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Sample records for extracellular calcium increases

  1. Extracellular calcium sensing and extracellular calcium signaling

    NASA Technical Reports Server (NTRS)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)

    2001-01-01

    , localized changes in Ca(o)(2+) within the ECF can originate from several mechanisms, including fluxes of calcium ions into or out of cellular or extracellular stores or across epithelium that absorb or secrete Ca(2+). In any event, the CaR and other receptors/sensors for Ca(o)(2+) and probably for other extracellular ions represent versatile regulators of numerous cellular functions and may serve as important therapeutic targets.

  2. Increased extracellular pressure stimulates tumor proliferation by a mechanosensitive calcium channel and PKC-β.

    PubMed

    Basson, Marc D; Zeng, Bixi; Downey, Christina; Sirivelu, Madhu P; Tepe, Jetze J

    2015-02-01

    Large tumors exhibit high interstitial pressure heightened by growth against the constraining stroma. Such pressures could stimulate tumor proliferation via a mechanosensitive ion channel. We studied the effects of 0-80 mmHg increased extracellular pressure for 24 h on proliferation of SW620, Caco-2, and CT-26 colon; MCF-7 breast; and MLL and PC3 prostate cancer cells, and delineated its mechanism in SW620 cells with specific inhibitors and siRNA. Finally, we compared NF-kB, phospho-IkB and cyclin D1 immunoreactivity in the high pressure centers and low pressure peripheries of human tumors. Pressure-stimulated proliferation in all cells. Pressure-driven SW620 proliferation required calcium influx via the T-type Ca(2+) channel Cav3.3, which stimulated PKC-β to invoke the IKK-IkB-NF-kB pathway to increase proliferation and S-phase fraction. The mitotic index and immunoreactivity of NF-kB, phospho-IkB, and cyclin D1 in the center of 28 large human colon, lung, and head and neck tumors exceeded that in tumor peripheries. Extracellular pressure increases [Ca(2+)]i via Cav3.3, driving a PKC-β- IKK- IkB-NF-kB pathway that stimulates cancer cell proliferation. Rapid proliferation in large stiff tumors may increase intratumoral pressure, activating this pathway to stimulate further proliferation in a feedback cycle that potentiates tumor growth. Targeting this pathway may inhibit proliferation in large unresectable tumors.

  3. Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin.

    PubMed

    Gonyar, Laura A; Gray, Mary C; Christianson, Gregory J; Mehrad, Borna; Hewlett, Erik L

    2017-06-01

    Pertussis (whooping cough), caused by Bordetella pertussis, is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species. Copyright © 2017 American Society for Microbiology.

  4. Role of membrane depolarization and extracellular calcium in increased complement receptor expression during neutrophil (PMN) activation

    SciTech Connect

    Berger, M.; Wetzler, E.; Birx, D.L.

    1986-03-05

    During PMN activation the surface expression of receptors (R) for C3b and C3bi increases rapidly. This is necessary for optimal cell adhesion, migration, and phagocytosis. Following stimulation with fMLP or LTB-4, the increased expression of C3bR depends only on the Ca/sup + +/ released from intracellular stores and is not inhibited by 5mM EDTA, while the increase in C3biR also requires extracellular Ca/sup + +/. CR expression also increases when the PMN are depolarized with 140 mM K/sup +/, but with this stimulus, EDTA inhibits C3bR by 67% and C3biR 100%, suggesting that intracellular Ca/sup + +/ stores may not be released. Pertussis toxin caused dose-dependent inhibition of both CR responses to fMLP and also inhibited the increases in both CR induced by K/sup +/. Membrane depolarization (monitored by di-O-C5 fluorescence) due to fMLP was similarly inhibited by toxin but the depolarization due to K/sup +/ was not. The dose of phorbol myristate acetate that maximally increased CR expression, 0.1 ng/ml, did not depolarize the membrane. These results suggest that membrane depolarization is neither necessary nor sufficient for increased CR expression. A Ca/sup + +/ and GTP binding protein-dependent enzyme such as phospholipase C is necessary to the amplify initial signals generated either by release of Ca/sup + +/ stores or by opening voltage dependent Ca/sup + +/ channels following membrane depolarization.

  5. Hypertrophy and transcriptional regulation induced in myogenic cell line L6-C5 by an increase of extracellular calcium.

    PubMed

    De Arcangelis, V; Coletti, D; Canato, M; Molinaro, M; Adamo, S; Reggiani, C; Naro, F

    2005-03-01

    Calcium plays a pivotal role in the establishment of the differentiated phenotype in myogenic cells but the involved molecular mechanisms are still matter of debate. Here we studied the effects of exposing L6-C5 myogenic cells to high extracellular Ca2+ concentration ([Ca2+]o), which induces an increase of intracellular calcium ([Ca2+]i) without involving Ca2+ release from the intracellular stores but exclusively due to plasma membrane influx (Naro et al., 2003). Exposure of L6-C5 cells to [Ca2+]o up to 20 mM for 30 min, before shifting them into a differentiative medium, induced the appearance of multinucleated, myosin-positive myotubes, much larger than in control cells with an increased protein/DNA ratio. These large myotubes showed nuclear accumulation of the hypertrophy marker GATA-2. The hypertrophic growth of these cells was blocked by cyclosporin A (CsA), FK506, or overexpression of a calcineurin-dominant negative protein, suggesting the involvement in this process of the Ca2+ responsive phosphatase calcineurin. Furthermore, transient exposure of L6-C5 cells to high [Ca2+]o increased the expression of luciferase reporter driven by myoglobin (Mb) and beta-MHC promoters but not IIB-MHC and MCK promoters. Luciferase transcription driven by CK promoter was, instead, enhanced by mobilizing Ca2+ from the intracellular stores. These data indicate that a transient increase of [Ca2+]i due to plasma-membrane influx is sufficient to induce a hypertrophic phenotype and an increased expression of slow-fiber genes but not fast-fiber genes. 2004 Wiley-Liss, Inc.

  6. [Pharmacology of the extracellular calcium ion receptor].

    PubMed

    Ruat, Martial

    2003-01-01

    The calcium sensing receptor (CaSR) belongs to family 3 of G-protein coupled receptors. The CaSR, expressed at the surface of the parathyroid cells, controls parathyroid hormone (PTH) secretion and is the main regulator of calcium homeostasis. Its activity is regulated by small changes in the physiological concentrations of calcium and magnesium ions present in the serum and extracellular fluids, leading to the stimulation of the phospholipases C and A2. Molecules that potentiate the effect of extracellular calcium are called calcimimetics. They reduce the PTH level in vivo and have been proposed to be of therapeutic benefit for the treatment of both primary and secondary hyperparathyroidism. The blocking of CaSR by a calcilytic molecule results in the increase in serum PTH and might be of interest in the treatment of osteoporosis. The CaSR is also expressed in the thyroid, kidney, bone and in neuronal and glial cell populations, where it should be involved in the complex responses associated with calcium and magnesium ions present in the extracellular fluids.

  7. Elevated extracellular calcium increases expression of bone morphogenetic protein-2 gene via a calcium channel and ERK pathway in human dental pulp cells

    SciTech Connect

    Tada, Hiroyuki; Nemoto, Eiji; Kanaya, Sousuke; Hamaji, Nozomu; Sato, Hisae; Shimauchi, Hidetoshi

    2010-04-16

    Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca{sup 2+}Ca{sub o}{sup 2+} has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca{sub o}{sup 2+} signaling in odontogenesis remains unclear. We found that elevated Ca{sub o}{sup 2+} increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca{sup 2+} increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca{sup 2+} channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca{sup 2+}, suggesting that the Ca{sup 2+} influx from Ca{sup 2+} channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca{sup 2+}-sensing receptors (CaSR) and only respond slightly to other cations such as Sr{sup 2+} and spermine, suggesting that dental pulp cells respond to Ca{sub o}{sup 2+} to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca{sub o}{sup 2+} among cations.

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

    PubMed

    Hofer, Aldebaran M

    2005-03-01

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

  9. Influence of extracellular calcium on allyl alcohol-induced hepatotoxicity.

    PubMed

    Strubelt, O; Younes, M; Pentz, R

    1986-07-01

    The role of calcium in allyl alcohol-induced hepatotoxicity was investigated in the isolated haemoglobin-free perfused rat liver. At a Ca++ concentration of 2.5 mmol/l in the perfusate, allyl alcohol (initial concentration 1.17 mmol/l) produced an enhanced release of GPT and SDH from the liver, an increase in the lactate/pyruvate ratio of the perfusate, a decrease in hepatic oxygen consumption and an increase of both hepatic calcium and malondialdehyde content. In the absence of Ca++ in the perfusate, no hepatic calcium accumulation occurred with allyl alcohol, but all other signs of hepatic damage were as severe as with 2.5 mmol/l Ca++. On the other hand, high extracellular Ca++ (5 mmol/l) alone led to a threefold increase of liver calcium but produced only marginal hepatotoxicity and only slightly enhanced the hepatotoxic effects of allyl alcohol. The concentrations of allyl alcohol in the perfusate were not altered at different Ca++ concentrations. In conclusion, the primary allyl alcohol-induced hepatotoxic injury does not appear to depend upon an influx of extracellular calcium.

  10. Grape seed extract triggers apoptosis in Caco-2 human colon cancer cells through reactive oxygen species and calcium increase: extracellular signal-regulated kinase involvement.

    PubMed

    Dinicola, Simona; Mariggiò, Maria Addolorata; Morabito, Caterina; Guarnieri, Simone; Cucina, Alessandra; Pasqualato, Alessia; D'Anselmi, Fabrizio; Proietti, Sara; Coluccia, Pierpaolo; Bizzarri, Mariano

    2013-09-14

    Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca2+ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca2+ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca2+ influx. ROS and Ca2+ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca2+ increase, eventually leading to apoptosis in cancer cells.

  11. The influence of extracellular and intracellular calcium on the secretion of renin

    PubMed Central

    Atchison, Douglas K.; Beierwaltes, William H.

    2012-01-01

    Changes in plasma, extracellular and intracellular calcium can affect renin secretion from the renal juxtaglomerular (JG) cells. Elevated intracellular calcium directly inhibits renin release from JG cells by decreasing the dominant second messenger intracellular cyclic adenosine monophosphate (cAMP) via actions on calcium-inhibitable adenylyl cyclases and calcium-activated phosphodiesterases. Increased extracellular calcium also directly inhibits renin release by stimulating the calcium-sensing receptor (CaSR) on JG cells, resulting in parallel changes in the intracellular environment and decreasing intracellular cAMP. In vivo, acutely elevated plasma calcium inhibits plasma renin activity (PRA) via parathyroid hormone-mediated elevations in renal cortical interstitial calcium that stimulate the JG cell CaSR. However, chronically elevated plasma calcium or CaSR activation may actually stimulate PRA. This elevation in PRA may be a compensatory mechanism resulting from calcium-mediated polyuria. Thus, changing the extracellular calcium in vitro or in vivo results in inversely related acute changes in cAMP, and therefore renin release, but chronic changes in calcium may result in more complex interactions dependent upon the duration of changes and the integration of the body’s response to these changes. PMID:22538344

  12. Effects of Extracellular Calcium on Cell Membrane Resealing during Sonoporation

    NASA Astrophysics Data System (ADS)

    Zhou, Yun; Cui, Jianmin; Deng, Cheri X.

    2006-05-01

    Sonoporation has been exploited as a novel strategy for intracellular drug and gene delivery. In sonoporation, ultrasound application generates transient pores or openings in the cell membrane that allow entry of extracellular agents normally not permeable to the cell membrane. In order to improve the sonoporation outcome, we seek to obtain improved understanding of the sonoporation mechanism and investigate the factors affecting sonoporation process. We established a voltage clamp technique for real time measurement of sonoporation at single cell level using Xenopus oocytes as a model system. As both cell survival and intracellular delivery efficiency of drug or genes depend on the sonoporation dynamic process, and Calcium plays important roles in cellular processes, we focus on studying of the effect of extracellular Calcium concentration on the formation, extension, and resealing of membrane pores in sonoporation. We obtained experimental results demonstrating that the cell membrane reseals in the order of seconds in the presence of physiological level of extracellular [Ca]. We measured the resealing as function of extracellular [Ca] (0-1.8mM) and observed that the resealing rate decreases as extracellular [Ca] decreases from normal physiological level. No resealing was demonstrated when 1mM EGTA was added in the extracellular medium to chelate the [Ca] extracellularly. Our experimental findings suggest that extracellular Calcium plays an important role in controlling membrane resealing in sonoporation and thus the sonoporation outcome such as cell survival and delivery efficiency.

  13. Calcium-Sensing Receptor: A Key Target for Extracellular Calcium Signaling in Neurons

    PubMed Central

    Jones, Brian L.; Smith, Stephen M.

    2016-01-01

    Though both clinicians and scientists have long recognized the influence of extracellular calcium on the function of muscle and nervous tissue, recent insights reveal that the mechanisms allowing changes in extracellular calcium to alter cellular excitability have been incompletely understood. For many years the effects of calcium on neuronal signaling were explained only in terms of calcium entry through voltage-gated calcium channels and biophysical charge screening. More recently however, it has been recognized that the calcium-sensing receptor is prevalent in the nervous system and regulates synaptic transmission and neuronal activity via multiple signaling pathways. Here we review the multiplicity of mechanisms by which changes in extracellular calcium alter neuronal signaling and propose that multiple mechanisms are required to describe the full range of experimental observations. PMID:27065884

  14. Extracellular calcium elicits a chemokinetic response from monocytes in vitro and in vivo

    NASA Technical Reports Server (NTRS)

    Olszak, I. T.; Poznansky, M. C.; Evans, R. H.; Olson, D.; Kos, C.; Pollak, M. R.; Brown, E. M.; Scadden, D. T.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    Recruitment of macrophages to sites of cell death is critical for induction of an immunologic response. Calcium concentrations in extracellular fluids vary markedly, and are particularly high at sites of injury or infection. We hypothesized that extracellular calcium participates in modulating the immune response, perhaps acting via the seven-transmembrane calcium-sensing receptor (CaR) on mature monocytes/macrophages. We observed a dose-dependent increase in monocyte chemotaxis in response to extracellular calcium or the selective allosteric CaR activator NPS R-467. In contrast, monocytes derived from mice deficient in CaR lacked the normal chemotactic response to a calcium gradient. Notably, CaR activation of monocytes bearing the receptor synergistically augmented the transmigration response of monocytes to the chemokine MCP-1 in association with increased cell-surface expression of its cognate receptor, CCR2. Conversely, stimulation of monocytes with MCP-1 or SDF-1alpha reciprocally increased CaR expression, suggesting a dual-enhancing interaction of Ca(2+) with chemokines in recruiting inflammatory cells. Subcutaneous administration in mice of Ca(2+), MCP-1, or (more potently) the combination of Ca(2+) and MCP-1, elicited an inflammatory infiltrate consisting of monocytes/macrophages. Thus extracellular calcium functions as an ionic chemokinetic agent capable of modulating the innate immune response in vivo and in vitro by direct and indirect actions on monocytic cells. Calcium deposition may be both consequence and cause of chronic inflammatory changes at sites of injury, infection, and atherosclerosis.

  15. Extracellular calcium elicits a chemokinetic response from monocytes in vitro and in vivo

    NASA Technical Reports Server (NTRS)

    Olszak, I. T.; Poznansky, M. C.; Evans, R. H.; Olson, D.; Kos, C.; Pollak, M. R.; Brown, E. M.; Scadden, D. T.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    Recruitment of macrophages to sites of cell death is critical for induction of an immunologic response. Calcium concentrations in extracellular fluids vary markedly, and are particularly high at sites of injury or infection. We hypothesized that extracellular calcium participates in modulating the immune response, perhaps acting via the seven-transmembrane calcium-sensing receptor (CaR) on mature monocytes/macrophages. We observed a dose-dependent increase in monocyte chemotaxis in response to extracellular calcium or the selective allosteric CaR activator NPS R-467. In contrast, monocytes derived from mice deficient in CaR lacked the normal chemotactic response to a calcium gradient. Notably, CaR activation of monocytes bearing the receptor synergistically augmented the transmigration response of monocytes to the chemokine MCP-1 in association with increased cell-surface expression of its cognate receptor, CCR2. Conversely, stimulation of monocytes with MCP-1 or SDF-1alpha reciprocally increased CaR expression, suggesting a dual-enhancing interaction of Ca(2+) with chemokines in recruiting inflammatory cells. Subcutaneous administration in mice of Ca(2+), MCP-1, or (more potently) the combination of Ca(2+) and MCP-1, elicited an inflammatory infiltrate consisting of monocytes/macrophages. Thus extracellular calcium functions as an ionic chemokinetic agent capable of modulating the innate immune response in vivo and in vitro by direct and indirect actions on monocytic cells. Calcium deposition may be both consequence and cause of chronic inflammatory changes at sites of injury, infection, and atherosclerosis.

  16. CADASIL patient with extracellular calcium deposits.

    PubMed

    Lewandowska, Eliza; Wierzba-Bobrowicz, Teresa; Buczek, Julia; Gromadzka, Grażyna; Dziewulska, Dorota

    2013-01-01

    We report the case of a 57-year-old male patient with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) diagnosed on the basis of ultrastructural and genetic examinations. Ultrastructurally, granular osmiophilic material (GOM) deposits, degeneration and loss of vascular smooth muscle cells (VSMC) and pericytes in small arterial and capillary vessels from skin-muscle biopsy typical of CADASIL were visible. Degeneration of pericytes and endothelial cells were often pronounced, which resulted in a complete disappearance of mural cells and extremely severe thickening of the basement membrane. Degenerative changes in blood vessels, especially evident in skeletal muscle arterioles, also included significant vacuolization of VSMC, misshapen nuclei both in vessel wall cells and skeletal muscle fibres, and deposits of a hyaline material and calcium in the vessel wall. Abundant calcium deposits were located in the vascular basement membrane and exhibited laminar morphology with abnormally arranged light and dark bands. In the basement membrane of the most severely affected microvessels, only clusters of calcium deposits and remnants of the mural cells were observed. Laminar calcifications were also observed within the basement membrane surrounding skeletal muscle fibres. Such abundant calcium deposits in CADASIL have not as yet been described. Morphological findings, described in this report, expand the spectrum of histopathological changes in this genetically determined angiopathy.

  17. Recent advances in understanding the extracellular calcium-sensing receptor

    PubMed Central

    Colella, Matilde; Gerbino, Andrea; Hofer, Aldebaran M.; Curci, Silvana

    2016-01-01

    The extracellular calcium-sensing receptor (CaR), a ubiquitous class C G-protein-coupled receptor (GPCR), is responsible for the control of calcium homeostasis in body fluids. It integrates information about external Ca 2+ and a surfeit of other endogenous ligands into multiple intracellular signals, but how is this achieved? This review will focus on some of the exciting concepts in CaR signaling and pharmacology that have emerged in the last few years. PMID:27803801

  18. Chondrocyte death in mechanically injured articular cartilage--the influence of extracellular calcium.

    PubMed

    Amin, Anish K; Huntley, James S; Bush, Peter G; Simpson, A Hamish R W; Hall, Andrew C

    2009-06-01

    Calcium is thought to be an important regulator of chondrocyte death associated with articular cartilage injury. Our objective was to determine the influence of extracellular calcium on chondrocyte death following mechanical injury. Using a surgically relevant model of sharp mechanical injury (with a scalpel) and confocal laser scanning microscopy (CLSM), in situ chondrocyte death was quantified within the full thickness of articular cartilage as a function of medium calcium concentration and time (2.5 h and 7 days). Exposure of articular cartilage to calcium-free media (approximately 0 mM) significantly reduced superficial zone chondrocyte death after mechanical injury compared with exposure to calcium-rich media (2-20 mM, ANOVA at 2.5 h, p = 0.002). In calcium-rich media, although the extent of chondrocyte death increased with increasing medium calcium concentration, cell death remained localized to the superficial zone of articular cartilage over 7 days (ANOVA, p < 0.05). However, in calcium-free media, there was an increase in chondrocyte death within deeper zones of articular cartilage over 7 days. The early (within hours) chondroprotective effect in calcium-free media suggests that the use of joint irrigation solutions without added calcium may decrease chondrocyte death from mechanical injury during articular surgery. The delayed (within days) increase in chondrocyte death in calcium-free media supports the use of calcium supplementation in media used during cartilage culture for tissue engineering or transplantation.

  19. Extracellular calcium and cholinergic stimulation of isolated canine parietal cells.

    PubMed Central

    Soll, A H

    1981-01-01

    The role of calcium gating in cholinergic stimulation of the function of parietal cells was studied using cells isolated from canine fundic mucosa by treatment with collagenase and EDTA and enriched by velocity separation in an elutriator rotor. Monitoring the accumulation of [14C[ aminopyrine as an index of parietal cell response, stimulation by carbachol, but not by histamine, was highly dependent upon the concentration of extracellular calcium. Incubation of parietal cells in 0-.1 mM calcium, rather than the usual 1.8 mM concentration, reduced the response to 100 microM carbachol by 92 +/- 2%, whereas histamine stimulation was impaired by 28 +/- 5%. A similar reduction in extracellular calcium suppressed the response to gastrin (100 nM) by 67 +/- 7%. The impairment of cholinergic stimulation found at low extracellular calcium concentrations was rapidly reversed with the readdition of calcium. Lanthanum, which blocks calcium movement across membranes, caused a similar pattern of effects on secretagogue stimulation of aminopyrine accumulation, with 100 microM lanthanum suppressing carbachol stimulation by 83 +/- 2%. This concentration of lanthanum suppressed gastrin stimulation by 40 +/- 7% and histamine stimulation by only 12 +/- 9%. Carbachol, but not histamine nor gastrin, stimulated 45Ca++ uptake. The magnitude of carbachol-stimulated calcium uptake correlated with the parietal cell content of the fractions examined (r = 0.88), and was dose responsive over carbachol concentrations from 1 microM to 1 mM. Atropine (100 nM) caused surmountable inhibition, and these effects of carbachol and atropine on calcium uptake correlated with their effects on oxygen consumption (r = 0.93) and [14C]-aminopyrine accumulation (r = 0.90). Cells preloaded with 45Ca++ lost cellular calcium in a time-dependent fashion; however, this rate of egress was not accelerated by treatment with histamine, gastrin, or carbachol, thus failing to implicate mobilization of intracellular calcium

  20. Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation.

    PubMed

    Quist, A P; Rhee, S K; Lin, H; Lal, R

    2000-03-06

    Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to increase in the volume of cells expressing gap-junctional proteins (connexins). Volume change did not occur in cells that were not expressing connexins. However, after the transient or stable transfection of connexin43, volume change did occur. The volume increase was accompanied by cytochalasin D-sensitive higher cell stiffness, which helped maintain cell integrity. These cellular physical changes were prevented by gap-junctional blockers, oleamide and beta-glycyrrhetinic acid, or were reversed by returning extracellular calcium to the normal level. We conclude that nongap-junctional hemichannels regulate cell volume in response to the change in extracellular physiological calcium in an otherwise isosmotic situation.

  1. The involvement of extracellular calcium in hypoxic injury to the isolated rat liver.

    PubMed

    Strubelt, O; Younes, M

    1988-09-01

    Isolated perfused livers from fasted rats were subjected to 30 min of hypoxia followed by 60 min of reoxygenation. At a calcium concentration of 1.25 mmol/l in the perfusate, hypoxia induced injury as evidenced by a marked release of GPT and SDH into the perfusate and by an accumulation of calcium in the livers. Omission of calcium from the perfusate attenuated hypoxia-induced enzyme release by about 50% and prevented the increase of hepatic calcium completely. A complete protection of the liver against hypoxic injury was attained in the absence of calcium when Na2 EDTA was added. An influx of calcium from the extracellular to the intracellular fluid seems to be involved in but is not the sole cause of hypoxia-induced hepatic injury.

  2. The effects of extracellular calcium on prairie dog gallbladder ion transport.

    PubMed

    Cates, J A; Saunders, K D; Abedin, M Z; Roslyn, J J

    1992-12-01

    Recent studies suggest that experimentally induced gallstone formation is associated with altered gallbladder absorptive function. Moreover, elevated biliary levels of calcium have been implicated in the pathogenesis of cholesterol gallstones. Nonetheless, the relationship between gallbladder ion transport and biliary calcium remains obscure. We tested the hypothesis that extracellular calcium modulates gallbladder ion transport. Prairie dog gallbladders were mounted in an Ussing chamber, and short-circuit current (Isc), transepithelial potential difference (Vms), and tissue resistance (Rt) were measured. Tissues were randomly exposed to physiologic salt solutions containing the following concentrations of calcium: 0.01, 1.3, 5, and 10 mmol/L. Exposure of gallbladder epithelium to increasing calcium concentrations resulted in concomitant increases in Isc and Vms (p < 0.001), without altering Rt. Regression analysis demonstrated a curvilinear correlation between calcium and Isc (Y = 167 + 22.5x - 1.4 x 26; p < 0.001). We conclude that extracellular calcium may be a modulator of gallbladder ion transport.

  3. Effects of extracellular calcium on calcium transport during hyperthermia of tumor cells.

    PubMed

    Anghileri, L J; Marcha, C; Crone-Escanyé, M C; Robert, J

    1985-08-01

    The effects of different concentrations of extracellular ion calcium on the transport of calcium by tumor cells have been studied by means of the uptake of radiocalcium. Tumor cells incubated at 45 degrees C take up 4-10 times the amount of radioactivity incorporated by cells incubated at 37 degrees C. The difference is still greater (up to 100 times) for the intracellular incorporation as assessed by elimination of the membrane-bound calcium by EGTA treatment. The possible mechanisms involved in this differential behavior are discussed.

  4. Extracellular calcium transients and action potential configuration changes related to post-stimulatory potentiation in rabbit atrium.

    PubMed

    Hilgemann, D W

    1986-05-01

    Extracellular calcium transients were monitored with 2 mM tetramethylmurexide at low calcium (250 microM total, 130 microM free), and action potentials were monitored together with developed tension at normal calcium (1.3 mM) during the production and decay of post-stimulatory potentiation in rabbit left atrial strips. At normal calcium, the contractile potentiation produced by a brief burst of 4 Hz stimulation is lost in three to five post-stimulatory excitations, which correlate with a negative staircase of the late action potential. At low calcium, stimulation at 4 Hz for 3-8 s results in a net extracellular calcium depletion of 5-15 microM. At the subsequent potentiated contraction (1-45 s rest), total extracellular calcium increases by 4-8 microM. The contractile response at a second excitation is greatly suppressed and results in little or no further calcium shift; the sequence can be repeated immediately thereafter. Reducing external sodium to 60 mM (sucrose replacement) enhances post-rest contractions, suppresses the late action potential, nearly eliminates loss of contractility and net calcium efflux at post-rest excitations, and markedly reduces extracellular calcium depletion during rapid stimulation. 4-Aminopyridine (1 mM) markedly suppresses the rapid early repolarization of this preparation at post-rest excitations and the loss of contractility at post-rest stimulation from the rested state; during a post-stimulatory potentiation sequence at low calcium, replenishment of extracellular calcium takes several post-stimulatory excitations. Ryanodine (10 nM to 5 microM) abolishes the post-stimulatory contraction at rest periods of greater than 5 s. If the initial repolarization is rapid, ryanodine suppresses the late action potential, calcium efflux during quiescence is greatly accelerated, and subsequent excitations do not result in an accumulation of extracellular calcium. A positive staircase of the early action potential correlates with the magnitude

  5. Calcium diffusion enhanced after cleavage of negatively charged components of brain extracellular matrix by chondroitinase ABC

    PubMed Central

    Hrabětová, Sabina; Masri, Daniel; Tao, Lian; Xiao, Fanrong; Nicholson, Charles

    2009-01-01

    The concentration of extracellular calcium plays a critical role in synaptic transmission and neuronal excitability as well as other physiological processes. The time course and extent of local fluctuations in the concentration of this ion largely depend on its effective diffusion coefficient (D*) and it has been speculated that fixed negative charges on chondroitin sulphate proteoglycans (CSPGs) and other components of the extracellular matrix may influence calcium diffusion because it is a divalent cation. In this study we used ion-selective microelectrodes combined with pressure ejection or iontophoresis of ions from a micropipette to quantify diffusion characteristics of neocortex and hippocampus in rat brain slices. We show that D* for calcium is less than the value predicted from the behaviour of the monovalent cation tetramethylammonium (TMA), a commonly used diffusion probe, but D* for calcium increases in both brain regions after the slices are treated with chondroitinase ABC, an enzyme that predominantly cleaves chondroitin sulphate glycans. These results suggest that CSPGs do play a role in determining the local diffusion properties of calcium in brain tissue, most likely through electrostatic interactions mediating rapid equilibrium binding. In contrast, chondroitinase ABC does not affect either the TMA diffusion or the extracellular volume fraction, indicating that the enzyme does not alter the structure of the extracellular space and that the diffusion of small monovalent cations is not affected by CSPGs in the normal brain ionic milieu. Both calcium and CSPGs are known to have many distinct roles in brain physiology, including brain repair, and our study suggests they may be functionally coupled through calcium diffusion properties. PMID:19546165

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

    PubMed

    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. Extracellular calcium sensing in rat aortic vascular smooth muscle cells

    SciTech Connect

    Smajilovic, Sanela; Hansen, Jakob Lerche; Christoffersen, Tue E.H.

    2006-10-06

    Extracellular calcium (Ca2+o) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca2+o stimulates proliferation of the cells. The effects of Ca2+o were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca2+o-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca2+o-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.

  8. Effects of extracellular calcium and of light adaptation on the response to dim light in honey bee drone photoreceptors.

    PubMed

    Raggenbass, M

    1983-11-01

    Light responses in honey bee drone photoreceptors were recorded with intracellular micro-electrodes in superfused slices of retina. The effects of changes in extracellular calcium on the size and the shape of the response to dim light were studied and compared with the effects of light adaptation. Dim light stimuli were used so that the amplitude of the response was linearly related to the number of the photons absorbed, the effects of voltage-dependent mechanisms were negligible and no detectable light adaptation was produced by the stimulus. Lowering the extracellular calcium concentration increased the amplitude and the duration of the response. Raising the extracellular calcium concentration produced the opposite effects. Changing the extracellular calcium concentration modified the response without altering either the linearity of the intensity--response relation or the resting membrane potential in the dark. Light adaptation decreased the amplitude and the duration of the response in a manner that could be quantitatively simulated, in the same photoreceptors, by an increase in the extracellular calcium concentration. Changing the extracellular calcium concentration, or light-adapting the preparation, modified the response without altering its early depolarizing phase. Lowering external calcium either did not affect, or slightly increased, the maximum rate of the light-induced depolarization; raising external calcium, or light-adapting the preparation, either did not affect, or slightly decreased, the maximum rate of the light-induced depolarization. The experimental data can be quantitatively described by a mathematical model with the basic assumption that calcium acts in the process of light adaptation by decreasing the mean open time of the light-activated channels.

  9. Effects of Extracellular Calcium and 1,25 dihydroxyvitamin D3 on Sebaceous Gland Cells In vitro and In vivo.

    PubMed

    Zouboulis, Christos C; Seltmann, Holger; Abdel-Naser, M Badawy; Hossini, Amir M; Menon, Gopinathan K; Kubba, Raj

    2017-03-10

    Calcium and 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) are promoters of epithelial cell functions; however their effects on sebaceous glands are unknown. In this study, morphology, ultrastructure, cell numbers, lipid synthesis and apoptosis of SZ95 sebocytes were assessed in vitro under different concentrations of extracellular calcium with or without 1,25(OH)2D3. Moreover, serum calcium and 1,25(OH)2D3 levels were assessed in acne and non-acne patients (controls). Under conditions of low extracellular calcium, lipogenesis and cell detachment were observed. Increasing extracellular calcium enhanced sebocyte numbers, induced epithelial morphology and reduced lipogenesis. Moreover, a reduction in extracellular calcium reduced E-cadherin and enhanced caspase 3/7 activity (apoptosis), whereas calcium chelation by EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) resulted in enhanced lipogenesis. 1,25(OH)2D3 decreased sebaceous lipogenesis, but also induced signs of autophagy. In the clinical study, patients and controls exhibited normal serum calcium levels. Younger acne patients presented lower 1,25(OH)2D3 levels than did older ones. In conclusion, extracellular calcium and 1,25(OH)2D3 regulate sebocyte morphology, increase cell numbers, decrease sebaceous lipogenesis and induce cell autophagy in vitro. The increased ionized calcium and the reduced 1,25(OH)2D3 levels detected in the serum of younger patients with acne may contribute respectively to increased sebaceous gland volume and enhanced lipogenesis.

  10. Calcium Nutrition and Extracellular Calcium Sensing: Relevance for the Pathogenesis of Osteoporosis, Cancer and Cardiovascular Diseases

    PubMed Central

    Peterlik, Meinrad; Kállay, Enikoe; Cross, Heide S.

    2013-01-01

    Through a systematic search in Pubmed for literature, on links between calcium malnutrition and risk of chronic diseases, we found the highest degree of evidence for osteoporosis, colorectal and breast cancer, as well as for hypertension, as the only major cardiovascular risk factor. Low calcium intake apparently has some impact also on cardiovascular events and disease outcome. Calcium malnutrition can causally be related to low activity of the extracellular calcium-sensing receptor (CaSR). This member of the family of 7-TM G-protein coupled receptors allows extracellular Ca2+ to function as a “first messenger” for various intracellular signaling cascades. Evidence demonstrates that Ca2+/CaSR signaling in functional linkage with vitamin D receptor (VDR)-activated pathways (i) promotes osteoblast differentiation and formation of mineralized bone; (ii) targets downstream effectors of the canonical and non-canonical Wnt pathway to inhibit proliferation and induce differentiation of colorectal cancer cells; (iii) evokes Ca2+ influx into breast cancer cells, thereby activating pro-apoptotic intracellular signaling. Furthermore, Ca2+/CaSR signaling opens Ca2+-sensitive K+ conductance channels in vascular endothelial cells, and also participates in IP3-dependent regulation of cytoplasmic Ca2+, the key intermediate of cardiomyocyte functions. Consequently, impairment of Ca2+/CaSR signaling may contribute to inadequate bone formation, tumor progression, hypertension, vascular calcification and, probably, cardiovascular disease. PMID:23340319

  11. Calcium-sensing receptor regulates stomatal closure through hydrogen peroxide and nitric oxide in response to extracellular calcium in Arabidopsis.

    PubMed

    Wang, Wen-Hua; Yi, Xiao-Qian; Han, Ai-Dong; Liu, Ting-Wu; Chen, Juan; Wu, Fei-Hua; Dong, Xue-Jun; He, Jun-Xian; Pei, Zhen-Ming; Zheng, Hai-Lei

    2012-01-01

    The Arabidopsis calcium-sensing receptor CAS is a crucial regulator of extracellular calcium-induced stomatal closure. Free cytosolic Ca(2+) (Ca(2+)(i)) increases in response to a high extracellular calcium (Ca(2+)(o)) level through a CAS signalling pathway and finally leads to stomatal closure. Multidisciplinary approaches including histochemical, pharmacological, fluorescent, electrochemical, and molecular biological methods were used to discuss the relationship of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) signalling in the CAS signalling pathway in guard cells in response to Ca(2+)(o). Here it is shown that Ca(2+)(o) could induce H(2)O(2) and NO production from guard cells but only H(2)O(2) from chloroplasts, leading to stomatal closure. In addition, the CASas mutant, the atrbohD/F double mutant, and the Atnoa1 mutant were all insensitive to Ca(2+)(o)-stimulated stomatal closure, as well as H(2)O(2) and NO elevation in the case of CASas. Furthermore, it was found that the antioxidant system might function as a mediator in Ca(2+)(o) and H(2)O(2) signalling in guard cells. The results suggest a hypothetical model whereby Ca(2+)(o) induces H(2)O(2) and NO accumulation in guard cells through the CAS signalling pathway, which further triggers Ca(2+)(i) transients and finally stomatal closure. The possible cross-talk of Ca(2+)(o) and abscisic acid signalling as well as the antioxidant system are discussed.

  12. Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption

    NASA Technical Reports Server (NTRS)

    Miyauchi, A.; Hruska, K. A.; Greenfield, E. M.; Duncan, R.; Alvarez, J.; Barattolo, R.; Colucci, S.; Zambonin-Zallone, A.; Teitelbaum, S. L.; Teti, A.

    1990-01-01

    The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.

