specifies that the store operation is to be performed without a cache coherence action, and cache coherence is maintained upon execution of the store-without-coherence-action instruction.

  8. 钙库调控性钙通道在肿瘤细胞中的矛盾作用及其机制%Contradictory effect and its mechanism of store-operated calcium channel in cancer cells

    Institute of Scientific and Technical Information of China (English)

    顾鹏; 阳东荣

    2013-01-01

    In most non-excitable cells,the store-operated calcium channel (SOCC) in the plasma membrane is the major calcium entry pathway.SOCC plays an important role in calcium signals that control many cancer cellular processes such as adhesion,secretion,movement,gene expression,proliferation,division and apoptosis.Stromal interaction molecule 1 (STIM1) and Orail are the two important parts of SOCC.So overexpression or suppression expression of STIM1/Orail can regulate SOCC activity and biological characteristics of cancer cells.However,SOCC plays a contradictory role in cancer cells,which can both promote tumor progression and inhibit tumor progression.SOCC is expected to become a new target for the treatment of tumors,but its complicated mechanism needs to consider both types of tumor cells and the external stimulators of tumor cells.%在非兴奋型细胞中,钙库调控性钙通道(SOCC)是胞外钙离子进入胞内的最主要途径,可调控多种肿瘤细胞的生物学特性,包括黏附、分泌、运动、基因表达、增殖、分裂与凋亡等.基质交联分子1(STIM1)和Orail是SOCC的重要组成部分,故过表达或抑制表达STIM1/Orail可调控SOCC活性和肿瘤细胞的生物学特性.而SOCC对肿瘤细胞的作用却自相矛盾,既可促进肿瘤进展,又可抑制肿瘤进展.SOCC有望成为治疗肿瘤的一个新靶点,但其机制复杂,既需考虑肿瘤细胞类型,还需考虑外界刺激因子对肿瘤细胞的作用.

  9. TRPC1 protein forms only one type of native store-operated channels in HEK293 cells.

    Science.gov (United States)

    Skopin, Anton; Shalygin, Alexey; Vigont, Vladimir; Zimina, Olga; Glushankova, Lyubov; Mozhayeva, Galina N; Kaznacheyeva, Elena

    2013-02-01

    TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I(max) channels, while the properties of I(min) and I(NS) channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current-voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I(max) channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I(max) channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I(max) channels but is not an essential subunit for other store-operated channel types in HEK293 cells.

  10. Store-operate-coherence-on-value

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Dong; Heidelberger, Philip; Kumar, Sameer; Ohmacht, Martin; Steinmacher-Burow, Burkhard

    2014-11-18

    A system, method and computer program product for performing various store-operate instructions in a parallel computing environment that includes a plurality of processors and at least one cache memory device. A queue in the system receives, from a processor, a store-operate instruction that specifies under which condition a cache coherence operation is to be invoked. A hardware unit in the system runs the received store-operate instruction. The hardware unit evaluates whether a result of the running the received store-operate instruction satisfies the condition. The hardware unit invokes a cache coherence operation on a cache memory address associated with the received store-operate instruction if the result satisfies the condition. Otherwise, the hardware unit does not invoke the cache coherence operation on the cache memory device.

  11. Complex actions of ionomycin in cultured cerebellar astrocytes affecting both calcium-induced calcium release and store-operated calcium entry

    DEFF Research Database (Denmark)

    Müller, Margit S; Obel, Linea Lykke Frimodt; Waagepetersen, Helle S;

    2013-01-01

    The polyether antibiotic ionomycin is a common research tool employed to raise cytosolic Ca(2+) in almost any cell type. Although initially thought to directly cause physicochemical translocation of extracellular Ca(2+) into the cytosol, a number of studies have demonstrated that the mechanism of...

  12. The foot structure from the type 1 ryanodine receptor is required for functional coupling to store-operated channels.

    Science.gov (United States)

    Sampieri, Alicia; Diaz-Muñoz, Mauricio; Antaramian, Anaid; Vaca, Luis

    2005-07-01

    In the present study we have explored structural determinants of the functional interaction between skeletal muscle ryanodine receptor (RyR1) and transient receptor potential channel 1 (TRPC1) channels expressed in Chinese hamster ovary cells. We have illustrated a functional interaction between TRPC1 channels and RyR1 for the regulation of store-operated calcium entry (SOCE) initiated after releasing calcium from a caffeine-sensitive intracellular calcium pool. RNA interference experiments directed to reduce the amount of TRPC1 protein indicate that RyR1 associates to at least two different types of store-operated channels (SOCs), one dependent and one independent of TRPC1. In contrast, bradykinin-induced SOCE is completely dependent on the presence of TRPC1 protein, as we have previously illustrated. Removing the foot structure from RyR1 results in normal caffeine-induced release of calcium from internal stores but abolishes the activation of SOCE, indicating that this structure is require for functional coupling to SOCs. The footless RyR1 protein shows a different cellular localization when compared with wild type RyR1. The later protein shows a higher percentage of colocalization with FM-464, a marker of plasma membrane. The implications of the foot structure for the functional and physical coupling to TRPC and SOCs is discussed.

  13. Silencing of the Charcot-Marie-Tooth disease-associated gene GDAP1 induces abnormal mitochondrial distribution and affects Ca2+ homeostasis by reducing store-operated Ca2+ entry.

    Science.gov (United States)

    Pla-Martín, David; Rueda, Carlos B; Estela, Anna; Sánchez-Piris, Maribel; González-Sánchez, Paloma; Traba, Javier; de la Fuente, Sergio; Scorrano, Luca; Renau-Piqueras, Jaime; Alvarez, Javier; Satrústegui, Jorgina; Palau, Francesc

    2013-07-01

    GDAP1 is an outer mitochondrial membrane protein that acts as a regulator of mitochondrial dynamics. Mutations of the GDAP1 gene cause Charcot-Marie-Tooth (CMT) neuropathy. We show that GDAP1 interacts with the vesicle-organelle trafficking proteins RAB6B and caytaxin, which suggests that GDAP1 may participate in the mitochondrial movement within the cell. GDAP1 silencing in the SH-SY5Y cell line induces abnormal distribution of the mitochondrial network, reduces the contact between mitochondria and endoplasmic reticulum (ER) and alters the mobilization of mitochondria towards plasma membrane upon depletion of ER-Ca(2+) stores. GDAP1 silencing does not affect mitochondrial Ca(2+) uptake, ER-Ca(2+), or Ca(2+) flow from ER to mitochondria, but reduces Ca(2+) inflow through store-operated Ca(2+) entry (SOCE) following mobilization of ER-Ca(2+) and SOCE-driven Ca(2+) entry in mitochondria. Our studies suggest that the pathophysiology of GDAP1-related CMT neuropathies may be associated with abnormal distribution and movement of mitochondria throughout cytoskeleton towards the ER and subplasmalemmal microdomains, resulting in a decrease in SOCE activity and impaired SOCE-driven Ca(2+) uptake in mitochondria.

  14. Visualizing the store-operated channel complex assembly in real time: identification of SERCA2 as a new member.

    Science.gov (United States)

    Sampieri, Alicia; Zepeda, Angelica; Asanov, Alexander; Vaca, Luis

    2009-05-01

    Depletion of intracellular calcium stores leads to the activation of calcium influx via the so-called store-operated channels (SOCs). Recent evidence positions Orai proteins as the putative channels responsible for this process. The stromal interacting molecule (STIM1) has been recently identified as the calcium sensor located at the endoplasmic reticulum (ER), and responsible for communicating the deplete state of calcium stores to Orai at the plasma membrane (PM). However, recent experimental findings suggest that Orai and STIM1 are only part of a larger molecular complex required to modulate store-operated calcium entry (SOCE). In the present study we describe the assembly of the several of the components from the SOC complex in real-time, utilizing a novel imaging method. Using FRET imaging we show that under resting conditions (with calcium stores replenished) STIM1 travels continuously through the ER associated to the microtubule tracking protein, EB1. Upon depletion of the ER STIM1 dissociates from EB1 and aggregates into macromolecular complexes at the ER which includes the microsomal calcium ATPase. This association follows the assembly of Orai into macromolecular aggregates at the PM. We show that STIM1-Orai association follows a similar time course as that of Orai aggregation at the PM. During this last step of the process, calcium-selective, whole-cell inward currents developed, simultaneously. We show that this process is fully reversible. Replenishing intracellular calcium stores induces STIM1-Orai complex dissociation and shuts down inward currents. Under these conditions STIM1 re-associates to EB1, and reinitiates its travel through the ER.

  15. Ethanol suppresses carbamylcholine-induced intracellular calcium oscillation in mouse pancreatic acinar cells.

    Science.gov (United States)

    Yoon, Mi Na; Kim, Min Jae; Koong, Hwa Soo; Kim, Dong Kwan; Kim, Se Hoon; Park, Hyung Seo

    2017-09-01

    Oscillation of intracellular calcium levels is closely linked to initiating secretion of digestive enzymes from pancreatic acinar cells. Excessive alcohol consumption is known to relate to a variety of disorders in the digestive system, including the exocrine pancreas. In this study, we have investigated the role and mechanism of ethanol on carbamylcholine (CCh)-induced intracellular calcium oscillation in murine pancreatic acinar cells. Ethanol at concentrations of 30 and 100 mM reversibly suppressed CCh-induced Ca(2+) oscillation in a dose-dependent manner. Pretreatment of ethanol has no effect on the store-operated calcium entry induced by 10 μM of CCh. Ethanol significantly reduced the initial calcium peak induced by low concentrations of CCh and therefore, the CCh-induced dose-response curve of the initial calcium peak was shifted to the right by ethanol pretreatment. Furthermore, ethanol significantly dose-dependently reduced inositol 1,4,5-trisphosphate-induced calcium release from the internal stores in permeabilized acinar cells. These results provide evidence that excessive alcohol intake could impair cytosolic calcium oscillation through inhibiting calcium release from intracellular stores in mouse pancreatic acinar cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. SOC and now also SIC: store-operated and store-inhibited channels.

    Science.gov (United States)

    Moreno, Claudia; Vaca, Luis

    2011-10-01

    There is a specialized form of calcium influx that involves a close communication between endoplasmic reticulum and the channels at the plasma membrane. In one side store depletion activates channels known as store-operated channels (SOC), which are responsible of the well-studied store-operated calcium entry (SOCE). SOC comprises two different types of channels. Orai, which is exclusively activated by store depletion being the channel responsible of the calcium release-activated calcium current, and transient receptor potential canonical channel, which in contrast, is activated by store depletion only under specific conditions and carries nonselective cationic currents. On the other hand, it has been recently shown that store depletion also inhibits calcium channels. The first member identified, of what we named as store-inhibited channels (SIC), is the L-type voltage-gated calcium channel. Stores control both SOC and SIC by means of the multifunctional protein STIM1. The identification of SOC and SIC opens a new scenario for the role of store depletion in the modulation of different calcium entry pathways, which may satisfy different cellular processes.

  17. CIF and other mysteries of the store-operated Ca2+-entry pathway.

    Science.gov (United States)

    Bolotina, Victoria M; Csutora, Peter

    2005-07-01

    The molecular mechanism of the store-operated Ca2+-entry (SOCE) pathway remains one of the most intriguing and long lasting mysteries of Ca2+ signaling. The elusive calcium influx factor (CIF) that is produced upon depletion of Ca2+ stores has attracted growing attention, triggered by new discoveries that filled the gap in the chain of reactions leading to activation of store-operated channels and Ca2+ entry. Ca2+-independent phospholipase A2 emerged as a target of CIF, and a major determinant of the SOCE mechanism. Here, we present our viewpoint on CIF and conformational-coupling models of SOCE from a historical perspective, trying to resolve some of the problem areas, and summarizing our present knowledge on how depletion of intracellular Ca2+ stores signals to plasma membrane channels to open and provide Ca2+ influx that is required for many important physiological functions.

  18. Inhibitors of arachidonate-regulated calcium channel signaling suppress triggered activity induced by the late sodium current.

    Science.gov (United States)

    Wolkowicz, Paul; Umeda, Patrick K; Sharifov, Oleg F; White, C Roger; Huang, Jian; Mahtani, Harry; Urthaler, Ferdinand

    2014-02-05

    Disturbances in myocyte calcium homeostasis are hypothesized to be one cause for cardiac arrhythmia. The full development of this hypothesis requires (i) the identification of all sources of arrhythmogenic calcium and (ii) an understanding of the mechanism(s) through which calcium initiates arrhythmia. To these ends we superfused rat left atria with the late sodium current activator type II Anemonia sulcata toxin (ATXII). This toxin prolonged atrial action potentials, induced early afterdepolarization, and provoked triggered activity. The calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphon-amide) suppressed ATXII triggered activity but its inactive congener KN-92 (2-[N-(4-methoxy benzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) did not. Neither drug affected normal atrial contractility. Calcium entry via L-type channels or calcium leakage from sarcoplasmic reticulum stores are not critical for this type of ectopy as neither verapamil ((RS)-2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl]-(methyl)amino}-2-prop-2-ylpentanenitrile) nor ryanodine affected ATXII triggered activity. By contrast, inhibitors of the voltage independent arachidonate-regulated calcium (ARC) channel and the store-operated calcium channel specifically suppressed ATXII triggered activity without normalizing action potentials or affecting atrial contractility. Inhibitors of cytosolic calcium-dependent phospholipase A2 also suppressed triggered activity suggesting that this lipase, which generates free arachidonate, plays a key role in ATXII ectopy. Thus, increased left atrial late sodium current appears to activate atrial Orai-linked ARC and store operated calcium channels, and these voltage-independent channels may be unexpected sources for the arrhythmogenic calcium that underlies triggered activity.

  19. Mitochondrial Ca²⁺ uniporter is critical for store-operated Ca²⁺ entry-dependent breast cancer cell migration.

    Science.gov (United States)

    Tang, Shihao; Wang, Xubu; Shen, Qiang; Yang, Xinyi; Yu, Changhui; Cai, Chunqing; Cai, Guoshuai; Meng, Xiaojing; Zou, Fei

    2015-02-27

    Metastasis of cancer cells is a complicated multistep process requiring extensive and continuous cytosolic calcium modulation. Mitochondrial Ca(2+) uniporter (MCU), a regulator of mitochondrial Ca(2+) uptake, has been implicated in energy metabolism and various cellular signaling processes. However, whether MCU contributes to cancer cell migration has not been established. Here we examined the expression of MCU mRNA in the Oncomine database and found that MCU is correlated to metastasis and invasive breast cancer. MCU inhibition by ruthenium red (RuR) or MCU silencing by siRNA abolished serum-induced migration in MDA-MB-231 breast cancer cells and reduced serum- or thapsigargin (TG)-induced store-operated Ca2+ entry (SOCE). Serum-induced migrations in MDA-MB-231 cells were blocked by SOCE inhibitors. Our results demonstrate that MCU plays a critical role in breast cancer cell migration by regulating SOCE.

  20. Induced calcium carbonate precipitation using Bacillus species.

    Science.gov (United States)

    Seifan, Mostafa; Samani, Ali Khajeh; Berenjian, Aydin

    2016-12-01

    Microbially induced calcium carbonate precipitation is an emerging process for the production of self-healing concrete. This study was aimed to investigate the effects and optimum conditions on calcium carbonate biosynthesis. Bacillus licheniformis, Bacillus sphaericus, yeast extract, urea, calcium chloride and aeration were found to be the most significant factors affecting the biomineralization of calcium carbonate. It was noticed that the morphology of microbial calcium carbonate was mainly affected by the genera of bacteria (cell surface properties), the viscosity of the media and the type of electron acceptors (Ca(2+)). The maximum calcium carbonate concentration of 33.78 g/L was achieved at the optimum conditions This value is the highest concentration reported in the literature.

  1. Mitochondrial modulation of store-operated Ca(2+) entry in model cells of Alzheimer's disease.

    Science.gov (United States)

    Ma, Tuo; Gong, Kai; Yan, Yufang; Song, Bo; Zhang, Xiufang; Gong, Yandao

    2012-09-21

    Mitochondrial malfunction and calcium dyshomeostasis are early pathological events considered as important features of the Alzheimer's disease (AD) brain. Recent studies have suggested mitochondrion as an active regulator of Ca(2+) signaling based on its calcium buffering capacity. Herein, we investigated the mitochondrial involvement in the modulation of store-operated calcium entry (SOCE) in neural 2a (N2a) transgenic AD model cells. Results showed that SOCE was significantly depressed in N2a cells transfected with wild-type human APP695 (N2a APPwt) compared with empty vector control (N2a WT) cells. Pharmacological manipulation with mitochondrial function blockers, such as FCCP, RuR, or antimycin A/oligomycin, could inhibit mitochondrial calcium handling, and then impair SOCE pathway in N2a WT cells. Furthermore, mitochondria of N2a APPwt cells exhibited more severe swelling in response to Ca(2+), which is an indication of mitochondrial membrane permeability transition (MPT), than the wild-type controls. Additionally, treatment with cyclosporin A, a potent inhibitor of cyclophilin D, which can block MPT, could significantly restore the attenuated SOCE in N2a APPwt cells. Therefore, inhibition of cyclophilin D might be a therapeutic strategy for Alzheimer's disease.

  2. Teaching Calcium-Induced Calcium Release in Cardiomyocytes Using a Classic Paper by Fabiato

    Science.gov (United States)

    Liang, Willmann

    2008-01-01

    This teaching paper utilizes the materials presented by Dr. Fabiato in his review article entitled "Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum." In the review, supporting evidence of calcium-induced calcium release (CICR) is presented. Data concerning potential objections to the CICR theory are discussed as well. In…

  3. STIM1/Orai1-mediated store-operated Ca2+ entry: the tip of the iceberg

    Directory of Open Access Journals (Sweden)

    F.R. Giachini

    2011-11-01

    Full Text Available Highly efficient mechanisms regulate intracellular calcium (Ca2+ levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1 was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.

  4. Designing dynamical output feedback controllers for store-operated Ca²+ entry.

    Science.gov (United States)

    Liu, Weijiu; Tang, Fusheng; Chen, Jingvoon

    2010-11-01

    Store-operated calcium entry (SOCE) has been proposed as the main process controlling Ca²+ entry in non-excitable cells. Although recent breakthroughs in experimental studies of SOCE have been made, its mathematical modeling has not been developed. In the present work, SOCE is viewed as a feedback control system subject to an extracellular agonist disturbance and an extracellular calcium input. We then design a dynamic output feedback controller to reject the disturbance and track Ca²+ resting levels in the cytosol and the endoplasmic reticulum (ER). The constructed feedback control system is validated by published experimental data and its global asymptotic stability is proved by using the LaSalle's invariance principle. We then simulate the dynamic responses of STIM1 and Orai1, two major components in the operation of the store-operated channels, to the depletion of Ca²+ in the ER with thapsigargin, which show that: (1) Upon the depletion of Ca²+ in the ER, the concentrations of activated STIM1 and STIM1-Orai1 cluster are elevated gradually, indicating that STIM1 is accumulating in the ER-PM junctions and that the cytosolic portion of the active STIM1 is binding to Orai1 and driving the opening of CRAC channels for Ca²+ entry; (2) after the extracellular Ca²+ addition, the concentrations of both STIM1 and STIM1-Orai1 cluster decrease but still much higher than the original levels. We also simulate the system responses to the agonist disturbance, which show that, when a sequence of periodic agonist pulses is applied, the system returns to its equilibrium after each pulse. This indicates that the designed feedback controller can reject the disturbance and track the equilibrium.

  5. Noise induced intercellular propagation of calcium waves

    Science.gov (United States)

    Nchange, A. K.; Kepseu, W. D.; Woafo, P.

    2008-04-01

    In this paper, we investigate the spatiotemporal dynamics of a bidirectional coupled chain of cells, in which a cell is subjected to an external noise. Noisy oscillations of calcium (Ca 2+), that is, a bursting-like phenomenon induced by noise with fluctuations in the baseline values of calcium, are induced in the first cell and propagated along the chain with noise suppression. This phenomenon of noise suppression is further investigated by computing the normalized fluctuation of pulse durations. It is therefore found that the noise induced coherence resonance phenomenon occurs at the cellular level. Coherence biresonance behaviour appears in the transmission of noise induced oscillations at appropriate noise intensity or noise coupling (for low noise intensity) and the information flow in each cell can be simultaneously optimized at the optimal value of noise or coupling.

  6. Mitochondrial Ca{sup 2+} uniporter is critical for store-operated Ca{sup 2+} entry-dependent breast cancer cell migration

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Shihao [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong (China); Guangzhou No.12 Hospital, Guangzhou (China); Wang, Xubu [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong (China); Shen, Qiang [Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Yang, Xinyi; Yu, Changhui; Cai, Chunqing [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong (China); Cai, Guoshuai [Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Meng, Xiaojing, E-mail: xiaojingmeng@smu.edu.cn [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong (China); Zou, Fei, E-mail: zoufei616@163.com [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong (China)

    2015-02-27

    Metastasis of cancer cells is a complicated multistep process requiring extensive and continuous cytosolic calcium modulation. Mitochondrial Ca{sup 2+} uniporter (MCU), a regulator of mitochondrial Ca{sup 2+} uptake, has been implicated in energy metabolism and various cellular signaling processes. However, whether MCU contributes to cancer cell migration has not been established. Here we examined the expression of MCU mRNA in the Oncomine database and found that MCU is correlated to metastasis and invasive breast cancer. MCU inhibition by ruthenium red (RuR) or MCU silencing by siRNA abolished serum-induced migration in MDA-MB-231 breast cancer cells and reduced serum- or thapsigargin (TG)-induced store-operated Ca2+ entry (SOCE). Serum-induced migrations in MDA-MB-231 cells were blocked by SOCE inhibitors. Our results demonstrate that MCU plays a critical role in breast cancer cell migration by regulating SOCE. - Highlights: • MCU is correlated to metastasis and invasive breast cancer. • MCU inhibition abolished serum-induced migration in MDA-MB-231 breast cancer cells and reduced serum- or TG-induced SOCE. • Serum-induced migrations in MDA-MB-231 cells were blocked by SOCE inhibitors. • MCU plays a critical role in MDA-MB-231 cell migration by regulating SOCE.

  7. Functional upregulation of STIM-1/Orai-1-mediated store-operated Ca2+ contributing to the hypertension development elicited by chronic EtOH consumption.

    Science.gov (United States)

    Bomfim, Guilherme Henrique Souza; Méndez-López, Iago; Arranz-Tagarro, Juan Alberto; Carbonel, Adriana Aparecida Ferraz; Roman-Campos, Danilo; Padín, Juan Fernando; García, Antonio García; Jurkiewicz, Aron; Jurkiewicz, Neide Hyppolito

    2017-02-01

    Chronic ethanol (EtOH) consumption has been associated with deleterious effects on the cardiovascular system by abnormal calcium (Ca²⁺) handling. Store-operated Ca2+ entry (SOCE) is related to cardiovascular remodeling which leads to the hypertension development, and the coupling between STIM-1 (ER Ca2+ sensor) and Orai-1 (channel pore) is a key mechanism to control SOCE through of store-operated Ca2+ channels (SOCCs). However, the role of STIM-1/Orai-1-mediated SOCE and its cross-talk with EtOH-triggered vascular remodeling and hypertension remain poorly understood. We address this subject in the present study by evaluating how chronic EtOH consumption induces alterations in Ca²⁺ handling via SOCE. Male Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats were subjected to the intake of increasing EtOH concentrations (5-20%, for 30 days). Systolic blood pressure (SBP) and EtOH concentration were measured; cardiovascular remodeling was assessed by histomorphometry; and function/expression of STIM-1/Orai-1-mediated Ca2+ influx were evaluated by isometric contraction and western blot experiments. Compared to the WKY-Control, our results show that: (1) chronic EtOH consumption caused a significant elevation of SBP in both strains; (2) cardiac hypertrophy and hypertrophic aortic wall remodeling much more pronounced in WKY-EtOH; (3) decreased capacity of ER to store and release Ca2+ ; (4) increased STIM-1/Orai-1-mediated SOCCs activation, which was selectively inhibited by YM-58483; and (5) increased expression of STIM-1 in WKY-EtOH and SHR-Control rats. These findings suggest that hypertrophic aortic remodeling and abnormal contraction triggered mainly by Ca2+ overload via STIM-1/Orai-1-mediated SOCE through SOCCs are involved hypertension developed by EtOH consumption.

  8. Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2.

    Directory of Open Access Journals (Sweden)

    Dan Zhang

    Full Text Available BACKGROUND: Antimicrobial peptides derived from the natural processing of chromogranin A (CgA are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. METHODOLOGY/PRINCIPAL FINDINGS: PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR or Catestatin (CAT induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate, a store operated channels (SOCs blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. CONCLUSIONS/SIGNIFICANCE: For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated i

  9. Effects of 2-APB on Store-operated Ca2+ Channel Currents of Hepatocytes after Hepatic Ischemia/Reperfusion Injury in Rats

    Institute of Scientific and Technical Information of China (English)

    HUANG Changzhou; ZHANG Zongming; QIU Fazu

    2005-01-01

    The effects of hepatic ischemia/reperfusion (I/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on storeoperated calcium current (Isoc) in isolated rat hepatocytes after hepaticischemia/reperfusion injuries were studied. Hepatic ischemia and reperfusion injury model was established and whole cell patch-clamp techniques were used to investigate the effects of 2-APB on Isoc. The results showed that ischemia/reperfusion injuries could significantly reduce hepatocellular viability and further increase Isoc in hepatocytes and 2-APB (20, 40, 60, 80, 100 μmol/L) produced a concentration-dependent decrease of Isoc with IC50 value of 64.63±10.56 μmol/L (n= 8). It was concluded that ischemia/reperfusion injuries could reduce hepatocellular viability, probably through increased Isoc in hepatocytes and 2-APB had a protective effect on ischemia/reperfusion-induced liver injury, probably though inhibiting Isoc.

  10. Gastrodin Inhibits Store-Operated Ca2+ Entry and Alleviates Cardiac Hypertrophy

    Directory of Open Access Journals (Sweden)

    Xiaoqiang Yao

    2017-04-01

    Full Text Available Cardiac hypertrophy is a major risk factor for heart failure, which are among the leading causes of human death. Gastrodin is a small molecule that has been used clinically to treat neurological and vascular diseases for many years without safety issues. In the present study, we examined protective effect of gastrodin against cardiac hypertrophy and explored the underlying mechanism. Phenylephrine and angiotensin II were used to induce cardiac hypertrophy in a mouse model and a cultured cardiomyocyte model. Gastrodin was found to alleviate the cardiac hypertrophy in both models. Mechanistically, gastrodin attenuated the store-operated Ca2+ entry (SOCE by reducing the expression of STIM1 and Orai1, two key proteins in SOCE, in animal models as well as in cultured cardiomyocyte model. Furthermore, suppressing SOCE by RO2959, Orai1-siRNAs or STIM1-siRNAs markedly attenuated the phenylephrine-induced hypertrophy in cultured cardiomyocyte model. Together, these results showed that gastrodin inhibited cardiac hypertrophy and it also reduced the SOCE via its action on the expression of STIM1 and Orai1. Furthermore, suppression of SOCE could reduce the phenylephrine-induced cardiomyocyte hypertrophy, suggesting that SOCE-STIM1-Orai1 is located upstream of hypertrophy.

  11. Functional and fluorochrome analysis of an exocytotic mutant yields evidence of store-operated Ca2+ influx in Paramecium.

    Science.gov (United States)

    Mohamed, I; Klauke, N; Hentschel, J; Cohen, J; Plattner, H

    2002-05-01

    A non-discharge mutant of Paramecium tetraurelia (nd12-35 degrees C, lacking exocytotic response upon stimulation with the nonpermeable polycationic secretagogue aminoethyldextran, AED), in the pawnA genetic context (d4-500r, lacking ciliary voltage-dependent Ca2+ influx), was shown to lack (45)Ca2+ entry from outside upon AED stimulation. In contrast, cells grown at 25 degrees C behave like the wildtype. To check the functional properties in more detail, fluorochrome-loaded 35 degrees C cells were stimulated, not only with AED (EC(100) = 10(-6) M in wildtype cells), but also with 4-chloro-meta-cresol, (4CmC, 0.5 mM), a permeable activator of ryanodine receptor-type Ca2+ release channels, usually at extracellular [Ca2+] of 50 microM, and eventually with a Ca2+ chelator added. We confirm that pwA-nd12(35 degrees C) cells lack any Ca2+ influx and any exocytosis of trichocysts in response to any stimulus. As we determined by x-ray microanalysis, total calcium content in alveolar sacs (subplasmalemmal stores) known to be mobilized upon exocytosis stimulation in wild-type cells, contain about the same total calcium in 35 degrees C as in 25 degrees C cells, and Ca2+ mobilization from alveoli by AED or 4CmC is also nearly the same. Due to the absence of any AED-induced Ca2+ influx in 35 degrees C cells and normal Ca2+ release from stores found by x-ray microanalysis one can exclude a "CICR"-type mechanism (Ca2+-induced Ca2+ release) and imply that normally a store-operated Ca2+ ("SOC") influx would occur (as in 25 degrees C cells). Furthermore, 35 degrees C cells display a significantly lower basal intracellular [Ca2+], so that any increase upon stimulation may be less expressed or even remain undetected. Under these conditions, any mobilization of Ca2+ from stores cannot compensate for the lack of Ca2+ influx, particularly since normally both components have to cooperate to achieve full exocytotic response. Also striking is our finding that 35 degrees C cells are unable

  12. Store-operated Ca2+ entry in hippocampal neurons: Regulation by protein tyrosine phosphatase PTP1B.

    Science.gov (United States)

    Koss, David J; Riedel, Gernot; Bence, Kendra; Platt, Bettina

    2013-02-01

    Store operated Ca(2+) entry (SOCE) replenishes intracellular Ca(2+) stores and activates a number of intracellular signalling pathways. Whilst several molecular components forming store operated Ca(2+) channels (SOCC) have been identified, their modulation in neurons remains poorly understood. Here, we extend on our previous findings and show that neuronal SOCE is modulated by tyrosine phosphorylation. Cyclopiazonic acid induced SOCE was characterised in hippocampal cultures derived from forebrain specific protein tyrosine phosphatase 1B knockout (PTP1B KO) mice and wild type (WT) litter mates using Fura-2 Ca(2+) imaging. PTP1B KO cultures expressed elevated SOCE relative to WT cultures without changes in cytoplasmic Ca(2+) homeostasis or depolarisation-induced Ca(2+) influx. WT and PTP1B KO cultures displayed similar pharmacological sensitivities towards the SOCE inhibitors gadolinium and 2-aminoethoxydiphenyl borate, as well as the tyrosine kinase inhibitor Ag126 indicating an augmentation of native SOCCs by PTP1B. Following store depletion WT culture homogenates showed heightened phospho-tyrosine levels, an increase in Src tyrosine kinase activation and two minor PTP1B species. These data suggest tyrosine phosphorylation gating SOCE, and implicate PTP1B as a key regulatory enzyme. The involvement of PTP1B in SOCE and its relation to SOCC components and mechanism of regulation are discussed.

  13. Rickets induced by calcium or phosphate depletion.

    OpenAIRE

    Abugassa, S.; Svensson, O.

    1990-01-01

    We studied the effects of calciopenia and phosphopenia on longitudinal growth, skeletal mineralization, and development of rickets in young Sprague-Dawley rats. At an age of 21 days, two experimental groups were given diets containing 0.02% calcium or 0.02% phosphorus; otherwise the diets were nutritionally adequate. After 7, 14, and 21 days, five animals from each group were randomly chosen. The animals were anaesthetized and blood samples were drawn for analysis of calcium, phosphorus, and ...

  14. The prion protein constitutively controls neuronal store-operated Ca2+ entry through Fyn kinase

    Science.gov (United States)

    De Mario, Agnese; Castellani, Angela; Peggion, Caterina; Massimino, Maria Lina; Lim, Dmitry; Hill, Andrew F.; Sorgato, M. Catia; Bertoli, Alessandro

    2015-01-01

    The prion protein (PrPC) is a cell surface glycoprotein mainly expressed in neurons, whose misfolded isoforms generate the prion responsible for incurable neurodegenerative disorders. Whereas PrPC involvement in prion propagation is well established, PrPC physiological function is still enigmatic despite suggestions that it could act in cell signal transduction by modulating phosphorylation cascades and Ca2+ homeostasis. Because PrPC binds neurotoxic protein aggregates with high-affinity, it has also been proposed that PrPC acts as receptor for amyloid-β (Aβ) oligomers associated with Alzheimer’s disease (AD), and that PrPC-Aβ binding mediates AD-related synaptic dysfunctions following activation of the tyrosine kinase Fyn. Here, use of gene-encoded Ca2+ probes targeting different cell domains in primary cerebellar granule neurons (CGN) expressing, or not, PrPC, allowed us to investigate whether PrPC regulates store-operated Ca2+ entry (SOCE) and the implication of Fyn in this control. Our findings show that PrPC attenuates SOCE, and Ca2+ accumulation in the cytosol and mitochondria, by constitutively restraining Fyn activation and tyrosine phosphorylation of STIM1, a key molecular component of SOCE. This data establishes the existence of a PrPC-Fyn-SOCE triad in neurons. We also demonstrate that treating cerebellar granule and cortical neurons with soluble Aβ(1–42) oligomers abrogates the control of PrPC over Fyn and SOCE, suggesting a PrPC-dependent mechanizm for Aβ-induced neuronal Ca2+ dyshomeostasis. PMID:26578881

  15. The prion protein constitutively controls neuronal store-operated Ca2+ entry through Fyn kinase

    Directory of Open Access Journals (Sweden)

    Agnese eDe Mario

    2015-10-01

    Full Text Available The prion protein (PrPC is a cell surface glycoprotein mainly expressed in neurons, whose misfolded isoforms generate the prion responsible for incurable neurodegenerative disorders. Whereas PrPC involvement in prion propagation is well established, PrPC physiological function is still enigmatic despite suggestions that it could act in cell signal transduction by modulating phosphorylation cascades and Ca2+ homeostasis. Because PrPC binds neurotoxic protein aggregates with high-affinity, it has also been proposed that PrPC acts as receptor for amyloid-β (Aβ oligomers associated with Alzheimer’s disease (AD, and that PrPC-Aβ binding mediates AD-related synaptic dysfunctions following activation of the tyrosine kinase Fyn.Here, use of gene-encoded Ca2+ probes targeting different cell domains in primary cerebellar granule neurons expressing, or not, PrPC allowed us to investigate whether PrPC regulates store-operated Ca2+ entry (SOCE and the implication of Fyn in this control. Our findings show that PrPC attenuates SOCE, and Ca2+ accumulation in the cytosol and mitochondria, by constitutively restraining Fyn activation and tyrosine phosphorylation of STIM1, a key molecular component of SOCE. This data establishes the existence of a PrPC-Fyn-SOCE triad in neurons.We also demonstrate that treating cerebellar granule and cortical neurons with soluble Aβ(1-42 oligomers abrogates the control of PrPC over Fyn and SOCE, suggesting a PrPC-dependent mechanism for Aβ-induced neuronal Ca2+ dyshomeostasis.

  16. Oyster shell calcium induced parotid swelling

    Directory of Open Access Journals (Sweden)

    Muthiah Palaniappan

    2014-01-01

    Full Text Available A 59 year old female consumer was started on therapy with oyster shell calcium in combination with vitamin D3 and she presented with swelling below the ear, after two doses. She stopped the drug by herself and the swelling disappeared in one day. She started the drug one day after recovery and again she developed the swelling. She was advised to stop the drug with a suggestion to take lemon to enhance parotid secretion and the swelling subsided. Calcium plays major role in salivary secretion and studies have shown reduced parotid secretion in rats, deficient of vitamin D. But in humans involvement of calcium and vitamin D3 in parotid secretion is unknown. However, the patient had no history of reaction though she had previously taken vitamin D3 with calcium carbonate which was not from oyster shell. Hence, we ruled out vitamin D3 in this reaction and suspecting oyster shell calcium as a culprit. This adverse drug reaction (ADR was assessed using World Health Organization (WHO causality assessment, Naranjo′s and Hartwig severity scales. As per WHO causality assessment scale, the ADR was classified as "certain". This reaction was analyzed as per Naranjo′s algorithm and was classified as probable. According to Hartwig′s severity scale the reaction was rated as mild. Our case is an example of a mild but rare adverse effect of oyster shell calcium carbonate which is widely used.

  17. Effects of modulation of calcium levels and calcium fluxes on ABA- induced gene expression in barley aleurone

    NARCIS (Netherlands)

    Meulen, R.M. van der; Visser, K.; Wang, M.

    1996-01-01

    We present data to elucidate the involvement of calcium ions in abscisic acid (ABA)-induced gene expression. Modulation of external calcium concentrations was able to affect ABA-induced specific RAB gene expression. At a constant ABA level with increasing extracellular calcium level, an increasing R

  18. Effects of modulation of calcium levels and calcium fluxes on ABA- induced gene expression in barley aleurone

    NARCIS (Netherlands)

    Meulen, R.M. van der; Visser, K.; Wang, M.

    1996-01-01

    We present data to elucidate the involvement of calcium ions in abscisic acid (ABA)-induced gene expression. Modulation of external calcium concentrations was able to affect ABA-induced specific RAB gene expression. At a constant ABA level with increasing extracellular calcium level, an increasing R

  19. Mitochondrial uncoupler FCCP activates proton conductance but does not block store-operated Ca(2+) current in liver cells.

    Science.gov (United States)

    To, Minh-Son; Aromataris, Edoardo C; Castro, Joel; Roberts, Michael L; Barritt, Greg J; Rychkov, Grigori Y

    2010-03-15

    Uncouplers of mitochondrial oxidative phosphorylation, including carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and carbonilcyanide m-cholorophenylhydrazone (CCCP), are widely used in experimental research to investigate the role of mitochondria in cellular function. Unfortunately, it is very difficult to interpret the results obtained in intact cells using FCCP and CCCP, as these agents not only inhibit mitochondrial potential, but may also affect membrane potential and cell volume. Here we show by whole-cell patch clamping that in primary rat hepatocytes and H4IIE liver cells, FCCP induced large proton currents across the plasma membrane, but did not activate any other observable conductance. In intact hepatocytes FCCP inhibits thapsigargin-activated store-operated Ca(2+) entry, but in patch clamping under the conditions of strong Ca(2+) buffering it has no effect on store-operated Ca(2+) current (I(SOC)). These results indicate that there is no direct connection between mitochondria and activation of I(SOC) in liver cells and support the notion of indirect regulation of I(SOC) by mitochondrial Ca(2+) buffering.

  20. 19 CFR 19.36 - Requirements for duty-free store operations.

    Science.gov (United States)

    2010-04-01

    ... reasonable assurance to the port director that conditionally duty-free merchandise purchased therein will be... 19 Customs Duties 1 2010-04-01 2010-04-01 false Requirements for duty-free store operations. 19.36 Section 19.36 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND...

  1. Cadmium induces transcription independently of intracellular calcium mobilization.

    Directory of Open Access Journals (Sweden)

    Brooke E Tvermoes

    Full Text Available BACKGROUND: Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca(2+](i and disrupting calcium-mediated intracellular signaling processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal. METHODOLOGY/PRINCIPAL FINDING: In the present report, the effects of cadmium on [Ca(2+](i under non-cytotoxic and cytotoxic conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60, which was stably expressed in HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium. Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca(2+](i mobilization or increase steady-state mRNA levels of calcium-responsive genes. In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered calcium-responsive gene expression. CONCLUSIONS/SIGNIFICANCE: These data indicate that at low levels, cadmium induces transcription independently of intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism. Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription.

  2. Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors.

    Science.gov (United States)

    Brailoiu, G Cristina; Deliu, Elena; Console-Bram, Linda M; Soboloff, Jonathan; Abood, Mary E; Unterwald, Ellen M; Brailoiu, Eugen

    2016-01-01

    Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum (ER). Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca(2+) entry (SOCE), a Ca(2+) influx mechanism promoted by depletion of intracellular Ca(2+) stores, in rat brain microvascular endothelial cells (RBMVEC). Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies indicate an unprecedented role for Sig-1R as a SOCE inhibitor.

  3. Sources of calcium in agonist-induced contraction of rat distal colon smooth muscle in vitro

    Institute of Scientific and Technical Information of China (English)

    Hua Zhou; De-Hu Kong; QunWan Pan; HaiHua Wang

    2008-01-01

    AIM:To study the origin of calcium necessary foragonist-induced contraction of the distal colon in rats.METHODS:The change in intracellular calcium concentration ([Ca2+]i)evoked by elevating external Ca2+was detected by fura 2/AM fluorescence.Contractile activity was measured with a force displacement transducer.Tension was continuously monitored and recorded using a Powerlab 4/25T data acquisition system with an ML110 bridge bioelectric physiographic amplifier.RESULTS:Store depletion induced Ca2+ influx had an effect on [Ca2+]i.In nominally Ca2+-free medium,the sarco-endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (1 μmol/L) increased [Ca2+]i from 68 to 241 nmol/L,and to 458 (P<0.01) and 1006 nmol/L (P<0.01),respectively,when 1.5 mmol/L and 3.0 mmol/L extracellular Ca2+ was reintroduced.Furthermore,the change in [Ca2+]1.was observed with verapamil (5 μmol/L),La3+(1 mmol/L) or KCI (40 mmol/L) in the bathing solution.These channels were sensitive to La3+(P<0.01),insensitive to verapamil,and voltage independent.In isolated distal colons we found that in normal Krebs solution,contraction induced by acetylcholine (ACh) was partially inhibited by verapamil,and the inhibitory rate was 41% (P<0.05).On the other hand,in Ca2+-free Krebs solution,ACh induced transient contraction due to Ca2+ release from the inLracellular stores.The transient contraction lasted until the Ca2+ store was depleted.Restoration of extracellular Ca2+ in the presence of atropine produced contraction,mainly due to Ca2+ influx.Such contraction was not inhibited by verapamil,but was decreased by La3+ (50 μmol/L) from 0.96 to 0.72 g (P<0.01).CONCLUSION:The predominant source of activator Ca2+ for the contractile response to agonist is extracellular Ca2+,and intracellular Caz+ has little role to play in mediating excitation-contraction coupling by agonists in rat distal colon smooth muscle in vitro.The influx of extracellular Ca2+ is mainly mediated through voltage-,receptor- and

  4. The influence of dihydropyridines calcium antagonists on 5-HT-induced intracellular calcium signal

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Confocal laser scanning microscopy (CLSM) was applied to detect the intracellular [Ca2+] variety of fluorescent intension, with Fluo-3/AM fluorescence loaded in SFSMC. The results show that 10 μmol/L Lacidipine can reduce the frequence which 10 μmol/L 5-HT induced [Ca2+] spark in SFSMC of calcium over loading to 50%, and amplitude to 50% or so. We can draw a conclusion that dihydropyridines cal-cium antagonists lacidipine can antagonize the release of intracellular [Ca2+] which 5-HT-induced in dose dependent manner.

  5. Proposal for the award of a contract for industrial support for logistics and stores operations

    CERN Document Server

    1999-01-01

    This document concerns the award of a contract for industrial support for logistics and stores operations. Following a market survey carried out among 59 firms in ten Member States, a call for tenders (IT-2401/SPL/Revised) was sent on 11 March 1999 to five firms and four consortia in five Member States. By the closing date, CERN had received eight tenders from five Member States. The Finance Committee is invited to agree to the negotiation of a contract with the consortium ISS GEBÄUDESERVICE (DE) - ISS SERVISYSTEM (CH) the lowest bidder complying with the specification, for industrial support for logistics and stores operations over a period of three years, starting on 1st January 2000, for a total amount of 3 830 000 Swiss francs, not subject to revision. The contract will include an option for two one-year extensions beyond the initial three-year period.

  6. Store-operated Ca2+ channels in airway epithelial cell function and implications for asthma

    Science.gov (United States)

    Samanta, Krishna; Parekh, Anant B.

    2016-01-01

    The epithelial cells of the lung are at the interface of a host and its environment and are therefore directly exposed to the inhaled air-borne particles. Rather than serving as a simple physical barrier, airway epithelia detect allergens and other irritants and then help organize the subsequent immune response through release of a plethora of secreted signals. Many of these signals are generated in response to opening of store-operated Ca2+ channels in the plasma membrane. In this review, we describe the properties of airway store-operated channels and their role in regulating airway epithelial cell function. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377718

  7. Homer regulates calcium signalling in growth cone turning

    Directory of Open Access Journals (Sweden)

    Thompson Michael JW

    2009-08-01

    Full Text Available Abstract Background Homer proteins are post-synaptic density proteins with known functions in receptor trafficking and calcium homeostasis. While they are key mediators of synaptic plasticity, they are also known to function in axon guidance, albeit by mechanisms that are yet to be elucidated. Homer proteins couple extracellular receptors – such as metabotropic glutamate receptors and the transient receptor potential canonical family of cation channels – to intracellular receptors such as inositol triphosphate and ryanodine receptors on intracellular calcium stores and, therefore, are well placed to regulate calcium dynamics within the neural growth cone. Here we used growth cones from dorsal root ganglia, a well established model in the field of axon guidance, and a growth cone turning assay to examine Homer1 function in axon guidance. Results Homer1 knockdown reversed growth cone turning from attraction to repulsion in response to the calcium-dependent guidance cues brain derived neurotrophic factor and netrin-1. Conversely, Homer1 knockdown had no effect on repulsion to the calcium-independent guidance cue Semaphorin-3A. This reversal of attractive turning suggested a requirement for Homer1 in a molecular switch. Pharmacological experiments confirmed that the operational state of a calcium-calmodulin dependent protein kinase II/calcineurin phosphatase molecular switch was dependent on Homer1 expression. Calcium imaging of motile growth cones revealed that Homer1 is required for guidance-cue-induced rise of cytosolic calcium and the attenuation of spontaneous cytosolic calcium transients. Homer1 knockdown-induced calcium transients and turning were inhibited by antagonists of store-operated channels. In addition, immunocytochemistry revealed the close association of Homer1 with the store-operated proteins TRPC1 and STIM1 within dorsal root ganglia growth cones. Conclusion These experiments provide evidence that Homer1 is an essential

  8. Astrocyte glycogenolysis is triggered by store-operated calcium entry and provides metabolic energy for cellular calcium homeostasis

    DEFF Research Database (Denmark)

    Müller, Margit S; Fox, Rebecca; Schousboe, Arne;

    2014-01-01

    Astrocytic glycogen, the only storage form of glucose in the brain, has been shown to play a fundamental role in supporting learning and memory, an effect achieved by providing metabolic support for neurons. We have examined the interplay between glycogenolysis and the bioenergetics of astrocytic...... cultures of murine cerebellar and cortical astrocytes, and determined glycogen content to investigate the effects of SOCE on glycogen metabolism. By blocking glycogenolysis, we tested energetic dependency of SOCE-related Ca(2+) dynamics on glycogenolytic ATP. Our results show that SOCE triggers astrocytic...... glycogenolysis. Upon inhibition of adenylate cyclase with 2',5'-dideoxyadenosine, glycogen content was no longer significantly different from that in unstimulated control cells, indicating that SOCE triggers astrocytic glycogenolysis in a cAMP-dependent manner. When glycogenolysis was inhibited in cortical...

  9. Fibroblast-like synoviocytes induce calcium mineral formation and deposition.

    Science.gov (United States)

    Sun, Yubo; Mauerhan, David R; Franklin, Atiya M; Zinchenko, Natalia; Norton, Harry James; Hanley, Edward N; Gruber, Helen E

    2014-01-01

    Calcium crystals are present in the synovial fluid of 65%-100% patients with osteoarthritis (OA) and 20%-39% patients with rheumatoid arthritis (RA). This study sought to investigate the role of fibroblast-like synoviocytes (FLSs) in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla) protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s). The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

  10. Fibroblast-Like Synoviocytes Induce Calcium Mineral Formation and Deposition

    Directory of Open Access Journals (Sweden)

    Yubo Sun

    2014-01-01

    Full Text Available Calcium crystals are present in the synovial fluid of 65%–100% patients with osteoarthritis (OA and 20%–39% patients with rheumatoid arthritis (RA. This study sought to investigate the role of fibroblast-like synoviocytes (FLSs in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s. The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

  11. Regulation of BMP2-induced intracellular calcium increases in osteoblasts.

    Science.gov (United States)

    Xu, Wenfeng; Liu, Bo; Liu, Xue; Chiang, Martin Y M; Li, Bo; Xu, Zichen; Liao, Xiaoling

    2016-10-01

    Although bone morphogenetic protein-2 (BMP2) is a well-characterized regulator that stimulates osteoblast differentiation, little is known about how it regulates intracellular Ca(2+) signaling. In this study, intracellular Ca(2+) concentration ([Ca(2+) ]i ) upon BMP2 application, focal adhesion kinase (FAK) and Src activities were measured in the MC3T3-E1 osteoblast cell line using fluorescence resonance energy transfer-based biosensors. Increase in [Ca(2+) ]i , FAK, and Src activities were observed during BMP2 stimulation. The removal of extracellular calcium, the application of membrane channel inhibitors streptomycin or nifedipine, the FAK inhibitor PF-573228 (PF228), and the alkaline phosphatase (ALP) siRNA all blocked the BMP2-stimulated [Ca(2+) ]i increase, while the Src inhibitor PP1 did not. In contrast, a gentle decrease of endoplasmic reticulum calcium concentration was found after BMP2 stimulation, which could be blocked by both streptomycin and PP1. Further experiments revealed that BMP2-induced FAK activation could not be inhibited by PP1, ALP siRNA or the calcium channel inhibitor nifedipine. PF228, but not PP1 or calcium channel inhibitors, suppressed ALP elevation resulting from BMP2 stimulation. Therefore, our results suggest that BMP2 can increase [Ca(2+) ]i through extracellular calcium influx regulated by FAK and ALP and can deplete ER calcium through Src signaling simultaneously. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1725-1733, 2016.

  12. Statins lower calcium-induced oxidative stress in isolated mitochondria.

    Science.gov (United States)

    Parihar, A; Parihar, M S; Zenebe, W J; Ghafourifar, P

    2012-04-01

    Statins are widely used cholesterol-lowering agents that exert cholesterol-independent effects including antioxidative. The present study delineates the effects of statins, atorvastatin, and simvastatin on oxidative stress and functions of mitochondria that are the primary cellular sources of oxidative stress. In isolated rat liver mitochondria, both the statins prevented calcium-induced cytochrome c release, lipid peroxidation, and opening of the mitochondrial membrane permeability transition (MPT). Both the statins decreased the activity of mitochondrial nitric oxide synthase (mtNOS), lowered the intramitochondrial ionized calcium, and increased the mitochondrial transmembrane potential. Our findings suggest that statins lower intramitochondrial ionized calcium that decreases mtNOS activity, lowers oxidative stress, prevents MPT opening, and prevents the release of cytochrome c from the mitochondria. These results provide a novel framework for understanding the antioxidative properties of statins and their effects on mitochondrial functions.

  13. Some assembly required: constructing the elementary units of store-operated Ca2+ entry.

    Science.gov (United States)

    Wu, Minnie M; Luik, Riina M; Lewis, Richard S

    2007-08-01

    The means by which Ca(2+) store depletion evokes the opening of store-operated Ca(2+) channels (SOCs) in the plasma membrane of excitable and non-excitable cells has been a longstanding mystery. Indirect evidence has supported local interactions between the ER and SOCs as well as long-range interactions mediated through a diffusible activator. The recent molecular identification of the ER Ca(2+) sensor (STIM1) and a subunit of the CRAC channel (Orai1), a prototypic SOC, has now made it possible to visualize directly the sequence of events that links store depletion to CRAC channel opening. Following store depletion, STIM1 moves from locations throughout the ER to accumulate in ER subregions positioned within 10-25nm of the plasma membrane. Simultaneously, Orai1 gathers at discrete sites in the plasma membrane directly opposite STIM1, resulting in local CRAC channel activation. These new studies define the elementary units of store-operated Ca(2+) entry, and reveal an unprecedented mechanism for channel activation in which the stimulus brings a channel and its activator/sensor together for interaction across apposed membrane compartments. We discuss the implications of this choreographic mechanism with regard to Ca(2+) dynamics, specificity of Ca(2+) signaling, and the existence of a specialized ER subset dedicated to the control of the CRAC channel.

  14. Calcium H & K Induced by Galaxy Halos

    CERN Document Server

    Zhu, Guangtun

    2013-01-01

    We present a measurement of the mean density profile of Ca II gas around galaxies out to ~ 200 kpc, traced by Fraunhofer's H & K absorption lines. The measurement is based on cross-correlating the positions of about one million foreground galaxies at z ~ 0.1 and the flux decrements induced in the spectra of about 10^5 background quasars from the Sloan Digital Sky Survey. This technique allows us to trace the total amount of Ca II absorption induced by the circumgalactic medium, including absorbers too weak to be detected in individual spectra. We can statistically measure Ca II rest equivalent widths down to several mA, corresponding to column densities of about 5x10^10 cm^{-2}. We find that the Ca II column density distribution follows N ~ rp^{-1.4} and the mean Ca II mass in the halo within 200 kpc is ~ 5x10^3 Msolar, averaged over the foreground galaxy sample with median mass ~ 10^10.3 Msolar. This is about an order-of-magnitude larger than the Ca II mass in the interstellar medium of the Milky Way, su...

  15. Cisplatin-induced peptic ulcers, vagotomy, adrenal and calcium modulation.

    Science.gov (United States)

    Aggarwal, S K; San Antonio, J D; Sokhansanj, A; Miller, C

    1994-04-01

    Cytochemical and autoradiographic studies in Wistar rats [Crl:(WI)BR] show that cisplatin treatment (9 mg/kg) inhibits the release of acetylcholine from the axonal endings of the stomach smooth muscle resulting in bloating of the stomach and ulceration. Cisplatin also induces corticosteroid release from the adrenal gland stimulating peptic ulceration. Vagotomy helps ameliorate the effect but not eliminate it. Calcium supplementation restores normal neuromuscular function to gastric smooth muscle, thereby eliminating the gastro-intestinal toxicity due to cisplatin.

  16. Calcium

    Science.gov (United States)

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

  17. Store operation with conditional push of a tag value to a queue

    Energy Technology Data Exchange (ETDEWEB)

    Heidelberger, Philip; Steinmacher-Burow, Burkhard

    2015-07-28

    According to one embodiment, a method for a store operation with a conditional push of a tag value to a queue is provided. The method includes configuring a queue that is accessible by an application, setting a value at an address in a memory device including a memory and a controller, receiving a request for an operation using the value at the address and performing the operation. The method also includes the controller writing a result of the operation to the address, thus changing the value at the address, the controller determining if the result of the operation meets a condition and the controller pushing a tag value to the queue based on the condition being met, where the tag value in the queue indicates to the application that the condition is met.

  18. Lithium- Sensitive Store-Operated Ca2+ Entry in the Regulation of FGF23 Release

    Directory of Open Access Journals (Sweden)

    Bingbing Zhang

    2015-12-01

    Full Text Available Background/Aims: Lithium, a widely used drug for the treatment of mood disorders, has previously been shown to stimulate the release of fibroblast growth factor FGF23, a powerful regulator of 1,25(OH2D3 formation and mineral metabolism. The cellular mechanisms involved have remained elusive. Lithium has been shown to modify Ca2+ signaling. In a wide variety of cells, Ca2+ entry is accomplished by the pore-forming Ca2+ channel subunit Orai1 and its regulator STIM, which stimulates Orai following Ca2+ depletion of intracellular stores. Transcription factors promoting Orai1 expression include NF-κB. The present study thus explored whether the effect of lithium on FGF23 involves and requires Ca2+ entry. Methods: Experiments were performed in UMR106 osteoblastic cells and immortalized primary osteoblasts (IPO. FGF23 and Orai1 transcript levels were estimated from qRT-PCR, cytosolic Ca2+ concentration ([Ca2+]i from Fura2 fluorescence and store-operated Ca2+ entry (SOCE from an increase in [Ca2+]i following store depletion by inhibition of the sarcoendoplasmatic Ca2+ ATPase (SERCA with thapsigargin (1 µM. Results: SOCE in UMR106 cells was enhanced by lithium treatment, an effect abrogated by Orai1 inhibitor 2-APB (50 µM. FGF23 transcript levels were increased by lithium and inhibited by Orai1 inhibitors 2-APB (50 µM and YM58483 (100 nM as well as NF-κB inhibitors wogonin (100 µM and withaferin A (500 nM. Moreover, Orai1 transcript levels were up-regulated by lithium, an effect attenuated by wogonin and withaferin A. Conclusion: Lithium stimulates FGF23 release at least in part by NF-κB dependent up-regulation of Orai1 transcription and store operated Ca2+ entry.

  19. Evidence for a distinct light-induced calcium-dependent potassium current in Hermissenda crassicornis.

    Science.gov (United States)

    Blackwell, K T

    2000-01-01

    A model of phototransduction is developed as a first step toward a model for investigating the critical interaction of light and turbulence stimuli within the type B photoreceptor of Hermissenda crassicronis. The model includes equations describing phototransduction, release of calcium from intracellular stores, and other calcium regulatory mechanisms, as well as equations describing ligand-gating of a rhabdomeric sodium current. The model is used to determine the sources of calcium in the soma, whether calcium or IP3 is a plausible ligand of the light-induced sodium current, and whether the light-induced potassium current is equivalent to the calcium-dependent potassium current activated by light-induced calcium release. Simulations show that the early light-induced calcium elevation is due to influx through voltage-dependent channels, whereas the later calcium elevation is due to release from intracellular stores. Simulations suggest that the ligand of the fast, light-induced sodium current is IP3 but that there is a smaller, prolonged component of the light-induced sodium current that is activated by calcium. In the model, the calcium-dependent potassium current, located in the soma, is activated only slightly by light-induced calcium elevation, leading to the prediction that a calcium-dependent potassium current, active at resting potential, is located in the rhabdomere and is responsible for the light-induced potassium current.

  20. [Therapy of glucocorticoid-induced osteoporosis with alfacalcidol/calcium and vitamin D/calcium].

    Science.gov (United States)

    Ringe, J D; Cöster, A; Meng, T; Schacht, E; Umbach, R

    2000-06-01

    Calcium/vitamin D supplementation is generally used as a first step treatment of glucocorticoid-induced osteoporosis (GIOP). The aim of this trial was to compare the efficacy of the D-hormone alfacalcidol with plain vitamin D in patients with established GIOP with or without vertebral fractures. Patients on long-term glucocorticoid-therapy were treated either with 1 microgram alfacalcidol plus 5000 mg calcium (group A: n = 43) or with 1000 IU vitamin D plus 500 mg calcium (group B: n = 42). The two groups were not different in respect to initial characteristics such as age, sex distribution, concomittant diseases, bone mineral density (mean T-score values at lumbar spine and femoral neck: -3.29 and -3.25 resp.), and in the number of prevalent vertebral and non-vertebral fractures. During the 3 years of treatment we found a significant increase in lumbar spine density in group A (+2.0%, p back pain only in group A (p vitamin D in GIOP.

  1. Treatment of glucocorticoid-induced osteoporosis with alfacalcidol/calcium versus vitamin D/calcium.

    Science.gov (United States)

    Ringe, J D; Cöster, A; Meng, T; Schacht, E; Umbach, R

    1999-10-01

    Vitamin D/calcium substitution is generally regarded as an effective first step treatment for glucocorticoid-induced osteoporosis (GIOP). The aim of our study was to evaluate the efficacy of the active vitamin D metabolite alfacalcidol (1alpha) compared with the native vitamin D(3) in patients with established GIOP with or without vertebral fractures. Patients on long-term corticoid therapy were given either 1 microg alfacalcidol plus 500 mg calcium per day (group A, n = 43) or 1000 IU vitamin D(3) plus 500 mg calcium (group B, n = 42). The two groups were alike in age range, sex ratio, percentages of underlying diseases, average initial bone density values (lumbar spine: mean T-score -3.28 and -3.25, respectively), and rates of vertebral and nonvertebral fractures. During the 3-year study we found a small but significant increase of lumbar spine density in group 1alpha (+2.0%, P back pain (P vitamin D group. We conclude that with the doses used in this trial, alfacalcidol is superior to vitamin D in the treatment of established GIOP.

  2. Outward potassium current oscillations in macrophage polykaryons: extracellular calcium entry and calcium-induced calcium release

    Directory of Open Access Journals (Sweden)

    Saraiva R.M.

    1997-01-01

    Full Text Available Outward current oscillations associated with transient membrane hyperpolarizations were induced in murine macrophage polykaryons by membrane depolarization in the absence of external Na+. Oscillations corresponded to a cyclic activation of Ca2+-dependent K+ currents (IKCa probably correlated with variations in intracellular Ca2+ concentration. Addition of external Na+ (8 mM immediately abolished the outward current oscillations, suggesting that the absence of the cation is necessary not only for their induction but also for their maintenance. Oscillations were completely blocked by nisoldipine. Ruthenium red and ryanodine reduced the number of outward current cycles in each episode, whereas quercetin prolonged the hyperpolarization 2- to 15-fold. Neither low molecular weight heparin nor the absence of a Na+ gradient across the membrane had any influence on oscillations. The evidence suggests that Ca2+ entry through a pathway sensitive to Ca2+ channel blockers is elicited by membrane depolarization in Na+-free medium and is essential to initiate oscillations, which are also dependent on the cyclic release of Ca2+ from intracellular Ca2+-sensitive stores; Ca2+ ATPase acts by reducing intracellular Ca2+, thus allowing slow deactivation of IKCa. Evidence is presented that neither a Na+/Ca2+ antiporter nor Ca2+ release from IP3-sensitive Ca2+ stores participate directly in the mechanism of oscillation

  3. Foeniculum vulgare Mill. increases cytosolic Ca(2+) concentration and inhibits store-operated Ca(2+) entry in vascular endothelial cells.