  13. Extracellular calcium and CaSR drive osteoinduction in mesenchymal stromal cells.

    PubMed

    González-Vázquez, Arlyng; Planell, Josep A; Engel, Elisabeth

    2014-06-01

    Bone is the main store of calcium and progenitor cells in the body. During the resorption process, the local calcium concentration reaches 8-40mM, and the surrounding cells are exposed to these fluctuations in calcium. This stimulus is a signal that is detected through the calcium sensing receptor (CaSR), which modulates chemotactic and proliferative G protein-dependent signaling pathways. The objective of the present work is to evaluate the roles of extracellular calcium ([Ca(2+)]o) and the CaSR in osteoinduction. Rat bone marrow mesenchymal stromal cells (rBMSCs) were stimulated with 10mM of Ca(2+). Several experiments were conducted to demonstrate the effect of [Ca(2+)]o on chemotaxis, proliferation and differentiation on the osteoblastic lineage. It was found that [Ca(2+)]o induces rBMSCs to migrate and proliferate in a concentration-dependent manner. Real-time polymerase chain reaction and immunofluorescence also revealed that 10mM Ca(2+) stimulates overexpression of osteogenic markers in rBMSCs, including alkaline phosphatase (ALP), bone sialoprotein, collagen Ia1 and osteocalcin. Functional assays determining ALP activity and mineralization tests both corroborate the increased expression of these markers in rBMSCs stimulated with Ca(2+). Moreover, CaSR blockage inhibited the cellular response to stimulation with high concentrations of [Ca(2+)]o, revealing that the CaSR is a key modulator of these cellular responses.

  14. Extracellular calcium sensing receptor stimulation in human colonic epithelial cells induces intracellular calcium oscillations and proliferation inhibition.

    PubMed

    Rey, Osvaldo; Young, Steven H; Jacamo, Rodrigo; Moyer, Mary P; Rozengurt, Enrique

    2010-10-01

    The extracellular Ca(2+)-sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However, the signaling mechanisms involved remain poorly defined. Here we examined signaling pathways activated by the CaR, including Ca(2+) oscillations, in individual human colon epithelial cells. Single cell imaging of colon-derived cells expressing the CaR, including SW-480, HT-29, and NCM-460 cells, shows that stimulation of this receptor by addition of aromatic amino acids or by an elevation of the extracellular Ca(2+) concentration promoted striking intracellular Ca(2+) oscillations. The intracellular calcium oscillations in response to extracellular Ca(2+) were of sinusoidal pattern and mediated by the phospholipase C/diacylglycerol/inositol 1,4,5-trisphosphate pathway as revealed by a biosensor that detects the accumulation of diacylglycerol in the plasma membrane. The intracellular calcium oscillations in response to aromatic amino acids were of transient type, that is, Ca(2+) spikes that returned to baseline levels, and required an intact actin cytoskeleton, a functional Rho, Filamin A and the ion channel TRPC1. Further analysis showed that re-expression and stimulation of the CaR in human epithelial cells derived from normal colon and from colorectal adenocarcinoma inhibits their proliferation. This inhibition was associated with the activation of the signaling pathway that mediates the generation of sinusoidal, but not transient, intracellular Ca(2+) oscillations. Thus, these results indicate that the CaR can function in two signaling modes in human colonic epithelial cells offering a potential link between gastrointestinal responses and food/nutrients uptake and metabolism.

  15. Calcium sensing receptor modulates extracellular calcium entry and proliferation via TRPC3/6 channels in cultured human mesangial cells.

    PubMed

    Meng, Kexin; Xu, Jia; Zhang, Chengwei; Zhang, Rui; Yang, He; Liao, Chang; Jiao, Jundong

    2014-01-01

    Calcium-sensing receptor (CaSR) has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions. A previous study indicated that CaSR is expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induces cell proliferation. However, the signaling cascades initiated by CaSR activation in MCs are currently unknown. In this study, our data demonstrate that CaSR mRNA and protein are expressed in a human mesangial cell line. Activating CaSR with high extracellular Ca2+ concentration ([Ca2+]o) or spermine induces a phospholipase C (PLC)-dependent increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx. This increase in Ca2+ was attenuated by treatment with a nonselective TRPC channel blocker but not by treatment with a voltage-gated calcium blocker or Na+/Ca2+ exchanger inhibitor. Furthermore, stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6 but not TRPC1 and TRPC4, and siRNA targeting TRPC3 and TRPC6 attenuated the CaSR activation-induced [Ca2+]i increase. Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase. Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores. Finally, our data indicate that pharmacological inhibition and knock down of TRPC3 and TRPC6 attenuates the CaSR activation-induced cell proliferation in human MCs. With these data, we conclude that Ca

  16. Calcium channel blockade reduces mechanical strain-induced extracellular matrix gene response in lamina cribrosa cells.

    PubMed

    Quill, B; Irnaten, M; Docherty, N G; McElnea, E M; Wallace, D M; Clark, A F; O'Brien, C J

    2015-07-01

    This study examines the effect of the L-type calcium channel blocker verapamil on mechanical strain-induced extracellular matrix genes in optic nerve head lamina cribrosa (LC) cells. Changes in LC cell intracellular calcium [Ca(2+)]i following hypotonic cell membrane stretch were measured with the fluorescent probe fura-2/AM. Fluorescence intensity was measured, after labelling, by calcium (Ca2+) imaging confocal microscopy. Confluent human LC cell cultures were serum starved for 24 h prior to exposure to cyclical mechanical strain (1 Hz, 15%) for 24 h in the presence or absence of verapamil (10 mm). Transforming growth factor-β 1 (TGF-β1), collagen 6A3 (COL6A3) and chondroitin sulfate proteoglycan 2 (CSPG2) mRNA expression levels were assessed by quantitative RT-PCR. Hypotonic cell membrane stretch of LC cells from normal donors significantly increased [Ca2+]i (p<0.05). Exposure to cyclical mechanical strain (15% strain) produced a statistically significant increase in the three matrix genes that were examined (TGF-β1, COL6A3 and CSPG2). This response in both cyclical and mechanical stretch was significantly reduced by pretreating LC cells with the L-type calcium channel blocker verapamil (p<0.05). This study provides evidence of a novel mechanotransduction pathway linking mechanical strain, cation channel function and the induction of LC cell matrix gene transcription. This highlights the potential involvement of calcium influx in the activation of matrix remodelling responses in the optic nerve head and supports the rationale that calcium channel blockers may attenuate disease progression in glaucoma. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  17. Extracellular NAD+ regulates intracellular calcium levels and induces activation of human granulocytes

    PubMed Central

    2005-01-01

    β-NAD+e (extracellular β-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the α and the β extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1–10 μM NAD+e. Thus extracellular β-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation. PMID:16225456

  18. Intracellular calcium movements of boar spermatozoa during 'in vitro' capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model.

    PubMed

    Yeste, M; Fernández-Novell, J M; Ramió-Lluch, L; Estrada, E; Rocha, L G; Cebrián-Pérez, J A; Muiño-Blanco, T; Concha, I I; Ramírez, A; Rodríguez-Gil, J E

    2015-07-01

    This work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca(2+) signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca(2+) labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca(2+) signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca(2+) . The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca(2+) -induced peak, O2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa. © 2015 American

  19. Transgenic plants with increased calcium stores

    NASA Technical Reports Server (NTRS)

    Wyatt, Sarah (Inventor); Tsou, Pei-Lan (Inventor); Robertson, Dominique (Inventor); Boss, Wendy (Inventor)

    2004-01-01

    The present invention provides transgenic plants over-expressing a transgene encoding a calcium-binding protein or peptide (CaBP). Preferably, the CaBP is a calcium storage protein and over-expression thereof does not have undue adverse effects on calcium homeostasis or biochemical pathways that are regulated by calcium. In preferred embodiments, the CaBP is calreticulin (CRT) or calsequestrin. In more preferred embodiments, the CaBP is the C-domain of CRT, a fragment of the C-domain, or multimers of the foregoing. In other preferred embodiments, the CaBP is localized to the endoplasmic reticulum by operatively associating the transgene encoding the CaBP with an endoplasmic reticulum localization peptide. Alternatively, the CaBP is targeted to any other sub-cellular compartment that permits the calcium to be stored in a form that is biologically available to the plant. Also provided are methods of producing plants with desirable phenotypic traits by transformation of the plant with a transgene encoding a CaBP. Such phenotypic traits include increased calcium storage, enhanced resistance to calcium-limiting conditions, enhanced growth and viability, increased disease and stress resistance, enhanced flower and fruit production, reduced senescence, and a decreased need for fertilizer production. Further provided are plants with enhanced nutritional value as human food or animal feed.

  20. Cyclosporine increases calcium in kidney medulla

    SciTech Connect

    Borowitz, J.L.

    1988-01-01

    Treatment of rats with 20, 50, or 100 mg/kg of cyclosporine p.o. markedly increased /sup 45/Ca accumulation in kidney slices especially in medulla. The effect was related to dose and duration of treatment, and was also observed in slices of kidney medulla from cyclosporine-treated mice. Total calcium was elevated in kidney medulla of cyclosporine-treated rats so that the effect is not merely an increased exchange but a build-up of calcium in the tissue. No histopathologic evidence of cyclosporine-related cell necrosis was present in mouse kidney, showing that calcium accumulation is not dystrophic in character. Accumulation of /sup 45/Ca in slices of rat heart, liver, or brain was not affected by cyclosporine pretreatment of the animals. It is suggested that cyclosporine-induced changes in calcium metabolism in kidney medulla may influence kidney function.

  1. Behavior of tricellulin during destruction and formation of tight junctions under various extracellular calcium conditions.

    PubMed

    Takasawa, Akira; Kojima, Takashi; Ninomiya, Takafumi; Tsujiwaki, Mitsuhiro; Murata, Masaki; Tanaka, Satoshi; Sawada, Norimasa

    2013-01-01

    Tricellulin is an important component of tricellular tight junctions (TJs) and is involved in the formation of tricellular contacts. However, little is known about its regulation during the assembly and disassembly of tricellular TJs. By using the well-differentiated pancreatic cancer cell line HPAC, which highly expresses tricellulin at tricellular contacts, we have investigated changes in the localization, expression and phosphorylation of tricellulin and in its TJ functions as a barrier and fence during the destruction and formation of TJs induced by changes in the extracellular calcium concentration. During both extracellular Ca(2+) depletion caused by EGTA treatment and Ca(2+) repletion after Ca(2+) starvation, the expression of tricellulin increased in whole lysates and in Triton-X-100-insoluble fractions without any change in its mRNA. The increases in immunoreactivity revealed by Western blotting were prevented by alkaline phosphatase treatment. Immunoprecipitation assays showed that tricellulin was phosphorylated on threonine residues when it increased after Ca(2+) depletion and repletion. In the early stage after Ca(2+) repletion, tricellulin was expressed not only at tricellular contacts but also in the cytoplasm and at bicellular borders. In confocal laser microscopy, tricellulin was observed at the apical-most regions and basolateral membranes of tricellular contacts after Ca(2+) repletion. Knockdown of tricellulin delayed the recovery of the barrier and fence functions after Ca(2+) repletion. Thus, the dynamic behavior of tricellulin during the destruction and formation of TJs under various extracellular calcium conditions seems to be closely associated with the barrier and fence functions of TJs.

  2. Effects of procaine and extracellular calcium concentration on response of rat stomach fundus muscle to acetylcholine and 5-hydroxytryptamine.

    PubMed Central

    Weinstock, M; Weiss, C

    1979-01-01

    1. When rat stomach fundus muscle was incubated for 30 min in Tyrode solution from which calcium chloride had been omitted, there was an almost complete abolition of the contractile response to 5-hydroxytryptamine (5-HT) while that to acetylcholine (ACh) was still present. 2. The maximum tension obtainable with ACh remained the same in external calcium concentrations ranging from 0.45 to 3.6 mM, but the pD2 value increased. 3. A concentration of at least 0.9 mM calcium was needed to maintain a maximum contraction with 5-HT, and the pD2 for this agent also increased significantly with increase in calcium content of the medium. 4. The effects of procaine on the responses of the muscle to 5-HT and ACh were similar to the respective changes induced by lowering the calcium concentration, and were reduced by the addition of calcium. 5. Concentrations of 2.2 x 10(-7) to 3.6 x 10(-5) M procaine reduced the effects of both 5-HT and KCl and suppressed the maximum responses. 6. The maximum responses to KCl and 5-HT were restored at higher concentrations of procaine (greater than 3.6 x 10(-4) M), while the effect of ACh was reduced. 7. It is suggested that 5-HT, like KCl, is almost entirely dependent on extracellular calcium for inducing muscle contraction, while ACh may utilize calcium from bound stores. PMID:435684

  3. Extracellular matrix production and calcium carbonate precipitation by coral cells in vitro

    PubMed Central

    Helman, Yael; Natale, Frank; Sherrell, Robert M.; LaVigne, Michèle; Starovoytov, Valentin; Gorbunov, Maxim Y.; Falkowski, Paul G.

    2008-01-01

    The evolution of multicellularity in animals required the production of extracellular matrices that serve to spatially organize cells according to function. In corals, three matrices are involved in spatial organization: (i) an organic ECM, which facilitates cell–cell and cell–substrate adhesion; (ii) a skeletal organic matrix (SOM), which facilitates controlled deposition of a calcium carbonate skeleton; and (iii) the calcium carbonate skeleton itself, which provides the structural support for the 3D organization of coral colonies. In this report, we examine the production of these three matrices by using an in vitro culturing system for coral cells. In this system, which significantly facilitates studies of coral cell physiology, we demonstrate in vitro excretion of ECM by primary (nondividing) tissue cultures of both soft (Xenia elongata) and hard (Montipora digitata) corals. There are structural differences between the ECM produced by X. elongata cell cultures and that of M. digitata, and ascorbic acid, a critical cofactor for proline hydroxylation, significantly increased the production of collagen in the ECM of the latter species. We further demonstrate in vitro production of SOM and extracellular mineralized particles in cell cultures of M. digitata. Inductively coupled plasma mass spectrometry analysis of Sr/Ca ratios revealed the particles to be aragonite. De novo calcification was confirmed by following the incorporation of 45Ca into acid labile macromolecules. Our results demonstrate the ability of isolated, differentiated coral cells to undergo fundamental processes required for multicellular organization. PMID:18162537

  4. Calcium binding domains and calcium-induced conformational transition of SPARC/BM-40/osteonectin, an extracellular glycoprotein expressed in mineralized and nonmineralized tissues

    SciTech Connect

    Engel, J.; Taylor, W.; Paulsson, M.; Sage, H.; Hogan, B.

    1987-11-03

    PSARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent M/sub r/ 43,000 (M/sub r/ 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, the authors analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acid region with clusters of glutamic acid residues. This region, although neither ..gamma..-carboxylated nor homologous, resembles the ..gamma..-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca/sup 2 +/ ions. This is accompanied by a 35% increase in ..cap alpha..-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extra-cellular matrix.

  5. Calcium binding domains and calcium-induced conformational transition of SPARC/BM-40/osteonectin, an extracellular glycoprotein expressed in mineralized and nonmineralized tissues.

    PubMed

    Engel, J; Taylor, W; Paulsson, M; Sage, H; Hogan, B

    1987-11-03

    SPARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent Mr 43,000 (Mr 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, we analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acidic region with clusters of glutamic acid residues. This region, although neither gamma-carboxylated nor homologous, resembles the gamma-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca2+ ions. This is accompanied by a 35% increase in alpha-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extracellular matrix.

  6. Alginate-coated magnetic nanoparticles for noninvasive MRI of extracellular calcium.

    PubMed

    Bar-Shir, Amnon; Avram, Liat; Yariv-Shoushan, Shani; Anaby, Debbie; Cohen, Smadar; Segev-Amzaleg, Niva; Frenkel, Dan; Sadan, Ofer; Offen, Daniel; Cohen, Yoram

    2014-07-01

    Nanoparticles (NPs) have great potential to increase the diagnostic capacity of many imaging modalities. MRI is currently regarded as the method of choice for the imaging of deep tissues, and metal ions, such as calcium ions (Ca(2+)), are essential ingredients for life. Despite the tremendous importance of Ca(2+) for the well-being of living systems, the noninvasive determination of the changes in Ca(2+) levels in general, and extracellular Ca(2+) levels in particular, in deep tissues remains a challenge. Here, we describe the preparation and contrast mechanism of a flexible easy to prepare and selective superparamagnetic iron oxide (SPIO) NPs for the noninvasive determination of changes in extracellular Ca(2+) levels using conventional MRI. We show that SPIO NPs coated with monodisperse and purified alginate, having a specific molecular weight, provide a tool to selectively determine Ca(2+) concentrations in the range of 250 µm to 2.5 mm, even in the presence of competitive ions. The alginate-coated magnetic NPs (MNPs) aggregate in the presence of Ca(2+) , which, in turn, affects the T2 relaxation of the water protons in their vicinity. The new alginate-coated SPIO NP formulations, which have no effect on cell viability for 24 h, allow the detection of Ca(2+) levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca(2+) levels in vivo. These results demonstrate that alginate-coated MNPs can be used, at least qualitatively, as a platform for the noninvasive MRI determination of extracellular Ca(2+) levels in myriad in vitro and in vivo biomedical applications.

  7. Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels

    PubMed Central

    Stephens, Robert F.; Guan, W.; Zhorov, Boris S.; Spafford, J. David

    2015-01-01

    How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel. PMID

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

    PubMed Central

    Gonçalves, A. P.; Monteiro, João; Lucchi, Chiara; Kowbel, David J.; Cordeiro, J. M.; Correia-de-Sá, Paulo; Rigden, Daniel J.; Glass, N. L.; Videira, Arnaldo

    2014-01-01

    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. PMID:28357255

  9. Chelation of extracellular calcium-induced cell death was prevented by glycogen synthase kinase-3 inhibitors in PC12 cells.

    PubMed

    Takadera, Tsuneo; Ohtsuka, Megumi; Aoki, Haruka

    2010-03-01

    Calcium ion is a secondary messenger that mediates a variety of physiological responses of neurons, including cell survival responses. To determine the role of calcium in regulating neuronal survival and death, we examined whether chelation of extracellular calcium with EGTA induces caspase-dependent apoptotic cell death and whether glycogen synthase kinase-3 is involved in EGTA-induced cell death in PC12 cells. EGTA increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation and fragmentation accompanied by caspase activation. EGTA increased GRP78 protein expression, suggesting that EGTA induces ER stress. Glycogen synthase kinase-3 inhibitors prevented EGTA-induced apoptosis. In addition, nerve growth factor and insulin growth factor-I completely blocked EGTA-induced cell death. Moreover, caspase-3 activation was inhibited by glycogen synthase kinase-3 inhibitors. These results suggest that chelation of extracellular calcium with EGTA induces caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.

  10. Extracellular sodium dependence of the conduction velocity-calcium relationship: evidence of ephaptic self-attenuation

    PubMed Central

    George, Sharon A.; Bonakdar, Mohammad; Zeitz, Michael; Davalos, Rafael V.; Smyth, James W.

    2016-01-01

    Our laboratory previously demonstrated that perfusate sodium and potassium concentrations can modulate cardiac conduction velocity (CV) consistent with theoretical predictions of ephaptic coupling (EpC). EpC depends on the ionic currents and intercellular separation in sodium channel rich intercalated disk microdomains like the perinexus. We suggested that perinexal width (WP) correlates with changes in extracellular calcium ([Ca2+]o). Here, we test the hypothesis that increasing [Ca2+]o reduces WP and increases CV. Mathematical models of EpC also predict that reducing WP can reduce sodium driving force and CV by self-attenuation. Therefore, we further hypothesized that reducing WP and extracellular sodium ([Na+]o) will reduce CV consistent with ephaptic self-attenuation. Transmission electron microscopy revealed that increasing [Ca2+]o (1 to 3.4 mM) significantly decreased WP. Optically mapping wild-type (WT) (100% Cx43) mouse hearts demonstrated that increasing [Ca2+]o increases transverse CV during normonatremia (147.3 mM), but slows transverse CV during hyponatremia (120 mM). Additionally, CV in heterozygous (∼50% Cx43) hearts was more sensitive to changes in [Ca2+]o relative to WT during normonatremia. During hyponatremia, CV slowed in both WT and heterozygous hearts to the same extent. Importantly, neither [Ca2+]o nor [Na+]o altered Cx43 expression or phosphorylation determined by Western blotting, or gap junctional resistance determined by electrical impedance spectroscopy. Narrowing WP, by increasing [Ca2+]o, increases CV consistent with enhanced EpC between myocytes. Interestingly, during hyponatremia, reducing WP slowed CV, consistent with theoretical predictions of ephaptic self-attenuation. This study suggests that serum ion concentrations may be an important determinant of cardiac disease expression. PMID:26945081

  11. Extracellular sodium dependence of the conduction velocity-calcium relationship: evidence of ephaptic self-attenuation.

    PubMed

    George, Sharon A; Bonakdar, Mohammad; Zeitz, Michael; Davalos, Rafael V; Smyth, James W; Poelzing, Steven

    2016-05-01

    Our laboratory previously demonstrated that perfusate sodium and potassium concentrations can modulate cardiac conduction velocity (CV) consistent with theoretical predictions of ephaptic coupling (EpC). EpC depends on the ionic currents and intercellular separation in sodium channel rich intercalated disk microdomains like the perinexus. We suggested that perinexal width (WP) correlates with changes in extracellular calcium ([Ca(2+)]o). Here, we test the hypothesis that increasing [Ca(2+)]o reduces WP and increases CV. Mathematical models of EpC also predict that reducing WP can reduce sodium driving force and CV by self-attenuation. Therefore, we further hypothesized that reducing WP and extracellular sodium ([Na(+)]o) will reduce CV consistent with ephaptic self-attenuation. Transmission electron microscopy revealed that increasing [Ca(2+)]o (1 to 3.4 mM) significantly decreased WP Optically mapping wild-type (WT) (100% Cx43) mouse hearts demonstrated that increasing [Ca(2+)]o increases transverse CV during normonatremia (147.3 mM), but slows transverse CV during hyponatremia (120 mM). Additionally, CV in heterozygous (∼50% Cx43) hearts was more sensitive to changes in [Ca(2+)]o relative to WT during normonatremia. During hyponatremia, CV slowed in both WT and heterozygous hearts to the same extent. Importantly, neither [Ca(2+)]o nor [Na(+)]o altered Cx43 expression or phosphorylation determined by Western blotting, or gap junctional resistance determined by electrical impedance spectroscopy. Narrowing WP, by increasing [Ca(2+)]o, increases CV consistent with enhanced EpC between myocytes. Interestingly, during hyponatremia, reducing WP slowed CV, consistent with theoretical predictions of ephaptic self-attenuation. This study suggests that serum ion concentrations may be an important determinant of cardiac disease expression. Copyright © 2016 the American Physiological Society.

  12. Effects of extracellular calcium on electrical bursting and intracellular and luminal calcium oscillations in insulin secreting pancreatic beta-cells.

    PubMed

    Chay, T R

    1997-09-01

    The extracellular calcium concentration has interesting effects on bursting of pancreatic beta-cells. The mechanism underlying the extracellular Ca2+ effect is not well understood. By incorporating a low-threshold transient inward current to the store-operated bursting model of Chay, this paper elucidates the role of the extracellular Ca2+ concentration in influencing electrical activity, intracellular Ca2+ concentration, and the luminal Ca2+ concentration in the intracellular Ca2+ store. The possibility that this inward current is a carbachol-sensitive and TTX-insensitive Na+ current discovered by others is discussed. In addition, this paper explains how these three variables respond when various pharmacological agents are applied to the store-operated model.

  13. Increased intra- and extracellular granzyme expression in patients with tuberculosis.

    PubMed

    Garcia-Laorden, M Isabel; Blok, Dana C; Kager, Liesbeth M; Hoogendijk, Arie J; van Mierlo, Gerard J; Lede, Ivar O; Rahman, Wahid; Afroz, Rumana; Ghose, Aniruddha; Visser, Caroline E; Md Zahed, Abu Shahed; Husain, Md Anwar; Alam, Khan Mashrequl; Chandra Barua, Pravat; Hassan, Mahtabuddin; Hossain, Ahmed; Tayab, Md Abu; Day, Nick; Dondorp, Arjen M; de Vos, Alex F; van der Poll, Tom

    2015-09-01

    Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Granzymes (gzms) are proteases mainly found in cytotoxic lymphocytes, but also extracellularly. While the role of gzms in target cell death has been widely characterized, considerable evidence points towards broader roles related to infectious and inflammatory responses. To investigate the expression of the gzms in TB, intracellular gzms A, B and K were measured by flow cytometry in lymphocyte populations from peripheral blood mononuclear cells from 18 TB patients and 12 healthy donors from Bangladesh, and extracellular levels of gzmA and B were measured in serum from 58 TB patients and 31 healthy controls. TB patients showed increased expression of gzmA in CD8(+) T, CD4(+) T and CD56(+) T, but not NK, cells, and of gzmB in CD8(+) T cells, when compared to controls. GzmK expression was not altered in TB patients in any lymphocyte subset. The extracellular levels of gzmA and, to a lesser extent, of gzmB, were increased in TB patients, but did not correlate with intracellular gzm expression in lymphocyte subsets. Our results reveal enhanced intra- and extracellular expression of gzmA and B in patients with pulmonary TB, suggesting that gzms are part of the host response to tuberculosis.

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

    PubMed Central

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

    2016-01-01

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

  15. Calcium carbonate mineralization: involvement of extracellular polymeric materials isolated from calcifying bacteria.

    PubMed

    Ercole, Claudia; Bozzelli, Paola; Altieri, Fabio; Cacchio, Paola; Del Gallo, Maddalena

    2012-08-01

    This study highlights the role of specific outer bacterial structures, such as the glycocalix, in calcium carbonate crystallization in vitro. We describe the formation of calcite crystals by extracellular polymeric materials, such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) isolated from Bacillus firmus and Nocardia calcarea. Organic matrices were isolated from calcifying bacteria grown on synthetic medium--in the presence or absence of calcium ions--and their effect on calcite precipitation was assessed. Scanning electron microscopy observations and energy dispersive X-ray spectrometry analysis showed that CPS and EPS fractions were involved in calcium carbonate precipitation, not only serving as nucleation sites but also through a direct role in crystal formation. The utilization of different synthetic media, with and without addition of calcium ions, influenced the biofilm production and protein profile of extracellular polymeric materials. Proteins of CPS fractions with a molecular mass between 25 and 70 kDa were overexpressed when calcium ions were present in the medium. This higher level of protein synthesis could be related to the active process of bioprecipitation.

  16. Effect of extracellular calcium on excitability of guinea pig airway vagal afferent nerves.

    PubMed

    Undem, Bradley J; Oh, Eun Joo; Lancaster, Eric; Weinreich, Daniel

    2003-03-01

    The effect of reducing extracellular calcium concentration ([Ca(2+)](o)) on vagal afferent excitability was analyzed in a guinea pig isolated vagally innervated trachea-bronchus preparation. Afferent fibers were characterized as either having low-threshold, rapidly adapting mechanosensors (Adelta fibers) or nociceptive-like phenotypes (Adelta and C fibers). The nociceptors were derived from neurons within the jugular ganglia, whereas the low-threshold mechanosensors were derived from neurons within the nodose ganglia. Reducing [Ca(2+)](o) did not affect the excitability of the low-threshold mechanosensors in the airway. By contrast, reducing [Ca(2+)](o) selectively increased the excitability of airway nociceptors as manifested by a substantive increase in action potential discharge in response to mechanical stimulation, and in a subset of fibers, by overtly evoking action potential discharge. This increase in the excitability of nociceptors was not mimicked by a combination of omega-conotoxin and nifedipine or tetraethylammonium. Whole cell patch recordings from airway-labeled and unlabeled neurons in the vagal jugular ganglia support the hypothesis that [Ca(2+)](o) inhibits a nonselective cation conductance in vagal nociceptors that may serve to regulate excitability of the nerve terminals within the airways.

  17. Low Extracellular Zinc Increases Neuronal Oxidant Production Through NADPH Oxidase and Nitric Oxide Synthase Activation

    PubMed Central

    Aimo, Lucila; Cherr, Gary N.; Oteiza, Patricia I.

    2012-01-01

    A decrease in zinc (Zn) levels increases the production of cell oxidants, affects the oxidant defense system and triggers oxidant sensitive signals in neuronal cells. However, the underlying mechanisms are still unclear. This work tested the hypothesis that the increase in neuronal oxidants that occurs when cellular Zn decreases is mediated by the activation of the NMDA receptor. Differentiated PC12 cells were cultured in control, Zn-deficient or Zn-repleted media. The incubation in Zn deficient media led to a rapid increase in cellular calcium levels, which was prevented by a NMDA receptor antagonist (MK-801). Cellular calcium accumulation was associated with NADPH oxidase and nitric oxide synthase (NOS) activation, an increase in cell oxidant levels, and an associated activation of a redox-sensitive signal (AP-1). In cells incubated in the Zn deficient medium, NADPH oxidase activation was prevented by MK-801 and by a protein kinase C inhibitor. The rise in cell oxidants was prevented by inhibitors of NADPH oxidase, of the NOS and by MK-801. A similar pattern of inhibitor action was observed for zinc deficiency-induced AP-1 activation. Results demonstrate that a decrease in extracellular Zn leads to an increase in neuronal oxidants through the activation of the NMDAR that leads to calcium influx and to a calcium-mediated activation of protein kinase C/NADPH oxidase and NOS. Changes in extracellular Zn concentrations can be sensed by neurons, which using reactive oxygen and nitrogen species as second messengers, can regulate signaling involved in neuronal development and function. PMID:20211250

  18. Direct Determination of Multiple Ligand Interactions with the Extracellular Domain of the Calcium-sensing Receptor*

    PubMed Central

    Zhang, Chen; Zhuo, You; Moniz, Heather A.; Wang, Shuo; Moremen, Kelley W.; Prestegard, James H.; Brown, Edward M.; Yang, Jenny J.

    2014-01-01

    Numerous in vivo functional studies have indicated that the dimeric extracellular domain (ECD) of the CaSR plays a crucial role in regulating Ca2+ homeostasis by sensing Ca2+ and l-Phe. However, direct interaction of Ca2+ and Phe with the ECD of the receptor and the resultant impact on its structure and associated conformational changes have been hampered by the large size of the ECD, its high degree of glycosylation, and the lack of biophysical methods to monitor weak interactions in solution. In the present study, we purified the glycosylated extracellular domain of calcium-sensing receptor (CaSR) (ECD) (residues 20–612), containing either complex or high mannose N-glycan structures depending on the host cell line employed for recombinant expression. Both glycosylated forms of the CaSR ECD were purified as dimers and exhibit similar secondary structures with ∼50% α-helix, ∼20% β-sheet content, and a well buried Trp environment. Using various spectroscopic methods, we have shown that both protein variants bind Ca2+ with a Kd of 3.0–5.0 mm. The local conformational changes of the proteins induced by their interactions with Ca2+ were visualized by NMR with specific 15N Phe-labeled forms of the ECD. Saturation transfer difference NMR approaches demonstrated for the first time a direct interaction between the CaSR ECD and l-Phe. We further demonstrated that l-Phe increases the binding affinity of the CaSR ECD for Ca2+. Our findings provide new insights into the mechanisms by which Ca2+ and amino acids regulate the CaSR and may pave the way for exploration of the structural properties of CaSR and other members of family C of the GPCR superfamily. PMID:25305020

  19. Role of low-voltage-activated calcium current and extracellular calcium in controlling the firing pattern of developing CA1 pyramidal neurons.

    PubMed

    Sánchez-Aguilera, Alberto; Sánchez-Alonso, José Luis; Vicente-Torres, María Ángeles; Colino, Asunción

    2017-03-06

    The firing pattern of individual neurons is an important element for information processing and storing. During the first weeks of development, there is a transitional period during which CA1 pyramidal neurons display burst-spiking behavior in contrast to the adult regular-firing pattern. Spike after-depolarizations (ADPs) constitute a major factor underlying burst-spiking behavior. Using current-clamp recordings, we studied ADP waveforms and firing patterns in CA1 pyramidal neurons of Wistar rats from 9 to 19 postnatal days (P9-19). The percentage of burst-spiking neurons increased up to P16, in correlation with the emergence of an active component in the ADP. The application of low-voltage-activated (LVA) calcium channel blockers such as nickel or mibefradil suppressed the generation of the active ADP component and burst-spiking behavior. In agreement with the development of the ADP waveform and burst-spiking behavior, voltage-clamp experiments in dissociated pyramidal neurons showed an increase in the LVA calcium current in P16-19 vs P9-12. Finally, we found that a reduction of extracellular calcium levels decreases the percentage of burst-spiking cells due to a reduction in the active component of the ADP. We conclude that a major contribution of LVA calcium channels to ADP determines the bursting capability of CA1 pyramidal neurons during a transitional postnatal period in contrast to adulthood.

  20. Do calcium supplements increase serum and urine calcium levels in post-menopausal women?

    PubMed

    Samozai, M N; Kulkarni, A K

    2015-05-01

    The frequent prescription of calcium supplements with vitamin D by health practitioners is a topic of concern globally. The present study was designed to find out whether the calcium supplements with vitamin D really affect serum and urinary calcium levels in post-menopausal women. The age-matched comparative study was performed among postmenopausal women who were already on and those who were not on calcium supplements with vitamin D for a period of time in relation to the estimation of the serum calcium and the urine calcium levels. Sixty healthy postmenopausal women were enrolled, with thirty among them forming the study group (SG) - who were on calcium supplements with vitamin D for a period of a month, three months, and up to twelve months - were studied and compared the results obtained with the age-matched control group (CG) of thirty post-menopausal women who were not on calcium supplements. The serum and the urinary calcium levels were estimated by using appropriate biochemical methods and the data were analysed using relevant statistical methods. The serum calcium levels did not vary significantly in SG in spite of consuming calcium supplements over a period of time, whereas the urinary calcium levels increased progressively (p value < 0.005) in those who have taken calcium supplements for a year compared to those who have taken for a month. Thus, the calcium supplements were of little significance on the serum calcium levels, but have a significant effect on the urinary calcium levels in post-menopausal women.

  1. Active vitamin D deficiency mediated by extracellular calcium and phosphorus results in male infertility in young mice.

    PubMed

    Sun, Weiwei; Chen, Lulu; Zhang, Wei; Wang, Rong; Goltzman, David; Miao, Dengshun

    2015-01-01

    We used mice with targeted deletion of 25-hydroxyvitamin D-1 α-hydroxylase [1α(OH)ase(-/-)] to investigate whether 1,25(OH)2D3 deficiency results in male infertility mediated by 1,25(OH)2D3 or extracellular calcium and phosphorus. Male 1α(OH)ase(-/-) and their wild-type littermates fed either a normal diet or a rescue diet from weaning were mated at 6-14 wk of age with female wild-type mice on the same diet. The fertility efficiency of females was analyzed, and the reproductive phenotypes of males were evaluated by histopathological and molecular techniques. Hypocalcemic and hypophosphatemic male 1α(OH)ase(-/-) mice on a normal diet developed infertility characterized by hypergonadotropic hypogonadism, with downregulation of testicular calcium channels, lower intracellular calcium levels, decreased sperm count and motility, and histological abnormalities of the testes. The proliferation of spermatogenic cells was decreased with downregulation of cyclin E and CDK2 and upregulation of p53 and p21 expression, whereas apoptosis of spermatogenic cells was increased with upregulation of Bax and p-caspase 3 expression and downregulation of Bcl-xl expression. When serum calcium and phosphorus were normalized by the rescue diet, the defective reproductive phenotype in the male 1α(OH)ase(-/-) mice, including the hypergonadotropic hypogonadism, decreased sperm count and motility, histological abnormalities of testis, and defective spermatogenesis, was reversed. These results indicate that the infertility seen in male 1,25(OH)2D3-deficient mice is not a direct effect of active vitamin D deficiency on the reproductive system but is an indirect effect mediated by extracellular calcium and phosphorus. Copyright © 2015 the American Physiological Society.

  2. Aristoyunnolin H attenuates extracellular matrix secretion in cardiac fibroblasts by inhibiting calcium influx.

    PubMed

    Chen, Shao-Rui; Zhang, Wen-Ping; Bao, Jing-Mei; Cheng, Zhong-Bin; Yin, Sheng

    2017-01-01

    Aristoyunnolin H is a novel aristophyllene sesquiterpenoid isolated from the traditional Chinese medicine Aristolochia yunnanensis Franch. The present research was designed to explore the anti-fibrotic effects of aristoyunnolin H in adult rat cardiac fibroblasts (CFs) stimulated with angiotensin II (Ang II). Western blot analysis data showed that aristoyunnolin H reduced the upregulation of fibronectin (FN), connective tissue growth factor and collagen I(Col I) production induced by Ang II in CFs. By studying the dynamic intracellular changes of Ca(2+), we further found that while aristoyunnolin H relieved the calcium influx, it has no effect on intracellular calcium store release. Meanwhile, aristoyunnolin H also inhibited the Ang II-stimulated phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II. In conclusion, aristoyunnolin H may attenuate extracellular matrix secretion in vitro by inhibiting Ang II-induced calcium signaling.

  3. Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor.