    Science.gov (United States)

    Han, A Young; Lee, Hui Su; Seol, Geun Hee

    2016-12-01

    This study assessed the effects of essential oil of Foeniculum vulgare Mill. (fennel oil) and of trans-anethole, the main component of fennel oil, on extracellular Ca(2+)-induced store-operated Ca(2+) entry (SOCE) into vascular endothelial (EA) cells and their mechanisms of action. Components of fennel oil were analyzed by gas chromatography-mass spectrometry. Cytosolic Ca(2+) concentration ([Ca(2+)]c) in EA cells was determined using Fura-2 fluorescence. In the presence of extracellular Ca(2+), fennel oil significantly increased [Ca(2+)]c in EA cells; this increase was significantly inhibited by the Ca(2+) channel blockers La(3+) and nifedipine. In contrast, fennel oil induced [Ca(2+)]c was significantly lower in Ca(2+)-free solution, suggesting that fennel oil increases [Ca(2+)]c mainly by enhancing Ca(2+) influx into EA cells. [Ca(2+)]c mobilization by trans-anethole was similar to that of fennel oil. Moreover, SOCE was suppressed by fennel oil and trans-anethole. SOCE was also attenuated by lanthanum (La(3+)), a non-selective cation channel (NSC) blocker; 2-aminoethoxydiphenyl borane (2-APB), an inositol 1,4,5-triphosphate (IP3) receptor inhibitor and SOCE blocker; and U73122, an inhibitor of phospholipase C (PLC). Further, SOCE was more strongly inhibited by La(3+) plus fennel oil or trans-anethole than by La(3+) alone. These findings suggest that fennel oil and trans-anethole significantly inhibit SOCE-induced [Ca(2+)]c increase in vascular endothelial cells and that these reactions may be mediated by NSC, IP3-dependent Ca(2+) mobilization, and PLC activation.

  4. Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

    Science.gov (United States)

    Betz, N. A.; Westhoff, B. A.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.

  5. Store-operated channels in the pulmonary circulation of high- and low-altitude neonatal lambs.

    Science.gov (United States)

    Parrau, Daniela; Ebensperger, Germán; Herrera, Emilio A; Moraga, Fernando; Riquelme, Raquel A; Ulloa, César E; Rojas, Rodrigo T; Silva, Pablo; Hernandez, Ismael; Ferrada, Javiera; Diaz, Marcela; Parer, Julian T; Cabello, Gertrudis; Llanos, Aníbal J; Reyes, Roberto V

    2013-04-15

    We determined whether store-operated channels (SOC) are involved in neonatal pulmonary artery function under conditions of acute and chronic hypoxia, using newborn sheep gestated and born either at high altitude (HA, 3,600 m) or low altitude (LA, 520 m). Cardiopulmonary variables were recorded in vivo, with and without SOC blockade by 2-aminoethyldiphenylborinate (2-APB), during basal or acute hypoxic conditions. 2-APB did not have effects on basal mean pulmonary arterial pressure (mPAP), cardiac output, systemic arterial blood pressure, or systemic vascular resistance in both groups of neonates. During acute hypoxia 2-APB reduced mPAP and pulmonary vascular resistance in LA and HA, but this reduction was greater in HA. In addition, isolated pulmonary arteries mounted in a wire myograph were assessed for vascular reactivity. HA arteries showed a greater relaxation and sensitivity to SOC blockers than LA arteries. The pulmonary expression of two SOC-forming subunits, TRPC4 and STIM1, was upregulated in HA. Taken together, our results show that SOC contribute to hypoxic pulmonary vasoconstriction in newborn sheep and that SOC are upregulated by chronic hypoxia. Therefore, SOC may contribute to the development of neonatal pulmonary hypertension. We propose SOC channels could be potential targets to treat neonatal pulmonary hypertension.

  6. Store-operated Ca2+ entry controls ameloblast cell function and enamel development

    Science.gov (United States)

    Eckstein, Miriam; Vaeth, Martin; Fornai, Cinzia; Vinu, Manikandan; Bromage, Timothy G.; Nurbaeva, Meerim K.; Sorge, Jessica L.; Coelho, Paulo G.; Idaghdour, Youssef; Feske, Stefan; Lacruz, Rodrigo S.

    2017-01-01

    Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release–activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel. PMID:28352661

  7. Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels

    OpenAIRE

    2008-01-01

    Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure plan...

  8. Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+ entry in mouse pancreatic acinar cells.

    Science.gov (United States)

    Rosado, Juan A; Redondo, Pedro C; Salido, Ginés M; Sage, Stewart O; Pariente, Jose A

    2005-01-01

    We recently reported that store-operated Ca(2+) entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca(2+) channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as "secretion-like coupling." As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca(2+) entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca(2+) influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca(2+) store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type.

  9. Store-operated Ca2+ entry does not control proliferation in primary cultures of human metastatic renal cellular carcinoma.

    Science.gov (United States)

    Dragoni, Silvia; Turin, Ilaria; Laforenza, Umberto; Potenza, Duilio Michele; Bottino, Cinzia; Glasnov, Toma N; Prestia, Martina; Ferulli, Federica; Saitta, Anna; Mosca, Alessandra; Guerra, Germano; Rosti, Vittorio; Luinetti, Ombretta; Ganini, Carlo; Porta, Camillo; Pedrazzoli, Paolo; Tanzi, Franco; Montagna, Daniela; Moccia, Francesco

    2014-01-01

    Store-operated Ca(2+) entry (SOCE) is activated following depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca(2+) pool to regulate proliferation in immortalized cell lines established from either primary or metastatic lesions. The molecular nature of SOCE may involve both Stim1, which senses Ca(2+) levels within the endoplasmic reticulum (ER) Ca(2+) reservoir, and a number of a Ca(2+)-permeable channels on the plasma membrane, including Orai1, Orai3, and members of the canonical transient receptor (TRPC1-7) family of ion channels. The present study was undertaken to assess whether SOCE is expressed and controls proliferation in primary cultures isolated from secondary lesions of heavily pretreated metastatic renal cell carcinoma (mRCC) patients. SOCE was induced following pharmacological depletion of the ER Ca(2+) store, but not by InsP3-dependent Ca(2+) release. Metastatic RCC cells express Stim1-2, Orai1-3, and TRPC1-7 transcripts and proteins. In these cells, SOCE was insensitive to BTP-2, 10 µM Gd(3+) and Pyr6, while it was inhibited by 100 µM Gd(3+), 2-APB, and carboxyamidotriazole (CAI). Neither Gd(3+) nor 2-APB or CAI impaired mRCC cell proliferation. Consistently, no detectable Ca(2+) signal was elicited by growth factor stimulation. Therefore, a functional SOCE is expressed but does not control proliferation of mRCC cells isolated from patients resistant to multikinase inhibitors.

  10. Store-Operated Ca2+ Entry Does Not Control Proliferation in Primary Cultures of Human Metastatic Renal Cellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Silvia Dragoni

    2014-01-01

    Full Text Available Store-operated Ca2+ entry (SOCE is activated following depletion of the inositol-1,4,5-trisphosphate (InsP3-sensitive Ca2+ pool to regulate proliferation in immortalized cell lines established from either primary or metastatic lesions. The molecular nature of SOCE may involve both Stim1, which senses Ca2+ levels within the endoplasmic reticulum (ER Ca2+ reservoir, and a number of a Ca2+-permeable channels on the plasma membrane, including Orai1, Orai3, and members of the canonical transient receptor (TRPC1–7 family of ion channels. The present study was undertaken to assess whether SOCE is expressed and controls proliferation in primary cultures isolated from secondary lesions of heavily pretreated metastatic renal cell carcinoma (mRCC patients. SOCE was induced following pharmacological depletion of the ER Ca2+ store, but not by InsP3-dependent Ca2+ release. Metastatic RCC cells express Stim1-2, Orai1–3, and TRPC1–7 transcripts and proteins. In these cells, SOCE was insensitive to BTP-2, 10 µM Gd3+ and Pyr6, while it was inhibited by 100 µM Gd3+, 2-APB, and carboxyamidotriazole (CAI. Neither Gd3+ nor 2-APB or CAI impaired mRCC cell proliferation. Consistently, no detectable Ca2+ signal was elicited by growth factor stimulation. Therefore, a functional SOCE is expressed but does not control proliferation of mRCC cells isolated from patients resistant to multikinase inhibitors.

  11. Store-operated Ca2+ Entry Modulates the Expression of Enamel Genes.

    Science.gov (United States)

    Nurbaeva, M K; Eckstein, M; Snead, M L; Feske, S; Lacruz, R S

    2015-10-01

    Dental enamel formation is an intricate process tightly regulated by ameloblast cells. The correct spatiotemporal patterning of enamel matrix protein (EMP) expression is fundamental to orchestrate the formation of enamel crystals, which depend on a robust supply of Ca2+. In the extracellular milieu, Ca2+ -EMP interactions occur at different levels. Despite its recognized role in enamel development, the molecular machinery involved in Ca2+ homeostasis in ameloblasts remains poorly understood. A common mechanism for Ca2+ influx is store-operated Ca2+ entry (SOCE). We evaluated the possibility that Ca2+ influx in enamel cells might be mediated by SOCE and the Ca2+ release-activated Ca2+ (CRAC) channel, the prototypical SOCE channel. Using ameloblast-like LS8 cells, we demonstrate that these cells express Ca2+ -handling molecules and mediate Ca2+ influx through SOCE. As a rise in the cytosolic Ca2+ concentration is a versatile signal that can modulate gene expression, we assessed whether SOCE in enamel cells had any effect on the expression of EMPs. Our results demonstrate that stimulating LS8 cells or murine primary enamel organ cells with thapsigargin to activate SOCE leads to increased expression of Amelx, Ambn, Enam, Mmp20. This effect is reversed when cells are treated with a CRAC channel inhibitor. These data indicate that Ca2+ influx in LS8 cells and enamel organ cells is mediated by CRAC channels and that Ca2+ signals enhance the expression of EMPs. Ca2+ plays an important role not only in mineralizing dental enamel but also in regulating the expression of EMPs.

  12. Cell type-specific glycosylation of Orai1 modulates store-operated Ca2+ entry.

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    Dörr, Kathrin; Kilch, Tatiana; Kappel, Sven; Alansary, Dalia; Schwär, Gertrud; Niemeyer, Barbara A; Peinelt, Christine

    2016-03-08

    N-glycosylation of cell surface proteins affects protein function, stability, and interaction with other proteins. Orai channels, which mediate store-operated Ca(2+) entry (SOCE), are composed of N-glycosylated subunits. Upon activation by Ca(2+) sensor proteins (stromal interaction molecules STIM1 or STIM2) in the endoplasmic reticulum, Orai Ca(2+) channels in the plasma membrane mediate Ca(2+) influx. Lectins are carbohydrate-binding proteins, and Siglecs are a family of sialic acid-binding lectins with immunoglobulin-like repeats. Using Western blot analysis and lectin-binding assays from various primary human cells and cancer cell lines, we found that glycosylation of Orai1 is cell type-specific. Ca(2+) imaging experiments and patch-clamp experiments revealed that mutation of the only glycosylation site of Orai1 (Orai1N223A) enhanced SOCE in Jurkat T cells. Knockdown of the sialyltransferase ST6GAL1 reduced α-2,6-linked sialic acids in the glycan structure of Orai1 and was associated with increased Ca(2+) entry in Jurkat T cells. In human mast cells, inhibition of sialyl sulfation altered the N-glycan of Orai1 (and other proteins) and increased SOCE. These data suggest that cell type-specific glycosylation influences the interaction of Orai1 with specific lectins, such as Siglecs, which then attenuates SOCE. In summary, the glycosylation state of Orai1 influences SOCE-mediated Ca(2+) signaling and, thus, may contribute to pathophysiological Ca(2+) signaling observed in immune disease and cancer.

  13. Molecular Determinants for STIM1 Activation During Store- Operated Ca2+ Entry.

    Science.gov (United States)

    Ma, G; Zheng, S; Ke, Y; Zhou, L; He, L; Huang, Y; Wang, Y; Zhou, Y

    2017-01-01

    STIM/ORAI-mediated store-operated Ca2+ entry (SOCE) mediates a myriad of Ca2+-dependent cellular activities in mammals. Genetic defects in STIM1/ORAI1 lead to devastating severe combined immunodeficiency; whereas gain-offunction mutations in STIM1/ORAI1 are intimately associated with tubular aggregate myopathy. At molecular level, a decrease in the Ca2+ concentrations within the lumen of endoplasmic reticulum (ER) initiates multimerization of the STIM1 luminal domain to switch on the STIM1 cytoplasmic domain to engage and gate ORAI channels, thereby leading to the ultimate Ca2+ influx from the extracellular space into the cytosol. Despite tremendous progress made in dissecting functional STIM1-ORAI1 coupling, the activation mechanism of SOCE remains to be fully characterized. Building upon a robust fluorescence resonance energy transfer assay designed to monitor STIM1 intramolecular autoinhibition, we aimed to systematically dissect the molecular determinants required for the activation and oligomerization of STIM1. Here we showed that truncation of the STIM1 luminal domain predisposes STIM1 to adopt a more active conformation. Replacement of the single transmembrane (TM) domain of STIM1 by a more rigid dimerized TM domain of glycophorin A abolished STIM1 activation. But this adverse effect could be partially reversed by disrupting the TM dimerization interface. Moreover, our study revealed regions that are important for the optimal assembly of hetero-oligomers composed of full-length STIM1 with its minimal STIM1-ORAI activating region, SOAR. Our study clarifies the roles of major STIM1 functional domains in maintaining a quiescent configuration of STIM1 to prevent preactivation of SOCE. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Calsequestrin-1 Regulates Store-Operated Ca2+ Entry by Inhibiting STIM1 Aggregation

    Directory of Open Access Journals (Sweden)

    Lane Zhang

    2016-05-01

    Full Text Available Background/Aims: Stromal interacting molecule-1 (STIM1 aggregation and redistribution to plasma membrane to interact with Orai1 constitute the core mechanism of store-operated Ca2+ entry (SOCE. Previous study has revealed that calsequestrin-1 (CSQ1 regulates SOCE in HEK293 cells through interacting with STIM1 and inhibiting STIM1/Orai1 interaction. Here, we further investigate how CSQ1/STIM1 interaction affects SOCE. Methods: Using confocal microscopy, STIM1 aggregation and co-localizations with CSQ1 or Orai1 upon Ca2+ store depletion by thapsigargin were measured and quantified by Imaris software in HeLa cells transfected with different CSQ1 mutants. The interactions of CSQ1/STIM1 and STIM1/Orai1, and internal Ca2+ changes were detected by co-immunoprecipitation and Fura2, respectively. Results: Wt-CSQ1 overexpression significantly reduced STIM1 clustering in the perimembrane and cytosolic regions, whereas over-expression of a C-terminal amino acid 362-396 deletion mutant (C35 did not. Consistently, a significant depression of SOCE, increased CSQ1 monomerization and CSQ1/STIM1 interaction, and a reduced STIM1/Orai1 association were observed in wt-CSQ1 but not in C35-transfected cells. Additionally, mutant lacking C-terminal AA 388-396 deletion exerted weaker potency in inhibiting STIM1 aggregation and association with Orai1 than wt-CSQ1. Conclusions: Our results demonstrate that CSQ1 monomers suppress SOCE by interacting with STIM1 and attenuating STIM1 aggregation via its C-terminal amino acid 362-396.

  15. CMT-linked loss-of-function mutations in GDAP1 impair store-operated Ca(2+) entry-stimulated respiration.

    Science.gov (United States)

    González-Sánchez, Paloma; Pla-Martín, David; Martínez-Valero, Paula; Rueda, Carlos B; Calpena, Eduardo; Del Arco, Araceli; Palau, Francesc; Satrústegui, Jorgina

    2017-02-21

    GDAP1 is an outer mitochondrial membrane protein involved in Charcot-Marie-Tooth (CMT) disease. Lack of GDAP1 gives rise to altered mitochondrial networks and endoplasmic reticulum (ER)-mitochondrial interactions resulting in a decreased ER-Ca(2+) levels along with a defect on store-operated calcium entry (SOCE) related to a misallocation of mitochondria to subplasmalemmal sites. The defect on SOCE is mimicked by MCU silencing or mitochondrial depolarization, which prevent mitochondrial calcium uptake. Ca(2+) release from de ER and Ca(2+) inflow through SOCE in neuroblastoma cells result in a Ca(2+)-dependent upregulation of respiration which is blunted in GDAP1 silenced cells. Reduced SOCE in cells with CMT recessive missense mutations in the α-loop of GDAP1, but not dominant mutations, was associated with smaller SOCE-stimulated respiration. These cases of GDAP1 deficiency also resulted in a decreased ER-Ca(2+) levels which may have pathological implications. The results suggest that CMT neurons may be under energetic constraints upon stimulation by Ca(2+) mobilization agonists and point to a potential role of perturbed mitochondria-ER interaction related to energy metabolism in forms of CMT caused by some of the recessive or null mutations of GDAP1.

  16. CMT-linked loss-of-function mutations in GDAP1 impair store-operated Ca2+ entry-stimulated respiration

    Science.gov (United States)

    González-Sánchez, Paloma; Pla-Martín, David; Martínez-Valero, Paula; Rueda, Carlos B.; Calpena, Eduardo; del Arco, Araceli; Palau, Francesc; Satrústegui, Jorgina

    2017-01-01

    GDAP1 is an outer mitochondrial membrane protein involved in Charcot-Marie-Tooth (CMT) disease. Lack of GDAP1 gives rise to altered mitochondrial networks and endoplasmic reticulum (ER)-mitochondrial interactions resulting in a decreased ER-Ca2+ levels along with a defect on store-operated calcium entry (SOCE) related to a misallocation of mitochondria to subplasmalemmal sites. The defect on SOCE is mimicked by MCU silencing or mitochondrial depolarization, which prevent mitochondrial calcium uptake. Ca2+ release from de ER and Ca2+ inflow through SOCE in neuroblastoma cells result in a Ca2+-dependent upregulation of respiration which is blunted in GDAP1 silenced cells. Reduced SOCE in cells with CMT recessive missense mutations in the α-loop of GDAP1, but not dominant mutations, was associated with smaller SOCE-stimulated respiration. These cases of GDAP1 deficiency also resulted in a decreased ER-Ca2+ levels which may have pathological implications. The results suggest that CMT neurons may be under energetic constraints upon stimulation by Ca2+ mobilization agonists and point to a potential role of perturbed mitochondria-ER interaction related to energy metabolism in forms of CMT caused by some of the recessive or null mutations of GDAP1. PMID:28220846

  17. Calcium phosphate bioceramics induce mineralization modulated by proteins.

    Science.gov (United States)

    Wang, Kefeng; Leng, Yang; Lu, Xiong; Ren, Fuzeng

    2013-08-01

    Proteins play an important role in the process of biomineralization, which is considered the critical process of new bone formation. The calcium phosphate (Ca-P) mineralization happened on hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) and biphasic calcium phosphate (BCP) when proteins presented were investigated systematically. The results reveal that the presence of protein in the revised simulated body fluid (RSBF) did not alter the shape and crystal structure of the precipitated micro-crystals in the Ca-P layer formed on the three types of bioceramics. However, the morphology of the Ca-P precipitates was regulated but the structure of Ca-P crystal was unchanged in vivo. The presence of proteins always inhibits Ca-P mineralization in RSBF and the degree of inhibitory effect is concentration dependent. Furthermore, Protein presence can increase the possibility of HA precipitation in vitro and in vivo. The results obtained in this study can be helpful for better understanding the mechanism of biomineralization induced by the Ca-P bioceramics.

  18. G protein-induced trafficking of voltage-dependent calcium channels.

    Science.gov (United States)

    Tombler, Eugene; Cabanilla, Nory Jun; Carman, Paul; Permaul, Natasha; Hall, John J; Richman, Ryan W; Lee, Jessica; Rodriguez, Jennifer; Felsenfeld, Dan P; Hennigan, Robert F; Diversé-Pierluissi, María A

    2006-01-20

    Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.

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

    Science.gov (United States)

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

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

  20. Store-operated Ca{sup 2+} entry in rhabdomyosarcoma cells

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    Schmid, Evi, E-mail: Evi.Schmid@med.uni-tuebingen.de [Department of Pediatric Surgery & Pediatric Urology, Eberhard-Karls-University, Hoppe-Seyler Straße 3, 72076, Tuebingen (Germany); Stagno, Matias Julian [Department of Pediatric Surgery & Pediatric Urology, Eberhard-Karls-University, Hoppe-Seyler Straße 3, 72076, Tuebingen (Germany); Yan, Jing [Department of Cardiology & Vascular Medicine and Physiology, Eberhard-Karls-University, Gmelinstr.5/Otfried-Mueller-Str.10, 72076, Tuebingen (Germany); Stournaras, Christos [Department of Biochemistry, University of Crete Medical School, GR-71003, Heraklion, Crete (Greece); Lang, Florian [Department of Cardiology & Vascular Medicine and Physiology, Eberhard-Karls-University, Gmelinstr.5/Otfried-Mueller-Str.10, 72076, Tuebingen (Germany); Fuchs, Jörg [Department of Pediatric Surgery & Pediatric Urology, Eberhard-Karls-University, Hoppe-Seyler Straße 3, 72076, Tuebingen (Germany); Seitz, Guido [Department of Pediatric Surgery & Pediatric Urology, Eberhard-Karls-University, Hoppe-Seyler Straße 3, 72076, Tuebingen (Germany); Department of Pediatric Surgery, University Hospital Marburg, Marburg (Germany)

    2016-08-12

    Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, has an intrinsic or early-acquisition of resistance to chemo- and radiation therapy. Molecular determinants pivotal for RMS migration, metastatic invasion, cell proliferation, and survival are incompletely identified. Migration and cell proliferation were shown to correlate with cytosolic Ca{sup 2+} activity ([Ca{sup 2+}]{sub i}). Store-operated Ca{sup 2+}-entry (SOCE) that increases intracellular [Ca{sup 2+}] is accomplished by Orai1, a pore-forming ion channel unit, the expression of which is stimulated by the transcription factor NFκB. The present study explored the expression of Orai1 and its regulators STIM1 and NFκB in human rhabdomyosarcoma cell lines and analyzed their impact on cell proliferation and migration. For the study human rhabdomyosarcoma cell lines RD (embryonal) and RH30 (alveolar) were analyzed for Orai1, STIM1, and NFκB transcription by RT-PCR and their corresponding proteins in Western blot. [Ca{sup 2+}]{sub i} was detected via Fura-2 fluorescence and SOCE – resulting from [Ca{sup 2+}]{sub i} increase following store depletion with extracellular Ca{sup 2+} removal and inhibition of the sarcoendoplasmatic reticular Ca{sup 2+} ATPase – detected with thapsigargin. Cell migration was analyzed in transwell and mitotic cell death with the clonogenic assay. In summary, Orai1, STIM1, and NFκB are expressed in embryonal (RD) and alveolar (RH30) rhabdomyosarcoma. SOCE inhibitor BTP2, Orai1 inhibitor 2-APB, or NFκB inhibitor wogonin virtually abrogated (BTP2, 2-APB) or significantly reduced (wogonin) SOCE. Moreover, SOCE inhibitors 2-APB and BTP2 and wogonin significantly inhibited migration and proliferation of both, RD and RH30 cells. These results suggest that Orai1 signaling is involved in SOCE into rhabdomyosarcoma cells thus contributing to migration, invasion and proliferation. - Highlights: • Orai1, STIM1, and NFκB are expressed in RD and RH30 rhabdomyosarcoma

  1. Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium.

    Science.gov (United States)

    Ok, Seong-Ho; Kwon, Seong-Chun; Kang, Sebin; Choi, Mun-Jeoung; Sohn, Ju-Tae

    2014-12-01

    Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca(2+)]i). However, the mechanism responsible for the mepivacaine-evoked [Ca(2+)]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca(2+)]i increment in isolated rat aorta. Isometric tension was measured in isolated rat aorta without endothelium. In addition, fura-2 loaded aortic muscle strips were illuminated alternately (48 Hz) at two excitation wavelengths (340 and 380 nm). The ratio of F340 to F380 (F340/F380) was regarded as an amount of [Ca(2+)]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd(3+)), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca(2+)]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca(2+)]i increment. Mepivacaine produced vasoconstriction and increased [Ca(2+)]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca(2+)]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca(2+)]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca(2+)]i increase. Gd(3+) had no effect on either vasoconstriction or the [Ca(2+)]i increment evoked by mepivacaine. The mepivacaine-evoked [Ca(2+)]i increment, which contributes to mepivacaine-evoked contraction, appears to be mediated mainly by calcium influx and partially by calcium released from the sarcoplasmic reticulum.

  2. CREB modulates calcium signaling in cAMP-induced bone marrow stromal cells (BMSCs).

    Science.gov (United States)

    Zhang, Linxia; Liu, Li; Thompson, Ryan; Chan, Christina

    2014-10-01

    Calcium signaling has a versatile role in many important cellular functions. Despite its importance, regulation of calcium signaling in bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) has not been explored extensively. Our previous study revealed that cyclic adenosine monophosphate (cAMP) enabled BMSCs to generate calcium signal upon stimulation by dopamine, KCl and glutamate. Concurrently, cAMP transiently activated the transcription factor cAMP response element binding protein (CREB) in BMSCs. Activity of CREB can be modulated by the calcium/calmodulin-dependent kinase signaling pathway, however, whether the calcium signaling observed in cAMP-induced BMSCs requires CREB has not been investigated. In an effort to uncover the role of CREB in the generation of calcium signaling in response to modulators such as dopamine and KCl, we knocked down CREB activity in BMSCs. Our study indicated that BMSCs, but not its close relative fibroblasts, are responsive to dopamine and KCl after cAMP treatment. Calcium signal elicited by dopamine depends, in part, on calcium influx whereas that elicited by KCl depends completely on calcium influx. Knock-down of CREB activity significantly reduced or abolished the cAMP-induced calcium response, and reintroducing a constitutively active CREB partially restored the calcium response.

  3. Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential

    Directory of Open Access Journals (Sweden)

    Brinton Roberta

    2008-12-01

    Full Text Available Abstract Background Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APα; 5α-pregnan-3α-ol-20-one promotes neural progenitor proliferation in vitro in cultures of rodent hippocampal and human cortical neural progenitors, and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APα-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation. Methods In the present study, we investigated the effect of APα on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging. Results Results indicate that APα rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC50 of 110 ± 15 nM and a maximal response occurring at three days in vitro. The stereoisomers 3β-hydroxy-5α-hydroxy-pregnan-20-one, and 3β-hydroxy-5β-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APα-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La3+, or the L-type calcium channel blocker nifedipine. Furthermore, the GABAA receptor blockers bicuculline and picrotoxin abolished APα-induced intracellular calcium concentration rise. Conclusion Collectively, these data indicate that APα promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABAA receptor and L-type calcium channel. These data suggest that APα-induced

  4. Fluoride induced endoplasmic reticulum stress and calcium overload in ameloblasts.

    Science.gov (United States)

    Zhang, Ying; Zhang, KaiQiang; Ma, Lin; Gu, HeFeng; Li, Jian; Lei, Shuang

    2016-09-01

    The aim of the study was to evaluate the involvement of endoplasmic reticulum stress and intracellular calcium overload on the development of dental fluorosis. We cultured and exposed rat ameloblast HAT-7 cells to various concentrations of fluoride and measured apoptosis with flow cytometry and intracellular Ca2+ changes using confocal microscopy, investigated the protein levels of GRP78, calreticulin, XBP1 and CHOP by western blotting, and their transcriptional levels with RT-PCR. We also created an in vivo model of dental fluorosis by exposing animals to various concentrations of fluoride. Subsequently, thin dental tissue slices were analyzed with H&E staining, immunohistochemical staining, and transmission electron microscopy, TUNEL assay was also performed on dental tissue slices for assessment of apoptosis. High fluoride concentration was associated with decreased ameloblast proliferation, elevated ameloblast apoptosis, and increased intracellular Ca2+ in vitro. The translation and transcription of the proteins associated with endoplasmic reticulum stress were significantly elevated with high concentrations of fluoride. Based on immunohistochemical staining, these proteins were also highly expressed in animals exposed to high fluoride concentrations. Histologically, we found significant fluorosis-like changes in tissues from animals exposed to high fluoride concentrations. Transmission electron microscopy cytology indicated significant apoptotic changes in tissues exposed to high concentrations of fluoride. These results indicate that exposure to high levels of fluoride led to endoplasmic reticulum stress which induced apoptosis in cultured ameloblasts and in vivo rat model, suggesting an important role of calcium overload and endoplasmic reticulum stress triggered by high concentrations of fluoride in the development of dental fluorosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Enhanced store-operated Ca2+ influx and ORAI1 expression in ventricular fibroblasts from human failing heart

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    Gracious R. Ross

    2017-03-01

    Full Text Available Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF. The human ventricular fibroblast (hVF remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for intracellular free Ca2+ in fibrosis. Therefore, we determined the association of differences in cellular Ca2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control hearts. Histology of left ventricle sections (Masson trichrome confirmed excessive fibrosis in HF versus normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 h of culture compared with control [85.9±7.4 µg/106 cells vs 58.5±8.8 µg/106 cells, P<0.05; (Sircol™ assay]. However, collagen gene expressions (COL1A1 and COL1A2; RT-PCR were not different. Ca2+ imaging (fluo-3 of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca2+ release capacity (control 16±1.4% vs HF 17±1.1%; however, Ca2+ influx via store-operated Ca2+ entry/Ca2+ release-activated channels (SOCE/CRAC was significantly (P≤0.05 greater in HF-hVFs (47±3% compared with non-failing (35±5%. Immunoblotting for ICRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly (P<0.01 greater in HF (0.5±0.01 than control (0.4±0.01 hVFs. The increase in collagen secretion of HF versus control hVFs was eliminated by incubation of hVFs with YM58483 (10 µM, a selective ICRAC inhibitor, for 48 h (66.78±5.87 µg/106 cells vs 55.81±7.09 µg/106 cells, P=0.27. In conclusion, hVFs from HF have increased collagen secretion capacity versus non-failing hearts and this is related to increase in Ca2

  6. Effects of dietary calcium on Helicobacter pylori-induced gastritis in Mongolian gerbils.

    Science.gov (United States)

    Iimuro, Masaki; Nakamura, Shiro; Arakawa, Tetsuo; Wakabayashi, Keiji; Mutoh, Michihiro

    2013-09-01

    Helicobacter pylori (Hp) infection causes gastritis and is considered a gastric cancer risk factor. We have previously reported that codfish meal markedly enhanced Hp-induced gastritis in Mongolian gerbils. In the present study, we sought the responsible components in codfish meal. Codfish were divided into three parts (meat, viscera and 'other parts', including bone), and administered to Hp-infected gerbils. Subsequently, cod bone, sardine bone and prawn shell were tested, along with major calcium components, hydroxyapatite and calcium carbonate, in bone and shell, respectively. 'Other parts' and cod bone enhanced Hp-induced gastritis, as was observed for whole codfish. Similarly, sardine bone and prawn shell, as well as 0.22-0.88% hydroxyapatite and calcium carbonate, enhanced gastritis. In contrast, administration of a higher dose of the calcium compounds exerted protective effects. Intake of calcium compounds may contribute to enhancement of Hp-induced gastritis.

  7. Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.

    Science.gov (United States)

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

    2010-03-10

    Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of "Ca(2+) spikes" (i.e., [Ca(2+)](c) transients having a bandwidth of 10 approximately 100 seconds) in SCN neurons, but it is unclear if these SCN Ca(2+) spikes are related to the slow circadian rhythms. We addressed this issue based on a Ca(2+) indicator dye (fluo-4) and a protein Ca(2+) sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca(2+) spikes in 18% of rat SCN cells in acute brain slices, but the Ca(2+) spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca(2+) spike was barely observed (fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca(2+) spikes was increased to 13 approximately 14%. Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca(2+) spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca(2+) spiking activity is caused by the Ca(2+) chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca(2+)](c) in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca(2+) spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca(2+) spikes in the function of SCN.

  8. Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.

    Directory of Open Access Journals (Sweden)

    Jin Hee Hong

    Full Text Available BACKGROUND: Circadian rhythms in spontaneous action potential (AP firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN. Also reported is the existence of "Ca(2+ spikes" (i.e., [Ca(2+](c transients having a bandwidth of 10 approximately 100 seconds in SCN neurons, but it is unclear if these SCN Ca(2+ spikes are related to the slow circadian rhythms. METHODOLOGY/PRINCIPAL FINDINGS: We addressed this issue based on a Ca(2+ indicator dye (fluo-4 and a protein Ca(2+ sensor (yellow cameleon. Using fluo-4 AM dye, we found spontaneous Ca(2+ spikes in 18% of rat SCN cells in acute brain slices, but the Ca(2+ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca(2+ spike was barely observed (<3%. When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca(2+ spikes was increased to 13 approximately 14%. CONCLUSIONS/SIGNIFICANCE: Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca(2+ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca(2+ spiking activity is caused by the Ca(2+ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca(2+](c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca(2+ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca(2+ spikes in the function of SCN.

  9. Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels.

    Science.gov (United States)

    Arispe, Nelson; Diaz, Juan Carlos; Simakova, Olga; Pollard, Harvey B

    2008-02-19

    Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure planar phospholipid bilayers. These digitoxin channels are blocked by Al(3+) and La(3+) but not by Mg(2+) or the classical l-type calcium channel blocker, nitrendipine. In bilayers, we find that the chemistry of the lipid affects the kinetics of the digitoxin channel activity, but not the cation selectivity. Antibodies against digitoxin promptly neutralize digitoxin channels in both cells and bilayers. We propose that these digitoxin calcium channels may be part of the mechanism by which digitoxin and other active cardiac glycosides, such as digoxin, exert system-wide actions at and above the therapeutic concentration range.

  10. Mitochondrial response and calcium ion change in apoptotic insect cells induced by SfaMNPV

    Institute of Scientific and Technical Information of China (English)

    XIU Meihong; PENG Jianxin; HONG Huazhu

    2005-01-01

    Mitochondrial responses and changes of calcium ions in apoptotic insect SL-1 cells induced by Syngrapha falcifera multiple nuclear polyhedrosis virus (SfaMNPV) are reported in this paper. By using Rhodamine 123 as a fluorescent labeling probe, flow cytometry analysis and confocal laser scanning microscope observation we observed that the mitochondrial transmembrane potential (△Ψm) began to decrease in SL-1 cells at 4 h post infection and △Ψm reduced continuously with the extension of virus infection. Western blotting indicated that the Bcl-2 level in the mitochondria gradually declined and was down- regulated. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria, which indicated that cytochrome c was released from mitochondria into cytosol. These results suggest that mitochondrion-mediated apoptotic signal transduction pathway exists in apoptotic insect cell induced by SfaMNPV. Cytosolic free calcium ([Ca2+]i) concentration rapidly increased after SfaMNPV infection and the elevated calcium was tested to come partly from extracelllular calcium ion influx. Flow cytometry analysis indicated that the apoptosis in SL-1 cells was not influenced by established cytosolic calcium clamped conditions and the EGTA inhibiting calcium influx. Therefore, neither the elevation of cytosolic calcium ion nor extracellular calcium entry was the inducing factor of apoptosis, which hinted that the depletion of ER Ca2+ store contributed to SL-1 cell apoptosis induced by SfaMNPV.

  11. Differential dependence of store-operated and excitation-coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1.

    Science.gov (United States)

    Lyfenko, Alla D; Dirksen, Robert T

    2008-10-15

    In non-excitable cells, agonist-induced depletion of intracellular Ca(2+) stores triggers Ca(2+) influx via a process termed store-operated Ca(2+) entry (SOCE). In T-lymphocytes, stromal interaction molecule 1 (STIM1) acts as the intra-store Ca(2+) sensor and Orai1 functions as the Ca(2+)-permeable SOCE channel activated by STIM1 following store depletion. Two functionally distinct Ca(2+) entry pathways exist in skeletal muscle; one activated by store depletion (SOCE) and a second by sustained/repetitive depolarization that does not require store depletion (excitation-coupled Ca(2+) entry, ECCE). However, the role of STIM1 and Orai1 in coordinating SOCE and ECCE activity in skeletal muscle and whether these two Ca(2+) entry pathways represent distinct molecular entities or two different activation mechanisms of the same channel complex is unknown. Here we address these issues using siRNA-mediated STIM1 knockdown, dominant-negative Orai1, and permeation-defective Orai1 to determine the role of STIM1 and Orai1 in store-operated and excitation-coupled Ca(2+) entry in skeletal myotubes. SOCE and ECCE activity were quantified from both intracellular Ca(2+) measurements and Mn(2+) quench assays. We found that STIM1 siRNA reduced STIM1 protein by more than 90% and abolished SOCE activity, while expression of siRNA-resistant hSTIM1 fully restored SOCE. SOCE was also abolished by dominant-negative Orai1 (E106Q) and markedly reduced by expression of a permeation-defective Orai1 (E190Q). In contrast, ECCE was unaffected by STIM1 knockdown, E106Q expression or E190Q expression. These results are the first to demonstrate that SOCE in skeletal muscle requires both STIM1 and Orai1 and that SOCE and ECCE represent two distinct molecular entities.

  12. Pressure induced reactions amongst calcium aluminate hydrate phases

    KAUST Repository

    Moon, Ju-hyuk

    2011-06-01

    The compressibilities of two AFm phases (strätlingite and calcium hemicarboaluminate hydrate) and hydrogarnet were obtained up to 5 GPa by using synchrotron high-pressure X-ray powder diffraction with a diamond anvil cell. The AFm phases show abrupt volume contraction regardless of the molecular size of the pressure-transmitting media. This volume discontinuity could be associated to a structural transition or to the movement of the weakly bound interlayer water molecules in the AFm structure. The experimental results seem to indicate that the pressure-induced dehydration is the dominant mechanism especially with hygroscopic pressure medium. The Birch-Murnaghan equation of state was used to compute the bulk modulus of the minerals. Due to the discontinuity in the pressure-volume diagram, a two stage bulk modulus of each AFm phase was calculated. The abnormal volume compressibility for the AFm phases caused a significant change to their bulk modulus. The reliability of this experiment is verified by comparing the bulk modulus of hydrogarnet with previous studies. © 2011 Elsevier Ltd. All rights reserved.

  13. Calcium and gibberellin-induced elongation of lettuce hypocotyl sections.

    Science.gov (United States)

    Moll, C; Jones, R L

    1981-08-01

    The relationship between calcium ions and gibberellic acid (GA3)-induced growth in the excised hypocotyl of lettuce (Lactuca sativa L.) was investigated. The short-term kinetics of growth responses were measured using a linear displacement transducer. Test solutions were added either as drops to the filter paper on which the hypocotyl stood ("non-flow-past") or by switching solution flowing past the base of hypocotyl ("flow-past"), resulting in differences in growth behavior. Drops of CaCl2 added at a high concentration (10 mM) inhibited growth within a few minutes. This inhibition was reversed by ethylenediaminetetraacetic acid (EDTA). Drops of EDTA or ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid caused a rapid increase in growth rate. Growth induced by EDTA was not further promoted by GA3. A continuous H2O flow resulted in growth rates comparable to those in response to GA3. Addition of CaCl2 to the flow-past medium inhibited growth and this inhibition was reversed by a decrease in CaCl2 concentration. The growth rate was found to be a function of CaCl2 concentration. When a constant CaCl2 concentration was maintained by the flow-past medium, a shift in pH from 5.5 to 4.25 had no obvious effect on hypocotyl elongation. Gibberellic acid was found to reverse the inhibitory effect of CaCl2, causing an increase in growth rate similar to that found previously when GA3 was added to hypocotyls grown in H2O under non-flow-past conditions. We propose that gibberellin controls extension growth in lettuce hypocotyl sections by regulating the uptake of Ca(2+) by the hypocotyl cells.

  14. Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast.

    Science.gov (United States)

    Busti, Stefano; Mapelli, Valeria; Tripodi, Farida; Sanvito, Rossella; Magni, Fulvio; Coccetti, Paola; Rocchetti, Marcella; Nielsen, Jens; Alberghina, Lilia; Vanoni, Marco

    2016-06-16

    Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and metabolism. Accumulation of unfolded proteins within the calcium-depleted lumen of the endoplasmic reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections linking disturbance of ER calcium equilibrium to ER stress and UPR signaling are evolutionary conserved and highlight the crucial role of metabolism in modulating the effects induced by calcium shortage.

  15. Mefloquine-Induced Disruption of Calcium Homeostasis in Mammalian Cells Is Similar to That Induced by Ionomycin▿

    Science.gov (United States)

    Caridha, D.; Yourick, D.; Cabezas, M.; Wolf, L.; Hudson, T. H.; Dow, G. S.

    2008-01-01

    In previous studies, we have shown that mefloquine disrupts calcium homeostasis in neurons by depletion of endoplasmic reticulum (ER) stores, followed by an influx of external calcium across the plasma membrane. In this study, we explore two hypotheses concerning the mechanism(s) of action of mefloquine. First, we investigated the possibility that mefloquine activates non-N-methyl-d-aspartic acid receptors and the inositol phosphate 3 (IP3) signaling cascade leading to ER calcium release. Second, we compared the disruptive effects of mefloquine on calcium homeostasis to those of ionomycin in neuronal and nonneuronal cells. Ionomycin is known to discharge the ER calcium store (through an undefined mechanism), which induces capacitative calcium entry (CCE). In radioligand binding assays, mefloquine showed no affinity for the known binding sites of several glutamate receptor subtypes. The pattern of neuroprotection induced by a panel of glutamate receptor antagonists was dissimilar to that of mefloquine. Both mefloquine and ionomycin exhibited dose-related and qualitatively similar disruptions of calcium homeostasis in both neurons and macrophages. The influx of external calcium was blocked by the inhibitors of CCE in a dose-related fashion. Both mefloquine and ionomycin upregulated the IP3 pathway in a manner that we interpret to be secondary to CCE. Collectively, these data suggest that mefloquine does not activate glutamate receptors and that it disrupts calcium homeostasis in mammalian cells in a manner similar to that of ionomycin. PMID:17999964

  16. Neutrophils and the calcium-binding protein MRP-14 mediate carrageenan-induced antinociception in mice

    Directory of Open Access Journals (Sweden)

    Rosana L. Pagano

    2002-01-01

    Full Text Available Background: We have previously shown that the calcium-binding protein MRP-14 secreted by neutrophils mediates the antinociceptive response in an acute inflammatory model induced by the intraperitoneal injection of glycogen in mice.

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

    Science.gov (United States)

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

    2016-04-01

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

  18. Modeling of progesterone-induced intracellular calcium signaling in human spermatozoa.

    Science.gov (United States)

    Li, Long-Fei; Xiang, Cheng; Zhu, Ya-Bing; Qin, Kai-Rong

    2014-06-21

    Calcium ion is a secondary messenger of mammalian spermatozoa. The dynamic change of its concentration plays a vital role in the process of sperm motility, capacitation, acrosome and fertilization. Progesterone released by the cumulus cells, as a potent stimulator of fertilization, can activate the calcium channels on the plasma membrane, which in turn triggers the dynamic change of intracellular calcium concentration. In this paper, a mathematical model of calcium dynamic response in mammalian spermatozoa induced by progesterone is proposed and numerical simulation of the dynamic model is conducted. The results show that the dynamic response of calcium concentration predicted by the model is in accordance with experimental evidence. The proposed dynamic model can be used to explain the phenomena observed in the experiments and predict new phenomena to be revealed by experimental investigations, which will provide the basis to quantitatively investigate the fluid mechanics and biochemistry for the sperm motility induced by progesterone.

  19. Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium

    OpenAIRE

    Ok, Seong-Ho; Kwon, Seong-Chun; Kang, Sebin; Choi, Mun-Jeoung; Sohn, Ju-Tae

    2014-01-01

    Background Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca2+]i). However, the mechanism responsible for the mepivacaine-evoked [Ca2+]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca2+]i increment in isolated rat aorta. Methods Isometric tension was measured ...

  20. Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention.

    Science.gov (United States)

    Kristian, Tibor; Pivovarova, Natalia B; Fiskum, Gary; Andrews, S Brian

    2007-08-01

    Both isolated brain mitochondria and mitochondria in intact neurons are capable of accumulating large amounts of calcium, which leads to formation in the matrix of calcium- and phosphorus-rich precipitates, the chemical composition of which is largely unknown. Here, we have used inhibitors of the mitochondrial permeability transition (MPT) to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial morphology, precipitate composition, and precipitate retention. Using isolated rat brain (RBM) or liver mitochondria (RLM) Ca(2+)-loaded by continuous cation infusion, precipitate composition was measured in situ in parallel with Ca(2+) uptake and mitochondrial swelling. In RBM, the endogenous MPT inhibitors adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) increased mitochondrial Ca(2+) loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form varies with the conditions of loading. In both RBM and RLM, the presence of cyclosporine A in addition to ADP increased the Ca(2+) capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca(2+) is rapid but incomplete; significant residual calcium in the form of precipitates is retained in damaged mitochondria for prolonged periods.

  1. Calcium regulation of EGF-induced ERK5 activation: role of Lad1-MEKK2 interaction.

    Directory of Open Access Journals (Sweden)

    Zhong Yao

    Full Text Available The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1.

  2. Effects of calcium channel on 3-morpholinosydnonimine-induced rat hippocampal neuronal apoptosis

    Institute of Scientific and Technical Information of China (English)

    Quanzhong Chang; Shuling Zhang; Yuanyin Zheng; Lijuan Xu; Jinbao Yin; Shining Cai

    2011-01-01

    Previous studies have demonstrated that increased chloride channel activity plays a role in nitric oxide-induced neuronal apoptosis in the rat hippocampus.The present study investigated the effects of the broad-spectrum calcium channel blocker CdC12 on survival rate, percentage of apoptosis, and morphological changes in hippocampal neurons cultured in vitro, as well as the effects of calcium channels on neuronal apoptosis.The chloride channel blockers 4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulfonic acid (SITS) or 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) increased the survival rate of 3-morpholinosydnonimine (SIN-1)-treated neurons and suppressed SIN-1-induced neuronal apoptosis.The calcium channel blocker CdC12 did not increase the survival rate of neurons and did not affect SIN-1-induced apoptosis or SITS- or DIDS-suppressed neuronal apoptosis.Results demonstrated that calcium channels did not significantly affect neuronal apoptosis.

  3. The Role of Mitochondria in the Activation/Maintenance of SOCE: Store-Operated Ca(2+) Entry and Mitochondria.

    Science.gov (United States)

    Spät, András; Szanda, Gergö

    2017-01-01

    Mitochondria extensively modify virtually all cellular Ca(2+) transport processes, and store-operated Ca(2+) entry (SOCE) is no exception to this rule. The interaction between SOCE and mitochondria is complex and reciprocal, substantially altering and, ultimately, fine-tuning both capacitative Ca(2+) influx and mitochondrial function. Mitochondria, owing to their considerable Ca(2+) accumulation ability, extensively buffer the cytosolic Ca(2+) in their vicinity. In turn, the accumulated ion is released back into the neighboring cytosol during net Ca(2+) efflux. Since store depletion itself and the successive SOCE are both Ca(2+)-regulated phenomena, mitochondrial Ca(2+) handling may have wide-ranging effects on capacitative Ca(2+) influx at any given time. In addition, mitochondria may also produce or consume soluble factors known to affect store-operated channels. On the other hand, Ca(2+) entering the cell during SOCE is sensed by mitochondria, and the ensuing mitochondrial Ca(2+) uptake boosts mitochondrial energy metabolism and, if Ca(2+) overload occurs, may even lead to apoptosis or cell death. In several cell types, mitochondria seem to be sterically excluded from the confined space that forms between the plasma membrane (PM) and endoplasmic reticulum (ER) during SOCE. This implies that high-Ca(2+) microdomains comparable to those observed between the ER and mitochondria do not form here. In the following chapter, the above aspects of the many-sided SOCE-mitochondrion interplay will be discussed in greater detail.

  4. Caffeine-Induced Suppression of GABAergic Inhibition and Calcium-Independent Metaplasticity

    Directory of Open Access Journals (Sweden)

    Masako Isokawa

    2016-01-01

    Full Text Available GABAergic inhibition plays a critical role in the regulation of neuron excitability; thus, it is subject to modulations by many factors. Recent evidence suggests the elevation of intracellular calcium ([Ca2+]i and calcium-dependent signaling molecules underlie the modulations. Caffeine induces a release of calcium from intracellular stores. We tested whether caffeine modulated GABAergic transmission by increasing [Ca2+]i. A brief local puff-application of caffeine to hippocampal CA1 pyramidal cells transiently suppressed GABAergic inhibitory postsynaptic currents (IPSCs by 73.2 ± 6.98%. Time course of suppression and the subsequent recovery of IPSCs resembled DSI (depolarization-induced suppression of inhibition, mediated by endogenous cannabinoids that require a [Ca2+]i rise. However, unlike DSI, caffeine-induced suppression of IPSCs (CSI persisted in the absence of a [Ca2+]i rise. Intracellular applications of BAPTA and ryanodine (which blocks caffeine-induced calcium release from intracellular stores failed to prevent the generation of CSI. Surprisingly, ruthenium red, an inhibitor of multiple calcium permeable/release channels including those of stores, induced metaplasticity by amplifying the magnitude of CSI independently of calcium. This metaplasticity was accompanied with the generation of a large inward current. Although ionic basis of this inward current is undetermined, the present result demonstrates that caffeine has a robust Ca2+-independent inhibitory action on GABAergic inhibition and causes metaplasticity by opening plasma membrane channels.

  5. Ca2+ signals induced from calcium stores in pancreatic islet β cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In single rat pancreatic β cells,using fura-2 microfluorometry to measure [Ca2+]i response upon different stimuli,the ways of calcium regulation have been studied.When the extracellular calcium concentration was 2.5 mmol/L,either 60 mmol/L KCl,20 mmol/L D-glucose or 0.1 mmol/L tolbutamide induced increase in [Ca2+]i.Such increase in [Ca2+]i was absent when the same stimuli were applied under zero extracellular calcium.These results indicate that the increase of [Ca2+]i is induced by the activation of voltage-dependent calcium channels in β cells.The manifold forms of [Ca2+]i change induced by glucose imply that the effects of glucose are complex.5 mmol/L caffeine or 5 mmol/L MCh increase the [Ca2+]i ,which is independent of the external calcium,suggesting that [Ca2+]i can be regulated by Ca2+ release from not only the IP3-sensitive but also the ryanodine sensitive calcium stores in β cells.The latency of Ca responses for IP3 pathway (5 s) is faster than that for ryanodine pathway (30 s).It is concluded that there are multiple calcium stores in rat pancreatic β cells.

  6. Noise induced calcium oscillations in a cell exposed to electromagnetic fields.

    Science.gov (United States)

    Zhang, Yuhong; Zhao, Yongli; Chen, Yafei; Yuan, Changqing; Zhan, Yong

    2015-01-01

    The effects of noise on the calcium oscillations in a cell exposed to electromagnetic fields are described by a dynamic model. Noise is a very important factor to be considered in the dynamic research on the calcium oscillations in a cell exposed to electromagnetic fields. Some meaningful results have been obtained here based on the discussion. The results show that the pattern of intracellular calcium oscillations exposure to electromagnetic fields can be influenced by noise. Furthermore, the intracellular calcium oscillations exposure to electromagnetic fields can also be induced by noise. And the work has also studied the relationships between the voltage sensitive calcium channel's open probability and electromagnetic field. The result can provide new insights into constructive roles and potential applications of selecting appropriate electromagnetic field frequency during the research of biological effect of electromagnetic field.

  7. Effect of calcium chelators on heat coagulation and heat-induced changes of concentrated micellar casein solutions: The role of calcium-ion activity and micellar integrity

    NARCIS (Netherlands)

    Kort, de E.J.P.; Minor, M.; Snoeren, T.A.L.; Hooijdonk, van A.C.M.; Linden, van der E.

    2012-01-01

    There is general consensus that calcium chelators enhance heat stability in milk. However, they increase the heat stability to considerably different extents. For this reason, the effect of various calcium chelators on heat coagulation and heat-induced changes of concentrated micellar casein

  8. Effect of calcium chelators on heat coagulation and heat-induced changes of concentrated micellar casein solutions: The role of calcium-ion activity and micellar integrity

    NARCIS (Netherlands)

    Kort, de E.J.P.; Minor, M.; Snoeren, T.A.L.; Hooijdonk, van A.C.M.; Linden, van der E.

    2012-01-01

    There is general consensus that calcium chelators enhance heat stability in milk. However, they increase the heat stability to considerably different extents. For this reason, the effect of various calcium chelators on heat coagulation and heat-induced changes of concentrated micellar casein solutio

  9. Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry.

    Directory of Open Access Journals (Sweden)

    Se-Young Choi

    Full Text Available Polychlorinated biphenyls (PCBs are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca2+ signaling in PC12 cells. We found that ortho-substituted 2,2',6-trichlorinated biphenyl (PCB19 caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling.

  10. Depletion of H2S during obesity enhances store-operated Ca2+ entry in adipose tissue macrophages to increase cytokine production.

    Science.gov (United States)

    Velmurugan, Gopal V; Huang, Huiya; Sun, Hongbin; Candela, Joseph; Jaiswal, Mukesh K; Beaman, Kenneth D; Yamashita, Megumi; Prakriya, Murali; White, Carl

    2015-12-15

    The increased production of proinflammatory cytokines by adipose tissue macrophages (ATMs) contributes to chronic, low-level inflammation during obesity. We found that obesity in mice reduced the bioavailability of the gaseous signaling molecule hydrogen sulfide (H2S). Steady-state, intracellular concentrations of H2S were lower in ATMs isolated from mice with diet-induced obesity than in ATMs from lean mice. In addition, the intracellular concentration of H2S in the macrophage cell line RAW264.7 was reduced during an acute inflammatory response evoked by the microbial product lipopolysaccharide (LPS). Reduced intracellular concentrations of H2S led to increased Ca(2+) influx through the store-operated Ca(2+) entry (SOCE) pathway, which was prevented by the exogenous H2S donor GYY4137. Furthermore, GYY4137 inhibited the Orai3 channel, a key component of the SOCE machinery. The enhanced production of proinflammatory cytokines by RAW264.7 cells and ATMs from obese mice was reduced by exogenous H2S or by inhibition of SOCE. Together, these data suggest that the depletion of macrophage H2S that occurs during acute (LPS-induced) or chronic (obesity) inflammation increases SOCE through disinhibition of Orai3 and promotes the production of proinflammatory cytokines.

  11. Depletion of H2S during obesity enhances store-operated Ca2+ entry in adipose tissue macrophages to increase cytokine production

    Science.gov (United States)

    Velmurugan, Gopal V.; Huang, Huiya; Sun, Hongbin; Candela, Joseph; Jaiswal, Mukesh K.; Beaman, Kenneth D.; Yamashita, Megumi; Prakriya, Murali; White, Carl

    2017-01-01

    The increased production of proinflammatory cytokines by adipose tissue macrophages (ATMs) contributes to chronic, low-level inflammation during obesity. We found that obesity in mice reduced the bioavailability of the gaseous signaling molecule hydrogen sulfide (H2S). Steady-state, intracellular concentrations of H2S were lower in ATMs isolated from mice with diet-induced obesity than in ATMs from lean mice. In addition, the intracellular concentration of H2S in the macrophage cell line RAW264.7 was reduced during an acute inflammatory response evoked by the microbial product lipopolysaccharide (LPS). Reduced intracellular concentrations of H2S led to increased Ca2+ influx through the store-operated Ca2+ entry (SOCE) pathway, which was prevented by the exogenous H2S donor GYY4137. Furthermore, GYY4137 inhibited the Orai3 channel, a key component of the SOCE machinery. The enhanced production of proinflammatory cytokines by RAW264.7 cells and ATMs from obese mice was reduced by exogenous H2S or by inhibition of SOCE. Together, these data suggest that the depletion of macrophage H2S that occurs during acute (LPS-induced) or chronic (obesity) inflammation increases SOCE through disinhibition of Orai3 and promotes the production of proinflammatory cytokines. PMID:26671149

  12. Wnt-induced calcium signaling mediates axon growth and guidance in the developing corpus callosum.

    Science.gov (United States)

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

    2012-01-10

    Wnt5a gradients guide callosal axons by repulsion through Ryk receptors in vivo. We recently found that Wnt5a repels cortical axons and promotes axon outgrowth through calcium signaling in vitro. Here, using cortical slices, we show that Wnt5a signals through Ryk to guide and promote outgrowth of callosal axons after they cross the midline. Calcium transient frequencies in callosal growth cones positively correlate with axon outgrowth rates in vitro. In cortical slices, calcium release through inositol 1,4,5-trisphosphate (IP(3)) receptors and calcium entry through transient receptor potential channels modulate axon growth and guidance. Knocking down Ryk inhibits calcium signaling in cortical axons, reduces rates of axon outgrowth subsequent to midline crossing, and causes axon guidance defects. Calcium- and calmodulin-dependent protein kinase II (CaMKII) is required downstream of Wnt-induced calcium signaling for postcrossing callosal axon growth and guidance. Taken together, these results suggest that growth and guidance of postcrossing callosal axons by Wnt-Ryk-calcium signaling involves axon repulsion through CaMKII.

  13. Inhibitory action of oestrogen on calcium-induced mitosis in rat bone marrow and thymus.

    Science.gov (United States)

    Smith, G R; Gurson, M L; Riddell, A J; Perris, A D

    1975-04-01

    In the male rat injections of CaCl-2 and MgCl-2 stimulated mitosis in bone marrow and thymus tissue. The magnesium salt was also mitogenic in the normal female, but calcium only exerted its mitogenic effect after ovariectomy. Oestradiol, but not progesterone replacement therpy abolished calcium-induced mitosis in the ovariectomized rat. The inability of calcium to stimulate cell division was also apparent in the thyroparathyroidectomized female rat, suggesting the oestradiol blockage did not operate via some indirect action on the calcium homeostatic hormones calcitonin or parathyroid hormone. When thymic lymphocytes derived from male or female rats were isolated and maintained in suspension, increased calcium or magnesium concentrations in the culture medium stimulated the entry of cells into mitosis. Addition of oestradiol to the culture medium abolished the mitogenic effect of increased calcium levels, but had no effect on magnesium-induced proliferation. These experiments suggested that oestradiol might act at the cell surface to prevent the influx of calcium but not magnesium ions into the interior of the cell and thus to block the sequence of biochemical events which led to the initiation of DNA synthesis and culminate in mitosis.

  14. [Calcium polystyrene sulfonate induced colonic necrosis in patient with chronic kidney disease].

    Science.gov (United States)

    Lee, Sung Hoa; Kim, Sung Jung; Kim, Go Eun; Lee, Woo Jin; Hong, Won Ki; Baik, Gwang Ho; Choi, Young Hee; Kim, Dong Joon

    2010-04-01

    A 63-year-old woman was admitted due to right upper quadrant abdominal pain. She was going through hemodialysis due to end stage renal disease and taking calcium polystyrene sulfonate orally and rectally due to hyperkalemia. Colonoscopy showed a circular ulcerative mass on the proximal ascending colon. Biopsy specimen from the mass showed inflammation and necrotic debris. It also revealed basophilic angulated crystals which were adherent to the ulcer bed and normal mucosa. These crystals were morphologically consistent with calcium polystyrene sulfonate. She was diagnosed with calcium polystyrene phosphate induced colonic necrosis and improved with conservative treatment.

  15. Calcium channel antagonists increase morphine-induced analgesia and antagonize morphine tolerance.

    Science.gov (United States)

    Contreras, E; Tamayo, L; Amigo, M

    1988-04-13

    The influence of calcium channel blockers on morphine-induced analgesia and on tolerance to the chronic administration of the opiate was investigated in mice. The effects of a test dose of morphine were significantly increased by the administration of diltiazem, flunarizine, nicardipine and verapamil. In contrast, nifedipine induced an antagonistic effect. The calcium channel antagonists did not change the reaction time to thermal stimulation in mice (hot plate test). The administration of nifedipine, flunarizine and verapamil reduced the intensity of the tolerance induced by a single dose of morphine administered in a slow release preparation. Diltiazem induced a non-significant decrease of the process. The present results are in accordance with the known interaction of acute and chronic morphine administration with the intracellular calcium concentration in neurones of the central nervous system.

  16. Plasma cytokine concentration changes induced by the antitumor agents dipterinyl calcium pentahydrate (DCP) and related calcium pterins.

    Science.gov (United States)

    Moheno, Phillip; Pfleiderer, Wolfgang; Fuchs, Dietmar

    2009-01-01

    Analysis of plasma cytokine concentration changes determined that oral dosing with the antitumor agent (1:4 mol:mol) calcium pterin (CaPterin) increased plasma IL-10, decreased plasma IL-6, and decreased plasma IFN-gamma concentrations in nude mice with MDA-MB-231 xenograph tumors [Moheno, P., Pfleiderer, W., Dipasquale, A.G., Rheingold, A.L., Fuchs, D., 2008. Cytokine and IDO metabolite changes effected by calcium pterin during inhibition of MDA-MB-231 xenograph tumors in nude mice. Int. J. Pharm. 355, 238-248]. A further analysis, reported here, of plasma cytokine concentration changes in nude mice with the same tumor xenographs treated with dipterinyl calcium pentahydrate (DCP), (1:2 mol:mol) calcium pterin, and CaCl(2).2H(2)O has been carried out. The measured cytokines included: IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-gamma, and TNF-alpha. The major preliminary findings from the analyses of these data are that (1) the overall relative tumor volumes for the treatments correlated significantly with a full study antitumor plasma cytokine pattern (fsAPCP), a composite measure consisting of decreased plasma IL-6 and increased IL-4 concentrations, and (2) DCP induces a significant threshold antitumor response strongly correlated to a derived DCP antitumor plasma cytokine pattern (DCP/APCP) consisting of plasma IL-12, IL-6, and IL-4 concentration changes. This DCP/APCP composite measure identifies plasma IL-12 concentration increases, plasma IL-6 concentration decreases, and plasma IL-4 concentration increases correlated to relative tumor volume decreases caused by DCP dosing. The finding that the novel calcium pterins and CaCl(2).2H(2)O treatments decrease plasma IL-6 concentrations corroborates the previous finding that CaPterin dosing decreases plasma IL-6 concentrations in this mouse/tumor system [Moheno, P., Pfleiderer, W., Dipasquale, A.G., Rheingold, A.L., Fuchs, D., 2008. Cytokine and IDO metabolite changes effected by calcium pterin during inhibition

  17. [Effect of calcium on medium alkalinization induced by salicylic acid in Salvia miltiorrhiza suspension cultures].

    Science.gov (United States)

    Liu, Liancheng; Wang, Cong; Dong, Juan'e; Su, Hui; Zhuo, Zequn; Xue, Yaxin

    2013-07-01

    We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism.

  18. Histamine release induced from rat mast cells by the ionophore A23187 in the absence of extracellular calcium

    DEFF Research Database (Denmark)

    Johansen, Torben

    1980-01-01

    Isolated rat mast cells were used to study whether ionophore A23187 could induce histamine release by mobilizing cellular calcium. The histamine release was a slow process which was completed after about 20 min incubation with A23187. The A23187-induced histamine release was inhibited after...... incubation of the cells with EDTA for 1 h in a 37 degrees C water bath in calcium-free medium. Reintroduction of calcium in excess of EDTA induced the release of histamine. The observations suggest that A23187 can induce histamine release by mobilizing a cellular pool of calcium....

  19. The study of serum calcium and serum magnesium in pregnancy induced hypertension and normal pregnancy

    Directory of Open Access Journals (Sweden)

    Jagannath Pairu

    2015-02-01

    Full Text Available Background: Preeclampsia along with its complications is one of the major causes of maternal and fetal mortality and morbidity. Association of calcium and magnesium with pregnancy induced hypertension is known since decades. Evidence of decreased serum calcium and decreased serum magnesium has been observed in patients with pregnancy induced hypertension and has been implicated in the etiopathogenesis of preeclampsia. Methods: The present study was undertaken in 100 pregnant women. Data for the study was collected from 50 normotensive pregnant women with more than 20 weeks of gestational age (control group and 50 pregnancy induced hypertension patients (study group attending for the antenatal care in department of obstetrics and gynaecology in Vanivilas hospital, Bowring and Lady Curzon hospital attached to Bangalore medical college and research institute. Cases and controls were matched. Serum calcium and serum magnesium levels were estimated by spectrophotometry method. Results: The mean serum calcium is significantly lower in pregnancy induced hypertension group (8.15 +/- 0.37 mg/dl compared to normal pregnancy (9.16 +/- 0.82 mg/dl. The mean serum magnesium is lower in pregnancy induced hypertension group (1.78 +/- 0.70 mEq/L than normal pregnancy (2.08 +/- 0.46 mEq/L which is moderately significant. Conclusions: The serum calcium and serum magnesium levels are decreased in pregnancy induced hypertension patients compared to normotensive normal pregnant women, suggesting the possible role of calcium and magnesium in etiopathophysiology of pregnancy induced hypertension. [Int J Reprod Contracept Obstet Gynecol 2015; 4(1.000: 30-34

  20. Polyamine triggering of exocytosis in Paramecium involves an extracellular Ca(2+)/(polyvalent cation)-sensing receptor, subplasmalemmal Ca-store mobilization and store-operated Ca(2+)-influx via unspecific cation channels.

    Science.gov (United States)

    Klauke, N; Blanchard, M; Plattner, H

    2000-03-15

    The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca(2+)] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca(2+) signals also occur when AED is applied in presence of the fast Ca(2+) chelator, BAPTA. (ii) Extracellular La(3+) application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca(2+)](i) transient (while injected La(3+) causes a sustained fluorescence signal). (iii) Simply increasing [Ca(2+)](o) causes a similar rapid, short-lived [Ca(2+)](i) transient. All these phenomena, (i-iii), are compatible with activation of an extracellular "Ca(2+)/(polyvalent cation)-sensing receptor" known from some higher eukaryotic systems, where this sensor (responding to Ca(2+), La(3+) and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores ("alveolar sacs") are physically linked to the cell membrane and they can also be activated by the Ca(2+) releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca(2+) signal also in absence of Ca(2+)(o) we largely exclude a "Ca(2+)-induced Ca(2+) release" (CICR) mechanism. Our finding of increased cortical Ca(2+) signals after store depletion and re-addition of extracellular Ca(2+) can be explained by a "store-operated Ca(2+) influx" (SOC), i.e., a Ca(2+) influx superimposing store activation. AED stimulation in presence of Mn(2+)(o) causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca(2+) influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca(2+)/polyamine-sensing receptor, (ii) release of Ca(2+) from

  1. Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells.

    Science.gov (United States)

    Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S

    2016-04-15

    The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.

  2. Alpha hemolysin induces an increase of erythrocytes calcium: a FLIM 2-photon phasor analysis approach.

    Directory of Open Access Journals (Sweden)

    Susana Sanchez

    Full Text Available α-Hemolysin (HlyA from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin, a group of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin cause the lysis of several cells such as erythrocytes, granulocytes, monocytes, endothelial and renal epithelial cells of different species. At low concentrations it induces the production of cytokines and apoptosis. Since many of the subcytolytic effects in other cells have been reported to be triggered by the increase of intracellular calcium, we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA. Calcium concentration was monitored using the calcium indicator Green 1, 2-photon excitation, and fluorescence lifetime imaging microscopy (FLIM. Data were analyzed using the phasor representation. In this report, we present evidence that, at sublytic concentrations, HlyA induces an increase of calcium concentration in rabbit erythrocytes in the first 10 s. Results are discussed in relation to the difficulties of measuring calcium concentrations in erythrocytes where hemoglobin is present, the contribution of the background and the heterogeneity of the response observed in individual cells.

  3. 5-Hydroxytryptamino-induced calcium sparks in cultured rat stomach fundus smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Xiaoling; (张小玲); YAN; Hongtao; (阎宏涛); YAN; Yang; (闫炀)

    2003-01-01

    With a new fluorescence probe of Ca2+, STDIn-AM, 5-hydroxytryptamino (5-HT)-induced spontaneous calcium release events (calcium sparks) in cultured rat stomach fundus smooth muscle cells (SFSMC) are investigated by laser scanning confocal microscope. The mechanisms of initiation of Ca2+ sparks, propagating Ca2+ waves and their relation to E-C coupling are discussed. After the extracellular [Ca2+] is increased to 10 mmol/L, addition of 5-HT causes hot spots throughout the cytoplasm, which is brighter near the plasmalemma. The amplitude of the event is at least two times greater than the standard deviation of fluorescence intensity fluctuations measured in the neighboring region and the duration of the Ca2+ signal is over 100 ms. The results suggest that 5-HT acts by the way of 5-HT2 receptors on SFSMC, then through 5-HT2 receptors couples IP3/Ca2+ and DG/PKC double signal transduction pathways to cause Ca2+ release from intracellular Ca2+ stores and followed Ca2+ influx possibly through calcium release-activated calcium influx. The acceptor of activated 5-HT2 can also cause membrane depolarization, which then stimulates the L-type Ca2+ channels leading to Ca2+ influx. Thenthe local Ca2+ entry mentioned above activates ryanodine-sensitive Ca2+ releasechannels (RyR) on sarcoplasmic reticulum (SR) to cause local Ca2+ release events (Ca2+ sparks) through calcium-induced calcium release (CICR).

  4. Preserved frontal lobe oxygenation following calcium chloride for treatment of anesthesia-induced hypotension

    DEFF Research Database (Denmark)

    Kitchen, Carl-Christian; Nissen, Peter; Secher, Niels H

    2014-01-01

    Vasopressor agents may affect cerebral oxygenation (rScO2) as determined by near-infrared spectroscopy on the forehead. This case series evaluated the effect of calcium chloride vs. α and β-adrenergic receptor agonists on rScO2 in patients (n = 47) undergoing surgery during i.v. anesthesia. Mean ...... to treat anesthesia-induced hypotension tissue oxygenation is reduced while the use of β-adrenergic agonists and calcium chloride preserve tissue oxygenation......./min), noradrenaline (3.8 ± 1.2 vs. 3.7 ± 0.7 l/min), or calcium chloride (4.0 ± 1.4 vs. 4.1 ± 1.5 l/min). Following administration of β-adrenergic agents and calcium chloride rScO2 was preserved while after administration of α-adrenergic drugs rScO2 was reduced by app. 2% (P

  5. Expanding the neuron's calcium signaling repertoire: intracellular calcium release via voltage-induced PLC and IP3R activation.

    Directory of Open Access Journals (Sweden)

    Stefanie Ryglewski

    2007-04-01

    Full Text Available Neuronal calcium acts as a charge carrier during information processing and as a ubiquitous intracellular messenger. Calcium signals are fundamental to numerous aspects of neuronal development and plasticity. Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert. This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons. These neurons possess a membrane voltage sensor that, independent of calcium influx, causes G-protein activation, which subsequently leads to calcium release from intracellular stores via phospholipase C and inositol 1,4,5-trisphosphate receptor activation. This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels.

  6. Effects of extracellular calcium and sodium on depolarization-induced automaticity in guinea pig papillary muscle.

    Science.gov (United States)

    Katzung, B G

    1975-07-01

    Regenerative discharge of action potentials is induced in mammalian papillary muscles by passage of small depolarizing currents. In this paper, the effects of various extracellular calcium and sodium concentrations and of tetrodotoxin on this phenomenon were studied in guinea pig papillary muscles in a sucrose gap chamber. Phase 4 diastolic depolarization was found to be associated with an increase in membrane resistance. The slope of phase 4 depolarization was decreased by reductions in extracellular calcium or sodium concentration. The range of maximum diastolic potentials and the thresholds from which regenerative potentials arose were reduced, especially at the positive limit of potentials, by a reduction in either ion. It was concluded that both calcium and sodium influence diastolic depolarization and participate in the regenerative action potentials of depolarization-induced ventricular automaticity.

  7. Time resolved laser-induced breakdown spectroscopy for calcium concentration detection in water

    Institute of Scientific and Technical Information of China (English)

    WU Jiang-lai; LU Yuan; Li Ying; CHENG Kai; GUO Jin-jia; ZHENG Rong-er

    2011-01-01

    @@ The laser induced breakdown spectroscopy (LIBS) is an element analysis technique with the advantages of real time detection, simultaneous multi-element identification, and in-situ and stand-off capacities.To evaluate its potential of ocean applications, in this paper, the time resolved laser-induced breakdown spectroscopy for calcium concentration detection in water is investigated.With the optimum experimental parameters, the plasma emission lifetime is determined to be about 500 ns with 532 nm laser excitation, and 1000 ns with 1064 nm laser excitation.The lowest detection concentration of 50ppm is achieved for calcium detection in CaC12 water solution using the 532 nm LIBS.Even better detection sensitivity is achieved using the 1064 nm LIBS, and the resulted lowest detection concentration of calcium is 25 ppm.The results suggest that it is feasible to develop LIBS as an on-line sensor for metal element monitoring in the sea.

  8. Calcium-induced patterns of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L. and Albizia julibrissin Durazz.

    Science.gov (United States)

    Borchert, R

    1985-08-01

    For experimental induction of crystal cells (=crystal idioblasts) containing calcium-oxalate crystals, the lower epidermis was peeled from seedling leaflets of Gleditsia triacanthos L., exposing the crystal-free mesophyll and minor veins to the experimental solutions on which leaflets were floated for up to 10 d under continous light. On 0.3-2.0 mM Ca-acetate, increasing numbers of crystals, appearing 96 h after peeling, were induced. The pattern of crystal distribution changed with Ca(2+)-concentration ([Ca(2+)]): at low [Ca(2+)], crystals formed only in the non-green bundlesheath cells surrounding the veins, believed to have a relatively low Ca(2+)-extrusion capacity; at higher [Ca(2+)], crystals developed in up to 90% of the mesophyll cells, and at supraoptimal [Ca(2+)], large extracellular crystals formed on the tissue surface. By sequential treatments with solutions of different [Ca(2+)], the following three phases were identified in the induction of crystal cells: (1) during the initial 24-h period (adaptive aging), Ca(2+) is not required and crystal induction is not possible; (2) during the following 48 h (induction period), exposure to 1-2 mM Ca-acetate induces the differentiation of mesophyll cells into crystal cells; (3) crystal growth begins 72 h after the start of induction. In intact leaflets of Albizia julibrissin Durazz., calcium-oxalate crystals are found exclusively in the bundle-sheath cells of the veins, but crystals were induced in the mesophyll of peeled leaflets floating on 1 mM Ca-acetate. Exposure to inductive [Ca(2+)] will thus trigger the differentiation of mature leaf cells into crystal cells; the spatial distribution of crystals is determined by the external [Ca(2+)] and by the structural and functional properties of the cells in the tissue.

  9. Fibroblast growth factor-23 negates 1,25(OH)2D3-induced intestinal calcium transport by reducing the transcellular and paracellular calcium fluxes.

    Science.gov (United States)

    Khuituan, Pissared; Wongdee, Kannikar; Jantarajit, Walailuk; Suntornsaratoon, Panan; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

    2013-08-01

    The calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has been known to stimulate intestinal calcium transport via both transcellular and paracellular pathways. Recently, we reported that the 1,25(OH)2D3-enhanced calcium transport in the mouse duodenum could be abolished by fibroblast growth factor (FGF)-23, but the targeted calcium transport pathway has been elusive. Herein, the 1,25(OH)2D3-enhanced calcium transport was markedly inhibited by FGF-23 and inhibitors of the basolateral calcium transporters, NCX1 and PMCA1b, suggesting the negative effect of FGF-23 on the transcellular calcium transport. Similar results could be observed in the intestinal epithelium-like Caco-2 monolayer. Although the Arrhenius plot indicated that FGF-23 decreased the potential barrier (e.g., activation energy) of the paracellular calcium movement, FGF-23 was found to modestly decrease the 1,25(OH)2D3-enhanced paracellular calcium transport and calcium permeability. Moreover, FGF-23 affected the 1,25(OH)2D3-induced change in duodenal water permeability as determined by tritiated water, but both 1,25(OH)2D3 and FGF-23 were without effects on the transepithelial fluxes of paracellular markers, (3)H-mannitol and (14)C-polyethylene glycol. It could be concluded that FGF-23 diminished the 1,25(OH)2D3-enhanced calcium absorption through the transcellular and paracellular pathways. Our findings have thus corroborated the presence of a bone-kidney-intestinal axis of FGF-23/vitamin D system in the regulation of calcium homeostasis.

  10. Electrically Induced Calcium Handling in Cardiac Progenitor Cells

    Science.gov (United States)

    Wagner, Mary B.

    2016-01-01

    For nearly a century, the heart was viewed as a terminally differentiated organ until the discovery of a resident population of cardiac stem cells known as cardiac progenitor cells (CPCs). It has been shown that the regenerative capacity of CPCs can be enhanced by ex vivo modification. Preconditioning CPCs could provide drastic improvements in cardiac structure and function; however, a systematic approach to determining a mechanistic basis for these modifications founded on the physiology of CPCs is lacking. We have identified a novel property of CPCs to respond to electrical stimulation by initiating intracellular Ca2+ oscillations. We used confocal microscopy and intracellular calcium imaging to determine the spatiotemporal properties of the Ca2+ signal and the key proteins involved in this process using pharmacological inhibition and confocal Ca2+ imaging. Our results provide valuable insights into mechanisms to enhance the therapeutic potential in stem cells and further our understanding of human CPC physiology.

  11. Dihydropyridine type calcium channel blocker-induced turbid dialysate in patients undergoing peritoneal dialysis.

    Science.gov (United States)

    Yoshimoto, K; Saima, S; Nakamura, Y; Nakayama, M; Kubo, H; Kawaguchi, Y; Nishitani, H; Nakamura, Y; Yasui, A; Yokoyama, K; Kuriyama, S; Shirai, D; Kugiyama, A; Hayano, K; Fukui, H; Horigome, I; Amagasaki, Y; Tsubakihara, Y; Kamekawa, T; Ando, R; Tomura, S; Okamoto, R; Miwa, S; Koyama, T; Echizen, H

    1998-08-01

    We previously reported that manidipine, a new dihydropyridine type calcium channel blocker, produced chylous peritoneal dialysate being visually indistinguishable from infective peritonitis in 5 patients undergoing continuous ambulatory peritoneal dialysis (CAPD) [Yoshimoto et al. 1993]. To study whether such an adverse drug reaction would also be elicited by other commonly prescribed calcium channel blockers in CAPD patients, we have conducted postal inquiry to 15 collaborating hospitals and an institutional survey in International Medical Center of Japan as to the possible occurrence of calcium channel blocker-associated non-infective, turbid peritoneal dialysate in CAPD patients. Our diagnostic criteria for drug-induced turbidity of dialysate as a) it developed within 48 h after the administration of a newly introduced calcium channel blocker to the therapeutic regimen, b) absence of clinical symptoms of peritoneal inflammation (i.e., pyrexia, abdominal pain, nausea or vomiting), c) the fluid containing normal leukocyte counts and being negative for bacterial and fungal culture of the fluid, and d) it disappeared shortly after the withdrawal of the assumed causative agent. Results showed that 19 out of 251 CAPD patients given one of the calcium channel blockers developed non-infective turbid peritoneal dialysis that fulfilled all the above criteria. Four calcium channel blockers were suspected to be associated with the events: benidipine [2 out of 2 (100%) patients given the drug], manidipine [15 out of 36 (42%) patients], nisoldipine [1 out of 11 (9%) patients] and nifedipine [1 out of 159 (0.6%)] in descending order of frequency. None of the patients who received nicardipine, nilvadipine, nitrendipine, barnidipine and diltiazem (25, 7, 2, 1 and 8 patients, respectively) exhibited turbid dialysate. In conclusion, we consider that certain dihydropyridine type calcium channel blockers would cause turbid peritoneal dialysate being similar to that observed in

  12. Calcium acetate induces calcium uptake and formation of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L.

    Science.gov (United States)

    Borchert, R

    1986-09-01

    During treatment of isolated, peeled leaflets of Gleditsia triacanthos with 0.5-2 mM [(45)Ca]acetate, saturation of the cell-wall free space with Ca(2+) occurred within 10 min and was followed by a period of 6-10 h during which there was no significant Ca-uptake into the protoplast, but apoplastic Ca(2+) was periodically released into the medium. Later, Ca(2+) was absorbed for 3-4 d at rates of up to 2.2 μmol Ca(2+)·h(-1)·(g FW)(-1) to final concentrations of 350 μmol Ca(2+)· (g FW)(-1). The distribution of absorbed Ca(2+) between cell wall, vacuole and Ca-oxalate crystals was determined during Ca-uptake. Wheras intact, cut leaflets deposited absorbed Ca(2+) as Ca-oxalate in the crystal cells, peeled leaflets lacking crystal cells accumulated at least 40-50 μmol·(g FW)(-1) soluble Ca(2+) before the absorbed Ca(2+) was precipitated as Ca-oxalate. These observations indicate that the mechanisms for the continuous uptake of Ca(2+), the synthesis of oxalate and the precipitation of Ca(2+) as Ca-oxalate are operational in the crystal cells of intact leaflets, but not in the mesophyll cells of peeled leaflets where they must be induced by exposure to Ca(2+). The precipitation of absorbed Ca(2+) as Ca-oxalate by the crystal cells of isolated Gleditsia leaflets illustrates the role of these cells in the excretion of surplus Ca(2+) which enters normal, attached leaves with the transpiration stream.In addition to acetate, only Ca-lactate and Ca-carbonate lead to Ca-uptake, but at rates well below those observed with Ca-acetate. Other small organic anions (citrate, glycolate, glyoxalate, malate) and inorganic anions (chloride, nitrate, sulfate) did not permit Ca-uptake. Acetate-(14)C was rapidly absorbed during Ca-uptake, but less than 20% was incorporated into Ca-oxalate; the rest remained mostly in the soluble fraction or was metabolized to CO2. Acetate, as a permeable weak acid, may enable rapid Ca-uptake by stimulating proton extrusion at the plasmalemma and by

  13. Spermidine-Induced Improvement of Reconsolidation of Memory Involves Calcium-Dependent Protein Kinase in Rats

    Science.gov (United States)

    Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello

    2016-01-01

    In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus.…

  14. Formation of ring calcium oxalate patterns induced by domains in DPPC Langmuir-Blodgett films

    Institute of Scientific and Technical Information of China (English)

    Yi Ming Liu; Sui Ping Deng; Hui Zheng; Jian Ming Ouyang

    2007-01-01

    The ring patterns of calcium oxalate crystals were induced by domains in Langmuir-Blodgett (LB) films of dipalmitoylpho-sphatidylcholine (DPPC). The result was explained by the defects at the ring boundaries of liquid condensed (LC) and liquid expanded (LE) phases of LB film. These boundaries could provide less free energy and much more nucleating sites for COM crystals.

  15. Multiple Modes of Calcium-Induced Calcium Release in Sympathetic Neurons II

    Science.gov (United States)

    Hongpaisan, Jarin; Pivovarova, Natalia B.; Colegrove, Stephen L.; Leapman, Richard D.; Friel, David D.; Andrews, S. Brian

    2001-01-01

    CICR from an intracellular store, here directly characterized as the ER, usually refers to net Ca2+ release that amplifies evoked elevations in cytosolic free calcium ([Ca2+]i). However, the companion paper (Albrecht, M.A., S.L. Colegrove, J. Hongpaisan, N.B. Pivovarova, S.B. Andrews, and D.D. Friel. 2001. J. Gen. Physiol. 118:83–100) shows that in sympathetic neurons, small [Ca2+]i elevations evoked by weak depolarization stimulate ER Ca accumulation, but at a rate attenuated by activation of a ryanodine-sensitive CICR pathway. Here, we have measured depolarization-evoked changes in total ER Ca concentration ([Ca]ER) as a function of [Ca2+]i, and found that progressively larger [Ca2+]i elevations cause a graded transition from ER Ca accumulation to net release, consistent with the expression of multiple modes of CICR. [Ca]ER is relatively high at rest (12.8 ± 0.9 mmol/kg dry weight, mean ± SEM) and is reduced by thapsigargin or ryanodine (5.5 ± 0.7 and 4.7 ± 1.1 mmol/kg, respectively). [Ca]ER rises during weak depolarization (to 17.0 ± 1.6 mmol/kg over 120s, [Ca2+]i less than ∼350 nM), changes little in response to stronger depolarization (12.1 ± 1.1 mmol/kg, [Ca2+]i ∼700 nM), and declines (to 6.5 ± 1.0 mmol/kg) with larger [Ca2+]i elevations (>1 μM) evoked by the same depolarization when mitochondrial Ca2+ uptake is inhibited (FCCP). Thus, net ER Ca2+ transport exhibits a biphasic dependence on [Ca2+]i. With mitochondrial Ca2+ uptake enabled, [Ca]ER rises after repolarization (to 16.6 ± 1.8 mmol/kg at 15 min) as [Ca2+]i falls within the permissive range for ER Ca accumulation over a period lengthened by mitochondrial Ca2+ release. Finally, although spatially averaged [Ca]ER is unchanged during strong depolarization, net ER Ca2+ release still occurs, but only in the outermost ∼5-μm cytoplasmic shell where [Ca2+]i should reach its highest levels. Since mitochondrial Ca accumulation occurs preferentially in peripheral cytoplasm, as demonstrated

  16. Renal Calcium Oxalate Deposits Induce a Pro-Atherosclerotic and Pro-Osteoporotic Response in Mice.

    Science.gov (United States)

    Kusumi, Kirsten; Barr-Beare, Evan; Saxena, Vijay; Safedi, Fayez; Schwaderer, Andrew

    2017-09-01

    Urinary stone disease (USD) is increasing in adult and pediatric populations. Adult and pediatric studies have demonstrated decreased bone mineral density and increased fracture rates. USD has also been independently linked to increased rates of myocardial infarction and cerebral vascular accidents. Although USD is a multisystem disorder involving the kidneys, bone, and vasculature, the molecular mechanisms linking these three organs remain unknown. Calcium oxalate nephropathy was induced in C57BL/6J mice with intra-peritoneal (ip) injection of sodium glyoxolate. Half of each kidney underwent Pizzalato staining and half was snap frozen for RNA extraction. RT(2) Profiler Mouse Atherosclerosis, Osteoporosis, and Calcium Signaling PCR Arrays (Qiagen) were performed. Only results that passed quality checks in PCR array reproducibility and genomic DNA contamination were included. Genes had to show at least fourfold differential expression and P 10-fold increase. All 10 have P ≤ 0.003. The calcium signaling array showed significant fourfold upregulation of 10 genes, four of which were ≥10-fold. All 10 have P ≤ 0.03. We have demonstrated that calcium oxalate nephropathy can induce upregulation of atherosclerotic, metabolic bone, and calcium homeostasis genes in a murine model. This may be and initial step in identifying the molecular mechanisms linking stone, bone, and cardiovascular disease. J. Cell. Biochem. 118: 2744-2751, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Proposal for the award of an industrial service contract for stores operations and relatedlogistics, in-house mail distribution and transport services on the CERN site

    CERN Document Server

    2006-01-01

    This document concerns the award of an industrial support contract for stores operations and related logistics, in-house mail distribution and transport services on the CERN site. The Finance Committee is invited to agree to the negotiation of a contract with the consortium ISS (CH) - ISS (ES), for stores operations and related logistics, in-house mail distribution and transport services on the CERN site for a period of three years for a total amount not exceeding 10 312 028 Swiss francs not subject to revision. The contract will include options for two one-year extensions beyond the initial three-year period.

  18. Multiple Modes of Calcium-Induced Calcium Release in Sympathetic Neurons I

    Science.gov (United States)

    Albrecht, Meredith A.; Colegrove, Stephen L.; Hongpaisan, Jarin; Pivovarova, Natalia B.; Andrews, S. Brian; Friel, David D.

    2001-01-01

    Many cells express ryanodine receptors (RyRs) whose activation is thought to amplify depolarization-evoked elevations in cytoplasmic Ca2+ concentration ([Ca2+]i) through a process of Ca2+-induced Ca2+ release (CICR). In neurons, it is usually assumed that CICR triggers net Ca2+ release from an ER Ca2+ store. However, since net ER Ca2+ transport depends on the relative rates of Ca2+ uptake and release via distinct pathways, weak activation of a CICR pathway during periods of ER Ca accumulation would have a totally different effect: attenuation of Ca2+ accumulation. Stronger CICR activation at higher [Ca2+]i could further attenuate Ca2+ accumulation or trigger net Ca2+ release, depending on the quantitative properties of the underlying Ca2+ transporters. This and the companion study (Hongpaisan, J., N.B. Pivovarova, S.L. Colgrove, R.D. Leapman, and D.D. Friel, and S.B. Andrews. 2001. J. Gen. Physiol. 118:101–112) investigate which of these CICR “modes” operate during depolarization-induced Ca2+ entry in sympathetic neurons. The present study focuses on small [Ca2+]i elevations (less than ∼350 nM) evoked by weak depolarization. The following two approaches were used: (1) Ca2+ fluxes were estimated from simultaneous measurements of [Ca2+]i and ICa in fura-2–loaded cells (perforated patch conditions), and (2) total ER Ca concentrations ([Ca]ER) were measured using X-ray microanalysis. Flux analysis revealed triggered net Ca2+ release during depolarization in the presence but not the absence of caffeine, and [Ca2+]i responses were accelerated by SERCA inhibitors, implicating ER Ca2+ accumulation, which was confirmed by direct [Ca]ER measurements. Ryanodine abolished caffeine-induced CICR and enhanced depolarization-induced ER Ca2+ accumulation, indicating that activation of the CICR pathway normally attenuates ER Ca2+ accumulation, which is a novel mechanism for accelerating evoked [Ca2+]i responses. Theory shows how such a low gain mode of CICR can operate

  19. Calcium-dependent and calcium-sensitizing pathways in the mature and immature ductus arteriosus.

    Science.gov (United States)

    Clyman, Ronald I; Waleh, Nahid; Kajino, Hiroki; Roman, Christine; Mauray, Francoise

    2007-10-01

    Studies performed in sheep and baboons have shown that after birth, the normoxic muscle media of ductus arteriosus (DA) becomes profoundly hypoxic as it constricts and undergoes anatomic remodeling. We used isolated fetal lamb DA (pretreated with inhibitors of prostaglandin and nitric oxide production) to determine why the immature DA fails to remain tightly constricted during the hypoxic phase of remodeling. Under normoxic conditions, mature DA constricts to 70% of its maximal active tension (MAT). Half of its normoxic tension is due to Ca(2+) entry through calcium L-channels and store-operated calcium (SOC) channels. The other half is independent of extracellular Ca(2+) and is unaffected by inhibitors of sarcoplasmic reticulum (SR) Ca(2+) release (ryanodine) or reuptake [cyclopiazonic acid (CPA)]. The mature DA relaxes slightly during hypoxia (to 60% MAT) due to decreases in calcium L-channel-mediated Ca(2+) entry. Inhibitors of Rho kinase and tyrosine kinase inhibit both Ca(2+)-dependent and Ca(2+)-independent DA tension. Although Rho kinase activity may increase during gestation, immature DA develop lower tensions than mature DA, primarily because of differences in the way they process Ca(2+). Calcium L-channel expression increases with advancing gestation. Under normoxic conditions, differences in calcium L-channel-mediated Ca(2+) entry account for differences in tension between immature (60% MAT) and mature (70% MAT) DA. Under hypoxic conditions, differences in both calcium L-channel-dependent and calcium L-channel-independent Ca(2+) entry, account for differences in tension between immature (33% MAT) and mature (60% MAT) DA. Stimulation of Ca(2+) entry through reverse-mode Na(+)/Ca(2+) exchange or CPA-induced SOC channel activity constrict the DA and eliminate differences between immature and mature DA during both hypoxia and normoxia.

  20. Generation of calcium waves in living cells induced by 1 kHz femtosecond laser protuberance microsurgery

    Science.gov (United States)

    Zhou, M.; Zhao, E. L.; Yang, H. F.; Gong, A. H.; di, J. K.; Zhang, Z. J.

    2009-07-01

    We have demonstrated that intracellular calcium waves in a living olfactory ensheathing cell (OEC) can be induced by femtosecond laser surgery on cellular protuberance. In this paper, calcium wave generation mechanisms are further investigated using different culture mediums and protuberance diameters. The protuberances of living OECs are cut by home-made 1 kHz femtosecond laser surgery system with 130 fs pulsewidth and 800 nm wavelength, and the average power of 200 μW is chosen for stable and effective cell surgery. Whether the cells are cultured in mediums with Ca2+ or not, intracellular calcium waves can be induced after cell surgery. The generation of calcium waves is independent on the dimension of protuberance diameter. Based on these results, we analyze generation mechanisms of calcium wave and conclude that shockwave-induced mechanical force and laser-induced cytoskeleton depolymerization are two key factors.

  1. Smad signaling pathway in pathogenesis of kidney injury induced by calcium oxalate stone in rats

    Institute of Scientific and Technical Information of China (English)

    Fan Zhang

    2016-01-01

    Objective:To investigate the involvement of Smad signaling pathway in the pathogenesis of kidney injury induced by calcium oxalate stone in rats to provide a reference for clinical treatment.Methods: Clean SD rats were randomly divided into 3 group, namely the control group, model group and pirfenidone group. Ethylene glycol + αhydroxy vitamin D3 was used as a stone-inducing agent to replicate the renal calcium oxalate stone model. Rats in the pirfenidone group were treated with pirfenidone intragastric administration. The serum Cr, BUN and 24-hour oxalate and calcium in renal tissues were assayed. The expressions of Bax/Bcl2 protein, Caspase3 protein, TGFβ, Smad1, Smad2 and Smad3 proteins were detected by the fluorescent quantitation PCR method.Results:Compared with the rats of the control group, the results showed that the levels of serum BUN, Cr and 24-hour oxalate in rats of the model group were increased greatly,BaxandCaspase3 mRNA also increased while the level ofBcl2 decreased significantly, and the expressions of TGFβ, Smad1, Smad2 and Smad3 proteins increased distinctly as well (P<0.01). These abnormal parameters could be normalized effectively by pirfenidone.Conclusions:Activated TGFβ/Smad signaling pathway is involved in the pathogenesis of kidney injury induced by calcium oxalate stone in rats.

  2. Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle.

    Science.gov (United States)

    Park, S; Scheffler, T L; Gunawan, A M; Shi, H; Zeng, C; Hannon, K M; Grant, A L; Gerrard, D E

    2009-01-01

    Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced GLUT-4 expression in skeletal muscle.

  3. Modelling biological and chemically induced precipitation of calcium phosphate in enhanced biological phosphorus removal systems.

    Science.gov (United States)

    Barat, R; Montoya, T; Seco, A; Ferrer, J

    2011-06-01

    The biologically induced precipitation processes can be important in wastewater treatment, in particular treating raw wastewater with high calcium concentration combined with Enhanced Biological Phosphorus Removal. Currently, there is little information and experience in modelling jointly biological and chemical processes. This paper presents a calcium phosphate precipitation model and its inclusion in the Activated Sludge Model No 2d (ASM2d). The proposed precipitation model considers that aqueous phase reactions quickly achieve the chemical equilibrium and that aqueous-solid change is kinetically governed. The model was calibrated using data from four experiments in a Sequencing Batch Reactor (SBR) operated for EBPR and finally validated with two experiments. The precipitation model proposed was able to reproduce the dynamics of amorphous calcium phosphate (ACP) formation and later crystallization to hydroxyapatite (HAP) under different scenarios. The model successfully characterised the EBPR performance of the SBR, including the biological, physical and chemical processes.

  4. The Appetite-Inducing Peptide, Ghrelin, Induces Intracellular Store-Mediated Rises in Calcium in Addiction and Arousal-Related Laterodorsal Tegmental Neurons in Mouse Brain Slices

    DEFF Research Database (Denmark)

    Hauberg, Katrine; Kohlmeier, Kristi Anne

    2015-01-01

    Ghrelin, a gut and brain peptide, has recently been shown to be involved in motivated behavior and regulation of the sleep and wakefulness cycle. The laterodorsal tegmental nucleus (LDT) is involved in appetitive behavior and control of the arousal state of an organism, and accordingly, behavioral...... this peptide has been shown in other cell types to lead to rises in calcium via release of calcium from intracellular stores. To determine whether ghrelin induced intracellular calcium rises in mouse LDT neurons, we conducted calcium imaging studies in LDT brain slices loaded with the calcium binding dye, Fura...

  5. Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

    Science.gov (United States)

    Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

    2013-05-07

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

  6. Effects of Arecoline on Calcium Channel Currents and Caffeine-induced Calcium Release in Isolated Single Ventricular Myocyte of Guinea Pig

    Institute of Scientific and Technical Information of China (English)

    林先明; 李真; 胡本容; 夏国瑾; 姚伟星; 向继洲

    2002-01-01

    Summary: The effects of Arecoline (Are) on calcium mobilization were investigated. In isolatedsingle ventricular myocyte of guinea pig, patch clamp whole cell recording techniques were used torecord the current of L-type calcium channel and cytosolic Ca2+ level ([Ca2+]i) labeled with fluo-rescence probe Fluo-3/AM was measured under a laser scanning confocal microscope. Results re-vealed that Are (3-100 μmol/L) could inhibit L-type calcium current in a concentration-depen-dent manner and the value of IC50 was 33. 73μmol/L (n= 5). In the absence of extracellular calci-um, the resting levels of [Ca2+]i was not affected by Are (n=6, P>0. 05), but pretreatmentwith Are (30 μmol/L) could significantly inhibit the [Ca2+]i elevation induced by caffeine (10mmol/L, n = 6, P < 0. 01). It was concluded that Are could inhibit not only calcium influxthrough L-type calcium channel but also calcium release from sarcoplasmic reticulum.

  7. Purinergically induced membrane fluidization in ciliary cells: characterization and control by calcium and membrane potential.

    Science.gov (United States)

    Alfahel, E; Korngreen, A; Parola, A H; Priel, Z

    1996-02-01

    To examine the role of membrane dynamics in transmembrane signal transduction, we studied changes in membrane fluidity in mucociliary tissues from frog palate and esophagus epithelia stimulated by extracellular ATP. Micromolar concentrations of ATP induced strong changes in fluorescence polarization, possibly indicating membrane fluidization. This effect was dosage dependent, reaching a maximum at 10-microM ATP. It was dependent on the presence of extracellular Ca2+ (or Mg2+), though it was insensitive to inhibitors of voltage-gated calcium channels. It was inhibited by thapsigargin and by ionomycin (at low extracellular Ca2+ concentration), both of which deplete Ca2+ stores. It was inhibited by the calcium-activated potassium channel inhibitors quinidine, charybdotoxin, and apamine and was reduced considerably by replacement of extracellular Na+ with K+. Hyperpolarization, or depolarization, of the mucociliary membrane induced membrane fluidization. The degree of membrane fluidization depended on the degree of hyperpolarization or depolarization of the ciliary membrane potential and was considerably lower than the effect induced by extracellular ATP. These results indicate that appreciable membrane fluidization induced by extracellular ATP depends both on an increase in intracellular Ca2+, mainly from its internal stores, and on hyperpolarization of the membrane. Calcium-dependent potassium channels couple the two effects. In light of recent results on the enhancement of ciliary beat frequency, it would appear that extracellular ATP-induced changes both in ciliary beat frequency and in membrane fluidity are triggered by similar signal transduction pathways.

  8. IP3-dependent, post-tetanic calcium transients induced by electrostimulation of adult skeletal muscle fibers

    Science.gov (United States)

    Casas, Mariana; Figueroa, Reinaldo; Jorquera, Gonzalo; Escobar, Matías; Molgó, Jordi

    2010-01-01

    Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors (IP3Rs) and related to transcriptional events. We searched for slow calcium signals in adult muscle fibers using isolated adult flexor digitorum brevis fibers from 5–7-wk-old mice, loaded with fluo-3. When stimulated with trains of 0.3-ms pulses at various frequencies, cells responded with a fast calcium signal associated with muscle contraction, followed by a slower signal similar to one previously described in cultured myotubes. Nifedipine inhibited the slow signal more effectively than the fast one, suggesting a role for DHPR in its onset. The IP3R inhibitors Xestospongin B or C (5 µM) also inhibited it. The amplitude of post-tetanic calcium transients depends on both tetanus frequency and duration, having a maximum at 10–20 Hz. At this stimulation frequency, an increase of the slow isoform of troponin I mRNA was detected, while the fast isoform of this gene was inhibited. All three IP3R isoforms were present in adult muscle. IP3R-1 was differentially expressed in different types of muscle fibers, being higher in a subset of fast-type fibers. Interestingly, isolated fibers from the slow soleus muscle did not reveal the slow calcium signal induced by electrical stimulus. These results support the idea that IP3R-dependent slow calcium signals may be characteristic of distinct types of muscle fibers and may participate in the activation of specific transcriptional programs of slow and fast phenotype. PMID:20837675

  9. Plasma Calcium, Inorganic Phosphate and Magnesium During Hypocalcaemia Induced by a Standardized EDTA Infusion in Cows

    Directory of Open Access Journals (Sweden)

    Enemark JMD

    2001-06-01

    Full Text Available The intravenous Na2EDTA infusion technique allows effective specific chelation of circulating Ca2+ leading to a progressive hypocalcaemia. Methods previously used were not described in detail and results obtained by monitoring total and free ionic calcium were not comparable due to differences in sampling and analysis. This paper describes a standardized EDTA infusion technique that allowed comparison of the response of calcium, phosphorus and magnesium between 2 groups of experimental cows. The concentration of the Na2EDTA solution was 0.134 mol/l and the flow rate was standardized at 1.2 ml/kg per hour. Involuntary recumbency occurred when ionised calcium dropped to 0.39 – 0.52 mmol/l due to chelation. An initial fast drop of ionized calcium was observed during the first 20 min of infusion followed by a fluctuation leading to a further drop until recumbency. Pre-infusion [Ca2+] between tests does not correlate with the amount of EDTA required to induce involuntary recumbence. Total calcium concentration measured by atomic absorption remained almost constant during the first 100 min of infusion but declined gradually when the infusion was prolonged. The concentration of inorganic phosphate declined gradually in a fluctuating manner until recumbency. Magnesium concentration remained constant during infusion. Such electrolyte responses during infusion were comparable to those in spontaneous milk fever. The standardized infusion technique might be useful in future experimental studies.

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

    Science.gov (United States)

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

    2016-03-15

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

  11. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

    Science.gov (United States)

    Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

    2013-08-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Calcium citrate improves the epithelial-to-mesenchymal transition induced by acidosis in proximal tubular cells

    Directory of Open Access Journals (Sweden)

    Maria José Rodriguez Cabalgante

    2012-12-01

    Full Text Available INTRODUCTION: Epithelial-to-mesenchymal transition (EMT is a key event in renal fibrosis. The aims of the study were to evaluate acidosis induced EMT, transforming-growth-factor (TGF β1 role and citrate effect on it. METHODS: HK2 cells (ATCC 2290 were cultured in DMEM/HAM F12 medium, pH 7.4. At 80% confluence, after 24 hr under serum free conditions, cells were distributed in three groups (24 hours: A Control: pH 7.4, B Acidosis: pH 7.0 and C Calcium citrate (0.2 mmol/L + pH 7.0. Change (Δ of intracellular calcium concentration, basal and after Angiotensin II (10-6M exposition, were measured to evaluate cellular performance. EMT was evaluated by the expression of α-smooth muscle actin (α-SMA and E-cadherin by immunocytochemistry and/or Western blot. TGF-β1 secretion was determined by ELISA in cell supernatant. RESULTS: At pH 7.0 HK2 cells significantly reduced E-cadherin and increased α-SMA expression (EMT. Supernatant TGF-β1 levels were higher than in control group. Calcium citrate decreased acidosis induced EMT and improved cells performance, without reduction of TGF-β production. CONCLUSIONS: Acidosis induces EMT and secretion of TGF-β1 in tubular proximal cells in culture and citrate improves cellular performance and ameliorates acidosis induced EMT.

  13. Calcium-acting drugs modulate expression and development of chronic tolerance to nicotine-induced antinociception in mice.

    Science.gov (United States)

    Damaj, M I

    2005-11-01

    Initial studies in our laboratory suggested that tolerance to nicotine is thought to involve neuronal adaptation not only at the level of the drug-receptor interaction but at postreceptor events such as calcium-dependent second messengers. The present study was undertaken to investigate the hypothesis that L-type calcium channels and calcium-dependent calmodulin protein kinase II are involved in the development and expression of nicotine tolerance. To that end, the effects of modulation of L-type calcium channels (through the use of inhibitors or activators) as well as calcium-dependent calmodulin protein kinase II inactivation were studied in a mouse model of tolerance where mice were infused with nicotine in minipumps (24 mg/kg/day) for 14 days. In addition, the activity of calcium-dependent calmodulin protein kinase II in the lumbar spinal cord region obtained from nicotine-tolerant mice was measured. Our data showed that chronic administration of L-type calcium channel antagonists nimodipine (1 and 5 mg/kg) and verapamil (10 mg/kg) prevented the development of tolerance to nicotine-induced antinociception. In contrast, chronic exposure of BAYK8644 [(+/-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester], a calcium channel activator, enhanced nicotine's tolerance. Moreover, a significant increase in both dependent and independent calcium-dependent calmodulin protein kinase II activity was seen in the spinal cord in nicotine-tolerant mice. Finally, spinal administration of 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-phenylpiperazine (KN-62), a calcium-dependent calmodulin protein kinase II antagonist, reduced the expression of tolerance to nicotine-induced antinociception in mice. In conclusion, our data indicate that calcium-dependent mechanisms such as L-type calcium channels and calcium-dependent calmodulin protein kinase II activation are involved in the expression and development of nicotine

  14. Effect of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    刘曜蓉

    2013-01-01

    Objective To investigate the impact of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells (HASMCs) .Methods Uremic serum was incubated at 37℃for 3days.Calcium phosphate crystals and uremic supernatant were isolated from uremic serum by ultracentrifugation.

  15. Role of Calcium Ion in Apoptosis of MD Cancer Cells Induced by Arsenic Trioxide

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jiuli; WANG Jintao; XU Shiwen

    2008-01-01

    In order to observe the role of calcium ion in apoptosis of MD cancer cells induced by arsenic trioxide, inhibition percentage was detected by MTT assay;morphology changes were examined by fluorescence microscope;apoptosis was examined by DNA Ladder;[Ca2+]i was investigated by spectrofluorimeter in vitro on MDCC-MSB1 cells. The results showed that As2O3 inhibited the proliferation of MDCC-MSB1 cells in concentration dependent manner (P<0.05 or P<0.01);typical apoptosis character was observed by fluorescence microscope;DNA Ladder was observed;the [Ca2+]i was elevated significantly after the treatment of As203 (P<0.05 or P<0.01) and showed a dose-dependent manner. It is concluded that the calcium may play an important role in apoptosis of MD cancer cells induced by arsenic trioxide.

  16. Calcium channel blocker prevents stress-induced activation of renin and aldosterone in conscious pig

    Energy Technology Data Exchange (ETDEWEB)

    Ceremuzynski, L.K.; Klos, J.; Barcikowski, B.; Herbaczynska-Cedro, K. (Department of Cardiology, Postgraduate Medical School, Warsaw (Poland))

    1991-06-01

    A considerable amount of data suggest the involvement of calcium-mediated processes in the activation of the renin-angiotensin-aldosterone (RAA) cascade. To investigate the effect of calcium-channel inhibition on the RAA system, the authors studied 21 conscious pigs. Blood renin and aldosterone levels increased by subjecting animals to 24 hours of immobilization stress. Renin and aldosterone levels were repeatedly measured by radioimmunoassay in blood samples taken periodically over 24 hours from a chronically implanted arterial cannula. Pretreatment of the animals (N = 11) with nisoldipine, 2 {times} 20 mg p.o. daily for 2 days before and on the day of immobilization, transiently attenuated the stress-induced increase of plasma renin activity and completely prevented the rise of aldosterone, as compared to nontreated controls (N = 10). The finding that nisoldipine suppresses RAA activation induced by a nonpharmacologic stimulus in the conscious intact animal may have clinical implications.

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

    Science.gov (United States)

    Singla, Neha; Dhawan, D K

    2014-10-01

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

  18. The role of calcium in endotoxin-induced release of calcitonin gene-related peptide (CGRP) from rat spinal cord

    Institute of Scientific and Technical Information of China (English)

    唐跃明; 韩启德; 王宪

    1997-01-01

    In the present study, the role of calcium in endotoxin-induced CGRP release was studied. 2 .5-50 μg/mL endotoxin and 1 -10 mmol/L caffeine caused concentration-dependent increase of CGRP release from rat spinal cord in vitro. However, no additive effect could he found when caffeine and endotoxin were concomitantly incubated. By using capsaicin, Ca2+-free medium, Omega-Conotoxin, nifedipine, W-7, ryanodine, MgCl2, Tris-ATP, rutheni-um red, the results indicate that the release of CGRP evoked by endotoxin from the sensory fibers of rat spinal cord is dependent on extracellular calcium. After entering into the cell through the N-type calcium channel, calcium binds to calmodulin, and triggers calcium release from intracellular calcium store by activating the caffeine-sensitive but ryan-odine-insensitive mechanism.

  19. The protective effect of supplemental calcium on colonic permeability depends on a calcium phosphate-induced increase in luminal buffering capacity

    NARCIS (Netherlands)

    Schepens, M.A.A.; Bruggencate, ten S.J.M.; Schonewille, A.J.; Brummer, R.J.M.; Meer, van der R.; Bovee-Oudenhoven, I.M.J.

    2012-01-01

    An increased intestinal permeability is associated with several diseases. Previously, we have shown that dietary Ca decreases colonic permeability in rats. This might be explained by a calcium-phosphate-induced increase in luminal buffering capacity, which protects against an acidic pH due to microb

  20. Novel Resorbable and Osteoconductive Calcium Silicophosphate Scaffold Induced Bone Formation

    Directory of Open Access Journals (Sweden)

    Patricia Ros-Tárraga

    2016-09-01

    Full Text Available This aim of this research was to develop a novel ceramic scaffold to evaluate the response of bone after ceramic implantation in New Zealand (NZ rabbits. Ceramics were prepared by the polymer replication method and inserted into NZ rabbits. Macroporous scaffolds with interconnected round-shaped pores (0.5–1.5 mm = were prepared. The scaffold acted as a physical support where cells with osteoblastic capability were found to migrate, develop processes, and newly immature and mature bone tissue colonized on the surface (initially and in the material’s interior. The new ceramic induced about 62.18% ± 2.28% of new bone and almost complete degradation after six healing months. An elemental analysis showed that the gradual diffusion of Ca and Si ions from scaffolds into newly formed bone formed part of the biomaterial’s resorption process. Histological and radiological studies demonstrated that this porous ceramic scaffold showed biocompatibility and excellent osteointegration and osteoinductive capacity, with no interposition of fibrous tissue between the implanted material and the hematopoietic bone marrow interphase, nor any immune response after six months of implantation. No histological changes were observed in the various organs studied (para-aortic lymph nodes, liver, kidney and lung as a result of degradation products being released.

  1. The Study on Intramyocardial Calcium Overload and Apoptosis Induced by Cosackievirus B3

    Institute of Scientific and Technical Information of China (English)

    HU; Xiufen; WANG; Hongwei; LU; Weiwei; DONG; Yongsui; CHENG; Peixuan

    2001-01-01

    The isolated cardiac myocytes of rats were immediately infected by cosackievirus B3(CVB3) to investigate the effects of such procedure on the cell cycle, apoptosis and intracellular ionized calcium (Ca2+ i) of cardiac myocytes. Newborn Balb/c murine cardiac myocytes were cultivated,then infected by CVB3. Intracellular Ca2+ i was measured by flow cytometer. The calcium in the medium for culturing cardiac myocytes was detected by using atom absorb spectrum test. It was found that CVB3 could markedly inhibit the differentiation and proliferation of the infected cardiac myocytes and induce the apoptosis. The intracellular Ca2+ i level in the infected group was significantly higher than in the control group (P<0. 01). The calcium concentration in the medium for culturing cardiac myocytes in the infected group was significantly lower than in the control group (P<0.05). It was suggested that the apoptosis and intracellular calcium overload of the CVB3-affected cardiac myocytes are likely to play an important role in the pathogenesis of viral myocarditis.

  2. High dietary calcium intake does not counteract disuse-induced bone loss

    Science.gov (United States)

    Baecker, N.; Boese, A.; Smith, S. M.; Heer, M.

    Reduction of mechanical stress on bone inhibits osteoblast-mediated bone formation, increases osteoclast-mediated bone resorption, and leads to what has been called disuse osteoporosis. Prolonged therapeutic bed rest, immobilization and space flight are common causes of disuse osteoporosis. There are sufficient data supporting the use of calcium in combination with vitamin D in the prevention and treatment of postmenopausal osteoporosis. In our study we examined the potential of high dietary calcium intake as a nutrition therapy for disuse-induced bone loss during head-down bed rest in healthy young men. In 2 identical metabolic ward, head-down bed rest (HDBR) experiments (crossover design), we studied the effect of high dietary calcium intake (2000 mg/d) in comparison to the recommended calcium intake of 1000 mg/d on markers of bone turnover. Experiment A (EA) was a 6-day randomized, controlled HDBR study. Experiment B (EB) was a 14-day randomized, controlled HDBR study. In both experiments, the test subjects stayed under well-controlled environmental conditions in our metabolic ward. Subjects' diets in the relevant study phases (HDBR versus Ambulatory Control) of EA and EB were identical except for the calcium intake. The subjects obtained 2000 mg/d Calcium in EA and 2000 mg/d in EB. Blood was drawn at baseline, before entering the relevant intervention period, on day 5 in study EA, and on days 6, 11 and 14 in study EB. Serum calcium, bone formation markers - Procollagen-I-C-Propeptide (PICP) and bone alkaline phosphatase (bAP) were analyzed in serum. 24h-urine was collected throughout the studies for determination of the excretion of calcium (UCaV) and a bone resorption marker, C-terminal telopeptide of collagen type I (UCTX). In both studies, serum calcium levels were unchanged. PICP tended to decrease in EA (p=0.08). In EB PICP decreased significantly over time (p=0.003) in both the control and HDBR periods, and tended to further decrease in the HDBR period (p

  3. Mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells: reversal by non-steroidal anti-inflammatory drugs.

    Science.gov (United States)

    Hernández-Morales, Miriam; Sobradillo, Diego; Valero, Ruth A; Muñoz, Eva; Ubierna, Daniel; Moyer, Mary P; Núñez, Lucía; Villalobos, Carlos

    2017-08-15

    Tumor cells undergo a critical remodeling of intracellular Ca(2+) homeostasis that contribute to important cancer hallmarks. Store-operated Ca(2+) entry (SOCE), a Ca(2+) entry pathway modulated by mitochondria, is dramatically enhanced in colon cancer cells. In addition, most cancer cells display the Warburg effect, a metabolic switch from mitochondrial metabolism to glycolysis that provides survival advantages. Accordingly, we investigated mitochondria control of store-operated currents (SOCs) in two cell lines previously selected for representing human normal colonic cells and colon cancer cells. We found that, in normal cells, mitochondria are important for SOCs activity but they are unable to prevent current inactivation. In contrast, in colon cancer cells, mitochondria are dispensable for SOCs activation but are able to prevent the slow, Ca(2+)-dependent inactivation of SOCs. This effect is associated to increased ability of tumor cell mitochondria to take up Ca(2+) due to increased mitochondrial potential (ΔΨ) linked to the Warburg effect. Consistently with this view, selected non-steroidal anti-inflammatory drugs (NSAIDs) depolarize mitochondria, inhibit mitochondrial Ca(2+) uptake and promote SOC inactivation, leading to inhibition of both SOCE and cancer cell proliferation. Thus, mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells and this effect is counteracted by selected NSAIDs providing a mechanism for cancer chemoprevention.

  4. Mitochondrial calcium uniporter protein MCU is involved in oxidative stress-induced cell death.

    Science.gov (United States)

    Liao, Yajin; Hao, Yumin; Chen, Hong; He, Qing; Yuan, Zengqiang; Cheng, Jinbo

    2015-06-01

    Mitochondrial calcium uniporter (MCU) is a conserved Ca(2+) transporter at mitochondrial in eukaryotic cells. However, the role of MCU protein in oxidative stress-induced cell death remains unclear. Here, we showed that ectopically expressed MCU is mitochondrial localized in both HeLa and primary cerebellar granule neurons (CGNs). Knockdown of endogenous MCU decreases mitochondrial Ca(2+) uptake following histamine stimulation and attenuates cell death induced by oxidative stress in both HeLa cells and CGNs. We also found MCU interacts with VDAC1 and mediates VDAC1 overexpression-induced cell death in CGNs. This finding demonstrates that MCU-VDAC1 complex regulates mitochondrial Ca(2+) uptake and oxidative stress-induced apoptosis, which might represent therapeutic targets for oxidative stress related diseases.

  5. Mechanism for Alternating Electric Fields Induced-Effects on Cytosolic Calcium

    Institute of Scientific and Technical Information of China (English)

    LUO Ming-Yan; SONG Kun; ZHANG Xu; LEE Imshik

    2009-01-01

    In this work, from the point of calcium ions in the cytosol, we extend a Vm-[Ca2+]cyt model to explain the changes of action potential Vm of the plasma membrane and the calcium concentration in the cytosol [Ca2+]cyt under an alternating electric field in cells. An alternating external electric field may exert an oscillating force to each of the free electrolytes, existing on both sides of the plasma membrane. The mechanism for the alternating electric field induced-effects on Vm and [Ca2+]cyt is elucidated. The simulation results show a correlation between the changes of [Ca2+]cyt and the alternating electric field. When the numerical ratio between the intensity Eo(mV/m) and the frequency ν, (Hz) of the field was about 1-2, the [Ca2+]cyt signal is changed dramatically. The bioactive changes of [Ca2+]cyt appear at low frequency, in the range of 0-100 Hz.

  6. PROTECTIVE EFFECTS OF CALCIUM ANTAGONIST ON VASCULAR SYSTEM AGAINST TOXICITY INDUCED BY MERCURIC CHLORIDE

    Institute of Scientific and Technical Information of China (English)

    马欣; 厉英倩; 白宇飞; 刘明

    2004-01-01

    Objective To explore the toxic effects of mercuric chloride (HgCl2) on vascular smooth muscle as well as its relationship to calcium antagonist. Methods By using isolated vascular tension methods, we studied the effect of HgCl2 on isolated rabbit aortic rings. Results HgCl2 (1-100μmol*L-1) caused a concentration-dependent contraction of rabbit aortic rings, which did not change with phentolamin or without endothelium. In KH solution with Ca2+ , the maximum contraction amplitude reduced by(61.2±3.3)%. Nifedipine produced a concentration-dependent decrease of the maximum contraction amplitude. Conclusion Calcium antagonist has protective effects on vascular smooth muscle against damage induced by HgCl2.

  7. The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa: differences from the zona pellucida

    Institute of Scientific and Technical Information of China (English)

    Julio C Chávez; Claudia L Trevi(n)o; Gerardo A de Blas; José L de la Vega-Beltrán; Takuya Nishigaki; Mayel Chirinos; María Elena González-González; Fernando Larrea; Alejandra Solís; Alberto Darszon

    2011-01-01

    The acrosome reaction(AR),an absolute requirement for spermatozoa and egg fusion,requires the influx of Ca2+into the spermatozoa through voltage-dependent Ca2+channels and store-operated channels.Maitotoxin(MTx),a Ca2+-mobilizing agent,has been shown to be a potent inducer of the mouse sperm AR,with a pharmacology similar to that of the zona pellucida(ZP),possibly suggesting a common pathway for both inducers.Using recombinant human ZP3(rhZP3),mouse ZP and two MTx channel blockers(U73122 and U73343),we investigated and compared the MTx-and ZP-induced ARs in human and mouse spermatozoa.Herein,we report that MTx induced AR and elevated intracellular Ca2+([Ca2+]1)in human spermatozoa,both of which were blocked by U73122 and U73343.These two compounds also inhibited the MTx-induced AR in mouse spermatozoa.In disagreement with our previous proposal,the AR triggered by rhZP3 or mouse ZP was not blocked by U73343,indicating that in human and mouse spermatozoa,the AR induction by the physiologicalligands or by MTx occurred through distinct pathways.U73122,but not U73343(inactive analogue),can block phospholipase C(PLC).Another PLC inhibitor,edelfosine,also blocked the rhZP3-and ZP-induced ARs.These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR.Notably,edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human,suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans.

  8. Pseudomonas, Pantoea and Cupriavidus isolates induce calcium carbonate precipitation for biorestoration of ornamental stone.

    Science.gov (United States)

    Daskalakis, M I; Magoulas, A; Kotoulas, G; Catsikis, I; Bakolas, A; Karageorgis, A P; Mavridou, A; Doulia, D; Rigas, F

    2013-08-01

    Bacterially induced calcium carbonate precipitation from various isolates was investigated aiming at developing an environmentally friendly technique for ornamental stone protection and restoration. Micro-organisms isolated from stone samples and identified using 16S rDNA and biochemical tests promoted calcium carbonate precipitation in solid and novel liquid growth media. Biomineral morphology was studied on marble samples with scanning electron microscopy. Most isolates demonstrated specimen weight increase, covering partially or even completely the marble surfaces mainly with vaterite. The conditions under which vaterite precipitated and its stability throughout the experimental runs are presented. A growth medium that facilitated bacterial growth of different species and promoted biomineralization was formulated. Most isolates induced biomineralization of CaCO3 . Micro-organisms may actually be a milestone in the investigation of vaterite formation facilitating our understanding of geomicrobiological interactions. Pseudomonas, Pantoea and Cupriavidus strains could be candidates for bioconsolidation of ornamental stone protection. Characterization of biomineralization capacity of different bacterial species improves understanding of the bacterially induced mineralization processes and enriches the list of candidates for biorestoration applications. Knowledge of biomineral morphology assists in differentiating mineral from biologically induced precipitates. © 2013 The Society for Applied Microbiology.

  9. Role of calcium in gonadotropin releasing hormone-induced luteinizing hormone secretion from the bovine pituitary

    Energy Technology Data Exchange (ETDEWEB)

    Kile, J.P.

    1986-01-01

    The hypothesis was tested that GnRH acts to release LH by increasing calcium uptake by gonadotroph which in turn stimulates calcium-calmodulin activity and results in LH release from bovine pituitary cells as it does in the rat. Pituitary glands of calves (4-10 months of age) were enzymatically dispersed (0.2% collagenase) and grown for 5 days to confluency in multiwell plates (3 x 10/sup 5//well). Cells treated with GnRH Ca/sup + +/ ionophore A23187, and ouabain all produced significant releases of LH release in a pronounced all or none fashion, while thorough washing of the cells with 0.5 mM EGTA in Ca/sup + +/-free media prevented the action of GnRH. GnRH caused a rapid efflux of /sup 45/Ca/sup + +/. Both GnRH-stimulated /sup 45/Ca efflux and LH release could be partially blocked by verapamil GnRH-induced LH release could also be blocked by nifedipine and tetrodotoxin, although these agents did not affect /sup 45/Ca efflux. The calmodulin antagonists calmidazolium and W7 were found to block GnRH induced LH release, as well as LH release induced by theophylline, KC PGE/sub 2/ and estradiol. These data indicated that: (1) calcium is required for GnRH action, but extracellular Ca/sup + +/ does not regulate LH release; (2) GnRH elevates intracellular Ca/sup + +/ by opening both voltage sensitive and receptor mediated Ca/sup + +/ channels; (3) activation of calmodulin is one mechanism involved in GnRH-induced LH release.

  10. Mitochondrial calcium uniporter inhibition attenuates mouse bone marrow-derived mast cell degranulation induced by beta-1,3-glucan.

    Science.gov (United States)

    Cuong, Dang Van; Kim, Hyoung Kyu; Marquez, Jubert; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo; Han, Jin

    2016-03-01

    Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca(2+), which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 µg/ml BG, 100 µg/ml peptidoglycan (PGN), or 10 µM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca(2+) uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca(2+) uniporter has an important regulatory role in BG-induced mast cell degranulation.

  11. The coupling of acetylcholine-induced BK channel and calcium channel in guinea pig saccular type II vestibular hair cells.