    PubMed

    Saidak, Zuzana; Boudot, Cedric; Abdoune, Rachida; Petit, Laurent; Brazier, Michel; Mentaverri, Romuald; Kamel, Said

    2009-07-15

    Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca2+. Here, we show that extracellular Ca2+ (Ca2+(o)) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca2+(o) compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca2+(o) in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cbeta (PLCbeta) led to an abolition of the Ca2+(o)-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca2+(o)-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca2+(o) concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca2+-rich environment.

  4. Ubiquitylation functions in the calcium carbonate biomineralization in the extracellular matrix.

    PubMed

    Fang, Dong; Pan, Cong; Lin, Huijuan; Lin, Ya; Xu, Guangrui; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2012-01-01

    Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes.

  5. Ubiquitylation Functions in the Calcium Carbonate Biomineralization in the Extracellular Matrix

    PubMed Central

    Fang, Dong; Pan, Cong; Lin, Huijuan; Lin, Ya; Xu, Guangrui; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2012-01-01

    Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes. PMID:22558208

  6. Calcium imaging in gentamicin ototoxicity: increased intracellular calcium relates to oxidative stress and late apoptosis.

    PubMed

    Chang, Jiwon; Yang, Ji Yun; Choi, June; Jung, Hak Hyun; Im, Gi Jung

    2011-12-01

    To estimate intracellular calcium changes in gentamicin (GM) ototoxicity using calcium imaging. To investigate GM-induced physiologic changes in auditory cells including cell viability, apoptosis, and oxidative stress. Varying concentrations of GM were applied to the HEI-OC1 cochlear cell line. Calcium imaging tracked changes in intracellular calcium concentration during GM cytotoxicity. Cell viability and intracellular reactive oxygen species (ROS) levels also were measured. Little change in calcium levels occurred in HEI-OC1 cells exposed to less than 35 mM GM. However, calcium rose continuously in cells exposed to more than 60 mM GM. With administration of intermediate concentrations of 40 or 50 mM GM, calcium increased variably in different cells, returning to baseline in some cases, or rising continuously in others. Upon increase of GM concentration, intracellular calcium concentration and ROS were increased, and cell viability was decreased due to late apoptosis. This study shows that GM increased intracellular calcium, ROS, and late apoptosis of HEI-OC1 cells derived from cochlear tissue. Increase of intracellular calcium is related to GM-induced apoptosis and oxidative stress. Calcium imaging can be used to determine change of intracellular calcium concentrations and apoptosis in GM ototoxicity. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  7. Acute uptake inhibition increases extracellular serotonin in the rat forebrain.

    PubMed

    Rutter, J J; Auerbach, S B

    1993-06-01

    The effect of acute uptake inhibition on serotonin (5-HT) in the rat central nervous system was monitored by using in vivo dialysis. Peripheral administration of the selective 5-HT uptake blocker, fluoxetine, caused a dose-dependent increase in extracellular 5-HT in both the diencephalon and the striatum. Administration of fluoxetine or sertraline, another selective 5-HT uptake inhibitor, caused a prolonged (24 hr) increase in 5-HT and decrease in 5-hydroxyindoleacetic acid. In addition, fluoxetine and sertraline attenuated the 5-HT releasing effect of fenfluramine administered 24 hr later. Local infusion of fluoxetine into the diencephalon caused an increase in 5-HT that was twice as large as the effect of peripheral injection. Peripheral fluoxetine, by enhancing extracellular 5-HT in the raphe, probably resulted in activation of somatodendritic autoreceptors and inhibition of 5-HT neuronal discharge. Thus, the increase in 5-HT in the diencephalon after peripheral fluoxetine presumably reflected a balance between decreased release and inhibition of reuptake. In support of this, after first infusing fluoxetine into the diencephalon to maximally block reuptake, peripheral injection of the uptake inhibitor caused a decrease in 5-HT.

  8. Expression profiling of colorectal cancer cells reveals inhibition of DNA replication licensing by extracellular calcium.

    PubMed

    Aggarwal, Abhishek; Schulz, Herbert; Manhardt, Teresa; Bilban, Martin; Thakker, Rajesh V; Kallay, Enikö

    2017-06-01

    Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca(2+)]o) in colon cancer cells. Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24h with 2mM [Ca(2+)]o identified significant changes in expression of 1571 probe sets (ANOVA, p<10(-5)). The main biological processes affected by [Ca(2+)]o were DNA replication, cell division, and regulation of transcription. All factors involved in DNA replication-licensing were significantly downregulated by [Ca(2+)]o. Furthermore, we show that the calcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet. We show that among the mechanisms behind the chemopreventive effect of [Ca(2+)]o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Extracellular zinc stimulates a calcium-activated chloride conductance through mobilisation of intracellular calcium in renal inner medullary collecting duct cells.

    PubMed

    Linley, J E; Simmons, N L; Gray, M A

    2007-01-01

    We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn(2+) on whole-cell Ca(2+)-activated Cl(-) currents (I (CLCA)) in mouse inner medullary collecting duct cells (mIMCD-3). I (CLCA) was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10-400 microM) to the bathing solution resulted in a dose-dependent increase in I (CLCA) with little change in Cl(-) selectivity or biophysical characteristics, whereas gadolinium chloride (30 microM) and lanthanum chloride (100 microM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn(2+) (400 microM) stimulated an increase in intracellular Ca(2+) to an elevated plateau. The Zn(2+)-stimulated [Ca(2+)](i) increase was inhibited by thapsigargin (200 nM), the IP(3) receptor antagonist 2-aminoethoxydiphenyl borate (10 microM) and removal of bath Ca(2+). Pre-exposure to Zn(2+) (400 microM) markedly attenuated the ATP (100 microM)-stimulated [Ca(2+)](i) increase. These data are consistent with the hypothesis that extracellular Zn(2+) stimulates an increase in [Ca(2+)](i) by a release of calcium from thapsigargin/IP(3) sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca(2+)-sensing receptor, in IMCD cells is discussed.

  10. Copper-induced activation of TRP channels promotes extracellular calcium entry, activation of CaMs and CDPKs, copper entry and membrane depolarization in Ulva compressa

    PubMed Central

    Gómez, Melissa; González, Alberto; Sáez, Claudio A.; Morales, Bernardo; Moenne, Alejandra

    2015-01-01

    In order to identify channels involved in membrane depolarization, Ulva compressa was incubated with agonists of TRP channels C5, A1 and V1, and the level of intracellular calcium was detected. Agonists of TRPC5, A1 and V1 induced increases in intracellular calcium at 4, 9, and 11 min of exposure, respectively, and antagonists of TRPC5, A1, and V1 corresponding to SKF-96365 (SKF), HC-030031 (HC), and capsazepin (CPZ), respectively, inhibited calcium increases indicating that functional TRPs exist in U. compressa. In addition, copper excess induced increases in intracellular calcium at 4, 9, and 12 min which were inhibited by SKF, HC, and CPZ, respectively, indicating that copper activate TRPC5, A1, and V1 channels. Moreover, copper-induced calcium increases were inhibited by EGTA, a non-permeable calcium chelating agent, but not by thapsigargin, an inhibitor of endoplasmic reticulum (ER) calcium ATPase, indicating that activation of TRPs leads to extracellular calcium entry. Furthermore, copper-induced calcium increases were not inhibited by W-7, an inhibitor of CaMs, and staurosporine, an inhibitor of CDPKs, indicating that extracellular calcium entry did not require activation of CaMs and CDPKs. In addition, copper induced membrane depolarization events at 4, 8, and 11 min and these events were inhibited by SKF, HC, CPZ, and bathocuproine, a specific copper chelating agent, indicating that copper entry through TRP channels leads to membrane depolarization. Moreover, membrane depolarization events were inhibited by W-7 and staurosporine, indicating that activation of CaMs and CDPKs is required to allow copper entry through TRPs. Interestingly, copper-induced calcium increases and depolarization events were light-dependent and were inhibited by DCMU, an inhibitor of photosystem II, and ATP-γ-S, a non-hydrolizable analog of ATP, suggesting that ATP derived from photosynthesis is required to activate TRPs. Thus, light-dependent copper-induced activation TRPC5, A1

  11. Copper-induced activation of TRP channels promotes extracellular calcium entry, activation of CaMs and CDPKs, copper entry and membrane depolarization in Ulva compressa.

    PubMed

    Gómez, Melissa; González, Alberto; Sáez, Claudio A; Morales, Bernardo; Moenne, Alejandra

    2015-01-01

    In order to identify channels involved in membrane depolarization, Ulva compressa was incubated with agonists of TRP channels C5, A1 and V1, and the level of intracellular calcium was detected. Agonists of TRPC5, A1 and V1 induced increases in intracellular calcium at 4, 9, and 11 min of exposure, respectively, and antagonists of TRPC5, A1, and V1 corresponding to SKF-96365 (SKF), HC-030031 (HC), and capsazepin (CPZ), respectively, inhibited calcium increases indicating that functional TRPs exist in U. compressa. In addition, copper excess induced increases in intracellular calcium at 4, 9, and 12 min which were inhibited by SKF, HC, and CPZ, respectively, indicating that copper activate TRPC5, A1, and V1 channels. Moreover, copper-induced calcium increases were inhibited by EGTA, a non-permeable calcium chelating agent, but not by thapsigargin, an inhibitor of endoplasmic reticulum (ER) calcium ATPase, indicating that activation of TRPs leads to extracellular calcium entry. Furthermore, copper-induced calcium increases were not inhibited by W-7, an inhibitor of CaMs, and staurosporine, an inhibitor of CDPKs, indicating that extracellular calcium entry did not require activation of CaMs and CDPKs. In addition, copper induced membrane depolarization events at 4, 8, and 11 min and these events were inhibited by SKF, HC, CPZ, and bathocuproine, a specific copper chelating agent, indicating that copper entry through TRP channels leads to membrane depolarization. Moreover, membrane depolarization events were inhibited by W-7 and staurosporine, indicating that activation of CaMs and CDPKs is required to allow copper entry through TRPs. Interestingly, copper-induced calcium increases and depolarization events were light-dependent and were inhibited by DCMU, an inhibitor of photosystem II, and ATP-γ-S, a non-hydrolizable analog of ATP, suggesting that ATP derived from photosynthesis is required to activate TRPs. Thus, light-dependent copper-induced activation TRPC5, A1

  12. Intracellular NAADP increase induced by extracellular NAADP via the P2Y11-like receptor.

    PubMed

    Djerada, Zoubir; Millart, Hervé

    2013-06-28

    The aim of the study was to identify a signalling pathway allowing NAADP-induced intracellular NAADP increase and involving the P2Y11-like receptor. P2Y11-like and β-adrenergic receptors may play important regulatory roles within the cardiovascular system. Both receptors have been shown to be involved in triggering myocardial preconditioning. Using a Langendorff model we report a positive inotropic response induced by extracellular NAADP via P2Y11-like receptor stimulation. In cardiomyocyte cultures, P2Y11-like receptor stimulation by extracellular NAADP ([NAADP]e) increased intracellular cADP-ribose and NAADP concentration as evidenced by direct measurements. NF546, a new selective P2Y11 receptor agonist, increased intracellular cAMP, cADP-ribose and NAADP concentration confirming the involvement of the P2Y11-like receptor in this signalling pathway. NF157, a P2Y11 receptor antagonist, suppressed the increase in intracellular cADPr, NAADP and NAAD induced by either [NAADP]e or NF546. The response profile for intracellular cADP-ribose and NAADP concentration following P2Y11-like stimulation with NF546 was similar to reported data relating β-adrenergic stimulation with isoprenaline. This response represents the signature of the Gs/ADP-ribosyl cyclase activity. Moreover, this study provides a signalling pathway: intracellular NAADP increase induced by extracellular NAADP via metabotropic activity of P2Y11-like receptor. This pathway implying P2Y11-like could take part in the intracellular calcium rise reported for extracellular NAADP.

  13. Electrical slow waves in the mouse oviduct are dependent on extracellular and intracellular calcium sources

    PubMed Central

    Dixon, Rose Ellen; Britton, Fiona C.; Baker, Salah A.; Hennig, Grant W.; Rollings, Christina M.; Sanders, Kenton M.

    2011-01-01

    Spontaneous contractions of the myosalpinx are critical for oocyte transport along the oviduct. Slow waves, the electrical events that underlie myosalpinx contractions, are generated by a specialized network of pacemaker cells called oviduct interstitial cells of Cajal (ICC-OVI). The ionic basis of oviduct pacemaker activity is unknown. Intracellular recordings and Ca2+ imaging were performed to examine the role of extracellular and intracellular Ca2+ sources in slow wave generation. RT-PCR was performed to determine the transcriptional expression of Ca2+ channels. Molecular studies revealed most isoforms of L- and T-type calcium channels (Cav1.2,1.3,1.4,3.1,3.2,3.3) were expressed in myosalpinx. Reduction of extracellular Ca2+ concentration ([Ca2+]o) resulted in the abolition of slow waves and myosalpinx contractions without significantly affecting resting membrane potential (RMP). Spontaneous Ca2+ waves spread through ICC-OVI cells at a similar frequency to slow waves and were inhibited by reduced [Ca2+]o. Nifedipine depolarized RMP and inhibited slow waves; however, pacemaker activity returned when the membrane was repolarized with reduced extracellular K+ concentration ([K+]o). Ni2+ also depolarized RMP but failed to block slow waves. The importance of ryanodine and inositol 1,4,5 trisphosphate-sensitive stores were examined using ryanodine, tetracaine, caffeine, and 2-aminoethyl diphenylborinate. Results suggest that although both stores are involved in regulation of slow wave frequency, neither are exclusively essential. The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor cyclopiazonic acid inhibited pacemaker activity and Ca2+ waves suggesting that a functional SERCA pump is necessary for pacemaker activity. In conclusion, results from this study suggest that slow wave generation in the oviduct is voltage dependent, occurs in a membrane potential window, and is dependent on extracellular calcium and functional SERCA pumps. PMID:21881003

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

    NASA Technical Reports Server (NTRS)

    Bjorkman, T.; Cleland, R. E.

    1991-01-01

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

  15. Enzymes in the extracellular matrix of Volvox: an inducible, calcium-dependent phosphatase with a modular composition.

    PubMed

    Hallmann, A

    1999-01-15

    The volvocine algae provide the unique opportunity for exploring development of an extracellular matrix. Volvox is the most advanced member of this family and represents the simplest multicellular organism, with differentiated cells, a complete division of labor, and a complex extracellular matrix, which serves structural and enzymatic functions. In Volvox carteri a glycosylated extracellular phosphatase was identified, which is partially released from the extracellular matrix into the growth medium. The phosphatase is synthesized in response to inorganic phosphate starvation and is strictly calcium-dependent. The metalloenzyme has been purified to homogeneity and characterized. Its gene and cDNA have been cloned. Comparisons of genomic and cDNA sequences revealed an extremely intron-rich gene (32 introns). With an apparent molecular mass of 160 kDa the Volvox extracellular phosphatase is the largest phosphatase cloned, with no sequence similarity to any other phosphatase. This enzyme exhibits a modular composition. There are two large domains and a small one. The large domains are highly homologous to each other and therefore most likely originated from gene duplication and fusion. At least one EF-hand motif for calcium binding was identified in this extracellular protein. Volvox extracellular phosphatase is the first calcium-dependent extracellular phosphatase to be cloned.

  16. Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro.

    PubMed

    Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Zhou, Ping; Zhang, Xiaolei; Zhang, Wei; Chen, Qingyu; Kou, Dongquan; Ying, Xiaozhou; Shen, Yue; Cheng, Xiaojie; Yu, Ziming; Peng, Lei; Lu, Chuanzhu

    2013-09-01

    Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.

  17. A protein involved in the assembly of an extracellular calcium storage matrix.

    PubMed

    Glazer, Lilah; Shechter, Assaf; Tom, Moshe; Yudkovski, Yana; Weil, Simy; Aflalo, Eliahu David; Pamuru, Ramachandra Reddy; Khalaila, Isam; Bentov, Shmuel; Berman, Amir; Sagi, Amir

    2010-04-23

    Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBank data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate.

  18. Expression of extracellular calcium (Ca2+o)-sensing receptor in human peripheral blood monocytes

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Olozak, I.; Chattopadhyay, N.; Butters, R. R.; Kifor, O.; Scadden, D. T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor playing key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone turnover and may play a role in the "reversal" phase of skeletal remodeling that follows osteoclastic resorption and precedes osteoblastic bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for such mononuclear cells present locally within the bone marrow microenvironment. Indeed, previous studies by other investigators have shown that raising Ca2+o either in vivo or in vitro stimulated the release of interleukin-6 (IL-6) from human peripheral blood monocytes, suggesting that these cells express a Ca2+o-sensing mechanism. In these earlier studies, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in peripheral blood monocytes. Since we recently found that non-specific esterase-positive, putative monocytes isolated from murine bone marrow express the CaR, we reevaluated the expression of this receptor in human peripheral blood monocytes. Immunocytochemistry, flow cytometry, and Western blot analysis, performed using a polyclonal antiserum specific for the CaR, detected CaR protein in human monocytes. In addition, the use of RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products, identified CaR transcripts in the cells. Therefore, taken together, our data show that human peripheral blood monocytes possess both CaR protein and mRNA very similar if not identical to those expressed in parathyroid and kidney that could mediate the previously described, direct effects of Ca2+o on these cells. Furthermore, since mononuclear cells isolated from bone marrow also express the CaR, the latter might play some role in

  19. Expression of extracellular calcium (Ca2+o)-sensing receptor in human peripheral blood monocytes

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Olozak, I.; Chattopadhyay, N.; Butters, R. R.; Kifor, O.; Scadden, D. T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor playing key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone turnover and may play a role in the "reversal" phase of skeletal remodeling that follows osteoclastic resorption and precedes osteoblastic bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for such mononuclear cells present locally within the bone marrow microenvironment. Indeed, previous studies by other investigators have shown that raising Ca2+o either in vivo or in vitro stimulated the release of interleukin-6 (IL-6) from human peripheral blood monocytes, suggesting that these cells express a Ca2+o-sensing mechanism. In these earlier studies, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in peripheral blood monocytes. Since we recently found that non-specific esterase-positive, putative monocytes isolated from murine bone marrow express the CaR, we reevaluated the expression of this receptor in human peripheral blood monocytes. Immunocytochemistry, flow cytometry, and Western blot analysis, performed using a polyclonal antiserum specific for the CaR, detected CaR protein in human monocytes. In addition, the use of RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products, identified CaR transcripts in the cells. Therefore, taken together, our data show that human peripheral blood monocytes possess both CaR protein and mRNA very similar if not identical to those expressed in parathyroid and kidney that could mediate the previously described, direct effects of Ca2+o on these cells. Furthermore, since mononuclear cells isolated from bone marrow also express the CaR, the latter might play some role in

  20. Activation of the calcium sensing receptor with cinacalcet increases serum gastrin levels in healthy older subjects

    USDA-ARS?s Scientific Manuscript database

    Gastric acidity is postulated to enhance calcium absorption since calcium is better dissolved at low pH. Extracellular calcium stimulates gastrin and gastric acid secretion in humans. Ex vivo studies indicate that the calcium sensing receptor (CaR), which is expressed on the surface of human G cells...

  1. Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism

    PubMed Central

    Campion, Katherine L.; McCormick, Wanda D.; Warwicker, Jim; Khayat, Mohd Ezuan Bin; Atkinson-Dell, Rebecca; Steward, Martin C.; Delbridge, Leigh W.; Mun, Hee-Chang; Conigrave, Arthur D.

    2015-01-01

    The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pHo) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pHo can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pHo from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca2+i) mobilization, whereas raising pHo to 7.6 potentiated responsiveness to extracellular calcium (Ca2+o). Similar pHo effects were observed for Ca2+o-induced extracellular signal-regulated kinase phosphorylation and actin polymerization and for L-Phe-induced Ca2+i mobilization. Intracellular pH was unaffected by acute 0.4-unit pHo changes, and the presence of physiologic albumin concentrations failed to attenuate the pHo-mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pHo sensitivity. Finally, pathophysiologic pHo elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pHo changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo. PMID:25556167

  2. Influence of Calcium in Extracellular DNA Mediated Bacterial Aggregation and Biofilm Formation

    PubMed Central

    Koop, Leena; Wong, Yie Kuan; Ahmed, Safia; Siddiqui, Khawar Sohail; Manefield, Mike

    2014-01-01

    Calcium (Ca2+) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca2+ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca2+ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca2+ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca2+ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca2+ to eDNA thereby mediating bacterial aggregation and biofilm formation. PMID:24651318

  3. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation.

    PubMed

    Das, Theerthankar; Sehar, Shama; Koop, Leena; Wong, Yie Kuan; Ahmed, Safia; Siddiqui, Khawar Sohail; Manefield, Mike

    2014-01-01

    Calcium (Ca(2+)) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+) and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+) binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+) had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+) at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca(2+) is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+) alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+) to eDNA thereby mediating bacterial aggregation and biofilm formation.

  4. [New insights into the pharmacology of the extracellular calcium sensing receptor].

    PubMed

    Ruat, Martial; Petrel, Christophe

    2004-11-01

    The extracellular calcium-sensing receptor (CaR) belongs to class III of G-protein coupled receptors. The CaR is expressed at the surface of the parathyroid cells and plays an essential role in the regulation of Ca2+ homeostasis through the control of parathyroid secretion. The CaR is activated by Ca2+ and Mg2+ present in the extracellular fluids, various di- and trivalent cations, L-aminoacids and charged molecules including several antibiotics. Calcimimetics potentiate the effect of Ca2+ and are proposed to be of therapeutic benefit for the treatment of both primary and secondary hyperparathyroidism. Calcilytics block the Ca2+-induced activation of the CaR. Three-dimensional models of the seven transmembrane domains of the human CaR have been used to identify specific residues implicated in the recognition of calcimimetics and calcilytics. These molecules should be useful for delineating the physiological roles played by the CaR in several tissues and for clarifying the direct effects attributed to extracellular Ca2+.

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

    PubMed

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

    2015-09-01

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

  6. Food restriction increases NMDA receptor-mediated calcium-calmodulin kinase II and NMDA receptor/extracellular signal-regulated kinase 1/2-mediated cyclic amp response element-binding protein phosphorylation in nucleus accumbens upon D-1 dopamine receptor stimulation in rats.

    PubMed

    Haberny, S L; Carr, K D

    2005-01-01

    Biological drive states exert homeostatic control in part by increasing the reinforcing effects of environmental incentive stimuli. An apparent by-product of this adaptive response is the enhanced acquisition of drug self-administration behavior in food-restricted (FR) animals. While previous research has demonstrated increased central sensitivity to rewarding effects of abused drugs and direct dopamine (DA) receptor agonists in FR subjects, the underlying neurobiology is not well understood. Recently, it was demonstrated that intracerebroventricular (i.c.v.) injection of the D-1 DA receptor agonist, SKF-82958 produces a stronger activation of striatal extracellular signal-regulated kinase (ERK) 1/2 and cyclic AMP response element-binding protein (CREB) in FR relative to ad libitum (AL) fed rats. The main purpose of the present study was to characterize the involvement and mechanisms of interaction between NMDA receptor function and the augmented cellular responses to D-1 DA receptor stimulation in nucleus accumbens (NAc) of FR rats. In experiment 1, Western immunoblotting was used to demonstrate that i.c.v. injection of SKF-82958 (20 microg) produces greater phosphorylation of the NMDA NR1 subunit and calcium-calmodulin kinase II (CaMK II) in NAc of FR as compared with AL rats. In experiment 2, pretreatment of subjects with the NMDA antagonist, MK-801 (1.0 mg/kg, i.p.) decreased SKF-82958-induced activation of CaMK II, ERK1/2 and CREB, and reversed the augmenting effect of FR on activation of all three proteins. In experiment 3, pretreatment with the mitogen-activated protein kinase/ERK kinase inhibitor SL-327 (60 mg/kg, i.p.) suppressed SKF-82958- induced activation of ERK1/2 and reversed the augmenting effect of FR on CREB activation. These results point to specific neuroadaptations in the NAc of FR rats whereby D-1 DA receptor stimulation leads to increased NMDA NR1 subunit phosphorylation and consequent increases in NMDA receptor-dependent CaMK II and ERK1

  7. Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source.

    PubMed

    Mellor, Liliana F; Mohiti-Asli, Mahsa; Williams, John; Kannan, Arthi; Dent, Morgan R; Guilak, Farshid; Loboa, Elizabeth G

    2015-09-01

    We have previously shown that elevating extracellular calcium from a concentration of 1.8 to 8 mM accelerates and increases human adipose-derived stem cell (hASC) osteogenic differentiation and cell-mediated calcium accretion, even in the absence of any other soluble osteogenic factors in the culture medium. However, the effects of elevated calcium on hASC chondrogenic differentiation have not been reported. The goal of this study was to determine the effects of varied calcium concentrations on chondrogenic differentiation of hASC. We hypothesized that exposure to elevated extracellular calcium (8 mM concentration) in a chondrogenic differentiation medium (CDM) would inhibit chondrogenesis of hASC when compared to basal calcium (1.8 mM concentration) controls. We further hypothesized that a full osteochondral construct could be engineered by controlling local release of calcium to induce site-specific chondrogenesis and osteogenesis using only hASC as the cell source. Human ASC was cultured as micromass pellets in CDM containing transforming growth factor-β1 and bone morphogenetic protein 6 for 28 days at extracellular calcium concentrations of either 1.8 mM (basal) or 8 mM (elevated). Our findings indicated that elevated calcium induced osteogenesis and inhibited chondrogenesis in hASC. Based on these findings, stacked polylactic acid nanofibrous scaffolds containing either 0% or 20% tricalcium phosphate (TCP) nanoparticles were electrospun and tested for site-specific chondrogenesis and osteogenesis. Histological assays confirmed that human ASC differentiated locally to generate calcified tissue in layers containing 20% TCP, and cartilage in the layers with no TCP when cultured in CDM. This is the first study to report the effects of elevated calcium on chondrogenic differentiation of hASC, and to develop osteochondral nanofibrous scaffolds using a single cell source and controlled calcium release to induce site-specific differentiation. This approach

  8. Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source

    PubMed Central

    Mellor, Liliana F.; Mohiti-Asli, Mahsa; Williams, John; Kannan, Arthi; Dent, Morgan R.; Guilak, Farshid

    2015-01-01

    We have previously shown that elevating extracellular calcium from a concentration of 1.8 to 8 mM accelerates and increases human adipose-derived stem cell (hASC) osteogenic differentiation and cell-mediated calcium accretion, even in the absence of any other soluble osteogenic factors in the culture medium. However, the effects of elevated calcium on hASC chondrogenic differentiation have not been reported. The goal of this study was to determine the effects of varied calcium concentrations on chondrogenic differentiation of hASC. We hypothesized that exposure to elevated extracellular calcium (8 mM concentration) in a chondrogenic differentiation medium (CDM) would inhibit chondrogenesis of hASC when compared to basal calcium (1.8 mM concentration) controls. We further hypothesized that a full osteochondral construct could be engineered by controlling local release of calcium to induce site-specific chondrogenesis and osteogenesis using only hASC as the cell source. Human ASC was cultured as micromass pellets in CDM containing transforming growth factor-β1 and bone morphogenetic protein 6 for 28 days at extracellular calcium concentrations of either 1.8 mM (basal) or 8 mM (elevated). Our findings indicated that elevated calcium induced osteogenesis and inhibited chondrogenesis in hASC. Based on these findings, stacked polylactic acid nanofibrous scaffolds containing either 0% or 20% tricalcium phosphate (TCP) nanoparticles were electrospun and tested for site-specific chondrogenesis and osteogenesis. Histological assays confirmed that human ASC differentiated locally to generate calcified tissue in layers containing 20% TCP, and cartilage in the layers with no TCP when cultured in CDM. This is the first study to report the effects of elevated calcium on chondrogenic differentiation of hASC, and to develop osteochondral nanofibrous scaffolds using a single cell source and controlled calcium release to induce site-specific differentiation. This approach

  9. Accumulation of extracellular calcium at the endplate of mouse diaphragm after ecothiopate in vitro.

    PubMed Central

    Burd, P. F.; Ferry, C. B.; Smith, J. W.

    1989-01-01

    1. Experiments were carried out to investigate the accumulation from the extracellular medium of 45Ca2+ by the endplate region of skeletal muscle. 2. Mouse diaphragm muscle was incubated in physiological saline labelled with 45Ca at 37 degrees C for periods of up to 1.5 h. 3. The muscle was divided into junctional and non-junctional portions and the Ca from the extracellular fluid accumulated at the endplate determined from the 45Ca content of the portions. 4. The accumulation of extracellular Ca at the endplate region of muscles incubated in pysiological saline alone was nil, but there was accumulation in the presence of the anticholinesterase ecothiopate iodide 0.5 x 10(-6) M (ECO). Stimulation of the phrenic nerve at 0.02 Hz caused no further increase in accumulation but reduced the amount of spontaneous fasciculation. In tetrodotoxin (TTX) 10(-6) M, the accumulation was halved, and in 3.5 mM Mg2+ the accumulation was nil. Carbachol 10(-4) M resulted in an accumulation of Ca similar to that in ECO. 5. It is concluded that there was an accumulation of extracellular Ca following excitation of the nerve by stimulation at a low frequency and during the spontaneous fasciculations, and about half of the accumulation of extracellular Ca after ECO in the experiments was due to the postsynaptic action of ACh released non-quantally from the nerve terminals. PMID:2804548

  10. Calcium efflux from Myxicola giant axons: effects of extracellular calcium and intracellular EGTA

    PubMed Central

    Abercrombie, Ronald F.; Sjodin, Raymond A.

    1980-01-01

    1. 45Ca efflux was examined in Myxicola giant axons injected with 45CaCl2 or various concentrations of 45Ca/EGTA buffers. In axons injected with 45CaCl2, the Cao-dependent Ca efflux in 1 mM-Cao was about half that in 10 mM-Cao. 2. Axons injected with 45Ca/EGTA buffers consistently showed two types of results: in one type (B type), K½ for Cao activation was less than 1 mM-Cao. In the other type of result (A type), there was an additional Ca activation of Ca efflux. This additional efflux exhibited a linear dependence on Cao when the Cao values ranged between 1 mM-Cao and 10 mM-Cao. 3. The B-type result remained unchanged when the injected Ca/EGTA concentrations were varied. The A type result, however, changed as a function of Ca/EGTAi in the following way: (a) at a constant ratio of Ca/EGTAi = 8/10, the megnitude of the linear component of the Cao-activated Ca efflux was reduced by increasing the intracellular concentration of (Ca/EGTA) buffer; and (b) at a ratio Ca/EGTA = 1/10, the linear component of the Cao-activated Ca efflux appeared to acquire a slower time response to changes in Cao. 4. Nao acts synergistically with Cao to produce the linear component of the Ca-activated Ca efflux seen in the A type result. 5. With axons containing 45Ca/EGTA buffers (total EGTAi = 1 mM), changing the Ca/EGTA ratio by repetitive injections of CaCl2 did not increase 45Ca efflux by as great an amount as would be predicted if Cai2+ were controlled by the EGTA buffer alone. 6. Cai2+ (measured by arsenazo III absorbance) is influenced by Cao irrespective of the presence of 1 mM-EGTA buffer inside the axon. There was a variability in the sensitivity of Cai to Cao that resembled the variability found in 45Ca efflux measurements. 7. 45Ca influx is not affected by the concentration of Ca/EGTA buffer injected into the cell and appears to be only slightly, if at all, affected by increasing ionized Cai2+ from 0·016 to 0·56 μM in the injection medium. 8. These results are consistent

  11. Calcium-dependent inactivation of calcium channels in the medial striatum increases at eye opening.

    PubMed

    Evans, R C; Herin, G A; Hawes, S L; Blackwell, K T

    2015-04-01

    Influx of calcium through voltage-gated calcium channels (VGCCs) is essential for striatal function and plasticity. VGCCs expressed in striatal neurons have varying kinetics, voltage dependences, and densities resulting in heterogeneous subcellular calcium dynamics. One factor that determines the calcium dynamics in striatal medium spiny neurons is inactivation of VGCCs. Aside from voltage-dependent inactivation, VGCCs undergo calcium-dependent inactivation (CDI): inactivating in response to an influx of calcium. CDI is a negative feedback control mechanism; however, its contribution to striatal neuron function is unknown. Furthermore, although the density of VGCC expression changes with development, it is unclear whether CDI changes with development. Because calcium influx through L-type calcium channels is required for striatal synaptic depression, a change in CDI could contribute to age-dependent changes in striatal synaptic plasticity. Here we use whole cell voltage clamp to characterize CDI over developmental stages and across striatal regions. We find that CDI increases at the age of eye opening in the medial striatum but not the lateral striatum. The developmental increase in CDI mostly involves L-type channels, although calcium influx through non-L-type channels contributes to the CDI in both age groups. Agents that enhance protein kinase A (PKA) phosphorylation of calcium channels reduce the magnitude of CDI after eye opening, suggesting that the developmental increase in CDI may be related to a reduction in the phosphorylation state of the L-type calcium channel. These results are the first to show that modifications in striatal neuron properties correlate with changes to sensory input.

  12. Calcium regulates estrogen increase in permeability of cultured CaSki epithelium by eNOS-dependent mechanism.

    PubMed

    Gorodeski, G I

    2000-11-01

    Estrogen increases baseline transepithelial permeability across CaSki cultures and augments the increase in permeability in response to hypertonic gradients. In estrogen-treated cells, lowering cytosolic calcium abrogated the hypertonicity-induced augmented increase in permeability and decreased baseline permeability to a greater degree than in estrogen-deprived cells. Steady-state levels of cytosolic calcium in estrogen-deprived cells were higher than in estrogen-treated cells. Increases in extracellular calcium increased cytosolic calcium more in estrogen-deprived cells than in estrogen-treated cells. However, in estrogen-treated cells, increasing cytosolic calcium was associated with greater increases in permeability in response to hypertonic gradients than in estrogen-deprived cells. Lowering cytosolic calcium blocked the estrogen-induced increase in nitric oxide (NO) release and in the in vitro conversion of L-[(3)H]arginine to L-[(3)H]citrulline. Treatment with estrogen upregulated mRNA of the NO synthase isoform endothelial nitric oxide synthase (eNOS). These results indicate that cytosolic calcium mediates the responses to estrogen and suggest that the estrogen increase in permeability and the augmented increase in permeability in response to hypertonicity involve an increase in NO synthesis by upregulation of the calcium-dependent eNOS.