    Science.gov (United States)

    Kong, Wei-Jia; Guo, Chang-Kai; Zhang, Xiao-Wen; Chen, Xiong; Zhang, Song; Li, Guan-Qiao; Li, Zhi-Wang; Van Cauwenberge, Paul

    2007-01-19

    Molecular biological studies and electrophysiological data have demonstrated that acetylcholine (ACh) is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type II vestibular hair cells (VHCs II) among mammalians are still unclear, with the exception of the well-known alpha9-containing nicotinic ACh receptor (alpha9-containing nAChR)-activated small conductance, calcium-dependent potassium current (SK) in cochlear hair cells and frog saccular hair cells. The activation of SK current was necessary for the calcium influx through the alpha9-containing nAChR. Recently, we have demonstrated that ACh-induced big conductance, calcium-dependent potassium current (BK) was present in VHCs II of the vestibular end-organ of guinea pig. In this study, the nature of calcium influx for the activation of ACh-induced BK current in saccular VHCs II of guinea pig was investigated. Following extracellular perfusion of ACh, saccular VHCs II displayed a sustained outward current, which was sensitive to iberiotoxin (IBTX). High concentration of apamin failed to inhibit the current amplitude of ACh-induced outward current. Intracellular application of Cs(+) completely abolished the current evoked by ACh. ACh-induced current was potently inhibited by nifedipine, nimodipine, Cd(2+) and Ni(2+), respectively. The inhibition potency of these four calcium channel antagonists was nimodipine>nifedipine>cadmium>nickel. The L-type Ca(2+) channels agonist, (-)-Bay-K 8644 mimicked the effect of ACh and activated an IBTX-sensitive current. In addition, partial VHCs II displayed a biphasic waveform. In conclusion, the present data showed that in the guinea pig saccular VHCs II, ACh-induced BK channel was coupled with the calcium channel, but not the receptor. The perfusion of ACh will drive the opening of calcium channels; the influx of calcium ions will then activate the BK current.

  12. Roscovitine in combination with calcium ionophore induces oocyte activation through reduction of M-phase promoting factor activity in mice.

    Science.gov (United States)

    Iba, Tomomi; Yano, Yuya; Umeno, Mayumi; Hinokio, Kenji; Kuwahara, Akira; Irahara, Minoru; Yamano, Shuji; Yasui, Toshiyuki

    2012-11-01

    The aim of the present study was to determine oocyte activation and change in M-phase promoting factor (MPF) activity induced by treatment with calcium ionophore and roscovitine in comparison with those induced by treatment with roscovitine alone and treatment with calcium ionophore and puromycin in mice. Freshly ovulated oocytes obtained from 6-8-week-old mice were divided into five groups (no activation treatment; 5 μM calcium ionophore A23187; 50 μM roscovitine; 5 μM calcium ionophore and 10 μg/ml puromycin; and 5 μM calcium ionophore and 50 μM roscovitine) and were incubated for 6 h. Oocyte activation, assessed by morphological changes, and changes in MPF activity in the five groups at 0, 2, 4 and 6 h of incubation were examined. Activated oocytes were defined as oocytes with at least one pronucleus. Oocytes treated with roscovitine alone were not activated during the 6-h incubation period. All of the oocytes in the calcium ionophore with puromycin group and in the calcium ionophore with roscovitine group were activated. The percentage activity of MPF in oocytes treated with roscovitine alone was decreased after 2 h and increased after 4 h of incubation. The percentage activity of MPF in oocytes treated with calcium ionophore and roscovitine was significantly decreased with suppression of MPF activity being maintained for 6 h, and this change was similar to that in oocytes treated with calcium ionophore and puromycin. Roscovitine with calcium ionophore is effective for induction of oocyte activation through suppression of MPF activity in mice.

  13. A model of calcium signaling and degranulation dynamics induced by laser irradiation in mast cells

    Institute of Scientific and Technical Information of China (English)

    SHI XiaoMin; ZHENG YuFan; LIU ZengRong; YANG WenZhong

    2008-01-01

    Recent experiments show that calcium signaling and degranulation dynamics induced by low power laser irradiation in mast cells must rely on extracellular Ca2+ influx. An analytical expression of Ca2+ flux through TRPV4 cation channel in response to interaction of laser photon energy and extracellular Ca2+ is deduced, and a model characterizing dynamics of calcium signaling and degranulation activated by laser irradiation in mast cells is established. The model indicates that the characteristics of calcium signaling and degranulation dynamics are determined by interaction between laser photon energy and Ca2+ influx. Extracellular Ca2+ concentration is so high that even small photon energy can activate mast cells, thus avoiding the possible injury caused by laser irradiation with shorter wavelengths. The model predicts that there exists a narrow parameter domain of photon energy and extracellular Ca2+ concentration of which results in cytosolic Ca2+ limit cycle oscillations, and shows that PKC activity is in direct proportion to the frequency of Ca2+ oscillations. With the model it is found that sustained and stable maximum plateau of cytosolic Ca2+ concentration can get optimal degranulation rate. Furthermore, the idea of introducing the realistic physical energy into model is applicable to modeling other physical signal transduction systems.

  14. Lead Poisoning Disturbs Oligodendrocytes Differentiation Involved in Decreased Expression of NCX3 Inducing Intracellular Calcium Overload

    Directory of Open Access Journals (Sweden)

    Teng Ma

    2015-08-01

    Full Text Available Lead (Pb poisoning has always been a serious health concern, as it permanently damages the central nervous system. Chronic Pb accumulation in the human body disturbs oligodendrocytes (OLs differentiation, resulting in dysmyelination, but the molecular mechanism remains unknown. In this study, Pb at 1 μM inhibits OLs precursor cells (OPCs differentiation via decreasing the expression of Olig 2, CNPase proteins in vitro. Moreover, Pb treatment inhibits the sodium/calcium exchanger 3 (NCX3 mRNA expression, one of the major means of calcium (Ca2+ extrusion at the plasma membrane during OPCs differentiation. Also addition of KB-R7943, NCX3 inhibitor, to simulate Pb toxicity, resulted in decreased myelin basic protein (MBP expression and cell branching. Ca2+ response trace with Pb and KB-R7943 treatment did not drop down in the same recovery time as the control, which elevated intracellular Ca2+ concentration reducing MBP expression. In contrast, over-expression of NCX3 in Pb exposed OPCs displayed significant increase MBP fluorescence signal in positive regions and CNPase expression, which recovered OPCs differentiation to counterbalance Pb toxicity. In conclusion, Pb exposure disturbs OLs differentiation via affecting the function of NCX3 by inducing intracellular calcium overload.

  15. Acetylcholine-induced calcium oscillation in isolated outer hair cells in guinea pig

    Institute of Scientific and Technical Information of China (English)

    XIE Ding-hua; XIAO Zi-an; YANG Shu

    2006-01-01

    Abstract Objective This study is to explore the relationship between acetylcholine (ACh)-induced calcium release from intracellular Ca2+ stores and function of outer hair cell (OHC) motors, in an attempt to elucidate the mechanism of OHC electromotility at resting state. Methods OHCs were isolated from adult guinea pig (200-300 g) cochlea and loaded with Fluo-3/AM. The cells were treated with ACh/dHBSS, ACh/HBSS, dHBSS only or HBSS only. Intracellular [Ca2+]i variations in cells under the four treatments were observed using an Ar-Kr laser scan confocal microscope. Results [Ca2+]i oscillations were recorded in five OHCs treated with ACh/dHBSS but not in other cells. This is the first time that Ach-excited [Ca2+]i oscillations are reported in guinea pig OHCs independent of extracellular calcium. Conclusions ACh-excited [Ca2+]i oscillations in OHCs originates from intracellular calcium release and may play a crucial role in maintaining active mechanical motility of the OHC at resting and modulating OHC electromotility.

  16. Beneficial effects of calcium oral coadministration in gentamicin-induced nephrotoxicity in rats.

    Science.gov (United States)

    Stojiljkovic, Nenad; Stoiljkovic, Milan; Mihailovic, Dragan; Randjelovic, Pavle; Ilic, Sonja; Gocmanac-Ignjatovic, Marija; Veljkovic, Milica

    2012-01-01

    Frequent therapeutical use of an aminoglycoside antibiotic gentamicin (GM) is limited by its nephrotoxic effects often characterized by both morphological and functional alterations of kidney leading to acute renal failure. The aim of this study was to examine the effect of dietary calcium supplementation on GM-induced nephrotoxicity in rats. Experiments were performed on 30 adult male Wistar rats divided into three groups of 10 animals each. G-group received GM intraperitoneally at a dose of 100 mg/kg; GCa-group received the same dose of GM concomitantly with 1 g/kg calcium carbonate given orally; and C-group, serving as control, received 1 mL/day of normal saline. All groups were treated during 8 consecutive days. Quantitative evaluation of GM-induced structural and functional changes of kidney was performed by histopathological, morphometrical, and biochemical analyses. Compared with control, G-group of rats were found to have diffusely and unequally thickened glomerular basement membrane with neutrophil cells infiltration. In addition, vacuolization of cytoplasm of proximal tubule cells with coagulation-type necrosis was observed. These GM-induced pathological lesions were significantly reduced in the rats of GCa-group. Morphometric analysis revealed statistically significant differences in the size of glomeruli (area, major and minor axes, perimeter), optical density, and roundness of glomeruli (p Biochemical analysis showed significant elevation in blood urea and serum creatinine concentrations, whereas potassium concentration was lowered in G-group compared with the other groups (p < 0.01). It is concluded that oral supplementation of calcium during treatment with GM resulted in significant reduction of morphological and functional kidney alterations.

  17. Changes of neuronal calcium channel following brain damage induced by injection of pertussis bacilli in rats

    Institute of Scientific and Technical Information of China (English)

    陈立华; 于嘉; 刘丽旭; 曹美鸿

    2002-01-01

    To explore changes of neuronal calcium channel following brain damage induced by injection of pertussis bacilli in rats, and to investigate the relationship between cytosolic free calcium concentration ( [ Ca2 + ] i ) in the synaptosome and Ca2 + -ATPase activities of mitochondria. Methods: The level of [ Ca2+ ]i in the synaptosome and Ca2+ -ATPase activities of mitochondria in the acute brain damage induced by injection of pertussis bacilli (PB)in rat was determined and nimodipine was administrated to show its effects on [ Ca2+ ]i in the synaptosome and on alteration of Ca2+ -ATPase activity in the mitochondria.Seventy-three rats were randomly divided into four groups,ie, normal control group (Group A ), sham-operation control group (Group B), PB group (Group C) and nimodipine treatment group (Group D). Results: The level of [ Ca2+ ]i was significantly increased in the PB-injected cerebral hemisphere in the Group C as compared with that in the Group A and the Group B at 30 minutes after injection of PB. The level of [ Ca2+ ]i was kept higher in the 4 hours and 24 hours subgroups after the injection in the Group C ( P < 0.05).In contrast, the Ca2+ -ATPase activities were decreased remarkably among all of the subgroups in the Group C.Nimodipine, which was administered after injection of PB,could significantly decrease the [ Ca2+ ]i and increase the activity of Ca2 + -ATPase ( P < 0.05 ). Conclusions: The neuronal calcium channel is opened after injection of PB. There is a negative correlation between activities of Ca2 +-ATPase and [ Ca2 + ]i.Nimodipine can reduce brain damage through stimulating the activities of Ca2+ -ATPase in the mitochondria, and decrease the level of [ Ca2+ ]i in the synaptosome.Treatment with nimodipine dramatically reduces the effects of brain damage induced by injection of PB.

  18. On the Pressure-Induced Loss of Crystallinity in Zinc- and Calcium-Phosphates

    Energy Technology Data Exchange (ETDEWEB)

    Shakhvorostov, D.; Mosey, N; Munoz-Paniagua, D; Pereira, G; Song, Y; Kasrai, M; Norton, P; Müser, M

    2008-01-01

    A recently suggested mechanism for the stress memory of various metal phosphates is investigated experimentally. Based on first-principles simulations [N. J. Mosey et al., Science 307, 1612 (2005)], it had been argued that atoms with flexible coordination, such as zinc or heavy-metal cations, act as network-forming agents, undergoing irreversible pressure-induced changes in bonding that lead to increased connectivity between phosphate anions. In the present study, orthophosphates of zinc and calcium were exposed to high pressures on surfaces and in diamond anvil cells. An additional set of first-principles simulations was accomplished on ?-orthophosphate of zinc, which suggested that this material was already cross-linked before compression but that it nevertheless underwent a reversible coordination change under pressure in agreement with the experimental results presented here. Raman spectra indicate an irreversible, pressure-induced loss of long-range crystallinity. The pressures required to induce these changes are around 7 GPa for the zinc phosphates, while they are close to 21 GPa for the calcium phosphates. Hydrogenation of the metal phosphate lowers the threshold pressure by approximately 2-3 GPa in both cases. Moreover, ?-orthophosphate of zinc could be partially amorphisized under nonisotropic pressure on copper foils.

  19. Calcium Flux between the Endoplasmic Reticulum and Mitochondrion Contributes to Poliovirus-Induced Apoptosis▿

    Science.gov (United States)

    Brisac, Cynthia; Téoulé, François; Autret, Arnaud; Pelletier, Isabelle; Colbère-Garapin, Florence; Brenner, Catherine; Lemaire, Christophe; Blondel, Bruno

    2010-01-01

    We show that poliovirus (PV) infection induces an increase in cytosolic calcium (Ca2+) concentration in neuroblastoma IMR5 cells, at least partly through Ca2+ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. This leads to Ca2+ accumulation in mitochondria through the mitochondrial Ca2+ uniporter and the voltage-dependent anion channel (VDAC). This increase in mitochondrial Ca2+ concentration in PV-infected cells leads to mitochondrial dysfunction and apoptosis. PMID:20861253

  20. Role of calcium signaling in down-regulation of aggrecan induced by cyclic tensile strain in annulus fibrosus cells

    Institute of Scientific and Technical Information of China (English)

    GUO Zhi-liang; ZHOU Yue; LI Hua-zhuang; CAO Guo-yong; TENG Hai-jun

    2006-01-01

    Objective:To study the role of intracellular calcium signal pathway in the down-regulation of aggrecan induced by cyclic tensile strain in the annulus fibrosus cells. Methods :The expression of aggrecan mRNA and core protein were respectively detected with RT-PCR and western blot after the channels transmitting calcium ions were blocked with EGTA, gadolinium and verapamil. Results:EGTA, gadolinium and verapamil partially prevented the effects of cyclic tensile strain on the expression of aggrecan in annulus fibrosus cells. Conclusion:The calcium signaling is involved in the down-regulation of proteoglycan resulting from cyclic tensile strain in the annulus fibrosus cells.

  1. Developmental Profile and Mechanisms of GABA-Induced Calcium Signaling in Hippocampal Astrocytes

    Institute of Scientific and Technical Information of China (English)

    SILKE D. MEIER; KARL W. KAFITZ; CHRISTINE R. ROSE

    2008-01-01

    γ-氨基丁酸(GABA)是具有双重作用的递质,它在产后发育的第1周对神经元具有兴奋作用,但在成年大脑中是主要的抑制性递质.GABA还能通过与离子型(GABAA)和代谢型(GABAB)受体结合来活化星形胶质细胞,导致胶质细胞钙升高及神经递质释放,GABA在神经元-胶质细胞相互作用中起重要的调节作用.本文采用全细胞膜片钳和比率钙成象分析出生后3~34 d的大鼠海马切片,星形胶质细胞GABAA和GABAB受体活化诱导的钙信号的发育特征及细胞机制.GABAA和GABAB受体都可介导胶质细胞的细胞内钙瞬对升高.在整个发育过程中,GABAA受体活化通过激活电压依赖性钙通道的钙流入引起大多数星形胶质细胞快速的钙瞬变.相反的是,GABAB受体活化导致细胞延迟的钙升高,并且这种作用能被细胞内钙库消耗和持久的异源三聚G蛋白活化所阻滞.GABAB受体介导的钙信号呈现明确的发育规律,即<10%的星形胶质细胞在出生后3 d或32~34 d有应答,大约60%的星形胶质细胞在出生后11~15 d有应答.本文提示,GABAB受体通过激活G蛋白,诱导细胞内钙库释放钙,导致细胞的钙瞬变.星形胶质细胞中GABAB受体介导的钙信号在出生后海马网络发育完成时优先出现.%GABA (γ-aminobutyric acid) is a transmitter with dual action. Whereas it excites neurons during the first week of postnatal development, it represents the major inhibitory transmitter in the mature brain. GABA also activates astrocytes by binding to ionotropic (GABAA) and metabotropic (GABAB) receptors. This results in glial calcium transients which can induce the release of gliotransmitters, rendering GABA an important mediator of neuron-glia interaction. Using whole-cell patch-clamp and ratiometric calcium imaging in hippocampal slices from rats at postnatal days 3~34, we have analyzed the developmental profile as well as the cellular mechanisms of calcium signals induced by

  2. Riboflavin and vitamin E increase brain calcium and antioxidants, and microsomal calcium-ATP-ase values in rat headache models induced by glyceryl trinitrate.

    Science.gov (United States)

    Bütün, Ayşe; Nazıroğlu, Mustafa; Demirci, Serpil; Çelik, Ömer; Uğuz, Abdulhadi Cihangir

    2015-04-01

    The essential use of riboflavin is the prevention of migraine headaches, although its effect on migraines is considered to be associated with the increased mitochondrial energy metabolism. Oxidative stress is also important in migraine pathophysiology. Vitamin E is a strong antioxidant in nature and its analgesic effect is not completely clear in migraines. The current study aimed to investigate the effects of glyceryl trinitrate (GTN)-sourced exogen nitric oxide (NO), in particular, and also riboflavin and/or vitamin E on involved in the headache model induced via GTN-sourced exogen NO on oxidative stress, total brain calcium levels, and microsomal membrane Ca(2+)-ATPase levels. GTN infusion is a reliable method to provoke migraine-like headaches in experimental animals and humans. GTN resulted in a significant increase in brain cortex and microsomal lipid peroxidation levels although brain calcium, vitamin A, vitamin C, and vitamin E, and brain microsomal-reduced glutathione (GSH), glutathione peroxidase (GSH-Px), and plasma-membrane Ca(2+)-ATPase values decreased through GTN. The lipid peroxidation, GSH, vitamin A, β-carotene, vitamin C, and vitamin E, and calcium concentrations, GSH-Px, and the Ca(2+)-ATPase activities were increased both by riboflavin and vitamin E treatments. Brain calcium and vitamin A concentrations increased through riboflavin only. In conclusion, riboflavin and vitamin E had a protective effect on the GTN-induced brain injury by inhibiting free radical production, regulation of calcium-dependent processes, and supporting the antioxidant redox system. However, the effects of vitamin E on the values seem more important than in riboflavin.

  3. Concurrence of replicative senescence and elevated expression of p16(INK4A) with subculture-induced but not calcium-induced differentiation in normal human oral keratinocytes.

    Science.gov (United States)

    Lee, G; Park, B S; Han, S E; Oh, J E; You, Y O; Baek, J H; Kim, G S; Min, B M

    2000-10-01

    Primary normal human oral keratinocytes (NHOKs) undergo differentiation in the presence of calcium concentrations higher than 0.15 mM in vitro, which is useful in investigating the mechanisms involved in the differentiation of epithelial cells. Serial subculture of NHOKs to the postmitotic stage also induces terminal differentiation. However, the detailed mechanisms of both differentiation processes remain substantially unknown. To investigate the molecular differences in these processes, NHOKs were induced to differentiate by exposure to 1.2 mM of calcium and by serial subculture to the postmitotic stage. To study whether the cells were induced to differentiate and to undergo replicative senescence, the amount of cellular involucrin and the expression of senescence-associated beta-galactosidase (SA-beta-gal) were measured respectively. The expression of replicative senescence-associated genes and the activity of telomerase from the differentiated cells were also determined. Both calcium treatment and serial subculture to the postmitotic stage notably elevated the cellular involucrin. The percentage of SA-beta-gal-positive cells was significantly elevated by the continued subculture, but such changes were not observed in keratinocytes exposed to calcium. The concentration of cellular p16(INK4A) protein was progressively increased by the continued subculture but was not changed by calcium treatment. On the other hand, the concentrations of cellular p53 were similar in both differentiation processes. However, telomerase activity was lost in NHOKs that had undergone differentiation by both calcium treatment and serial subculture. The results indicate that calcium-induced differentiation of NHOKs has similar characteristics to their serial subculture-induced differentiation, but that the differentiation processes are not identical, because calcium-induced differentiation does not concur with either replicative senescence or the gradually increased concentration of p16

  4. Opposing regulation of histamine-induced calcium signaling by sodium selenite and ebselen via alterations of thiol redox status.

    Science.gov (United States)

    Zhang, Huihui; Zhong, Liangwei

    2010-01-25

    Elevated blood histamine plays a role in the pathogenesis of atherosclerosis. Calcium signaling mediates histamine action in endothelial cells. Selenium (Se) is a dietary essential trace element for humans. Se compounds in different oxidation states were found to exhibit an opposing effect on the histamine-induced calcium signaling in the ECV304 cell line. When Se in the form of sodium selenite was added in the cell culture, the reactivity of the histamine H(1)-receptor was increased as reported in our previous paper. We here show that as a culture supplement, sodium selenite enhanced the activity of selenoprotein thioredoxin reductase (TrxR) and the calcium response to histamine stimulation, which were reversed by treating the cells with gold thioglucose, a nucleophilic drug that selectively modifies thiolate/selenolate groups. Sodium selenite most likely caused a reductive shift in the thiol/disulfide redox balance through increasing TrxR activity. In contrast, when the cells were treated with Se in the form of ebselen, a thiol oxidant with peroxidase-like activity, histamine-induced calcium release and calcium entry were significantly suppressed. This effect appeared related to the thiol-directed modification rather than the peroxidase-like activity of ebselen, because this inhibitory effect was not replicated by increasing cellular peroxidase activity. Thus, the opposing effects of sodium selenite and ebselen on histamine-induced calcium signaling are achieved, at least in part, through their opposite actions in modulating the thiol/disulfide redox state.

  5. Image-based Modeling of Biofilm-induced Calcium Carbonate Precipitation

    Science.gov (United States)

    Connolly, J. M.; Rothman, A.; Jackson, B.; Klapper, I.; Cunningham, A. B.; Gerlach, R.

    2013-12-01

    Pore scale biological processes in the subsurface environment are important to understand in relation to many engineering applications including environmental contaminant remediation, geologic carbon sequestration, and petroleum production. Specifically, biofilm induced calcium carbonate precipitation has been identified as an attractive option to reduce permeability in a lasting way in the subsurface. This technology may be able to replace typical cement-based grouting in some circumstances; however, pore-scale processes must be better understood for it to be applied in a controlled manor. The work presented will focus on efforts to observe biofilm growth and ureolysis-induced mineral precipitation in micro-fabricated flow cells combined with finite element modelling as a tool to predict local chemical gradients of interest (see figure). We have been able to observe this phenomenon over time using a novel model organism that is able to hydrolyse urea and express a fluorescent protein allowing for non-invasive observation over time with confocal microscopy. The results of this study show the likely existence of a wide range of local saturation indices even in a small (1 cm length scale) experimental system. Interestingly, the locations of high predicted index do not correspond to the locations of higher precipitation density, highlighting the need for further understanding. Figure 1 - A micro-fabricated flow cell containing biofilm-induced calcium carbonate precipitation. (A) Experimental results: Active biofilm is in green and dark circles are calcium carbonate crystals. Note the channeling behavior in the top of the image, leaving a large hydraulically inactive area in the biofilm mass. (B) Finite element model: The prediction of relative saturation of calcium carbonate (as calcite). Fluid enters the system at a low saturation state (blue) but areas of high supersaturation (red) are predicted within the hydraulically inactive area in the biofilm. If only effluent

  6. Calcium Carbonate Storage in Amorphous Form and Its Template-Induced Crystallization

    Energy Technology Data Exchange (ETDEWEB)

    Han, T Y; Aizenberg, J

    2007-08-31

    Calcium carbonate crystallization in organisms often occurs through the transformation from the amorphous precursor. It is believed that the amorphous phase could be temporarily stabilized and stored, until its templated transition to the crystalline form is induced. Here we develop a bio-inspired crystallization strategy that is based on the above mechanism. Amorphous calcium carbonate (ACC) spherulitic particles are formed and stabilized on a self-assembled monolayer (SAM) of hydroxy-terminated alkanethiols on Au surface. The ACC is stored as a reservoir for ions and is induced to crystallize on command by introducing a secondary surface that is functionalized with carboxylic acid-terminated SAM. This secondary surface acts as a template for oriented and patterned nucleation. Various oriented crystalline arrays and micropatterned films are formed. We also show that the ACC phase can be doped with foreign ions (e.g. Mg) and organic molecules (e.g. dyes) and that these dopants later function as growth modifiers of calcite crystals and become incorporated into the crystals during the transformation process of ACC to calcite. We believe that our strategy opens the way of using a stabilized amorphous phase as a versatile reservoir system that can be converted in a highly controlled fashion to a crystalline form upon contacting the nucleating template.

  7. Deposition of calcium carbonate films by a polymer-induced liquid-precursor (PILP) process

    Science.gov (United States)

    Gower, Laurie B.; Odom, Damian J.

    2000-03-01

    A polypeptide additive has been used to transform the solution crystallization of calcium carbonate to a solidification process of a liquid-phase mineral precursor. In situ observations reveal that polyaspartate induces liquid-liquid phase separation of droplets of a mineral precursor. The droplets deposit on the substrate and coalesce to form a coating, which then solidifies into calcitic tablets and films. Transition bars form during the amorphous to crystalline transition, leading to sectorization of calcite tablets, and the defect textures and crystal morphologies are atypical of solution grown crystals. The formation of nonequilibrium crystal morphologies using an acidic polypeptide may have implications in the field of biomineralization, and the environmentally friendly aspects of this polymer-induced liquid-precursor (PILP) process may offer new techniques for aqueous-based processing of ceramic films, coatings, and particulates.

  8. Chiral acidic amino acids induce chiral hierarchical structure in calcium carbonate

    Science.gov (United States)

    Jiang, Wenge; Pacella, Michael S.; Athanasiadou, Dimitra; Nelea, Valentin; Vali, Hojatollah; Hazen, Robert M.; Gray, Jeffrey J.; McKee, Marc D.

    2017-04-01

    Chirality is ubiquitous in biology, including in biomineralization, where it is found in many hardened structures of invertebrate marine and terrestrial organisms (for example, spiralling gastropod shells). Here we show that chiral, hierarchically organized architectures for calcium carbonate (vaterite) can be controlled simply by adding chiral acidic amino acids (Asp and Glu). Chiral, vaterite toroidal suprastructure having a `right-handed' (counterclockwise) spiralling morphology is induced by L-enantiomers of Asp and Glu, whereas `left-handed' (clockwise) morphology is induced by D-enantiomers, and sequentially switching between amino-acid enantiomers causes a switch in chirality. Nanoparticle tilting after binding of chiral amino acids is proposed as a chiral growth mechanism, where a `mother' subunit nanoparticle spawns a slightly tilted, consequential `daughter' nanoparticle, which by amplification over various length scales creates oriented mineral platelets and chiral vaterite suprastructures. These findings suggest a molecular mechanism for how biomineralization-related enantiomers might exert hierarchical control to form extended chiral suprastructures.

  9. Rhinovirus-induced calcium flux triggers NLRP3 and NLRC5 activation in bronchial cells.

    Science.gov (United States)

    Triantafilou, Kathy; Kar, Satwik; van Kuppeveld, Frank J M; Triantafilou, Martha

    2013-12-01

    Human rhinoviruses have been linked with underlying lung disorders, such as asthma and chronic obstructive pulmonary disease, in children and adults. However, the mechanism of virus-induced airway inflammation is poorly understood. In this study, using virus deletion mutants and silencing for nucleotide-binding oligomerization domain-like receptors (NLRs), we show that the rhinovirus ion channel protein 2B triggers NLRP3 and NLRC5 inflammasome activation and IL-1β secretion in bronchial cells. 2B protein targets the endoplasmic reticulum and Golgi and induces Ca(2+) reduction in these organelles, thereby disturbing the intracellular calcium homeostasis. NLRP3 and NLRC5 act in a cooperative manner during the inflammasome assembly by sensing intracellular Ca(2+) fluxes and trigger IL-1β secretion. These results reveal for the first time that human rhinovirus infection in primary bronchial cells triggers inflammasome activation.

  10. Beef meat promotion of dimethylhydrazine-induced colorectal carcinogenesis biomarkers is suppressed by dietary calcium.

    Science.gov (United States)

    Pierre, Fabrice; Santarelli, Raphaëlle; Taché, Sylviane; Guéraud, Françoise; Corpet, Denis E

    2008-05-01

    Red meat consumption is associated with increased risk of colorectal cancer. We have previously shown that haemin, Hb and red meat promote carcinogen-induced preneoplastic lesions: aberrant crypt foci (ACF) and mucin-depleted foci (MDF) in rats. We have also shown that dietary Ca, antioxidant mix and olive oil inhibit haemin-induced ACF promotion, and normalize faecal lipoperoxides and cytotoxicity. Here we tested if these strategies are effective also against red meat promotion in dimethylhydrazine-induced rats. Three diets with 60 % beef meat were supplemented with calcium phosphate (31 g/kg), antioxidant agents (rutin and butylated hydroxyanisole, 0.05 % each) and olive oil (5 %). ACF, MDF, faecal water cytotoxicity, thiobarbituric acid reactive substances (TBARS) and urinary 1,4-dihydroxynonane mercapturic acid (DHN-MA) were measured. Beef meat diet increased the number of ACF (+30 %) and MDF (+100 %) (P Promotion was associated with increased faecal water TBARs ( x 4) and cytotoxicity ( x 2), and urinary DHN-MA excretion ( x 15). Ca fully inhibited beef meat-induced ACF and MDF promotion, and normalized faecal TBARS and cytotoxicity, but did not reduce urinary DHN-MA. Unexpectedly, high-calcium control diet-fed rats had more MDF and ACF in the colon than low-Ca control diet-fed rats. Antioxidant mix and olive oil did not normalize beef meat promotion nor biochemical factors. The results confirm that haem causes promotion of colon carcinogenesis by red meat. They suggest that Ca can reduce colorectal cancer risk in meat-eaters. The results support the concept that toxicity associated with the excess of a useful nutrient may be prevented by another nutrient.

  11. The calcium-sensing receptor participates in testicular damage in streptozotocin-induced diabetic rats.

    Science.gov (United States)

    Kong, Wei-Yuan; Tong, Li-Quan; Zhang, Hai-Jun; Cao, Yong-Gang; Wang, Gong-Chen; Zhu, Jin-Zhi; Zhang, Feng; Sun, Xue-Ying; Zhang, Tie-Hui; Zhang, Lin-Lin

    2016-01-01

    Male infertility caused by testicular damage is one of the complications of diabetes mellitus. The calcium-sensing receptor (CaSR) is expressed in testicular tissues and plays a pivotal role in calcium homeostasis by activating cellular signaling pathways, but its role in testicular damage induced by diabetes remains unclear. A diabetic model was established by a single intraperitoneal injection of streptozotocin (STZ, 40 mg kg-1 ) in Wistar rats. Animals then received GdCl 3 (an agonist of CaSR, 8.67 mg kg-1 ), NPS-2390 (an antagonist of CaSR, 0.20 g kg-1 ), or a combination of both 2 months after STZ injection. Diabetic rats had significantly lower testes weights and serum levels of testosterone compared to healthy rats, indicating testicular damage and dysfunction in STZ-induced diabetic rats. Compared with healthy controls, the testicular tissues of diabetic rats overexpressed the CaSR protein and had higher levels of malondialdehyde (MDA), lower superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, and higher numbers of apoptotic germ cells. The testicular tissues from diabetic rats also expressed lower levels of Bcl-2 and higher levels of Bax and cleaved caspase-3 in addition to higher phosphorylation rates of c-Jun NH 2 -terminal protein kinase (JNK), p38, and extracellular signaling-regulated kinase (ERK) 1/2. The above parameters could be further increased or aggravated by the administration of GdCl 3 , but could be attenuated by injection of NPS-2390. In conclusion, the present results indicate that CaSR activation participates in diabetes-induced testicular damage, implying CaSR may be a potential target for protective strategies against diabetes-induced testicular damage and could help to prevent infertility in diabetic men.

  12. The calcium-sensing receptor participates in testicular damage in streptozotocin-induced diabetic rats

    Directory of Open Access Journals (Sweden)

    Wei-Yuan Kong

    2016-01-01

    Full Text Available Male infertility caused by testicular damage is one of the complications of diabetes mellitus. The calcium-sensing receptor (CaSR is expressed in testicular tissues and plays a pivotal role in calcium homeostasis by activating cellular signaling pathways, but its role in testicular damage induced by diabetes remains unclear. A diabetic model was established by a single intraperitoneal injection of streptozotocin (STZ, 40 mg kg−1 in Wistar rats. Animals then received GdCl 3 (an agonist of CaSR, 8.67 mg kg−1 , NPS-2390 (an antagonist of CaSR, 0.20 g kg−1 , or a combination of both 2 months after STZ injection. Diabetic rats had significantly lower testes weights and serum levels of testosterone compared to healthy rats, indicating testicular damage and dysfunction in STZ-induced diabetic rats. Compared with healthy controls, the testicular tissues of diabetic rats overexpressed the CaSR protein and had higher levels of malondialdehyde (MDA, lower superoxide dismutase (SOD and glutathione peroxidase (GSH-Px activity, and higher numbers of apoptotic germ cells. The testicular tissues from diabetic rats also expressed lower levels of Bcl-2 and higher levels of Bax and cleaved caspase-3 in addition to higher phosphorylation rates of c-Jun NH 2 -terminal protein kinase (JNK, p38, and extracellular signaling-regulated kinase (ERK 1/2. The above parameters could be further increased or aggravated by the administration of GdCl 3 , but could be attenuated by injection of NPS-2390. In conclusion, the present results indicate that CaSR activation participates in diabetes-induced testicular damage, implying CaSR may be a potential target for protective strategies against diabetes-induced testicular damage and could help to prevent infertility in diabetic men.

  13. Mechanism of histamine release from rat mast cells induced by the ionophore A23187: effects of calcium and temperature

    DEFF Research Database (Denmark)

    Johansen, Torben

    1978-01-01

    1 The mechanism of histamine release from a pure population of rat mast cells induced by the lipid soluble antibiotic, A23187, has been studied and compared with data for anaphylactic histamine release reported in the literature. 2 Histamine release induced by A23187 in the presence of calcium 10......(-3) mol/l was completed in 10 minutes. By preincubation of the mast cells with A23187 for 10 min in the absence of calcium the histamine release induced by calcium, 10(-3) mol/l or 5 x 10(-3) mol/l, was completed in 90 s and 45 s, respectively. 3 A23187-induced histamine release was maximal with calcium...... 10(-3) mol/l when the cells were incubated at 33 to 39 degrees C for 10 minutes. 4 The cellular mechanism, which was stimulated by A23187 and calcium for the release of histamine, was irreversibly inactivated by incubation at 45 degrees C. 5 An inhibition of energy metabolism was excluded...

  14. Calcium/calmodulin-dependent protein kinase IV mediates acute nicotine-induced antinociception in acute thermal pain tests.

    Science.gov (United States)

    Jackson, Kia J; Damaj, Mohamad I

    2013-12-01

    Calcium-activated second messengers such as calcium/calmodulin-dependent protein kinase II have been implicated in drug-induced antinociception. The less abundant calcium-activated second messenger, calcium/calmodulin-dependent protein kinase IV (CaMKIV), mediates emotional responses to pain and tolerance to morphine analgesia but its role in nicotine-mediated antinociception is currently unknown. The goal of this study was to evaluate the role of CaMKIV in the acute effects of nicotine, primarily acute nicotine-induced antinociception. CaMKIV knockout (-/-), heterozygote (+/-), and wild-type (+/+) mice were injected with various doses of nicotine and evaluated in a battery of tests, including the tail-flick and hot-plate tests for antinociception, body temperature, and locomotor activity. Our results show a genotype-dependent reduction in tail-flick and hot-plate latency in CaMKIV (+/-) and (-/-) mice after acute nicotine treatment, whereas no difference was observed between genotypes in the body temperature and locomotor activity assessments. The results of this study support a role for CaMKIV in acute nicotine-induced spinal and supraspinal pain mechanisms, and further implicate involvement of calcium-dependent mechanisms in drug-induced antinociception.

  15. The role of calcium in growth induced by indole-3-acetic acid and gravity in the leaf-sheath pulvinus of oat (Avena sativa)

    Science.gov (United States)

    Brock, T. G.; Burg, J.; Ghosheh, N. S.; Kaufman, P. B.

    1992-01-01

    Leaf-sheath pulvini of excised segments from oat (Avena sativa L.) were induced to grow by treatment with 10 micromoles indole-3-acetic acid (IAA), gravistimulation, or both, and the effects of calcium, EGTA, and calcium channel blockers on growth were evaluated. Unilaterally applied calcium (10 mM CaCl2) significantly inhibited IAA-induced growth in upright pulvini but had no effect on growth induced by either gravity or gravity plus IAA. Calcium alone had no effect on upright pulvini. The calcium chelator EGTA alone (10 mM) stimulated growth in upright pulvini. However, EGTA had no effect on either IAA- or gravity-induced growth but slightly diminished growth in IAA-treated gravistimulated pulvini. The calcium channel blockers lanthanum chloride (25 mM), verapamil (2.5 mM), and nifedipine (2.5 mM) greatly inhibited growth as induced by IAA (> or = 50% inhibition) or IAA plus gravity (20% inhibition) but had no effect on gravistimulated pulvini. Combinations of channel blockers were similar in effect on IAA action as individual blockers. Since neither calcium ions nor EGTA significantly affected the graviresponse of pulvini, we conclude that apoplastic calcium is unimportant in leaf-sheath pulvinus gravitropism. The observation that calcium ions and calcium channel blockers inhibit IAA-induced growth, but have no effect on gravistimulated pulvini, further supports previous observations that gravistimulation alters the responsiveness of pulvini to IAA.

  16. Calcium paradox induces apoptosis in the isolated perfused Rana ridibunda heart: involvement of p38-MAPK and calpain.

    Science.gov (United States)

    Aggeli, Ioanna-Katerina; Zacharias, Triantafyllos; Papapavlou, Georgia; Gaitanaki, Catherine; Beis, Isidoros

    2013-12-01

    "Calcium paradox" as a term describes the deleterious effects conferred to a heart perfused with a calcium-free solution followed by repletion, including loss of mechanical activity and sarcomere disruption. Given that the signaling mechanisms triggered by calcium paradox remain elusive, in the present study, we tried to investigate them in the isolated perfused heart from Rana ridibunda. Calcium paradox was found to markedly activate members of the MAPKs (p43-ERK, JNKs, p38-MAPK). In addition to lactate dehydrogenase (LDH) release in the perfusate (indicative of necrosis), we also confirmed the occurrence of apoptosis by using the TUNEL assay and identifying poly(ADP-ribose) polymerase (PARP) fragmentation and upregulated Bax expression. Furthermore, using MDL28170 (a selective calpain inhibitor), a role for this protease was revealed. In addition, various divalent cations were shown to exert a protective effect against the calcium paradox. Interestingly, SB203580, a p38-MAPK inhibitor, alleviated calcium-paradox-conferred apoptosis. This result indicates that p38-MAPK plays a pro-apoptotic role, contributing to the resulting myocardial dysfunction and cell death. To our knowledge, this is the first time that the calcium paradox has been shown to induce apoptosis in amphibians, with p38-MAPK and calpain playing significant roles.

  17. Role of arachidonic acid in hyposmotic membrane stretch-induced increase in calcium-activated potassium currents in gastric myocytes

    Institute of Scientific and Technical Information of China (English)

    Meng YANG; Wen-xie XU; Xing-lan LI; Hui-ying XU; Jia-bin SUN; Bin MEI; Hai-feng ZHENG; Lian-hua PIAO; De-gang XING; Zhai-liu LI

    2005-01-01

    Aim: To study effects of arachidonic acid (AA) and its metabolites on the hyposmotic membrane stretch-induced increase in calcium-activated potassium currents (IKCa) in gastric myocytes. Methods: Membrane currents were recorded by using a conventional whole cell patch-clamp technique in gastric myocytes isolated with collagenase. Results: Hyposmotic membrane stretch and AA increased both IK(Ca) and spontaneous transient outward currents significantly.Exogenous AA could potentiate the hyposmotic membrane stretch-induced increase in IK(Ca). The hyposmotic membrane stretch-induced increase in IK(Ca) was significantly suppressed by dimethyleicosadienoic acid (100 μmol/L in pipette solution), an inhibitor of phospholipase A2. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly suppressed AA and hyposmotic membrane stretch-induced increases in IK(Ca). External calcium-free or gadolinium chloride, a blocker of stretch-activated channels, blocked the AA-induced increase in IK(Ca) significantly, but it was not blocked by nicardipine, an L-type calcium channel blocker. Ryanodine, a calcium-induced calcium release agonist, completely blocked the AA-induced increase in IK(Ca); however, heparin, a potent inhibitor of inositol triphosphate receptor, did not block the AA-induced increase in IK(Ca). Conclusion:Hyposmotic membrane stretch may activate phospholipase A2, which hydrolyzes membrane phospholipids to ultimately produce AA; AA as a second messenger mediates Ca2+ influx, which triggers Ca2+-induced Ca2+ release and elicits activation of IK(Ca) in gastric antral circular myocytes of the guinea pig.

  18. Correlation between oxidative stress and alteration of intracellular calcium handling in isoproterenol-induced myocardial infarction.

    Science.gov (United States)

    Díaz-Muñoz, Mauricio; Alvarez-Pérez, Marco Antonio; Yáñez, Lucía; Vidrio, Susana; Martínez, Lidia; Rosas, Gisele; Yáñez, Mario; Ramírez, Sotero; de Sánchez, Victoria Chagoya

    2006-09-01

    Myocardial Ca(2+) overload and oxidative stress are well documented effects associated to isoproterenol (ISO)-induced myocardial necrosis, but information correlating these two issues is scarce. Using an ISO-induced myocardial infarction model, 3 stages of myocardial damage were defined: pre-infarction (0-12 h), infarction (12-24 h) and post-infarction (24-96 h). Alterations in Ca(2+) homeostasis and oxidative stress were studied in mitochondria, sarcoplasmic reticulum and plasmalemma by measuring the Ca(2+) content, the activity of Ca(2+) handling proteins, and by quantifying TBARs, nitric oxide (NO) and oxidative protein damage (changes in carbonyl and thiol groups). Free radicals generated system, antioxidant enzymes and oxidative stress (GSH/GSSG ratio) were also monitored at different times of ISO-induced cardiotoxicity. The Ca(2+) overload induced by ISO was counterbalanced by a diminution in the ryanodine receptor activity and the Na(+)-Ca(+2) exchanger as well as by the increase in both calcium ATPases activities (vanadate- and thapsigargine-sensitive) and mitochondrial Ca(2+) uptake during pre-infarction and infarction stages. Pro-oxidative reactions and antioxidant defences during the 3 stages of cardiotoxicity were observed, with maximal oxidative stress during the infarction. Significant correlations were found among pro-oxidative reactions with plasmalemma and sarcoplasmic reticulum Ca(2+) ATPases, and ryanodine receptor activities at the onset and development of ISO-induced infarction. These findings could be helpful in the design of antioxidant therapies in this pathology.

  19. Structural basis for calcium-induced inhibition of rhodopsin kinase by recoverin.

    Science.gov (United States)

    Ames, James B; Levay, Konstantin; Wingard, Jennifer N; Lusin, Jacqueline D; Slepak, Vladlen Z

    2006-12-01

    Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca(2+)-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca(2+)-bound recoverin in the absence of target (<1.8A root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca(2+) is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca(2+)-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca(2+)-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca(2+).

  20. Identification and characterization of calcium sparks in cardiomyocytes derived from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Guang Qin Zhang

    Full Text Available INTRODUCTION: Ca2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C coupling in mature cardiomyocytes. Human induced pluripotent stem cell (hiPSC-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. However, it is unclear if they share similar calcium handling property. We hypothesized that Ca2+ sparks in human induced pluripotent stem cell (hiPSCs-derived cardiomyocytes (hiPSC-CMs may display unique structural and functional properties than mature adult cardiomyocytes. METHODS AND RESULTS: Ca2+ sparks in hiPSC-CMs were recorded with Ca2+ imaging assay with confocal laser scanning microscopy. Those sparks were stochastic with a tendency of repetitive occurrence at the same site. Nevertheless, the spatial-temporal properties of Ca2+ spark were analogous to that of adult CMs. Inhibition of L-type Ca2+ channels by nifedipine caused a 61% reduction in calcium spark frequency without affecting amplitude of those sparks and magnitude of caffeine releasable sarcoplasmic reticulum (SR Ca2+ content. In contrast, high extracellular Ca2+ and ryanodine increased the frequency, full width at half maximum (FWHM and full duration at half maximum (FDHM of spontaneous Ca2+ sparks. CONCLUSIONS: For the first time, spontaneous Ca2+ sparks were detected in hiPSC-CMs. The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR mechanism. However, focal repetitive sparks originated from the same intracellular organelle could reflect an immature status of the hiPSC-CMs.

  1. High Glucose Enhances Isoflurane-Induced Neurotoxicity by Regulating TRPC-Dependent Calcium Influx.

    Science.gov (United States)

    Liu, ZhongJie; Ma, ChangQing; Zhao, Wei; Zhang, QingGuo; Xu, Rui; Zhang, HongFei; Lei, HongYi; Xu, ShiYuan

    2017-01-06

    Isoflurane is a commonly used inhalational anesthetic that can induce neurotoxicity via elevating cytosolic calcium (Ca(2+)). High glucose regulates the expression of a family of non-selective cation channels termed transient receptor potential canonical (TRPC) channels that may contribute to Ca(2+) influx. In the present study, we investigated whether high glucose enhances isoflurane-induced neurotoxicity by regulating TRPC-dependent Ca(2+) influx. First, we evaluated toxic damage in mice primary cultured hippocampal neurons and human neuroblastoma cells (SH-SY5Y cells) after hyperglycemia and isoflurane exposure. Next, we investigated cytosolic Ca(2+) concentrations, TRPC mRNA expression levels and tested the effect of the TRPC channel blocker SKF96365 on cytosolic Ca(2+) levels in cells treated with high glucose or/and isoflurane. Finally, we employed knocked down TRPC6 to demonstrate the role of TRPC in high glucose-mediated enhancement of isoflurane-induced neurotoxicity. The results showed that high glucose could enhance isoflurane-induecd toxic damage in primary hippocampal neurons and SH-SY5Y cells. High glucose enhanced the isoflurane-induced increase of cytosolic Ca(2+) in SH-SY5Y cells. High glucose elevated TRPC mRNA expression, especially that of TRPC6. SKF96365 and knock down of TRPC6 were able to inhibit the high glucose-induced increase of cytosolic Ca(2+) and decrease isoflurane-induced neurotoxicity in SH-SY5Y cells cultured with high glucose. Our findings indicate that high glucose could elevate TRPC expression, thus increasing Ca(2+) influx and enhancing isoflurane-induced neurotoxicity.

  2. Calcium-induced folding and stabilization of the Pseudomonas aeruginosa alkaline protease.

    Science.gov (United States)

    Zhang, Liang; Conway, James F; Thibodeau, Patrick H

    2012-02-03

    Pseudomonas aeruginosa is an opportunistic pathogen that contributes to the mortality of immunocompromised individuals and patients with cystic fibrosis. Pseudomonas infection presents clinical challenges due to its ability to form biofilms and modulate host-pathogen interactions through the secretion of virulence factors. The calcium-regulated alkaline protease (AP), a member of the repeats in toxin (RTX) family of proteins, is implicated in multiple modes of infection. A series of full-length and truncation mutants were purified for structural and functional studies to evaluate the role of Ca(2+) in AP folding and activation. We find that Ca(2+) binding induces RTX folding, which serves to chaperone the folding of the protease domain. Subsequent association of the RTX domain with an N-terminal α-helix stabilizes AP. These results provide a basis for the Ca(2+)-mediated regulation of AP and suggest mechanisms by which Ca(2+) regulates the RTX family of virulence factors.

  3. Monitoring of hardening and hygroscopic induced strains in a calcium phosphate bone cement using FBG sensor.

    Science.gov (United States)

    Bimis, A; Karalekas, D; Bouropoulos, N; Mouzakis, D; Zaoutsos, S

    2016-07-01

    This study initially deals with the investigation of the induced strains during hardening stage of a self-setting calcium phosphate bone cement using fiber-Bragg grating (FBG) optical sensors. A complementary Scanning Electron Microscopy (SEM) investigation was also conducted at different time intervals of the hardening period and its findings were related to the FBG recordings. From the obtained results, it is demonstrated that the FBG response is affected by the microstructural changes taking place when the bone cement is immersed into the hardening liquid media. Subsequently, the FBG sensor was used to monitor the absorption process and hygroscopic response of the hardened and dried biocement when exposed to a liquid/humid environment. From the FBG-based calculated hygric strains as a function of moisture concentration, the coefficient of moisture expansion (CME) of the examined bone cement was obtained, exhibiting two distinct linear regions.

  4. Bradykinin and histamine-induced cytosolic calcium increase in capillary endothelial cells of bovine adrenal medulla.

    Science.gov (United States)

    Vinet, Raúl; Cortés, Magdalena P; Alvarez, Rocío; Delpiano, Marco A

    2014-09-01

    We have assessed the effect of bradykinin and histamine on the cytosolic free calcium concentration ([Ca(2+)]i ) of bovine adrenal medulla capillary endothelial cells (BAMCECs). To measure [Ca(2+)]i changes in BAMCECs the intracellular fluorescent probe, fluo-3 AM, was used. Bradykinin (3 µM) produced a transient monophasic increase in [Ca(2+)]i , which was depressed by B1650 (0.1 µM), a B2-bradykinin receptor antagonist (D-Arg-[Hyp(3), Thi(5,8) , D-Phe(7)]-Bradykinin). Similarly, increase in [Ca(2+)]i induced by histamine was also depressed by tripolidine (0.1 µM), an H1-histamine receptor antagonist. [Ca(2+)]i increase induced by both agonists was unaffected in the absence of extracellular Ca(2+) or presence of antagonists of voltage operated Ca(2+) channels (VOCCs). Thapsigargin (1 µM) did not abolish the increase of [Ca(2+)]i produced by bradykinin, but abolished that of histamine. In contrast, caffeine (100 µM), abolished the [Ca(2+)]i response induced by bradykinin (3 µM), but did not affect the [Ca(2+)]i increase induced by histamine (100 µM). The results indicate the presence of B2 bradykinin- and H1 histamine-receptors in BAMCECs. Liberation of Ca(2+) induced by both agonists occurs through 2 different intracellular mechanisms. While bradykinin activates a sarco(endo) plasmic reticulum (SER) containing a SER Ca(2+) -ATPase (SERCA) thapsigargin-insensitive, histamine activates a SER containing a SERCA thapsigargin-sensitive. We suggest that the increase in [Ca(2+)]i induced by bradykinin and histamine could be of physiological relevance, modulating adrenal gland microcirculation.

  5. G Protein-induced Trafficking of Voltage-dependent Calcium Channels

    National Research Council Canada - National Science Library

    Eugene Tombler; Nory Jun Cabanilla; Paul Carman; Natasha Permaul; John J. Hall; Ryan W. Richman; Jessica Lee; Jennifer Rodriguez; Dan P. Felsenfeld; Robert F. Hennigan; María A. Diversé-Pierluissi

    2006-01-01

    .... Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane...

  6. Long-term prolactin exposure differentially stimulated the transcellular and solvent drag-induced calcium transport in the duodenum of ovariectomized rats.

    Science.gov (United States)

    Tudpor, Kukiat; Charoenphandhu, Narattaphol; Saengamnart, Wasana; Krishnamra, Nateetip

    2005-12-01

    Prolactin, having been shown to stimulate transcellular active and solvent drag-induced calcium transport in the duodenum of female rats, was postulated to improve duodenal calcium transport in estrogen-deficient rats. The aim of the present study was, therefore, to demonstrate the effects of long-term prolactin exposure produced by anterior pituitary (AP) transplantation on the duodenal calcium transport in young (9-week-old) and adult (22-week-old) ovariectomized rats. We found that ovariectomy did not alter the transcellular active duodenal calcium transport in young and adult rats fed normal calcium diet (1.0% w/w Ca) but decreased the solvent drag-induced duodenal calcium transport from 75.50 +/- 10.12 to 55.75 +/- 4.77 nmol.hr(-1).cm(-2) (P calcium transport in young and adult AP-grafted ovariectomized rats fed with normal calcium diet by more than 2-fold from 7.56 +/- 0.79 to 16.54 +/- 2.05 (P calcium transport in young rats was enhanced by prolactin from 95.51 +/- 10.64 to 163.20 +/- 18.03 nmol.hr(-1).cm(-2) (P calcium supplement has been widely used to improve calcium balance in estrogen-deficient animals, the effect of a high-calcium diet (2.0% w/w Ca) was also investigated. The results showed that stimulatory action of long-term prolactin on the transcellular active duodenal calcium transport in both young and adult rats was diminished after being fed a high-calcium diet. The same diet also abolished prolactin-enhanced solvent drag-induced duodenal calcium transport in young and further decreased that in adult AP-grafted ovariectomized rats. We concluded that the solvent drag-induced duodenal calcium transport in adult rats was decreased after ovariectomy. Long-term prolactin exposure stimulated the transcellular active duodenal calcium transport in both young and adult rats whereas enhancing the solvent drag-induced duodenal calcium transport only in young rats. Effects of prolactin were abolished by a high-calcium diet.

  7. Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast

    DEFF Research Database (Denmark)

    Busti, Stefano; Mapelli, Valeria; Tripodi, Farida

    2016-01-01

    Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regula...

  8. Blockade of L-type calcium channel in myocardium and calcium-induced contractions of vascular smooth muscle by CPU 86017.

    Science.gov (United States)

    Dai, De-zai; Hu, Hui-juan; Zhao, Jing; Hao, Xue-mei; Yang, Dong-mei; Zhou, Pei-ai; Wu, Cai-hong

    2004-04-01

    To assess the blockade by CPU 86017 on the L-type calcium channels in the myocardium and on the Ca(2+)-related contractions of vascular smooth muscle. The whole-cell patch-clamp was applied to investigate the blocking effect of CPU 86017 on the L-type calcium current in isolated guinea pig myocytes and contractions by KCl or phenylephrine (Phe) of the isolated rat tail arteries were measured. Suppression of the L-type current of the isolated myocytes by CPU 86017 was moderate, in time- and concentration-dependent manner and with no influence on the activation and inactivation curves. The IC(50) was 11.5 micromol/L. Suppressive effect of CPU 86017 on vaso-contractions induced by KCl 100 mmol/L, phenylephrine 1 micromol/L in KH solution (phase 1), Ca(2+) free KH solution ( phase 2), and by addition of CaCl(2) into Ca(2+)-free KH solution (phase 3) were observed. The IC(50) to suppress vaso-contractions by calcium entry via the receptor operated channel (ROC) and voltage-dependent channel (VDC) was 0.324 micromol/L and 16.3 micromol/L, respectively. The relative potency of CPU 86017 to suppress vascular tone by Ca(2+) entry through ROC and VDC is 1/187 of prazosin and 1/37 of verapamil, respectively. The blocking effects of CPU 86017 on the L-type calcium channel of myocardium and vessel are moderate and non-selective. CPU 86017 is approximately 50 times more potent in inhibiting ROC than VDC.

  9. Blockade of L-type calcium channel in myocardium and calcium-induced contractions of vascular smooth muscle by by CPU 86017

    Institute of Scientific and Technical Information of China (English)

    De-zai DAI; Hui-juan HU; Jing ZHAO; Xue-mei HAO; Dong-mei YANG; Pei-ai ZHOU; Cai-hong WU

    2004-01-01

    AIM: To assess the blockade by CPU 86017 on the L-type calcium channels in the myocardium and on the Ca2+related contractions of vascular smooth muscle. METHODS: The whole-cell patch-clamp was applied to investigate the blocking effect of CPU 86017 on the L-type calcium current in isolated guinea pig myocytes and contractions by KC1 or phenylephrine (Phe) of the isolated rat tail arteries were measured. RESULTS: Suppression of the L-type current of the isolated myocytes by CPU 86017 was moderate, in time- and concentration-dependent manner and with no influence on the activation and inactivation curves. The IC50 was 11.5 μmol/L. Suppressive effect of CPU 86017 on vaso-contractions induced by KC1 100 mmol/L, phenylephrine I μmol/Lin KH solution (phase 1),Ca2+ free KH solution ( phase 2), and by addition of CaCI2 into Ca2+-free KH solution (phase 3) were observed. The IC50 to suppress vaso-contractions by calcium entry via the receptor operated channel (ROC) and Voltage-dependent channel (VDC) was 0.324 μmol/L and 16.3 μmol/L, respectively. The relative potency of CPU 86017 to suppress vascular tone by Ca2+ entry through ROC and VDC is 1/187 of prazosin and 1/37 of verapamil, respectively.CONCLUSION: The blocking effects of CPU 86017 on the L-type calcium channel of myocardium and vessel are moderate and non-selective. CPU 86017 is approximately 50 times more potent in inhibiting ROC than VDC.

  10. Allicin Induces Calcium and Mitochondrial Dysregulation Causing Necrotic Death in Leishmania.

    Directory of Open Access Journals (Sweden)

    María J Corral

    2016-03-01

    Full Text Available Allicin has shown antileishmanial activity in vitro and in vivo. However the mechanism of action underlying its antiproliferative effect against Leishmania has been virtually unexplored. In this paper, we present the results obtained in L.infantum and a mechanistic basis is proposed.Exposure of the parasites to allicin led to high Ca2+ levels and mitochondrial reactive oxygen species (ROS, collapse of the mitochondrial membrane potential, reduced production of ATP and elevation of cytosolic ROS. The incubation of the promastigotes with SYTOX Green revealed that decrease of ATP was not associated with plasma membrane permeabilization. Annexin V and propidium iodide (PI staining indicated that allicin did not induce phospholipids exposure on the plasma membrane. Moreover, DNA agarose gel electrophoresis and TUNEL analysis demonstrated that allicin did not provoke DNA fragmentation. Analysis of the cell cycle with PI staining showed that allicin induced cell cycle arrest in the G2/M phase.We conclude that allicin induces dysregulation of calcium homeostasis and oxidative stress, uncontrolled by the antioxidant defense of the cell, which leads to mitochondrial dysfunction and a bioenergetic catastrophe leading to cell necrosis and cell cycle arrest in the premitotic phase.

  11. Circular patterns of calcium oxalate crystals induced by defective Langmuir-Blodgett film

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The injury of the renal epithelial cell membrane can promote the nucleation of nascent crystals, as well as adhesion of crystals on it. It thus accelerates the formation of renal calculi. In this paper, the defective Langmuir-Blodgett(LB)films were used as a model system to simulate the injured renal epithelial cell membrane. The microcosmic structure of the defective LB film and the molecular mechanism of the effect of this film on nucleation, growth, deposited patterns and adhesion of calcium oxalate monohydrate(COM)were investigated. The circular defective domains were formed in dipalmitoylphosphatidylcholine(DPPC)LB film after the film was treated by potassium oxalate. These domains could induce ring-shaped patterns of COM crystals. In comparison, the LB film without pretreatment by potassium oxalate only induced random growth of hexagonal COM crystals. As the crystallization time increased, the size of COM crystals in the patterns increased, the crystal patterns changed from empty circles to solid circles, and the number of the circular patterns with small size(5-20μm)increased. The results would shed light on the molecular mechanism of urolithiasis induced by injury of the renal epithelial membrane at the molecular and supramolecular level.

  12. Circular patterns of calcium oxalate crystals induced by defective Langmuir-Blodgett film

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The injury of the renal epithelial cell membrane can promote the nucleation of nascent crystals, as well as adhesion of crystals on it. It thus accelerates the formation of renal calculi. In this paper, the defective Langmuir-Blodgett (LB) films were used as a model system to simulate the injured renal epithelial cell membrane. The microcosmic structure of the defective LB film and the molecular mechanism of the effect of this film on nucleation, growth, deposited patterns and adhesion of calcium oxalate monohydrate (COM) were investigated. The circular defective domains were formed in dipalmitoylphosphatidylcholine (DPPC) LB film after the film was treated by potassium oxalate. These domains could induce ring-shaped patterns of COM crystals. In comparison, the LB film without pretreatment by potassium oxalate only induced random growth of hexagonal COM crystals. As the crystallization time increased, the size of COM crystals in the patterns increased, the crystal patterns changed from empty circles to solid circles, and the number of the circular patterns with small size (5-20 μm) increased. The results would shed light on the molecular mechanism of urolithiasis induced by injury of the renal epithelial membrane at the molecular and supramolecular level.

  13. Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

    Energy Technology Data Exchange (ETDEWEB)

    Kozuka, M.; Ito, T.; Hirose, S.; Takahashi, K.; Hagiwara, H.

    1989-02-28

    Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using /sup 125/I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.

  14. Dopamine Induces LTP Differentially in Apical and Basal Dendrites through BDNF and Voltage-Dependent Calcium Channels

    Science.gov (United States)

    Navakkode, Sheeja; Sajikumar, Sreedharan; Korte, Martin; Soong, Tuck Wah

    2012-01-01

    The dopaminergic modulation of long-term potentiation (LTP) has been studied well, but the mechanism by which dopamine induces LTP (DA-LTP) in CA1 pyramidal neurons is unknown. Here, we report that DA-LTP in basal dendrites is dependent while in apical dendrites it is independent of activation of L-type voltage-gated calcium channels (VDCC).…

  15. Dopamine Induces LTP Differentially in Apical and Basal Dendrites through BDNF and Voltage-Dependent Calcium Channels

    Science.gov (United States)

    Navakkode, Sheeja; Sajikumar, Sreedharan; Korte, Martin; Soong, Tuck Wah

    2012-01-01

    The dopaminergic modulation of long-term potentiation (LTP) has been studied well, but the mechanism by which dopamine induces LTP (DA-LTP) in CA1 pyramidal neurons is unknown. Here, we report that DA-LTP in basal dendrites is dependent while in apical dendrites it is independent of activation of L-type voltage-gated calcium channels (VDCC).…

  16. Creation of reduced fat foods: influence of calcium-induced droplet aggregation on microstructure and rheology of mixed food dispersions.

    Science.gov (United States)

    Wu, Bi-cheng; Degner, Brian; McClements, David Julian

    2013-12-15

    The impact of calcium-induced fat droplet aggregation on the microstructure and physicochemical properties of model mixed colloidal dispersions was investigated. These systems consisted of 2 wt% whey protein-coated fat droplets and 4 wt% modified starch granules heated to induce starch swelling (pH 7). Optical and confocal microscopy showed that the fat droplets were dispersed within the interstitial region between the swollen starch granules. The structural organisation of the fat droplets within these interstitial regions could be modulated by controlling the calcium concentration: (i) at a low calcium concentration the droplets were evenly distributed; (ii) at an intermediate calcium concentration they formed a layer around the starch granules; (iii) at a high calcium concentration they formed a network of aggregated droplets. Paste-like materials were produced when the fat droplets formed a three-dimensional network in the interstitial region. The properties of fat droplet-starch granule suspensions can be modulated by altering the electrostatic interactions to alter microstructure.

  17. Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels.

    Science.gov (United States)

    Yang, Xinhui; Jiang, Jiangtao; Yang, Xinyan; Han, Jichun; Zheng, Qiusheng

    2016-07-01

    Licochalcone A (LCA) has been reported to significantly inhibit cell proliferation, increase reactive oxygen species (ROS) levels, and induce apoptosis of T24 human bladder cancer cells via mitochondria and endoplasmic reticulum (ER) stress-triggered signaling pathways. Based on these findings, the present study aimed to investigate the mechanisms by which LCA induces apoptosis of T24 cells. Cultured T24 cells were treated with LCA, and cell viability was measured using the sulforhodamine B assay. Apoptosis was detected by flow cytometry with Annexin V/propidium iodide staining, and by fluorescent microscopy with Hoechst 33258 staining. The levels of intracellular free calcium ions were determined using Fluo-3 AM dye marker. Intracellular ROS levels were assessed using the 2',7'-dichlorodihydrofluorescein diacetate probe assay. The mitochondrial membrane potential was measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide. Furthermore, the mRNA expression levels of B‑cell lymphoma (Bcl)‑extra large, Bcl‑2‑associated X protein, Bcl‑2‑interacting mediator of cell death, apoptotic protease activating factor‑1 (Apaf‑1), calpain 2, cysteinyl aspartate specific proteinase (caspase)‑3, caspase‑4 and caspase‑9 were determined using reverse transcription semiquantitative and quantitative polymerase chain reaction analyses. Treatment with LCA inhibited proliferation and induced apoptosis of T24 cells, and increased intracellular Ca2+ levels and ROS production. Furthermore, LCA induced mitochondrial dysfunction, decreased mitochondrial membrane potential, and increased the mRNA expression levels of Apaf‑1, caspase‑9 and caspase‑3. Exposure of T24 cells to LCA also triggered calpain 2 and caspase‑4 activation, resulting in apoptosis. These findings indicated that LCA increased intracellular Ca2+ levels, which may be associated with mitochondrial dysfunction. In addition, the ER stress pathway may be

  18. Octopamine and Dopamine differentially modulate the nicotine-induced calcium response in Drosophila Mushroom Body Kenyon Cells.

    Science.gov (United States)

    Leyton, V; Goles, N I; Fuenzalida-Uribe, N; Campusano, J M

    2014-02-07

    In Drosophila associative olfactory learning, an odor, the conditioned stimulus (CS), is paired to an unconditioned stimulus (US). The CS and US information arrive at the Mushroom Bodies (MB), a Drosophila brain region that processes the information to generate new memories. It has been shown that olfactory information is conveyed through cholinergic inputs that activate nicotinic acetylcholine receptors (nAChRs) in the MB, while the US is coded by biogenic amine (BA) systems that innervate the MB. In this regard, the MB acts as a coincidence detector. A better understanding of the properties of the responses gated by nicotinic and BA receptors is required to get insights on the cellular and molecular mechanisms responsible for memory formation. In recent years, information has become available on the properties of the responses induced by nAChR activation in Kenyon Cells (KCs), the main neuronal MB population. However, very little information exists on the responses induced by aminergic systems in fly MB. Here we have evaluated some of the properties of the calcium responses gated by Dopamine (DA) and Octopamine (Oct) in identified KCs in culture. We report that exposure to BAs induces a fast but rather modest increase in intracellular calcium levels in cultured KCs. The responses to Oct and DA are fully blocked by a VGCC blocker, while they are differentially modulated by cAMP. Moreover, co-application of BAs and nicotine has different effects on intracellular calcium levels: while DA and nicotine effects are additive, Oct and nicotine induce a synergistic increase in calcium levels. These results suggest that a differential modulation of nicotine-induced calcium increase by DA and Oct could contribute to the events leading to learning and memory in flies.

  19. Acidosis and Urinary Calcium Excretion

    DEFF Research Database (Denmark)

    Alexander, R Todd; Cordat, Emmanuelle; Chambrey, Régine

    2016-01-01

    Metabolic acidosis is associated with increased urinary calcium excretion and related sequelae, including nephrocalcinosis and nephrolithiasis. The increased urinary calcium excretion induced by metabolic acidosis predominantly results from increased mobilization of calcium out of bone and inhibi...

  20. Calcium homeostasis and cone signaling are regulated by interactions between calcium stores and plasma membrane ion channels.

    Directory of Open Access Journals (Sweden)

    Tamas Szikra

    Full Text Available Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca(2+ entry (SOCE to Ca(2+ homeostasis and its role in regulation of neurotransmission at cone synapses. Mn(2+ quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca(2+ influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca(2+ stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca(2+ channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca(2+ entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca(2+ channels. Exposure to MRS 1845 resulted in approximately 40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca(2+ currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca(2+ homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse.

  1. R-type calcium channels are crucial for semaphorin 3A-induced DRG axon growth cone collapse.

    Directory of Open Access Journals (Sweden)

    Rimantas Treinys

    Full Text Available Semaphorin 3A (Sema3A is a secreted protein involved in axon path-finding during nervous system development. Calcium signaling plays an important role during axonal growth in response to different guidance cues; however it remains unclear whether this is also the case for Sema3A. In this study we used intracellular calcium imaging to figure out whether Sema3A-induced growth cone collapse is a Ca2+ dependent process. Intracellular Ca2+ imaging results using Fura-2 AM showed Ca2+ increase in E15 mice dorsal root ganglia neurons upon Sema3A treatment. Consequently we analyzed Sema3A effect on growth cones after blocking or modifying intracellular and extracellular Ca2+ channels that are expressed in E15 mouse embryos. Our results demonstrate that Sema3A increased growth cone collapse rate is blocked by the non-selective R- and T- type Ca2+ channel blocker NiCl2 and by the selective R-type Ca2+ channel blocker SNX482. These Ca2+ channel blockers consistently decreased the Sema3A-induced intracellular Ca2+ concentration elevation. Overall, our results demonstrate that Sema3A-induced growth cone collapses are intimately related with increase in intracellular calcium concentration mediated by R-type calcium channels.

  2. Calcium Uptake via Mitochondrial Uniporter Contributes to Palmitic Acid-induced Apoptosis in Mouse Podocytes.

    Science.gov (United States)

    Yuan, Zeting; Cao, Aili; Liu, Hua; Guo, Henjiang; Zang, Yingjun; Wang, Yi; Wang, Yunman; Wang, Hao; Yin, Peihao; Peng, Wen

    2017-02-09

    Podocytes are component cells of the glomerular filtration barrier, and their loss by apoptosis is the main cause of proteinuria that leads to diabetic nephropathy (DN). Therefore, insights into podocyte apoptosis mechanism would allow a better understanding of DN pathogenesis and thus help develop adequate therapeutic strategies. Here, we investigated the molecular mechanism of palmitic acid-inhibited cell death in mouse podocytes, and found that palmitic acid increased cell death in a dose- and time-dependent manner. Palmitic acid induces apoptosis in podocytes through up-regulation of cytosolic and mitochondrial Ca(2+) , mitochondrial membrane potential (MMP), cytochrome c release and depletion of endoplasmic reticulum (ER) Ca(2+) , The intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N, N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), partially prevented this up-regulation whereas 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor (IP3R) inhibitor; dantrolene, a ryanodine receptor (RyR) inhibitor; and 4,4'-diisothiocyanatostibene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, had no effect. Interestingly, ruthenium red and Ru360, both inhibitors of the mitochondrial Ca(2+) uniporter (MCU), blocked palmitic acid-induced mitochondrial Ca(2+) elevation, cytochrome c release from mitochondria to cytosol, and apoptosis. siRNA to MCU markedly reduced curcumin-induced apoptosis. These data indicate that Ca(2+) uptake via mitochondrial uniporter contributes to palmitic acid-induced apoptosis in mouse podocytes. This article is protected by copyright. All rights reserved.

  3. Involvement of mouse and porcine PLCζ-induced calcium oscillations in preimplantation development of mouse embryos

    Energy Technology Data Exchange (ETDEWEB)

    Yoneda, Akihiro, E-mail: ayoneda@sci.hokudai.ac.jp [Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University (Japan); Division of Molecular Therapeutics, Center for Food & Medical Innovation, Hokkaido University (Japan); Watanabe, Tomomasa [Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University (Japan)

    2015-05-01

    In mammals, phospholipase Cζ (PLCζ) has the ability to trigger calcium (Ca{sup 2+}) oscillations in oocytes, leading to oocyte activation. Although there is a species-specific difference in the PLCζ-induced Ca{sup 2+} oscillatory pattern, whether PLCζ-induced Ca{sup 2+} oscillations affect preimplantation embryonic development remains unclear. Here, we show that Ca{sup 2+} oscillations in mouse PLCζ cRNA-injected oocytes stopped just before pronuclear formation, while that in porcine PLCζ cRNA-injected oocytes continued for several hours after pronuclei had been formed. This difference of Ca{sup 2+} oscillations in oocytes after pronuclear formation was dependent on the difference in the nuclear localization signal (NLS) sequence of PLCζ between the mouse and pig. However, mouse and porcine PLCζ cRNA-injected oocytes parthenogenetically developed to blastocysts regardless of the absence or presence of Ca{sup 2+} oscillations after pronuclear formation. Furthermore, the developmental rate of mouse or porcine PLCζ-activated oocytes injected with round spermatids to the blastocyst stage was not significantly different from that of strontium-activated oocytes injected with round spermatids. These results suggest that the PLCζ-induced Ca{sup 2+} oscillatory pattern in mouse oocytes is dependent on the NLS sequence of PLCζ and injection of PLCζ may be a useful method for activation of round spermatid-injected and somatic nuclear transferred oocytes. - Highlights: • Porcine PLCζ-induced Ca{sup 2+} oscillations continued after pronuclear formation. • The Ca{sup 2+} oscillatory pattern was dependent on the difference in the NLS sequence of PLCζ. • PLCζ-activated oocytes parthenogenetically developed to blastocysts. • PLCζ-activated oocytes injected with round spermatids developed to blastocysts.

  4. Impaired Compensation for Salt-Induced Urinary Calcium Loss in a Space Flight Model

    Science.gov (United States)

    Navidi, Meena; Harper, J. S.; Evans, J.; Fung, P.; Wolinsky, I.; Arnaud, S. B.; Wade, Charles E. (Technical Monitor)

    1994-01-01

    The loss of urinary calcium (UCa) induced by high sodium (HiNa) diets is compensated for by an increase in net intestinal Ca absorption (abs.). To determine the capacity of the intestine to absorb Ca in a space flight model in which the formation of 1,25-dihydroxyvitamin D (1,25-D) is suppressed, we induced Ca loss with HiNa diets (8%) and restricted dietary Ca (0.2%). In 200 g rats with hind limbs unloaded by tail suspension (S), we examined intestinal Ca abs. by direct measurement in the duodenum (everted gut sac or S/M), vitamin D receptors (VDR) and Ca balance. We also measured serum ionized calcium (ICa), pH, parathyroid hormone (PTH) and 1,25D. PTH was related to ICa (r = -0.44, p is less than 0.02), pH (r = -0.47, p is less than 0.02) and %Ca abs. (r = -0.40, p is less than 0.05). 1,25-D was related to %Ca abs. (r = 0.60, p is less than 0.001) but not VDR or S/M. Effects of the model were lower serum 1,25-D (110 +/- 59 vs. 199 +/- 80 pg/ml, p is less than 0.005), %Ca abs. (83 +/- 6.9 vs. 93 +/- 3.2, p is less than 0.03) and Ca balance (27 +/- 0.2 vs. 30 +/- 0.3 mg/d, p is less than 0.001) in S than controls (C). The HiNa diet increased UCa excretion from 2 to 13% of dietary Ca. Responses to HiNa diets, compared to normal Na, revealed no differences in 1,25-D, Ca abs. or VDR. Ca balances were lower in HiNa (27 +/- 0.3 vs. 30 +/- 0.4 mg/d, p is less than 0.001) in spite of higher Ca intakes. The failure of S rats fed HiNa diets to increase Ca abs. in response to Na-induced Ca loss appears to be related to suppressed 1,25-D in the space flight model, the cause of which remains obscure.

  5. Structure and Property Changes in Self-Assembled Lubricin Layers Induced by Calcium Ion Interactions.

    Science.gov (United States)

    Greene, George W; Thapa, Rajiv; Holt, Stephen A; Wang, Xiaoen; Garvey, Christopher J; Tabor, Rico F

    2017-03-14

    Lubricin (LUB) is a "mucin-like" glycoprotein found in synovial fluids and coating the cartilage surfaces of articular joints, which is now generally accepted as one of the body's primary boundary lubricants and antiadhesive agents. LUB's superior lubrication and antiadhesion are believed to derive from its unique interfacial properties by which LUB molecules adhere to surfaces (and biomolecules, such as hyaluronic acid and collagen) through discrete interactions localized to its two terminal end domains. These regionally specific interactions lead to self-assembly behavior and the formation of a well-ordered "telechelic" polymer brush structure on most substrates. Despite its importance to biological lubrication, detailed knowledge on the LUB's self-assembled brush structure is insufficient and derived mostly from indirect and circumstantial evidence. Neutron reflectometry (NR) was used to directly probe the self-assembled LUB layers, confirming the polymer brush architecture and resolving the degree of hydration and level of surface coverage. While attempting to improve the LUB contrast in the NR measurements, the LUB layers were exposed to a 20 mM solution of CaCl2, which resulted in a significant change in the polymer brush structural parameters consisting of a partial denaturation of the surface-binding end-domain regions, partial dehydration of the internal mucin-domain "loop", and collapse of the outer mucin-domain surface region. A series of atomic force microscopy measurements investigating the LUB layer surface morphology, mechanical properties, and adhesion forces in phosphate-buffered saline and CaCl2 solutions reveal that the structural changes induced by calcium ion interactions also significantly alter key properties, which may have implications to LUB's efficacy as a boundary lubricant and wear protector in the presence of elevated calcium ion concentrations.

  6. Chronic calcium pyrophosphate crystal inflammatory arthritis induced by extreme hypomagnesemia in short bowel syndrome

    Directory of Open Access Journals (Sweden)

    Hahn Markus

    2012-09-01

    Full Text Available Abstract Background Short bowel syndrome (SBS may induce a plethora of clinical symptoms ranging from underweight to nutrient-, vitamin- and electrolyte deficiencies. The objective of this case report is to illustrate how demanding the management of a 60 year old patient with SBS and recurrent joint attacks was for different medical disciplines. Case presentation The patient with SBS presented with a body mass index of 16.5 kg/m2 after partial jejunoileal resection of the small intestine with a six year long history of recurrent pain attacks in multiple peripheral joints, chronic diarrhoea and food intolerances. Pain attacks occurred 4–5 times a week with a median consumption of 15 mg prednisone per day. The interdisciplinary workup after several gastroenterologic, rheumatologic, radiologic, psychiatric and orthopedic consultations is shown including successful treatment steps. Clinical diagnosis revealed no systemic inflammatory disease, but confirmed extreme hypomagnesemia (0.2 mmol/l after reproducible pathological magnesium resorption tests as causative for chronic calcium pyrophosphate crystal inflammatory arthritis (pseudogout, chondrocalcinosis. Multidisciplinary treatment included application of colchicines, parenteral nutrition and magnesium substitution, antiperistaltic agents and avoidance of intolerant foods. Normalization of magnesium levels and a marked remission of joint attacks were achieved after six months with significant reduction of prednisone to 1.5 mg/day. Conclusion Despite the rarity of this condition, it is important to know that hypomagnesaemia may be associated with calcium pyrophosphate crystal inflammatory arthritis (chondrocalcinosis and that SBS patients may be prone to develop extreme hypomagnesaemia causing recurrent joint attacks without systemic inflammation.

  7. The protective effect of supplemental calcium on colonic permeability depends on a calcium phosphate-induced increase in luminal buffering capacity.

    Science.gov (United States)

    Schepens, Marloes A A; ten Bruggencate, Sandra J M; Schonewille, Arjan J; Brummer, Robert-Jan M; van der Meer, Roelof; Bovee-Oudenhoven, Ingeborg M J

    2012-04-01

    An increased intestinal permeability is associated with several diseases. Previously, we have shown that dietary Ca decreases colonic permeability in rats. This might be explained by a calcium-phosphate-induced increase in luminal buffering capacity, which protects against an acidic pH due to microbial fermentation. Therefore, we investigated whether dietary phosphate is a co-player in the effect of Ca on permeability. Rats were fed a humanised low-Ca diet, or a similar diet supplemented with Ca and containing either high, medium or low phosphate concentrations. Chromium-EDTA was added as an inert dietary intestinal permeability marker. After dietary adaptation, short-chain fructo-oligosaccharides (scFOS) were added to all diets to stimulate fermentation, acidify the colonic contents and induce an increase in permeability. Dietary Ca prevented the scFOS-induced increase in intestinal permeability in rats fed medium- and high-phosphate diets but not in those fed the low-phosphate diet. This was associated with higher faecal water cytotoxicity and higher caecal lactate levels in the latter group. Moreover, food intake and body weight during scFOS supplementation were adversely affected by the low-phosphate diet. Importantly, luminal buffering capacity was higher in rats fed the medium- and high-phosphate diets compared with those fed the low-phosphate diet. The protective effect of dietary Ca on intestinal permeability is impaired if dietary phosphate is low. This is associated with a calcium phosphate-induced increase in luminal buffering capacity. Dragging phosphate into the colon and thereby increasing the colonic phosphate concentration is at least part of the mechanism behind the protective effect of Ca on intestinal permeability.

  8. STIM1 and STIM2 differently regulate endogenous Ca(2+) entry and promote TGF-β-induced EMT in breast cancer cells.

    Science.gov (United States)

    Zhang, Siheng; Miao, Yutian; Zheng, Xianchong; Gong, Yong; Zhang, Jinxin; Zou, Fei; Cai, Chunqing

    2017-06-17

    The Ca(2+) sensor proteins STIM1 and STIM2 are crucial elements of store-operated calcium entry (SOCE) in breast cancer cells. Increased SOCE activity may contribute to epithelial-mesenchymal transitions (EMT) and increase cell migration and invasion. However, the roles of STIM1 and STIM2 in TGF-β-induced EMT are still unclear. In this study, we demonstrate roles of STIMs in TGF-β-induced EMT in breast cancer cells. In particular, STIM1 and STIM2 expression affected TGF-β-induced EMT by mediating SOCE in MDA-MB-231 and MCF-7 breast cancer cells. The specific SOCE inhibitor YM58483 blocked TGF-β-induced EMT, and differing effects of STIM1 and STIM2 on TGF-β-induced EMT correlated with differing roles in SOCE. Finally, we showed that STIM2 is associated with non-store-operated calcium entry (non-SOCE) during TGF-β-induced EMT, whereas STIM1 is not. What's more, non-SOCE have a large possibility to be ROCE. In conclusion, STIM1 and STIM2 proteins play important roles in TGF-β-induced EMT and these effects are related to both SOCE and non-SOCE. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Spine calcium transients induced by synaptically-evoked action potentials can predict synapse location and establish synaptic democracy.

    Directory of Open Access Journals (Sweden)

    David C Sterratt

    Full Text Available CA1 pyramidal neurons receive hundreds of synaptic inputs at different distances from the soma. Distance-dependent synaptic scaling enables distal and proximal synapses to influence the somatic membrane equally, a phenomenon called "synaptic democracy". How this is established is unclear. The backpropagating action potential (BAP is hypothesised to provide distance-dependent information to synapses, allowing synaptic strengths to scale accordingly. Experimental measurements show that a BAP evoked by current injection at the soma causes calcium currents in the apical shaft whose amplitudes decay with distance from the soma. However, in vivo action potentials are not induced by somatic current injection but by synaptic inputs along the dendrites, which creates a different excitable state of the dendrites. Due to technical limitations, it is not possible to study experimentally whether distance information can also be provided by synaptically-evoked BAPs. Therefore we adapted a realistic morphological and electrophysiological model to measure BAP-induced voltage and calcium signals in spines after Schaffer collateral synapse stimulation. We show that peak calcium concentration is highly correlated with soma-synapse distance under a number of physiologically-realistic suprathreshold stimulation regimes and for a range of dendritic morphologies. Peak calcium levels also predicted the attenuation of the EPSP across the dendritic tree. Furthermore, we show that peak calcium can be used to set up a synaptic democracy in a homeostatic manner, whereby synapses regulate their synaptic strength on the basis of the difference between peak calcium and a uniform target value. We conclude that information derived from synaptically-generated BAPs can indicate synapse location and can subsequently be utilised to implement a synaptic democracy.

  10. Spine Calcium Transients Induced by Synaptically-Evoked Action Potentials Can Predict Synapse Location and Establish Synaptic Democracy

    Science.gov (United States)

    Meredith, Rhiannon M.; van Ooyen, Arjen

    2012-01-01

    CA1 pyramidal neurons receive hundreds of synaptic inputs at different distances from the soma. Distance-dependent synaptic scaling enables distal and proximal synapses to influence the somatic membrane equally, a phenomenon called “synaptic democracy”. How this is established is unclear. The backpropagating action potential (BAP) is hypothesised to provide distance-dependent information to synapses, allowing synaptic strengths to scale accordingly. Experimental measurements show that a BAP evoked by current injection at the soma causes calcium currents in the apical shaft whose amplitudes decay with distance from the soma. However, in vivo action potentials are not induced by somatic current injection but by synaptic inputs along the dendrites, which creates a different excitable state of the dendrites. Due to technical limitations, it is not possible to study experimentally whether distance information can also be provided by synaptically-evoked BAPs. Therefore we adapted a realistic morphological and electrophysiological model to measure BAP-induced voltage and calcium signals in spines after Schaffer collateral synapse stimulation. We show that peak calcium concentration is highly correlated with soma-synapse distance under a number of physiologically-realistic suprathreshold stimulation regimes and for a range of dendritic morphologies. Peak calcium levels also predicted the attenuation of the EPSP across the dendritic tree. Furthermore, we show that peak calcium can be used to set up a synaptic democracy in a homeostatic manner, whereby synapses regulate their synaptic strength on the basis of the difference between peak calcium and a uniform target value. We conclude that information derived from synaptically-generated BAPs can indicate synapse location and can subsequently be utilised to implement a synaptic democracy. PMID:22719238

  11. Sphingomyelin-induced inhibition of the plasma membrane calcium ATPase causes neurodegeneration in type A Niemann-Pick disease.

    Science.gov (United States)

    Pérez-Cañamás, A; Benvegnù, S; Rueda, C B; Rábano, A; Satrústegui, J; Ledesma, M D

    2017-05-01

    Niemann-Pick disease type A (NPA) is a rare lysosomal storage disorder characterized by severe neurological alterations that leads to death in childhood. Loss-of-function mutations in the acid sphingomyelinase (ASM) gene cause NPA, and result in the accumulation of sphingomyelin (SM) in lysosomes and plasma membrane of neurons. Using ASM knockout (ASMko) mice as a NPA disease model, we investigated how high SM levels contribute to neural pathology in NPA. We found high levels of oxidative stress both in neurons from these mice and a NPA patient. Impaired activity of the plasma membrane calcium ATPase (PMCA) increases intracellular calcium. SM induces PMCA decreased activity, which causes oxidative stress. Incubating ASMko-cultured neurons in the histone deacetylase inhibitor, SAHA, restores PMCA activity and calcium homeostasis and, consequently, reduces the increased levels of oxidative stress. No recovery occurs when PMCA activity is pharmacologically impaired or genetically inhibited in vitro. Oral administration of SAHA prevents oxidative stress and neurodegeneration, and improves behavioral performance in ASMko mice. These results demonstrate a critical role for plasma membrane SM in neuronal calcium regulation. Thus, we identify changes in PMCA-triggered calcium homeostasis as an upstream mediator for NPA pathology. These findings can stimulate new approaches for pharmacological remediation in a disease with no current clinical treatments.

  12. Mechanisms of cholecystokinin-induced calcium mobilization in gastric antral interstitial cells of Cajal

    Institute of Scientific and Technical Information of China (English)

    Yao-Yao Gong; Xin-Min Si; Lin Lin; Jia Lu

    2012-01-01

    AIM:To investigate the effect of sulfated cholecystokinin-8 (CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal (ICC) and its possible mechanisms.METHODS:ICC were isolated from the gastric antrum of mice and cultured.Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC.The responsiveness of ICC to CCK-8S was measured using Fluo-3/AM based digital microfluorimetric measurement of intracellular Ca2+ concentration ([Ca2+]i).A confocal laser scanning microscope was used to monitor [Ca2+]i changes.The selective CCK1 receptor antagonist lorglumide,the intracellular Ca2+-ATPase inhibitor thapsigargin,the type Ⅲ inositol 1,4,5-triphosphate (InsP3) receptor blocker xestospongin C and the L-type voltage-operated Ca2+ channel inhibitor nifedipine were used to examine the mechanisms of [Ca2+]i elevation caused by CCK-8S.Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type Ⅲ InsP3 receptor (InsP3R3) in ICC.Protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca2+]i increment of ICC.RESULTS:ICC were successfully isolated from the gastric antrum of mice and cultured.Cultured ICC were identified by immunofluorescence staining.When given 80 nmol/L or more than 80 nmol/L CCK-8S,the [Ca2+]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca2+]i by 59.30% ± 4.85% (P <0.01).Pretreatment of ICC with 5 μmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca2+]i increment from 59.30% ± 4.85% to 14.97% ± 9.05% (P < 0.01),suggesting a CCK1R-mediated event.Emptying of intracellular calcium stores by thapsigargin (5 μmol/L)prevented CCK-8S (100 nmol/L) from inducing a [Ca2+]i increase.Moreover,pretreatment with xestospongin C (1 μmol/L) could also abolish the CCK-8S-induced effect

  13. Osteogenic differentiation of osteoblasts induced by calcium silicate and calcium silicate/β-tricalcium phosphate composite bioceramics.

    Science.gov (United States)

    Fei, Lisha; Wang, Chen; Xue, Yang; Lin, Kaili; Chang, Jiang; Sun, Jiao

    2012-07-01

    In this study, calcium silicate (CS) and CS/β-tricalcium phosphate (CS/β-TCP) composites were investigated on their mechanism of osteogenic proliferation and differentiation through regulating osteogenic-related gene and proteins. Osteoblast-like cells were cultured in the extracts of these CS-based bioceramics and pure β-TCP, respectively. The main ionic content in extracts was analyzed by inductively coupled plasma-atomic emission spectroscopy. The cell viability, mineralization, and differentiation were evaluated by MTT assay, Alizarin Red-S staining and alkaline phosphatase (ALP) activity assay. The expressions of BMP-2, transforming growth factor-β (TGF-β), Runx2, ALP, and osteocalcin (OCN) at both gene and protein level were detected by real-time polymerase chain reaction analysis and Western blot. The result showed that the extracts of CS-based bioceramics promoted cells proliferation, differentiation, and mineralization when compared with pure β-TCP. Accordingly, pure CS and CS/β-TCP composites stimulated osteoblast-like cells to express BMP-2/TGF-β gene and proteins, and further regulate the expression of Runx2 gene and protein, and ultimately affect the ALP activity and OCN deposition. This study suggested that the CS-based bioceramics could not only promote the expression of osteogenic-related genes but also enhance the genes to encode the corresponding proteins, which could finally control osteoblast-like cells proliferation and differentiation.

  14. Calcium, zinc and vitamin E ameliorate cadmium-induced renal oxidative damage in albino Wistar rats

    Directory of Open Access Journals (Sweden)

    Pradeepkiran Jangampalli Adi

    2016-01-01

    Full Text Available This study was aimed to examine the protective effects of supplementation with calcium + zinc (Ca + Zn or vitamin E (Vit-E on Cd-induced renal oxidative damage. Young albino Wistar rats (180 ± 10 g (n = 6 control rats, Cd, Cd + Ca + Zn, and Cd + Vit-E experimental groups and the experimental period was 30 days. Rats were exposed to Cd (20 mg/kg body weight alone treated as Cd treated group and the absence or presence of Ca + Zn (2 mg/kg each or Vit-E (20 mg/kg body weight supplementation treated as two separate groups. The activities of the stress marker enzymes superoxide dismutase (SOD, catalase (CAT, glutathione reductase (GR, glutathione peroxidase (GPx, glutathione-S-transferase (GST and lipid peroxidase (LPx were determined in renal mitochondrial fractions of experimental rats. We observed quantitative changes in SOD isoenzymatic patterns by non-denaturing PAGE analysis, and quantified band densities. These results showed that Cd exposure leads to decreases in SOD, CAT, GR, and GPx activities and a concomitant increase in LPx and GST activities. Ca + Zn and Vit-E administration with Cd significantly reversed Cd-induced perturbations in oxidative stress marker enzymes. However, Vit-E showed more inhibitory activity against Cd than did Ca + Zn, and it protected against Cd-induced nephrotoxicity.

  15. Calcium, zinc and vitamin E ameliorate cadmium-induced renal oxidative damage in albino Wistar rats.

    Science.gov (United States)

    Adi, Pradeepkiran Jangampalli; Burra, Siva Prasad; Vataparti, Amardev Rajesh; Matcha, Bhaskar

    2016-01-01

    This study was aimed to examine the protective effects of supplementation with calcium + zinc (Ca + Zn) or vitamin E (Vit-E) on Cd-induced renal oxidative damage. Young albino Wistar rats (180 ± 10 g) (n = 6) control rats, Cd, Cd + Ca + Zn, and Cd + Vit-E experimental groups and the experimental period was 30 days. Rats were exposed to Cd (20 mg/kg body weight) alone treated as Cd treated group and the absence or presence of Ca + Zn (2 mg/kg each) or Vit-E (20 mg/kg body weight) supplementation treated as two separate groups. The activities of the stress marker enzymes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and lipid peroxidase (LPx) were determined in renal mitochondrial fractions of experimental rats. We observed quantitative changes in SOD isoenzymatic patterns by non-denaturing PAGE analysis, and quantified band densities. These results showed that Cd exposure leads to decreases in SOD, CAT, GR, and GPx activities and a concomitant increase in LPx and GST activities. Ca + Zn and Vit-E administration with Cd significantly reversed Cd-induced perturbations in oxidative stress marker enzymes. However, Vit-E showed more inhibitory activity against Cd than did Ca + Zn, and it protected against Cd-induced nephrotoxicity.

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

    Directory of Open Access Journals (Sweden)

    Jingjing Ye

    2016-01-01

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

  17. L-Type Calcium Channels Do Not Play a Critical Role in Chest Blow Induced Ventricular Fibrillation: Commotio Cordis

    Directory of Open Access Journals (Sweden)

    Christopher Madias

    2016-01-01

    Full Text Available Background. In a commotio cordis swine model, ventricular fibrillation (VF can be induced by a ball blow to the chest believed secondary to activation of mechanosensitive ion channels. The purpose of the current study is to evaluate whether stretch induced activation of the L-type calcium channel may cause intracellular calcium overload and underlie the VF in commotio cordis. Method and Results. Anesthetized juvenile swine received 6 chest wall strikes with a 17.9 m/s lacrosse ball timed to the vulnerable period for VF induction. Animals were randomized to IV verapamil (n=6 or placebo (n=6. There was no difference in the observed frequency of VF between verapamil (19/26: 73% and placebo (20/36: 56% treated animals (p=0.16. There was also no significant difference in the combined endpoint of VF or nonsustained VF (21/26: 81% in verapamil versus 24/36: 67% in controls, p=0.22. Conclusions. In this experimental model of commotio cordis, verapamil did not prevent VF induction. Thus, in commotio cordis it is unlikely that stretch activation of the L-type calcium channel with resultant intracellular calcium overload plays a prominent role.

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

    Science.gov (United States)

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

    2009-01-01

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

  19. Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation

    Energy Technology Data Exchange (ETDEWEB)

    Bergemann, Claudia [University Medical Center Rostock, Cell Biology, Schillingallee 69, D-18057 Rostock (Germany); Cornelsen, Matthias [University of Rostock, Fluid Technology and Microfluidics, Justus-von-Liebig Weg 6, D-18059 Rostock (Germany); Quade, Antje [Leibniz-Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Str. 2, D-17489 Greifswald (Germany); Laube, Thorsten; Schnabelrauch, Matthias [INNOVENT e.V., Biomaterials Department, Pruessingstrasse 27B, D-07745 Jena (Germany); Rebl, Henrike [University Medical Center Rostock, Cell Biology, Schillingallee 69, D-18057 Rostock (Germany); Weißmann, Volker [Institute for Polymer Technologies (IPT) e.V., Alter Holzhafen 19, D-23966 Wismar (Germany); Seitz, Hermann [University of Rostock, Fluid Technology and Microfluidics, Justus-von-Liebig Weg 6, D-18059 Rostock (Germany); Nebe, Barbara, E-mail: barbara.nebe@med.uni-rostock.de [University Medical Center Rostock, Cell Biology, Schillingallee 69, D-18057 Rostock (Germany)

    2016-02-01

    The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(L-lactide-co-D,L-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA — improvement of compressive strength of calcium phosphate scaffolds – is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10 mm hybrid scaffold were dynamically cultivated for 14 days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts. - Highlights: • Mechanical stabilization of β-tricalcium phosphate scaffolds by PLA infiltration • Hybrid scaffolds with higher cell attraction due to plasma polymerized allylamine • 3D perfusion in vitro model for observation of cell migration inside scaffolds • Enhanced cell migration within plasma polymer coated TCP hybrid scaffolds.

  20. Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations.

    Science.gov (United States)

    Zheng, Hewen; Korendovych, Ivan V; Luk, Yan-Yeung

    2016-01-01

    For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa.

  1. Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation.

    Science.gov (United States)

    Bergemann, Claudia; Cornelsen, Matthias; Quade, Antje; Laube, Thorsten; Schnabelrauch, Matthias; Rebl, Henrike; Weißmann, Volker; Seitz, Hermann; Nebe, Barbara

    2016-02-01

    The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA - improvement of compressive strength of calcium phosphate scaffolds - is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa

    Directory of Open Access Journals (Sweden)

    A. Pedro Gonçalves

    2014-08-01

    Full Text Available Alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the Neurospora crassa response to staurosporine. We highlight the importance of the extracellular availability of Ca2+ for this response. Limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ROS. Conversely, an approximately 30-fold excess of external Ca2+ leads to increased drug tolerance and lower ROS generation. In line with this, distinct staurosporine-induced cytosolic Ca2+ signaling profiles were observed in the absence or presence of excessive external Ca2+. High-throughput RNA sequencing revealed that different concentrations of extracellular Ca2+ define distinct transcriptional programs. Our transcriptional profiling also pointed to two putative novel Ca2+-binding proteins, encoded by the NCU08524 and NCU06607 genes, and provides a reference dataset for future investigations on the role of Ca2+ in fungal biology.

  3. Comparison of Tooth Discoloration Induced by Calcium-Enriched Mixture and Mineral Trioxide Aggregate

    Science.gov (United States)

    Rouhani, Armita; Akbari, Majid; Farhadi-faz, Aida

    2016-01-01

    Introduction: The aim of this in vitro study was to evaluate the tooth discoloration induced by calcium-enriched mixture (CEM) cement and mineral trioxide aggregate (MTA). Methods and Materials: Forty five endodontically treated human maxillary central incisors were selected and divided into three groups (n=15) after removing the coronal 3 mm of the obturating materials. In the MTA group, white MTA plug was placed in pulp chamber and coronal zone of the root canal. In CEM cement group, CEM plug was placed in the tooth in the same manner. In both groups, a wet cotton pellet was placed in the access cavity and the teeth were temporarily sealed. After 24 h the teeth were restored with resin composite. In the negative control group the teeth were also restored with resin composite. The color change in the cervical third of teeth was measured with a colorimeter and was repeated 3 times for each specimen. The teeth were kept in artificial saliva for 6 months. After this period, the color change was measured again. Data were collected by Commission International de I'Eclairage's L*a*b color values, and corresponding ΔE values were calculated. The results were analyzed using the one-way ANOVA and post-hoc Tukey’s test with the significance level defined as 0.05. Results: There was no significant differences between CEM group and control group in mean discoloration. The mean tooth discoloration in MTA group was significantly greater than CEM and control groups (P<0.05). Conclusion: According to the result of the present study CEM cement did not induce tooth discoloration after six months. Therefore it can be used in vital pulp therapy of esthetically sensitive teeth. PMID:27471526

  4. The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects.

    LENUS (Irish Health Repository)

    Lyng, F M

    2006-04-01

    Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of

  5. Potassium-induced contraction in the lamb proximal urethra: Involvement of norepinephrine and different calcium entry pathways

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Pascual, A.; Costa, G.; Isla, M.; Jimenez, E.; Garcia-Sacristan, A. (Universidad Complutense de Madrid (Spain))

    1991-01-01

    The purpose of this work was to investigate the mechanisms involved in the peculiar biphasic response of the lamb urethral smooth muscle to high K+ solutions. The relative amplitude of the phasic and tonic components of the contraction and its reproducibility were dependent on the concentration of K+ used. Only concentrations higher than 80 mM (i.e., 120 mM) showed a tonic component greater in amplitude than the phasic one and manifested a tachyphylactic effect. Phentolamine (10(-6) M), prazosin (10(-6) M) and chemical denervation with 6-hydroxydopamine significantly inhibited the tonic component of the K+ (120 mM)-induced contraction, modifying its morphology. Reproducible contractions to K+ (120 mM) could be obtained in the presence of prazosin (10(-6) M) or cocaine (10(-6) M). The preparations were also shown to accumulate (3H)noradrenaline and release it upon depolarization with K+ (60 and 120 mM). Calcium removal inhibited the K+ (120 mM)-induced contraction. After addition of calcium (0.5-5 mM) the contractile activity was restored. Nifedipine (10(-6) M) and verapamil (10(-6) M) but not sodium nitroprusside (10(-6) M) significantly blocked the contractile response for calcium as well as the phasic component of the K+ contraction in calcium-containing medium. In preparations treated with prazosin (10(-6) M) the tonic component of the K+ (120 mM) contraction was more sensitive to nifedipine and removal of extracellular calcium than the phasic one.

  6. Roles of store-operated Ca2+ channels in regulating cell cycling and migration of human cardiac c-kit+ progenitor cells.

    Science.gov (United States)

    Che, Hui; Li, Gang; Sun, Hai-Ying; Xiao, Guo-Sheng; Wang, Yan; Li, Gui-Rong

    2015-11-15

    Cardiac c-kit(+) progenitor cells are important for maintaining cardiac homeostasis and can potentially contribute to myocardial repair. However, cellular physiology of human cardiac c-kit(+) progenitor cells is not well understood. The present study investigates the functional store-operated Ca(2+) entry (SOCE) channels and the potential role in regulating cell cycling and migration using confocal microscopy, RT-PCR, Western blot, coimmunoprecipitation, cell proliferation, and migration assays. We found that SOCE channels mediated Ca(2+) influx, and TRPC1, STIM1, and Orai1 were involved in the formation of SOCE channels in human cardiac c-kit(+) progenitor cells. Silencing TRPC1, STIM1, or Orai1 with the corresponding siRNA significantly reduced the Ca(2+) signaling through SOCE channels, decreased cell proliferation and migration, and reduced expression of cyclin D1, cyclin E, and/or p-Akt. Our results demonstrate the novel information that Ca(2+) signaling through SOCE channels regulates cell cycling and migration via activating cyclin D1, cyclin E, and/or p-Akt in human cardiac c-kit(+) cells.

  7. Muscarinic receptor activation determines the effects of store-operated Ca(2+)-entry on excitability and energy metabolism in pyramidal neurons.

    Science.gov (United States)

    Kann, Oliver; Taubenberger, Nando; Huchzermeyer, Christine; Papageorgiou, Ismini E; Benninger, Felix; Heinemann, Uwe; Kovács, Richard

    2012-01-01

    In various cell types, depletion of intracellular Ca(2+)-stores results in store-operated Ca(2+)-entry (SOCE) across the cellular membrane. However, the effects of SOCE on neuronal membrane excitability and mitochondrial functions in central neurons are not well defined. We investigated such cellular downstream effects in pyramidal neurons of rat organotypic hippocampal slice cultures by applying electrophysiological and fluorescence imaging techniques. We report that SOCE is associated with (i) elevations of Ca(2+)-concentration in individual neuronal mitochondria ([Ca(2+)](m)). In addition, SOCE can result in (ii) hyperpolarizing neuronal membrane currents, (iii) increase in extracellular K(+)-concentration ([K(+)](o)), (iv) mitochondrial membrane depolarization, and (v) changes in intracellular redox state (NAD(P)H and FAD fluorescence), the latter reflecting responses of energy metabolism. These additional downstream effects of SOCE required concomitant muscarinic receptor activation by carbachol or acetylcholine, and were suppressed by agonist washout or application of antagonist, atropine. We conclude that muscarinic receptor activation determines the downstream effects of SOCE on neuronal membrane excitability and energy metabolism. This mechanism might have significant impact on information processing and neurometabolic coupling in central neurons. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. In vitro aging promotes endoplasmic reticulum (ER)-mitochondria Ca(2+) cross talk and loss of store-operated Ca(2+) entry (SOCE) in rat hippocampal neurons.

    Science.gov (United States)

    Calvo-Rodríguez, María; García-Durillo, Mónica; Villalobos, Carlos; Núñez, Lucía

    2016-11-01

    Aging is associated to cognitive decline and susceptibility to neuron death, two processes related recently to subcellular Ca(2+) homeostasis. Memory storage relies on mushroom spines stability that depends on store-operated Ca(2+) entry (SOCE). In addition, Ca(2+) transfer from endoplasmic reticulum (ER) to mitochondria sustains energy production but mitochondrial Ca(2+) overload promotes apoptosis. We have addressed whether SOCE and ER-mitochondria Ca(2+) transfer are influenced by culture time in long-term cultures of rat hippocampal neurons, a model of neuronal aging. We found that short-term cultured neurons show large SOCE, low Ca(2+) store content and no functional coupling between ER and mitochondria. In contrast, in long-term cultures reflecting aging neurons, SOCE is essentially lost, Stim1 and Orai1 are downregulated, Ca(2+) stores become overloaded, Ca(2+) release is enhanced, expression of the mitochondrial Ca(2+) uniporter (MCU) increases and most Ca(2+) released from the ER is transferred to mitochondria. These results suggest that neuronal aging is associated to increased ER-mitochondrial cross talking and loss of SOCE. This subcellular Ca(2+) remodeling might contribute to cognitive decline and susceptibility to neuron cell death in the elderly. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Shangfu Li

    2016-10-01

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

  10. Protective Effect of Carvedilol on Abnormality of L-type Calcium Current Induced by Oxygen Free Radical in Cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    刘念; 喻荣辉; 阮燕菲; 周强; 卜军; 李泱

    2004-01-01

    The protective effect of carvedilol on abnormality of L-type calcium current induced by oxygen free radical in single guinea pig ventricular myocytes was studied. Whole-cell patch clamp technique was used to study the effect of H2 O2 (0.5 mmol/L) on L-type calcium current in single guinea pig ventricular myocytes and the action of pretreatment with carvedilol (0.5 μmol/L). 0.5μmol/L carvedilol had no significant effect on ICa,L and its channel dynamics. In the presence of 0.5 mmol/L H2O2, peak current of ICa,L was reduced significantly (P<0.001), the I-V curve of Ica,L was shifted upward, steady-state activation curve and steady-state deactivation curve of ICa,L were shifted left and recovery time of ICa,L was delayed significantly (P<0. 001). 0. 5 μmol/L carvedilol significantly alleviated the inhibitory effect of H2O2 on ICa,L as compared with that in H2O2 group (P<0.01). In addition, carvedilol reversed the changes of dynamics of ICa,L induced by H2O2. It was concluded that carvedilol could alleviate the abnormality of L-type calcium current induced by oxygen free radical in cardiomyocytes. It shows partly the possible mechanism of the special availability of carvedilol in chronic heart failure.

  11. Nuclear calcium signaling induces expression of the synaptic organizers Lrrtm1 and Lrrtm2.

    Science.gov (United States)

    Hayer, Stefanie N; Bading, Hilmar

    2015-02-27

    Calcium transients in the cell nucleus evoked by synaptic activity in hippocampal neurons function as a signaling end point in synapse-to-nucleus communication. As an important regulator of neuronal gene expression, nuclear calcium is involved in the conversion of synaptic stimuli into functional and structural changes of neurons. Here we identify two synaptic organizers, Lrrtm1 and Lrrtm2, as targets of nuclear calcium signaling. Expression of both Lrrtm1 and Lrrtm2 increased in a synaptic NMDA receptor- and nuclear calcium-dependent manner in hippocampal neurons within 2-4 h after the induction of action potential bursting. Induction of Lrrtm1 and Lrrtm2 occurred independently of the need for new protein synthesis and required calcium/calmodulin-dependent protein kinases and the nuclear calcium signaling target CREB-binding protein. Analysis of reporter gene constructs revealed a functional cAMP response element in the proximal promoter of Lrrtm2, indicating that at least Lrrtm2 is regulated by the classical nuclear Ca(2+)/calmodulin-dependent protein kinase IV-CREB/CREB-binding protein pathway. These results suggest that one mechanism by which nuclear calcium signaling controls neuronal network function is by regulating the expression of Lrrtm1 and Lrrtm2.

  12. Lack of GDAP1 induces neuronal calcium and mitochondrial defects in a knockout mouse model of charcot-marie-tooth neuropathy.

    Science.gov (United States)

    Barneo-Muñoz, Manuela; Juárez, Paula; Civera-Tregón, Azahara; Yndriago, Laura; Pla-Martin, David; Zenker, Jennifer; Cuevas-Martín, Carmen; Estela, Anna; Sánchez-Aragó, María; Forteza-Vila, Jerónimo; Cuezva, José M; Chrast, Roman; Palau, Francesc

    2015-04-01

    Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria-endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

  13. Lack of GDAP1 induces neuronal calcium and mitochondrial defects in a knockout mouse model of charcot-marie-tooth neuropathy.

    Directory of Open Access Journals (Sweden)

    Manuela Barneo-Muñoz

    2015-04-01

    Full Text Available Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2, axonal dominant (CMT2K and demyelinating recessive (CMT4A forms of Charcot-Marie-Tooth (CMT neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE upon mobilization of endoplasmic reticulum (ER Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria-endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

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

    Science.gov (United States)

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

    2015-10-01

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

  15. Energetics of discrete selectivity bands and mutation-induced transitions in the calcium-sodium ion channels family

    CERN Document Server

    Kaufman, I; Tindjong, R; McClintock, P V E; Eisenberg, R S

    2013-01-01

    We use Brownian dynamics simulations to study the permeation properties of a generic electrostatic model of a biological ion channel as a function of the fixed charge Q_f at its selectivity filter. We reconcile the recently-discovered discrete calcium conduction bands M0 (Q_f=1e), M1 (3e), M2 (5e) with the set of sodium conduction bands L0 (0.5-0.7e), L1 (1.5-2e) thereby obtaining a completed pattern of conduction and selectivity bands v Q_f for the sodium-calcium channels family. An increase of Q_f leads to an increase of calcium selectivity: L0 (sodium selective, non-blocking channel) -> M0 (non-selective channel) -> L1 (sodium selective channel with divalent block) -> M1 (calcium selective channel exhibiting the anomalous mole fraction effect). We create a consistent identification scheme where the L1 band is identified with the eukaryotic (DEKA) sodium channel, and L0 (speculatively) with the bacterial NaChBac channel. The scheme created is able to account for the experimentally observed mutation-induced ...

  16. Increased pressure-induced tone in rat parenchymal arterioles vs. middle cerebral arteries: role of ion channels and calcium sensitivity.

    Science.gov (United States)

    Cipolla, Marilyn J; Sweet, Julie; Chan, Siu-Lung; Tavares, Matthew J; Gokina, Natalia; Brayden, Joseph E

    2014-07-01

    Brain parenchymal arterioles (PAs) are high-resistance vessels that branch off pial arteries and perfuse the brain parenchyma. PAs are the target of cerebral small vessel disease and have been shown to have greater pressure-induced tone at lower pressures than pial arteries. We investigated mechanisms by which brain PAs have increased myogenic tone compared with middle cerebral arteries (MCAs), focusing on differences in vascular smooth muscle (VSM) calcium and ion channel function. The amount of myogenic tone and VSM calcium was measured using Fura 2 in isolated and pressurized PAs and MCAs. Increases in intraluminal pressure caused larger increases in tone and cytosolic calcium in PAs compared with MCAs. At 50 mmHg, myogenic tone was 37 ± 5% for PAs vs. 6.5 ± 4% for MCAs (P channel (VDCC) inhibitor nifedipine than MCAs (EC50 for PAs was 3.5 ± 0.4 vs. 82.1 ± 2.1 nmol/l for MCAs;P channel inhibitor iberiotoxin, whereas MCAs constricted ∼15%. Thus increased myogenic tone in PAs appears related to differences in ion channel activity that promotes VSM membrane depolarization but not to a direct sensitization of the contractile apparatus to calcium.

  17. External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel.

    Science.gov (United States)

    Kiang, Juliann G; Ives, John A; Jonas, Wayne B

    2005-03-01

    External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca2+], was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca2+]i in Jurkat T cells was 70 +/- 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca2+]i to 52 +/- 2 nM (n = 23), indicating that Ca2+ entry from the external source is important for maintaining the basal level of [Ca2+]i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca2+]i by 30 +/- 5% (P EBE did not attenuate [Ca2+]i responsiveness to EBE. Removal of external Ca2+ or Na+, but not Mg2+, inhibited the EBE-induced increase in [Ca2+]i. Dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, also inhibited the EBE-induced increase in [Ca2+]i in a concentration-dependent manner with an IC50 of 0.11 +/- 0.02 nM. When external [K+] was increased from 4.5 mM to 25 mM, EBE decreased [Ca2+]i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca2+ channel blocker. These results suggest that the EBE-induced [Ca2+]i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca2+]i is mediated by activation of Na+/Ca2+ exchangers and opening of L-type voltage-gated Ca2+ channels.

  18. The differentiation inducer, dimethyl sulfoxide, transiently increases the intracellular calcium ion concentration in various cell types.

    Science.gov (United States)

    Morley, P; Whitfield, J F

    1993-08-01

    Dimethyl sulfoxide (DMSO) initiates a coordinated differentiation program in various cell types but the mechanism(s) by which DMSO does this is not understood. In this study, the effect of DMSO on intracellular calcium ion concentration ([Ca2+]i) was determined in primary cultures of chicken ovarian granulosa cells from the two largest preovulatory follicles of laying hens, and in three cell lines: undifferentiated P19 embryonal carcinoma cells, 3T3-L1 fibroblasts, and Friend murine erythroleukemia (MEL) cells. [Ca2+]i was measured in cells loaded with the Ca(2+)-specific fluoroprobe Fura-2. There was an immediate (i.e., within 5 sec), transient, two to sixfold increase in [Ca2+]i after exposing all cell types to 1% DMSO. DMSO was effective between 0.2 and 1%. The prompt DMSO-induced [Ca2+]i spike in all of the cell types was not prevented by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating them with the Ca(2+)-channel blockers methoxyverapamil (D600; 100 microM), nifedipine (20 microM), or cobalt (5 mM). However, when granulosa cells, 3T3-L1 cells, or MEL cells were pretreated with lanthanum (La3+; 1 mM), which blocks both Ca2+ channels and membrane Ca2+ pumps, there was a sustained increase in [Ca2+]i in response to 1% DMSO. By contrast, pretreating P19 cells with La3+ (1 mM) did not prolong the DMSO-triggered [Ca2+]i transient. In all cases, the DMSO-induced [Ca2+]i surge was unaffected by pretreating the cells with the inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) or U-73, 122 (2.5 microM). These results suggest that DMSO almost instantaneously triggers the release of Ca2+ from intracellular stores through a common mechanism in cells in primary cultures and in cells of a variety of established lines, but this release is not mediated through phosphoinositide breakdown. This large, DMSO-induced Ca2+ spike may play a role in the induction of cell differentiation by DMSO.

  19. Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille;

    2012-01-01

    Electroporation of cells with short, high-voltage pulses causes a transient permeabilization of cell membranes that permits passage of otherwise nonpermeating ions and molecules. In this study, we illustrate how electroporation with isotonic calcium can achieve highly effective cancer cell kill...... in vivo. Calcium electroporation elicited dramatic antitumor responses in which 89% of treated tumors were eliminated. Histologic analyses indicated complete tumor necrosis. Mechanistically, calcium electroporation caused acute ATP depletion likely due to a combination of increased cellular use of ATP......, decreased production of ATP due to effects on the mitochondria, as well as loss of ATP through the permeabilized cell membrane. Taken together, our findings offer a preclinical proof of concept for the use of electroporation to load cancer cells with calcium as an efficient anticancer treatment...

  20. Interleukin-1 inhibits osmotically-induced calcium signaling and volume regulation in articular chondrocytes

    Science.gov (United States)

    Pritchard, Scott; Votta, Bartholomew J.; Kumar, Sanjay; Guilak, Farshid

    2011-01-01

    OBJECTIVE Articular chondrocytes respond to osmotic stress with transient changes in cell volume and the intracellular concentration of calcium ion ([Ca2+]i). The goal of this study was to examine the hypothesis that interleukin-1 (IL-1), a pro-inflammatory cytokine associated with osteoarthritis, influences osmotically-induced Ca2+ signaling. METHODS Fluorescence ratio imaging was used to measure [Ca2+]i and cell volume in response to hypo- or hyper-osmotic stress in isolated porcine chondrocytes, with or without pre-exposure to 10 ng/ml IL-1α. Inhibitors of IL-1 (IL-1 receptor antagonist, IL-Ra), Ca2+ mobilization (thapsigargin, an inhibitor of Ca-ATPases), and cytoskeletal remodeling (Toxin B, an inhibitor of the Rho family of small GTPases) were used to determine the mechanisms involved in increased [Ca2+]i, F-actin remodeling, volume adaptation and active volume recovery. RESULTS In response to osmotic stress, chondrocytes exhibited transient increases in [Ca2+]i, generally followed by decaying oscillations. Pre-exposure to IL-1 significantly inhibited regulatory volume decrease following hypo-osmotic swelling and reduced the change in cell volume and the time to peak [Ca2+]i in response to hyper-osmotic stress, but did not affect the peak magnitudes of [Ca2+]i in those cells that did response. Co-treatment with IL-1Ra, thapsigargin, or Toxin B restored these responses to control levels. The effects were associated with alterations in F-actin organization. CONCLUSIONS IL-1 alters the normal volumetric and Ca2+ signaling response of chondrocytes to osmotic stress through mechanisms involving F-actin remodeling via small Rho GTPases. These findings provide further insights into the mechanisms by which IL-1 may interfere with normal physiologic processes in the chondrocyte, such as the adaptation or regulatory responses to mechanical and osmotic loading. PMID:18495501

  1. Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Roby Kanichay

    2003-01-01

    Full Text Available Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs composed of polar lipid fraction E (PLFE from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998. In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr for vesicle aggregation at 25 and 40 °C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 °C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was –22.0 and –13.2 mV at 25 and 40 °C, respectively, at pH 6.6, as determined by 2-(p-toluidinylnaphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.

  2. Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Kanichay, Roby; Boni, Lawrence T; Cooke, Peter H; Khan, Tapan K; Chong, Parkson Lee-Gau

    2003-10-01

    Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs) composed of polar lipid fraction E (PLFE) from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998). In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks) with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr) for vesicle aggregation at 25 and 40 degrees C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 degrees C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was -22.0 and -13.2 mV at 25 and 40 degrees C, respectively, at pH 6.6, as determined by 2-(p-toluidinyl)naphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.

  3. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells.

    Science.gov (United States)

    Abdulnour-Nakhoul, Solange; Brown, Karen L; Rabon, Edd C; Al-Tawil, Youhanna; Islam, Mohammed T; Schmieg, John J; Nakhoul, Nazih L

    2015-11-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell-cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell-cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett's, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin-catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells.

  4. Induced Pluripotent Stem Cell-derived Mesenchymal Stem Cell Seeding on Biofunctionalized Calcium Phosphate Cements

    Institute of Scientific and Technical Information of China (English)

    WahWah TheinHan; Jun Liu; Minghui Tang; Wenchuan Chen; Linzhao Cheng; Hockin H. K. Xu

    2013-01-01

    Induced pluripotent stem cells (iPSCs) have great potential due to their proliferation and differentiation capability. The objectives of this study were to generate iPSC-derived mesenchymal stem cells (iPSC-MSCs), and investigate iPSC-MSC proliferation and osteogenic differentiation on calcium phosphate cement (CPC) containing biofunctional agents for the first time. Human iPSCs were derived from marrow CD34+ cells which were reprogrammed by a single episomal vector. iPSCs were cultured to form embryoid bodies (EBs), and MSCs migrated out of EBs. Five biofunctional agents were incorporated into CPC:RGD (Arg-Gly-Asp) peptides, fibronectin (Fn), fibronectin-like engineered polymer protein (FEPP), extracellular matrix Geltrex, and platelet concentrate. iPSC-MSCs were seeded on five biofunctionalized CPCs:CPC-RGD, CPC-Fn, CPC-FEPP, CPC-Geltrex, and CPC-Platelets. iPSC-MSCs on biofunctional CPCs had enhanced proliferation, actin fiber expression, osteogenic differentiation and mineralization, compared to control. Cell proliferation was greatly increased on biofunctional CPCs. iPSC-MSCs underwent osteogenic differentiation with increased alkaline phosphatase, Runx2 and collagen-I expressions. Mineral synthesis by iPSC-MSCs on CPC-Platelets was 3-fold that of CPC control. In conclusion, iPSCs showed high potential for bone engineering. iPSC-MSCs on biofunctionalized CPCs had cell proliferation and bone mineralization that were much better than traditional CPC. iPSC-MSC-CPC constructs are promising to promote bone regeneration in craniofacial/orthopedic repairs.

  5. Rapid reversal of life-threatening diltiazem-induced tetany with calcium chloride.

    Science.gov (United States)

    Vinson, D R; Burke, T F; Sung, H M

    1999-11-01

    We describe a patient who developed tetany with sudden respiratory arrest after the infusion of intravenous diltiazem. The administration of calcium chloride rapidly resolved the patient's tetany with prompt recovery of respiratory function, averting the need for more aggressive airway management and ventilatory support. The emergency physician should be aware that life-threatening tetany may accompany the administration of intravenous diltiazem and that calcium chloride may be a rapid and effective remedy.

  6. Inhibition of 4NQO-Induced Oral Carcinogenesis by Dietary Oyster Shell Calcium.

    Science.gov (United States)

    Chen, Ying; Jiang, Yi; Liao, Liyan; Zhu, Xiaoxin; Tang, Shengan; Yang, Qing; Sun, Lihua; Li, Yujie; Gao, Shuangrong; Xie, Zhongjian

    2016-03-01

    Oyster has gained much attention recently for its anticancer activity but it is unclear whether calcium, the major antitumor ingredient in oyster shell, is responsible for the anticarcinogenic role of the oyster. To address this issue, C57BL/6 mice were fed with the carcinogen 4-nitroquinoline-1-oxide (4NQO, 50 µg/mL) and normal diet or a diet containing oyster powder, oyster calcium, or calcium depleted oyster powder. The tongue tissue specimens isolated from these mice were histologically evaluated for hyperplasia, dysplasia, and papillary lesions, and then analyzed for proliferation and differentiation markers by immunohistochemistry. The results showed that mice on the diet containing oyster calcium significantly reduced rates of tumors in the tongue and proliferation and enhanced differentiation in the oral epithelium compared with the diet containing calcium depleted oyster powder. These results suggest that calcium in oyster plays a critical role in suppressing formation of oral squamous cell carcinoma and proliferation and promoting differentiation of the oral epithelium.

  7. [The effect and mechanism of endothelin-1-induced intracellular free calcium in human lung adenocarcinoma cells SPC-A1.].