  13. Poliovirus protein 2BC increases cytosolic free calcium concentrations.

    PubMed Central

    Aldabe, R; Irurzun, A; Carrasco, L

    1997-01-01

    Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis. PMID:9223520

  14. Serum Calcium Increase Correlates With Worsening of Lipid Profile

    PubMed Central

    Gallo, Luigia; Faniello, Maria C.; Canino, Giovanni; Tripolino, Cesare; Gnasso, Agostino; Cuda, Giovanni; Costanzo, Francesco S.; Irace, Concetta

    2016-01-01

    Abstract Despite the well-documented role of calcium in cell metabolism, its role in the development of cardiovascular disease is still under heavy debate. Several studies suggest that calcium supplementation might be associated with an increased risk of coronary heart disease, whereas others underline a significant effect on lowering high blood pressure and hyperlipidemia. The purpose of this study was to investigate, in a large nonselected cohort from South Italy, if serum calcium levels correlate with lipid values and can therefore be linked to higher individual cardiovascular risk. Eight-thousand-six-hundred-ten outpatients addressed to the Laboratory of Clinical Biochemistry, University of Magna Græcia, Catanzaro, Italy from January 2012 to December 2013 for routine blood tests, were enrolled in the study. Total HDL-, LDL- and non-HDL colesterol, triglycerides, and calcium were determined with standard methods. We observed a significant association between total cholesterol, LDL-cholesterol, HDL-cholesterol, non-HDL cholesterol, triglycerides, and serum calcium in men and postmenopause women. Interestingly, in premenopause women, we only found a direct correlation between serum calcium, total cholesterol, and HDL-cholesterol. Calcium significantly increased while increasing total cholesterol and triglycerides in men and postmenopause women. Our results confirm that progressive increase of serum calcium level correlates with worsening of lipid profile in our study population. Therefore, we suggest that a greater caution should be used in calcium supplement prescription particularly in men and women undergoing menopause, in which an increase of serum lipids is already known to be associated with a higher cardiovascular risk. PMID:26937904

  15. Expression of OsCAS (Calcium-Sensing Receptor) in an Arabidopsis Mutant Increases Drought Tolerance.

    PubMed

    Zhao, Xin; Xu, Mengmeng; Wei, Rongrong; Liu, Yang

    2015-01-01

    The calcium-sensing receptor (CaS), which is localized in the chloroplasts, is a crucial regulator of extracellular calcium-induced stomatal closure in Arabidopsis. It has homologs in Oryza sativa and other plants. These sequences all have a rhodanese-like protein domain, which has been demonstrated to be associated with specific stress conditions. In this study, we cloned the Oryza sativa calcium-sensing receptor gene (OsCAS) and demonstrated that OsCAS could sense an increase of extracellular Ca2+ concentration and mediate an increase in cytosolic Ca2+ concentration. The OsCAS gene was transformed into an Arabidopsis CaS knockout mutant (Salk) and overexpressed in the transgenic plants. OsCAS promoted stomatal closure. We screened homozygous transgenic Arabidopsis plants and determined physiological indices such as the oxidative damage biomarker malondialdehyde (MDA), relative membrane permeability (RMP), proline content, and chlorophyll fluorescence parameters, after 21 days of drought treatment. Our results revealed lower RMP and MDA contents and a higher Proline content in transgenic Arabidopsis plants after drought stress, whereas the opposite was observed in Salk plants. With respect to chlorophyll fluorescence, the electron transport rate and effective PSII quantum yield decreased in all lines under drought stress; however, in the transgenic plants these two parameters changed fewer and were higher than those in wild-type and Salk plants. The quantum yield of regulated energy dissipation and nonregulated energy dissipation in PSII were higher in Salk plants, whereas these values were lower in the transgenic plants than in the wild type under drought stress. The above results suggest that the transgenic plants showed better resistance to drought stress by decreasing damage to the cell membrane, increasing the amount of osmoprotectants, and maintaining a relatively high photosynthetic capacity. In conclusion, OsCAS is an extracellular calcium-sensing receptor

  16. Calcium carbonate formation on mica supported extracellular polymeric substance produced by Rhodococcus opacus

    NASA Astrophysics Data System (ADS)

    Szcześ, Aleksandra; Czemierska, Magdalena; Jarosz-Wilkołazka, Anna

    2016-10-01

    Extracellular polymeric substance (EPS) extracted from Rhodococcus opacus bacterial strain was used as a matrix for calcium carbonate precipitation using the vapour diffusion method. The total exopolymer and water-soluble exopolymer fraction of different concentrations were spread on the mica surface by the spin-coating method. The obtained layers were characterized using the atomic force microscopy measurement and XPS analysis. The effects of polymer concentration, initial pH of calcium chloride solution and precipitation time on the obtained crystals properties were investigated. Raman spectroscopy and scanning electron microscopy were used to characterize the precipitated minerals. It was found that the type of precipitated CaCO3 polymorph and the crystal size depend on the kind of EPS fraction. The obtained results indicates that the water soluble fraction favours vaterite dissolution and calcite growth, whereas the total EPS stabilizes vaterite and this effect is stronger at basic pH. It seems to be due to different contents of the functional group of EPS fractions.

  17. Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

    PubMed Central

    Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

    2017-01-01

    Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

  18. Regulation of mouse lung development by the extracellular calcium-sensing receptor, CaR.

    PubMed

    Finney, Brenda A; del Moral, Pierre M; Wilkinson, William J; Cayzac, Sebastien; Cole, Martin; Warburton, David; Kemp, Paul J; Riccardi, Daniela

    2008-12-15

    Postnatal lung function is critically dependent upon optimal embryonic lung development. As the free ionized plasma calcium concentration ([Ca(2+)](o)) of the fetus is higher than that of the adult, the process of lung development occurs in a hypercalcaemic environment. In the adult, [Ca(2+)](o) is monitored by the G-protein coupled, extracellular calcium-sensing receptor (CaR), but neither its ontogeny nor its potential role in lung development are known. Here, we demonstrate that CaR is expressed in the mouse lung epithelium, and that its expression is developmentally regulated, with a peak of expression at embryonic day 12.5 (E12.5) and a subsequent decrease by E18, after which the receptor is absent. Experiments carried out using the lung explant culture model in vitro show that lung branching morphogenesis is sensitive to [Ca(2+)](o), being maximal at physiological adult [Ca(2+)](o) (i.e. 1.0-1.3 mM) and lowest at the higher, fetal (i.e. 1.7 mM) [Ca(2+)](o). Administration of the specific CaR positive allosteric modulator, the calcimimetic R-568, mimics the suppressive effects of high [Ca(2+)](o) on branching morphogenesis while both phospholipase C and PI3 kinase inhibition reverse these effects. CaR activation suppresses cell proliferation while it enhances intracellular calcium signalling, lung distension and fluid secretion. Conditions which are restrictive either to branching or to secretion can be rescued by manipulating [Ca(2+)](o) in the culture medium. In conclusion, fetal Ca(2+)(o), acting through a developmentally regulated CaR, is an important extrinsic factor that modulates the intrinsic lung developmental programme. Our observations support a novel role for the CaR in preventing hyperplastic lung disease in utero.

  19. Critical role of extracellular calcium in vanadate-induced renal vasoconstriction

    SciTech Connect

    Benabe, J.E.; Cruz-Soto, M.A.; Martinez-Maldonado, M.

    1984-03-01

    Intra-arterial infusion of vanadate (VO/sub 4/) in dogs produces a reduction in renal blood flow (RBF), glomerular filtration rate (GFR), urine flow (V), and the fractional excretion of sodium (FE/sub Na/sup +//). To evaluate the role of Ca/sup 2 +/ in these changes VO/sub 4/ was infused into the renal artery in the presence of the calcium antagonists trifluoperazine (TFP), verapamil, or EGTA. TFP inhibited the effect of VO/sub 4/ on RBF (TFP + VO/sub 4/: 64.1, VO/sub 4/: 38.5 ml/min; P < 0.05), GFR (TFP + VO/sub 4/: 22.9, VO/sub 4/: 9.3, ml/min; P < 0.05) and V (TFP + VO/sub 4/: 0.80, VO/sub 4/: 0.38 ml/min; P < 0.05) without changing FE/sub Na/sup +// (TFP + VO/sub 4/: 3.8, VO/sub 4/: 3.2%). Similar changes were obtained with verapamil as well as with EGTA. Furthermore thyroparathyroidectomy (TPTX) decreased serum calcium (control: 8.78, TPTX: 4.98 mg/100ml; P < 0.05) and blunted the effects of VO/sub 4/ on renal hemodynamics. Reestablishing normal serum Ca/sup 2 +/ by an intra-arterial infusion of CaCl/sub 2/ elicited the VO/sub 4/ effects of vasoconstriction and decreased GFR; V was not affected and FE/sub Na/sup +// rose. The data support the idea that influx of extracellular calcium into smooth muscle cells mediates the hemodynamic effects of VO/sub 4/ in the dog. 34 references.

  20. Release of calcium from endolysosomes increases calcium influx through N-type calcium channels: Evidence for acidic store-operated calcium entry in neurons.

    PubMed

    Hui, Liang; Geiger, Nicholas H; Bloor-Young, Duncan; Churchill, Grant C; Geiger, Jonathan D; Chen, Xuesong

    2015-12-01

    Neurons possess an elaborate system of endolysosomes. Recently, endolysosomes were found to have readily releasable stores of intracellular calcium; however, relatively little is known about how such 'acidic calcium stores' affect calcium signaling in neurons. Here we demonstrated in primary cultured neurons that calcium released from acidic calcium stores triggered calcium influx across the plasma membrane, a phenomenon we have termed "acidic store-operated calcium entry (aSOCE)". aSOCE was functionally distinct from store-operated calcium release and calcium entry involving endoplasmic reticulum. aSOCE appeared to be governed by N-type calcium channels (NTCCs) because aSOCE was attenuated significantly by selectively blocking NTCCs or by siRNA knockdown of NTCCs. Furthermore, we demonstrated that NTCCs co-immunoprecipitated with the lysosome associated membrane protein 1 (LAMP1), and that aSOCE is accompanied by increased cell-surface expression levels of NTCC and LAMP1 proteins. Moreover, we demonstrated that siRNA knockdown of LAMP1 or Rab27a, both of which are key proteins involved in lysosome exocytosis, attenuated significantly aSOCE. Taken together our data suggest that aSOCE occurs in neurons, that aSOCE plays an important role in regulating the levels and actions of intraneuronal calcium, and that aSOCE is regulated at least in part by exocytotic insertion of N-type calcium channels into plasma membranes through LAMP1-dependent lysosome exocytosis.

  1. Release of O2- and LTC4 by murine eosinophils: role of intra- and extracellular calcium.

    PubMed Central

    de Andres, B; del Pozo, V; Martin, E; Palomino, P; Lahoz, C

    1990-01-01

    Using an experimental model of mouse peritoneal eosinophilia, we investigated the role of Ca2+ in the in vitro activation of these cells challenged with specific Mesocestoides corti antigen. We have detected LTC4, a metabolite derived from arachidonic acid by way of 5'lipo-oxygenase and superoxide anion from the oxidative burst, as inflammatory mediators produced by activated eosinophils. Preincubation with hyperimmune mice serum increases the amount of LTC4 and superoxide anion in response to the antigenic extract. Release of O2- is inhibited by Verapamil (a voltage-gated calcium channel) and Quin 2 (an intracellular trapped chelator of calcium). Also, LTC4 produced by preincubated eosinophils challenged with M. corti is dramatically inhibited by Quin 2. Our results suggest an intact mechanism for calcium control for the release of these inflammatory mediators by eosinophils, after specific antigenic stimulation. PMID:1689695

  2. Vertebrate extracellular calcium-sensing receptor evolution: selection in relation to life history and habitat.

    PubMed

    Herberger, Amanda L; Loretz, Christopher A

    2013-03-01

    Ionic calcium (Ca(2+)) supports essential functions within physiological systems, and consequently its concentration is homeostatically regulated within narrow bounds in the body fluids of animals through endocrine effects at ion-transporting osmoregulatory tissues. In vertebrates, extracellular Ca(2+) is detected at the cell surface by the extracellular calcium-sensing receptor (CaSR), a member of the G protein-coupled receptor (GPCR) superfamily. Interestingly, the taxonomic distribution of CaSRs is restricted to vertebrates, with some CaSR-like receptors apparently present in non-vertebrate chordates. Since bone is a known Ca(2+) storage site and is characteristically restricted to the vertebrate lineage, we hypothesized a functional association of CaSR with vertebrate skeleton that may have an ancient origin. Protein sequence alignment and phylogenetic analysis of vertebrate CaSRs and related GPCRs of the glutamate receptor-like family expose similarities and indel differences among these receptors, and reveal the evolutionary history of CaSRs. Evolutionary selection was tested statistically by evaluating the relationship between non-synonymous (replacement, dN) versus synonymous (silent, dS) amino acid substitution rates (as dN/dS) of protein-coding DNA sequences among branches of the estimated protein phylogeny. On a background of strong purifying selection (dN/dS<1) in the CaSR phylogeny, statistical evidence for adaptive evolution (dN/dS>1) was detected on some branches to major clades in the CaSR phylogeny, especially to the tetrapod vertebrate CaSRs and chordate CaSR-like branches. Testing also revealed overall purifying selection at the codon level. At some sites relaxation from strong purifying selection was seen, but evidence for adaptive evolution was not detected for individual sites. The results suggest purifying selection of CaSRs, and of adaptive evolution among some major vertebrate clades, reflecting clade specific differences in natural history

  3. Calcium intake increases risk of prostate cancer among Singapore Chinese

    PubMed Central

    Butler, Lesley M.; Wong, Alvin S.; Koh, Woon-Puay; Wang, Renwei; Yuan, Jian-Min; Yu, Mimi C.

    2010-01-01

    Consumption of dairy products, the primary source of calcium in Western diets, has been found to be positively associated with prostate cancer. In an Asian diet, non-dairy foods are the major contributors of calcium. Thus, a study of dietary calcium and prostate cancer in Asians can better inform on whether calcium, as opposed to other dairy components is responsible for the dairy foods-prostate cancer association. We examined calcium intake and prostate cancer risk among 27,293 men of the Singapore Chinese Health Study that was established between 1993 and 1998. As of December 31, 2007, 298 incident prostate cancer cases had been diagnosed among the cohort members. Diet was assessed at baseline with a validated 165-item food frequency questionnaire. It is hypothesized that there is greater net absorption of calcium in smaller individuals. Therefore, the calcium-prostate cancer association was also assessed in stratified analyses by median body mass index (BMI). Vegetables were the largest contributor of daily calcium intake in the study population. Overall, we observed a modest, statistically nonsignificant 25% increase in prostate cancer risk for the 4th (median = 659 mg/day) versus 1st (median=211 mg/day) quartiles of calcium intake after adjustment for potential confounders. The association became considerably stronger and achieved statistical significance (hazard ratio=2.03; 95% confidence interval: 1.23, 3.34; P for trend=0.01) for men with below median (22.9 kg/m2) BMI. Dietary calcium may be a risk factor for prostate cancer even at relatively low intake. PMID:20516117

  4. Wind-induced plant motion immediately increases cytosolic calcium.

    PubMed Central

    Knight, M R; Smith, S M; Trewavas, A J

    1992-01-01

    Wind is one of the most unusual and more dramatic of the environmental signals to modify plant development. Wind-stimulated crops are also known to experience considerable reductions in growth and subsequent yield. There is at present no experimental data to suggest how wind signals are perceived and transduced by plant cells. We have genetically transformed Nicotiana plumbaginifolia to express aequorin and thus produced luminous plants that directly report cytosolic calcium by emitting blue light. With these plants we have found wind stimulation to cause immediate increases in cytosolic calcium and our evidence, based on the use of specific inhibitors, suggests that this calcium is mobilized from organelle sources. Our data further suggest that wind-induced movement of tissues, by mechanically stimulating and stressing constituent plant cells, is responsible for the immediate elevation of cytosolic calcium; increases occur only when the plant tissue is actually in motion. Repeated wind stimulation renders the cells refractory to further calcium signaling but responsiveness is rapidly recovered when stimulation is subsequently diminished. Our data suggest that mechanoperception in plant cells may possibly be transduced through intracellular calcium. Since mechanoperception and transduction are considered crucial to plant morphogenesis, our observations suggest that calcium could be central in the control and generation of plant form. Images PMID:11536497

  5. Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression.

    PubMed

    Gómez-Sánchez, Rubén; Gegg, Matthew E; Bravo-San Pedro, José M; Niso-Santano, Mireia; Alvarez-Erviti, Lydia; Pizarro-Estrella, Elisa; Gutiérrez-Martín, Yolanda; Alvarez-Barrientos, Alberto; Fuentes, José M; González-Polo, Rosa Ana; Schapira, Anthony H V

    2014-02-01

    Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinson's disease (PD). This gene encodes a mitochondrial serine/threonine kinase, which is partly localized to mitochondria, and has been shown to play a role in protecting neuronal cells from oxidative stress and cell death, perhaps related to its role in mitochondrial dynamics and mitophagy. In this study, we report that increased mitochondrial PINK1 levels observed in human neuroblastoma SH-SY5Y cells after carbonyl cyanide m-chlorophelyhydrazone (CCCP) treatment were due to de novo protein synthesis, and not just increased stabilization of full length PINK1 (FL-PINK1). PINK1 mRNA levels were significantly increased by 4-fold after 24h. FL-PINK1 protein levels at this time point were significantly higher than vehicle-treated, or cells treated with CCCP for 3h, despite mitochondrial content being decreased by 29%. We have also shown that CCCP dissipated the mitochondrial membrane potential (Δψm) and induced entry of extracellular calcium through L/N-type calcium channels. The calcium chelating agent BAPTA-AM impaired the CCCP-induced PINK1 mRNA and protein expression. Furthermore, CCCP treatment activated the transcription factor c-Fos in a calcium-dependent manner. These data indicate that PINK1 expression is significantly increased upon CCCP-induced mitophagy in a calcium-dependent manner. This increase in expression continues after peak Parkin mitochondrial translocation, suggesting a role for PINK1 in mitophagy that is downstream of ubiquitination of mitochondrial substrates. This sensitivity to intracellular calcium levels supports the hypothesis that PINK1 may also play a role in cellular calcium homeostasis and neuroprotection.

  6. Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression☆

    PubMed Central

    Gómez-Sánchez, Rubén; Gegg, Matthew E.; Bravo-San Pedro, José M.; Niso-Santano, Mireia; Alvarez-Erviti, Lydia; Pizarro-Estrella, Elisa; Gutiérrez-Martín, Yolanda; Alvarez-Barrientos, Alberto; Fuentes, José M.; González-Polo, Rosa Ana; Schapira, Anthony H.V.

    2014-01-01

    Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinson's disease (PD). This gene encodes a mitochondrial serine/threonine kinase, which is partly localized to mitochondria, and has been shown to play a role in protecting neuronal cells from oxidative stress and cell death, perhaps related to its role in mitochondrial dynamics and mitophagy. In this study, we report that increased mitochondrial PINK1 levels observed in human neuroblastoma SH-SY5Y cells after carbonyl cyanide m-chlorophelyhydrazone (CCCP) treatment were due to de novo protein synthesis, and not just increased stabilization of full length PINK1 (FL-PINK1). PINK1 mRNA levels were significantly increased by 4-fold after 24 h. FL-PINK1 protein levels at this time point were significantly higher than vehicle-treated, or cells treated with CCCP for 3 h, despite mitochondrial content being decreased by 29%. We have also shown that CCCP dissipated the mitochondrial membrane potential (Δψm) and induced entry of extracellular calcium through L/N-type calcium channels. The calcium chelating agent BAPTA-AM impaired the CCCP-induced PINK1 mRNA and protein expression. Furthermore, CCCP treatment activated the transcription factor c-Fos in a calcium-dependent manner. These data indicate that PINK1 expression is significantly increased upon CCCP-induced mitophagy in a calcium-dependent manner. This increase in expression continues after peak Parkin mitochondrial translocation, suggesting a role for PINK1 in mitophagy that is downstream of ubiquitination of mitochondrial substrates. This sensitivity to intracellular calcium levels supports the hypothesis that PINK1 may also play a role in cellular calcium homeostasis and neuroprotection. PMID:24184327

  7. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium.

    PubMed Central

    Mason, S. J.; Paradiso, A. M.; Boucher, R. C.

    1991-01-01

    1 The role of extracellular nucleotides in regulation of ion transport activities (short circuit current, Isc) of human respiratory epithelia was studied. 2 Application of nucleotides to the apical or basolateral membrane of human nasal epithelium induced a concentration-dependent increase in Isc. 3 The rank order of potency of purine- or pyrimidine-induced changes in Isc of normal human nasal epithelium when applied to the apical membrane (UTP greater than or equal to ATP greater than ATP gamma S greater than 2MeSATP greater than ADP beta S much greater than beta gamma MeATP greater than or equal to alpha beta MeATP) or basolateral membrane (2MeSATP greater than UTP greater than ATP greater than ATP gamma S greater than alpha beta MeATP greater than beta gamma MeATP) is consistent with involvement of a P2 purinoceptor. A similar rank order of potencies was observed for nucleotide effects on intracellular calcium measured by Fura-2 fluorescence using microspectrofluorimetry. 4 Similar nucleotide potency in the regulation of ion transport and intracellular calcium in cystic fibrosis (CF) airway epithelium (UTP greater than or equal to ATP) was observed, suggesting purinoceptors might be used to stimulate ion transport processes that would promote hydration of airway secretions and facilitate their clearance from CF lungs. 5 These data provide evidence for the regulation of ion transport by P2 purinoceptors in normal and cystic fibrosis human airway epithelium. PMID:1718521

  8. Extracellular calcium acts as a “third messenger” to regulate enzyme and alkaline secretion

    PubMed Central

    Caroppo, Rosa; Gerbino, Andrea; Fistetto, Gregorio; Colella, Matilde; Debellis, Lucantonio; Hofer, Aldebaran M.; Curci, Silvana

    2004-01-01

    It is generally assumed that the functional consequences of stimulation with Ca2+-mobilizing agonists are derived exclusively from the second messenger action of intracellular Ca2+, acting on targets inside the cells. However, during Ca2+ signaling events, Ca2+ moves in and out of the cell, causing changes not only in intracellular Ca2+, but also in local extracellular Ca2+. The fact that numerous cell types possess an extracellular Ca2+ “sensor” raises the question of whether these dynamic changes in external [Ca2+] may serve some sort of messenger function. We found that in intact gastric mucosa, the changes in extracellular [Ca2+] secondary to carbachol-induced increases in intracellular [Ca2+] were sufficient and necessary to elicit alkaline secretion and pepsinogen secretion, independent of intracellular [Ca2+] changes. These findings suggest that extracellular Ca2+ can act as a “third messenger” via Ca2+ sensor(s) to regulate specific subsets of tissue function previously assumed to be under the direct control of intracellular Ca2+. PMID:15240573

  9. Roscovitine increases intracellular calcium release and capacitative calcium entry in PC12 cells.

    PubMed

    Choi, Ho Sook; Chung, Sul-Hee

    2010-01-18

    Cyclin-dependent kinase 5 (Cdk5), which is activated by the non-cyclin regulator p35 or p39, is a proline-directed serine/threonine kinase that is implicated in many physiological and pathological processes. Here, we studied calcium signaling using the fluorescent cytosolic calcium indicator, Fura-4, in NGF-differentiated PC12 cells treated with roscovitine, a Cdk5 inhibitor. As compared to the control cells, the roscovitine-treated cells significantly potentiated intracellular calcium release by membrane depolarization (high K(+)) or through thapsigargin. In addition, roscovitine increased the magnitude of capacitative calcium entry (CCE), i.e., a calcium influx mechanism triggered by the depletion of intracellular calcium stores. Notably, roscovitine markedly slowed the rate of Ca(2+) removal from the plasma membrane. These results suggest that Cdk5 regulates intracellular calcium homeostasis and that the dysregulation of Cdk5 may contribute to disease pathogenesis by perturbing cellular Ca(2+) signaling. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

  10. Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium

    PubMed Central

    1990-01-01

    Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from

  11. Mechanical stimulation evokes rapid increases in extracellular adenosine concentration in the prefrontal cortex.

    PubMed

    Ross, Ashley E; Nguyen, Michael D; Privman, Eve; Venton, B Jill

    2014-07-01

    Mechanical perturbations can release ATP, which is broken down to adenosine. In this work, we used carbon-fiber microelectrodes and fast-scan cyclic voltammetry to measure mechanically stimulated adenosine in the brain by lowering the electrode 50 μm. Mechanical stimulation evoked adenosine in vivo (average: 3.3 ± 0.6 μM) and in brain slices (average: 0.8 ± 0.1 μM) in the prefrontal cortex. The release was transient, lasting 18 ± 2 s. Lowering a 15-μm-diameter glass pipette near the carbon-fiber microelectrode produced similar results as lowering the actual microelectrode. However, applying a small puff of artificial cerebral spinal fluid was not sufficient to evoke adenosine. Multiple stimulations within a 50-μm region of a slice did not significantly change over time or damage cells. Chelating calcium with EDTA or blocking sodium channels with tetrodotoxin significantly decreased mechanically evoked adenosine, signifying that the release is activity dependent. An alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, did not affect mechanically stimulated adenosine; however, the nucleoside triphosphate diphosphohydrolase 1,2 and 3 (NTDPase) inhibitor POM-1 significantly reduced adenosine so a portion of adenosine is dependent on extracellular ATP metabolism. Thus, mechanical perturbations from inserting a probe in the brain cause rapid, transient adenosine signaling which might be neuroprotective. We have discovered immediate changes in adenosine concentration in the prefrontal cortex following mechanical stimulation. The adenosine increase lasts only about 20 s. Mechanically stimulated adenosine was activity dependent and mostly because of extracellular ATP metabolism. This rapid, transient increase in adenosine may help protect tissue and would occur during implantation of any electrode, such as during deep brain stimulation.

  12. Understanding spatial and temporal patterning of astrocyte calcium transients via interactions between network transport and extracellular diffusion

    NASA Astrophysics Data System (ADS)

    Shtrahman, E.; Maruyama, D.; Olariu, E.; Fink, C. G.; Zochowski, M.

    2017-02-01

    Astrocytes form interconnected networks in the brain and communicate via calcium signaling. We investigate how modes of coupling between astrocytes influence the spatio-temporal patterns of calcium signaling within astrocyte networks and specifically how these network interactions promote coordination within this group of cells. To investigate these complex phenomena, we study reduced cultured networks of astrocytes and neurons. We image the spatial temporal patterns of astrocyte calcium activity and quantify how perturbing the coupling between astrocytes influences astrocyte activity patterns. To gain insight into the pattern formation observed in these cultured networks, we compare the experimentally observed calcium activity patterns to the patterns produced by a reduced computational model, where we represent astrocytes as simple units that integrate input through two mechanisms: gap junction coupling (network transport) and chemical release (extracellular diffusion). We examine the activity patterns in the simulated astrocyte network and their dependence upon these two coupling mechanisms. We find that gap junctions and extracellular chemical release interact in astrocyte networks to modulate the spatiotemporal patterns of their calcium dynamics. We show agreement between the computational and experimental findings, which suggests that the complex global patterns can be understood as a result of simple local coupling mechanisms.

  13. The extracellular calcium-sensing receptor regulates human fetal lung development via CFTR.

    PubMed

    Brennan, Sarah C; Wilkinson, William J; Tseng, Hsiu-Er; Finney, Brenda; Monk, Bethan; Dibble, Holly; Quilliam, Samantha; Warburton, David; Galietta, Luis J; Kemp, Paul J; Riccardi, Daniela

    2016-02-25

    Optimal fetal lung growth requires anion-driven fluid secretion into the lumen of the developing organ. The fetus is hypercalcemic compared to the mother and here we show that in the developing human lung this hypercalcaemia acts on the extracellular calcium-sensing receptor, CaSR, to promote fluid-driven lung expansion through activation of the cystic fibrosis transmembrane conductance regulator, CFTR. Several chloride channels including TMEM16, bestrophin, CFTR, CLCN2 and CLCA1, are also expressed in the developing human fetal lung at gestational stages when CaSR expression is maximal. Measurements of Cl(-)-driven fluid secretion in organ explant cultures show that pharmacological CaSR activation by calcimimetics stimulates lung fluid secretion through CFTR, an effect which in humans, but not mice, was also mimicked by fetal hypercalcemic conditions, demonstrating that the physiological relevance of such a mechanism appears to be species-specific. Calcimimetics promote CFTR opening by activating adenylate cyclase and we show that Ca(2+)-stimulated type I adenylate cyclase is expressed in the developing human lung. Together, these observations suggest that physiological fetal hypercalcemia, acting on the CaSR, promotes human fetal lung development via cAMP-dependent opening of CFTR. Disturbances in this process would be expected to permanently impact lung structure and might predispose to certain postnatal respiratory diseases.

  14. Vitamin D supplementation increases calcium absorption without a threshold effect

    USDA-ARS?s Scientific Manuscript database

    The maximal calcium absorption in response to vitamin D has been proposed as a biomarker for vitamin D sufficiency. Our objective was to determine whether there is a threshold beyond which increasing doses of vitamin D, or concentrations of serum 25-hydroxyvitamin D [25(OH)D], no longer increase cal...

  15. [Calcium supplementation and the possible increase in cardiovascular risk].

    PubMed

    Montero Sáez, Abelardo; Formiga, Francesc; Pujol Farriols, Ramón

    2013-01-01

    The primary goal of osteoporosis treatment is to prevent the occurrence of fragility fractures, and thereby reduce morbidity and mortality. Among the various approaches to the treatment of this disease include ensuring proper calcium intake and to obtain adequate levels of vitamin D. Virtually all clinical trials with drugs used to treat osteoporosis systematically include calcium and vitamin D supplements. In light of the recent publication of clinical trials and meta-analyses, a possible increase in cardiovascular risk, particularly in the form of a myocrdial infarction, is hypothesised in patients taking calcium supplements. However, data published to date are inconclusive. Until the development of new scientific evidence, it seems reasonable to recommend, whenever practicable and individualized for each patient, increasing calcium intake with food and reserve supplements for patients with very low calcium intake in the diet. It would also be advisable for the administration of total daily dose to be fractionated throughout the day and with meals, and to obtain appropriate levels of vitamin D (25-hydroxycholecalciferol or calcidiol), along with the basic treatment for osteoporosis that is decided to be prescribed to patients.

  16. P2X7 receptor-pannexin 1 hemichannel association: effect of extracellular calcium on membrane permeabilization.

    PubMed

    Poornima, V; Madhupriya, M; Kootar, S; Sujatha, G; Kumar, Arvind; Bera, Amal Kanti

    2012-03-01

    Activation of P2X(7) receptor (P2X(7)R) and pannexin have been implicated in membrane permeabilization associated with ischemic cell death and many other inflammatory processes. P2X(7)R has a unique property of forming large pore upon repeated or prolonged application of agonist like ATP or 2', 3'-(4-benzoyl) benzoyl ATP. It has been proposed that pannexin 1 (panx1) hemichannel associates with P2X(7)R to form large pore, though the actual mechanism is not yet understood. Calcium concentration in extracellular milieu drops in many patho-physiological conditions, e.g. ischemia, when P2X(7)R/pannexin is also known to be activated. Therefore, we hypothesize that extracellular calcium ([Ca(2+)](o)) plays an important role in the coupling of P2X(7)R-panx1 and subsequent membrane permeabilization. In this study we show that membrane permeability of the P2X(7)R and panx1 expressing N2A cell increases in ([Ca(2+)](o))-free solution. In [Ca(2+)](o)-free solution, fluorescent dye calcein trapped cells exhibited time-dependent dye leakage resulting in about 50% decrease of fluorescence intensity in 30 min. Control cells in 2 mM [Ca(2+)](o) did not show such leakage. Like N2A cells, mixed culture of neuron and glia, derived from hippocampal progenitor cells showed similar dye leakage. Dye leakage was blocked either by pannexin-specific blocker, carbenoxolone or P2X(7)R antagonists, Brilliant Blue G, and oxidized ATP. Furthermore P2X(7)R and panx1 were co-immunoprecipitated. The amount of P2X(7)R protein pulled-down with panx1, increased by twofold when cells were incubated 30 min in [Ca(2+)](o)-free buffer. Taken together, the results of this study demonstrate the activation and association of P2X(7)R-panx1, triggered by the removal of [Ca(2+)](o).

  17. Cell cavities increase tortuosity in brain extracellular space.

    PubMed

    Tao, A; Tao, L; Nicholson, C

    2005-06-21

    Brain extracellular space (ECS) forms hindered pathways for molecular diffusion in chemical signaling and drug delivery. Hindrance is quantified by the tortuosity lambda; the tortuosity obtained from simulations using uniformly spaced convex cells is significantly lower than that measured experimentally. To attempt to account for the difference in results, this study employed a variety of ECS models based on an array of cubic cells containing open rectangular cavities that provided the ECS with dead-space microdomains. Monte Carlo simulations demonstrated that, in such ECS models, lambda can equal or exceed the typical experimental value of about 1.6. The simulations further revealed that lambda is relatively independent of cavity shape and the number of cavities per cell. It mainly depends on the total ECS volume fraction alpha, the cavity volume fraction alpha(c), and whether the cavity is located at the center of a cell face or formed at the junction of multiple cells. To describe the results from the different ECS models, an expression was obtained that related lambda to alpha, alpha(c), and an empirical exit factor beta that correlated with the ease with which a molecule could leave a cavity and its vicinity.

  18. Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Ye, C.; Vassilev, P. M.; Sanders, J. L.; Brown, E. M.

    2001-01-01

    We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

  19. Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Ye, C.; Vassilev, P. M.; Sanders, J. L.; Brown, E. M.

    2001-01-01

    We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

  20. Increased Cortical Extracellular Adenosine Correlates with Seizure Termination

    PubMed Central

    Van Gompel, Jamie J.; Bower, Mark R.; Worrell, Gregory A.; Stead, Matt; Chang, Su-Youne; Goerss, Stephan J.; Kim, Inyong; Bennet, Kevin E.; Meyer, Fredric B.; Marsh, W. Richard; Blaha, Charles D.; Lee, Kendall H.

    2014-01-01

    Objective Seizures are currently defined by their electrographic features. However, neuronal networks are intrinsically dependent upon neurotransmitters of which little is known regarding their peri-ictal dynamics. Evidence supports adenosine as having a prominent role in seizure termination, as its administration can terminate and reduce seizures in animal models. Further, microdialysis studies in humans suggest adenosine is elevated peri-ictally, but the relationship to the seizure is obscured by its temporal measurement limitations. Because electrochemical techniques can provide vastly superior temporal resolution, we test the hypothesis that extracellular adenosine concentrations rise during seizure termination in an animal model and humans using electrochemistry. Methods White farm swine (n=45) were used in an acute cortical model of epilepsy and 10 human epilepsy patients were studied during intraoperative electrocorticography (Ecog). Wireless Instantaneous Neurotransmitter Concentration Sensor (WINCS) based fast scan cyclic voltametry (FSCV) and fixed potential amperometry were obtained utilizing an adenosine specific triangular waveform or biosensors respectively. Results Simultaneous Ecog and electrochemistry demonstrated an average adenosine rise of 260% compared to baseline at 7.5 ± 16.9 seconds with amperometry (n=75 events) and 2.6 ± 11.2 seconds with FSCV (n=15 events) prior to electrographic seizure termination. In agreement with these animal data, adenosine elevation prior to seizure termination in a human patient utilizing FSCV was also seen. Significance Simultaneous Ecog and electrochemical recording supports the hypothesis that adenosine rises prior to seizure termination, suggesting that adenosine itself may be responsible for seizure termination. Future work using intraoperative WINCS based FSCV recording may help to elucidate the precise relationship between adenosine and seizure termination. PMID:24483230

  1. Increasing of Mechanical Parameters of Clay soil Using Calcium Chloride

    NASA Astrophysics Data System (ADS)

    Beheshty, Seyyed Amir Hossein; Moosa Aniran, Mir; Firoozfar, Alireza; Kiamehr, Ramin

    2017-04-01

    Research on roads to increase the resistance of weak soils to build structures on it has been increased in recent years. The present article provide the effects of different mixtures containing calcium chloride solution and clay soil on mechanical parameters such as, compressibility, compressive strength, shear strength and durability characteristic. In this study also is investigated evaluation the effect of road subgrade based on proposed material. The used clay soil in this research was obtained from zanjan city where is located in northwestern of Iran. The obtained results show that the calcium chloride solution could play a major role in reducing the cost and required time for building roads and also building foundation on these types of soils.

  2. Does extracellular calcium determine what pool of GABA is the target for alpha-latrotoxin?

    PubMed

    Storchak, L G; Linetska, M V; Himmelreich, N H

    2002-04-01

    Presynaptic neurotoxin alpha-latrotoxin, from the venom of Latrodectus mactans tredecimguttatus, causes massive [(3)H]GABA release from rat brain synaptosomes, irrespective of calcium presence in the extracellular medium. Whether the binding of alpha-latrotoxin to Ca(2+)-dependent (neurexin 1 alpha) or to Ca(2+)-independent (latrophilin) receptor triggers [(3)H]GABA release by the same mechanisms or different ones, inducing either exocytotic process or outflow by mobile membrane GABA transporter, is unknown. We examined alpha-latrotoxin-evoked [(3)H]GABA release from synaptosomes which cytosolic [(3)H]GABA pool was depleted either by applying competitive inhibitors of the GABA transporter, nipecotic acid and 2,4-diaminobutyric acid, or by permeation with digitonin. We also compared the effect of the GABA transporter inhibitors on depolarisation-evoked and alpha-latrotoxin-evoked [(3)H]GABA release using as depolarising agents 4-aminopyridine and high KCl in the Ca(2+)-containing and in Ca(2+)-free medium, respectively. Incubation of synaptosomes with nipecotic acid induced the essential acceleration of unstimulated [(3)H]GABA release and deep inhibition of high KCl-evoked Ca(2+)-independent [(3)H]GABA release. In contrast, at the similar conditions the effect of alpha-latrotoxin was greatly augmented with respect to the control response. Another way to assay what GABA pool was involved in alpha-latrotoxin-induced release lays in an analysis of the effects of depolarisation and alpha-latrotoxin in consecutive order. The preliminary 4-aminopyridine-stimulated [(3)H]GABA release attenuated the toxin effect. But when depolarisation occurred in Ca(2+)-free medium, no influence on alpha-latrotoxin effect was revealed. Employing digitonin-permeated synaptosomes, we have shown that alpha-latrotoxin could stimulate [3H]GABA release in the medium with 1mM EGTA, this effect of the toxin was blocked by concanavalin A and was ATP-dependent. The latter suggests that alpha

  3. The extracellular calcium-sensing receptor is required for cholecystokinin secretion in response to L-phenylalanine in acutely isolated intestinal I cells.