    Science.gov (United States)

    Zhou, Juan; Zhang, Weimin; Ye, Qianjun; Jia, Gang

    2008-08-20

    Endothelin-1 (ET-1) is a potent mitogen involved in cell growth in human lung adenocarcinoma cells SPC-A1. The increase in intracellular free calcium ([Ca(2+)]i) plays a great role in this process. The aim of this study is to investigate the ET-1-induced [Ca(2+)]i responses in SPC-A1 cells and to explore its cellular mechanism. [Ca(2+)]i was measured by Fura-2/AM fluorescent assay. Endothelin receptors antagonists, calcium channel blockers and intracellular signal transduction blockers were used to study the underlying mechanism of ET-1-induced [Ca(2+)]i responses in SPC-A1 cells. At the concentration of 1*10(-15) mol/L-1*10(-8) mol/L, ET-1 caused a dose-dependent increase of [Ca(2+)]i in SPC-A1 cells (P 0.05), a highly selective endothelin receptor B (ETBR) antagonist. Depletion of extracellular Ca(2+) with free Ca(2+) solution and 0.1mmol/L ethyleneglycol bis (2-aminoethyl ether) tetraacetic acid (EGTA) or blockade of voltage dependent calcium channel with nifedipine at 1*10(-6) mol/L significantly reduced the ET-1-induced increase of [Ca(2+)]i. The ET-1-induced (1*10(-10) mol/L) increase of [Ca(2+)]i was also significantly attenuated by U73122 at 1*10(-5) mol/L (P <0.05), a phospholipase C inhibitor, and by Ryanodine at 50*10(-6) mol/L. However, Staurosporine (2*10(-9) mol/L), a protein kinas C inhibitor, exerted no significant effect on the ET-1-induced (1*10(-10) mol/L) increase of [Ca(2+)]i. ET-1 elevates [Ca(2+)]i via activation of ETA receptor. Both phospholipase C/Ca(2+) pathway and Ca(2+) influx through voltage dependent Ca(2+) channel activate by ETAR contribute to this process.

  8. Hydrogen sulfide-induced itch requires activation of Cav3.2 T-type calcium channel in mice

    Science.gov (United States)

    Wang, Xue-Long; Tian, Bin; Huang, Ya; Peng, Xiao-Yan; Chen, Li-Hua; Li, Jun-Cheng; Liu, Tong

    2015-01-01

    The contributions of gasotransmitters to itch sensation are largely unknown. In this study, we aimed to investigate the roles of hydrogen sulfide (H2S), a ubiquitous gasotransmitter, in itch signaling. We found that intradermal injection of H2S donors NaHS or Na2S, but not GYY4137 (a slow-releasing H2S donor), dose-dependently induced scratching behavior in a μ-opioid receptor-dependent and histamine-independent manner in mice. Interestingly, NaHS induced itch via unique mechanisms that involved capsaicin-insensitive A-fibers, but not TRPV1-expressing C-fibers that are traditionally considered for mediating itch, revealed by depletion of TRPV1-expressing C-fibers by systemic resiniferatoxin treatment. Moreover, local application of capsaizapine (TRPV1 blocker) or HC-030031 (TRPA1 blocker) had no effects on NaHS-evoked scratching. Strikingly, pharmacological blockade and silencing of Cav3.2 T-type calcium channel by mibefradil, ascorbic acid, zinc chloride or Cav3.2 siRNA dramatically decreased NaHS-evoked scratching. NaHS induced robust alloknesis (touch-evoked itch), which was inhibited by T-type calcium channels blocker mibefradil. Compound 48/80-induced itch was enhanced by an endogenous precursor of H2S (L-cysteine) but attenuated by inhibitors of H2S-producing enzymes cystathionine γ-lyase and cystathionine β-synthase. These results indicated that H2S, as a novel nonhistaminergic itch mediator, may activates Cav3.2 T-type calcium channel, probably located at A-fibers, to induce scratching and alloknesis in mice. PMID:26602811

  9. Regulation of store-operated Ca{sup 2+} entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kito, Hiroaki [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto (Japan); Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2015-04-10

    Store-operated Ca{sup 2+} entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca{sup 2+} influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. - Highlights: • Orai1 is essential for SOCE activity in brain capillary endothelial cells (BCECs). • Cell cycle independent expression of Orai1 regulated SOCE and cell proliferation. • Orai2 was up-regulated only at G2/M phase and this consequently reduced SOCE. • Orai2 as well as Orai1 is a key player controlling SOCE and proliferation in BCECs.

  10. Ionomycin-induced calcium influx induces neurite degeneration in mouse neuroblastoma cells: analysis of a time-lapse live cell imaging system.

    Science.gov (United States)

    Nakamura, Saki; Nakanishi, Ayumi; Takazawa, Minami; Okihiro, Shunsuke; Urano, Shiro; Fukui, Koji

    2016-01-01

    Reactive oxygen species induce neuronal cell death. However, the detailed mechanisms of cell death have not yet been elucidated. Previously, we reported neurite degeneration before the induction of cell death. Here, we attempted to elucidate the mechanisms of neurite degeneration before the induction of cell death using the neuroblastoma N1E-115 cell line and a time-lapse live cell imaging system. Treatment with the calcium ionophore ionomycin induced cell death and neurite degeneration in a concentration- and time-dependent manner. Treatment with a low concentration of ionomycin immediately produced a significant calcium influx into the intracellular region in N1E-115 cells. After 1-h incubation with ionomycin, the fluorescence emission of MitoSOX(TM) increased significantly compared to the control. Finally, analysis using a new mitochondrial specific fluorescence dye, MitoPeDPP, indicated that treatment with ionomycin significantly increased the mitochondrial lipid hydroperoxide production in N1E-115 cells. The fluorescence emissions of Fluo-4 AM and MitoPeDPP were detected in the cell soma and neurite regions in ionomycin-treated N1E-115 cells. However, the emissions of neurites were much lower than those of the cell soma. TBARS values of ionomycin-treated cells significantly increased compared to the control. These results indicate that ionomycin induces calcium influx into the intracellular region and reactive oxygen species production in N1E-115 cells. Lipid hydroperoxide production was induced in ionomycin-treated N1E-115 cells. Calcium influx into the intracellular region is a possible activator of neurite degeneration.

  11. Contribution of α4β2 nAChR in nicotine-induced intracellular calcium response and excitability of MSDB neurons.

    Science.gov (United States)

    Wang, Jiangang; Wang, Yali; Wang, Yang; Wang, Ran; Zhang, Yunpeng; Zhang, Qian; Lu, Chengbiao

    2014-12-10

    The neurons of medial septal diagonal band of broca (MSDB) project to hippocampus and play an important role in MSDB-hippocampal synaptic transmission, plasticity and network oscillation. Nicotinic acetylcholine receptor (nAChR) subunits, α4β2 and α7 nAChRs, are expressed in MSDB neurons and permeable to calcium ions, which may modulate the function of MSDB neurons. The aims of this study are to determine the roles of selective nAChR activation on the calcium responses and membrane currents in MSDB neurons. Our results showed that nicotine increased calcium responses in the majority of MSDB neurons, pre-treatment of MSDB slices with a α4β2 nAChR antagonist, DhβE but not a α7 nAChR antagonist, MLA prevented nicotine-induced calcium responses. The whole cell patch clamp recordings showed that nicotine-induced inward current and acetylcholine (ACh) induced-firing activity can be largely reduced or prevented by DhβE in MSDB neurons. Surprisingly, post-treatment of α4β2 or α7 nAChR antagonists failed to block nicotine׳s role, they increased calcium responses instead. Application of calcium chelator EGTA reduced calcium responses in all neurons tested. These results suggest that there was a subtype specific modulation of nAChRs on calcium signaling and membrane currents in MSDB neurons and nAChR antagonists were also able to induce calcium responses involving a distinct mechanism.

  12. Lymphocytes prime activation is required for nicotine-induced calcium waves.

    Science.gov (United States)

    Landais, Emilie; Liautaud-Roger, Francoise; Antonicelli, Frank

    2010-06-01

    Lymphocytes are reported to express nicotinic acetylcholine receptors (nAChR). However, no data are available on the expression of these nAChR on activated lymphocyte relatively to resting lymphocytes. In this study, we examined nAChR subunits expression in PHA-stimulated versus un-stimulated lymphocytes, and four leukemic cell lines. Cell stimulation with nicotine triggered calcium responses only in some experiments conducted with PHA-stimulated lymphocytes. Likewise, only the Jurkat and HL-60 cell lines displayed calcium waves upon nicotine stimulation, whereas the Raji and CCRF-CEM did not. All responding cells displayed an active form of the nicotinic a-7 nAChR. Indeed, use of 2 different sets of primers for the corresponding mRNA showed that expression of the full-length a-7 subunit mRNA was only present in PHA-stimulated lymphocytes for which calcium waves had been evidenced. Microscopy analysis of lymphocytes structure showed a direct relationship between their size, their a-7 nAChR expression, and calcium release upon nicotine stimulation. Then, this relationship suggested that lymphocytes need a prime activation to express the a-7 nAChR, and therefore to release calcium in response to nicotine.

  13. Calcium induces long-term legacy effects in a subalpine ecosystem.

    Directory of Open Access Journals (Sweden)

    Urs Schaffner

    Full Text Available Human activities have transformed a significant proportion of the world's land surface, with profound effects on ecosystem processes. Soil applications of macronutrients such as nitrate, phosphorus, potassium or calcium are routinely used in the management of croplands, grasslands and forests to improve plant health or increase productivity. However, while the effects of continuous fertilization and liming on terrestrial ecosystems are well documented, remarkably little is known about the legacy effect of historical fertilization and liming events in terrestrial ecosystems and of the mechanisms involved. Here, we show that more than 70 years after the last application of lime on a subalpine grassland, all major soil and plant calcium pools were still significantly larger in limed than in unlimed plots, and that the resulting shift in the soil calcium/aluminium ratio continues to affect ecosystem services such as primary production. The difference in the calcium content of the vegetation and the topmost 10 cm of the soil in limed vs. unlimed plots amounts to approximately 19.5 g m(-2, equivalent to 16.3% of the amount that was added to the plots some 70 years ago. In contrast, plots that were treated with nitrogen-phosphorus-potassium fertilizer in the 1930s did not differ from unfertilized plots in any of the soil and vegetation characteristics measured. Our findings suggest that the long-term legacy effect of historical liming is due to long-term storage of added calcium in stable soil pools, rather than a general increase in nutrient availability. Our results demonstrate that single applications of calcium in its carbonated form can profoundly and persistently alter ecosystem processes and services in mountain ecosystems.

  14. Efficacy of Methylene Blue in an Experimental Model of Calcium Channel Blocker Induced Shock

    Science.gov (United States)

    Jang, David H.; Donovan, Sean; Nelson, Lewis S.; Bania, Theodore C.; Hoffman, Robert S.; Chu, Jason

    2014-01-01

    BACKGROUND Calcium channel blocker poisonings account for a substantial number of reported deaths from cardiovascular drugs. While supportive care is the mainstay of treatment, experimental therapies such as high dose insulin-euglycemia and lipid emulsion have been studied in animal models and used in humans. In the most severe cases even aggressive care is inadequate and deaths occur. In both experimental models and clinical cases of vasodilatory shock, methylene blue improves hemodynamic measures. Methylene blue acts as both a nitric oxide scavenger and inhibits guanylate cyclase that is responsible for the production of cGMP. Excessive cGMP production is associated with refractory vasodilatory shock in sepsis and anaphylaxis. The aim of this study was to determine the efficacy of methylene blue in an animal model of amlodipine-induced shock. METHODS Sprague-Dawley rats were anesthetized, ventilated and instrumented for continuous blood pressure and heart rate monitoring. The dose of amlodipine that produced death within 60 minutes was 17 mg/kg/hour (LD50). Rats were divided into 2 groups: amlodipine followed by methylene blue or amlodipine followed by normal saline (NS) with 15 rats in each group. Rats received methylene blue at 2 mg/kg over 5 mins or an equivalent amount of NS in three intervals from the start of the protocol: Minute 5, 30, and 60. The animals were observed for a total of 2 hours after the start of the protocol. Mortality risk and survival time were analyzed using Fisher’s exact test and Kaplan Meier survival analysis with the log rank test. RESULTS Overall, 1/15 (7%) rats in the saline-treated group survived to 120 minutes compared with 5/15 (33%) rats in the methylene blue-treated group (difference −26%, 95% CI –54%, 0.3%). The median survival time for the NS group was 42 min (95% CI, 28.1,55.9) and the methylene blue group was 109 min (95% CI, 93.9,124.1). Heart rate and MAP differences between groups were analyzed until 60 minutes

  15. Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation

    Science.gov (United States)

    Gilbert, Daniel F.; Stebbing, Martin J.; Kuenzel, Katharina; Murphy, Robyn M.; Zacharewicz, Evelyn; Buttgereit, Andreas; Stokes, Leanne; Adams, David J.; Friedrich, Oliver

    2016-01-01

    Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+ entry (ROCE). Although store-operated Ca2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 μM) also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca2+ oscillations (P2X4) and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological insights into

  16. STORE-OPERATED CA2+ ENTRY (SOCE AND PURINERGIC RECEPTOR-MEDIATED CA2+ HOMEOSTASIS IN MURINE BV2 MICROGLIA CELLS: EARLY CELLULAR RESPONSES TO ATP-MEDIATED MICROGLIA ACTIVATION

    Directory of Open Access Journals (Sweden)

    Daniel F. Gilbert

    2016-10-01

    Full Text Available Microglia activation is a neuro-inflammatory response to parenchymal damage with release of intracellular metabolites, e.g. purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y and ionotropic (P2X cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuro-inflammation (so-called M1/M2 polarization. ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induce receptor-operated Ca2+ entry (ROCE. Although store-operated Ca2+ entry (SOCE was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 µM also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120 were tested on their profile to act on Ca2+ oscillations (P2X4 and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological

  17. Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.

    Directory of Open Access Journals (Sweden)

    Chia-Lin Ho

    Full Text Available Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

  18. Pore-scale network modeling of microbially induced calcium carbonate precipitation: Insight into scale dependence of biogeochemical reaction rates

    Science.gov (United States)

    Qin, Chao-Zhong; Hassanizadeh, S. Majid; Ebigbo, Anozie

    2016-11-01

    The engineering of microbially induced calcium carbonate precipitation (MICP) has attracted much attention in a number of applications, such as sealing of CO2 leakage pathways, soil stabilization, and subsurface remediation of radionuclides and toxic metals. The goal of this work is to gain insight into pore-scale processes of MICP and scale dependence of biogeochemical reaction rates. This will help us develop efficient field-scale MICP models. In this work, we have developed a comprehensive pore-network model for MICP, with geochemical speciation calculated by the open-source PHREEQC module. A numerical pseudo-3-D micromodel as the computational domain was generated by a novel pore-network generation method. We modeled a three-stage process in the engineering of MICP including the growth of biofilm, the injection of calcium-rich medium, and the precipitation of calcium carbonate. A number of test cases were conducted to illustrate how calcite precipitation was influenced by different operating conditions. In addition, we studied the possibility of reducing the computational effort by simplifying geochemical calculations. Finally, the effect of mass transfer limitation of possible carbonate ions in a pore element on calcite precipitation was explored.

  19. Fas mRNA expression and calcium influx change in H2O2-induced apoptotic hepatocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    Qi-Ping Lu; Lei Tian

    2005-01-01

    AIM: To investigate the relationship between Fas gene expression and calcium influx change in peroxide-induced apoptotic hepatocytes and the possible molecular mechanism of Rxa in protecting hepatocytes.METHODS: Single-cell Fas mRNA expression in H2O2-exposed L02 hepatocytes with or without treatment of Rxa,an extract from an anti-peroxidant, Radix Salviae Miltiorrhizae,was determined by all-cell patch clamp and single-cell reverse transcriptase polymerase chain reaction (RT-PCR). Transient calcium influx change ([Ca2+]i) in the cells was evaluated with all-cell patch clamp micro-fluorescence single-cytosolic free Ca2+ concentration technique. Fas protein expression, early apoptotic index (annexin-V+) and cell membrane change inthe cells were evaluated by immunohistochemistry, flow cytometry (FCM) and scan electron microscopy respectively.RESULTS: In cells exposed to H2O2 for 2 h, the specific lane for Fas mRNA was vivid on electrophoresis, with increased Fas protein expression, [Ca2+]i (from 143.66±34.21 to 1115.28±227.16), annexin-V+ index (from 4.00±0.79 to 16.18±0.72) and membrane vesicle formation. However, in cells exposed to H2O2 but pre-treated with Rxa, there was no increase in Fas mRNA or protein expression and [Ca2+]i (103.56±28.92). Annexin-V+ index (8.92±1.44) was lower than the controls (P<0.01), and the cell membrane was intact.CONCLUSION: H2O2 induces apoptosis of L02 cells by increasing cytosolic [Ca2+]i, and inducing Fas mRNA and protein expression. Rxa protects the L02 cells from apoptosis through anti-peroxidation, inhibition of calcium overloading and prevention of the activation of cytosolic Fas signal pathway.

  20. Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

    Directory of Open Access Journals (Sweden)

    Junjie Zhang

    2015-03-01

    Full Text Available G protein-coupled receptors (GPCRs constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR and cytomegalovirus (US28 shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA, which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

  1. Nitric oxide-induced calcium release: activation of type 1 ryanodine receptor, a calcium release channel, through non-enzymatic posttranslational modification by nitric oxide

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    Sho eKakizawa

    2013-10-01

    Full Text Available Nitric oxide (NO is a typical gaseous messenger involved in a wide range of biological processes. In our classical knowledge, effects of NO are largely achieved by activation of soluble guanylyl cyclase to form cyclic guanosine-3’, 5’-monophosphate. However, emerging evidences have suggested another signaling mechanism mediated by NO: S-nitrosylation of target proteins.S-nitrosylation is a covalent addition of an NO group to a cysteine thiol/sulfhydryl (RSH, and categorized into non-enzymatic posttranslational modification of proteins, contrasted to enzymatic posttranslational modification of proteins, such as phosphorylation mediated by various protein kinases.Very recently, we found novel intracellular calcium (Ca2+ mobilizing mechanism, NO-induced Ca2+ release (NICR in cerebellar Purkinje cells. NICR is mediated by type 1 ryanodine receptor (RyR1, a Ca2+ release channel expressed in endoplasmic-reticular membrane. Furthermore, NICR is indicated to be dependent on S-nitrosylation of RyR1, and involved in synaptic plasticity in the cerebellum. In this review, molecular mechanisms and functional significance of NICR, as well as non-enzymatic posttranslational modification of proteins by gaseous signals, are described.

  2. Effect of subclinical, clinical and supraclinical doses of calcium channel blockers on models of drug-induced hepatotoxicity in rats.

    Science.gov (United States)

    Okwa, Iniviefien B; Akindele, Abidemi J; Agbaje, Esther O; Oshinuga, Oladoyin T; Anunobi, Chidozie C; Adeyemi, Olufunmilayo O

    2013-01-01

    Drug-related hepatotoxicity is the leading cause of acute liver failure, and hepatic problems are responsible for a significant number of liver transplantations and deaths worldwide. Calcium has been associated with various metabolic processes that lead to cell death and apoptosis, and increased cytosolic Ca(2+) has been implicated in hepatotoxicity. This study was designed to investigate the effects of calcium channel blockers (CCBs) on isoniazid-rifampicin, zidovudine and erythromycin-induced hepatotoxicity in rats. Treatment groups comprised control, hepatotoxicant, hepatotoxicant along with each of silymarin, nifedipine, verapamil and diltiazem at subclinical, clinical and supraclinical doses. A day to the end of treatment for each model, rats were subjected to the hexobarbitone-induced hypnosis test. On the last days of treatment, blood samples were collected and serum was analyzed for relevant biochemical parameters. Animals were sacrificed after blood collection and livers were harvested, and samples obtained for in vivo antioxidant indices assay and histopathology. The hepatotoxicants significantly increased serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), as well as duration of sleep in the hypnosis test. These drugs significantly reduced the hepatic levels of reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and increased the level of malondialdehyde (MDA). The CCBs at the various doses significantly reversed the effects of isoniazid-rifampicin, zidovudine and erythromycin. The results obtained in this study suggest that the CCBs possess hepatoprotective activity in drug-induced hepatotoxicity and may be beneficial at the subclinical and clinical doses.

  3. Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels.

    Science.gov (United States)

    Guo, Kai; Wang, Xianming; Gao, Guofeng; Huang, Congxin; Elmslie, Keith S; Peterson, Blaise Z

    2010-11-01

    We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149-1159, 2010). The short 17 amino acid extracellular NH(2)-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate Ca(V)1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with Ca(V)1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on Ca(V)1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca(2+) dynamics in the heart.

  4. Probing calcium and sulfur distribution and pattern in hairs using micro-proton induced X-ray emission (MPIXE)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    More than 20 hair strands obtained from several people are analyzed by non-invasive micro-proton induced X-ray emission (PIXE) for probing calcium and sulfur distribution and pattern. The sulfur content in woman's hair along the longitudinal axis shows a monthly rhythm whereas the level of Ca is much higher in the medulla core and exhibits a daily rhythm both in male and female hair. The Ca content gradually decreases from the tip to the root position in the shedding process and more Ca can be found in black hair compared to white hair.

  5. Rapid changes in skeletal muscle calcium uptake induced in vitro by 1,25-dihydroxyvitamin D/sub 3/ are suppressed by calcium channel blockers

    Energy Technology Data Exchange (ETDEWEB)

    de Boland, A.R.; Boland, R.L.

    1987-05-01

    Previous investigations have shown that 1,25-dihydroxyvitamin D/sub 3/ (1,25-(OH)/sub 2/D/sub 3/) stimulates muscle Ca uptake through a nuclear mechanism. The possibility that 1,25-(OH)/sub 2/D/sub 3/ would induce rapid changes in muscle Ca fluxes independent of de novo protein synthesis was investigated in the present work. In vitro preparations of soleus muscles obtained from vitamin D-deficient chicks were used. A significant increase in /sup 45/Ca labeling of the tissue was already observed after 3-min treatment with 2.4 X 10(-10) M 1,25-(OH)/sub 2/D/sub 3/. This early stimulation in muscle Ca uptake became maximal at 10-15 min. Cycloheximide (50 microM) did not block the effect of the metabolite at 15 and 30 min. However, the antibiotic effectively blocked the increase in Ca uptake induced by 1,25-(OH)/sub 2/D/sub 3/ after 1-h treatment. The rapid 1,25-(OH)/sub 2/D/sub 3/-dependent stimulation of /sup 45/Ca labeling of soleus muscle was not associated to changes in lipid synthesis as assessed by measurements of /sup 3/H-glycerol incorporation into the tissue lipids. However, the calcium antagonists verapamil and nifedipine (50 microM) abolished the stimulation in Ca uptake produced by 1,25-(OH)/sub 2/D/sub 3/ in 5 min. These results suggest that 1,25-(OH)/sub 2/D/sub 3/ can act directly at the muscle membrane level affecting Ca fluxes through Ca channels.

  6. Natural Products Induce a G Protein-Mediated Calcium Pathway Activating p53 in Cancer Cells

    Science.gov (United States)

    van Ginkel, Paul R.; Yan, Michael B.; Bhattacharya, Saswati; Polans, Arthur S.; Kenealey, Jason D.

    2015-01-01

    Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. PMID:26341291

  7. Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells.

    Science.gov (United States)

    van Ginkel, Paul R; Yan, Michael B; Bhattacharya, Saswati; Polans, Arthur S; Kenealey, Jason D

    2015-11-01

    Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death.

  8. Mechanisms of pyrethroid insecticide-induced stimulation of calcium influx in neocortical neurons

    Science.gov (United States)

    Pyrethroid insecticides bind to voltage-gated sodium channels (VGSCs) and modify their gating kinetics, thereby disrupting neuronal function. Pyrethroids have also been reported to alter the function of other channel types, including activation of voltage-gated Ca2+ calcium chann...

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

    Science.gov (United States)

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

    2013-01-01

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

  10. Types of voltage—dependent calcium channels involved in high potassium depolarization—induced amylase secretion in the exocrine pancreatic tumour cell line AR4—2J

    Institute of Scientific and Technical Information of China (English)

    CUIZONGJIE

    1998-01-01

    In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium,Attached cells when stimulated with high potassium secreted large amount of amylase.High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation.High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel anatagonists with an order of potency as follows:nifedipine>ω-agatoxin IVA>ω-conotoxin GVIA.In contrast,the L-type calcium channel anatagonist nifedipine almost completely inhibited potassium-induced amylase secretion,whereas the N-type channel antagonist ω-conotoxin GVIA was without effect.The P-type channel antagonist ω-agatoxin IVA had a small inhibitory effect,but this inhibition was not significant at the level of amylase secretion.In conclusion,the AR4-2J cell line posesses different voltage-dependent calcium channels(L,P,N)with the L-type predominantly involved in depolarization induced amylase secretion.

  11. Capsaicin mimics mechanical load-induced intracellular signaling events: involvement of TRPV1-mediated calcium signaling in induction of skeletal muscle hypertrophy.

    Science.gov (United States)

    Ito, Naoki; Ruegg, Urs T; Kudo, Akira; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi

    2013-01-01

    Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca ( 2+) ]i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca ( 2+) ]i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.

  12. Intra-articular lentivirus-mediated gene therapy targeting CRACM1 for the treatment of collagen-induced arthritis

    Directory of Open Access Journals (Sweden)

    Shuang Liu

    2017-03-01

    Full Text Available Abnormal store-operated calcium uptake has been observed in peripheral T lymphocytes of rheumatoid arthritis (RA patients, and sustained intracellular calcium signalling is known to mediate the functions of many types of immune cells. Thus, it is hypothesized that regulating calcium entry through CRACM1 (the pore-forming subunit of calcium release-activated calcium (CRAC channels; also known as ORAI1 may be beneficial for the management of RA. Localized CRACM1 knockdown in the joints and draining lymph nodes (DLNs of mice with collagen-induced arthritis (CIA was achieved via lentiviral-based delivery of shRNA targeting mouse CRACM1. Consistent with CRACM1 knockdown, calcium influx in synovial cells and the histopathological features of CIA were reduced. These effects were also associated with reduced levels of several notable inflammatory cytokines, such as IL-6, IL-17A, and IFN-γ, in the joints. Additionally, CRACM1-shRNA reduced the number of bone marrow-derived osteoclasts in vitro as well as osteoclasts in CIA joints, which was associated with reduced RANKL levels in the serum and joints. In summary, inhibiting calcium entry by CRACM1 knockdown suppressed arthritis development and may be therapeutically beneficial for RA patients.

  13. Glibenclamide decreases ATP-induced intracellular calcium transient elevation via inhibiting reactive oxygen species and mitochondrial activity in macrophages.

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    Duo-ling Li

    Full Text Available Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP-induced intracellular calcium ([Ca(2+]i handling in Raw 264.7 macrophages. In the present study, [Ca(2+]i transient, reactive oxygen species (ROS and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K(+ channel blockers had no effect on the resting [Ca(2+]i of Raw 264.7 cells. Extracellular ATP (100 µM induced [Ca(2+]i transient elevation independent of extracellular Ca(2+. The transient elevation was inhibited by an ROS scavenger (tiron and mitochondria inhibitor (rotenone. Glibenclamide and 5-hydroxydecanoate (5-HD also decreased ATP-induced [Ca(2+]i transient elevation, but pinacidil and other unselective K(+ channel blockers had no effect. Glibenclamide also decreased the peak of [Ca(2+]i transient induced by extracellular thapsigargin (Tg, 1 µM. Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca(2+]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca(2+]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.

  14. Role for the magnetic field in the radiation-induced efflux of calcium ions from brain tissue in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Blackman, C.F.; Benane, S.G.; Rabinowitz, J.R.; House, D.E.; Joines, W.T.

    1985-01-01

    Two independent laboratories have demonstrated that specific frequencies of electromagnetic radiation can cause a change in the efflux of calcium ions from brain tissue in vitro. Under a static magnetic field intensity of 38 microTesla (microT) due to the earth's magnetic field, 15- and 45-Hz electromagnetic signals (40 Vp-p/m in air) have been shown to induce a change in the efflux of calcium ions from the exposed tissues, while 1- and 30-Hz signals do not. The authors now show that the effective 15-Hz signal can be rendered ineffective when the net static magnetic field is reduced to 19 microT with Helmholtz coils. In addition, the ineffective 30-Hz signal becomes effective when the static magnetic field is changed to + or - 25.3 microT or to + or - 76 microT. These results demonstrate that the net intensity of the static magnetic field is an important variable. The results appear to describe a resonance-like relationship in which the extremely-low-frequency electromagnetic field that can induce a change in efflux is proportional to a product of the net magnetic field intensity and an index, 2n+1, where n=0,1.

  15. Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes.

    Science.gov (United States)

    Granqvist, Emma; Sun, Jongho; Op den Camp, Rik; Pujic, Petar; Hill, Lionel; Normand, Philippe; Morris, Richard J; Downie, J Allan; Geurts, Rene; Oldroyd, Giles E D

    2015-08-01

    Plants that form root-nodule symbioses are within a monophyletic 'nitrogen-fixing' clade and associated signalling processes are shared with the arbuscular mycorrhizal symbiosis. Central to symbiotic signalling are nuclear-associated oscillations in calcium ions (Ca(2+) ), occurring in the root hairs of several legume species in response to the rhizobial Nod factor signal. In this study we expanded the species analysed for activation of Ca(2+) oscillations, including nonleguminous species within the nitrogen-fixing clade. We showed that Ca(2+) oscillations are a common feature of legumes in their association with rhizobia, while Cercis, a non-nodulating legume, does not show Ca(2+) oscillations in response to Nod factors from Sinorhizobium fredii NGR234. Parasponia andersonii, a nonlegume that can associate with rhizobia, showed Nod factor-induced calcium oscillations to S. fredii NGR234 Nod factors, but its non-nodulating sister species, Trema tomentosa, did not. Also within the nitrogen-fixing clade are actinorhizal species that associate with Frankia bacteria and we showed that Alnus glutinosa induces Ca(2+) oscillations in root hairs in response to exudates from Frankia alni, but not to S. fredii NGR234 Nod factors. We conclude that the ability to mount Ca(2+) oscillations in response to symbiotic bacteria is a common feature of nodulating species within the nitrogen-fixing clade.

  16. Acrolein induces Hsp72 via both PKCdelta/JNK and calcium signaling pathways in human umbilical vein endothelial cells.

    Science.gov (United States)

    Misonou, Yoshiko; Takahashi, Motoko; Park, Yong Seek; Asahi, Michio; Miyamoto, Yasuhide; Sakiyama, Haruhiko; Cheng, Xinyao; Taniguchi, Naoyuki

    2005-05-01

    Acrolein is a highly electrophilic alpha,beta-unsaturated aldehydes to which humans are exposed in a variety of environment situations and is also a product of lipid peroxidation. Increased levels of unsaturated aldehydes play an important role in the pathogenesis of a number of human diseases such as Alzheimer's disease, atherosclerosis and diabetes. A number of studies have reported that acrolein evokes downstream signaling via an elevation in cellular oxidative stress. Here, we report that low concentrations of acrolein induce Hsp72 in human umbilical vein endothelial cells (HUVEC) and that both the PKCdelta/JNK pathway and calcium pathway were involved in the induction. The findings confirm that the production of reactive oxygen species (ROS) is not directly involved in the pathway. The induction of Hsp72 was not observed in other cells such as smooth muscle cells (SMC) or COS-1 cells. The results suggest that HUVEC have a unique defense system against cell damage by acrolein in which Hsp72 is induced via activation of both the PKCd/JNK and the calcium pathway.

  17. Rapid Inhibition of the Glutamate-induced Increase of Intracellular Free Calcium by Magnesium in Rat Hippocampal Neurons

    Institute of Scientific and Technical Information of China (English)

    张蕲; 胡波; 孙圣刚; 邓学军; 梅元武; 童萼塘

    2004-01-01

    By using Fura-2/AM, the effects of magnesium (Mg2+) on the glutamate-induced increase of intracellular free calcium ([Ca2+]i) in the cultured hippocampal neurons and the features were investigated by integrated photoelectric detecting system. The experiments were designed to three groups (The drug was spit to the cells for 20 s): Group A receiving 1×10-5 mol/L glutamate; Group B receiving 1 × 10-5 mol/L glutamate and1× 10-5 mol/L Mg2+ simultaneously; Group C receiving 1 × 10-5 mol/L glutamate again after [Ca2+]i in group B back to the baseline. The results showed that in group A, [Ca2+]i was obviously increased. In group B, the changes in [Ca2+]i and the peak value were significantly decreased. Moreover, the elevation of Phase 1 was slowed down and Phase 2 was shortened to some extent, and the plateau phase between them was relatively prolonged. In group C, calcium oscillation similar to that in group A occurred, but both the Phase 1 and Phase 2 were shortened and the △[Ca2+]i was slightly decreased. It was suggested that Mg2+ could quickly inhibit the rise of [Ca2+]i induced by glutamate in the cultured hippocampal neurons in rats.

  18. Quantitative analysis of soil calcium by laser-induced breakdown spectroscopy using addition and addition-internal standardizations

    Science.gov (United States)

    Shirvani-Mahdavi, Hamidreza; Shafiee, Parisa

    2016-12-01

    Matrix mismatching in the quantitative analysis of materials through calibration-based laser-induced breakdown spectroscopy (LIBS) is a serious problem. In this paper, to overcome the matrix mismatching, two distinct approaches named addition standardization (AS) and addition-internal combinatorial standardization (A-ICS) are demonstrated for LIBS experiments. Furthermore, in order to examine the efficiency of these methods, the concentration of calcium in ordinary garden soil without any fertilizer is individually measured by each of the two procedures. To achieve this purpose, ten standard samples with different concentrations of calcium (as the analyte) and copper (as the internal standard) are prepared in the form of cylindrical tablets, so that the soil plays the role of the matrix in all of them. The measurements indicate that the relative error of concentration compared to a certified value derived by induced coupled plasma optical emission spectroscopy is 3.97% and 2.23% for AS and A-ICS methods, respectively. Furthermore, calculations related to standard deviation indicates that A-ICS method may be more accurate than AS one.

  19. Efficacy of methylene blue in an experimental model of calcium channel blocker-induced shock.

    Science.gov (United States)

    Jang, David H; Donovan, Sean; Nelson, Lewis S; Bania, Theodore C; Hoffman, Robert S; Chu, Jason

    2015-04-01

    Calcium channel blocker poisonings account for a substantial number of reported deaths from cardiovascular drugs. Although supportive care is the mainstay of treatment, experimental therapies such as high-dose insulin-euglycemia and lipid emulsion have been studied in animal models and used in humans. In the most severe cases, even aggressive care is inadequate and deaths occur. In both experimental models and clinical cases of vasodilatory shock, methylene blue improves hemodynamic measures. It acts as a nitric oxide scavenger and inhibits guanylate cyclase that is responsible for the production of cyclic guanosine monophosphate (cGMP). Excessive cGMP production is associated with refractory vasodilatory shock in sepsis and anaphylaxis. The aim of this study is to determine the efficacy of methylene blue in an animal model of amlodipine-induced shock. Sprague-Dawley rats were anesthetized, ventilated, and instrumented for continuous blood pressure and pulse rate monitoring. The dose of amlodipine that produced death within 60 minutes was 17 mg/kg per hour (LD50). Rats were divided into 2 groups: amlodipine followed by methylene blue or amlodipine followed by normal saline solution, with 15 rats in each group. Rats received methylene blue at 2 mg/kg during 5 minutes or an equivalent amount of normal saline solution in 3 intervals from the start of the protocol: minutes 5, 30, and 60. The animals were observed for a total of 2 hours after the start of the protocol. Mortality risk and survival time were analyzed with Fisher's exact test and Kaplan-Meier survival analysis with the log rank test. Overall, 1 of 15 rats (7%) in the saline solution-treated group survived to 120 minutes compared with 5 of 15 (33%) in the methylene blue-treated group (difference -26%; 95% confidence interval [CI] -54% to 0.3%). The median survival time for the normal saline solution group was 42 minutes (95% CI 28.1 to 55.9 minutes); for the methylene blue group, 109 minutes (95% CI 93.9 to

  20. Expression of genes encoding the calcium signalosome in cellular and transgenic models of Huntington’s disease

    Directory of Open Access Journals (Sweden)

    Magdalena eCzeredys

    2013-11-01

    Full Text Available Huntington’s disease (HD is a hereditary neurodegenerative disease caused by the expansion of a polyglutamine stretch in the huntingtin (HTT protein and characterized by dysregulated calcium homeostasis. We investigated whether these disturbances are correlated with changes in the mRNA level of the genes that encode proteins involved in calcium homeostasis and signaling (i.e., the calciosome. Using custom-made TaqMan low-density arrays containing probes for 96 genes, we quantified mRNA in the striatum in YAC128 mice, a model of HD, and wildtype mice. HTT mutation caused the increased expression of some components of the calcium signalosome, including calretinin, presenilin 2, and calmyrin 1, and the increased expression of genes indirectly involved in calcium homeostasis, such as huntingtin-associated protein 1 and calcyclin-binding protein. To verify these findings in a different model, we used PC12 cells with an inducible expression of mutated full-length HTT. Using single-cell imaging with Fura-2AM, we found that store-operated Ca2+ entry but not endoplasmic reticulum store content was changed as a result of the expression of mutant HTT. Statistically significant downregulation of the Orai calcium channel subunit 2, calmodulin, and septin 4 was detected in cells that expressed mutated HTT. Our data indicate that the dysregulation of calcium homeostasis correlates with changes in the gene expression of members of the calciosome. These changes, however, differed in the two models of HD used in this study. Our results indicate that each HD model exhibits distinct features that may only partially resemble the human disease.

  1. Crambescidin 816 induces calcium influx though glutamate receptors in primary cultures of cortical neurons

    Directory of Open Access Journals (Sweden)

    Víctor Martín Vázquez

    2014-06-01

    In summary, our data suggest that the cytotoxic effect of 10 μM Cramb816 in cortical neurons may be related to an increase in the cytosolic calcium concentration elicited by the toxin, which is shown to be mediated by glutamate receptor activation. Further studies analyzing the effect of glutamate receptor blockers on the cytotoxic effect of Cramb816 are needed to confirm this hypothesis.

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

    Directory of Open Access Journals (Sweden)

    Per eSvenningsen

    2013-10-01

    Full Text Available ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC and AQP2. Recently, we have shown that connexin (Cx 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30-/- mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca2+]i signaling in the CD. Cortical CDs (CCDs from wild type and Cx30-/- mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca2+]i in wild type CCDs. This response was blunted in Cx30-/- CCDs ([Ca2+]i increased only 1.2-fold, p

  3. Nonlinear time series analysis of nodulation factor induced calcium oscillations: evidence for deterministic chaos?

    Directory of Open Access Journals (Sweden)

    Saul Hazledine

    Full Text Available Legume plants form beneficial symbiotic interactions with nitrogen fixing bacteria (called rhizobia, with the rhizobia being accommodated in unique structures on the roots of the host plant. The legume/rhizobial symbiosis is responsible for a significant proportion of the global biologically available nitrogen. The initiation of this symbiosis is governed by a characteristic calcium oscillation within the plant root hair cells and this signal is activated by the rhizobia. Recent analyses on calcium time series data have suggested that stochastic effects have a large role to play in defining the nature of the oscillations. The use of multiple nonlinear time series techniques, however, suggests an alternative interpretation, namely deterministic chaos. We provide an extensive, nonlinear time series analysis on the nature of this calcium oscillation response. We build up evidence through a series of techniques that test for determinism, quantify linear and nonlinear components, and measure the local divergence of the system. Chaos is common in nature and it seems plausible that properties of chaotic dynamics might be exploited by biological systems to control processes within the cell. Systems possessing chaotic control mechanisms are more robust in the sense that the enhanced flexibility allows more rapid response to environmental changes with less energetic costs. The desired behaviour could be most efficiently targeted in this manner, supporting some intriguing speculations about nonlinear mechanisms in biological signaling.

  4. Role of the Mitochondrial Calcium Uniporter in Rat Hippocampal Neuronal Death After Pilocarpine-Induced Status Epilepticus.

    Science.gov (United States)

    Wang, Cui; Xie, Nanchang; Wang, Yunlong; Li, Yulin; Ge, Xinjie; Wang, Menglu

    2015-08-01

    The mitochondrial calcium uniporter (MCU) is reportedly involved in oxidative stress, apoptosis, and many neurological diseases. However, the role of the MCU in epilepsy remains unknown. In this study, we found that the MCU inhibitor Ru360 significantly attenuated neuronal death and exerted an anti-apoptotic effect on rat hippocampal neurons after pilocarpine-induced status epilepticus (SE), while the MCU activator spermine increased seizure-induced neuronal death and apoptosis. In addition, Ru360 decreased the level of seizure-induced reactive oxygen species (ROS) in mitochondria isolated from rat hippocampi. Moreover, Ru360 restored the altered mitochondrial membrane potential and cytochrome c (CytC) release in epileptic hippocampi. However, spermine treatment exerted an opposite effect on seizure-induced ROS production and mitochondrial membrane potential alteration and CytC release compared with Ru360 treatment. Altogether, the findings of this study suggest that MCU inhibition exerts a neuroprotective effect on seizure-induced brain injury possibly through the mitochondria/ROS/CytC pathway.

  5. Evaluating the function of calcium antagonist on the Cd-induced stress in sperm of Russian sturgeon, Acipenser gueldenstaedtii.

    Science.gov (United States)

    Li, Zhi-Hua; Li, Ping; Rodina, Marek; Randak, Tomas

    2010-11-15

    In the current study, the sperm of Russian sturgeon (Acipenser gueldenstaedtii) was used to evaluate the roles of Verapamil (VRP), a calcium channel blocker, against cadmium (Cd)-induced stress. Sturgeon sperm were exposed for 2h at 50μg/L VRP, 5.0μg/L Cd, the mixture of 50μg/L VRP+5.0μg/L Cd, 50μg/L Cd and the mixture of 50μg/L VRP+50μg/L Cd. After exposure, the sperm motility parameters (motility and velocity), oxidative stress levels (lipid peroxidation [LPO] and carbonyl protein [CP]) and antioxidant enzyme activities (superoxide dismutase [SOD], glutathione reductase [GR], glutathione peroxidase [GPx]) were measured in sturgeon sperm. Compared to the control, Cd-induced stress was apparent as reflected by depressed motility parameters, induced oxidative stress and inhibited antioxidant enzyme activities at both Cd concentrations. In the presence of VRP, Cd-induced stress was reduced in sturgeon sperm, especially all the measured parameters in the sperm exposed at 5.0μg/L Cd returned to control levels, expect for the sperm motility. The present results indicate that VRP can reduce the Cd-induced stress in sturgeon sperm and suggest that using of sperm in vitro assays may provide a novel and efficient means for evaluating the effects of residual metals in the aquatic environment of sturgeon. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Evaluating the function of calcium antagonist on the Cd-induced stress in sperm of Russian sturgeon, Acipenser gueldenstaedtii

    Energy Technology Data Exchange (ETDEWEB)

    Li Zhihua, E-mail: zhihuali06@yahoo.com [University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zatisi 728/II, 389 25 Vodnany (Czech Republic); Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Jingzhou 434000 (China); Li Ping [University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zatisi 728/II, 389 25 Vodnany (Czech Republic); Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Jingzhou 434000 (China); Rodina, Marek; Randak, Tomas [University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zatisi 728/II, 389 25 Vodnany (Czech Republic)

    2010-11-15

    In the current study, the sperm of Russian sturgeon (Acipenser gueldenstaedtii) was used to evaluate the roles of Verapamil (VRP), a calcium channel blocker, against cadmium (Cd)-induced stress. Sturgeon sperm were exposed for 2 h at 50 {mu}g/L VRP, 5.0 {mu}g/L Cd, the mixture of 50 {mu}g/L VRP + 5.0 {mu}g/L Cd, 50 {mu}g/L Cd and the mixture of 50 {mu}g/L VRP + 50 {mu}g/L Cd. After exposure, the sperm motility parameters (motility and velocity), oxidative stress levels (lipid peroxidation [LPO] and carbonyl protein [CP]) and antioxidant enzyme activities (superoxide dismutase [SOD], glutathione reductase [GR], glutathione peroxidase [GPx]) were measured in sturgeon sperm. Compared to the control, Cd-induced stress was apparent as reflected by depressed motility parameters, induced oxidative stress and inhibited antioxidant enzyme activities at both Cd concentrations. In the presence of VRP, Cd-induced stress was reduced in sturgeon sperm, especially all the measured parameters in the sperm exposed at 5.0 {mu}g/L Cd returned to control levels, expect for the sperm motility. The present results indicate that VRP can reduce the Cd-induced stress in sturgeon sperm and suggest that using of sperm in vitro assays may provide a novel and efficient means for evaluating the effects of residual metals in the aquatic environment of sturgeon.

  7. Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid depend on intracellular calcium levels in primary cultures of human adipocytes

    DEFF Research Database (Denmark)

    Kennedy, Arion; Martinez, Kristina; Chung, Soonkyu

    2010-01-01

    10,12 CLA-mediated production of reactive oxygen species (ROS), activation of ERK1/2 and cJun-NH2-terminal kinase (JNK), and induction of inflammatory genes. 10,12 CLA-mediated binding of NFkappaB to the promoters of interleukin (IL)-8 and cyclooxygenase (COX)-2 and induction of calcium......We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) induced inflammation and insulin resistance in primary human adipocytes by activating nuclear factor kappaB (NFkappaB) and extracellular signal-related kinase (ERK) signaling. In this study, we demonstrated...... that the initial increase in intracellular calcium ([Ca2+]i) mediated by 10,12 CLA was attenuated by TMB-8, an inhibitor of calcium release from the endoplasmic reticulum (ER), by BAPTA, an intracellular calcium chelator, and by D609, a phospholipase C (PLC) inhibitor. Moreover, BAPTA, TMB-8, and D609 attenuated...

  8. 脲解型微生物诱导碳酸钙沉积研究%Investigation on ureolytic microbiologically-induced calcium carbonate precipitation

    Institute of Scientific and Technical Information of China (English)

    徐晶; 杜雅莉; 白慧莉

    2016-01-01

    研究了钙源种类对脲解型微生物诱导碳酸钙沉积的生物-化学过程的影响。利用电位分析法实时测试了沉积过程中钙离子、铵离子及p H 值的变化,并利用显微计数对细菌浓度进行了监测。采用扫描电镜(SEM)、X射线衍射(XRD)和分析红外光谱(IR)对沉积产物进行了研究。结果表明,不同钙源环境下脲解型微生物诱导矿化沉积都存在化学沉淀、微生物诱导矿化沉积和沉淀完全3个阶段;有机钙源环境下细菌的产矿动力比在无机钙源中高,且两种钙源所获得的方解石晶体沉积物在形貌上差异显著。%The effects of calcium source type on the bio-chemical processes of ureolytic microbiologically-induced calcium carbonate precipitation was investigated in this paper.By using potentiometric analysis,calcium ions concentration,ammonium ions concentration,and pH value were measured.Cell density was also monitored by microscopic counting method.Sediments were characterized by scanning electron microscopy,X-ray diffraction, and infrared spectroscopy.The results showed that the processes of microbiologically-induced calcium carbonate precipitation consists the abiotic precipitation stage,the microbiologically-induced calcium carbonate precipitati-on stage,and the calcium ions depletion stage,regardless of the calcium source type.The efficiency of bacterial-ly-induce mineralized precipitation in organic calcium source environment is higher than that in inorganic calcium source environment.Significant morphological difference of precipitated calcites from two types of calcium sources was detected.

  9. Sound Waves Induce Neural Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells via Ryanodine Receptor-Induced Calcium Release and Pyk2 Activation.

    Science.gov (United States)

    Choi, Yura; Park, Jeong-Eun; Jeong, Jong Seob; Park, Jung-Keug; Kim, Jongpil; Jeon, Songhee

    2016-10-01

    Mesenchymal stem cells (MSCs) have shown considerable promise as an adaptable cell source for use in tissue engineering and other therapeutic applications. The aims of this study were to develop methods to test the hypothesis that human MSCs could be differentiated using sound wave stimulation alone and to find the underlying mechanism. Human bone marrow (hBM)-MSCs were stimulated with sound waves (1 kHz, 81 dB) for 7 days and the expression of neural markers were analyzed. Sound waves induced neural differentiation of hBM-MSC at 1 kHz and 81 dB but not at 1 kHz and 100 dB. To determine the signaling pathways involved in the neural differentiation of hBM-MSCs by sound wave stimulation, we examined the Pyk2 and CREB phosphorylation. Sound wave induced an increase in the phosphorylation of Pyk2 and CREB at 45 min and 90 min, respectively, in hBM-MSCs. To find out the upstream activator of Pyk2, we examined the intracellular calcium source that was released by sound wave stimulation. When we used ryanodine as a ryanodine receptor antagonist, sound wave-induced calcium release was suppressed. Moreover, pre-treatment with a Pyk2 inhibitor, PF431396, prevented the phosphorylation of Pyk2 and suppressed sound wave-induced neural differentiation in hBM-MSCs. These results suggest that specific sound wave stimulation could be used as a neural differentiation inducer of hBM-MSCs.

  10. Changes in root gravitropism, ultrastructure, and calcium balance of pea root statocytes induced by A23187

    Science.gov (United States)

    Belyavskaya, N.

    The role for calcium in the regulation of a wide variety of cellular events in plants is well known. Calcium signaling has been implicated in plant gravitropism. A carboxylic acid antibiotic A23187 (calcimycin) has been widely used in biological studies since it can translocate calcium across membranes. Seedlings of Pisum sativum L. cv. Uladovsky germinated in a vertically oriented cylinder of moist filter paper soaked in water during 4.5 day had been treated with 10-5 M A23187 for 12 hr. Tips of primary roots of control and A23187-treated pea seedlings were fixed for electron microscopy and electron cytochemistry. Experiments with Pisum sativum 5- day seedlings placed horizontally for 4 h after treatment with 10 μM A23187 during 12 h found that the graviresponsiveness of their primary roots was lost completely (91 % of roots) or inhibited (24 +/- 6° in comparison with 88 +/- 8° in control). At ultrastructural level, there were observed distribution of amyloplasts around the nucleus, remarkable lengthening of statocytes, advanced vacuolization, changes in dictyosome structure, ER fragmentation, cell wall thinning in A23187-treated statocytes. Cytochemical study has indicated that statocytes exposed to calcimycin have contained a number of Ca-pyroantimonate granules detected Ca 2 + ions in organelles and hyaloplasm (unlike the control ones). The deposits were mainly associated with the plasma membrane. Among organelles, mitochondria were notable for their ability to accumulate Ca 2 +. In amyloplasts, a fine precipitate was predominately located in their stroma and envelope lumens. In cell walls, deposits of the reaction product were observed along the periphery and in the median zone. Localization of electron-dense granules of lead phosphate, which indicated Ca 2 +- ATPase activities in pea statocytes exposed to A23187, was generally consistent with that in untreated roots. Apart from plasma membrane, chromatin, and nucleolus components, the cytochemical reaction

  11. Prevention of copper-induced calcium influx and cell death by prion-derived peptide in suspension-cultured tobacco cells.

    Science.gov (United States)

    Kagenishi, Tomoko; Yokawa, Ken; Kuse, Masaki; Isobe, Minoru; Bouteau, François; Kawano, Tomonori

    2009-01-01

    Impact of copper on the oxidative and calcium signal transductions leading to cell death in plant cells and the effects of the copper-binding peptide derived from the human prion protein (PrP) as a novel plant-protecting agent were assessed using a cell suspension culture of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species (ROS), such as hydroxyl radicals, and stimulation of calcium channel opening, allowing a transient increase in cytosolic calcium concentrations. The former was proven by the action of specific ROS scavengers blocking the calcium responses and the latter was proven by an increase in aequorin luminescence and its inhibition by specific channel blockers. Following these early events completed within 10 min, the development of copper-induced cell death was observed during additional 1 h in a dose-dependent manner. Addition of a synthetic peptide (KTNMKHMA) corresponding to the neurotoxic sequence in human PrP, prior to the addition of copper, effectively blocked both calcium influx and cell death induced by copper. Lastly, a possible mechanism of peptide action and future applications of this peptide in the protection of plant roots from metal toxicity or in favour of phytoremediation processes are discussed.

  12. Cortical overexpression of neuronal calcium sensor-1 induces functional plasticity in spinal cord following unilateral pyramidal tract injury in rat.

    Directory of Open Access Journals (Sweden)

    Ping K Yip

    Full Text Available Following trauma of the adult brain or spinal cord the injured axons of central neurons fail to regenerate or if intact display only limited anatomical plasticity through sprouting. Adult cortical neurons forming the corticospinal tract (CST normally have low levels of the neuronal calcium sensor-1 (NCS1 protein. In primary cultured adult cortical neurons, the lentivector-induced overexpression of NCS1 induces neurite sprouting associated with increased phospho-Akt levels. When the PI3K/Akt signalling pathway was pharmacologically inhibited the NCS1-induced neurite sprouting was abolished. The overexpression of NCS1 in uninjured corticospinal neurons exhibited axonal sprouting across the midline into the CST-denervated side of the spinal cord following unilateral pyramidotomy. Improved forelimb function was demonstrated behaviourally and electrophysiologically. In injured corticospinal neurons, overexpression of NCS1 induced axonal sprouting and regeneration and also neuroprotection. These findings demonstrate that increasing the levels of intracellular NCS1 in injured and uninjured central neurons enhances their intrinsic anatomical plasticity within the injured adult central nervous system.

  13. G protein-coupled calcium-sensing receptor is a crucial mediator of MTA-induced biological activities.

    Science.gov (United States)

    Kim, Jin Man; Choi, Seulki; Kwack, Kyu Hwan; Kim, Sun-Young; Lee, Hyeon-Woo; Park, Kyungpyo

    2017-05-01

    Mineral trioxide aggregate (MTA) is a calcium silicate-based bioactive material that has been extensively used in dentistry. MTA has been highlighted in its diverse biological functions and excellent clinical outcomes. However, limited insight into the intracellular signaling pathways has been provided to explain the biological activities of MTA. Here, we firstly elucidate that the extracellular calcium-sensing receptor (CaSR) is a major signaling mediator of MTA-induced biological reactions through versatile live imaging techniques of human dental pulp cells (hDPCs). We found that MTA activates diverse CaSR downstream pathways; notably, CaSR activation essentially requires dual modulation of extracellular Ca(2+) and pH via MTA. Among the CaSR downstream pathways, Ca(2+) mobilization from intracellular stores by the phospholipase C pathway plays an important role in osteogenic differentiation of hDPCs by regulating transcriptional activity. Our findings shed light on the signal transduction mechanism of MTA, thus providing a crucial molecular basis for the use of MTA in regenerative dental therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Power density, field intensity, and carrier frequency determinants of RF-energy-induced calcium-ion efflux from brain tissue

    Energy Technology Data Exchange (ETDEWEB)

    Joines, W.T.; Blackman, C.F.

    1980-01-01

    To explain a carrier frequency dependence reported for radiofrequency (RF)-induced calcium-ion efflux from brain tissue, a chick-brain hemisphere bathed in buffer solution is modeled as a sphere within the uniform field of the incident electromagnetic wave. Calculations on a spherical model show that the average electric-field intensity within the sample remains the same at different carrier frequencies if the incident power density (Pi) is adjusted by an amount that compensates for the change in complex permittivity (epsilon *r) and the change of wavelength, as a function of carrier frequency. The resulting formula for transforming Pi is seen to follow the pattern of both positive and negative demonstrations of calcium-ion efflux that have been observed at carrier frequencies of 50, 147, and 450 MHz. Indeed, all results obtained at these three frequencies, when related by Pi's that produce the same average electric-field intensity within the sample, are seen to be in agreement; no prediction is contradicted by an experiment.

  15. DC electric fields direct breast cancer cell migration, induce EGFR polarization, and increase the intracellular level of calcium ions.

    Science.gov (United States)

    Wu, Dan; Ma, Xiuli; Lin, Francis

    2013-01-01

    Migration of cancer cells leads to invasion of primary tumors to distant organs (i.e., metastasis). Growing number of studies have demonstrated the migration of various cancer cell types directed by applied direct current electric fields (dcEF), i.e., electrotaxis, and suggested its potential implications in metastasis. MDA-MB-231 cell, a human metastatic breast cancer cell line, has been shown to migrate toward the anode of dcEF. Further characterizations of MDA-MB-231 cell electrotaxis and investigation of its underlying signaling mechanisms will lead to a better understanding of electrically guided cancer cell migration and metastasis. Therefore, we quantitatively characterized MDA-MB-231 cell electrotaxis and a few associated signaling events. Using a microfluidic device that can create well-controlled dcEF, we showed the anode-directing migration of MDA-MB-231 cells. In addition, surface staining of epidermal growth factor receptor (EGFR) and confocal microscopy showed the dcEF-induced anodal EGFR polarization in MDA-MB-231 cells. Furthermore, we showed an increase of intracellular calcium ions in MDA-MB-231 cells upon dcEF stimulation. Altogether, our study provided quantitative measurements of electrotactic migration of MDA-MB-231 cells, and demonstrated the electric field-mediated EGFR and calcium signaling events, suggesting their involvement in breast cancer cell electrotaxis.

  16. Cinnamaldehyde and cinnamaldehyde-containing micelles induce relaxation of isolated porcine coronary arteries: role of nitric oxide and calcium

    Directory of Open Access Journals (Sweden)

    Raffai G

    2014-05-01

    Full Text Available Gábor Raffai,1 Byungkuk Kim,1 Sanga Park,1 Gilson Khang,1 Dongwon Lee,1 Paul M Vanhoutte1,21World Class University, Department of BIN Fusion Technology, Chonbuk National University, Jeonju, Jeonbuk, South Korea; 2Department of Pharmacology and Pharmacy and State Key Laboratory for Pharmaceutical Biotechnology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, Special Administrative Region, ChinaBackground and purpose: Cinnamaldehyde, a major component of cinnamon, induces the generation of reactive oxygen species and exerts vasodilator and anticancer effects, but its short half-life limits its clinical use. The present experiments were designed to compare the acute relaxing properties of cinnamaldehyde with those of self-assembling polymer micelles either loaded with cinnamaldehyde or consisting of a polymeric prodrug [poly(cinnamaldehyde] that incorporates the compound in its backbone.Methods: Rings of porcine coronary arteries were contracted with the thromboxane A2 receptor agonist U46619 or 40 mM KCl, and changes in isometric tension were recorded.Results: Cinnamaldehyde induced concentration-dependent but endothelium-independent, nitric oxide synthase (NOS-independent, cyclooxygenase-independent, soluble guanylyl cyclase (sGC-independent, calcium-activated potassium-independent, and TRPA1 channel-independent relaxations. Cinnamaldehyde also inhibited the contractions induced by 40 mM KCl Ca2+ reintroduction in 40 mM KCl Ca2+-free solution or by the Ca2+ channel opener Bay K8644. Cinnamaldehyde-loaded control micelles induced complete, partly endothelium-dependent relaxations sensitive to catalase and inhibitors of NOS or sGC, but not cyclooxygenase or TRPA1, channels. Cinnamaldehyde-loaded micelles also inhibited contractions induced by 40 mM KCl Ca2+ reintroduction or Bay K8644. Poly(cinnamaldehyde micelles induced only partial, endothelium-dependent relaxations that were reduced by inhibitors of NOS or sGC and by

  17. Raised Intracellular Calcium Contributes to Ischemia-Induced Depression of Evoked Synaptic Transmission.

    Directory of Open Access Journals (Sweden)

    Shirin Jalini

    Full Text Available Oxygen-glucose deprivation (OGD leads to depression of evoked synaptic transmission, for which the mechanisms remain unclear. We hypothesized that increased presynaptic [Ca2+]i during transient OGD contributes to the depression of evoked field excitatory postsynaptic potentials (fEPSPs. Additionally, we hypothesized that increased buffering of intracellular calcium would shorten electrophysiological recovery after transient ischemia. Mouse hippocampal slices were exposed to 2 to 8 min of OGD. fEPSPs evoked by Schaffer collateral stimulation were recorded in the stratum radiatum, and whole cell current or voltage clamp recordings were performed in CA1 neurons. Transient ischemia led to increased presynaptic [Ca2+]i, (shown by calcium imaging, increased spontaneous miniature EPSP/Cs, and depressed evoked fEPSPs, partially mediated by adenosine. Buffering of intracellular Ca2+ during OGD by membrane-permeant chelators (BAPTA-AM or EGTA-AM partially prevented fEPSP depression and promoted faster electrophysiological recovery when the OGD challenge was stopped. The blocker of BK channels, charybdotoxin (ChTX, also prevented fEPSP depression, but did not accelerate post-ischemic recovery. These results suggest that OGD leads to elevated presynaptic [Ca2+]i, which reduces evoked transmitter release; this effect can be reversed by increased intracellular Ca2+ buffering which also speeds recovery.

  18. Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation.

    Science.gov (United States)

    Dame, Michael K; Veerapaneni, Indiradevi; Bhagavathula, Narasimharao; Naik, Madhav; Varani, James

    2011-01-01

    We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the present study, we used intact human colon tissue in organ culture to compare responses initiated by Ca(2+) supplementation versus the multi-mineral extract. Normal human colon tissue was treated for 2 d in culture with various concentrations of calcium or the mineral-rich extract. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for levels of type I procollagen and type I collagen. At higher Ca(2+) concentrations or with the mineral-rich extract, proliferation of epithelial cells at the base and walls of the mucosal crypts was suppressed, as visualized by reduced Ki67 staining. E-cadherin, a marker of differentiation, was more strongly expressed at the upper third of the crypt and at the luminal surface. Treatment with Ca(2+) or with the multi-mineral extract influenced collagen turnover, with decreased procollagen and increased type I collagen. These data suggest that calcium or mineral-rich extract has the capacity to (1) promote differentiation in human colon tissue in organ culture and (2) modulate stromal function as assessed by increased levels of type I collagen. Taken together, these data suggest that human colon tissue in organ culture (supporting in vivo finding in mice) will provide a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.

  19. Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca(2+) release during the quasi-steady level of release in twitch fibers from frog skeletal muscle.

    Science.gov (United States)

    Fénelon, Karine; Lamboley, Cédric R H; Carrier, Nicole; Pape, Paul C

    2012-10-01

    Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits

  20. Different NaCl-Induced Calcium Signatures in the Arabidopsis thaliana Ecotypes Col-0 and C24

    KAUST Repository

    Schmöckel, Sandra M.

    2015-02-27

    A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca2+]cyt), which allow the specific and rapid transmission of stress signals through a plant after exposure to a stress, such as salinity. Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca2+]cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca2+]cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.

  1. Different NaCl-induced calcium signatures in the Arabidopsis thaliana ecotypes Col-0 and C24.

    Science.gov (United States)

    Schmöckel, Sandra M; Garcia, Alexandre F; Berger, Bettina; Tester, Mark; Webb, Alex A R; Roy, Stuart J

    2015-01-01

    A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca2+]cyt), which allow the specific and rapid transmission of stress signals through a plant after exposure to a stress, such as salinity. Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca2+]cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca2+]cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.

  2. Calcium paradox induces apoptosis in the isolated perfused Rana ridibunda heart: involvement of p38-MAPK and calpain

    National Research Council Canada - National Science Library

    Aggeli, Ioanna-Katerina; Zacharias, Triantafyllos; Papapavlou, Georgia; Gaitanaki, Catherine; Beis, Isidoros

    2013-01-01

    "Calcium paradox" as a term describes the deleterious effects conferred to a heart perfused with a calcium-free solution followed by repletion, including loss of mechanical activity and sarcomere disruption...

  3. Role for the magnetic field in the radiation-induced efflux of calcium ions from brain tissue in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Blackman, C.F.; Benane, S.G.; Rabinowitz, J.R.; House, D.E.; Joines, W.T.

    1985-01-01

    Two independent laboratories have demonstrated that electromagnetic radiation at specific frequencies can cause a change in the efflux of calcium ions from brain tissue in vitro. In a local geomagnetic field (LGF) at a density of 38 microTesla (microT), 15- and 45-Hz electromagnetic signals (40 Vp-p/m in air) have been shown to induce a change in the efflux of calcium ions from the exposed tissues, whereas 1- and 30-Hz signals do not. We now show that the effective 15-Hz signal can be rendered ineffective when the LGF is reduced to 19 microT with Helmholtz coils. In addition, the ineffective 30-Hz signal becomes effective when the LGF is changed to +/- 25.3 microT or to +/- 76 microT. These results demonstrate that the net intensity of the LGF is an important variable. The results appear to describe a resonance-like relationship in which the frequency of the electromagnetic field that can induce a change in efflux is proportional to a product of LGF density and an index, 2n + 1, where n = 0,1. These phenomenological findings may provide a basis for evaluating the apparent lack of reproducibility of biological effects caused by low-intensity extremely-low-frequency (ELF) electromagnetic signals. In future investigations of this phenomenon, the LGF vector should be explicitly described. If the underlying mechanism involves a general property of tissue, then research conducted in the ambient electromagnetic environment (50/60 Hz) may be subjected to unnoticed and uncontrolled influences, depending on the density of the LGF.

  4. Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts.

    Science.gov (United States)

    Hadjicharalambous, Chrystalleni; Kozlova, Diana; Sokolova, Viktoriya; Epple, Matthias; Chatzinikolaidou, Maria

    2015-12-01

    Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells.

  5. Subcellular propagation of calcium waves in Müller glia does not require autocrine/paracrine purinergic signaling.

    Science.gov (United States)

    Phuong, Tam T T; Yarishkin, Oleg; Križaj, David

    2016-09-02

    The polarized morphology of radial glia allows them to functionally interconnect different layers of CNS tissues including the retina, cerebellum, and cortex. A likely mechanism involves propagation of transcellular Ca(2+) waves which were proposed to involve purinergic signaling. Because it is not known whether ATP release is required for astroglial Ca(2+) wave propagation we investigated this in mouse Müller cells, radial astroglia-like retinal cells in which in which waves can be induced and supported by Orai/TRPC1 (transient receptor potential isoform 1) channels. We found that depletion of endoplasmic reticulum (ER) stores triggers regenerative propagation of transcellular Ca(2+) waves that is independent of ATP release and activation of P2X and P2Y receptors. Both the amplitude and kinetics of transcellular, depletion-induced waves were resistant to non-selective purinergic P2 antagonists such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Thus, store-operated calcium entry (SOCE) is itself sufficient for the initiation and subcellular propagation of calcium waves in radial glia.

  6. Calcium polycarbophil, a water absorbing polymer, increases bowel movement and prevents sennoside-induced diarrhea in dogs.

    Science.gov (United States)

    Saito, T; Yamada, T; Iwanaga, Y; Morikawa, K; Nagata, O; Kato, H; Mizumoto, A; Itoh, Z

    2000-07-01

    The effects of calcium polycarbophil (CP), a water-absorbing polymer, on bowel movement were examined in comparison with known laxatives and anti-diarrheal agents in dogs, a species that resembles humans for stool output. CP increased stool frequency, fecal water content and fecal weight in a dose-dependent manner, but did not induce diarrhea. Sennoside and carboxymethylcellulose sodium (CMC-Na) increased fecal water content and induced diarrhea at lower doses than that which enhanced stool frequency. Trimebutine decreased stool frequency, fecal weight and fecal water content, resulting in inhibition rather than stimulation of defecation. In sennoside-induced diarrhea, loperamide and CP improved stool consistency and this was accompanied by reduced fecal moisture and frequency of diarrhea. In contrast, CMC-Na aggravated stool consistency with increased fecal water content and frequency of diarrhea, and trimebutine had little noticeable effect apart from reducing fecal weight. Our results show that CP has both laxative and anti-diarrheal effects in dogs and differed from conventional laxatives and anti-diarrheal agents. CP may be a suitable agent for treatment of idiopathic constipation, secretory diarrhea and irritable bowel syndrome with alternating constipation and diarrhea and with either predominating in terms of less side effects such as diarrhea or constipation.

  7. Effects of calcium and nifedipine on noradrenaline- and PGF-2 alpha-induced activity of the ampullary-isthmic junction of the human oviduct in vitro.

    Science.gov (United States)

    Forman, A; Andersson, K E; Ulmsten, U

    1983-03-01

    From 22 women undergoing hysterectomy at various stages of the menstrual cycle, strip preparations were dissected from the outer, longitudinal and the inner, circular smooth muscle layers of the ampullary-isthmic junction (AIJ). The strips were mounted in organ baths, and isometric tension was recorded. Spontaneous contractions were recorded mainly in circular muscle strips. Contractions were elicited by 127 mM-K+, 10(-6) M-noradrenaline and 10(-6) M-PGF-2 alpha. Potassium induced biphasic responses that were slightly different in the two tissues. In circular muscle strips, noradrenaline and PGF-2 alpha induced phasic contractions superimposed on a rise in tone. In longitudinal muscle specimens, the two compounds produced tonic responses. All types of mechanical activity were inhibited by removal of extracellular calcium. K+-induced responses and phasic contractions produced by noradrenaline and PGF-2 alpha could be abolished by 10(-6) M-nifedipine whereas the tonic contractions in the circular and longitudinal muscle were more resistant to the calcium antagonist. The results suggest that K+-induced responses in circular and longitudinal muscle of the human AIJ, and the phasic contractions in circular muscle, depend on calcium influx via potential-sensitive membrane channels. Receptor-operated calcium channels seem to be involved in the tonic contractions observed mainly in the longitudinal smooth muscle.

  8. The effect of a calcium phosphate mouth rinse on (chemo) radiation induced oral mucositis in head and neck cancer patients : a prospective study

    NARCIS (Netherlands)

    Stokman, M. A.; Burlage, F. R.; Spijkervet, F. K. L.

    2012-01-01

    Objectives: Promising results of a calcium phosphate (CP) mouth rinse on reduced severity of oral mucositis have been reported. The aim of this study was to determine the effect of a CP mouth rinse on the frequency, duration and severity of (chemo) radiation induced oral mucositis in patients with h

  9. The effect of a calcium phosphate mouth rinse on (chemo) radiation induced oral mucositis in head and neck cancer patients : a prospective study

    NARCIS (Netherlands)

    Stokman, M. A.; Burlage, F. R.; Spijkervet, F. K. L.

    2012-01-01

    Objectives: Promising results of a calcium phosphate (CP) mouth rinse on reduced severity of oral mucositis have been reported. The aim of this study was to determine the effect of a CP mouth rinse on the frequency, duration and severity of (chemo) radiation induced oral mucositis in patients with h

  10. Calcium electroporation in three cell lines; a comparison of bleomycin and calcium, calcium compounds, and pulsing conditions

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille

    2013-01-01

    BACKGROUND: Electroporation with calcium (calcium electroporation) can induce ATP depletion-associated cellular death. In the clinical setting, the cytotoxic drug bleomycin is currently used with electroporation (electrochemotherapy) for palliative treatment of tumors. Calcium electroporation...... offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions, as is the case with cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing calcium...

  11. Blocking Cyclic Adenosine Diphosphate Ribose-mediated Calcium Overload Attenuates Sepsis-induced Acute Lung Injury in Rats

    Institute of Scientific and Technical Information of China (English)

    Qian-yi Peng; Yu Zou; Li-Na Zhang; Mei-Lin Ai; Wei Liu; Yu-Hang Ai

    2016-01-01

    Background:Acute lung injury (ALI) is a common complication of sepsis that is associated with high mortality.Intracellular Ca2+ overload plays an important role in the pathophysiology of sepsis-induced ALI,and cyclic adenosine diphosphate ribose (cADPR) is an important regulator of intracellular Ca2+ mobilization.The cluster of differentiation 38 (CD38)/cADPR pathway has been found to play roles in multiple inflammatory processes but its role in sepsis-induced ALI is still unknown.This study aimed to investigate whether the CD38/cADPR signaling pathway is activated in sepsis-induced ALI and whether blocking cADPR-mediated calcium overload attenuates ALI.Methods:Septic rat models were established by cecal ligation and puncture (CLP).Rats were divided into the sham group,the CLP group,and the CLP+ 8-bromo-cyclic adenosine diphosphate ribose (8-Br-cADPR) group.Nicotinamide adenine dinucleotide (NAD+),cADPR,CD38,and intracellular Ca2+ levels in the lung tissues were measured at 6,12,24,and 48 h after CLP surgery.Lung histologic injury,tumor necrosis factor (TNF)-α,malondialdehyde (MDA) levels,and superoxide dismutase (SOD) activities were measured.Results:NAD+,cADPR,CD38,and intracellular Ca2+ levels in the lungs of septic rats increased significantly at 24 h after CLP surgery.Treatment with 8-Br-cADPR,a specific inhibitor of cADPR,significantly reduced intracellular Ca2+ levels (P =0.007),attenuated lung histological injury (P =0.023),reduced TNF-α and MDA levels (P < 0.001 and P =0.002,respectively) and recovered SOD activity (P =0.031) in the lungs of septic rats.Conclusions:The CD38/cADPR pathway is activated in the lungs of septic rats,and blocking cADPR-mediated calcium overload with 8-Br-cADPR protects against sepsis-induced ALI.

  12. Inositol trisphosphate and calcium signalling

    Science.gov (United States)

    Berridge, Michael J.

    1993-01-01

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

  13. Cellular uptake of {sup 99m}TcN-NOET in human leukaemic HL-60 cells is related to calcium channel activation and cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Guillermet, Stephanie; Vuillez, Jean-Philippe; Caravel, Jean-Pierre; Marti-Batlle, Daniele; Fagret, Daniel [Universite de Grenoble, Radiopharmaceutiques Biocliniques, La Tronche (France); Fontaine, Eric [Universite de Grenoble, Laboratoire de Bioenergetique Fondamentale et Appliquee, Grenoble (France); Pasqualini, Roberto [Cis Bio International Schering SA, Gif-sur-Yvette (France)

    2006-01-01

    A major goal of nuclear oncology is the development of new radiolabelled tracers as proliferation markers. Intracellular calcium waves play a fundamental role in the course of the cell cycle. These waves occur in non-excitable tumour cells via store-operated calcium channels (SOCCs). Bis(N-ethoxy, N-ethyldithiocarbamato) nitrido technetium (V)-99m ({sup 99m}TcN-NOET) has been shown to interact with L-type voltage-operated calcium channels (VOCCs) in cultured cardiomyocytes. Considering the analogy between VOCCs and SOCCs, we sought to determine whether {sup 99m}TcN-NOET also binds to activated SOCCs in tumour cells in order to clarify the potential value of this tracer as a proliferation marker. Uptake kinetics of {sup 99m}TcN-NOET were measured in human leukaemic HL-60 cells over 60 min and the effect of several calcium channel modulators on 1-min tracer uptake was studied. The uptake kinetics of {sup 99m}TcN-NOET were compared both with the variations of cytosolic free calcium concentration measured by indo-1/AM and with the variations in the SG{sub 2}M cellular proliferation index. All calcium channel inhibitors significantly decreased the cellular uptake of {sup 99m}TcN-NOET whereas the activator thapsigargin induced a significant 10% increase. In parallel, SOCC activation by thapsigargin, as measured using the indo-1/AM probe, was inhibited by nicardipine. These results indicate that the uptake of {sup 99m}TcN-NOET is related to the activation of SOCCs. Finally, a correlation was observed between the tracer uptake and variations in the proliferation index SG{sub 2}M. The uptake of {sup 99m}TcN-NOET seems to be related to SOCC activation and to cell proliferation in HL-60 cells. These results indicate that {sup 99m}TcN-NOET might be a marker of cell proliferation. (orig.)

  14. Identification of calcium stress induced genes in amaranth leaves through suppression subtractive hybridization.

    Science.gov (United States)

    Aguilar-Hernández, Hugo S; Santos, Leticia; León-Galván, Fabiola; Barrera-Pacheco, Alberto; Espitia-Rangel, Eduardo; De León-Rodríguez, Antonio; Guevara-González, Ramón G; Barba de la Rosa, Ana P

    2011-11-15

    Calcium (Ca(2+)) is a critical ion for the growth and development of plants and plays an important role in signal transduction pathways in response to biotic and abiotic stresses. We investigated the Ca(2+) stress responsive-genes in amaranth leaves by using the suppression subtractive hybridization technique. Screening of the libraries generated 420 up-regulated transcripts and 199 down-regulated transcripts. The differentially expressed transcripts were associated with general stress response, transcription factors, gene regulation, signal transduction, and some other with unknown function. Selected genes were used to study their differential regulation by sqRT-PCR. Among the up-regulated transcripts, a fragment containing the motif of C3HC4-type RING-Zinc family was further characterized. The ORF of amaranth zinc finger protein (AhZnf) has a closer relationship with its ortholog from Ricinus communis while is distantly related to the Arabidopsis thaliana C3HC4-type ortholog. We have identified a novel putative zinc finger protein along with other novel proteins such as the wall associated kinase, phosphoinositide binding protein, and rhomboid protease involved in response to Ca(2+) stress in amaranth leaves.

  15. Calcium fluoride nanoparticles induced suppression of Streptococcus mutans biofilm: an in vitro and in vivo approach.

    Science.gov (United States)

    Kulshrestha, Shatavari; Khan, Shakir; Hasan, Sadaf; Khan, M Ehtisham; Misba, Lama; Khan, Asad U

    2016-02-01

    Biofilm formation on the tooth surface is the root cause of dental caries and periodontal diseases. Streptococcus mutans is known to produce biofilm which is one of the primary causes of dental caries. Acid production and acid tolerance along with exopolysaccharide (EPS) formation are major virulence factors of S. mutans biofilm. In the current study, calcium fluoride nanoparticles (CaF2-NPs) were evaluated for their effect on the biofilm forming ability of S. mutans in vivo and in vitro. The in vitro studies revealed 89 % and 90 % reduction in biofilm formation and EPS production, respectively. Moreover, acid production and acid tolerance abilities of S. mutans were also reduced considerably in the presence of CaF2-NPs. Confocal laser scanning microscopy and transmission electron microscopy images were in accordance with the other results indicating inhibition of biofilm without affecting bacterial viability. The qRT-PCR gene expression analysis showed significant downregulation of various virulence genes (vicR, gtfC, ftf, spaP, comDE) associated with biofilm formation. Furthermore, CaF2-NPs were found to substantially decrease the caries in treated rat groups as compared to the untreated groups in in vivo studies. Scanning electron micrographs of rat's teeth further validated our results. These findings suggest that the CaF2-NPs may be used as a potential antibiofilm applicant against S. mutans and may be applied as a topical agent to reduce dental caries.

  16. Stable cavitation induces increased cytoplasmic calcium in L929 fibroblasts exposed to 1-MHz pulsed ultrasound.

    Science.gov (United States)

    Tsukamoto, Akira; Higashiyama, Satoru; Yoshida, Kenji; Watanabe, Yoshiaki; Furukawa, Katsuko S; Ushida, Takashi

    2011-12-01

    An increase in cytoplasmic calcium (Ca(2+) increase) is a second messenger that is often observed under ultrasound irradiation. We hypothesize that cavitation is a physical mechanism that underlies the increase in Ca(2+) in these experiments. To control the presence of cavitation, the wave type was controlled in a sonication chamber. One wave type largely contained a traveling wave (wave type A) while the other wave type largely contained a standing wave (wave type B). Fast Fourier transform (FFT) analysis of a sound field produced by the wave types ascertained that stable cavitation was present only under wave type A ultrasound irradiation. Under the two controlled wave types, the increase in Ca(2+) in L929 fibroblasts was observed with fluorescence imaging. Under wave type A ultrasound irradiation, an increase in Ca(2+) was observed; however, no increase in Ca(2+) was observed under wave type B ultrasound irradiation. We conclude that stable cavitation is involved in the increase of Ca(2+) in cells subjected to pulsed ultrasound.

  17. Nanosqueezed light for probing mitochondria and calcium-induced membrane swelling for study of neuroprotectants

    Science.gov (United States)

    Gourley, Paul L.; Chen, P.; Copeland, R. G.; Hendricks, Judy K.; McDonald, Anthony E.; Keep, M. E.; Karlsson, J. R.

    2004-01-01

    We report a new bioMEMs nanolaser technique for measuring characteristics of small organelles. We have initially applied the method to study mitochondria, a very small (500nm to 1um) organelle containing the respiration apparatus for animal cells. Because the mitochondria are so tiny, it has been difficult to study them using standard light microscope or flow cytometry techniques. We employ a recently discovered a nano-optical transduction method for high-speed analysis of submicron organelles. This ultrasensitive detection of submicron particles uses nano-squeezing of light into photon modes imposed by the ultrasmall organelle dimensions in a submicron laser cavity. In this paper, we report measurements of mitochondria spectra under normal conditions and under high calcium ion gradient conditions that upset membrane homeostasis and lead to organelle swelling and lysis, similar to that observed in the diseased state. The measured spectra are compared with our calculations of the electromagnetic modes in normal and distended mitochondria using multiphysics finite element methods.

  18. Inhibition of nitrite-induced toxicity in channel catfish by calcium chloride and sodium chloride

    Science.gov (United States)

    Tommasso J.R., Wright; Simco, B.A.; Davis, K.B.

    1980-01-01

    Environmental chloride has been shown to inhibit methemoglobin formation in fish, thereby offering a protective effect against nitrite toxicity. Channel catfish (Ictalurus punctatus) were simultaneously exposed to various environmental nitrite and chloride levels (as either CaCl2 or NaCl) in dechlorinated tap water (40 mg/L total hardness, 47 mg/L alkalinity, 4 mg/L chloride, pH = 6.9-7.1, and temperature 21-24°C). Methemoglobin levels in fish simultaneously exposed to 2.5 mg/L nitrite and up to 30 mg/L chloride as either CaCl2 or NaCl were similar but significantly lower than in unprotected fish. Exposure to 10 mg/L nitrite and 60 mg/L chloride resulted in methemoglobin levels similar to those of the controls; most unprotected fish died. Fish exposed to 10 mg/L nitrite had significantly lower methemoglobin levels when protected with 15.0 mg/L chloride as CaCl2 than with NaCl. Fish exposed to nitrite in the presence of 60 mg/L chloride (as either CaCl2 or NaCl) had similar 24-h LC50 values that were significantly elevated above those obtained in the absence of chloride. Calcium had little effect on tolerance to nitrite toxicity in channel catfish in contrast to its large effect reported in steelhead trout (Salmo gairdneri).

  19. Influence of artificially-induced porosity on the compressive strength of calcium phosphate bone cements.

    Science.gov (United States)

    Mouzakis, Dionysios; Zaoutsos, Stefanos Polymeros; Bouropoulos, Nikolaos; Rokidi, Stamatia; Papanicolaou, George

    2016-07-01

    The biological and mechanical nature of calcium phosphate cements (CPC's) matches well with that of bone tissues, thus they can be considered as an appropriate environment for bone repair as bone defect fillers. The current study focuses on the experimental characterization of the mechanical properties of CPCs that are favorably used in clinical applications. Aiming on evaluation of their mechanical performance, tests in compression loading were conducted in order to determine the mechanical properties of the material under study. In this context, experimental results occurring from the above mechanical tests on porous specimens that were fabricated from three different porous additives, namely albumin, gelatin and sodium alginate, are provided, while assessment of their mechanical properties in respect to the used porous media is performed. Additionally, samples reinforced with hydroxyapatite crystals were also tested in compression and the results are compared with those of the above tested porous CPCs. The knowledge obtained allows the improvement of their biomechanical properties by controlling their structure in a micro level, and finds a way to compromise between mechanical and biological response.

  20. Extracellular calcium is involved in egg yolk-induced head-to-head agglutination of bull sperm.

    Science.gov (United States)

    Yang, D H; McMillan, A G; Standley, N T; Shannon, P; Xu, Z Z

    2012-10-15

    Head-to-head agglutination of bull sperm occurs when semen is highly diluted in an egg yolk-citrate diluent without streptomycin. The objectives were to investigate causes of sperm agglutination and the underlying mechanism. Aliquots of bull semen were diluted in a base diluent (BD) supplemented with various test components and the percentage of agglutinated sperm (% AggSp) was quantified at 1, 5, 24, 48, and 72 h of incubation. When sperm were incubated at 22 °C, no agglutination was observed in BD for up to 72 h, whereas the % AggSp was 5.0, 41.7, 72.2, 91.1, and 92.8% in BD + 5% egg yolk (BD + EY) at 1, 5, 24, 48 and 72 h, respectively. However, no sperm agglutination was observed in BD + EY if incubation temperature was 37 °C. Addition of 5 or 10 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid to BD + EY reduced the % AggSp from 95% to sperm agglutination in the absence of egg yolk, implicating calcium and other factors in egg yolk. Addition of the citrate-soluble fraction (CSF) of egg yolk to BD induced sperm agglutination similar to whole egg yolk, whereas water- and saline-soluble fractions of egg yolk were ineffective. The sperm-agglutinating efficacy of CSF (the % AggSp = 95% at 72 h) was reduced by dialysis (20%; P sperm agglutination, whereas addition of 2 mM of a cAMP analogue, dbcAMP, to a nonagglutinating diluent failed to induce sperm agglutination. Agglutination status had no effect on sperm plasma membrane/acrosome status and mitochondrial membrane potential. In conclusion, calcium and other component(s) in the CSF of egg yolk induced head-to-head agglutination of bull sperm in a time- and temperature-dependent manner. Although the mechanism of agglutination was not determined, the cAMP- protein kinase A signaling pathway was not involved.

  1. Digoxin-specific antibody fragments and a calcium antagonist for reversal of digoxin-induced mesenteric vasoconstriction.

    Science.gov (United States)

    Hess, T; Scholtysik, G; Salzmann, R; Riesen, W

    1983-10-01

    The effect of digoxin-specific antibody fragments on glycoside-induced mesenteric vasoconstriction were investigated. Digoxin caused a sustained contraction of strips of isolated feline mesenteric artery lasting for several hours, while in anaesthetized cats it produced a significant decrease in blood flow and increase in resistance in the mesenteric artery. In-vitro, digoxin's contractile effect was inhibited by 'prophylactic' addition of antibody to the organ bath, but the clinical use for prophylaxis is not a practical proposition. When the antibodies were added with the contraction of the arterial strip in response to digoxin already established, the tone of the preparation decreased significantly over 3 h, but the effect of the glycoside was not fully reversible. In-vivo, control animals not treated with antibodies developed arrhythmias, mesenteric blood flow fell by more than 50% and resistance increased by more than 80% relative to the initial values. These animals died of ventricular fibrillation before the end of the experiment. Animals treated with digoxin-specific antibody fragments after receiving digoxin injections showed no further decrease in mesenteric blood flow and 90 min after the last dose of digoxin, the flow was recovering and mesenteric resistance decreasing. Furthermore, all the animals that had received antibodies remained in sinus rhythm to the end of the experiment. In view of the latent period to onset of action of the antibodies, valuable time may be lost in impaired mesenteric blood flow. To bridge the gap or, indeed, as primary treatment, calcium antagonists merit consideration; in our experiments mesenteric vasoconstriction was abolished within a few minutes by application of the dihydropyridine calcium antagonist 4-(2,1,3-benzo-oxadiazol-4-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic aid, diethyl ester (PY 108-068).

  2. Aluminum-induced dreierketten chain cross-links increase the mechanical properties of nanocrystalline calcium aluminosilicate hydrate

    Science.gov (United States)

    Geng, Guoqing; Myers, Rupert J.; Li, Jiaqi; Maboudian, Roya; Carraro, Carlo; Shapiro, David A.; Monteiro, Paulo J. M.

    2017-03-01

    The incorporation of Al and increased curing temperature promotes the crystallization and cross-linking of calcium (alumino)silicate hydrate (C-(A-)S-H), which is the primary binding phase in most contemporary concrete materials. However, the influence of Al-induced structural changes on the mechanical properties at atomistic scale is not well understood. Herein, synchrotron radiation-based high-pressure X-ray diffraction is used to quantify the influence of dreierketten chain cross-linking on the anisotropic mechanical behavior of C-(A-)S-H. We show that the ab-planar stiffness is independent of dreierketten chain defects, e.g. vacancies in bridging tetrahedra sites and Al for Si substitution. The c-axis of non-cross-linked C-(A-)S-H is more deformable due to the softer interlayer opening but stiffens with decreased spacing and/or increased zeolitic water and Ca2+ of the interlayer. Dreierketten chain cross-links act as ‘columns’ to resist compression, thus increasing the bulk modulus of C-(A-)S-H. We provide the first experimental evidence on the influence of the Al-induced atomistic configurational change on the mechanical properties of C-(A-)S-H. Our work advances the fundamental knowledge of C-(A-)S-H on the lowest level of its hierarchical structure, and thus can impact the way that innovative C-(A-)S-H-based cementitious materials are developed using a ‘bottom-up’ approach.

  3. Endogenous and Exogenous Calcium Involved in the Betulin Production from Submerged Culture of Phellinus linteus Induced by Hydrogen Sulfide.

    Science.gov (United States)

    Fan, Guizhi; Jian, Duan; Sun, Meiling; Zhan, Yaguang; Sun, Feifei

    2016-02-01

    Using pharmacological and biochemical approaches, Ca(2+) involved in the betulin production in mycelia of Phellinus linteus induced by hydrogen sulfide (H2S) were investigated. The results showed that 2 mM H2S donor NaHS or 10 mM CaCl2 was found to enhance the betulin content in the mycelia of Phellinus to the maximum, which were 112.43 and 93.24% higher than that in the control, respectively. Further, NaHS and CaCl2 co-treatment also showed positive outcome, which were 128.95 or 24.52% higher than that in the control or NaHS treatment. At the same time, NaHS also enhanced the content of Ca(2+) and CaM. But, the above positive inductive effects for Ca(2+), CaM, and betulin production can be blocked with either Ca(2+) channel blocker (LaCl3, 2-aminoethoxydiphenyl borate) or Ca(2+) chelator (ethylenediaminetetraacetic acid (EDTA)). Among of them, betulin content was reduced 35.06% by NaHS and EGTA to the minimum, and this reduction could be reversed by the application of CaCl2 (NaHS + EGTA + CaCl2). From above results, it can be concluded that endogenous and exogenous calcium involved in the betulin production from submerged culture of P. linteus induced by hydrogen sulfide.

  4. Zirconium, calcium, and strontium contents in magnesium based biodegradable alloys modulate the efficiency of implant-induced osseointegration.

    Science.gov (United States)

    Mushahary, Dolly; Sravanthi, Ragamouni; Li, Yuncang; Kumar, Mahesh J; Harishankar, Nemani; Hodgson, Peter D; Wen, Cuie; Pande, Gopal

    2013-01-01

    Development of new biodegradable implants and devices is necessary to meet the increasing needs of regenerative orthopedic procedures. An important consideration while formulating new implant materials is that they should physicochemically and biologically mimic bone-like properties. In earlier studies, we have developed and characterized magnesium based biodegradable alloys, in particular magnesium-zirconium (Mg-Zr) alloys. Here we have reported the biological properties of four Mg-Zr alloys containing different quantities of strontium or calcium. The alloys were implanted in small cavities made in femur bones of New Zealand White rabbits, and the quantitative and qualitative assessments of newly induced bone tissue were carried out. A total of 30 experimental animals, three for each implant type, were studied, and bone induction was assessed by histological, immunohistochemical and radiological methods; cavities in the femurs with no implants and observed for the same period of time were kept as controls. Our results showed that Mg-Zr alloys containing appropriate quantities of strontium were more efficient in inducing good quality mineralized bone than other alloys. Our results have been discussed in the context of physicochemical and biological properties of the alloys, and they could be very useful in determining the nature of future generations of biodegradable orthopedic implants.

  5. Time-dependent subcellular structure injuries induced by nano-/micron-sized calcium oxalate monohydrate and dihydrate crystals.

    Science.gov (United States)

    Sun, Xin-Yuan; Yu, Kai; Ouyang, Jian-Ming

    2017-10-01

    Comparative studies were conducted to investigate the time effect of cell injury induced by nano-sized (50nm) and micron-sized (10μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The effects of nano-/micron-sized COM and COD exposure on Vero cells were investigated by detecting the cell viability, cell morphology, LDH release, reactive oxygen species, mitochondrial membrane potential, cell cycle, and cell apoptosis, as well as the intracellular and extracellular crystal distribution. Nano-/micron-sized COM and COD exposure lead to subcellular organelle injury in varying degrees, but the injury sequence of various organelles differed. The time sequence of organelle injury presenting significant variation was described as follows: cell membrane injury (1h)crystals lead organelle injury faster than micron-sized crystals, and COM crystals showed more obvious time-dependent effects than the same-sized COD crystals. This study may provide insights into the damage to renal epithelial cells induced by urinary crystals and the formation mechanism of kidney stones. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Inhibition of ATP-induced calcium influx in HT4 cells by glucocorticoids: involvement of protein kinase A

    Institute of Scientific and Technical Information of China (English)

    Jian-zhong HAN; Wen LIN; Yi-zhang CHEN

    2005-01-01

    Aim: In our previous observations, adenosine triphosphate (ATP) was found to evoke immediate elevations in intracellular free calcium concentration ([Ca2+]i) in HT4 neuroblastoma cells of mice. We tried to see if a brief pretreatment of glucocorticoids could inhibit the Ca2+ response and reveal the underlying signal ing mechanism. Methods: Measurement of [Ca2+]i was carried out using the dual-wavelength fluorescence method with Fura-2 as the indicator. Results: Pre incubation of HT4 cells for 5 min with corticosterone (B) or bovine serum albumin conjugated corticosterone (B-BSA) inhibited the peak [Ca2+]i increments in a concentration-dependent manner. Cortisol and dexamethasone had a similar action, while deoxycorticosterone and cholesterol were ineffective. Both extracellular Ca2+ influx and internal Ca2+ release contributed to ATP-induced [Ca2+]i elevation. The brief treatment with only B attenuated Ca2+ influx. Furthermore, the [Ca2+]i elevation induced by the P2X receptor agonist adenosine 5'-(β,γ-methylene) triphosphate (β,γ-meATP) was also suppressed. The rapid inhibitory effect of B can be reproduced by forskolin 1 mmol/L and blocked by H89 20 mmol/L. Neither nuclear glucocorticoid receptor antagonist mifepristone nor protein kinase C in hibitors influenced the rapid action of B. Conclusion: Our results suggest that glucocorticoids modulate P2X receptor-medicated Ca2+ influx through a membrane-initiated, non-genomic and PKA-dependent pathway in HT4 cells.

  7. Calcium ionophore (A-23187 induced peritoneal eicosanoid biosynthesis: a rapid method to evaluate inhibitors of arachidonic acid metabolism in vivo

    Directory of Open Access Journals (Sweden)

    T. S. Rao

    1993-01-01

    Full Text Available The present investigation characterizes calcium ionophore (A-23187 induced peritoneal eicosanoid biosynthesis in the rat. Intraperitoneal injection of A-23187 (20 μg/rat stimulated marked biosynthesis of 6-keto-PGF1α (6-KPA, TxB2, LTC4 and LTB4, with no detectable changes on levels of PGE2. Levels of all eicosanoids decreased rapidly after a peak which was seen as early as 5 min. Enzyme markers of cellular contents of neutrophils and mononuclear cells, MPO and NAG respectively, decreased rapidly after ionophore injection; this was followed by increases after 60 min. Indomethacin, a selective cyclooxygenase inhibitor, and zileuton and ICI D-2138, two selective 5-lipoxygenase inhibitors attenuated prostaglandin and leukotriene pathways respectively. Oral administration of zileuton (20 mg/kg, p.o. inhibited LTB4 biosynthesis for up to 6 h suggesting a long duration of pharmacological activity in the rats consistent with its longer half-life. The rapid onset and the magnitude of increases in levels of eicosanoids render the ionophore induced peritoneal eicosanoid biosynthesis a useful model to evaluate pharmacological profiles of inhibitors of eicosanoid pathways in vivo.

  8. Modulation of MAA-induced apoptosis in male germ cells: role of Sertoli cell P/Q-type calcium channels

    Directory of Open Access Journals (Sweden)

    Aguanno Salvatore

    2005-04-01

    Full Text Available Abstract Spontaneous germ cell death by apoptosis occurs during normal spermatogenesis in mammals and is thought to play a role in the physiological mechanism limiting the clonal expansion of such cell population in the male gonad. In the prepubertal rat testis, the most conspicuous dying cells are pachytene spermatocytes, which are also the primary target of the apoptosis experimentally induced by the methoxyacetic acid (MAA. Since we have recently reported that Sertoli cells, the somatic component of the seminiferous epithelium, regulate not only germ cell viability and differentiation but also their death, we have further investigated the mechanism involved in such a control. In this paper we have used the protein clusterin, produced by Sertoli cells and associated with tissue damage or injury, as indicator of germ cell apoptosis in rat seminiferous tubules treated with MAA in the presence or in the absence of omega-agatoxin, a specific inhibitor of P/Q type voltage-operated calcium channels (VOCC's. We performed both a qualitative analysis of clusterin content and germ cell apoptosis by immunofluorescence experiments and a quantitative analysis by in situ end labelling of apoptotic germ cells followed by flow cytometry. The results obtained demonstrate that Sertoli cells modulate germ cell apoptosis induced by methoxyacetic acid also throughout the P/Q-type VOCC's.

  9. Estimation of calcified tissues hardness via calcium and magnesium ionic to atomic line intensity ratio in laser induced breakdown spectra

    Energy Technology Data Exchange (ETDEWEB)

    Abdel-Salam, Z.A.; Galmed, A.H. [National Institute of Laser Enhanced Science (NILES), Cairo University, Giza (Egypt); Tognoni, E. [Applied Laser Spectroscopy Laboratory, IPCF/CNR, Pisa (Italy); Harith, M.A. [National Institute of Laser Enhanced Science (NILES), Cairo University, Giza (Egypt)], E-mail: mharithm@niles.edu.eg

    2007-12-15

    Calcified tissues representing three different matrices, namely enamel of human teeth, shells and eggshell, have been studied via Laser Induced Breakdown Spectroscopy (LIBS) technique. The experimental CaII/CaI and MgII/MgI ratios have been measured, in view of the expected correlation between the extent of ionization caused by the laser induced shock wave (SW) and the hardness of the target. The ratio CaII/CaI between the ionic calcium line at 373.69 nm and the neutral line at 428.9 nm is obtained for enamel, shells and eggshell spectra, as well as the ratio MgII/MgI between the ionic magnesium line at 280.26 nm and the neutral line at 285.22 nm. The results show that such spectral lines intensities ratio differs for different matrices and is indeed related to the target materials hardness. It is also found that the MgII/MgI ratio is preferable as an indicator of hardness since these lines are less affected by self absorption. The SW front speed has been measured in the three cases and the obtained values confirm the proportionality to the target hardness. The results here obtained suggest the feasibility of the quantitative estimation of hardness for any other calcified tissues.

  10. Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium.

    Science.gov (United States)

    Morero, R D; Viñals, A L; Bloj, B; Farías, R N

    1985-04-01

    Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.

  11. The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase-D toxin.

    Science.gov (United States)

    Chaves-Moreira, Daniele; Souza, Fernanda N; Fogaça, Rosalvo T H; Mangili, Oldemir C; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Chaim, Olga M; Veiga, Silvio S

    2011-09-01

    -mediated rather than leak-promoted because the influx was inhibited by L-type calcium channel inhibitors but not by a T-type calcium channel blocker, sodium channel inhibitor or a specific inhibitor of calcium activated potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase-D treatment occurred in a concentration-dependent manner in the presence of L-type calcium channel blockers such as nifedipine and verapamil. The data provided herein, suggest that the brown spider venom phospholipase-D-induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as SM and LPC, generating bioactive products that stimulate a calcium influx into red blood cells mediated by the L-type channel.

  12. Calcium-Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide-Induced Apoptosis in Tobacco Protoplasts

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In the present study, we focused on whether intracellular free Ca2+ ([Ca2+]i) regulates the formation of mitochondrial permeability transition pore (MPTP) in H2O2-induced apoptosis in tobacco protoplasts. It was shown that the decrease in mitochondrial membrane potential (△Ψm) preceded the appearance of H2O2-induced apoptosis;pretreatment with the specific MPTP inhibitor cyclosporine A, which also inhibits Ca2+ cycling by the mitochondria,effectively retarded apoptosis and the decrease in △Ψm. Apoptosis and decreased △Ψm were exacerbated by CaCl2, whereas the plasma membrane voltage-dependent Ca2+ channel blocker lanthanum chloride (LaCl3)attentuated these responses. Chelation of extracellular Ca2+ with EGTA almost totally inhibited apoptosis and the decrease in △Ψm induced by H2O2. The time-course of changes in [Ca2+]i in apoptosis was detected using the Ca2+ probe Fluo-3 AM. These studies showed that [Ca2+]i was increased at the very early stage of H2O2-induced apoptosis. The EGTA evidently inhibited the increase in [Ca2+]i induced by H2O2, whereas it was only partially inhibited by LaCl3. The results suggest that H2O2 may elevate cytoplasmic free Ca2+ concentrations in tobacco protoplasts, which mainly results from the entry of extracellular Ca2+, to regulate mitochondrial permeability transition. The signaling pathway of [Ca2+]i-mediated mitochondrial permeability transition was associated with H2O2-induced apoptosis in tobacco protoplasts.

  13. Calcium Influx Inhibition is Involved in the Hypotensive and Vasorelaxant Effects Induced by Yangambin

    Directory of Open Access Journals (Sweden)

    Islania Giselia Albuquerque Araújo

    2014-05-01

    Full Text Available The pharmacological effects on the cardiovascular system of yangambin, a lignan isolated from Ocotea duckei Vattimo (Lauraceae, were studied in rats using combined functional and biochemical approaches. In non-anaesthetized rats, yangambin (1, 5, 10, 20, 30 mg/kg, i.v. induced hypotension (−3.5 ± 0.2; −7.1 ± 0.8; −8.9 ± 1.3; −14 ± 2.3, −25.5% ± 2.6%, respectively accompanied by tachycardia (5.9 ± 0.5; 5.9 ± 1.6; 8.8 ± 1.4; 11.6, 18.8% ± 3.4%, respectively. In isolated rat atria, yangambin (0.1 µM–1 mM had very slight negative inotropic (Emax = 35.6% ± 6.4% and chronotropic effects (Emax = 10.2% ± 2.9%. In endothelium-intact rat mesenteric artery, yangambin (0.1 µM–1 mM induced concentration-dependent relaxation (pD2 = 4.5 ± 0.06 of contractions induced by phenylephrine and this effect was not affected by removal of the endothelium. Interestingly, like nifedipine, the relaxant effect induced by yangambin was more potent on the contractile response induced by KCl 80 mM (pD2 = 4.8 ± 0.05 when compared to that induced by phenylephrine. Furthermore, yangambin inhibited CaCl2-induced contractions in a concentration-dependent manner. This lignan also induced relaxation (pD2 = 4.0 ± 0.04 of isolated arteries pre-contracted with S(−-Bay K 8644. In fura-2/AM-loaded myocytes of rat mesenteric arteries, yangambin inhibited the Ca2+ signal evoked by KCl 60 mM. In conclusion, these results suggest that the hypotensive effect of yangambin is probably due to a peripheral vasodilatation that involves, at least, the inhibition the Ca2+ influx through voltage-gated Ca2+ channels.

  14. Calcium-dependent potassium current following penicillin-induced epileptiform discharges in the hippocampal slice.

    Science.gov (United States)

    Domann, R; Dorn, T; Witte, O W

    1989-01-01

    Penicillin-induced paroxysmal depolarization shifts (PDS) are followed by prolonged afterhyperpolarizations of about 2 seconds duration. Intracellular injection of EGTA blocked a late component of the afterhyperpolarizations; an early one lasting up to one second was only slightly reduced by EGTA. It is concluded that afterhyperpolarizations following penicillin-induced PDS comprise different components: an initial one lasting up to one second which is not Ca2+-dependent and a slow one lasting up to two seconds which is caused by a Ca2+-dependent K+ current.

  15. Chemoprevention with Acetylsalicylic Acid, Vitamin D and Calcium Reduces Risk of Carcinogen-induced Lung Tumors

    DEFF Research Database (Denmark)

    Pommergaard, Hans-Christian; Burcharth, Jakob; Rosenberg, J

    2013-01-01

    Background/Aim: Research has shown that chemoprevention may be effective against the development of lung cancer. The purpose of the present study was to evaluate the effect of oral chemoprevention in a mouse model of tobacco carcinogen-induced lung tumor.......Background/Aim: Research has shown that chemoprevention may be effective against the development of lung cancer. The purpose of the present study was to evaluate the effect of oral chemoprevention in a mouse model of tobacco carcinogen-induced lung tumor....

  16. Endogenous nitric oxide mediates alleviation of cadmium toxicity induced by calcium in rice seedlings

    Institute of Scientific and Technical Information of China (English)

    Long Zhang; Zhen Chen; Cheng Zhu

    2012-01-01

    The effect of calcium chloride (CaCl2) on rice seedling growth under cadmium chloride (CdCl2) stress,as well as the possible role of endogenous nitric oxide (NO) in this process,was studied.The growth of rice seedlings was seriously inhibited by CdCl2,and the inhibition was significantly mitigated by CaCl2.However,hemoglobin (Hb) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline1-oxyl-3-oxide (cPTIO) weakened the promotion effect of CaCl2.The resuhs of NO fluorescence localization suggest that growth accelerated by CaCl2 might be associated with elevated NO levels.The content of Cd,protein thiols (PBT),and nonprotein thiols (NPT) in cell walls,cell organelles,and soluble fractions,respectively,of rice seedlings decreased considerably in the presence of CaCl2,whereas the content of pectin,hemicellulose 1 (HC1),and hemicellulose 2 (HC2) increased significantly.Elimination of endogenous NO in Cd+Ca treatment could promote the transportation of Cd2+ to cell organelles and soluble fractions and increase the content of NPT and PBT in leaves.In addition,transportation of Cd2+ to cell organelles and soluble fractions was retarded in roots,the content of NPT increased,and the content of PBT decreased.With elimination of endogenous NO in Cd+Ca treatment,the content of pectin,HC 1,and HC2 decreased significantly.Thus,Ca may alleviate Cd toxicity via endogenous NO with variation in the levels of NPT,PBT,and matrix polysaccharides.

  17. Comparative analysis of MAMP-induced calcium influx in Arabidopsis seedlings and protoplasts.

    Science.gov (United States)

    Maintz, Jens; Cavdar, Meltem; Tamborski, Janina; Kwaaitaal, Mark; Huisman, Rik; Meesters, Christian; Kombrink, Erich; Panstruga, Ralph

    2014-10-01

    Rapid transient elevation of cytoplasmic calcium (Ca(2+)) levels in plant cells is an early signaling event triggered by many environmental cues including abiotic and biotic stresses. Cellular Ca(2+) levels and their alterations can be monitored by genetically encoded reporter systems such as the bioluminescent protein, aequorin. Employment of proteinaceous Ca(2+) sensors is usually performed in transgenic lines that constitutively express the reporter construct. Such settings limit the usage of these Ca(2+) biosensors to particular reporter variants and plant genetic backgrounds, which can be a severe constraint in genetic pathway analysis. Here we systematically explored the potential of Arabidopsis thaliana leaf mesophyll protoplasts, either derived from a transgenic apoaequorin-expressing line or transfected with apoaequorin reporter constructs, as a complementary biological resource to monitor cytoplasmic changes of Ca(2+) levels in response to various biotic stress elicitors. We tested a range of endogenous and pathogen-derived elicitors in seedlings and protoplasts of the corresponding apoaequorin-expressing reporter line. We found that the protoplast system largely reflects the Ca(2+) signatures seen in intact transgenic seedlings. Results of inhibitor experiments including the calculation of IC50 values indicated that the protoplast system is also suitable for pharmacological studies. Moreover, analyses of Ca(2+)signatures in mutant backgrounds, genetic complementation of the mutant phenotypes and expression of sensor variants targeted to different subcellular localizations can be readily performed. Thus, in addition to the prevalent use of seedlings, the leaf mesophyll protoplast setup represents a versatile and convenient tool for the analysis of Ca(2+) signaling pathways in plant cells.

  18. Effects of calcium signaling on coagulation factor VIIa-induced proliferation and migration of the SW620 colon cancer cell line.

    Science.gov (United States)

    Wu, Ying; Wang, Jing; Zhou, Hong; Yu, Xiaoyan; Hu, Lichao; Meng, Fanlu; Jiang, Shuanghong

    2014-12-01

    Tissue factor (TF)/VIIa/protease‑activated receptor 2 (PAR2) has been shown to trigger the ERK1/2 signaling pathway. This was shown to be closely associated with the proliferation and migration of SW620 colon cancer cells; however, the detailed mechanisms remain unclear. The aim of the present study was to elucidate the effects of calcium signaling on the proliferation and migration of SW620 cells induced by coagulation factor VIIa. The results demonstrated that VIIa and PAR2 agonist PAR2‑AP increased [Ca2+]i in SW620 cells. In addition, VIIa‑and PAR2‑AP‑induced ERK1/2 activation was inhibited by thapsigargin (TG)‑induced depletion of intracellular Ca2+ stores and EGTA‑mediated removal of extracellular Ca2+. It was also identified that VIIa and PAR2‑AP‑induced proliferation and migration of SW620 cells was modulated by EGTA and TG. Taken together, the present results indicate that VIIa triggers calcium signaling in SW620 cells, in a TF‑dependent manner, which is critical for VIIa‑induced ERK1/2 activation in SW620 cells. These results suggested that calcium signaling had a vital role in the proliferation and migration of SW620 cells.

  19. Effect of calcium on nicotine-induced current expressed by an atypical alpha-bungarotoxin-insensitive nAChR2.

    Science.gov (United States)

    Thany, Steeve H; Courjaret, Raphael; Lapied, Bruno

    2008-06-27

    Two distinct native alpha-bungarotoxin (alpha-Bgt)-insensitive nicotinic acetylcholine receptors (nAChRs), named nAChR1 and nAChR2, were identified in the cockroach Periplaneta americana dorsal unpaired median (DUM) neurons. They differed in their electrophysiological, pharmacological properties and intracellular regulation pathways. nAChR2 being an atypical nicotinic receptor closed upon agonist application and its current-voltage relationship resulted from a reduction in potassium conductance. In this study, using whole-cell patch-clamp technique, we demonstrated that calcium modulated nAChR2-mediated nicotine response. Under 0.5 microM alpha-Bgt and 20 mM d-tubocurarine, the nicotine-induced inward current amplitude was strongly reduced in the presence of intracellularly applied BAPTA or bath application of calcium-free solution. In addition, using cadmium chloride, we showed that nicotine response was modulated by extracellular calcium through plasma membrane calcium channels. Moreover, extracellular application of caffeine and thapsigargin reduced nAChR2-mediated response. Together these experiments revealed a complex calcium-dependent regulation of nAChR2.

  20. Induced Bone by Calcium Ohosphate(Ca-P) Ceramics after 2-years Implantation

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhiyong; KURITA Hiroshi; KOBAYASHI Hiroichi; KURASHINA Kenji

    2009-01-01

    The changes of induced bone with four kinds of Ca-P ceramics after 2 years im-plantation in the dorsal muscles of rabbits were investigated.After 2 years implantation,mature bone (with a lamellar structure after the remodeling process)with bone marrow was observed in HA1100 (HA,micro+macro porous),HA900(HA,micro+macro porous)and BCP(HA/TCP=7:3,mi-cro+macro porous),and no bone formation was observed in HA1200(HA,macro porous)ceramics. The induced bone neither disappeared nor grew uncontrollably,and was seen inside the pores of the implants or on the outer surface under the fibrous layer after 2 years.No bone formation was ob-served in the soft tissues distant from the implants.These results indicate that heterotopic bone for-mation induced by Ca-P materials did not give rise to uncontrolled growth over time,and the induced bone is limited to the inside or around the implants.Moreover,ectopic bone formation on the outer surface of the implants was detected in rabbits after 2 years.

  1. Laser-frequency locking using light-pressure-induced spectroscopy in a calcium beam

    NARCIS (Netherlands)

    Mollema, A. K.; Wansbeek, L. W.; Willmann, L.; Jungmann, K.; Timmermans, R. G. E.; Hoekstra, R.

    We demonstrate a spectroscopy method that can be applied in an atomic beam, light-pressure-induced spectroscopy (LiPS). A simple pump and probe experiment yields a dispersivelike spectroscopy signal that can be utilized for laser frequency stabilization. The underlying principles are discussed and

  2. Chemoprevention with Acetylsalicylic Acid, Vitamin D and Calcium Reduces Risk of Carcinogen-induced Lung Tumors

    DEFF Research Database (Denmark)

    Pommergaard, Hans-Christian; Burcharth, Jakob; Rosenberg, J

    2013-01-01

    Background/Aim: Research has shown that chemoprevention may be effective against the development of lung cancer. The purpose of the present study was to evaluate the effect of oral chemoprevention in a mouse model of tobacco carcinogen-induced lung tumor....

  3. Attenuation of endothelin-1-induced calcium response by tyrosine kinase inhibitors in vascular smooth muscle cells.

    Science.gov (United States)

    Liu, C Y; Sturek, M

    1996-06-01

    Although tyrosine kinases play an important role in cell growth and have been implicated in regulation of smooth muscle contraction, their role in agonist-induced myoplasmic Ca2+ responses is unclear. We examined effects of the tyrosine kinase inhibitors genistein and methyl 2,5-dihydroxycinnamate (MDHC) on the endothelin-1 (ET-1)-induced Ca2+ response and determined underlying mechanisms for the effects. Freshly isolated smooth muscle cells from porcine coronary arteries were loaded with fura 2 ester, and myoplasmic free Ca2+ (Ca2+ (m)) concentration was estimated with fura 2 microfluorometry. Both genistein and MDHC inhibited the initial transient Cam2+ response to ET by 54 and 81%, respectively (P latent period from ET-1 application to the beginning of the Cam2+ response being increased from 1.08 +/- 0.17 to 2.65 +/- 0.52 min (P < 0.05). In the absence of extracellular Ca2+, genistein inhibited the ET-1-induced Cam2+ response by 93% (P < 0.05). The Cam2+ responses to caffeine (5 mM) or inositol trisphosphate (IP3) applied intracellularly via a patch-clamp pipette were not affected by genistein. Both genistein and MDHC also abolished the sustained Cam2+ response to ET-1. However, the Cam2+ response to depolarization by 80 mM K+ was not inhibited by MDHC and only inhibited 22% by genistein (P < 0.05). These results indicate that 1) activation of tyrosine kinases is an important regulatory mechanism for the ET-1-induced Cam2+ response in vascular smooth muscle and 2) tyrosine kinases mediate ET-1-induced Ca2+ release with no direct effect on IP3-mediated Ca2+ release. Thus ET-1-mediated signaling upstream of IP3 interaction with the Ca2+ stores is regulated by tyrosine kinases.

  4. Role of heterotrimeric G protein and calcium in cardiomyocyte hypertrophy induced by IGF-1.

    Science.gov (United States)

    Carrasco, Loreto; Cea, Paola; Rocco, Paola; Peña-Oyarzún, Daniel; Rivera-Mejias, Pablo; Sotomayor-Flores, Cristian; Quiroga, Clara; Criollo, Alfredo; Ibarra, Cristian; Chiong, Mario; Lavandero, Sergio

    2014-04-01

    In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and β-myosin heavy chain (β-MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin. © 2013 Wiley Periodicals, Inc.

  5. Orai1 and Ca2+-independent phospholipase A2 are required for store-operated Icat-SOC current, Ca2+ entry, and proliferation of primary vascular smooth muscle cells.

    Science.gov (United States)

    Yang, Bo; Gwozdz, Tomasz; Dutko-Gwozdz, Joanna; Bolotina, Victoria M

    2012-03-01

    Store-operated Ca(2+) entry (SOCE) is important for multiple functions of vascular smooth muscle cells (SMC), which, depending of their phenotype, can resemble excitable and nonexcitable cells. Similar to nonexcitable cells, Orai1 was found to mediate Ca(2+)-selective (CRAC-like) current and SOCE in dedifferentiated cultured SMC and smooth muscle-derived cell lines. However, the role of Orai1 in cation-selective store-operated channels (cat-SOC), which are responsible for SOCE in primary SMC, remains unclear. Here we focus on primary SMC, and assess the role of Orai1 and Ca(2+)-independent phospholipase A(2) (iPLA(2)β, or PLA2G6) in activation of cat-SOC current (I(cat-SOC)), SOCE, and SMC proliferation. Using molecular, electrophysiological, imaging, and functional approaches, we demonstrate that molecular knockdown of either Orai1 or iPLA(2)β leads to similar inhibition of the whole cell cat-SOC current and SOCE in primary aortic SMC and results in significant reduction in DNA synthesis and impairment of SMC proliferation. This is the first demonstration that Orai1 and iPLA(2)β are equally important for cat-SOC, SOCE, and proliferation of primary aortic SMC.

  6. Vitamin D3-induced calcemic and phosphatemic responses in the freshwater mud eel Amphipnous cuchia maintained in different calcium environments

    Directory of Open Access Journals (Sweden)

    Ajai K. Srivastav

    1997-11-01

    Full Text Available Vitamin D3 (100 ng 100 g body weight-l day-l was administered intraperitoneally (ip to the freshwater mud eel Amphipnous cuchia kept in artificial freshwater, calcium-free freshwater, low-calcium freshwater (0.2 mmol/l CaCl2 or calcium-rich freshwater (13.4 mmol/l CaCl2 for 15 days. Analyses of serum calcium and phosphate levels were performed on days 1, 3, 5, 10 and 15 after the beginning of the experiment (six eels from each group at each interval. Administration of vitamin D3 elevated the serum calcium [maximum elevation occurred at day 10 in artificial freshwater (vehicle: 10.55 ± 0.298, vitamin D: 13.90 ± 0.324, low-calcium freshwater (vehicle: 11.17 ± 0.220, vitamin D: 12.98 ± 0.297 and calcium-rich freshwater (vehicle: 11.24 ± 0.373, vitamin D: 14.24 ± 0.208 whereas it occurred at day 5 (vehicle: 8.42 ± 0.253, vitamin D: 11.07 ± 0.328 in calcium-free freshwater] and phosphate levels [maximum elevation at day 15 in artificial freshwater (vehicle: 4.39 ± 0.105, vitamin D: 5.37 ± 0.121, calcium-free freshwater (vehicle: 4.25 ± 0.193, vitamin D: 5.12 ± 0.181, low-calcium freshwater (vehicle: 3.93 ± 0.199, vitamin D: 5.28 ± 0.164 and calcium-rich freshwater (vehicle: 3.77 ± 0.125, vitamin D: 5.46 ± 0.151] of the fish maintained in the above mentioned environmental media, but the responses were more pronounced in the fish kept in calcium-rich media

  7. Effects of lipoxin A4 on store-operated caldron channel and production of reactive oxygen species in macrophages%脂氧素A4对小鼠巨噬细胞钙池操纵的钙通道及活性氧的影响

    Institute of Scientific and Technical Information of China (English)

    金胜威; 连庆泉; 梅虹霞; 应斌宇; 程碧环; 叶笃筠; 姚尚龙

    2008-01-01

    目的 研究脂氧素A4对LPS诱导的小鼠RAW 264.7巨噬细胞钙池操纵的钙通道活化及活性氧产生的影响,探讨脂氧素A4保护内毒素性肺损伤的具体机制.方法 小鼠RAW 264.7巨噬细胞,随机分为对照组、LPS组、Thapsigargin组、脂氧素A4+LPs组、脂氧素A4+Thaosigargin组、2-Aminoethoxydiphenylborate+Thapsigargin组.采用细胞内钙离子特异性荧光探针Fluo3/AM和活性氧特异性荧光探针DCFH-DA标记小鼠巨噬细胞.激光共聚焦显微镜动态观察巨噬细胞游离钙浓度变化,流式细胞仪测定活性氧产生变化.结果 脂多糖呈剂量依赖升高细胞内游离钙浓度和活性氧的产生.脂氧素A4抑制脂多糖引起的钙内流(抑制率为93%),脂氧素A4同时可抑制Thapsigargin激活钙池操纵的钙通道引起的钙内流(抑制率为75%).脂多糖诱导巨噬细胞产生大量活性氧(阳性细胞百分比为28.87%±4.5%),脂氧素A4抑制脂多糖诱导巨噬细胞产生活性氧(阳性细胞百分比11.16%±2.5%,P<0.05).结论 脂多糖通过促进内钙释放而开放钙池操纵的钙通道,大量细胞外钙内流引起钙超载,活性氧大量产生,组织细胞损伤.脂氧素A4可能通过抑制钙池操纵的钙通道,使外钙内流减少,保持细胞钙稳态,减少活性氧的生成而起到抗炎促炎症消退作用.%Objective To investigate the effects of lipoxin A4 on store-operated calcium channel (SOC) and production of reactive oxygen species in macrophages induced by hpopolysaccharide (LPS).Method Macrophages were randomly assigned Io one of the following six groups:control group,LPS group,Thapsigargin group,lipoxin A4+LPS group,lipoxin A4+Thapsigargin group,2-Aminoethoxydiphenylborate+Thapsigargin group.The intracellular[Ca2+]iwas analyzed by eonfoeal laser microscopy.The production of reactive oxygen specips(ROS) was assayed by flow cytometry.Results LPS increased intracellular[Ca2+]i and reactive oxygen species in a dose-dependent manner

  8. Dietary fructose inhibits lactation-induced adaptations in rat 1,25-(OH)2D3 synthesis and calcium transport

    Science.gov (United States)

    Douard, Veronique; Suzuki, Takuji; Sabbagh, Yves; Lee, Jacklyn; Shapses, Sue; Lin, Sheldon; Ferraris, Ronaldo P.

    2012-01-01

    We recently showed that excessive fructose consumption, already associated with numerous metabolic abnormalities, reduces rates of intestinal Ca2+ transport. Using a rat lactation model with increased Ca2+ requirements, we tested the hypothesis that mechanisms underlying these inhibitory effects of fructose involve reductions in renal synthesis of 1,25-(OH)2D3. Pregnant and virgin (control) rats were fed isocaloric fructose or, as controls, glucose, and starch diets from d 2 of gestation to the end of lactation. Compared to virgins, lactating dams fed glucose or starch had higher rates of intestinal transcellular Ca2+ transport, elevated intestinal and renal expression of Ca2+ channels, Ca2+-binding proteins, and CaATPases, as well as increased levels of 25-(OH)D3 and 1,25-(OH)2D3. Fructose consumption prevented almost all of these lactation-induced increases, and reduced vitamin D receptor binding to promoter regions of Ca2+ channels and binding proteins. Changes in 1,25-(OH)2D3 level were tightly correlated with alterations in expression of 1α-hydroxylase but not with levels of parathyroid hormone and of 24-hydroxylase. Bone mineral density, content, and mechanical strength each decreased with lactation, but then fructose exacerbated these effects. When Ca2+ requirements increase during lactation or similar physiologically challenging conditions, excessive fructose consumption may perturb Ca2+ homeostasis because of fructose-induced reductions in synthesis of 1,25-(OH)2D3.—Douard, V., Suzuki, T., Sabbagh, Y., Lee, J., Shapses, S., Lin, S., Ferraris, R. P. Dietary fructose inhibits lactation-induced adaptations in rat 1,25-(OH)2D3 synthesis and calcium transport. PMID:22038050

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

    Science.gov (United States)

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

    2010-10-01

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

  10. STIM and calcium channel complexes in cancer.

    Science.gov (United States)

    Jardin, Isaac; Rosado, Juan A

    2016-06-01

    The ion Ca(2+) is a ubiquitous second messenger that mediates a variety of cellular functions. Dysfunction of the mechanisms involved in Ca(2+) homeostasis underlies a number of pathological processes, including cancer. Store-operated Ca(2+) entry (SOCE) is a major mechanism for Ca(2+) entry modulated by the intracellular Ca(2+) stores. The Ca(2+)-selective store-operated current (ICRAC) is mediated by the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and the store-operated Ca(2+) (SOC) channel Orai1, while other non-selective cation currents (ISOC) involves the participation of members of the canonical transient receptor potential (TRPC) channel family, including TRPC1. Distinct isoforms of the key components of SOCE have been described in mammalian cells, STIM1 and 2, Orai1-3 and TRPC1-7. In cancer cells, SOCE has been reported to play an important role in cell cycle progression and proliferation, migration, metastasis and evasion of apoptosis. Changes in the expression of the key elements of SOCE and Ca(2+) homeostasis remodeling have been account to play important roles in the phenotypic changes observed in transformed cells. Despite there are differences in the expression level of the molecular components of SOCE, as well as in the relevance of the STIM, Orai and TRPC isoforms in SOCE and tumorigenesis among cancer cell types, there is a body of evidence supporting an important role for SOCE underlying the phenotypic modifications of cancer cells that propose STIM and the SOC channels as suitable candidate targets for future prognostic or therapeutic strategies. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

  11. Calcium antagonism and the vasorelaxation of the rat aorta induced by rotundifolone

    Directory of Open Access Journals (Sweden)

    Guedes D.N.