    PubMed

    Liou, Alice P; Sei, Yoshitatsu; Zhao, Xilin; Feng, Jianying; Lu, Xinping; Thomas, Craig; Pechhold, Susanne; Raybould, Helen E; Wank, Stephen A

    2011-04-01

    The extracellular calcium-sensing receptor (CaSR) has recently been recognized as an L-amino acid sensor and has been implicated in mediating cholecystokinin (CCK) secretion in response to aromatic amino acids. We investigated whether direct detection of L-phenylalanine (L-Phe) by CaSR results in CCK secretion in the native I cell. Fluorescence-activated cell sorting of duodenal I cells from CCK-enhanced green fluorescent protein (eGFP) transgenic mice demonstrated CaSR gene expression. Immunostaining of fixed and fresh duodenal tissue sections confirmed CaSR protein expression. Intracellular calcium fluxes were CaSR dependent, stereoselective for L-Phe over D-Phe, and responsive to type II calcimimetic cinacalcet in CCK-eGFP cells. Additionally, CCK secretion by an isolated I cell population was increased by 30 and 62% in response to L-Phe in the presence of physiological (1.26 mM) and superphysiological (2.5 mM) extracellular calcium concentrations, respectively. While the deletion of CaSR from CCK-eGFP cells did not affect basal CCK secretion, the effect of L-Phe or cinacalcet on intracellular calcium flux was lost. In fact, both secretagogues, as well as superphysiological Ca(2+), evoked an unexpected 20-30% decrease in CCK secretion compared with basal secretion in CaSR(-/-) CCK-eGFP cells. CCK secretion in response to KCl or tryptone was unaffected by the absence of CaSR. The present data suggest that CaSR is required for hormone secretion in the specific response to L-Phe by the native I cell, and that a receptor-mediated mechanism may inhibit hormone secretion in the absence of a fully functional CaSR.

  4. Increased calcium bioavailability in mice fed genetically engineered plants lacking calcium oxalate

    USDA-ARS?s Scientific Manuscript database

    Bioavailable calcium affects bone formation and calcification. Here we investigate how a single gene mutation altering calcium partitioning in the model forage crop Medicago truncatula affects calcium bioavailability. Previously, the cod5 M. truncatula mutant was identified which contains identical ...

  5. Increased serum serotonin improves parturient calcium homeostasis in dairy cows.

    PubMed

    Hernández-Castellano, Lorenzo E; Hernandez, Laura L; Weaver, Samantha; Bruckmaier, Rupert M

    2017-02-01

    Hypocalcemia in dairy cows is caused by the sudden increase in calcium demand by the mammary gland for milk production at the onset of lactation. Serotonin (5-HT) is a key factor for calcium homeostasis, modulating calcium concentration in blood. Therefore, it is hypothesized that administration of 5-hydroxy-l-tryptophan (5-HTP), a 5-HT precursor, can increase 5-HT concentrations in blood and, in turn, induce an increase in blood calcium concentration. In this study, 20 Holstein dairy cows were randomly assigned to 2 experimental groups. Both groups received a daily i.v. infusion of 1 L of either 0.9% NaCl (C group; n = 10) or 0.9% NaCl containing 1 mg of 5-HTP/kg of BW (5-HTP group, n = 10). Infusions started d 10 before the estimated parturition and ceased the day of parturition, resulting in at least 4 d of infusion (8.37 ± 0.74 d of infusion). Until parturition, blood samples were collected every morning before the infusions, after parturition samples were taken daily until d 7, and a final sample was collected on d 30. Milk yield was recorded during this period. No differences between groups were observed for blood glucose, magnesium, and β-hydroxybutyrate. Cows receiving the 5-HTP infusion showed an increase in fatty acid concentrations from d -3 to -1 before parturition. Serum 5-HT concentrations were increased at d -4 related to parturition until d 5 postpartum in the 5-HTP group compared with the C group. In addition, cows from the 5-HTP group had increased 5-HT concentrations in colostrum, but not in mature milk, on d 7 postpartum. Serum calcium concentrations decreased in both groups around parturition; however, calcium remained higher in the 5-HTP group than in controls, with a significant difference between groups on d 1 (1.62 ± 0.08 vs. 1.93 ± 0.09 mmol/L in control and 5-HTP groups, respectively) and d 2 (1.83 ± 0.06 vs. 2.07 ± 0.07 mmol/L in control and 5-HTP groups, respectively). Additionally, colostrum yield (first milking) was lower in the

  6. Calciotropic hormones and lipolysis of human adipose tissue: role of extracellular calcium as conditioning but not regulating factor.

    PubMed

    Ziegler, R; Jobst, W; Minne, H; Faulhaber, J D

    1980-01-01

    The influences of different calcium concentrations (0, 0.924 and 2.772 mMol/l) on lipolysis of in vitro incubated human adipose tissue slices or adipocytes were studied under the conditions of stimulation with isoproterenol and parathyroid hormone preparations or inhibition by insulin. Extractive bovine PTH (as well as synthetic PTH 1--34) stimulated glycerol release in a biphasic pattern similarly to isoproterenol; PTH was about half as potent as isoproterenol. The optimal conditions for lipolysis were observed using a calcium concentration of 0.924 mMol/l, whereas lipolysis was distinctly impaired at concentrations of 0 or 2.772 mMol/l; this was true for basal as well as isoproterenol- and PTH stimulated lipolysis or the inhibitory effect of insulin. In contrast to partially purified extractive calcitonin, pure synthetic calcitonin did not inhibit lipolysis. Isoproterenol- and PTH-administrations led to cAMP accumulation in the adipose tissue, this process was also diminished at the non-optimal calcium concentrations. The results suggest a conditioning, but not a regulating significance of extracellular calcium for lipolysis, whereas the importance of the lipolytic potency of PTH remains to be elucidated.

  7. β-phenylethylamine Requires the Dopamine Transporter to Increase Extracellular Dopamine in C. elegans Dopaminergic Neurons

    PubMed Central

    Hossain, Murad; Wickramasekara, Rochelle N.; Carvelli, Lucia

    2013-01-01

    β-phenylethylamine (βPEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by βPEA, however the mechanism through which βPEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the βPEA-induced increase of extracellular DA required DAT-1. Our data show that βPEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the βPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by βPEA. These results suggest that βPEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that βPEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of βPEA to increase extracellular DA. Finally, our electrophysiology data show that βPEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, βPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA. PMID:24161617

  8. β-Phenylethylamine requires the dopamine transporter to increase extracellular dopamine in Caenorhabditis elegans dopaminergic neurons.

    PubMed

    Hossain, Murad; Wickramasekara, Rochelle N; Carvelli, Lucia

    2014-07-01

    β-Phenylethylamine (βPEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by βPEA, however the mechanism through which βPEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the βPEA-induced increase of extracellular DA required DAT-1. Our data show that βPEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the βPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by βPEA. These results suggest that βPEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that βPEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of βPEA to increase extracellular DA. Finally, our electrophysiology data show that βPEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, βPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium

    PubMed Central

    London, Fredda S.; Marcinkiewicz, Mariola; Walsh, Peter N.

    2008-01-01

    We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 seconds and positive factor IXa binding by 2 minutes, with calcium transients sustained for 45 minutes. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365 or LaCl3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5 to 20 minutes before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (Kd 160 nM) had minimal effects, 100 μM 5, 5′-dimethylBAPTA/AM (Kd 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients. PMID:16752917

  10. Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes.

    PubMed

    Micallef, Ludovic; Belaubre, Françoise; Pinon, Aline; Jayat-Vignoles, Chantal; Delage, Christiane; Charveron, Marie; Simon, Alain

    2009-02-01

    The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro, the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time-period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 mm. In NHK, addition of calcium-induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2-M phases. Furthermore, RT-PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium-induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.

  11. Cholinergic modulation of extracellular ATP-induced cytoplasmic calcium concentrations in cochlear outer hair cells.

    PubMed

    Wikström, M A; Lawoko, G; Heilbronn, E

    1998-01-01

    Outer hair cells (OHC) of the mammalian cochlea modulate the inner hair cell (IHC) mechanoelectrical transduction of sound. They are contacted by synapsing efferent neurons from the CNS, their main efferent neurotransmitter being acetylcholine (ACh). OHC function and in particular their control of [Ca2+]i is highly important and is modulated by ACh and also by other substances including extracellular (EC) ATP. OHC carry at their efferent synapse a not yet completely identified neuronal type of ionotropic ACh receptor (AChR), with an unusual pharmacology, which is, in vivo and in vitro, reversibly blocked by alpha-bungarotoxin (alpha-bgtx). The AChR mediates a fast influx of Ca2+ into OHC which, in turn, activates a closeby located outwardly-directed Ca(2+)-dependent K(+)-channel, thus shortly hyperpolarizing the cell. A cloned homomeric alpha 9 nAChR mimicks the function and pharmacology of this receptor. We here report results from a study designed to observe only slower effects triggered by EC ATP and the ACh-AChR system. EC presence of ATP at OHC increases [Ca2+]i by activating both P2x and P2y purinoceptors and also by indirect activation of OHC L-type Ca(2+)-channels. The L-type channel activation is responsible for a large part of the [Ca2+]i increase. Simultaneous EC presence of ACh and ATP at OHC was found to depress ATP-induced effects on OHC [Ca2+]i, an effect that is completely blocked in the presence of alpha-bgtx. Our observations suggest that the ACh-AChR system is involved in the modulation of the observed EC ATP-triggered events; possibly the OHC AChR is able to act both in its well known rapid ionotropic way, but also, perhaps after modification in a slower, metabotropic way interfering with the EC ATP-induced [Ca2+]i increase.

  12. Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKKβ-AMPK-SIRT1 Signaling Pathway

    PubMed Central

    Zhu, Yuanfeng; Yang, Yongjun; Chen, Xiaoli; Fan, Shijun; Chen, Qian; Zheng, Jiang

    2016-01-01

    Activated macrophages are the primary sources of IL-12, a key cytokine bridging innate and adaptive immunity. However, macrophages produce low amounts of IL-12 upon stimulation and the underlying regulatory mechanism remains unclear. In this study, we found a new calcium-dependent mechanism that controlled IL-12 production in LPS-treated murine macrophages. First, LPS was demonstrated to induce extracellular calcium entry in murine peritoneal macrophages and inhibition of calcium influx resulted in marked enhancement in IL-12 production. Then, withdrawal of extracellular calcium was found to suppress CaMKKβ and AMPK activation triggered by LPS while chemical inhibition or genetic knockdown of these two kinases augmented LPS induced IL-12 production. AMPK activation increased the NAD+/NADH ratio and activated Sirtuin 1 (SIRT1), a NAD+-dependent deacetylating enzyme and negative regulator of inflammation. Chemical inhibitor or siRNA of SIRT1 enhanced IL-12 release while its agonist suppressed IL-12 production. Finally, it was found that SIRT1 selectively affected the transcriptional activity of NF-κB which thereby inhibited IL-12 production. Overall, our study demonstrates a new role of transmembrane calcium mobilization in immunity modulation such that inhibition of calcium influx leads to impaired activation of CaMKKβ-AMPK-SIRT1 signaling pathway which lifts restriction on NF-κB activation and results in enhanced IL-12 production. PMID:27313401

  13. Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

    SciTech Connect

    Feng, Shan-Li; Sun, Ming-Rui; Li, Ting-Ting; Yin, Xin; Xu, Chang-Qing; Sun, Yi-Hua

    2011-03-11

    Research highlights: {yields} Calcium-sensing receptor (CaR) activation stimulates TRP channels. {yields} CaR promoted transient receptor potential C3 (TRPC3) expression. {yields} Adult rat ventricular myocytes display capacitative calcium entry (CCE), which was operated by TRPCs. {yields} TRPC channels activation induced by CaR activator sustained the increased [Ca{sup 2+}]{sub i} to evoke cardiomyocytes apoptosis. -- Abstract: Transient receptor potential (TRP) channels are expressed in cardiomyocytes, which gate a type of influx of extracellular calcium, the capacitative calcium entry. TRP channels play a role in mediating Ca{sup 2+} overload in the heart. Calcium-sensing receptors (CaR) are also expressed in rat cardiac tissue and promote the apoptosis of cardiomyocytes by Ca{sup 2+} overload. However, data about the link between CaR and TRP channels in rat heart are few. In this study, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of the TRP canonical proteins TRPC1 and TRPC3 in adult and neonatal rat cardiomyocytes. Laser scan confocal microscopy was used to detect intracellular [Ca{sup 2+}]{sub i} levels in isolated adult rat ventricular myocytes. The results showed that, in adult rat cardiomyocytes, the depletion of Ca{sup 2+} stores in the endoplasmic/sarcoplasmic reticulum (ER/SR) by thapsigargin induced a transient increase in [Ca{sup 2+}]{sub i} in the absence of [Ca{sup 2+}]{sub o} and the subsequent restoration of [Ca{sup 2+}]{sub o} sustained the increased [Ca{sup 2+}]{sub i} for a few minutes, whereas, the persisting elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of the TRPC inhibitor SKF96365. The stimulation of CaR by its activator gadolinium chloride (GdCl{sub 3}) or spermine also resulted in the same effect and the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of [Ca{sup 2+}]{sub o}. In adult and neonatal rat cardiomyocytes, GdCl{sub 3

  14. Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells

    SciTech Connect

    Ziegelstein, Roy C.; Xiong Yali; He Chaoxia; Hu Qinghua . E-mail: qinghuaa@jhmi.edu

    2006-03-31

    Extracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub o}) regulates the functions of many cell types through a G protein-coupled [Ca{sup 2+}]{sub o}-sensing receptor (CaR). Whether the receptor is functionally expressed in vascular endothelial cells is largely unknown. In cultured human aortic endothelial cells (HAEC), RT-PCR yielded the expected 555-bp product corresponding to the CaR, and CaR protein was demonstrated by fluorescence immunostaining and Western blot. RT-PCR also demonstrated the expression in HAEC of alternatively spliced variants of the CaR lacking exon 5. Although stimulation of fura 2-loaded HAEC by several CaR agonists (high [Ca{sup 2+}]{sub o}, neomycin, and gadolinium) failed to increase intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}), the CaR agonist spermine stimulated an increase in [Ca{sup 2+}]{sub i} that was diminished in buffer without Ca{sup 2+} and was abolished after depletion of an intracellular Ca{sup 2+} pool with thapsigargin or after blocking IP{sub 3}- and ryanodine receptor-mediated Ca{sup 2+} release with xestospongin C and with high concentration ryanodine, respectively. Spermine stimulated an increase in DAF-FM fluorescence in HAEC, consistent with NO production. Both the increase in [Ca{sup 2+}]{sub i} and in NO production were reduced or absent in HAEC transfected with siRNA specifically targeted to the CaR. HAEC express a functional CaR that responds to the endogenous polyamine spermine with an increase in [Ca{sup 2+}]{sub i}, primarily due to release of IP{sub 3}- and ryanodine-sensitive intracellular Ca{sup 2+} stores, leading to the production of NO. Expression of alternatively spliced variants of the CaR may result in the absence of a functional response to other known CaR agonists in HAEC.

  15. Extracellular norepinephrine in the medial hypothalamus increases during feeding in chicks: a microdialysis study.

    PubMed

    Tachibana, T; Utimura, D; Kato, H; Kubo, T; Sugahara, K

    2000-11-01

    Norepinephrinergic function in the medial hypothalamus is important for the regulation of feeding behavior in chicks as well as in rats. This study was conducted to clarify the variation of extracellular norepinephrine (NE) in the medial hypothalamus, including the paraventricular nucleus (PVN) and the ventromedial hypothalamic nucleus (VMN), during feeding behavior of layer-type chicks. To measure extracellular NE and 4-hydroxy-3-methoxyphenylglycol (MHPG), a major metabolite of NE, we used microdialysis and high-pressure liquid chromatography (HPLC) with electrochemical detection. After the collection of baseline samples, food-deprived animals were allowed access to the food for 3 h. Extracellular NE significantly increased during the first hour of access to food, and then returned to baseline levels. MHPG also increased during the feeding, but its increase continued throughout the remainder of the experiment. This study suggests that the variation of NE in the medial hypothalamus may be involved in the control of feeding in layer-type chicks.

  16. Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels.

    PubMed

    Zeng, Xu-Hui; Xia, Xiao-Ming; Lingle, Christopher J

    2003-06-01

    An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.

  17. T-type calcium channels contribute to calcium disturbances in brain during hyponatremia.

    PubMed

    Odackal, John; Sherpa, Ang D; Patel, Nisha; Colbourn, Robert; Hrabetova, Sabina

    2015-11-01

    Disturbance of calcium homeostasis is implicated in the normal process of aging and brain pathology prevalent in the elderly such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. Previous studies demonstrated that applying a hyponatremic iso-osmotic (low-NaCl) artificial cerebrospinal fluid (ACSF) to rodent hippocampus causes extracellular calcium to rapidly decrease. Restoring normonatremia after low-NaCl treatment causes a rapid increase in extracellular calcium that overshoots baseline. This study examined the amplitude, timing, and mechanism of these surprising calcium changes. We also tested whether hyponatremia increased calcium entry into brain cells or calcium binding to chondroitin sulfate (CS), a negatively charged constituent of the extracellular matrix (ECM) that may be occupied by sodium during normonatremia. We report three major findings. First we show that CS does not contribute to extracellular calcium changes during low-NaCl treatments. Second, we show that the time to minimum extracellular calcium during low-NaCl treatment is significantly shorter than the time to maximum extracellular calcium in recovery from low-NaCl treatment. Third, we show that the decrease in extracellular calcium observed during hyponatremia is attenuated by ML 218, a highly selective T-type calcium channel blocker. Together these data suggest that calcium rapidly enters cells at the onset of low-NaCl treatment and is extruded from cells when normonatremia is restored. Calcium binding to CS does not significantly contribute to calcium changes in brain during hyponatremia. Differences in timing suggest that extracellular calcium changes during and in recovery from hyponatremia occur by distinct mechanisms or by a multistep process. Finally, partial block of extracellular calcium influx by ML 218 suggests that T-type channels are involved in calcium entering cells during hyponatremia. Given the high prevalence of hyponatremia among elderly patients and the

  18. Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α*

    PubMed Central

    Jiang, Jason Y.; Nagaraju, Mulpuri; Meyer, Rebecca C.; Zhang, Li; Hamelberg, Donald; Hall, Randy A.; Brown, Edward M.; Conn, P. Jeffrey; Yang, Jenny J.

    2014-01-01

    Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca2+ enhanced mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca2+ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α. PMID:24280223

  19. Calcium fertilization increases the concentration of calcium in sapwood and calcium oxalate in foliage of red spruce

    Treesearch

    Kevin T. Smith; Walter C. Shortle; Jon H. Connolly; Rakesh Minocha; Jody Jellison

    2009-01-01

    Calcium cycling plays a key role in the health and productivity of red spruce forests in the northeastern US. A portion of the flowpath of calcium within forests includes translocation as Ca2+ in sapwood and accumulation as crystals of calcium oxalate in foliage. Concentrations of Ca in these tree tissues have been used as markers of...

  20. Acetylcholine increases intracellular calcium of arterial chemoreceptor cells of adult cats.

    PubMed

    Shirahata, M; Fitzgerald, R S; Sham, J S

    1997-11-01

    Acetylcholine increases intracellular calcium of arterial chemoreceptor cells of adult cats. J. Neurophysiol. 78: 2388-2395, 1997. Several neurotransmitters have been reported to play important roles in the chemoreception of the carotid body. Among them acetylcholine (ACh) appears to be involved in excitatory processes in the cat carotid body. As one of the steps to elucidate possible roles of ACh in carotid body chemoreception in the cat, we examined the effect of ACh on intracellular calcium concentration ([Ca2+]i) of cultured carotid body cells. The carotid body from adult cats was dissociated and cultured for up to 2 wk. [Ca2+]i was measured from clusters of cells with a microfluorometric technique using Indo-1 AM. Experiments were performed at 37 degrees C, and cells were continuously superfused with modified Krebs solutions equilibrated with 5% CO2-16% O2-79% N2. ACh (100 mu M) caused a marked increase in [Ca2+]i in approximately 70% of clusters, and the responses to 1-300 mu M of ACh were concentration dependent. The magnitude and kinetics of the ACh response were mimicked by the application of nicotine, whereas muscarinic agonists, pilocarpine, and muscarine failed to evoke a similar response. ACh-induced increase in [Ca2+]i was dependent on extracellular Ca2+: it was greatly reduced or completely abolished by a transient removal of extracellular Ca2+. The response was consistently but only partially reduced by caffeine (5 mM) or nifedipine (10 mu M). The effect of mecamylamine (100 mu M) was inhibitory but small. Moreover, the increase in [Ca2+]i in response to ACh was also observed in some clusters that did not respond to high K (100 mM) Krebs. These results suggest that ACh increases [Ca2+]i of cultured carotid body cells by activating neuronal nicotinic ACh receptors, leading to Ca2+ influx via nicotinic channels. In addition, other pathways such as Ca2+ influx through L-type calcium channels, perhaps secondary to membrane depolarization, and Ca2

  1. Adolescence: How do we increase intestinal calcium absorption to allow for bone mineral mass accumulation?

    USDA-ARS?s Scientific Manuscript database

    An increase in calcium absorptive efficiency (fractional absorption of dietary calcium) during adolescence is associated with a rapid increase in total body bone mineral mass (BMM) accumulation. This increase occurs across a range of calcium intakes. It appears to be principally mediated by hormonal...

  2. Arabidopsis histone methylase CAU1/PRMT5/SKB1 acts as an epigenetic suppressor of the calcium signaling gene CAS to mediate stomatal closure in response to extracellular calcium.

    PubMed

    Fu, Yan-Lei; Zhang, Guo-Bin; Lv, Xin-Fang; Guan, Yuan; Yi, Hong-Ying; Gong, Ji-Ming

    2013-08-01

    Elevations in extracellular calcium ([Ca(2+)]o) are known to stimulate cytosolic calcium ([Ca(2+)]cyt) oscillations to close stomata. However, the underlying mechanisms regulating this process remain largely to be determined. Here, through the functional characterization of the calcium underaccumulation mutant cau1, we report that the epigenetic regulation of CAS, a putative Ca(2+) binding protein proposed to be an external Ca(2+) sensor, is involved in this process. cau1 mutant plants display increased drought tolerance and stomatal closure. A mutation in CAU1 significantly increased the expression level of the calcium signaling gene CAS, and functional disruption of CAS abolished the enhanced drought tolerance and stomatal [Ca(2+)]o signaling in cau1. Map-based cloning revealed that CAU1 encodes the H4R3sme2 (for histone H4 Arg 3 with symmetric dimethylation)-type histone methylase protein arginine methytransferase5/Shk1 binding protein1. Chromatin immunoprecipitation assays showed that CAU1 binds to the CAS promoter and modulates the H4R3sme2-type histone methylation of the CAS chromatin. When exposed to elevated [Ca(2+)]o, the protein levels of CAU1 decreased and less CAU1 bound to the CAS promoter. In addition, the methylation level of H4R3sme2 decreased in the CAS chromatin. Together, these data suggest that in response to increases in [Ca(2+)]o, fewer CAU1 protein molecules bind to the CAS promoter, leading to decreased H4R3sme2 methylation and consequent derepression of the expression of CAS to mediate stomatal closure and drought tolerance.

  3. Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells

    SciTech Connect

    Bosche, Bert; Schäfer, Matthias; Graf, Rudolf; Härtel, Frauke V.; Schäfer, Ute; Noll, Thomas

    2013-05-03

    Highlights: •We investigate free calcium as a central signalling element in endothelial cells. •Inhibition of glycolysis with 2-deoxy-D-glucose reduces cellular ATP. •This manoeuvre leads to a biphasic increase and overload of free calcium. •Pre-treatment with lithium for 24 h abolishes both phases of the calcium increase. •This provides a new strategy to protect endothelial calcium homeostasis and barrier function. -- Abstract: Cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca{sup 2+}]{sub i} overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca{sup 2+}]{sub i} overload can be prevented by lithium treatment. [Ca{sup 2+}]{sub i} and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-D-glucose (5 mM; 2-DG) plus sodium cyanide (5 mM; NaCN) caused a significant decrease in cellular ATP content (14 ± 1 nmol/mg protein vs. 18 ± 1 nmol/mg protein in the control, n = 6 culture dishes, P < 0.05), an increase in [Ca{sup 2+}]{sub i} (278 ± 24 nM vs. 71 ± 2 nM in the control, n = 60 cells, P < 0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10 mM 2-DG led to a similar decrease in ATP content (14 ± 2 nmol/mg vs. 18 ± 1 nmol/mg in the control, P < 0.05) with a delay of 5 min. The [Ca{sup 2+}]{sub i} response of EC was biphasic with a peak after 1 min (183 ± 6 nM vs. 71 ± 1 nM, n = 60 cells, P < 0.05) followed by a sustained increase in [Ca{sup 2+}]{sub i}. A 24-h pre-treatment with 10 mM of lithium

  4. Histamine release by exocytosis from rat mast cells on reduction of extracellular sodium: a secretory response inhibited by calcium, strontium, barium or magnesium.

    PubMed Central

    Cochrane, D E; Douglas, W W

    1976-01-01

    1. Histamine release from peritoneal mast cells of the rat was stimulated when the cells were exposed for 10 min to sodium-deficient media where all NaCl had been replaced by KC1, RbC1, glucose, sucrose, mannitol, or Tris, provided calcium was less than about 0-5 mM. 2. Light and electron microscopy showed the response to be exocytosis. 3. The chelating agents, EDTA and EGTA, abolished the response to sodium lack and their inhibitory effects were reversed by re-incubating cells with calcium but not magnesium. 4. The response was inhibited by dinitrophenol combined with glucose-deprivation. 5. The response was inversely related to the concentrations of sodium and calcium below 137-5 and 0-5 mM respectively. 6. The related alkaline earth metals, barium, strontium, and magnesium, resembled calcium in inhibiting the response to sodium lack. 7. No secretory response was seen when the cells were exposed for 10 min to calcium-free medium in which lithium replaced sodium. Exposure to this medium for 60 min, however, elicited secretion. 8. It is concluded that when extracellular calcium is low, a reduction in extracellular sodium induces a conventional exocytotic secretory response dependent on energy and cellular calcium. It is suggested that sodium lack may mobilize calcium from a cellular site possibly the inner aspect of the plasma membrane. Images A B C D E F G H PMID:59804

  5. Effect of glutamate and extracellular calcium on uptake of inorganic lead (Pb2+) in immortalized mouse hypothalamic GT1-7 neuronal cells.

    PubMed

    Loikkanen, J; Naarala, J; Vähäkangas, K H; Savolainen, K M

    2006-01-25

    We have previously shown that although glutamate alone has no effects on viability of mouse hypothalamic GT1-7 cells, it clearly enhances Pb2+-induced cytotoxicity. It is likely that Pb2+ must enter cells to exert most of its toxic effects. Pb2+ is known to substitute for Ca2+ in many cellular processes. Therefore, we studied the uptake mechanisms of Pb2+ into GT1-7 neuronal cells with a special focus on the role of extracellular calcium (Ca2+), voltage-sensitive calcium channels (VSCCs) and glutamate. Basal uptake of Pb2+ (1 microM or 10 microM), i.e. without any external stimulus, clearly increased in nominally Ca2+-free buffer and was partially abolished by 13 mM Ca2+ when compared to uptake in the presence of a physiological concentration of extracellular Ca2+ (1.3 mM). Depolarization by 25 mM K+, or antagonists of VSCCs, verapamil (10 microM) or flunarizine (10 microM) had no clear effect on basal Pb2+ uptake. Glutamate (1 mM) increased Pb2+ uptake, but only when cells were treated with 1 microM Pb2+ in the presence of 1.3 mM Ca2+. Our data suggest that Pb2+ competes for the same cellular uptake pathways with Ca2+, although not via VSCCs. In addition, enhancement of Pb2+-induced neurotoxicity by glutamate may be due to increased neuronal uptake of Pb2+.

  6. Participation of extracellular calcium in α-hederin-induced contractions of rat isolated stomach strips.

    PubMed

    Mendel, Marta; Chłopecka, Magdalena; Dziekan, Natalia; Karlik, Wojciech; Wiechetek, Maria

    2013-03-07

    The dry extract of Hedera helix leaves, due to its secretolytic and antispasmodic effects, is commonly used to produce pharmaceuticals applied in case of cough and other respiratory symptoms. The results of some in vitro studies as well as the clinical signs of poisoning caused by Hedera helix suggest however strong contractile effect on smooth muscle. In order to clarify the impact of α-hederin (the main active agent of ivy extract) on smooth muscle, the origin of activated calcium involved in α-hederin-induced contraction of gastric smooth muscle preparations was studied. The study was carried out on rat isolated stomach corpus and fundus strips, under isotonic conditions. The effect of α-hederin (100 μM) on smooth muscle preparations was measured before and after the treatment with verapamil during the incubation in modified Krebs-Henseleit solution (M K-HS). Besides, the effect of saponin was measured during the incubation of preparation in Ca2+-free modified Krebs-Henseleit solution or Ca2+-free EGTA-containing modified Krebs-Henseleit solution. The obtained results revealed that the application of verapamil significantly inhibited the reaction evoked by α-hederin. The incubation of stomach strips in calcium-free modified Krebs-Henseleit solution did not change the force of the observed contraction in comparison to the reaction of the preparations incubated in regular incubation solution (M K-HS). In contrary, the replacement of M K-HS by calcium-free chelator-containing solution inhibited totally the reaction to α-hederin. The results indicated that α-hederin-induced contraction results from the influx of calcium which is located in intercellular spaces or bound to the outside of the cell membrane. The Ca2+ influx occurs predominantly through voltage-dependent calcium channels of L-type. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  7. The common inhaled anesthetic isoflurane increases aggregation of huntingtin and alters calcium homeostasis in a cell model of Huntington's disease

    SciTech Connect

    Wang Qiujun; Liang Ge; Yang Hui; Wang Shouping; Eckenhoff, Maryellen F.; Wei Huafeng

    2011-02-01

    Isoflurane is known to increase {beta}-amyloid aggregation and neuronal damage. We hypothesized that isoflurane will have similar effects on the polyglutamine huntingtin protein and will cause alterations in intracellular calcium homeostasis. We tested this hypothesis in striatal cells from the expanded glutamine huntingtin knock-in mouse (STHdh{sup Q111/Q111}) and wild type (STHdh{sup Q7/Q7}) striatal neurons. The primary cultured neurons were exposed for 24 h to equipotent concentrations of isoflurane, sevoflurane, and desflurane in the presence or absence of extracellular calcium and with or without xestospongin C, a potent endoplasmic reticulum inositol 1,4,5-trisphosphate (InsP{sub 3}) receptor antagonist. Aggregation of huntingtin protein, cell viability, and calcium concentrations were measured. Isoflurane, sevoflurane, and desflurane all increased the aggregation of huntingtin in STHdh{sup Q111/Q111} cells, with isoflurane having the largest effect. Isoflurane induced greater calcium release from the ER and relatively more cell damage in the STHdh{sup Q111/Q111} huntingtin cells than in the wild type STHdh{sup Q7/Q7} striatal cells. However, sevoflurane and desflurane caused less calcium release from the ER and less cell damage. Xestospongin C inhibited the isoflurane-induced calcium release from the ER, aggregation of huntingtin, and cell damage in the STHdh{sup Q111/Q111} cells. In summary, the Q111 form of huntingtin increases the vulnerability of striatal neurons to isoflurane neurotoxicity through combined actions on the ER IP{sub 3} receptors. Calcium release from the ER contributes to the anesthetic induced huntingtin aggregation in STHdh{sup Q111/Q111} striatal cells.

  8. Calcium-independent activation of extracellular signal-regulated kinases 1 and 2 by cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Sumpio, B. E.

    1998-01-01

    We have previously demonstrated that cyclic strain induces extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in endothelial cells (EC). The aim of this study was to investigate the effect of Ca2+ on the activation of ERK1/2. Bovine aortic EC were pretreated with a chelator of extracellular Ca2+, ethylaneglycol-bis(aminoethylether)-tetra-acetate (EGTA), a depleter of Ca2+ pools, 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ), or a Ca2+ channel blocker, GdCl3, and subjected to an average 10 % strain at a rate of 60 cycles/min for 10 min. BHQ and GdCl3 did not inhibit the strain-induced ERK1/2 activation. Chelation of normal extracellular Ca2+ (1.8 mM) medium with EGTA (3 mM) acutely stimulated baseline phosphorylation and activation of ERK1/2, thereby obscuring any strain-induced activation of ERK1/2. However, in EC preincubated for 24 hours in Ca2+-free medium, elevated baseline phosphorylation was minimally activated by EGTA (200 microM) such that cyclic strain stimulated ERK1/2 in the presence or absence of BHQ. These results suggest a Ca2+ independence of the ERK1/2 signaling pathway by cyclic strain. Copyright 1998 Academic Press.

  9. Calcium-independent activation of extracellular signal-regulated kinases 1 and 2 by cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Sumpio, B. E.

    1998-01-01

    We have previously demonstrated that cyclic strain induces extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in endothelial cells (EC). The aim of this study was to investigate the effect of Ca2+ on the activation of ERK1/2. Bovine aortic EC were pretreated with a chelator of extracellular Ca2+, ethylaneglycol-bis(aminoethylether)-tetra-acetate (EGTA), a depleter of Ca2+ pools, 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ), or a Ca2+ channel blocker, GdCl3, and subjected to an average 10 % strain at a rate of 60 cycles/min for 10 min. BHQ and GdCl3 did not inhibit the strain-induced ERK1/2 activation. Chelation of normal extracellular Ca2+ (1.8 mM) medium with EGTA (3 mM) acutely stimulated baseline phosphorylation and activation of ERK1/2, thereby obscuring any strain-induced activation of ERK1/2. However, in EC preincubated for 24 hours in Ca2+-free medium, elevated baseline phosphorylation was minimally activated by EGTA (200 microM) such that cyclic strain stimulated ERK1/2 in the presence or absence of BHQ. These results suggest a Ca2+ independence of the ERK1/2 signaling pathway by cyclic strain. Copyright 1998 Academic Press.

  10. Extracellular signal-regulated kinase activation during cardiac hypertrophy reduces sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) transcription

    PubMed Central

    Huang, Haiyan; Joseph, Leroy C.; Gurin, Michael I.; Thorp, Edward B.; Morrow, John P.

    2014-01-01

    Pathologic cardiac hypertrophy can lead to heart failure, but the mechanisms involved are poorly understood. SERCA2 is critical for normal cardiac calcium handling and function and SERCA2 mRNA and protein levels are reduced by cardiac hypertrophy. We hypothesized that Extracellular Signal-regulated Kinase (ERK) 1/2 activation during hypertrophy reduced SERCA2 transcription. Using a neonatal rat ventricular myocyte model of hypertrophy, we found that pharmacologic inhibitors of ERK activation preserve SERCA2 mRNA levels during hypertrophy. ERK activation is sufficient to reduce SERCA2 mRNA. We determined that ERK represses SERCA2 transcription via nuclear factor-kappaB (NFkB), and activation of NFkB is sufficient to reduce SERCA2 mRNA in cardiomyocytes. This work establishes novel connections between ERK, NFkB, and SERCA2 repression during cardiac hypertrophy. This mechanism may have implications for the progression of hypertrophy to heart failure. PMID:25008120

  11. Increasing Soil Calcium Availability Alters Forest Soil Carbon Stocks

    NASA Astrophysics Data System (ADS)

    Melvin, A.; Goodale, C. L.

    2011-12-01

    Acid deposition in the Northeastern U.S. has been linked to a loss of soil base cations, especially calcium (Ca). While much research has addressed the effects of Ca depletion on soil and stream acidification, few studies have investigated its effects on ecosystem carbon (C) balance. We studied the long-term effects of increased Ca availability on C cycling in a northern hardwood forest in the Adirondack Park, NY. In 1989, calcium carbonate (lime) was added to ~ 100 ha of the Woods Lake Watershed to ameliorate the effects of soil Ca depletion. An additional 100 ha were maintained as controls. We hypothesized that the lime addition would improve forest health and that this improvement would be evident in increased tree biomass, leaf litter, and fine root production. Within the forest floor, we anticipated that the increased pH associated with liming would stimulate microbial activity resulting in increased decomposition and basal soil respiration, and reduced C stocks. Additionally, we hypothesized that increased Ca availability could enhance Ca-OM complexation in the upper mineral soils, leading to increased C stocks in these horizons. Eighteen years after liming, soil pH and exchangeable Ca pools remained elevated in the forest floor and upper mineral soil of the limed plots. Forest floor C stocks were significantly larger in limed plots (68 vs. 31 t C ha-1), and were driven primarily by greater C accumulation in the forest floor Oa horizon. Mineral soil C stocks did not differ between limed and control soils. Liming did not affect tree growth, however a net decline in biomass was observed across the entire watershed. There was a trend for larger fine root and foliar litter inputs in limed plots relative to controls, but the observed forest floor accumulation appears to be driven primarily by a suppression of decomposition. Liming reduced basal soil respiration rates by 17 and 43 % in the Oe and Oa horizons, respectively. This research suggests that Ca may

  12. Low extracellular Ca2+ conditions induce an increase in brain endothelial permeability that involves intercellular Ca2+ waves.

    PubMed

    De Bock, Marijke; Culot, Maxime; Wang, Nan; da Costa, Anaelle; Decrock, Elke; Bol, Mélissa; Bultynck, Geert; Cecchelli, Romeo; Leybaert, Luc

    2012-12-03

    The intracellular calcium concentration ([Ca(2+)](i)) is an important factor determining the permeability of endothelial barriers including the blood-brain barrier (BBB). However, nothing is known concerning the effect of spatially propagated intercellular Ca(2+) waves (ICWs). The propagation of ICWs relies in large part on channels formed by connexins that are present in endothelia. We hypothesized that ICWs may result in a strong disturbance of endothelial function, because the [Ca(2+)](i) changes are coordinated and involve multiple cells. Thus, we aimed to investigate the effect of ICWs on endothelial permeability. ICW activity was triggered in immortalized and primary brain endothelial cells by lowering the extracellular Ca(2+) concentration. Low extracellular Ca(2+) increased the endothelial permeability and this was significantly suppressed by buffering [Ca(2+)](i) with BAPTA-AM, indicating a central role of [Ca(2+)](i) changes. The endothelial permeability increase was furthermore inhibited by the connexin channel blocking peptide Gap27, which also blocked the ICWs, and by inhibiting protein kinase C (PKC), Ca(2+)/calmodulin-dependent kinase II (CaMKII) and actomyosin contraction. We compared these observations with the [Ca(2+)](i) changes and permeability alterations provoked by the inflammatory agent bradykinin (BK), which triggers oscillatory [Ca(2+)](i) changes without wave activity. BK-associated [Ca(2+)](i) changes and the endothelial permeability increase were significantly smaller than those associated with ICWs, and the permeability increase was not influenced by inhibition of PKC, CaMKII or actomyosin contraction. We conclude that ICWs significantly increase endothelial permeability and therefore, the connexins that underlie wave propagation form an interesting target to limit BBB alterations. This article is part of a Special Issue entitled Electrical Synapses. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Modulation of cytosolic free calcium levels by extracellular phosphate and lanthanum

    SciTech Connect

    Korc, M.; Schoeni, M.H.

    1987-03-01

    The effects of extracellular phosphate and lanthanum on cytosolic free Ca/sup 2 +/ ((Ca/sup 2 +/)/sub i/) levels were studied in isolated rat pancreatic acini. In the presence of 1.28 mM Ca/sup 2 +/ and 1.0 mM phosphate, the mean resting (Ca/sup 2 +/)/sub i/ level was 120 nM. Omission of phosphate from incubation medium significantly lowered this value to 94 nM. The gastrointestinal hormone cholecystokinin octapeptide (CCK-8) rapidly enhanced both (Ca/sup 2 +/)/sub i/ levels and /sup 45/Ca/sup 2 +/ efflux, irrespective of the presence or absence of phosphate. Lanthanum (0.1 mM), a compound known to block transmembrane Ca/sup 2 +/ fluxes, attenuated both actions of CCK-8, but only in the absence of extracellular phosphate. There was a concomitant decrease in amylase secretion induced by 0.1 nM CCK-8 but not by 10 nM CCK-8, without a significant change in cellular ATP levels. The inhibitory actions of lanthanum on CCK-8-stimulated (Ca/sup 2 +/)/sub i/ levels were very rapid and were mimicked only by prolonged incubation of acini in Ca/sup 2 +/-free medium supplemented with EGTA. Omission of phosphate from incubation medium also lowered basal (Ca/sup 2 +/)/sub i/ levels in IM-9 lymphocytes. These findings suggest that extracellular phosphate may modulate resting (Ca/sup 2 +/)/sub i/ levels in pancreatic acini and other cell types and that mobilization of intracellular Ca/sup 2 +/ may partly depend on the availability of a lanthanum-sensitive pool of cell-surface Ca/sup 2 +/ that is not readily removed by EGTA.

  14. Increased calcium bioavailability in mice fed genetically engineered plants lacking calcium oxalate.

    PubMed

    Morris, Jay; Nakata, Paul A; McConn, Michele; Brock, Amanda; Hirschi, Kendal D

    2007-07-01

    Bioavailable calcium affects bone formation and calcification. Here we investigate how a single gene mutation altering calcium partitioning in the model forage crop Medicago truncatula affects calcium bioavailability. Previously, the cod5 M. truncatula mutant was identified which contains identical calcium concentrations to wild-type, but contains no oxalate crystals. In this study, equal number of male and female mice were randomly grouped and then fed one of four 45Ca-containing diets: M. truncatula extrinsically or intrinsically labeled, and cod5 extrinsically or intrinsically labeled. Absorption of the tracer was determined in the legs one day after consumption. The absorption was similar in the M. truncatula and cod5 extrinsically labeled diets; however, in the intrinsically labeled diets, calcium absorption was 22.87% (P < 0.001) higher in mice fed cod5. Our study presents the first genetic evidence demonstrating the nutritional impact of removing oxalate crystals from foods.

  15. Calcium-containing crystals enhance receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor-mediated osteoclastogenesis via extracellular-signal-regulated kinase and p38 pathways.

    PubMed

    Chang, Chi-Ching; Tsai, Yu-Hui; Liu, Yu; Lin, Shyr-Yi; Liang, Yu-Chih

    2015-10-01

    Diseases associated with calcium-containing crystal deposition can lead to local bone erosion. We aimed to determine whether calcium-containing crystal-hydroxyapatite, β-tricalcium phosphate and CPPD enhanced osteoclastogenesis and to define underlying mechanisms of action. Osteoclastogenesis was studied by culturing murine RAW 264.7 osteoclast precursor cells with RANK ligand (RANKL)/ M-CSF and/or calcium-containing crystals, and observing the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity. Resorption pit formation was used to evaluate osteoclast activity. Real-time RT-PCR analysis revealed osteoclast marker genes, including TRAP, cathepsin K and calcitonin receptor (CTR). Western blotting was used to analyse the phosphorylation levels of signal transduction molecules. Three kinds of calcium-containing crystal significantly enhanced RANKL/M-CSF-induced osteoclastogenesis in RAW 264.7 cells, as evidenced by the increased number of TRAP-positive multinucleated cells, TRAP activity and resorption pit formation in a dose-dependent manner. Hydroxyapatite, β-tricalcium phosphate and CPPD treatments significantly enhanced RANKL/M-CSF-induced mRNA expression of TRAP, cathepsin K and CTR. Moreover, the three kinds of calcium-containing crystal enhanced the phosphorylation of extracellular-signal-regulated kinase and p38 in RANKL/M-CSF-treated cells. We concluded that calcium-containing crystals can promote osteoclastogenesis and bone resorption through the extracellular-signal-regulated kinase and p38 pathways. Together with synovial activation, this mechanism may be important in the pathogenesis of destructive arthropathies triggered by calcium-containing crystals. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Controversy regarding the association of high calcium intake and increased risk for cardiovascular disease.

    PubMed

    Chrysant, Steven G; Chrysant, George S

    2014-08-01

    Calcium intake has been shown to be associated with beneficial effects regarding hypertension, coronary heart disease (CHD), vascular disease, and stroke by several prospective cohort studies. However, recent studies have questioned the beneficial cardiovascular effects of calcium intake and instead have shown that high calcium intake is associated with an increased risk for CHD and stroke. These findings have created controversy and concern among physicians, because calcium is consumed by a large number of older men and women to prevent osteoporosis and bone fractures. Based on the methods of patient self-reporting of calcium intake and cardiovascular events, the conclusions drawn from the studies may not be entirely valid. Therefore, until more confirmatory data are available, physicians should not be dissuaded from prescribing calcium supplements to their patients. The best candidates are patients with low calcium intake, but their calcium supplementation should not exceed the recommended 1200 mg/d to 1500 mg/d. © 2014 Wiley Periodicals, Inc.

  17. Dependence of the carbon-tetrachloride--induced death of cultured hepatocytes on the extracellular calcium concentration

    SciTech Connect

    Casini, A.F.; Farber, J.L.

    1981-01-01

    The role of extracellular Ca/sup 2/+ ions in the killing of liver cells by CCl/sub 4/ was studied in primary cultures of rat hepatocytes. The dependence of in vitro cell killing on the metabolism of CCl/sub 4/ was first examined in order to document the similarity between the action of CCl/sub 4/ on cultured hepatocytes and the action of CCl/sub 4/ on liver cells in the intact animal. Cells prepared from male rats pretreated with phenobarbital were more sensitive to CCl/sub 4/ than cells prepared from either male or female rats. The killing of hepatocytes by CCl/sub 4/ was prevented by addition of SKF 525A to the culture medium. This protection was accompanied by evidence of decreased CCl/sub 4/ metabolism as assessed by the extent of covalent binding of 14C-CCl/sub 4/ metabolites to total cellular lipids and proteins, and by the extent of formation of conjugated dienes accompanying the peroxidation of phospholipids isolated from total cell lipids. The extent of killing of the hepatocytes by CCl/sub 4/ was dependent on the Ca/sup 2/+ concentration in the tissue culture medium. The results of this study indicate that it is the presence of extracellular Ca/sup 2/+ that converts initially nonlethal cell injury into irreversible cell injury in CCl/sub 4/-treated cells. This action of Ca/sup 2/+ most likely represents an influx into the cell across an injured permeability barrier at the plasma membrane, in accord with the accumulation of large quantities of Ca/sup 2/+ in CCl/sub 4/-intoxicated liver cells in the intact animal. The relation between this alteration in Ca/sup 2/+ homeostasis and the metabolism of CCl/sub 4/ is discussed.

  18. Calcium

    MedlinePlus

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

  19. Phosphate decreases urine calcium and increases calcium balance: A meta-analysis of the osteoporosis acid-ash diet hypothesis

    PubMed Central

    Fenton, Tanis R; Lyon, Andrew W; Eliasziw, Michael; Tough, Suzanne C; Hanley, David A

    2009-01-01

    Background The acid-ash hypothesis posits that increased excretion of "acidic" ions derived from the diet, such as phosphate, contributes to net acidic ion excretion, urine calcium excretion, demineralization of bone, and osteoporosis. The public is advised by various media to follow an alkaline diet to lower their acidic ion intakes. The objectives of this meta-analysis were to quantify the contribution of phosphate to bone loss in healthy adult subjects; specifically, a) to assess the effect of supplemental dietary phosphate on urine calcium, calcium balance, and markers of bone metabolism; and to assess whether these affects are altered by the b) level of calcium intake, c) the degree of protonation of the phosphate. Methods Literature was identified through computerized searches regarding phosphate with surrogate and/or direct markers of bone health, and was assessed for methodological quality. Multiple linear regression analyses, weighted for sample size, were used to combine the study results. Tests of interaction included stratification by calcium intake and degree of protonation of the phosphate supplement. Results Twelve studies including 30 intervention arms manipulated 269 subjects' phosphate intakes. Three studies reported net acid excretion. All of the meta-analyses demonstrated significant decreases in urine calcium excretion in response to phosphate supplements whether the calcium intake was high or low, regardless of the degree of protonation of the phosphate supplement. None of the meta-analyses revealed lower calcium balance in response to increased phosphate intakes, whether the calcium intake was high or low, or the composition of the phosphate supplement. Conclusion All of the findings from this meta-analysis were contrary to the acid ash hypothesis. Higher phosphate intakes were associated with decreased urine calcium and increased calcium retention. This meta-analysis did not find evidence that phosphate intake contributes to demineralization

  20. Phosphate decreases urine calcium and increases calcium balance: a meta-analysis of the osteoporosis acid-ash diet hypothesis.

    PubMed

    Fenton, Tanis R; Lyon, Andrew W; Eliasziw, Michael; Tough, Suzanne C; Hanley, David A

    2009-09-15

    The acid-ash hypothesis posits that increased excretion of "acidic" ions derived from the diet, such as phosphate, contributes to net acidic ion excretion, urine calcium excretion, demineralization of bone, and osteoporosis. The public is advised by various media to follow an alkaline diet to lower their acidic ion intakes. The objectives of this meta-analysis were to quantify the contribution of phosphate to bone loss in healthy adult subjects; specifically, a) to assess the effect of supplemental dietary phosphate on urine calcium, calcium balance, and markers of bone metabolism; and to assess whether these affects are altered by the b) level of calcium intake, c) the degree of protonation of the phosphate. Literature was identified through computerized searches regarding phosphate with surrogate and/or direct markers of bone health, and was assessed for methodological quality. Multiple linear regression analyses, weighted for sample size, were used to combine the study results. Tests of interaction included stratification by calcium intake and degree of protonation of the phosphate supplement. Twelve studies including 30 intervention arms manipulated 269 subjects' phosphate intakes. Three studies reported net acid excretion. All of the meta-analyses demonstrated significant decreases in urine calcium excretion in response to phosphate supplements whether the calcium intake was high or low, regardless of the degree of protonation of the phosphate supplement. None of the meta-analyses revealed lower calcium balance in response to increased phosphate intakes, whether the calcium intake was high or low, or the composition of the phosphate supplement. All of the findings from this meta-analysis were contrary to the acid ash hypothesis. Higher phosphate intakes were associated with decreased urine calcium and increased calcium retention. This meta-analysis did not find evidence that phosphate intake contributes to demineralization of bone or to bone calcium excretion

  1. Comparative expression of the extracellular calcium-sensing receptor in the mouse, rat, and human kidney.

    PubMed

    Graca, J A Z; Schepelmann, M; Brennan, S C; Reens, J; Chang, W; Yan, P; Toka, H; Riccardi, D; Price, S A

    2016-03-15

    The calcium-sensing receptor (CaSR) was cloned over 20 years ago and functionally demonstrated to regulate circulating levels of parathyroid hormone by maintaining physiological serum ionized calcium concentration ([Ca(2+)]). The receptor is highly expressed in the kidney; however, intrarenal and intraspecies distribution remains controversial. Recently, additional functions of the CaSR receptor in the kidney have emerged, including parathyroid hormone-independent effects. It is therefore critical to establish unequivocally the localization of the CaSR in the kidney to relate this to its proposed physiological roles. In this study, we determined CaSR expression in mouse, rat, and human kidneys using in situ hybridization, immunohistochemistry (using 8 different commercially available and custom-made antibodies), and proximity ligation assays. Negative results in mice with kidney-specific CaSR ablation confirmed the specificity of the immunohistochemistry signal. Both in situ hybridization and immunohistochemistry showed CaSR expression in the thick ascending limb, distal tubule, and collecting duct of all species, with the thick ascending limb showing the highest levels. Within the collecting ducts, there was significant heterogeneity of expression between cell types. In the proximal tubule, lower levels of immunoreactivity were detected by immunohistochemistry and proximity ligation assays. Proximity ligation assays were the only technique to demonstrate expression within glomeruli. This study demonstrated CaSR expression throughout the kidney with minimal discrepancy between species but with significant variation in the levels of expression between cell and tubule types. These findings clarify the intrarenal distribution of the CaSR and enable elucidation of the full physiological roles of the receptor within this organ.

  2. Intrastriatal taurine increases striatal extracellular dopamine in a tetrodotoxin-sensitive manner in rats.

    PubMed

    Ruotsalainen, M; Ahtee, L

    1996-07-19

    In vivo effects of locally administered taurine on striatal dopamine release and metabolism were studied by microdialysis in freely moving rats. Concentrations of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in striatal dialysates were quantified by high pressure liquid chromatography (HPLC) using electrochemical detection. Infusion of 150 mM taurine into the striatum for 2 h induced a 2.5-fold increase in the extracellular dopamine concentration. Extracellular DOPAC concentration increased nearly 2-fold. Taurine infusion initially decreased HVA to 70% but afterwards increased it to 140% of the control. When taurine was infused simultaneously with 1 microM tetrodotoxin starting 60 min after tetrodotoxin, the output of dopamine did not differ from that in the presence of tetrodotoxin alone. Tetrodotoxin abolished the effects of taurine on dopamine metabolites as well. Tetrodotoxin-sensitivity of the effects of taurine on dopamine and its metabolites suggests that intrastriatal taurine elevates extracellular dopamine by releasing it from neuronal pool.

  3. Acute isoproterenol induces anxiety-like behavior in rats and increases plasma content of extracellular vesicles.

    PubMed

    Leo, Giuseppina; Guescini, Michele; Genedani, Susanna; Stocchi, Vilberto; Carone, Chiara; Filaferro, Monica; Sisti, Davide; Marcoli, Manuela; Maura, Guido; Cortelli, Pietro; Guidolin, Diego; Fuxe, Kjell; Agnati, Luigi Francesco

    2015-04-01

    Several clinical observations have demonstrated a link between heart rate and anxiety or panic disorders. In these patients, β-adrenergic receptor function was altered. This prompted us to investigate whether the β-adrenergic receptor agonist isoproterenol, at a dose that stimulates peripheral β-adrenergic system but has no effects at the central nervous system, can induce anxiety-like behavior in rats. Moreover, some possible messengers involved in the peripheral to brain communication were investigated. Our results showed that isoproterenol (5 mg kg(-1) i.p.) increased heart rate, evoked anxiety-like behavior, did not result in motor impairments and increased extracellular vesicle content in the blood. Plasma corticosterone level was unmodified as well as vesicular Hsp70 content. Vesicular miR-208 was also unmodified indicating a source of increased extracellular vesicles different from cardiomyocytes. We can hypothesize that peripheral extracellular vesicles might contribute to the β-adrenergic receptor-evoked anxiety-like behavior, acting as peripheral signals in modulating the mental state.

  4. Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

    PubMed Central

    Levy, D N; Refaeli, Y; Weiner, D B

    1995-01-01

    The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics. PMID:7815499

  5. Increased digitalis-like activity in human cerebrospinal fluid after expansion of the extracellular fluid volume

    SciTech Connect

    Halperin, J.A.; Martin, A.M.; Malave, S.

    1985-08-12

    The present study was designed to determine whether acute expansion of the extracellular fluid volume influenced the digitalis-like activity of human cerebrospinal fluid (CSF), previously described. Human CSF samples, drawn before and 30 minutes after the intravenous infusion of 1 liter of either saline or glucose solutions, were assayed for digitalis-like activity by inhibition of either the /sup 86/Rb/sup +/ uptake into human erythrocytes or by the activity of a purified Na/sup +/-K/sup +/ ATPase. The CSF inhibitory activity on both systems significantly increased after the infusion of sodium solutions but did not change after the infusion of glucose. These results indicate that the digitalis-like factor of human CSF might be involved in the regulation of the extracellular fluid volume and electrolyte content and thereby in some of the physiological responses to sodium loading. 31 references, 2 figures, 1 table.

  6. Modafinil enhances extracellular levels of dopamine in the nucleus accumbens and increases wakefulness in rats.

    PubMed

    Murillo-Rodríguez, Eric; Haro, Reyes; Palomero-Rivero, Marcela; Millán-Aldaco, Diana; Drucker-Colín, René

    2007-01-25

    Modafinil (MOD) is a wakefulness-promoting drug that improves the alertness levels in narcolepsy; however, the molecular mechanism of action remains to be elucidated. We found that after a single icv injection of MOD (10 microg/5 microl) the extracellular levels of dopamine (DA) and l-DOPA collected from the nucleus accumbens were increased and decreased, respectively. Separately, the icv administration of MOD (10 microg/5 microl) to rats enhanced wakefulness (W) whereas diminished sleep during 4h. Lastly, the alertness induced by MOD was partially antagonized by the sleep-inducing endocannabinoid anandamide (ANA). We conclude that MOD enhances the extracellular levels of DA, promotes W and its effects on sleep are partially blocked by ANA.

  7. Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger

    DOE PAGES

    Liao, Jun; Marinelli, Fabrizio; Lee, Changkeun; ...

    2016-05-16

    Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3:1Na+/Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamicsmore » and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. Lastly, these calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.« less

  8. Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger

    PubMed Central

    Lee, ChangKeun; Huang, Yihe; Faraldo-Gómez, José D.; Jiang, Youxing

    2016-01-01

    Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. PMID:27183196

  9. Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger

    SciTech Connect

    Liao, Jun; Marinelli, Fabrizio; Lee, Changkeun; Huang, Yihe; Faraldo-Gomez, Jose D.; Jiang, Youxing

    2016-05-16

    Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3:1Na+/Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. Lastly, these calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.

  10. Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger

    SciTech Connect

    Liao, Jun; Marinelli, Fabrizio; Lee, Changkeun; Huang, Yihe; Faraldo-Gomez, Jose D.; Jiang, Youxing

    2016-05-16

    Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3:1Na+/Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. Lastly, these calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.

  11. The Extracellular Calcium-Sensing Receptor in the Intestine: Evidence for Regulation of Colonic Absorption, Secretion, Motility, and Immunity

    PubMed Central

    Tang, Lieqi; Cheng, Catherine Y.; Sun, Xiangrong; Pedicone, Alexandra J.; Mohamadzadeh, Mansour; Cheng, Sam X.

    2016-01-01

    Different from other epithelia, the intestinal epithelium has the complex task of providing a barrier impeding the entry of toxins, food antigens, and microbes, while at the same time allowing for the transfer of nutrients, electrolytes, water, and microbial metabolites. These molecules/organisms are transported either transcellularly, crossing the apical and basolateral membranes of enterocytes, or paracellularly, passing through the space between enterocytes. Accordingly, the intestinal epithelium can affect energy metabolism, fluid balance, as well as immune response and tolerance. To help accomplish these complex tasks, the intestinal epithelium has evolved many sensing receptor mechanisms. Yet, their roles and functions are only now beginning to be elucidated. This article explores one such sensing receptor mechanism, carried out by the extracellular calcium-sensing receptor (CaSR). In addition to its established function as a nutrient sensor, coordinating food digestion, nutrient absorption, and regulating energy metabolism, we present evidence for the emerging role of CaSR in the control of intestinal fluid homeostasis and immune balance. An additional role in the modulation of the enteric nerve activity and motility is also discussed. Clearly, CaSR has profound effects on many aspects of intestinal function. Nevertheless, more work is needed to fully understand all functions of CaSR in the intestine, including detailed mechanisms of action and specific pathways involved. Considering the essential roles CaSR plays in gastrointestinal physiology and immunology, research may lead to a translational opportunity for the development of novel therapies that are based on CaSR's unique property of using simple nutrients such as calcium, polyamines, and certain amino acids/oligopeptides as activators. It is possible that, through targeting of intestinal CaSR with a combination of specific nutrients, oral solutions that are both inexpensive and practical may be

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

    PubMed

    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.

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

    PubMed Central

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

    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 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 [Ca2+]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd3+), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca2+]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca2+]i increment. Results Mepivacaine produced vasoconstriction and increased [Ca2+]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca2+]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca2+]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca2+]i increase. Gd3+ had no effect on either vasoconstriction or the [Ca2+]i increment evoked by mepivacaine. Conclusions The mepivacaine-evoked [Ca2+]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. PMID:25558341

  14. Transcranial direct-current stimulation increases extracellular dopamine levels in the rat striatum

    PubMed Central

    Tanaka, Tomoko; Takano, Yuji; Tanaka, Satoshi; Hironaka, Naoyuki; Kobayashi, Kazuto; Hanakawa, Takashi; Watanabe, Katsumi; Honda, Manabu

    2013-01-01

    Background: Transcranial direct-current stimulation (tDCS) is a non-invasive procedure that achieves polarity-dependent modulation of neuronal membrane potentials. It has recently been used as a functional intervention technique for the treatment of psychiatric and neurological diseases; however, its neuronal mechanisms have not been fully investigated in vivo. Objective/Hypothesis: To investigate whether the application of cathodal or anodal tDCS affects extracellular dopamine and serotonin levels in the rat striatum. Methods: Stimulation and in vivo microdialysis were carried out under urethane anesthesia, and microdialysis probes were slowly inserted into the striatum. After the collection of baseline fractions in the rat striatum, cathodal or anodal tDCS was applied continuously for 10 min with a current intensity of 800 μA from an electrode placed on the skin of the scalp. Dialysis samples were collected every 10 min until at least 400 min after the onset of stimulation. Results: Following the application of cathodal, but not anodal, tDCS for 10 min, extracellular dopamine levels increased for more than 400 min in the striatum. There were no significant changes in extracellular serotonin levels. Conclusion: These findings suggest that tDCS has a direct and/or indirect effect on the dopaminergic system in the rat basal ganglia. PMID:23596399

  15. The extracellular calcium-sensing receptor reciprocally regulates the secretion of BMP-2 and the BMP antagonist Noggin in colonic myofibroblasts.

    PubMed

    Peiris, Dinithi; Pacheco, Ivan; Spencer, Craig; MacLeod, R John

    2007-03-01

    To understand whether postprandial extracellular Ca(2+) (Ca(o)(2+)) changes were related to intestinal epithelial homeostasis, we performed array analysis on extracellular calcium-sensing receptor (CaSR)-expressing colonic myofibroblasts (18Co cells) and observed increases in bone morphogenetic protein (BMP)-2 transcripts. The present experiments demonstrated that regulated secretion of BMP-2 occurs in response to CaSR activation of these cells and revealed a new property of BMP-2 on the intestinal barrier. Activation by Ca(o)(2+), spermine, GdCl(3), or neomycin sulfate of 18Co cells or primary isolates of myofibroblasts from the normal human colon stimulated both the synthesis (RT-PCR) and secretion (ELISA) of BMP-2. Transient transfection with short interfering RNA against CaSR completely inhibited BMP-2 secretion. Transient transfection with dominant negative CaSR (R185Q) increased the EC(50) of Ca(o)(2+) (5.7 vs. 2.3 mM). Upregulation of BMP-2 transcript and secretion occurring within 3 h of CaSR activation was prevented by actinomycin D. CaSR-mediated BMP-2 synthesis and secretion required phosphatidylinositol 3-kinase activation (as assessed by phospho-Akt generation). Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells accelerated restitution in wounded postconfluent Caco-2 cells. Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells increased the transepithelial resistance of low- and high-resistance T-84 epithelial monolayers. CaSR stimulation of T-84 epithelia and colonic myofibroblasts downregulated the BMP family antagonist Noggin, as assessed by RT-PCR and Western blot analysis. Together, our data suggest that the CaSR mediates the effective concentration of BMP-2 in the intestine, which leads to enhanced repair and barrier development.

  16. Increased extracellular concentrations of norepinephrine in cortex and hippocampus following vagus nerve stimulation in the rat.

    PubMed

    Roosevelt, Rodney W; Smith, Douglas C; Clough, Richard W; Jensen, Robert A; Browning, Ronald A

    2006-11-13

    The vagus nerve is an important source of afferent information about visceral states and it provides input to the locus coeruleus (LC), the major source of norepinephrine (NE) in the brain. It has been suggested that the effects of electrical stimulation of the vagus nerve on learning and memory, mood, seizure suppression, and recovery of function following brain damage are mediated, in part, by the release of brain NE. The hypothesis that left vagus nerve stimulation (VNS) at the cervical level results in increased extracellular NE concentrations in the cortex and hippocampus was tested at four stimulus intensities: 0.0, 0.25, 0.5, and 1.0 mA. Stimulation at 0.0 and 0.25 mA had no effect on NE concentrations, while the 0.5 mA stimulation increased NE concentrations significantly in the hippocampus (23%), but not the cortex. However, 1.0 mA stimulation significantly increased NE concentrations in both the cortex (39%) and hippocampus (28%) bilaterally. The increases in NE were transient and confined to the stimulation periods. VNS did not alter NE concentrations in either structure during the inter-stimulation baseline periods. No differences were observed between NE levels in the initial baseline and the post-stimulation baselines. These findings support the hypothesis that VNS increases extracellular NE concentrations in both the hippocampus and cortex.

  17. Increased Extracellular Concentrations of Norepinephrine in Cortex and Hippocampus Following Vagus Nerve Stimulation in the Rat.

    PubMed Central

    Roosevelt, Rodney W.; Smith, Douglas C.; Clough, Richard W.; Jensen, Robert A.; Browning, Ronald A.

    2006-01-01

    The vagus nerve is an important source of afferent information about visceral states and it provides input to the locus coeruleus (LC), the major source of norepinephrine (NE) in the brain. It has been suggested that the effects of electrical stimulation of the vagus nerve on learning and memory, mood, seizure suppression, and recovery of function following brain damage are mediated, in part, by the release of brain NE. The hypothesis that left vagus nerve stimulation (VNS) at the cervical level results in increased extracellular NE concentrations in the cortex and hippocampus was tested at four stimulus intensities 0.0, 0.25, 0.5, and 1.0 mA. Stimulation at 0.0 and 0.25 mA had no effect on NE concentrations, while the 0.5 mA stimulation increased NE concentrations significantly in the hippocampus (23%), but not the cortex. However, 1.0 mA stimulation significantly increased NE concentrations in both the cortex (39%) and hippocampus (28%) bilaterally. The increases in NE were transient and confined to the stimulation periods. VNS did not alter NE concentrations in either structure during the inter-stimulation baseline periods. No differences were observed between NE levels in the initial baseline and the post-stimulation baselines. These findings support the hypothesis that VNS increases extracellular NE concentrations in both the hippocampus and cortex. PMID:16962076

  18. Vitamin D does not increase calcium absorption in young women: a randomized clinical trial.

    PubMed

    Gallagher, J Christopher; Jindal, Prachi S; Smith, Lynette M

    2014-01-01

    It is commonly said that vitamin D should be used to increase calcium absorption. We tested this statement in a dose-response study of vitamin D on calcium absorption. A total of 198 white and African American women, aged 25 to 45 years, with vitamin D insufficiency, serum 25-hydroxyvitamin D (25OHD) <20 ng/mL, were randomized in a double-blind study to vitamin D3 400, 800, 1600, 2400 IU, or placebo. A calcium supplement was given to increase mean calcium intake at baseline from 706 mg/d to 1031 mg/d. Calcium absorption was measured at baseline and after 12 months using a single isotope method with radiocalcium45 and 100 mg of calcium. Mean baseline serum 25OHD was 13.4 ng/mL (33.5 nmol/L) and increased to 40 ng/mL (100 nmol/L) on the highest dose of 2400 IU. Using a multivariate regression analysis with significant predictors, baseline absorption, calcium intake, and weight, there was no increase in 12-month calcium absorption compared with baseline on any dose of vitamin D in either whites or African Americans. There was no significant relationship between 12-month calcium absorption and final serum 25OHD. In an analysis of calcium absorption and serum 25OHD at baseline, serum 25OHD levels were divided into groups: 0 to 5, 6 to 10, 11 to 15, or 16 to 20 ng/mL. There was no evidence of a threshold decrease in calcium absorption or serum 1,25 dihydroxyvitamin D (1,25(OH)2 D) amongst the lowest groups. Vitamin D doses up to 2400 IU daily did not increase calcium absorption. No threshold level of serum 25OHD for calcium absorption was found at baseline or in the longitudinal study, suggesting that active transport of calcium is saturated at very low serum 25OHD levels <5 ng/mL. There is no need to recommend vitamin D for increasing calcium absorption in normal subjects. Very efficient calcium absorption at very low levels of serum 25OHD explains why people do not develop osteomalacia provided that dietary intakes of calcium and phosphorus are adequate.

  19. Intracellular renin increases the inward calcium current in smooth muscle cells of mesenteric artery of SHR. Implications for hypertension and vascular remodeling.

    PubMed

    De Mello, Walmor

    2016-10-01

    The influence of intracellular renin on the inward calcium current in isolated smooth muscle cells from SHR mesenteric arteries was investigated. Measurements of calcium current were performed using the whole cell configuration of pCLAMP. The results indicated that: 1) renin (100nM) dialyzed into smooth muscle cells, increased the inward calcium current; 2) verapamil (10-9M) administered to the bath inhibited the effect of renin on the inward calcium current; 3) concurrently with the increase of calcium current a depolarization of 6.8+/-2.1mV (n=16)(P<0.05) was found in cells dialyzed with renin; 4) intracellular dialysis of renin (100nM) into smooth muscle cells isolated from mesenteric arteries of normal Wystar Kyoto rats showed no significant change on calcium current; 5) aliskiren (10-9M) dialyzed into the cell together with renin (100nM) abolished the effect of the enzyme on the calcium current in SHR; 6) Ang II (100nM) dialyzed into the smooth muscle cell from mesenteric artery of SHR in absence of renin, decreased the calcium current-an effect greatly reduced by valsartan (10-9M) added to the cytosol; 7) administration of renin (100nM) plus angiotensinogen (100nM) into the cytosol of muscles cells from SHR rats reduced the inward calcium current; 8) extracellular administration of Ang II (100nM) increased the inward calcium current in mesenteric arteries of SHR. intracellular renin in vascular resistance vessels from SHR due to internalization or expression, contributes to the regulation of vascular tone and control of peripheral resistance-an effect independently of Ang II. Implications for hypertension and vascular remodeling are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Effects of acidification and increased extracellular potassium on dynamic muscle contractions in isolated rat muscles.

    PubMed

    Overgaard, Kristian; Højfeldt, Grith Westergaard; Nielsen, Ole Bækgaard

    2010-12-15

    Since accumulation of both H(+) and extracellular K(+) have been implicated in the reduction in dynamic contractile function during intense exercise, we investigated the effects of acidification and high K(+) on muscle power and the force-velocity relation in non-fatigued rat soleus muscles. Contractions were elicited by supramaximal electrical stimulation at 60 Hz. Force-velocity (FV) curves were obtained by fitting data on force and shortening velocity at different loads to the Hill equation. Acidification of the muscles by incubation with up to 24 mm lactic acid produced no significant changes in maximal power (P(max)) at 30 °C. More pronounced acidification, obtained by increasing CO(2) levels in the equilibration gas from 5% to 53%, markedly decreased P(max) and maximal isometric force (F(max)), increased the curvature of the FV relation, but left maximal shortening velocity (V(max)) unchanged. Increase of extracellular K(+) from 4 to 10 mm caused a depression of 58% in P(max) and 52% in F(max), but had no significant effect on V(max) or curvature of the FV curve. When muscles at 10 mM K(+) were acidified by 20 mm lactic acid, P(max) and F(max) recovered completely to the initial control level at 4 mm K(+). CO(2) acidification also induced significant recovery of dynamic contractions, but not entirely to control levels. These results demonstrate that in non-fatigued muscles severe acidification can be detrimental to dynamic contractile function, but in muscles depolarised by exposure to high extracellular [K(+)], approaching the [K(+)] level seen during intense fatiguing exercise, acidification can have positive protective effects on dynamic muscle function.

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

    PubMed

    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.

  2. Increased intracellular free calcium and sensitivity to angiotensin II in platelets of preeclamptic women.

    PubMed

    Haller, H; Oeney, T; Hauck, U; Distler, A; Philipp, T

    1989-04-01

    Preeclampsia is characterized by a generalized vasoconstriction and increased vascular sensitivity to angiotensin II. Intracellular free calcium, implicated in vascular smooth muscle contraction, has been found to be elevated in platelets of other hypertensive disorders. We therefore measured intracellular free calcium concentrations by using the fluorescent probe quin-2 in platelets of six patients with preeclampsia and compared them to measurements in ten normotensive pregnant women and ten age-matched nonpregnant women. Intracellular free calcium was also determined in the preeclamptic women after delivery. We found that intracellular free calcium was slightly elevated in normal pregnancy (102 +/- 13 nmol/L v 87 +/- 17 nmol/L) but was markedly increased in preeclampsia (138 +/- 13 nmol/L, P less than .05). This increase disappeared six weeks after delivery (84 + 10 nmol/L, P less than .01). To investigate whether the increased intracellular free calcium was related to angiotensin II, the platelets were exposed to thrombin and angiotensin II in vitro. Exposure to thrombin and angiotensin II caused a dose-dependent increase in intracellular free calcium. The intracellular response to thrombin was not significantly different in the three groups. However, stimulation with angiotensin II revealed an increased response in intracellular free calcium in preeclampsia (P less than .05) that disappeared after delivery. Our findings show a sustained increase in platelet intracellular free calcium in preeclampsia and suggest a functional alteration of the angiotensin II receptor in this disease.

  3. Calcium

    MedlinePlus

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

  4. Calcium

    MedlinePlus

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

  5. Activation of endothelial nitric oxide synthase by proanthocyanidin-rich fraction from Croton celtidifolius (Euphorbiaceae): involvement of extracellular calcium influx in rat thoracic aorta.