    2004-01-01

    Full Text Available The vasorelaxing activity of rotundifolone (ROT, a major constituent (63.5% of the essential oil of Mentha x villosa, was tested in male Wistar rats (300-350 g. In isolated rat aortic rings, increasing ROT concentrations (0.3, 1, 10, 100, 300, and 500 µg/ml inhibited the contractile effects of 1 µM phenylephrine and of 80 or 30 mM KCl (IC50 values, reported as means ± SEM = 184 ± 6, 185 ± 3 and 188 ± 19 µg/ml, N = 6, respectively. In aortic rings pre-contracted with 1 µM phenylephrine, the smooth muscle-relaxant activity of ROT was inhibited by removal of the vascular endothelium (IC50 value = 235 ± 7 µg/ml, N = 6. Furthermore, ROT inhibited (pD2 = 6.04, N = 6 the CaCl2-induced contraction in depolarizing medium in a concentration-dependent manner. In Ca2+-free solution, ROT inhibited 1 µM phenylephrine-induced contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (20 mM. In conclusion, in the present study we have shown that ROT produces an endothelium-independent vasorelaxing effect in the rat aorta. The results further indicated that in the rat aorta ROT is able to induce vasorelaxation, at least in part, by inhibiting both: a voltage-dependent Ca² channels, and b intracellular Ca2+ release selectively due to inositol 1,4,5-triphosphate activation. Additional studies are required to elucidate the mechanisms underlying ROT-induced relaxation.

  12. NF-κB-Dependent Upregulation of NCX1 Induced by Angiotensin II Contributes to Calcium Influx in Rat Aortic Smooth Muscle Cells.

    Science.gov (United States)

    Liu, Bei; Yang, Lixia; Zhang, Bin; Kuang, Chenwei; Huang, Shiliang; Guo, Ruiwei

    2016-11-01

    The reverse mode of Na(+)/Ca(2+) exchanger (NCX) 1 can transport Ca(2+) into cells and is involved in the contractile regulation of vascular smooth muscle cells (VSMCs) and the development of hypertension. We hypothesized that upregulation of NCX1 expression induced by angiotensin II (Ang II) could be dependent on activation of nuclear factor-kappa B (NF-κB) and contributes to Ca(2+) influx in VSMCs. An osmotic minipump was implanted for administration of Ang II in Sprague-Dawley rats, and blood pressure, as well as NCX1 expression, in the aorta was measured. VSMCs were cultured to verify that Ang II-upregulated NCX1 expression is dependent on activation of NF-κB and contributes to Ca(2+) influx. Ang II-upregulated NCX1 expression in rat aortas (2.1-fold at day 6) and VSMCs (1.7-fold at 24 hours) and NF-κB knockdown and p38 mitogen-activated protein kinase (MAPK) inhibitor resulted in 2.1- and 2.0-fold decreases in Ang II-upregulated NCX1 expression in VSMCs. KB-R7943 (an inhibitor of NCX1 reversal) and NCX1 knockdown decreased Ang II-induced Ca(2+) influx 1.4- and 1.3- fold, respectively. KB-R7943 and removal of extracellular Na(+) decreased Ang II-initiated store depletion-mediated Ca(2+) entry by 1.5- and 1.3-fold, respectively. Moreover, NF-κB knockdown and use of a p38 MAPK inhibitor resulted in about 1.3-fold decreases in Ang II-induced Ca(2+) influx through activation of reverse-mode NCX1. Ang II upregulates NCX1 expression through p38 MAPK and NF-κB pathways, and reverse-mode NCX1 plays an important part in Ang II-induced Ca(2+) influx in VSMCs, which may be associated with Ang II-initiated store-operated channel entry. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

  13. Nitric oxide and L-type calcium channel influences the changes in arterial blood pressure and heart rate induced by central angiotesin II

    Directory of Open Access Journals (Sweden)

    Guarda Ismael FMS

    2008-05-01

    Full Text Available Abstract We study the voltage dependent calcium channels and nitric oxide involvement in angiotensin II-induced pressor effect. The antipressor action of L-Type calcium channel antagonist, nifedipine, has been studied when it was injected into the third ventricle prior to angiotensin II. The influence of nitric oxide on nifedipine antipressor action has also been studied by utilizing NW-nitro-L-arginine methyl ester (LNAME (40 μg/0.2 μl a nitric oxide synthase inhibitor and L-arginine (20 μg/0.2 μl, a nitric oxide donor agent. Adult male Holtzman rats weighting 200–250 g, with cannulae implanted into the third ventricle were injected with angiotensin II. Angiotensin II produced an elevation in mean arterial pressure and a decreased in heart rate. Such effects were potentiated by the prior injection of LNAME. L-arginine and nifedipine blocked the effects of angiotensin II. These data showed the involvement of L-Type calcium channel and a free radical gas nitric oxide in the central control of angiotensin II-induced pressor effect. This suggested that L-Type calcium channel of the circunventricular structures of central nervous system participated in both short and long term neuronal actions of ANG II with the influence of nitrergic system.

  14. Particulate air pollution induces arrhythmia via oxidative stress and calcium calmodulin kinase II activation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin-Bae [The Division of Cardiology, Kyung Hee University College of Medicine, 1 Hoegi-dong, Dongdaemun-Gu, Seoul (Korea, Republic of); Kim, Changsoo [The Department of Preventive Medicine, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Choi, Eunmi [Cardiovascular Research Institute and Severance Biomedical Science Institute, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Park, Sanghoon; Park, Hyelim; Pak, Hui-Nam; Lee, Moon-Hyoung [The Division of Cardiology, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Shin, Dong Chun [The Department of Preventive Medicine, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Hwang, Ki-Chul [Cardiovascular Research Institute and Severance Biomedical Science Institute, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); The Division of Cardiology, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of); Joung, Boyoung, E-mail: cby6908@yuhs.ac [The Division of Cardiology, Yonsei University College of Medicine, 250 Seungsanno, Seodaemun-gu, Seoul (Korea, Republic of)

    2012-02-15

    Ambient particulate matter (PM) can increase the incidence of arrhythmia. However, the arrhythmogenic mechanism of PM is poorly understood. This study investigated the arrhythmogenic mechanism of PM. In Sprague–Dawley rats, QT interval was increased from 115.0 ± 14.0 to 142.1 ± 18.4 ms (p = 0.02) after endotracheal exposure of DEP (200 μg/ml for 30 min, n = 5). Ventricular premature contractions were more frequently observed after DEP exposure (100%) than baseline (20%, p = 0.04). These effects were prevented by pretreatment of N-acetylcysteine (NAC, 5 mmol/L, n = 3). In 12 Langendorff-perfused rat hearts, DEP infusion of 12.5 μg/ml for 20 min prolonged action potential duration (APD) at only left ventricular base increasing apicobasal repolarization gradients. Spontaneous early afterdepolarization (EAD) and ventricular tachycardia (VT) were observed in 8 (67%) and 6 (50%) hearts, respectively, versus no spontaneous triggered activity or VT in any hearts before DEP infusion. DEP-induced APD prolongation, EAD and VT were successfully prevented with NAC (5 mmol/L, n = 5), nifedipine (10 μmol/L, n = 5), and active Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) blockade, KN 93 (1 μmol/L, n = 5), but not by thapsigargin (200 nmol/L) plus ryanodine (10 μmol/L, n = 5) and inactive CaMKII blockade, KN 92 (1 μmol/L, n = 5). In neonatal rat cardiomyocytes, DEP provoked ROS generation in dose dependant manner. DEP (12.5 μg/ml) induced apoptosis, and this effect was prevented by NAC and KN 93. Thus, this study shows that in vivo and vitro exposure of PM induced APD prolongation, EAD and ventricular arrhythmia. These effects might be caused by oxidative stress and CaMKII activation. -- Highlights: ► The ambient PM consistently prolonged repolarization. ► The ambient PM induced triggered activity and ventricular arrhythmia. ► These effects were prevented by antioxidants, I{sub CaL} blockade and CaMKII blockade. ► The ambient PM can induce

  15. STIM1 Phosphorylation at Y361 Recruits Orai1 to STIM1 Puncta and Induces Ca2+ Entry

    Science.gov (United States)

    Yazbeck, Pascal; Tauseef, Mohammad; Kruse, Kevin; Amin, Md-Ruhul; Sheikh, Rayees; Feske, Stefan; Komarova, Yulia; Mehta, Dolly

    2017-01-01

    Store-operated Ca2+ entry (SOCE) mediates the increase in intracellular calcium (Ca2+) in endothelial cells (ECs) that regulates several EC functions including tissue-fluid homeostasis. Stromal-interaction molecule 1 (STIM1), upon sensing the depletion of (Ca2+) from the endoplasmic reticulum (ER) store, organizes as puncta that trigger store-operated Ca2+ entry (SOCE) via plasmalemmal Ca2+-selective Orai1 channels. While the STIM1 and Orai1 binding interfaces have been mapped, signaling mechanisms activating STIM1 recruitment of Orai1 and STIM1-Orai1 interaction remains enigmatic. Here, we show that ER Ca2+-store depletion rapidly induces STIM1 phosphorylation at Y361 via proline-rich kinase 2 (Pyk2) in ECs. Surprisingly, the phospho-defective STIM1-Y361F mutant formed puncta but failed to recruit Orai1, thereby preventing. SOCE Furthermore, studies in mouse lungs, expression of phosphodefective STIM1-Y361F mutant in ECs prevented the increase in vascular permeability induced by the thrombin receptor, protease activated receptor 1 (PAR1). Hence, Pyk2-dependent phosphorylation of STIM1 at Y361 is a critical phospho-switch enabling recruitment of Orai1 into STIM1 puncta leading to SOCE. Therefore, Y361 in STIM1 represents a novel target for limiting SOCE-associated vascular leak. PMID:28218251

  16. Involvement of transient receptor potential melastatin-8 (TRPM8) in menthol-induced calcium entry, reactive oxygen species production and cell death in rheumatoid arthritis rat synovial fibroblasts.

    Science.gov (United States)

    Zhu, Shuyan; Wang, Yuxiang; Pan, Leiting; Yang, Shuang; Sun, Yonglin; Wang, Xinyu; Hu, Fen

    2014-02-15

    Rheumatoid arthritis is most prominently characterized by synoviocyte hyperplasia which therefore serves as an important target for clinical therapy. In the present study, it was observed that menthol, the specific agonist of transient receptor potential melastatin subtype 8 (TRPM8), could induce sustained increases of cytosolic calcium concentration ([Ca(2+)]c) in synoviocytes isolated from collagen-induced arthritis rats in dose-dependent manner, which was evidently blocked by applying an extracellular Ca(2+)-free buffer. Menthol-induced [Ca(2+)]c increase was also significantly inhibited by potent TRPM8 antagonist capsazepine (CZP), indicating that this [Ca(2+)]c elevation was mostly attributed to TRPM8-mediated Ca(2+) entry. Besides, RT-PCR indeed demonstrated presence of TRPM8 in the synoviocytes. Meanwhile, it was found that menthol evoked production of intracellular reactive oxygen species, which could be abolished by Ca(2+) free solutions or CZP. Further experiments showed that menthol reduced the cell numbers and survival of synoviocytes. This reduction was associated with apoptosis as suggested by mitochondrial membrane depolarization, nuclear condensation and a caspase 3/7 apoptotic assay. Menthol-induced death and apoptosis of synoviocytes both were obviously inhibited by CZP, intracellular calcium chelator BAPTA-AM, and reactive oxygen species inhibitor diphenylene iodonium, respectively. Taken together, our data indicated that menthol resulted in synoviocyte death associated with apoptosis via calcium entry and reactive oxygen species production depending on TRPM8 activation.

  17. Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells.

    Science.gov (United States)

    Yoon, Yeo Cho; Kim, Sung-Hee; Kim, Min Jung; Yang, Hye Jeong; Rhyu, Mee-Ra; Park, Jae-Ho

    2015-01-01

    This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC-MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells.

  18. High vitamin D and calcium intakes increase bone mineral (Ca and P) content in high-fat diet-induced obese mice.

    Science.gov (United States)

    Song, Qingming; Sergeev, Igor N

    2015-02-01

    Vitamin D and calcium are essential for bone formation, mineralization, and remodeling. Recent studies demonstrated that an increased body mass can be detrimental to bone health. However, whether an increase in dietary vitamin D and calcium intakes in obesity is beneficial to bone health has not been established. The aim of this study was to examine the effects of increased vitamin D and calcium intakes, alone or in combination, on bone status in a high-fat diet-induced obesity (DIO) mouse model. We hypothesized that DIO in growing mice affects bone mineral status and that high vitamin D and calcium intakes will promote mineralization of the growing bone in obesity via Ca(2+) regulatory hormones, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH). Male mice were fed high vitamin D3 (10 000 IU/kg), high calcium (1.2%), or high vitamin D3 plus high-calcium diets containing 60% energy as fat for 10 weeks. Bone weight, specific gravity, mineral (Ca and P), and collagen (hydroxyproline) content were measured in the femur and the tibia. Regulators of Ca(2+) metabolism and markers of bone status (PTH, 25-hydroxyvitamin D [25(OH)D], 1,25(OH)2D3, and osteocalcin) were measured in blood plasma. Diet-induced obese mice exhibited lower bone Ca and P content and relative bone weight compared with the normal-fat control mice, whereas collagen (hydroxyproline) content was not different between the two groups. High vitamin D3 and calcium intakes significantly increased bone Ca and P content and relative bone weight in DIO mice, which was accompanied by an increase in 1,25(OH)2D3 and a decrease in PTH and osteocalcin concentrations in blood. The findings obtained indicate that increased vitamin D and calcium intakes are effective in increasing mineral (Ca and P) content in the growing bone of obese mice and that the hormonal mechanism of this effect may involve the vitamin D-PTH axis. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Mepivacaine-induced contraction involves increased calcium sensitization mediated via Rho kinase and protein kinase C in endothelium-denuded rat aorta.

    Science.gov (United States)

    Ok, Seong-Ho; Kwon, Seong-Chun; Yeol Han, Jeong; Yu, Jongsun; Shin, Il-Woo; Lee, Heon-Keun; Chung, Young-Kyun; Choi, Mun-Jeoung; Sohn, Ju-Tae

    2014-01-15

    Mepivacaine is an aminoamide local anesthetic that produces vasoconstriction in vivo and in vitro. The goals of this in vitro study were to determine whether mepivacaine-induced contraction involves calcium sensitization in isolated endothelium-denuded aortas, and to investigate the specific protein kinases involved. The effects of mepivacaine and potassium chloride on intracellular calcium concentrations ([Ca(2+)]i) and tension in the presence or absence of Y-27632 or GF 109203X were measured simultaneously using the acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: Rho kinase inhibitor Y-27632, protein kinase C (PKC) inhibitor GF 109203X, extracellular signal-regulated kinase (ERK) inhibitor PD 98059, c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, and p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580. Phosphorylation of PKC and MAPK, and membrane translocation of Rho kinase were detected in vascular smooth muscle cells by Western blotting. The slope of the mepivacaine-induced [Ca(2+)]i-tension curve was higher than that of the KCl-induced [Ca(2+)]i-tension curve. Pretreatment with Y-27632 or GF 109203X shifted the mepivacaine-induced [Ca(2+)]i-tension curve to the lower right. Pretreatment with Y-27632, GF 109203X, PD 98059, or SP600125 attenuated mepivacaine-induced contraction in a concentration-dependent manner. Y-27632 and GF 109203X attenuated mepivacaine-induced Rho kinase membrane translocation and PKC phosphorylation, respectively. PD 98059 and SP600125 attenuated mepivacaine-induced ERK and JNK phosphorylation, respectively. Taken together, these results indicate that mepivacaine-induced contraction involves increased calcium sensitization mediated by Rho kinase and PKC. Such contraction mainly involves activation of ERK- and JNK-mediated pathways. © 2013 Published by Elsevier B.V.

  20. L-type calcium channel blockers enhance 5-HTP-induced antinociception in mice

    Institute of Scientific and Technical Information of China (English)

    Jian-hui LIANG; Jun-xu LI; Xu-hua WANG; Bi CHEN; Ying LU; Pan ZHANG; Rong HAN; Xiang-feng YE

    2004-01-01

    AIM: To investigate the involvement of L-type Ca2+ channels in antinociceptive action induced by the 5-HT precursor,5-hydroxytryptophan (5-HTP). METHODS: Female Kunming mice were treated with either 5-HTP (20-80 mg/kg,ip) alone, or the combination of 5-HTP and fluoxetine (2-8 mg/kg, ip), pargyline (15-60 mg/kg, ip), nimodipine (2.5-10 mg/kg, ip), nifedipine (2.5-10 mg/kg, ip), verapamil (2.5-10 mg/kg, ip), CaC12 (5-20 mmol/L, icv), or EGTA (0.5-3 mmol/L, icv) prior to the hot-plate test (55 ℃, hind-paw licking latency). In addition, locomotor activity in mice treated with 5-HTP alone was measured using an ambulometer with five activity boxes. RESULTS: Ip injection of 5-HTP alone had no influence on the spontaneous locomotor activity, whereas dose-dependently increased the latency to licking hind-paw in the hot-plate test in mice. The inhibitory effects of 5-HTP on nociceptive response were significantly enhanced by fluoxetine in the mouse hot-plate test. At a sub-effective dose, pargyline could cause a leftward shift in the dose-response curve of 5-HTP-induced antinociception. Co-administration with 5-HTP and nimodipine, nifedipine, or verapamil obviously potentiated the antinociceptive effects elicited by 5-HTP.Interestingly, 5-HTP-induced antinociception was antagonized by CaC12 and enhanced by EGTA injected icv in the mouse hot-plate test. CONCLUSION: These findings suggest that systemic administration of 5-HTP may yield the antinociceptive effects, which are related to Ca2+ influx from extracellular fluid through L-type Ca2+ channels.

  1. Role of calcium and EPAC in norepinephrine-induced ghrelin secretion.

    Science.gov (United States)

    Mani, Bharath K; Chuang, Jen-Chieh; Kjalarsdottir, Lilja; Sakata, Ichiro; Walker, Angela K; Kuperman, Anna; Osborne-Lawrence, Sherri; Repa, Joyce J; Zigman, Jeffrey M

    2014-01-01

    Ghrelin is an orexigenic hormone secreted principally from a distinct population of gastric endocrine cells. Molecular mechanisms regulating ghrelin secretion are mostly unknown. Recently, norepinephrine (NE) was shown to enhance ghrelin release by binding to β1-adrenergic receptors on ghrelin cells. Here, we use an immortalized stomach-derived ghrelin cell line to further characterize the intracellular signaling pathways involved in NE-induced ghrelin secretion, with a focus on the roles of Ca(2+) and cAMP. Several voltage-gated Ca(2+) channel (VGCC) family members were found by quantitative PCR to be expressed by ghrelin cells. Nifedipine, a selective L-type VGCC blocker, suppressed both basal and NE-stimulated ghrelin secretion. NE induced elevation of cytosolic Ca(2+) levels both in the presence and absence of extracellular Ca(2+). Ca(2+)-sensing synaptotagmins Syt7 and Syt9 were also highly expressed in ghrelin cell lines, suggesting that they too help mediate ghrelin secretion. Raising cAMP with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated ghrelin secretion, although such a cAMP-mediated effect likely does not involve protein kinase A, given the absence of a modulatory response to a highly selective protein kinase A inhibitor. However, pharmacological inhibition of another target of cAMP, exchange protein-activated by cAMP (EPAC), did attenuate both basal and NE-induced ghrelin secretion, whereas an EPAC agonist enhanced basal ghrelin secretion. We conclude that constitutive ghrelin secretion is primarily regulated by Ca(2+) influx through L-type VGCCs and that NE stimulates ghrelin secretion predominantly through release of intracellular Ca(2+). Furthermore, cAMP and its downstream activation of EPAC are required for the normal ghrelin secretory response to NE.

  2. L-type calcium channel blockers enhance 5-HTP-induced antinociception in mice.

    Science.gov (United States)

    Liang, Jian-hui; Li, Jun-xu; Wang, Xu-hua; Chen, Bi; Lu, Ying; Zhang, Pan; Han, Rong; Ye, Xiang-feng

    2004-05-01

    To investigate the involvement of L-type Ca(2+) channels in antinociceptive action induced by the 5-HT precursor, 5-hydroxytryptophan (5-HTP). Female Kunming mice were treated with either 5-HTP (20-80 mg/kg, ip) alone, or the combination of 5-HTP and fluoxetine (2-8 mg/kg, ip), pargyline (15-60 mg/kg, ip), nimodipine (2.5-10 mg/kg, ip), nifedipine (2.5-10 mg/kg, ip), verapamil (2.5-10 mg/kg, ip), CaCl(2) (5-20 mmol/L, icv), or EGTA (0.5-3 mmol/L, icv) prior to the hot-plate test (55 degree, hind-paw licking latency). In addition, locomotor activity in mice treated with 5-HTP alone was measured using an ambulometer with five activity boxes. Ip injection of 5-HTP alone had no influence on the spontaneous locomotor activity, whereas dose-dependently increased the latency to licking hind-paw in the hot-plate test in mice. The inhibitory effects of 5-HTP on nociceptive response were significantly enhanced by fluoxetine in the mouse hot-plate test. At a sub-effective dose, pargyline could cause a leftward shift in the dose-response curve of 5-HTP-induced antinociception. Co-administration with 5-HTP and nimodipine, nifedipine, or verapamil obviously potentiated the antinociceptive effects elicited by 5-HTP. Interestingly, 5-HTP-induced antinociception was antagonized by CaCl(2) and enhanced by EGTA injected icv in the mouse hot-plate test. These findings suggest that systemic administration of 5-HTP may yield the antinociceptive effects, which are related to Ca(2+) influx from extracellular fluid through L-type Ca(2+) channels.

  3. Influence of calcium preconditioning and streptomycin on ventricular dilation-induced arrhythmias in isolated rat hearts

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate the mechanism of ventricular dilation-induced arrhythmias by dilating isolated rat hearts. Methods Isolated rat hearts were perfused by Langerdorff method. After equilibration, 80 hearts were randomly divided into four groups as follows: (1) control group (n=20), (2) Ca2+ preconditioning (CPC) group (n=20), (3) streptomycin group (n=20), and (4) CPC + streptomycin group (n=20). A latex balloon which can be filled with fluid was anchored in the left ventricle through the left atrium and mitral valve. Epicardial ECG of the left ventricle, left ventricular pressure, coronary flow and heart rate were recorded before and during ventricular dilation by injecting fluid into the latex balloon. The rate and duration of ventricular dilation-induced arrhythmias were recorded. Results Under the same increase in ventricular end-diastolic pressure made by inflation of the balloon, the rate of arrhythmias was 100% and duration of arrhythmias was 2.56±0.46 s in the control group. Both the rates of premature ventricular beat (90 %) and ventricular tachycardia 70 % ) were high. Compared with the control group, the total rate (60 % ) of arrhythmias was lower, and duration (1.67±0.61 s ) of arrhythmias was shorter in the CPC group. Both the rates of premature ventricular beat (60%) and ventricular tachycardia (40%) were low comparatively. The rate of arrhythmias (45 %) was lower and duration ( 1.64±0.42 s)of arrhythmias was shorter, and the rates of premature ventricular beat (30 % ) or ventricular tachycardia (35 %) were lower in the streptomycin group than in the control one. The least ventricular dilation-induced arrhythmias occurred in the CPC + streptomycin group. The rate of arrhythmias (10%) was the lowest and duration (1.01±0.37s) of arrhythmias was the shortest; both the rates of premature ventricular beat (5%) and ventricular tachycardia (10%) were the lowest. Conclusions Ventricular dilation may induce arrhythmias in isolated rat hearts. Stretch

  4. Renal tubular injury induced by ischemia promotes the formation of calcium oxalate crystals in rats with hyperoxaluria.

    Science.gov (United States)

    Cao, Yanwei; Liu, Wanpeng; Hui, Limei; Zhao, Jianjun; Yang, Xuecheng; Wang, Yonghua; Niu, Haitao

    2016-10-01

    Hyperoxaluria and cell injury are key factors in urolithiasis. Oxalate metabolism may be altered by renal dysfunction and therefore, impact the deposition of calcium oxalate (CaOx) crystals. We investigated the relationship of renal function, oxalate metabolism and CaOx crystal deposition in renal ischemia. One hundred male Sprague-Dawley rats were randomly divided into four groups. Hyperoxaluria model (Group A and B) was established by feeding rats with 0.75 % ethylene glycol (EG). The left renal pedicle was clamped for 30 min to establish renal ischemia Groups (B and C), while Groups A and D underwent sham operation. Then, serum and urine oxalate (Ox), creatinine (Cr) and urea nitrogen (UN) levels were evaluated by liquid chromatography mass spectrometry (LCMS) and ion mass spectrum (IMS) at days 0, 2, 4, 7, and 14. CaOx crystallization was assessed by transmission electron microscope (TEM). A temporal and significant increase of serum Cr and UN levels was observed in Groups B and C compared to values obtained for Groups A and D (P renal tissue. Our results indicated that renal tubular injury induced by renal ischemia might not affect Ox levels but could promote CaOx crystal retention under hyperoxaluria.

  5. 1H NMR and Rheological Studies of the Calcium Induced Gelation Process in Aqueous Low Methoxyl Pectin Solutions

    Science.gov (United States)

    Dobies, M.; Kuśmia, S.; Jurga, S.

    2006-07-01

    The 1H NMR relaxometry in combination with water proton spin-spin relaxation time measurements and rheometry have been applied to study the ionic gelation of 1% w/w aqueous low methoxyl pectin solution induced by divalent Ca2+ cations from a calcium chloride solution. The model-free approach to the analysis of 1H NMR relaxometry data has been used to separate the information on the static (β) and dynamic () behaviour of the systems tested. The 1H NMR results confirm that the average mobility of both water and the pectin molecules is largely dependent on the concentration of the cross-linking agent. The character of this dependency (β, and T2 vs. CaCl2 concentration) is consistent with the two-stage gelation process of low methoxyl pectin, in which the formation of strongly linked dimer associations (in the range of 0-2.5 mM CaCl2) is followed by the appearance of weak inter-dimer aggregations (for CaCl2≥ 3.5 mM). The presence of the weak gel structure for the sample with 3.5 mM CaCl2 has been confirmed by rheological measurements. Apart from that, the T1 and T2 relaxation times have been found to be highly sensitive to the syneresis phenomenon, which can be useful to monitor the low methoxyl pectin gel network stability.

  6. Rheological and physical properties of camel and cow milk gels enriched with phosphate and calcium during acid-induced gelation.

    Science.gov (United States)

    Kamal, Mohammad; Foukani, Mohammed; Karoui, Romdhane

    2017-02-01

    The rheological properties of acid-induced coagulation of camel and cow milk gels following the addition of calcium chloride (CaCl2) and hydrogen phosphate dehydrate (Na2HPO4*2H2O) were investigated using a dynamic low amplitude oscillatory rheology. For a considered condition, the final values of storage modulus (G') and loss modulus (G″) of camel milk gels were significantly lower than those of cow milk gels. The increase of the added CaCl2 levels improved significantly the gelation properties of camel and cow milk gels, since a reduction in the gelation time and an increase in the gel firmness were observed. Following the addition of Na2HPO4*2H2O at 10 and 20 mM, no significant effect on the gelation rate and the firmness of camel milk gels was observed, while, a significant decrease in the gelation rate and firmness were observed for cow milk gels.

  7. Calcium H & K and sodium D absorption induced by the interstellar and circumgalactic media of the Milky Way

    CERN Document Server

    Murga, Maria; Ménard, Brice; Lan, Ting-Wen

    2015-01-01

    We map out calcium II & sodium I absorption (Fraunhofer H, K & D lines) induced by both the interstellar medium and the circumgalactic medium of the Milky Way. Our measurements cover more than $9000$ deg$^2$ and make use of about $300,000$ extragalactic spectra from the Sloan Digital Sky Survey. We present absorption maps for these two species and then compare their distributions to those of neutral hydrogen and dust. We show that the abundance of Na I with respect to neutral hydrogen stays roughly constant in different environments, while that of Ca II decreases with hydrogen column density. Studying how these tracers vary as a function of velocity, we show that, on average, the N(Na I)/N(Ca II) ratio decreases at higher velocity with respect to the local standard of rest, similar to the local Routly-Spitzer effect but seen on Galactic scale. We show that it is likely caused by higher gas/dust density at lower velocity. Finally, we show that Galactic Ca II and Na I absorption needs to be taken into a...

  8. A lipochito-oligosaccharide, Nod factor, induces transient calcium influx in soybean suspension-cultured cells.

    Science.gov (United States)

    Yokoyama, T; Kobayashi, N; Kouchi, H; Minamisawa, K; Kaku, H; Tsuchiya, K

    2000-04-01

    Lipochito-oligosaccharides (Nod factors) produced by Rhizobium or Bradyrhizobium are the key signal molecules for eliciting nodulation in their corresponding host legumes. To elucidate the signal transduction events mediated by Nod factors, we investigated the effects of Nod factors on the cytosolic [Ca2+] of protoplasts prepared from roots and suspension-cultured cells of soybean (Glycine max and G. soja) using a fluorescent Ca2+ indicator, Fura-PE3. NodBj-V (C18:1, MeFuc), which is a major component of Nod factors produced by Bradyrhizobium japonicum, induces transient elevation of cytosolic [Ca2+] in the cells of soybean within a few minutes. This effect is specific to soybean cells and was not observed in the tobacco BY-2 cells. Furthermore, NodBj-V without MeFuc did not induce any cytosolic [Ca2+] elevation in soybean cells. Exclusion of Ca2+ from the medium, as well as pre-treatment of the cells with an external Ca2+ chelator or with a plasma membrane voltage-dependent Ca2+ channel inhibitor, suppressed the Nod factor-dependent cytosolic [Ca2+] elevation. These results indicate that transient Ca2+ influx from extracellular fluid is one of the earliest responses of soybean cells to NodBj-V (C18:1, MeFuc) in a host-specific manner.

  9. Zirconium, calcium, and strontium contents in magnesium based biodegradable alloys modulate the efficiency of implant-induced osseointegration

    Directory of Open Access Journals (Sweden)

    Mushahary D

    2013-08-01

    Full Text Available Dolly Mushahary,1,2 Ragamouni Sravanthi,2 Yuncang Li,2 Mahesh J Kumar,1 Nemani Harishankar,4 Peter D Hodgson,1 Cuie Wen,3 Gopal Pande2 1Institute for Frontier Materials, Deakin University, Geelong, Australia; 2CSIR- Centre for Cellular and Molecular Biology, Hyderabad, India; 3Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, Australia; 4National Institute of Nutrition (ICMR, Tarnaka, Hyderabad, India Abstract: Development of new biodegradable implants and devices is necessary to meet the increasing needs of regenerative orthopedic procedures. An important consideration while formulating new implant materials is that they should physicochemically and biologically mimic bone-like properties. In earlier studies, we have developed and characterized magnesium based biodegradable alloys, in particular magnesium-zirconium (Mg-Zr alloys. Here we have reported the biological properties of four Mg-Zr alloys containing different quantities of strontium or calcium. The alloys were implanted in small cavities made in femur bones of New Zealand White rabbits, and the quantitative and qualitative assessments of newly induced bone tissue were carried out. A total of 30 experimental animals, three for each implant type, were studied, and bone induction was assessed by histological, immunohistochemical and radiological methods; cavities in the femurs with no implants and observed for the same period of time were kept as controls. Our results showed that Mg-Zr alloys containing appropriate quantities of strontium were more efficient in inducing good quality mineralized bone than other alloys. Our results have been discussed in the context of physicochemical and biological properties of the alloys, and they could be very useful in determining the nature of future generations of biodegradable orthopedic implants. Keywords: osteoblasts, bone mineralization, corrosion, osseointegration, surface energy, peri-implant

  10. Changes in mitochondrial morphology induced by calcium or rotenone in primary astrocytes occur predominantly through ros-mediated remodeling.

    Science.gov (United States)

    Deheshi, Samineh; Dabiri, Bahram; Fan, Susu; Tsang, Michelle; Rintoul, Gordon L

    2015-06-01

    Morphological changes in mitochondria have been primarily attributed to fission and fusion, while the more pliable transformations of mitochondria (remodeling, rounding, or stretching) have been largely overlooked. In this study, we quantify the contributions of fission and remodeling to changes in mitochondrial morphology induced by the Ca(2+) ionophore 4Br-A23187 and the metabolic toxin rotenone. We also examine the role of reactive oxygen species (ROS) in the regulation of mitochondrial remodeling. In agreement with our previous studies, mitochondrial remodeling, not fission, is the primary contributor to Ca(2+) -mediated changes in mitochondrial morphology induced by 4Br-A23187 in rat cortical astrocytes. Treatment with rotenone produced similar results. In both paradigms, remodeling was selectively blocked by antioxidants whereas fission was not, suggesting a ROS-mediated mechanism for mitochondrial remodeling. In support of this hypothesis, inhibition of endogenous ROS by overnight incubation in antioxidants resulted in elongated reticular networks of mitochondria. Examination of inner and outer mitochondrial membranes revealed that they largely acted in concert during the remodeling process. While mitochondrial morphology is traditionally ascribed to a net output of fission and fusion processes, in this study we provide evidence that the acute pliability of mitochondria can be a dominant factor in determining their morphology. More importantly, our results suggest that the remodeling process is independently regulated through a ROS-signaling mechanism. Mitochondrial morphology is traditionally ascribed to a balance of fission and fusion processes. We have shown that mitochondria can undergo more pliable transformations; remodeling, rounding, or stretching. We demonstrate that remodeling, not fission, is the primary contributor to calcium mediated changes in mitochondrial morphology in primary astrocytes. Others have shown fission is mediated by calcineurin

  11. Aluminum-induced dreierketten chain cross-links increase the mechanical properties of nanocrystalline calcium aluminosilicate hydrate

    Science.gov (United States)

    Geng, Guoqing; Myers, Rupert J.; Li, Jiaqi; Maboudian, Roya; Carraro, Carlo; Shapiro, David A.; Monteiro, Paulo J. M.

    2017-01-01

    The incorporation of Al and increased curing temperature promotes the crystallization and cross-linking of calcium (alumino)silicate hydrate (C-(A-)S-H), which is the primary binding phase in most contemporary concrete materials. However, the influence of Al-induced structural changes on the mechanical properties at atomistic scale is not well understood. Herein, synchrotron radiation-based high-pressure X-ray diffraction is used to quantify the influence of dreierketten chain cross-linking on the anisotropic mechanical behavior of C-(A-)S-H. We show that the ab-planar stiffness is independent of dreierketten chain defects, e.g. vacancies in bridging tetrahedra sites and Al for Si substitution. The c-axis of non-cross-linked C-(A-)S-H is more deformable due to the softer interlayer opening but stiffens with decreased spacing and/or increased zeolitic water and Ca2+ of the interlayer. Dreierketten chain cross-links act as ‘columns’ to resist compression, thus increasing the bulk modulus of C-(A-)S-H. We provide the first experimental evidence on the influence of the Al-induced atomistic configurational change on the mechanical properties of C-(A-)S-H. Our work advances the fundamental knowledge of C-(A-)S-H on the lowest level of its hierarchical structure, and thus can impact the way that innovative C-(A-)S-H-based cementitious materials are developed using a ‘bottom-up’ approach. PMID:28281635

  12. Protection of cortical cells by equine estrogens against glutamate-induced excitotoxicity is mediated through a calcium independent mechanism

    Directory of Open Access Journals (Sweden)

    Perrella Joel

    2005-05-01

    Full Text Available Abstract Background High concentrations of glutamate can accumulate in the brain and may be involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. This form of neurotoxicity involves changes in the regulation of cellular calcium (Ca2+ and generation of free radicals such as peroxynitrite (ONOO-. Estrogen may protect against glutamate-induced cell death by reducing the excitotoxic Ca2+ influx associated with glutamate excitotoxicity. In this study, the inhibition of N-methyl-D-aspartate (NMDA receptor and nitric oxide synthase (NOS along with the effect of 17β-estradiol (17β-E2 and a more potent antioxidant Δ8, 17β-estradiol (Δ8, 17β-E2 on cell viability and intracellular Ca2+ ([Ca2+]i, following treatment of rat cortical cells with glutamate, was investigated. Results Primary rat cortical cells were cultured for 7–12 days in Neurobasal medium containing B27 supplements. Addition of glutamate (200 μM decreased cell viability to 51.3 ± 0.7% compared to control. Treatment with the noncompetitive NMDAR antagonist, MK-801, and the NOS inhibitor, L-NAME, completely prevented cell death. Pretreatment (24 hrs with 17β-E2 and Δ8, 17β-E2 (0.01 to 10 μM significantly reduced cell death. 17β-E2 was more potent than Δ8, 17β-E2. Glutamate caused a rapid 2.5 fold increase in [Ca2+]i. Treatment with 0.001 to 10 μM MK-801 reduced the initial Ca2+ influx by 14–41% and increased cell viability significantly. Pretreatment with 17β-E2 and Δ8, 17β-E2 had no effect on Ca2+ influx but protected the cortical cells against glutamate-induced cell death. Conclusion Glutamate-induced cell death in cortical cultures can occur through NMDAR and NOS-linked mechanisms by increasing nitric oxide and ONOO-. Equine estrogens: 17β-E2 and Δ8, 17β-E2, significantly protected cortical cells against glutamate-induced excitotoxicity by a mechanism that appears to be independent of Ca2+ influx. To our knowledge, this is a first

  13. Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells

    Science.gov (United States)

    Yoon, Mi Na; Kim, Dong Kwan; Kim, Se Hoon

    2017-01-01

    Intracellular calcium (Ca2+) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H2O2) on intracellular Ca2+ accumulation in mouse pancreatic acinar cells. Perfusion of H2O2 at 300 µM resulted in additional elevation of intracellular Ca2+ levels and termination of oscillatory Ca2+ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca2+. Antioxidants, catalase or DTT, completely prevented H2O2-induced additional Ca2+ increase and termination of Ca2+ oscillation. In Ca2+-free medium, H2O2 still enhanced CCh-induced intracellular Ca2+ levels and thapsigargin (TG) mimicked H2O2-induced cytosolic Ca2+ increase. Furthermore, H2O2-induced elevation of intracellular Ca2+ levels was abolished under sarco/endoplasmic reticulum Ca2+ ATPase-inactivated condition by TG pretreatment with CCh. H2O2 at 300 µM failed to affect store-operated Ca2+ entry or Ca2+ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca2+ uniporter blocker, failed to attenuate H2O2-induced intracellular Ca2+ elevation. These results provide evidence that excessive generation of H2O2 in pathological conditions could accumulate intracellular Ca2+ by attenuating refilling of internal Ca2+ stores rather than by inhibiting Ca2+ extrusion to extracellular fluid or enhancing Ca2+ mobilization from extracellular medium in mouse pancreatic acinar cells.

  14. Molecular evolution and functional divergence of the Ca(2+ sensor protein in store-operated Ca(2+ entry: stromal interaction molecule.

    Directory of Open Access Journals (Sweden)

    Xinjiang Cai

    Full Text Available Receptor-mediated Ca(2+ signaling in many non-excitable cells initially induces Ca(2+ release from intracellular Ca(2+ stores, followed by Ca(2+ influx across the plasma membrane. Recent findings have suggested that stromal interaction molecules (STIMs function as the Ca(2+ sensor to detect changes of Ca(2+ content in the intracellular Ca(2+ stores. Human STIMs and invertebrate STIM share several functionally important protein domains, but diverge significantly in the C-terminus. To better understand the evolutionary significance of STIM activity, phylogenetic analysis of the STIM protein family was conducted after extensive database searching. Results from phylogeny and sequence analysis revealed early adaptation of the C-terminal divergent domains in Urochordata, before the expansion of STIMs in Vertebrata. STIMs were subsequently subjected to one round of gene duplication as early as in the Euteleostomi lineage in vertebrates, with a second round of fish-specific gene duplication. After duplication, STIM-1 and STIM-2 molecules appeared to have undergone purifying selection indicating strong evolutionary constraints within each group. Furthermore, sequence analysis of the EF-hand Ca(2+ binding domain and the SAM domain, together with functional divergence studies, identified critical regions/residues likely underlying functional changes, and provided evidence for the hypothesis that STIM-1 and STIM-2 might have developed distinct functional properties after duplication.

  15. Molecular evolution and functional divergence of the Ca(2+) sensor protein in store-operated Ca(2+) entry: stromal interaction molecule.

    Science.gov (United States)

    Cai, Xinjiang

    2007-07-11

    Receptor-mediated Ca(2+) signaling in many non-excitable cells initially induces Ca(2+) release from intracellular Ca(2+) stores, followed by Ca(2+) influx across the plasma membrane. Recent findings have suggested that stromal interaction molecules (STIMs) function as the Ca(2+) sensor to detect changes of Ca(2+) content in the intracellular Ca(2+) stores. Human STIMs and invertebrate STIM share several functionally important protein domains, but diverge significantly in the C-terminus. To better understand the evolutionary significance of STIM activity, phylogenetic analysis of the STIM protein family was conducted after extensive database searching. Results from phylogeny and sequence analysis revealed early adaptation of the C-terminal divergent domains in Urochordata, before the expansion of STIMs in Vertebrata. STIMs were subsequently subjected to one round of gene duplication as early as in the Euteleostomi lineage in vertebrates, with a second round of fish-specific gene duplication. After duplication, STIM-1 and STIM-2 molecules appeared to have undergone purifying selection indicating strong evolutionary constraints within each group. Furthermore, sequence analysis of the EF-hand Ca(2+) binding domain and the SAM domain, together with functional divergence studies, identified critical regions/residues likely underlying functional changes, and provided evidence for the hypothesis that STIM-1 and STIM-2 might have developed distinct functional properties after duplication.

  16. Synergistic effect of fluconazole and doxycycline against Candida albicans biofilms resulting from calcium fluctuation and downregulation of fluconazole-inducible efflux pump gene overexpression.

    Science.gov (United States)

    Gao, Yuan; Li, Hui; Liu, Shuyuan; Zhang, Xiang; Sun, Shujuan

    2014-07-01

    Candida albicans biofilms are intrinsically resistant to antimicrobial agents. Previous work demonstrated that the antifungal activity of fluconazole against C. albicans biofilms is notably enhanced by doxycycline. In order to explore the synergistic mechanism of fluconazole and doxycycline, we investigated the changes of efflux pump gene expression, intracellular calcium concentration and cell cycle distribution after drug intervention in this study. The expression levels of CDR1, CDR2 and MDR1 were determined by real-time PCR, and the results showed that fluconazole alone could stimulate the high expression of CDR1, CDR2 and MDR1, and the combination of doxycycline and fluconazole downregulated the gene overexpression induced by fluconazole. Intracellular calcium concentration was determined using Fluo-3/AM by observing the fluorescence with flow cytometry. A calcium fluctuation, which started 4 h and peaked 8 h after the treatment with fluconazole, was observed. The combined drugs also initiated a calcium fluctuation after 4 h treatment and showed a peak at 16 h, and the peak was higher than that stimulated by fluconazole alone. The cell cycle was measured using flow cytometry. Fluconazole alone and the combined drugs both induced a reduction in the percentages of S-phase cells and an elevation in the percentages of cells in the G2/M phase. The results of this research showed that the synergism of fluconazole and doxycycline against C. albicans biofilms is associated with blockade of the efflux pump genes CDR1, CDR2 and MDR1, and stimulation of high intracellular calcium concentration. The findings of this study are of great significance in the search for new antifungal mechanisms.

  17. Different Roles for Contracture and Calpain in Calcium Paradox-Induced Heart Injury

    Science.gov (United States)

    Zhang, Jian-Ying; Bi, Sheng-Hui; Xu, Ming; Jin, Zhen-Xiao; Yang, Yang; Jiang, Xiao-Fan; Zhou, Jing-Jun

    2012-01-01

    The Ca2+ paradox represents a good model to study Ca2+ overload injury in ischemic heart diseases. We and others have demonstrated that contracture and calpain are involved in the Ca2+ paradox-induced injury. This study aimed to elucidate their roles in this model. The Ca2+ paradox was elicited by perfusing isolated rat hearts with Ca2+-free KH media for 3 min or 5 min followed by 30 min of Ca2+ repletion. The LVDP was measured to reflect contractile function, and the LVEDP was measured to indicate contracture. TTC staining and the quantification of LDH release were used to define cell death. Calpain activity and troponin I release were measured after Ca2+ repletion. Ca2+ repletion of the once 3-min Ca2+ depleted hearts resulted in almost no viable tissues and the disappearance of contractile function. Compared to the effects of the calpain inhibitor MDL28170, KB-R7943, an inhibitor of the Na+/Ca2+ exchanger, reduced the LVEDP level to a greater extent, which was well correlated with improved contractile function recovery and tissue survival. The depletion of Ca2+ for 5 min had the same effects on injury as the 3-min Ca2+ depletion, except that the LVEDP in the 5-min Ca2+ depletion group was lower than the level in the 3-min Ca2+ depletion group. KB-R7943 failed to reduce the level of LVEDP, with no improvement in the LVDP recovery in the hearts subjected to the 5-min Ca2+ depletion treatment; however, KB-R7943 preserved its protective effects in surviving tissue. Both KB-R7943 and MDL28170 attenuated the Ca2+ repletion-induced increase in calpain activity in 3 min or 5 min Ca2+ depleted hearts. However, only KB-R7943 reduced the release of troponin I from the Ca2+ paradoxic heart. These results provide evidence suggesting that contracture is the main cause for contractile dysfunction, while activation of calpain mediates cell death in the Ca2+ paradox. PMID:23284963

  18. Different roles for contracture and calpain in calcium paradox-induced heart injury.

    Directory of Open Access Journals (Sweden)

    Jian-Ying Zhang

    Full Text Available The Ca(2+ paradox represents a good model to study Ca(2+ overload injury in ischemic heart diseases. We and others have demonstrated that contracture and calpain are involved in the Ca(2+ paradox-induced injury. This study aimed to elucidate their roles in this model. The Ca(2+ paradox was elicited by perfusing isolated rat hearts with Ca(2+-free KH media for 3 min or 5 min followed by 30 min of Ca(2+ repletion. The LVDP was measured to reflect contractile function, and the LVEDP was measured to indicate contracture. TTC staining and the quantification of LDH release were used to define cell death. Calpain activity and troponin I release were measured after Ca(2+ repletion. Ca(2+ repletion of the once 3-min Ca(2+ depleted hearts resulted in almost no viable tissues and the disappearance of contractile function. Compared to the effects of the calpain inhibitor MDL28170, KB-R7943, an inhibitor of the Na(+/Ca(2+ exchanger, reduced the LVEDP level to a greater extent, which was well correlated with improved contractile function recovery and tissue survival. The depletion of Ca(2+ for 5 min had the same effects on injury as the 3-min Ca(2+ depletion, except that the LVEDP in the 5-min Ca(2+ depletion group was lower than the level in the 3-min Ca(2+ depletion group. KB-R7943 failed to reduce the level of LVEDP, with no improvement in the LVDP recovery in the hearts subjected to the 5-min Ca(2+ depletion treatment; however, KB-R7943 preserved its protective effects in surviving tissue. Both KB-R7943 and MDL28170 attenuated the Ca(2+ repletion-induced increase in calpain activity in 3 min or 5 min Ca(2+ depleted hearts. However, only KB-R7943 reduced the release of troponin I from the Ca(2+ paradoxic heart. These results provide evidence suggesting that contracture is the main cause for contractile dysfunction, while activation of calpain mediates cell death in the Ca(2+ paradox.

  19. Membrane depolarization induces calcium-dependent secretion of tissue plasminogen activator.

    Science.gov (United States)

    Gualandris, A; Jones, T E; Strickland, S; Tsirka, S E

    1996-04-01

    Tissue plasminogen activator (tPA), a serine protease that converts inactive plasminogen to active plasmin, is produced in the rat and mouse hippocampus and participates in neuronal plasticity. To help define the role of tPA in the nervous system, we have analyzed the regulation of its expression in the neuronal cell line PC12. In control cultures, tPA activity is exclusively cell-associated, and no activity is measurable in the culture medium. When the cells are treated with depolarizing agents, such as KCI, tPA activity becomes detectable in the medium. The increased secreted tPA activity is not accompanied by an increase in tPA mRNA levels, and it is not blocked by protein synthesis inhibitors. In contrast, tPA release is abolished by Ca2+ channel blockers, suggesting that chemically induced membrane depolarization stimulates the secretion of preformed enzyme. Moreover, KCI has a similar effect in vivo when administered to the murine brain via an osmotic pump: tPA activity increases along the CA2-CA3 regions and dentate gyrus of the hippocampal formation. These results demonstrate a neuronal activity-dependent secretory mechanism that can rapidly increase the amount of tPA in neuronal tissue.

  20. Mitochondrial dysfunction induced by frataxin deficiency is associated with cellular senescence and abnormal calcium metabolism

    Directory of Open Access Journals (Sweden)

    Arantxa eBolinches-Amorós

    2014-05-01

    Full Text Available Friedreich ataxia is considered a neurodegenerative disorder involving both the peripheral and central nervous systems. Dorsal root ganglia (DRG are the major target tissue structures. This neuropathy is caused by mutations in the FXN gene that encodes frataxin. Here, we investigated the mitochondrial and cell consequences of frataxin depletion in a cellular model based on frataxin silencing in SH-SY5Y human neuroblastoma cells, a cell line that has been used widely as in vitro models for studies on neurological diseases. We showed that the reduction of frataxin induced mitochondrial dysfunction due to a bioenergetic deficit and abnormal Ca2+ homeostasis in the mitochondria that were associated with oxidative and endoplasmic reticulum stresses. The depletion of frataxin did not cause cell death but increased autophagy, which may have a cytoprotective effect against cellular insults such as oxidative stress. Frataxin silencing provoked slow cell growth associated with cellular senescence, as demonstrated by increased SA-βgal activity and cell cycle arrest at the G1 phase. We postulate that cellular senescence might be related to a hypoplastic defect in the DRG during neurodevelopment, as suggested by necropsy studies.

  1. Quantitative determination of calcium, magnesium, and zinc in fingernails by laser-induced breakdown spectroscopy.

    Science.gov (United States)

    Rusak, David A; Zeleniak, Ann E; Obuhosky, Jillian L; Holdren, Scott M; Noldy, Craig A

    2013-12-15

    Quantitative determination of Ca, Mg, and Zn in fingernails was performed with laser-induced breakdown spectroscopy. Two different methods of producing solid standards for calibration were explored - preparation of keratin pellets and deposition of aqueous solutions to filter papers. Measurements of the temperature and electron density of the plasma produced on keratin pellets, filter paper, and nails were performed, and it was determined that the standards prepared on filter paper gave plasma temperatures and electron densities closer to those observed on the nails. The ablation rate of the filter paper was also more similar to that of the nails. Using calibration curves produced using these filter paper standards, Ca, Mg, and Zn were determined in fingernails of 11 subjects. For comparison, the same samples were digested and atomic absorption was used to determine the same three elements. The differences in results are discussed in light of sample homogeneity and instrumental precision; the best agreement was obtained for determination of Zn. The work suggests that the filter paper method of standard preparation may be appropriate for LIBS analysis of other samples that give relatively low temperature, low electron density plasmas (i.e., polymers). © 2013 Elsevier B.V. All rights reserved.

  2. FATE1 antagonizes calcium- and drug-induced apoptosis by uncoupling ER and mitochondria.

    Science.gov (United States)

    Doghman-Bouguerra, Mabrouka; Granatiero, Veronica; Sbiera, Silviu; Sbiera, Iuliu; Lacas-Gervais, Sandra; Brau, Frédéric; Fassnacht, Martin; Rizzuto, Rosario; Lalli, Enzo

    2016-09-01

    Several stimuli induce programmed cell death by increasing Ca(2+) transfer from the endoplasmic reticulum (ER) to mitochondria. Perturbation of this process has a special relevance in pathologies as cancer and neurodegenerative disorders. Mitochondrial Ca(2+) uptake mainly takes place in correspondence of mitochondria-associated ER membranes (MAM), specialized contact sites between the two organelles. Here, we show the important role of FATE1, a cancer-testis antigen, in the regulation of ER-mitochondria distance and Ca(2+) uptake by mitochondria. FATE1 is localized at the interface between ER and mitochondria, fractionating into MAM FATE1 expression in adrenocortical carcinoma (ACC) cells under the control of the transcription factor SF-1 decreases ER-mitochondria contact and mitochondrial Ca(2+) uptake, while its knockdown has an opposite effect. FATE1 also decreases sensitivity to mitochondrial Ca(2+)-dependent pro-apoptotic stimuli and to the chemotherapeutic drug mitotane. In patients with ACC, FATE1 expression in their tumor is inversely correlated with their overall survival. These results show that the ER-mitochondria uncoupling activity of FATE1 is harnessed by cancer cells to escape apoptotic death and resist the action of chemotherapeutic drugs. © 2016 The Authors.

  3. Downregualtion of dynamin-related protein 1 attenuates glutamate-induced excitotoxicity via regulating mitochondrial function in a calcium dependent manner in HT22 cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Chi; Yuan, Xian-rui; Li, Hao-yu; Zhao, Zi-jin; Liao, Yi-wei; Wang, Xiang-yu; Su, Jun; Sang, Shu-shan; Liu, Qing, E-mail: xiangyaliuqing@163.com

    2014-01-03

    Highlights: •Downregulation of Drp-1 attenuates glutamate-induced excitotoxicity. •Downregulation of Drp-1 inhibits glutamate-induced apoptosis. •Downregulation of Drp-1 reduces glutamate-induced mitochondrial dysfunction. •Downregulation of Drp-1 preserves intracellular calcium homeostasis. -- Abstract: Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Dynamin related protein 1 (Drp-1), one of the GTPase family of proteins that regulate mitochondrial fission and fusion balance, is associated with apoptotic cell death in cancer and neurodegenerative diseases. Here we investigated the effect of downregulating Drp-1 on glutamate excitotoxicity-induced neuronal injury in HT22 cells. We found that downregulation of Drp-1 with specific small interfering RNA (siRNA) increased cell viability and inhibited lactate dehydrogenase (LDH) release after glutamate treatment. Downregulation of Drp-1 also inhibited an increase in the Bax/Bcl-2 ratio and cleavage of caspase-9 and caspase-3. Drp-1 siRNA transfection preserved the mitochondrial membrane potential (MMP), reduced cytochrome c release, enhanced ATP production, and partly prevented mitochondrial swelling. In addition, Drp-1 knockdown attenuated glutamate-induced increases of cytoplasmic and mitochondrial Ca{sup 2+}, and preserved the mitochondrial Ca{sup 2+} buffering capacity after excitotoxicity. Taken together, these results suggest that downregulation of Drp-1 protects HT22 cells against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the preservation of mitochondrial function through regulating intracellular calcium homeostasis.

  4. Exogenous calcium induces tolerance to atrazine stress in Pennisetum seedlings and promotes photosynthetic activity, antioxidant enzymes and psbA gene transcripts.

    Science.gov (United States)

    Erinle, Kehinde Olajide; Jiang, Zhao; Ma, Bingbing; Li, Jinmei; Chen, Yukun; Ur-Rehman, Khalil; Shahla, Andleeb; Zhang, Ying

    2016-10-01

    Calcium (Ca) has been reported to lessen oxidative damages in plants by upregulating the activities of antioxidant enzymes. However, atrazine mediated reactive oxygen species (ROS) reduction by Ca is limited. This study therefore investigated the effect of exogenously applied Ca on ROS, antioxidants activity and gene transcripts, the D1 protein (psbA gene), and chlorophyll contents in Pennisetum seedlings pre-treated with atrazine. Atrazine toxicity increased ROS production and enzyme activities (ascorbate peroxidase APX, peroxidase POD, Superoxide dismutase SOD, glutathione-S-transferase GST); but decreased antioxidants (APX, POD, and Cu/Zn SOD) and psbA gene transcripts. Atrazine also decreased the chlorophyll contents, but increased chlorophyll (a/b) ratio. Contrarily, Ca application to atrazine pre-treated seedlings lowered the harmful effects of atrazine by reducing ROS levels, but enhancing the accumulation of total chlorophyll contents. Ca-protected seedlings in the presence of atrazine manifested reduced APX and POD activity, whereas SOD and GST activity was further increased with Ca application. Antioxidant gene transcripts that were down-regulated by atrazine toxicity were up-regulated with the application of Ca. Calcium application also resulted in up-regulation of the D1 protein. In conclusion, ability of calcium to reverse atrazine-induced oxidative damage and calcium regulatory role on GST in Pennisetum was presented. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Disruption of Calcium Signaling in Fibroblasts and Attenuation of Bleomycin-Induced Fibrosis by Nifedipine.

    Science.gov (United States)

    Mukherjee, Subhendu; Ayaub, Ehab A; Murphy, James; Lu, Chao; Kolb, Martin; Ask, Kjetil; Janssen, Luke J

    2015-10-01

    Fibrotic lung disease afflicts millions of people; the central problem is progressive lung destruction and remodeling. We have shown that external growth factors regulate fibroblast function not only through canonical signaling pathways but also through propagation of periodic oscillations in Ca(2+). In this study, we characterized the pharmacological sensitivity of the Ca(2+)oscillations and determined whether a blocker of those oscillations can prevent the progression of fibrosis in vivo. We found Ca(2+) oscillations evoked by exogenously applied transforming growth factor β in normal human fibroblasts were substantially reduced by 1 μM nifedipine or 1 μM verapamil (both L-type blockers), by 2.7 μM mibefradil (a mixed L-/T-type blocker), by 40 μM NiCl2 (selective at this concentration against T-type current), by 30 mM KCl (which partially depolarizes the membrane and thereby fully inactivates T-type current but leaves L-type current intact), or by 1 mM NiCl2 (blocks both L- and T-type currents). In our in vivo study in mice, nifedipine prevented bleomycin-induced fibrotic changes (increased lung stiffness, overexpression of smooth muscle actin, increased extracellular matrix deposition, and increased soluble collagen and hydroxyproline content). Nifedipine had little or no effect on lung inflammation, suggesting its protective effect on lung fibrosis was not due to an antiinflammatory effect but rather was due to altering the profibrotic response to bleomycin. Collectively, these data show that nifedipine disrupts Ca(2+) oscillations in fibroblasts and prevents the impairment of lung function in the bleomycin model of pulmonary fibrosis. Our results provide compelling proof-of-principle that interfering with Ca(2+) signaling may be beneficial against pulmonary fibrosis.

  6. The effects of calcium channel blockade on agouti-induced obesity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Han; Moustaid, N.; Zemel, M.B. [Univ. of Tennessee, Knoxville, TN (United States)] [and others

    1996-12-01

    We have previously observed that obese viable yellow (A{sup vy}/a) mice exhibit increased intracellular Ca{sup 2+} ([Ca{sup 2+}]i) and fatty acid synthase (FAS) gene expression; further, recombinant agouti protein increases in cultured adipocytes and these effects are inhibited by Ca{sup 2+} channel blockade. Accordingly, we determined the effect of Ca{sup 2+} channel blockade (nifedipine for 4 wk) on FAS and obesity in transgenic mice expressing the agouti gene in a ubiquitous manner. The transgenic mice initially were significantly heavier (30.5 {+-} 0.6 vs. 27.3 {+-} 0.3 g; P<0.001) and exhibited a 0.81{degrees}C lower initial core temperature (P<0.0005), an approximately twofold increase in fat pad weights (P=0.002), a sevenfold increase in adipose FAS activity (P=0.009), and a twofold increase in plasma insulin level (P<0.05) compared to control mice. Nifedipine treatment resulted in an 18% decrease in fat pad weights (P<0.007) and a 74% decrease in adipose FAS activity (P=0.03), normalized circulating insulin levels and insulin sensitivity (P,0.05), and transiently elevated core temperature in the transgenic mice, but was without effect in the control mice. These data suggest that agouti regulates FAS, fat storage, and possibly thermogenesis, at least partially, via a [Ca{sup 2+}]{sub i}-dependent mechanism, and that Ca{sup 2+} channel blockade may partially attenuate agouti-induced obesity. 42 refs., 4 figs., 1 tab.

  7. Parathyroid hormone-related protein overexpression protects goat mammary gland epithelial cells from calcium-sensing receptor activation-induced apoptosis.

    Science.gov (United States)

    Li, Hui; Sun, Yongsen; Zheng, Huiling; Li, Lihui; Yu, Qian; Yao, Xiaotong

    2015-01-01

    Normal mammary gland epithelial cells and breast cancer cells express the calcium-sensing receptor (CaSR), which is the master regulator of systemic calcium metabolism. During lactation, activation of the CaSR in mammary epithelial cells downregulates parathyroid hormone-related protein (PTHrP) levels in milk and in the circulation, and increases calcium transport into milk. However, very little information is available on the role of CaSR in goat mammary gland epithelial cells (GMECs) apoptosis. In this investigation, the full-length cDNA of CaSR from Xinong Saanen dairy goats was cloned, which contains an open-reading frame of 3,258 bp encoding 1,085 amino acids with a predicted molecular weight of 121.0 kDa and an isoelectric point of 5.65. The amino acid sequence is highly homologous with sheep, and the goat CaSR gene is mapped to chromosome 1. Quantitative real-time PCR suggested that CaSR was predominantly expressed in the heart, kidney and mammary gland. Then, we found the stimulation of CaSR with its activator gadolinium chloride (GdCl3) contributed to increase CaSR mRNA levels in GMECs and simultaneously promoted cell apoptosis, and these effects were abrogated partially by NPS2390 which is an inhibitor of CaSR. We also demonstrated that Ca(2+) increased CaSR mRNA levels and induced GMECs apoptosis and restrained cell proliferation. In contrast, PTHrP overexpression protected GMECs from calcium-induced apoptosis, and promoted cell proliferation. In conclusion, these results suggest that PTHrP overexpression protects GMECs from CaSR activation-induced apoptosis.

  8. Differentiation inducement of calcium phosphate / calcium silicate / bismutite cement to dental pulp cells in vitro%新型复合盖髓材料对人牙髓细胞分化的体外诱导效果

    Institute of Scient