    PubMed

    DalBó, Silvia; Goulart, Suelen; Horst, Heros; Pizzolatti, Moacir Geraldo; Ribeiro-do-Valle, Rosa Maria

    2008-06-01

    The present study investigates the mechanisms related to the endogenous nitric oxide synthase (eNOS) activation in the relaxant effects of a proanthocyanidin-rich fraction (PRF), obtained from Croton celtidifolius Baill barks, in rat thoracic aorta rings with endothelium. In vessels pre-contracted with phenylephrine (Phe), PRF (0.1 - 100 microg/mL) induced a concentration-dependent relaxation. This effect was significantly reduced by endothelium denudation, by N(omega)-nitro-L-arginine, and by 1H[1,2,3]oxadiazolo[4,3-alpha]quinoxalin. However, the vasorelaxant effect was not altered by indomethacin, atropine, tetraethylammonium, and charybdotoxin plus apamin. In thoracic aorta rings pre-contracted with phorbol-12,13-dibuyrate, PRF also induced a concentration-dependent relaxation. The PRF-induced relaxation disappeared in the absence of extracellular calcium in the medium and decreased significantly in the presence of lanthanum. A sulfhydryl alkylating agent, N-ethylmaleimide, and a phospholipase C (PLC) blocker, neomycin, significantly decreased PRF-induced vasorelaxation. In vessels pre-contracted with Phe, the PRF-induced vasorelaxant effect was not altered by quinacrine and ONO-RS-082, genistein and thyrphostin A-23, GF109203, and pertussis toxin and cholera toxin. The results suggest that the PRF-induced vasorelaxant effect is endothelium-dependent and involves the NO/cGMP pathway. We hypothesize that the activation of eNOS is due to an increase of intracellular calcium derived from PLC activation and an N-ethylmaleimide sensitive pathway.

  6. Extracellular Volume Overload and Increased Vasoconstriction in Patients With Recurrent Intradialytic Hypertension

    PubMed Central

    Van Buren, Peter Noel; Zhou, Yunyun; Neyra, Javier A.; Xiao, Guanghua; Vongpatanasin, Wanpen; Inrig, Jula; Toto, Robert

    2016-01-01

    Background/Aims Intradialytic hypertension (IH) occurs frequently in some hemodialysis patients and increases mortality risk. We simultaneously compared pre-dialysis, post-dialysis and changes in extracellular volume and hemodynamics in recurrent IH patients and controls. Methods We performed a case-control study among prevalent hemodialysis patients with recurrent IH and hypertensive hemodialysis controls. We used bioimpedance spectroscopy and impedance cardiography to compare pre-dialysis, post-dialysis, and intradialytic change in total body water (TBW) and extracellular water (ECW), as well as cardiac index (CI) and total peripheral resistance index (TPRI). Results The ECW/TBW was 0.453 (0.05) pre-dialysis and 0.427 (0.04) post-dialysis in controls vs. 0.478 (0.03) and 0.461 (0.03) in IH patients (p=0.01 post-dialysis). The ECW/TBW change was −0.027 (0.03) in controls and −0.013 (0.02) in IH patients (p=0.1). In controls, pre- and post-dialysis TPRI were 3254 (994) and 2469 (529) dynes/sec/cm2/m2 vs. 2983 (747) and 3408 (980) dynes/sec/cm2/m2 in IH patients (p=0.002 post-dialysis). There were between-group differences in TPRI change (0=0.0001), but not CI (p=0.09). Conclusions Recurrent intradialytic hypertension is associated with higher post-dialysis extracellular volume and TPRI. Intradialytic TPRI surges account for the vasoconstrictive state post-dialysis, but intradialytic fluid shifts may contribute to post-hemodialysis volume expansion. PMID:27832647

  7. Extracellular Self-DNA (esDNA), but Not Heterologous Plant or Insect DNA (etDNA), Induces Plasma Membrane Depolarization and Calcium Signaling in Lima Bean (Phaseolus lunatus) and Maize (Zea mays).

    PubMed

    Barbero, Francesca; Guglielmotto, Michela; Capuzzo, Andrea; Maffei, Massimo E

    2016-09-29

    Extracellular self-DNA (esDNA) is produced during cell and tissue damage or degradation and has been shown to induce significant responses in several organisms, including plants. While the inhibitory effects of esDNA have been shown in conspecific individuals, little is known on the early events involved upon plant esDNA perception. We used electrophysiology and confocal laser scanning microscopy calcium localization to evaluate the plasma membrane potential (Vm) variations and the intracellular calcium fluxes, respectively, in Lima bean (Phaseolus lunatus) and maize (Zea mays) plants exposed to esDNA and extracellular heterologous DNA (etDNA) and to etDNA from Spodoptera littoralis larvae and oral secretions. In both species, esDNA induced a significant Vm depolarization and an increased flux of calcium, whereas etDNA was unable to exert any of these early signaling events. These findings confirm the specificity of esDNA to induce plant cell responses and to trigger early signaling events that eventually lead to plant response to damage.

  8. Extracellular Self-DNA (esDNA), but Not Heterologous Plant or Insect DNA (etDNA), Induces Plasma Membrane Depolarization and Calcium Signaling in Lima Bean (Phaseolus lunatus) and Maize (Zea mays)

    PubMed Central

    Barbero, Francesca; Guglielmotto, Michela; Capuzzo, Andrea; Maffei, Massimo E.

    2016-01-01

    Extracellular self-DNA (esDNA) is produced during cell and tissue damage or degradation and has been shown to induce significant responses in several organisms, including plants. While the inhibitory effects of esDNA have been shown in conspecific individuals, little is known on the early events involved upon plant esDNA perception. We used electrophysiology and confocal laser scanning microscopy calcium localization to evaluate the plasma membrane potential (Vm) variations and the intracellular calcium fluxes, respectively, in Lima bean (Phaseolus lunatus) and maize (Zea mays) plants exposed to esDNA and extracellular heterologous DNA (etDNA) and to etDNA from Spodoptera littoralis larvae and oral secretions. In both species, esDNA induced a significant Vm depolarization and an increased flux of calcium, whereas etDNA was unable to exert any of these early signaling events. These findings confirm the specificity of esDNA to induce plant cell responses and to trigger early signaling events that eventually lead to plant response to damage. PMID:27690017

  9. PDGF-mediated protection of SH-SY5Y cells against Tat toxin involves regulation of extracellular glutamate and intracellular calcium

    SciTech Connect

    Zhu Xuhui; Yao Honghong; Peng Fuwang; Callen, Shannon; Buch, Shilpa

    2009-10-15

    The human immunodeficiency virus (HIV-1) protein Tat has been implicated in mediating neuronal apoptosis, one of the hallmark features of HIV-associated dementia (HAD). Mitigation of the toxic effects of Tat could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study we demonstrated that Tat-induced neurotoxicity was abolished by NMDA antagonist-MK801, suggesting the role of glutamate in this process. Furthermore, we also found that pretreatment of SH-SY5Y cells with PDGF exerted protection against Tat toxicity by decreasing extracellular glutamate levels. We also demonstrated that extracellular calcium chelator EGTA was able to abolish PDGF-mediated neuroprotection, thereby underscoring the role of calcium signaling in PDGF-mediated neuroprotection. We also showed that Erk signaling pathway was critical for PDGF-mediated protection of cells. Additionally, blocking calcium entry with EGTA resulted in suppression of PDGF-induced Erk activation. These findings thus underscore the role of PDGF-mediated calcium signaling and Erk phosphorylation in the protection of cells against HIV Tat toxicity.

  10. ICP/OES application for assessing cadmium uptake (or toxicity) in glomerular cells: influence of extracellular calcium.

    PubMed

    Ohayon-Courtès, Céline; Passagne, Isabelle; De Portal, Caroline; Pouvreau, Carole; Cambar, Jean; L'Azou, Béatrice

    2007-05-01

    The risks of metals for health are highlighted by their chemical stability and their persistence in the environment. Chronic exposure to low cadmium (Cd) concentrations results in renal dysfunction mainly. Cd has been regarded primarily as a renal tubular toxicant, but glomerular structures may also be affected. Since the cellular environment may influence metal toxicity, differences concerning Cd uptake and toxicity were evaluated according to calcium (Ca) medium concentrations. An optimized inductively coupled plasma emission spectrometry method (ICP/OES) was developed under defined conditions, as a selective analytical tool to determine cadmium uptake in glomerular mesangial cells. The performance characteristics of the analytical system were evaluated for both Cd and Ca by calibration (50 to 250 microg/L and 1 to 5 mg/L), linearity (r2 .9968 and .9943), limits of detection (1 microg/L and 0.1 mg/L) and quantitation (3 microg/L and 0.3 mg/L), accuracy with spiking, and repeatability (1.2 and 2.9%) with matrix matched standards. Total intracellular Cd content was significantly threefold lower in 0.175 mM Ca medium (Ca-free Eagle's minimum essential medium [EMEM] medium with 5% fetal bovine serum [FBS]) than in EMEM medium (1.8 mM Ca) with respectively 0.16 and 0.37 microg/mg proteins after 24 h of Cd (1 microM) exposure. Similar differences were obtained in cytotoxicity studies with a fourfold reduction in the mortality index (IC50). Complementary assays using Ca-spiked medium reinforced that Cd cytotoxicity and uptake were significantly dependent on the concentration of extracellular Ca. These findings suggest direct link between Cd uptake and toxicity, underlining the relevance of the analytical method.

  11. Stimulation by leukotriene D4 of increases in the cytosolic concentration of calcium in dimethylsulfoxide-differentiated HL-60 cells.

    PubMed Central

    Baud, L; Goetzl, E J; Koo, C H

    1987-01-01

    The C6-sulfidopeptide leukotrienes C4 (LTC4) and D4 (LTD4) evoked increases in the cytosolic concentration of intracellular calcium ([Ca+2]i) in dimethylsulfoxide-differentiated HL-60 cells, as assessed by the fluorescence of quin-2. The increases in [Ca+2]i reached a peak within 15-90 s, attained 50% of the maximum level at 1.2 nM LTD4 and 60 nM LTC4, were greater in maximal magnitude for LTD4 than LTC4, and subsided in 5-7 min. Flow cytometric evaluation of the LTD4-induced increases in [Ca+2]i, reflected in increases in the fluorescence of intracellular indo-1, revealed that a mean of 77% of differentiated HL-60 cells responded, as contrasted with lesser increases in only 50% of undifferentiated HL-60 cells. The capacity of pretreatment of HL-60 cells with LTD4 to prevent subsequent responses of [Ca+2]i to LTC4 and LTD4, and the finding that the serine-borate inhibitor of conversion of LTC4 to LTD4 suppressed concurrently both LTC4-induced rises in [Ca+2]i and increases in adherence to Sephadex G-25 indicated that the responses of HL-60 cells to LTC4 required conversion to LTD4. That pertussis toxin and a chemical antagonist of LTD4 reduced the [Ca+2]i response suggested a dependence on LTD4 receptors. The LTD4-induced increases in [Ca+2]i were dependent on extracellular calcium and diminished by lanthanum, but not affected by nifedipine nor associated with changes in membrane potential, as measured with the fluorescent probe 3,3'-dipentyloxacarbocyanine. Thus, the increase in [Ca+2]i in HL-60 cells, which is coupled to an increase in adherence, appears to involve LTD4 receptor-specific and voltage-independent calcium channels in the plasma membrane. PMID:3477571

  12. Calcium Supplementation Increases Blood Creatinine Concentration in a Randomized Controlled Trial

    PubMed Central

    Barry, Elizabeth L.; Mott, Leila A.; Melamed, Michal L.; Rees, Judith R.; Ivanova, Anastasia; Sandler, Robert S.; Ahnen, Dennis J.; Bresalier, Robert S.; Summers, Robert W.; Bostick, Roberd M.; Baron, John A.

    2014-01-01

    Background Calcium supplements are widely used among older adults for osteoporosis prevention and treatment. However, their effect on creatinine levels and kidney function has not been well studied. Methods We investigated the effect of calcium supplementation on blood creatinine concentration in a randomized controlled trial of colorectal adenoma chemoprevention conducted between 2004–2013 at 11 clinical centers in the United States. Healthy participants (N = 1,675) aged 45–75 with a history of colorectal adenoma were assigned to daily supplementation with calcium (1200 mg, as carbonate), vitamin D3 (1000 IU), both, or placebo for three or five years. Changes in blood creatinine and total calcium concentration were measured after one year of treatment and multiple linear regression was used to estimate effects on creatinine concentrations. Results After one year of treatment, blood creatinine was 0.013±0.006 mg/dL higher on average among participants randomized to calcium compared to placebo after adjustment for other determinants of creatinine (P = 0.03). However, the effect of calcium treatment appeared to be larger among participants who consumed the most alcohol (2–6 drinks/day) or whose estimated glomerular filtration rate (eGFR) was less than 60 ml/min/1.73 m2 at baseline. The effect of calcium treatment on creatinine was only partially mediated by a concomitant increase in blood total calcium concentration and was independent of randomized vitamin D treatment. There did not appear to be further increases in creatinine after the first year of calcium treatment. Conclusions Among healthy adults participating in a randomized clinical trial, daily supplementation with 1200 mg of elemental calcium caused a small increase in blood creatinine. If confirmed, this finding may have implications for clinical and public health recommendations for calcium supplementation. Trial Registration ClinicalTrials.gov NCT00153816 PMID:25329821

  13. TOL plasmid carriage enhances biofilm formation and increases extracellular DNA content in Pseudomonas putida KT2440.

    PubMed

    D'Alvise, Paul W; Sjøholm, Ole R; Yankelevich, Tatiana; Jin, Yujie; Wuertz, Stefan; Smets, Barth F

    2010-11-01

    Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid-specific stains revealed differences in the production of extracellular polymeric substances: TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNase I treatment. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads to increased biofilm formation by production of eDNA. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  14. Bromelain Surface Modification Increases the Diffusion of Silica Nanoparticles in the Tumor Extracellular Matrix

    PubMed Central

    2015-01-01

    Tumor extracellular matrix (ECM) represents a major obstacle to the diffusion of therapeutics and drug delivery systems in cancer parenchyma. This biological barrier limits the efficacy of promising therapeutic approaches including the delivery of siRNA or agents intended for thermoablation. After extravasation due to the enhanced penetration and retention effect of tumor vasculature, typical nanotherapeutics are unable to reach the nonvascularized and anoxic regions deep within cancer parenchyma. Here, we developed a simple method to provide mesoporous silica nanoparticles (MSN) with a proteolytic surface. To this extent, we chose to conjugate MSN to Bromelain (Br–MSN), a crude enzymatic complex, purified from pineapple stems, that belongs to the peptidase papain family. This surface modification increased particle uptake in endothelial, macrophage, and cancer cell lines with minimal impact on cellular viability. Most importantly Br–MSN showed an increased ability to digest and diffuse in tumor ECM in vitro and in vivo. PMID:25119793

  15. Bromelain surface modification increases the diffusion of silica nanoparticles in the tumor extracellular matrix.

    PubMed

    Parodi, Alessandro; Haddix, Seth G; Taghipour, Nima; Scaria, Shilpa; Taraballi, Francesca; Cevenini, Armando; Yazdi, Iman K; Corbo, Claudia; Palomba, Roberto; Khaled, Sm Z; Martinez, Jonathan O; Brown, Brandon S; Isenhart, Lucas; Tasciotti, Ennio

    2014-10-28

    Tumor extracellular matrix (ECM) represents a major obstacle to the diffusion of therapeutics and drug delivery systems in cancer parenchyma. This biological barrier limits the efficacy of promising therapeutic approaches including the delivery of siRNA or agents intended for thermoablation. After extravasation due to the enhanced penetration and retention effect of tumor vasculature, typical nanotherapeutics are unable to reach the nonvascularized and anoxic regions deep within cancer parenchyma. Here, we developed a simple method to provide mesoporous silica nanoparticles (MSN) with a proteolytic surface. To this extent, we chose to conjugate MSN to Bromelain (Br-MSN), a crude enzymatic complex, purified from pineapple stems, that belongs to the peptidase papain family. This surface modification increased particle uptake in endothelial, macrophage, and cancer cell lines with minimal impact on cellular viability. Most importantly Br-MSN showed an increased ability to digest and diffuse in tumor ECM in vitro and in vivo.

  16. Hypotonic medium increases calcium permeant channels activity in human normal and dystrophic myotubes.

    PubMed

    Vandebrouck, Clarisse; Duport, Gérard; Raymond, Guy; Cognard, Christian

    2002-05-03

    Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin and an elevated intracellular calcium level. Single-channel recordings were performed with the cell-attached configuration of the patch-clamp technique. The present study shows, on human co-cultured normal and dystrophic muscle cells, the evidence for an increased activity of calcium permeant cationic mechano-sensitive channels under hypotonic medium stimulation. This activity was particularly enhanced in DMD cells. The hypotonic medium induced drastic changes in the single-channel activity characteristics, which are: a large increase of the calcium over potassium permeability ratio; and a great enhancement of the quantity of current crossing through these channels. These channels could contribute to a significant calcium entry, which could participate in the abnormal calcium homeostasis observed in DMD muscle.

  17. Hypoxic increase in nitric oxide generation of rat sensory neurons requires activation of mitochondrial complex II and voltage-gated calcium channels.

    PubMed

    Henrich, M; Paddenberg, R; Haberberger, R V; Scholz, A; Gruss, M; Hempelmann, G; Kummer, W

    2004-01-01

    Recently, we have demonstrated that sensory neurons of rat lumbar dorsal root ganglia (DRG) respond to hypoxia with an activation of endothelial nitric oxide (NO) synthase (eNOS) resulting in enhanced NO production associated with mitochondria which contributes to resistance against hypoxia. Extracellular calcium is essential to this effect. In the present study on rat DRG slices, we set out to determine what types of calcium channels operate under hypoxia, and which upstream events contribute to their activation, thereby focusing upon mitochondrial complex II. Both the metallic ions Cd2+ and Ni2+, known to inhibit voltage-gated calcium channels and T-type channels, respectively, and verapamil and nifedipine, typical blocker of L-type calcium channels completely prevented the hypoxic neuronal NO generation. Inhibition of complex II by thenoyltrifluoroacetone at the ubiquinon binding site or by 3-nitropropionic acid at the substrate binding site largely diminished hypoxic-induced NO production while having an opposite effect under normoxia. An additional blockade of voltage-gated calcium channels entirely abolished the hypoxic response. The complex II inhibitor malonate inhibited both normoxic and hypoxic NO generation. These data show that complex II activity is required for increased hypoxic NO production. Since succinate dehydrogenase activity of complex II decreased at hypoxia, as measured by histochemistry and densitometry, we propose a hypoxia-induced functional switch of complex II from succinate dehydrogenase to fumarate reductase, which subsequently leads to activation of voltage-gated calcium channels resulting in increased NO production by eNOS.

  18. Co-occurring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excess.

    PubMed

    Gonzalez, Alberto; Vera, Jeannette; Castro, Jorge; Dennett, Geraldine; Mellado, Macarena; Morales, Bernardo; Correa, Juan A; Moenne, Alejandra

    2010-10-01

    In order to analyse copper-induced calcium release and (reactive oxygen species) ROS accumulation and their role in antioxidant and defense enzymes activation, the marine alga Ulva compressa was exposed to 10 µM copper for 7 d. The level of calcium, extracellular hydrogen peroxide (eHP), intracellular hydrogen peroxide (iHP) and superoxide anions (SA) as well as the activities of ascorbate peroxidase (AP), glutathione reductase (GR), glutathione-S-transferase (GST), phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) were determined. Calcium release showed a triphasic pattern with peaks at 2, 3 and 12 h. The second peak was coincident with increases in eHP and iHP and the third peak with the second increase of iHP. A delayed wave of SA occurred after day 3 and was not accompanied by calcium release. The accumulation of iHP and SA was mainly inhibited by organellar electron transport chains inhibitors (OETCI), whereas calcium release was inhibited by ryanodine. AP activation ceased almost completely after the use of OETCI. On the other hand, GR and GST activities were partially inhibited, whereas defense enzymes were not inhibited. In contrast, PAL and LOX were inhibited by ryanodine, whereas AP was not inhibited. Thus, copper stress induces calcium release and organellar ROS accumulation that determine the differential activation of antioxidant and defense enzymes.

  19. Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

    NASA Technical Reports Server (NTRS)

    Wyatt, Sarah E.; Tsou, Pei-Lan; Robertson, Dominique; Brown, C. S. (Principal Investigator)

    2002-01-01

    Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca2+-transport and storage pathways that include Ca2+ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca2+ binding characteristics of the C-domain of calreticulin to selectively increase Ca2+ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20-50 moles of Ca2+ per mole of protein and has been shown to be the major site of Ca2+ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9-35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca2+ stores, and that this Ca2+ reserve can be used by the plant in times of stress.

  20. Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

    NASA Technical Reports Server (NTRS)

    Wyatt, Sarah E.; Tsou, Pei-Lan; Robertson, Dominique; Brown, C. S. (Principal Investigator)

    2002-01-01

    Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca2+-transport and storage pathways that include Ca2+ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca2+ binding characteristics of the C-domain of calreticulin to selectively increase Ca2+ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20-50 moles of Ca2+ per mole of protein and has been shown to be the major site of Ca2+ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9-35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca2+ stores, and that this Ca2+ reserve can be used by the plant in times of stress.

  1. The role of calcium and nitric oxide during liver enzyme release induced by increased physical forces as evidenced in partially hepatectomized rats.

    PubMed

    Díaz-Juárez, Julieta; Hernández-Muñoz, Rolando

    2011-03-01

    Although increased plasma enzyme activities could be diagnostic for tissue damage, the mechanisms controlling cellular enzyme release remain poorly understood. We found a selective and drastic elevation of serum enzyme activities accompanying rat liver regeneration after partial hepatectomy (PH), apparently controlled by a mechanism dependent on flow-bearing physical forces. In fact, this study assesses a putative role of calcium mobilization and nitric oxide (NO) production underlying rat liver enzyme release. The role of increased shear stress (by enhancing viscosity during perfusion) and the participation of cell calcium and NO were tested in isolated livers subjected to increasing flow rate. After PH, there was a drastic elevation of serum activities for liver enzyme markers, clearly predominating those of mitochondrial localization. Liver enzyme release largely depended on extracellular calcium entry, probably mediated by stretch-sensitive calcium channels, as well as by increasing NO production. However, these effects were differentially observed when comparing liver enzymes from cytoplasmic or mitochondrial compartments. Moreover, a possible role for cell-mediated mechanotransduction in liver enzyme release was suggested by increasing shear stress (high viscosity), which also selectively affected the release of the enzymes tested. Therefore, we show, for the first time, that flow-induced shear stress can control the amount of hepatic enzymes released into the bloodstream, which is largely regulated through modifications in cell calcium mobilization and production of liver NO, events markedly elevated in the proliferating rat liver. Copyright © 2011 American Association for the Study of Liver Diseases.

  2. A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

    PubMed

    Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

    2000-02-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

  3. Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells.

    PubMed

    Naro, Fabio; De Arcangelis, Vania; Coletti, Dario; Molinaro, Mario; Zani, Bianca; Vassanelli, Stefano; Reggiani, Carlo; Teti, Anna; Adamo, Sergio

    2003-04-01

    Cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) variation is a key event in myoblast differentiation, but the mechanism by which it occurs is still debated. Here we show that increases of extracellular Ca(2+) concentration ([Ca(2+)](o)) produced membrane hyperpolarization and a concentration-dependent increase of [Ca(2+)](i) due to Ca(2+) influx across the plasma membrane. Responses were not related to inositol phosphate turnover and Ca(2+)-sensing receptor. [Ca(2+)](o)-induced [Ca(2+)](i) increase was inhibited by Ca(2+) channel inhibitors and appeared to be modulated by several kinase activities. [Ca(2+)](i) increase was potentiated by depletion of intracellular Ca(2+) stores and depressed by inactivation of the Na(+)/Ca(2+) exchanger. The response to arginine vasopressin (AVP), which induces inositol 1,4,5-trisphosphate-dependent [Ca(2+)](i) increase in L6-C5 cells, was not modified by high [Ca(2+)](o). On the contrary, AVP potentiated the [Ca(2+)](i) increase in the presence of elevated [Ca(2+)](o). Other clones of the L6 line as well as the rhabdomyosarcoma RD cell line and the satellite cell-derived C2-C12 line expressed similar responses to high [Ca(2+)](o), and the amplitude of the responses was correlated with the myogenic potential of the cells.

  4. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators

    SciTech Connect

    Noah, T.L.; Paradiso, A.M.; Madden, M.C.; McKinnon, K.P.; Devlin, R.B. )

    1991-11-01

    Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. The authors therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of their data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

  5. Amantadine increases L-DOPA-derived extracellular dopamine in the striatum of 6-hydroxydopamine-lesioned rats.

    PubMed

    Arai, Akira; Kannari, Kazuya; Shen, Huo; Maeda, Tetsuya; Suda, Toshihiro; Matsunaga, Muneo

    2003-05-16

    We investigated the effect of amantadine on L-DOPA-derived extracellular dopamine (DA) levels and aromatic L-amino acid decarboxylase (AADC) activity in the striatum of rats with nigrostriatal dopaminergic denervation by 6-hydroxydopamine (6-OHDA). Pretreatment with 30 mg/kg amantadine increased the cumulative amount of extracellular DA in the striatum of 6-OHDA-lesioned rats treated with 10 mg/kg benserazide and 50 mg/kg L-DOPA to 250% of that without amantadine (P<0.01). Under pretreatment with 10 mg/kg benserazide, AADC activity after 30 mg/kg amantadine administration was reduced to 43% of controls (P<0.01). Amantadine-induced increase in L-DOPA-derived extracellular DA provides the basis for the clinical usefulness of amantadine in combination with L-DOPA. However, the effect of amantadine on L-DOPA-derived extracellular DA may not be caused by changes in AADC activity.

  6. Sertraline increases extracellular levels not only of serotonin, but also of dopamine in the nucleus accumbens and striatum of rats.

    PubMed

    Kitaichi, Yuji; Inoue, Takeshi; Nakagawa, Shin; Boku, Shuken; Kakuta, Aya; Izumi, Takeshi; Koyama, Tsukasa

    2010-11-25

    Selective serotonin reuptake inhibitors (SSRIs) are a first-line treatment for depression. Recent reports in the literature describe differences in antidepressant effects among SSRIs. Although each SSRI apparently has different pharmacological actions aside from serotonin reuptake inhibition, the relations between antidepressant effects and unique pharmacological properties in respective SSRIs remain unclear. This study was designed to compare abilities of three systemically administered SSRIs to increase the extracellular levels of serotonin, dopamine, and noradrenaline acutely in three brain regions of male Sprague-Dawley rats. We examined effects of sertraline, fluvoxamine, and paroxetine on extracellular serotonin, dopamine, and noradrenaline levels in the medial prefrontal cortex, nucleus accumbens and striatum of rats using in vivo microdialysis. Dialysate samples were collected in sample vials every 20 min for 460 min. Extracellular serotonin, dopamine, and noradrenaline levels were determined using high-performance liquid chromatography with electrochemical detection. All SSRI administrations increased extracellular serotonin levels in all regions. Only sertraline administration increased extracellular dopamine concentrations in the nucleus accumbens and striatum. All SSRI administrations increased extracellular noradrenaline levels in the nucleus accumbens, although fluvoxamine was less effective. These results suggest that neurochemical differences account for the differences in clinical antidepressant effects among SSRIs. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Data on calcium increases depending on stretch in dystrophic cardiomyocytes.

    PubMed

    Aguettaz, E; Lopez, J J; Krzesiak, A; Constantin, B; Cognard, C; Sebille, S

    2016-09-01

    In this data article, intracellular Ca(2+) concentration ([Ca(2+)]i) was measured in isolated ventricular Wild Type (WT) and mdx cardiomyocytes in two different conditions: at rest and during the application of an axial stretch. Using a carbon microfibers technique, axial stretch was applied to mimic effects of physiological conditions of ventricular filling. A study of cation entry with the same experimental model and the manganese quenching method reported (i) a constitutive cation entry in mdx cardiomyocytes and (ii) the involvement of TRPV2 channels in axial-stretch dependant cation entry, "Axial stretch-dependent cation entry in dystrophic cardiomyopathy: involvement of several TRPs channels" (Aguettaz et al., 2016) [1]. Here, the Ca(2+) dye fluo-8 was used for [Ca(2+)]i measurement, in both resting and stretching conditions, using a perfusion protocol starting initially with a calcium free Tyrode solution followed by the perfusion of 1.8 mM Ca(2+) Tyrode solution. The variation of [Ca(2+)]i was found higher in mdx cardiomyocytes.

  8. Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

    PubMed

    Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

    2011-12-01

    The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology.

  9. Extracellular superoxide dismutase increased the therapeutic potential of human mesenchymal stromal cells in radiation pulmonary fibrosis.

    PubMed

    Wei, Li; Zhang, Jing; Yang, Zai-Liang; You, Hua

    2017-05-01

    Pulmonary fibrosis induced by irradiation is a significant problem of radiotherapy in cancer patients. Extracellular superoxide dismutase (SOD3) is found to be predominantly and highly expressed in the extracellular matrix of lung and plays a pivotal role against oxidative damage. Early administration of mesenchymal stromal cells (MSCs) has been demonstrated to reduce fibrosis of damaged lung. However, injection of MSCs at a later stage would be involved in fibrosis development. The present study aimed to determine whether injection of human umbilical cord-derived MSCs (UC-MSCs) over-expressing SOD3 at the established fibrosis stage would have beneficial effects in a mice model of radiation pulmonary fibrosis. Herein, pulmonary fibrosis in mice was induced using Cobalt-60 ((60)Co) irradiator with 20 Gy, followed by intravenous injection of UC-MSCs, transduced or not to express SOD3 at 2 h (early delivery) and 60 day (late delivery) post-irradiation, respectively. Our results demonstrated that the early administration of UC-MSCs could attenuate the microscopic damage, reduce collagen deposition, inhibit (myo)fibroblast proliferation, reduce inflammatory cell infiltration, protect alveolar type II (AE2) cell injury, prevent oxidative stress and increase antioxidant status, and reduce pro-fibrotic cytokine level in serum. Furthermore, the early treatment with SOD3-infected UC-MSCs resulted in better improvement. However, we failed to observe the therapeutic effects of UC-MSCs, transduced to express SOD3, during established fibrosis. Altogether, our results demonstrated that the early treatment with UC-MSCs alone significantly reduced radiation pulmonary fibrosis in mice through paracrine effects, with further improvement by administration of SOD3-infected UC-MSCs, suggesting that SOD3-infected UC-MSCs may be a potential cell-based gene therapy to treat clinical radiation pulmonary fibrosis. Copyright © 2017 International Society for Cellular Therapy. Published by

  10. Increased dependency of cardiac pacemaker activity on extracellular Ca after adrenergic blockade in the frog heart.

    PubMed

    Fukuda, Y

    1986-01-01

    The frog sinus venosus shows spontaneous regular pacemaker activity, even in the absence of extracellular Ca2+. When an alpha-adrenergic blocking agent (phentolamine) is applied, the rate of pacemaker activity, height of action potential, rate of slow diastolic depolarization, and the maximum diastolic potential become strongly dependent upon the extracellular Ca2+ concentration.

  11. Risperidone attenuates the increase of extracellular nitric oxide and glutamate levels in serotonin syndrome animal models.

    PubMed

    Shioda, Katsutoshi; Nisijima, Koichi; Kasai, Makiko; Yoshino, Tatsuki; Kato, Satoshi

    2012-10-18

    Serotonin (5-hydroxytryptamine; 5-HT) syndrome is a potentially life-threatening neurotoxic condition provoked by pharmacologically induced excess serotonergic activity. Several studies report that nitric oxide (NO) and glutamate play a role in psychostimulant-induced hyperthermia related to neurotoxicity. In the present study, the involvement of NO and glutamate, as well as the effect of risperidone, a potent 5-HT(2A) and D(2) (and a less potent D(1)) receptor antagonist, were investigated in animal models of 5-HT syndrome. Two 5-HT syndrome animal models were utilized. The first model was induced by administration of tranylcypromine, a nonselective monoamine oxidase (MAO) inhibitor, and fluoxetine, a selective 5-HT reuptake inhibitor. The second model was induced by the administration of clorgyline, an MAO-A inhibitor, and 5-hydroxy-l-tryptophan, a precursor of 5-HT. Changes in the level of NO metabolites and glutamate in the anterior hypothalamus were measured using microdialysis. In both models, NO metabolite levels significantly increased, and this increase was significantly attenuated by risperidone pretreatment. Extracellular levels of glutamate were increased only in the tranylcypromine and fluoxetine model, and this increase was significantly attenuated by risperidone pretreatment. These results indicate that NO and glutamate may be involved in the development of 5-HT syndrome and that risperidone may be effective against neurotransmitter abnormalities in 5-HT syndrome.

  12. Evidence that depletion of internal calcium stores sensitive to noradrenaline elicits a contractile response dependent on extracellular calcium in rat aorta.

    PubMed Central

    Noguera, M. A.; D'Ocon, M. P.

    1993-01-01

    1. Noradrenaline 1 microM induced a contractile response in rat isolated aorta in the presence or in the absence of extracellular Ca2+ with depletion of intracellular Ca2+ stores. Thereafter, during incubation in the presence of Ca2+, an increase in the resting tone was observed. Such a contractile response did not occur after exposure to caffeine or 5-hydroxytryptamine. 2. This increase in tension was inhibited in a concentration-dependent manner by alpha-adrenoceptor antagonists (prazosin, phentolamine and yohimbine), the non-specific relaxing compound, papaverine and by the Ca(2+)-entry blocker, nifedipine. Therefore, this contractile process is related to depletion of Ca2+ stores sensitive to noradrenaline and is linked to Ca2+ entry through voltage-operated Ca2+ channels and alpha-adrenoceptors. 3. Phentolamine and yohimbine did not block the Ca2+ refill pathway; prazosin and nifedipine inhibited the reuptake of Ca2+ by an internal store sensitive only to noradrenaline; papaverine inhibited the refilling of caffeine- and noradrenaline-sensitive Ca(2+)-stores. PMID:7902180

  13. Calcium increases Xylella fastidiosa surface attachment, biofilm formation, and twitching motility.

    PubMed

    Cruz, Luisa F; Cobine, Paul A; De La Fuente, Leonardo

    2012-03-01

    Xylella fastidiosa is a plant-pathogenic bacterium that forms biofilms inside xylem vessels, a process thought to be influenced by the chemical composition of xylem sap. In this work, the effect of calcium on the production of X. fastidiosa biofilm and movement was analyzed under in vitro conditions. After a dose-response study with 96-well plates using eight metals, the strongest increase of biofilm formation was observed when medium was supplemented with at least 1.0 mM CaCl(2). The removal of Ca by extracellular (EGTA, 1.5 mM) and intracellular [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), 75 μM] chelators reduced biofilm formation without compromising planktonic growth. The concentration of Ca influenced the force of adhesion to the substrate, biofilm thickness, cell-to-cell aggregation, and twitching motility, as shown by assays with microfluidic chambers and other assays. The effect of Ca on attachment was lost when cells were treated with tetracycline, suggesting that Ca has a metabolic or regulatory role in cell adhesion. A double mutant (fimA pilO) lacking type I and type IV pili did not improve biofilm formation or attachment when Ca was added to the medium, while single mutants of type I (fimA) or type IV (pilB) pili formed more biofilm under conditions of higher Ca concentrations. The concentration of Ca in the medium did not significantly influence the levels of exopolysaccharide produced. Our findings indicate that the role of Ca in biofilm formation may be related to the initial surface and cell-to-cell attachment and colonization stages of biofilm establishment, which rely on critical functions by fimbrial structures.

  14. Electrical field stimulation (EFS)-induced relaxations turn into contractions upon removal of extracellular calcium in rat mesenteric artery.

    PubMed

    Ozkan, Melike Hacer; Ozturk, Elif Inci; Uma, Serdar

    2013-04-01

    In the present study, we aimed to examine the effect of blockade of L-type Ca(2+) channels (LTCC) and in addition the removal of extracellular Ca(2+), on EFS-induced relaxations in rings of rat mesenteric artery. EFS applied to the tissues precontracted with phenylephrine caused relaxations which were markedly inhibited by nifedipine (10(-7)M) and tetraethylammonium (TEA) (1mM). Addition of LTCC opener BAY K 8644 (10(-7)M) failed to enhance the relaxations. Upon removal of Ca(2+), EFS with the same stimulation parameters produced frequency-dependent transient contractions. Tetrodotoxin (10(-6)M), capsaicin (10(-5)M) and removal of endothelium did not alter these contractions suggesting that they were not neural in origin and endothelium-derived contracting factors were unlikely to be involved. However, they were increased by nearly 40% in response to BAY K 8644 (10(-7)M) and were inhibited by nifedipine (10(-7)M), indicating that activation of the LTCCs was essential. Inositol triphosphate (InsP3) receptor antagonist 2-APB (10(-4)M) significantly reduced, and high concentration of caffeine (20mM) almost totally suppressed the contractions. These results suggest that in the absence of extracellular Ca(2+) EFS through membrane depolarization, evokes the opening of the LTCCs which subsequently leads to the release of Ca(2+) from internal stores via InsP3 receptors, a phenomenon known as Ca(2+) channel-induced Ca(2+) release (CCICR), to trigger vasoconstriction. That activation of LTCCs causes arterial relaxation or contraction depending on the Ca(2+) status apparently exemplifies how the same messenger fulfils opposing physiological functions in a given cell.

  15. Extracellular calcium stimulates DNA synthesis in synergism with zinc, insulin and insulin-like growth factor I in fibroblasts.

    PubMed

    Huang, J S; Mukherjee, J J; Chung, T; Crilly, K S; Kiss, Z

    1999-12-01

    In serum-starved mouse NIH 3T3 fibroblasts cultured in 1.8 mM Ca2+-containing medium, addition of 0.75-2 mM extra Ca2+ stimulated DNA synthesis in synergism with zinc (15-60 microM), insulin and insulin-like growth factor I. Extra Ca2+ stimulated phosphorylation/activation of p42/p44 mitogen-activated protein kinases by an initially (10 min) zinc-independent mechanism; however, insulin, and particularly zinc, significantly prolonged Ca2+-induced mitogen-activated protein kinase phosphorylation. In addition, extra Ca2+ activated p70 S6 kinase by a zinc-dependent mechanism and enhanced the stimulatory effect of zinc on choline kinase activity. Insulin and insulin-like growth factor I also commonly increased both p70 S6 kinase and choline kinase activities. In support of the role of the choline kinase product phosphocholine in the mediation of mitogenic Ca2+ effects, cotreatments with the choline kinase substrate choline (250 microM) and the choline kinase inhibitor hemicholinium-3 (2 mM) enhanced and inhibited, respectively, the combined stimulatory effect of extra Ca2+ (3.8 mM total) and zinc on DNA synthesis. In various human skin fibroblast lines, 1-2 mM extra Ca2+ also stimulated DNA synthesis in synergism with zinc and insulin. The results show that in various fibroblast cultures, high concentrations of extracellular Ca2+ can collaborate with zinc and certain growth factors to stimulate DNA synthesis. Considering the high concentration of extracellular Ca2+ in the dermal layer, Ca2+ may promote fibroblast growth during wound healing in concert with zinc, insulin growth factor-I insulin, and perhaps other growth factors.

  16. N-Cadherin Extracellular Repeat 4 Mediates Epithelial to Mesenchymal Transition and Increased Motility

    PubMed Central

    Kim, Jae-Beom; Islam, Shahidul; Kim, Young J.; Prudoff, Ryan S.; Sass, Kristin M.; Wheelock, Margaret J.; Johnson, Keith R.

    2000-01-01

    E- and N-cadherin are members of the classical cadherin family of proteins. E-cadherin plays an important role in maintaining the normal phenotype of epithelial cells. Previous studies from our laboratory and other laboratories have shown that inappropriate expression of N-cadherin by tumor cells derived from epithelial tissue results in conversion of the cell to a more fibroblast-like cell, with increased motility and invasion. Our present study was designed to determine which domains of N-cadherin make it different from E-cadherin, with respect to altering cellular behavior, such as which domains are responsible for the epithelial to mesenchymal transition and increased cell motility and invasion. To address this question, we constructed chimeric cadherins comprised of selected domains of E- and N-cadherin. The chimeras were transfected into epithelial cells to determine their effect on cell morphology and cellular behavior. We found that a 69–amino acid portion of EC-4 of N-cadherin was necessary and sufficient to promote both an epithelial to mesenchymal transition in squamous epithelial cells and increased cell motility. Here, we show that different cadherin family members promote different cellular behaviors. In addition, we identify a novel activity that can be ascribed to the extracellular domain of N-cadherin. PMID:11121435

  17. Intrinsic stiffness of extracellular matrix increases with age in skeletal muscles of mice.

    PubMed

    Wood, Lauren K; Kayupov, Erdan; Gumucio, Jonathan P; Mendias, Christopher L; Claflin, Dennis R; Brooks, Susan V

    2014-08-15

    Advanced age is associated with increases in muscle passive stiffness, but the contributors to the changes remain unclear. Our purpose was to determine the relative contributions of muscle fibers and extracellular matrix (ECM) to muscle passive stiffness in both adult and old animals. Passive mechanical properties were determined for isolated individual muscle fibers and bundles of muscle fibers that included their associated ECM, obtained from tibialis anterior muscles of adult (8-12 mo old) and old (28-30 mo old) mice. Maximum tangent moduli of individual muscle fibers from adult and old muscles were not different at any sarcomere length tested. In contrast, the moduli of bundles of fibers from old mice was more than twofold greater than that of fiber bundles from adult muscles at sarcomere lengths >2.5 μm. Because ECM mechanical behavior is determined by the composition and arrangement of its molecular constituents, we also examined the effect of aging on ECM collagen characteristics. With aging, muscle ECM hydroxyproline content increased twofold and advanced glycation end-product protein adducts increased threefold, whereas collagen fibril orientation and total ECM area were not different between muscles from adult and old mice. Taken together, these findings indicate that the ECM of tibialis anterior muscles from old mice has a higher modulus than the ECM of adult muscles, likely driven by an accumulation of densely packed extensively crosslinked collagen.

  18. Enhancement of rat bladder contraction by artificial sweeteners via increased extracellular Ca{sup 2+} influx

    SciTech Connect

    Dasgupta, Jaydip; Elliott, Ruth A. . E-mail: rae5@leicester.ac.uk; Doshani, Angie; Tincello, Douglas G.

    2006-12-01

    Introduction: Consumption of carbonated soft drinks has been shown to be independently associated with the development of overactive bladder symptoms (OR 1.62, 95% CI 1.18, 2.22) [Dallosso, H.M., McGrother, C.W., Matthews, R.J., Donaldson, M.M.K., 2003. The association of diet and other lifestyle factors with overactive bladder and stress incontinence: a longitudinal study in women. BJU Int. 92, 69-77]. We evaluated the effects of three artificial sweeteners, acesulfame K, aspartame and sodium saccharin, on the contractile response of isolated rat detrusor muscle strips. Methods: Strips of detrusor muscle were placed in an organ bath and stimulated with electrical field stimulation (EFS) in the absence and presence of atropine, and with {alpha},{beta} methylene ATP, potassium, calcium and carbachol. Results: Sweeteners 10{sup -7} M to 10{sup -2} M enhanced the contractile response to 10 Hz EFS compared to control (p < 0.01). The atropine-resistant response to EFS was marginally increased by acesulfame K 10{sup -6} M, aspartame 10{sup -7} M and sodium saccharin 10{sup -7} M. Acesulfame K 10{sup -6} M increased the maximum contractile response to {alpha},{beta} methylene ATP by 35% ({+-} 9.6%) (p < 0.05) and to KCl by 12% ({+-} 3.1%) (p < 0.01). Sodium saccharin also increased the response to KCl by 37% ({+-} 15.2%) (p < 0.05). These sweeteners shifted the calcium concentration-response curves to the left. Acesulfame K 10{sup -6} M increased the log EC{sub 5} from -2.79 ({+-} 0.037) to -3.03 ({+-} 0.048, p < 0.01) and sodium saccharin 10{sup -7} M from -2.74 ({+-} 0.03) to 2.86 ({+-} 0.031, p < 0.05). The sweeteners had no significant effect on the contractile response to carbachol but they did increase the amplitude of spontaneous bladder contractions. Discussion: These results suggest that low concentrations of artificial sweeteners enhanced detrusor muscle contraction via modulation of L-type Ca{sup +2} channels.

  19. Regulation by thapsigargin and carbachol of the intracellular calcium concentration in rat submandibular glands.

    PubMed

    Métioui, M; Grosfils, K; Dehaye, J P

    1994-11-01

    1. Carbachol and thapsigargin both increased the intracellular calcium concentration in rat submandibular cells in the presence and in the absence of extracellular calcium. Depletion of intracellular calcium pools with thapsigargin prevented the response to carbachol. 2. The two agents also increased the influx of calcium. The muscarinic agonist stimulated the efflux of calcium outside the cell. 3. From these results it is concluded that submandibular cells possess several intracellular calcium pools sensitive to thapsigargin, among which some are sensitive to IP3. Depletion of these pools increase the uptake of extracellular calcium.

  20. Intracellular Modulation, Extracellular Disposal and Serum Increase of MiR-150 Mark Lymphocyte Activation

    PubMed Central

    de Candia, Paola; Torri, Anna; Gorletta, Tatiana; Fedeli, Maya; Bulgheroni, Elisabetta; Cheroni, Cristina; Marabita, Francesco; Crosti, Mariacristina; Moro, Monica; Pariani, Elena; Romanò, Luisa; Esposito, Susanna; Mosca, Fabio; Rossetti, Grazisa; Rossi, Riccardo L.; Geginat, Jens; Casorati, Giulia; Dellabona, Paolo; Pagani, Massimiliano; Abrignani, Sergio

    2013-01-01

    Activated lymphocytes release nano-sized vesicles (exosomes) containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of lymphocyte-specific microRNAs. We found that, upon activation in vitro, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. In vivo, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation. PMID:24205408

  1. Absence of K-Ras Reduces Proliferation and Migration But Increases Extracellular Matrix Synthesis in Fibroblasts.

    PubMed

    Muñoz-Félix, José M; Fuentes-Calvo, Isabel; Cuesta, Cristina; Eleno, Nélida; Crespo, Piero; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-10-01

    The involvement of Ras-GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H- and N-Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K-Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K-Ras knockout (KO) mouse embryonic fibroblasts (K-ras(-/-) ) stimulated with transforming growth factor-β1 (TGF-β1) exhibited reduced proliferation and impaired mobility than wild-type fibroblasts. Moreover, an increase on ECM production was observed in K-Ras KO fibroblasts in basal conditions. The absence of K-Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF-β1-induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K-ras but reduced migration and ECM proteins expression only in wild-type fibroblasts, while the PI3K-AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K-Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K-Ras may be a crucial mediator in TGF-β1-mediated effects in this cell type. J. Cell. Physiol. 231: 2224-2235, 2016. © 2016 Wiley Periodicals, Inc.

  2. Extracellular matrix metalloproteinase inducer is increased in smokers' bronchoalveolar lavage fluid.

    PubMed

    Betsuyaku, Tomoko; Tanino, Mishie; Nagai, Katsura; Nasuhara, Yasuyuki; Nishimura, Masaharu; Senior, Robert M

    2003-07-15

    Extracellular matrix metalloproteinase inducer (EMMPRIN), also called basigin, is present in the lung during development, but its expression in normal adult lung is minimal. Increases of EMMPRIN have been found in various forms of experimental lung injury. To determine whether EMMPRIN might be involved in alveolar injury/repair associated with smoking, we developed an ELISA for EMMPRIN and applied it to bronchoalveolar lavage fluids from never-smokers (n = 7), former smokers (n = 16), and current smokers (n = 58). The smoker groups included subjects with emphysema, as determined by high-resolution chest computed tomography. EMMPRIN levels were significantly elevated in current and former smokers (315 +/- 20 and 175 +/- 15 pg/ml SEM, respectively, compared with 31 +/- 7 pg/ml in never-smokers), but the EMMPRIN levels of smokers with emphysema were not different from smokers without emphysema. Immunohistochemistry of smokers' lung tissue showed EMMPRIN in bronchiolar epithelium and alveolar macrophages, but EMMPRIN mRNA in alveolar macrophages was not different between current and never-smokers. Matrix metalloproteinase-1 was also detectable in the bronchoalveolar lavage fluid from some smokers but not in never-smokers. These findings indicate that smoking is associated with increased intrapulmonary EMMPRIN. Whether EMMPRIN is involved in smoking-induced lung pathology remains to be determined.

  3. Computational modeling of neurons: intensity-duration relationship of extracellular electrical stimulation for changes in intracellular calcium.

    PubMed

    Adams, Robert D; Willits, Rebecca K; Harkins, Amy B

    2016-01-01

    In many instances of extensive nerve damage, the injured nerve never adequately heals, leaving lack of nerve function. Electrical stimulation (ES) has been shown to increase the rate and orient the direction of neurite growth, and is a promising therapy. However, the mechanism in which ES affects neuronal growth is not understood, making it difficult to compare existing ES protocols or to design and optimize new protocols. We hypothesize that ES acts by elevating intracellular calcium concentration ([Ca(2+)]i) via opening voltage-dependent Ca(2+) channels (VDCCs). In this work, we have created a computer model to estimate the ES Ca(2+) relationship. Using COMSOL Multiphysics, we modeled a small dorsal root ganglion (DRG) neuron that includes one Na(+) channel, two K(+) channels, and three VDCCs to estimate [Ca(2+)]i in the soma and growth cone. As expected, the results show that an ES that generates action potentials (APs) can efficiently raise the [Ca(2+)]i of neurons. More interestingly, our simulation results show that sub-AP ES can efficiently raise neuronal [Ca(2+)]i and that specific high-voltage ES can preferentially raise [Ca(2+)]i in the growth cone. The intensities and durations of ES on modeled growth cone calcium rise are consistent with directionality and orientation of growth cones experimentally shown by others. Finally, this model provides a basis to design experimental ES pulse parameters, including duration, intensity, pulse-train frequency, and pulse-train duration to efficiently raise [Ca(2+)]i in neuronal somas or growth cones.

  4. Computational modeling of neurons: intensity-duration relationship of extracellular electrical stimulation for changes in intracellular calcium

    PubMed Central

    Adams, Robert D.; Willits, Rebecca K.

    2015-01-01

    In many instances of extensive nerve damage, the injured nerve never adequately heals, leaving lack of nerve function. Electrical stimulation (ES) has been shown to increase the rate and orient the direction of neurite growth, and is a promising therapy. However, the mechanism in which ES affects neuronal growth is not understood, making it difficult to compare existing ES protocols or to design and optimize new protocols. We hypothesize that ES acts by elevating intracellular calcium concentration ([Ca2+]i) via opening voltage-dependent Ca2+ channels (VDCCs). In this work, we have created a computer model to estimate the ES Ca2+ relationship. Using COMSOL Multiphysics, we modeled a small dorsal root ganglion (DRG) neuron that includes one Na+ channel, two K+ channels, and three VDCCs to estimate [Ca2+]i in the soma and growth cone. As expected, the results show that an ES that generates action potentials (APs) can efficiently raise the [Ca2+]i of neurons. More interestingly, our simulation results show that sub-AP ES can efficiently raise neuronal [Ca2+]i and that specific high-voltage ES can preferentially raise [Ca2+]i in the growth cone. The intensities and durations of ES on modeled growth cone calcium rise are consistent with directionality and orientation of growth cones experimentally shown by others. Finally, this model provides a basis to design experimental ES pulse parameters, including duration, intensity, pulse-train frequency, and pulse-train duration to efficiently raise [Ca2+]i in neuronal somas or growth cones. PMID:26510759

  5. Intracellular calcium elevation induced by extracellular application of cyclic-ADP-ribose or oxytocin is temperature-sensitive in rodent NG108-15 neuronal cells with or without exogenous expression of human oxytocin receptors.

    PubMed

    Amina, S; Hashii, M; Ma, W-J; Yokoyama, S; Lopatina, O; Liu, H-X; Islam, M S; Higashida, H

    2010-05-01

    ADP-ribosyl cyclase and/or CD38 are activated after oxytocin receptor stimulation in the hypothalamus and pituitary in adult mice, leading to facilitation of oxytocin secretion. Although cyclic adenosine 5'-diphosphoribose (cADPR) primarily acts as an intracellular second messenger, it has been suggested that extracellular cADPR stimulates intracellular ryanodine receptors after internalisation via the nucleotide-transporting capacity of CD38 in fibroblasts and astrocytes. However, little is known about whether extracellular cADPR activates neurones. To address this question, we used a model neuronal cell line, NG108-15 mouse neuroblastoma x rat glioma hybrid cells possessing CD38 but not oxytocin receptors, and measured cytosolic free calcium concentrations ([Ca(2+)](i)). Extracellular application of cADPR to NG108-15 cells elevated [Ca(2+)](i) at 35 degrees C. The elevation was significantly enhanced when measured at 40 degrees C. The cADPR and heat-induced [Ca(2+)](i) increase were blocked under extracellular Ca(2+)-free conditions and by 2-aminoethoxydiphenyl borate, an antagonist of melastatin-related transient receptor potential channel 2 (TRPM2) cation channels. Reverse transcriptation-polymerase chain reaction analyses indicated that TRPM2 channels were expressed in NG108-15 cells. Application of oxytocin elevated [Ca(2+)](i) in NG108-15 cells transformed to transiently express cloned human oxytocin receptors. The oxytocin-induced [Ca(2+)](i) response was also enhanced by heat. These results indicate that the extracellular application of cADPR, together with heat, activates cation influx downstream of oxytocin receptor signalling in NG108-15 neuronal cells, and suggest the possible involvement of TRPM2 channels in oxytocin release in the mammalian brain.

  6. Calcium overload increases oxidative stress in old rat gastrocnemius muscle.

    PubMed

    Capel, F; Demaison, L; Maskouri, F; Diot, A; Buffiere, C; Patureau Mirand, P; Mosoni, L

    2005-09-01

    In order to challenge in vivo muscle Ca2+ homeostasis and analyze consequences on mitochondrial H2O2 release (MHR) and sarcopenia, we injected Ca2+ ionophore A23187 (200 microg/kg, ip) in adult and old rats and measured gastrocnemius mass and mitochondrial Ca2+ content (MCC) using radioactive Ca2+ 48 h after injection. In a second experiment performed in old rats, we measured isocitrate dehydrogenase (ICDH) activity as an index of MCC, MHR, mitochondrial respiration, citrate synthase, COX and antioxydant enzyme activities 24 h after a 150 microg/kg injection. In adult rats, muscle mass and MCC were unchanged by A23187. In old rats, MCC increased 24 h after injection as reflected by a significant increase in ICDH activity; measured MCC tended to increase at 48 h. MHR and Mn-SOD activity were significantly increased at 24 h, and GPX activity was reduced. Muscle mass was unchanged but was negatively correlated with MCC in control and treated old rats. In conclusion, in old rats, A23187 probably induced a mitochondrial Ca2+ overload responsible for the observed increase in MHR without leading to muscle atrophy on a short term basis.

  7. Canine distemper virus infection of primary hippocampal cells induces increase in extracellular glutamate and neurodegeneration.

    PubMed

    Brunner, Jean-Marc; Plattet, Philippe; Majcherczyk, Paul; Zurbriggen, Andreas; Wittek, Riccardo; Hirling, Harald

    2007-11-01

    The canine distemper virus (CDV) belongs to the Morbillivirus genus which includes important human pathogens like the closely related measles virus. CDV infection can reach the nervous system where it causes serious malfunctions. Although this pathology is well described, the molecular events in brain infection are still poorly understood. Here we studied infection in vitro by CDV using a model of dissociated cell cultures from newborn rat hippocampus. We used a recombinant CDV closely related to the neurovirulent A75/17 which also expresses the enhanced green fluorescent protein. We found that infected neurons and astrocytes could be clearly detected, and that infection spreads only slowly to neighboring cells. Interestingly, this infection causes a massive cell death of neurons, which includes also non-infected neurons. Antagonists of NMDA-type or alpha-amino-3-hydroxy-5-methylisoxazole-4-propinate (AMPA)-type glutamate receptors could slow down this neuron loss, indicating an involvement of the glutamatergic system in the induction of cell death in infected and non-infected cells. Finally, we show that, following CDV infection, there is a steady increase in extracellular glutamate in infected cultures. These results indicate that CDV infection induces excitotoxic insults on neurons via glutamatergic signaling.

  8. Exposure to repeated immobilization stress inhibits cocaine-induced increase in dopamine extracellular levels in the rat ventral tegmental area.

    PubMed

    Sotomayor-Zárate, Ramón; Abarca, Jorge; Araya, Katherine A; Renard, Georgina M; Andrés, María E; Gysling, Katia

    2015-11-01

    A higher vulnerability to drug abuse has been observed in human studies of individuals exposed to chronic or persistent stress, as well as in animal models of drug abuse. Here, we explored the effect of repeated immobilization stress on cocaine-induced increase in dopamine extracellular levels in VTA and its regulation by corticotropin-releasing factor (CRF) and GABA systems. Cocaine (10mg/Kg i.p.) induced an increase of VTA DA extracellular levels in control rats. However, this effect was not observed in repeated stress rats. Considering the evidence relating stress with CRF, we decided to perfuse CRF and CP-154526 (selective antagonist of CRF1 receptor) in the VTA of control and repeated stress rats, respectively. We observed that perfusion of 20μM CRF inhibited the increase of VTA DA extracellular levels induced by cocaine in control rats. Interestingly, we observed that in the presence of 10μM CP-154526, cocaine induced a significant increase of VTA DA extracellular levels in repeated stress rats. Regarding the role of VTA GABA neurotransmission, cocaine administration induced a significant increase in VTA GABA extracellular levels only in repeated stress rats. Consistently, cocaine was able to increase VTA DA extracellular levels in repeated stress rats when 100μM bicuculline, an antagonist of GABAA receptor, was perfused intra VTA. Thus, both CRF and GABA systems are involved in the lack of response to cocaine in the VTA of repeated stress rats. It is tempting to suggest that the loss of response in VTA dopaminergic neurons to cocaine, after repeated stress, is due to an interaction between CRF and GABA systems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Calcium amendment may increase hydraulic efficiency and forest evapotranspiration

    Treesearch

    Kevin T. Smith; Walter C. Shortle

    2013-01-01

    Green et al. (1) report 2 y of increased evapotranspiration (ET; calculated as the difference between total precipitation and total runoff) and decreased water yield following watershed-scale amendment of soil with wollastonite (CaSiO3) at the Hubbard Brook Experimental Forest in the White Mountains of New Hampshire. The...

  10. Extracellular calcium ions modify the effects of Anemonia sulcata toxin (ATX II) in guinea-pig papillary muscles.

    PubMed

    Ravens, U

    1983-08-15

    In isolated guinea-pig papillary muscle ATX II prolonged the action potential duration to a lesser extent at high extracellular Ca++-concentrations. This is interpreted as an interference of Ca++ with ATX II-binding sites.

  11. Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

    PubMed

    Chapman, Mark A; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David; Lieber, Richard L

    2017-02-01

    Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell.

  12. ALK1 heterozygosity increases extracellular matrix protein expression, proliferation and migration in fibroblasts.

    PubMed

    Muñoz-Félix, José M; Perretta-Tejedor, Nuria; Eleno, Nélida; López-Novoa, José M; Martínez-Salgado, Carlos

    2014-06-01

    Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1(+/+) and ALK1(+/-) mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.

  13. Increasing extracellular potassium results in subthalamic neuron activity resembling that seen in a 6-hydroxydopamine lesion.

    PubMed

    Strauss, Ulf; Zhou, Fu-Wen; Henning, Jeannette; Battefeld, Arne; Wree, Andreas; Köhling, Rüdiger; Haas, Stefan Jean-Pierre; Benecke, Reiner; Rolfs, Arndt; Gimsa, Ulrike

    2008-06-01

    Abnormal neuronal activity in the subthalamic nucleus (STN) plays a crucial role in the pathophysiology of Parkinson's disease (PD). Although altered extracellular potassium concentration ([K+]o) and sensitivity to [K+]o modulates neuronal activity, little is known about the potassium balance in the healthy and diseased STN. In vivo measurements of [K+]o using ion-selective electrodes demonstrated a twofold increase in the decay time constant of lesion-induced [K+]o transients in the STN of adult Wistar rats with a unilateral 6-hydroxydopamine (6-OHDA) median forebrain bundle lesion, employed as a model of PD, compared with nonlesioned rats. Various [K+]o concentrations (1.5-12.5 mM) were applied to in vitro slice preparations of three experimental groups of STN slices from nonlesioned control rats, ipsilateral hemispheres, and contralateral hemispheres of lesioned rats. The majority of STN neurons of nonlesioned rats and in slices contralateral to the lesion fired spontaneously, predominantly in a regular pattern, whereas those in slices ipsilateral to the lesion fired more irregularly or even in bursts. Experimentally increased [K+]o led to an increase in the number of spontaneously firing neurons and action potential firing rates in all groups. This was accompanied by a decrease in the amplitude of post spike afterhyperpolarization (AHP) and the amplitude and duration of the posttrain AHP. Lesion effects in ipsilateral neurons at physiological [K+]o resembled the effects of elevated [K+]o in nonlesioned rats. Our data suggest that changed potassium sensitivity due to conductivity alterations and delayed clearance may be critical for shaping STN activity in parkinsonian states.

  14. Alpha Hemolysin Induces an Increase of Erythrocytes Calcium: A FLIM 2-Photon Phasor Analysis Approach

    PubMed Central

    Sanchez, Susana; Bakás, Laura; Gratton, Enrico; Herlax, Vanesa

    2011-01-01

    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. PMID:21698153

  15. Acetate transiently inhibits myocardial contraction by increasing mitochondrial calcium uptake.

    PubMed

    Schooley, James F; Namboodiri, Aryan M A; Cox, Rachel T; Bünger, Rolf; Flagg, Thomas P

    2014-12-09

    There is a close relationship between cardiovascular disease and cardiac energy metabolism, and we have previously demonstrated that palmitate inhibits myocyte contraction by increasing Kv channel activity and decreasing the action potential duration. Glucose and long chain fatty acids are the major fuel sources supporting cardiac function; however, cardiac myocytes can utilize a variety of substrates for energy generation, and previous studies demonstrate the acetate is rapidly taken up and oxidized by the heart. In this study, we tested the effects of acetate on contractile function of isolated mouse ventricular myocytes. Acute exposure of myocytes to 10 mM sodium acetate caused a marked, but transient, decrease in systolic sarcomere shortening (1.49 ± 0.20% vs. 5.58 ± 0.49% in control), accompanied by a significant increase in diastolic sarcomere length (1.81 ± 0.01 μm vs. 1.77 ± 0.01 μm in control), with a near linear dose response in the 1-10 mM range. Unlike palmitate, acetate caused no change in action potential duration; however, acetate markedly increased mitochondrial Ca(2+) uptake. Moreover, pretreatment of cells with the mitochondrial Ca(2+) uptake blocker, Ru-360 (10 μM), markedly suppressed the effect of acetate on contraction. Lehninger and others have previously demonstrated that the anions of weak aliphatic acids such as acetate stimulate Ca(2+) uptake in isolated mitochondria. Here we show that this effect of acetate appears to extend to isolated cardiac myocytes where it transiently modulates cell contraction.

  16. Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells.

    PubMed

    Lavi, Ronit; Shainberg, Asher; Friedmann, Harry; Shneyvays, Vladimir; Rickover, Ophra; Eichler, Maor; Kaplan, Doron; Lubart, Rachel

    2003-10-17

    Low energy visible light (LEVL) irradiation has been shown to exert some beneficial effects on various cell cultures. For example, it increases the fertilizing capability of sperm cells, promotes cell proliferation, induces sprouting of neurons, and more. To learn about the mechanism of photobiostimulation, we studied the relationship between increased intracellular calcium ([Ca2+]i) and reactive oxygen species production following LEVL illumination of cardiomyocytes. We found that visible light causes the production of O2. and H2O2 and that exogenously added H2O2 (12 microm) can mimic the effect of LEVL (3.6 J/cm2) to induce a slow and transient increase in [Ca2+]i. This [Ca2+]i elevation can be reduced by verapamil, a voltage-dependent calcium channel inhibitor. The kinetics of [Ca2+]i elevation and morphologic damage following light or addition of H2O2 were found to be dose-dependent. For example, LEVL, 3.6 J/cm2, which induced a transient increase in [Ca2+]i, did not cause any cell damage, whereas visible light at 12 J/cm2 induced a linear increase in [Ca2+]i and damaged the cells. The linear increase in [Ca2+]i resulting from high energy doses of light could be attenuated into a non-linear small rise in [Ca2+]i by the presence of extracellular catalase during illumination. We suggest that the different kinetics of [Ca2+]i elevation following various light irradiation or H2O2 treatment represents correspondingly different adaptation levels to oxidative stress. The adaptive response of the cells to LEVL represented by the transient increase in [Ca2+]i can explain LEVL beneficial effects.

  17. Modulation of iron metabolism by iron chelation regulates intracellular calcium and increases sensitivity to doxorubicin

    PubMed Central

    Yalcintepe, Leman; Halis, Emre

    2016-01-01

    Increased intracellular iron levels can both promote cell proliferation and death, as such; iron has a “two-sided effect” in the delicate balance of human health. Though the role of iron in the development of cancer remains unclear, investigations of iron chelators as anti-tumor agents have revealed promising results. Here, we investigated the influence of iron and desferrioxamine (DFO), the iron chelating agent on intracellular calcium in a human leukemia cell line, K562. Iron uptake is associated with increased reactive oxygen species (ROS) generation. Therefore, we showed that iron also caused dose-dependent ROS generation in K562 cells. The measurement of intracellular calcium was determined using Furo-2 with a fluorescence spectrophotometer. The iron delivery process to the cytoplasmic iron pool was examined by monitoring the fluorescence of cells loaded with calcein-acetoxymethyl. Our data showed that iron increased intracellular calcium, and this response was 8 times higher when cells were incubated with DFO. K562 cells with DFO caused a 3.5 times increase of intracellular calcium in the presence of doxorubicin (DOX). In conclusion, DFO induces intracellular calcium and increases their sensitivity to DOX, a chemotherapeutic agent. PMID:26773173

  18. Glibenclamide prevents increased extracellular matrix formation induced by high glucose concentration in mesangial cells.

    PubMed

    Giannico, Giovanna; Cortes, Pedro; Baccora, Mohammed H; Hassett, Clare; Taube, David W; Yee, Jerry

    2007-01-01

    Other than stimulation of cell contractility, little is known about the potential metabolic effects induced by sulfonylureas, independently of insulin action. Previous studies from our laboratory demonstrated complete abrogation of glomerulosclerosis in an experimental model of type 1 diabetes chronically (9 mo) treated with low-dose sulfonylureas (Biederman JI, Vera E, Pankhaniya R, Hassett C, Giannico G, Yee J, Cortes P. Kidney Int 67: 554-565, 2005). Therefore, the effects of glibenclamide (Glib) on net collagen I, collagen IV, and fibronectin medium net secretion and cell layer collagen I deposition were investigated in mesangial cells continuously exposed to 25 mM glucose for 8 wk and treated with predetermined increasing concentrations of Glib for the same period. Clinically relevant concentrations (0.01 microM) of Glib fully suppressed the high glucose-enhanced accumulation of collagen I, collagen IV, and fibronectin in the medium and inhibited collagen I deposition in the cell layer. These effects occurred while transforming growth factor (TGF)-beta1 medium concentration remained elevated and glucose uptake was increased to levels above those in 25 mM glucose-incubated cultures. The decreased collagen I accumulation occurred simultaneously with enhanced collagen I mRNA expression in concert with marked suppression of plasminogen inhibitor type-1 (PAI-1) mRNA and protein expression. This strongly suggests an accelerated matrix turnover favoring breakdown. Glib-induced effects demonstrated a biphasic pattern, being absent or reversed in cells treated with higher Glib concentrations (0.1 or 1 microM). Therefore, chronic Glib treatment at low concentrations markedly diminishes the high glucose-induced enhanced accumulation of extracellular matrix components by suppression of steady-state PAI-1 transcriptional activity. These results and those previously reported in vivo suggest that long-term Glib treatment may prevent glomerulosclerosis in insulin

  19. Extracellular matrix hyaluronan signals via its CD44 receptor in the increased responsiveness to mechanical stimulation.

    PubMed

    Ferrari, L F; Araldi, D; Bogen, O; Levine, J D

    2016-06-02

    We propose that the extracellular matrix (ECM) signals CD44, a hyaluronan receptor, to increase the responsiveness to mechanical stimulation in the rat hind paw. We report that intradermal injection of hyaluronidase induces mechanical hyperalgesia, that is inhibited by co-administration of a CD44 receptor antagonist, A5G27. The intradermal injection of low (LMWH) but not high (HMWH) molecular weight hyaluronan also induces mechanical hyperalgesia, an effect that was attenuated by pretreatment with HMWH or A5G27. Pretreatment with HMWH also attenuated the hyperalgesia induced by hyaluronidase. Similarly, intradermal injection of A6, a CD44 receptor agonist, produced hyperalgesia that was inhibited by HMWH and A5G27. Inhibitors of protein kinase A (PKA) and Src, but not protein kinase C (PKC), significantly attenuated the hyperalgesia induced by both A6 and LMWH. Finally, to determine if CD44 receptor signaling is involved in a preclinical model of inflammatory pain, we evaluated the effect of A5G27 and HMWH on the mechanical hyperalgesia associated with the inflammation induced by carrageenan. Both A5G27 and HMWH attenuated carrageenan-induced mechanical hyperalgesia. Thus, while LMWH acts at its cognate receptor, CD44, to induce mechanical hyperalgesia, HMWH acts at the same receptor as an antagonist. That the local administration of HMWH or A5G27 inhibits carrageenan-induced hyperalgesia supports the suggestion that carrageenan produces changes in the ECM that contributes to inflammatory pain. These studies define a clinically relevant role for signaling by the hyaluronan receptor, CD44, in increased responsiveness to mechanical stimulation.

  20. Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-β1.

    PubMed

    Mewhort, Holly E M; Lipon, Brodie D; Svystonyuk, Daniyil A; Teng, Guoqi; Guzzardi, David G; Silva, Claudia; Yong, V Wee; Fedak, Paul W M

    2016-03-15

    Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P < 0.01) and increased local ECM remodeling quantified by confocal microscopy. Under coculture conditions that allow indirect cellular interaction via paracrine factors but prevent direct cell-cell contact, monocytes had minimal effects on myofibroblast activity (17.9 ± 11.1% vs. 6.4 ± 7.0% increase, respectively; P < 0.01). When cells were cultured under direct contact conditions, multiplex analysis of the coculture media revealed an increase in the paracrine factors TGF-β1 and matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P < 0.001). TGF-β blockade abolished the monocyte-induced increase in cardiac myofibroblast activity. These data suggest that direct cell-cell interaction between monocytes and cardiac myofibroblasts stimulates TGF-β-mediated myofibroblast activity and increases remodeling of local matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1

  1. 2,4,6-Trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide increases calcium influx in lipopolisaccharide-pre-treated arteries

    PubMed Central

    Grześk, Elżbieta; Szadujkis-Szadurska, Katarzyna; Bloch-Bogusławska, Elżbieta; Wiciński, Michał; Malinowski, Bartosz; KołTan, Sylwia; Tejza, Barbara; Pujanek, Maciej; GrześK, Grzegorz

    2017-01-01

    It has been demonstrated that 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide (m-3M3FBS) activates phospholipase C (PLC) and stimulates apoptosis in smooth muscle cells, which may increase vascular reactivity. The primary aim of the present study was to evaluate the physiological effects of the direct stimulation of PLC by m-3M3FBS on vascular smooth muscle reactivity in arteries pre-treated with lipopolysaccharides (LPS) as a model of septic shock. Experiments were performed on isolated and perfused tail arteries of Wistar rats. The contraction force in the model was measured by assessing increases in perfusion pressure at a constant flow. Parameters describing the concentration-response curves (CRCs) obtained for phenylephrine and arginine-vasopressin in the presence of LPS confirmed a decrease in vessels reactivity. In comparison with the controls, m-3M3FBS treatment caused a significant increase in LPS-untreated as well as pre-treated arteries. Furthermore, in the presence of m-3M3FBS, calcium influx from intra- as well as extracellular calcium stores was significantly higher for LPS-untreated and pre-treated arteries. The results of the present study suggested that m-3M3FBS significantly increased the reactivity of vascular smooth muscle cells pre-treated with LPS by increasing the calcium influx from intra- and extracellular calcium stores. Further studies investigating this mechanism are required to evaluate whether this pathway may be a potential therapeutic strategy to treat sepsis. PMID:28352364

  2. Calcium.

    PubMed

    Williams, Robert J P

    2002-01-01

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

  3. A calcium-dependent potassium current is increased by a single-gene mutation in Paramecium.

    PubMed

    Hennessey, T M; Kung, C

    1987-01-01

    The membrane currents of wild type Paramecium tetraurelia and the behavioral mutant teaA were analyzed under voltage clamp. The teaA mutant was shown to have a greatly increased outward current which was blocked completely by the combined use of internally delivered Cs+ and external TEA+. This, along with previous work (Satow, Y., Kung, C., 1976, J. Exp. Biol. 65:51-63) identified this as a K+ current. It was further found to be a calcium-activated K+ current since this increased outward K+ current cannot be elicited when the internal calcium is buffered with injected EGTA. The mutation pwB, which blocks the inward calcium current, also blocks this increased outward K+ current in teaA. This shows that this mutant current is activated by calcium through the normal depolarization-sensitive calcium channel. While tail current decay kinetic analysis showed that the apparent inactivation rates for this calcium-dependent K+ current are the same for mutant and wild type, the teaA current activates extremely rapidly. It is fully activated within 2 msec. This early activation of such a large outward current causes a characteristic reduction in the amplitude of the action potential of the teaA mutant. The teaA mutation had no effect on any of the other electrophysiological parameters examined. The phenotype of the teaA mutant is therefore a general decrease in responsiveness to depolarizing stimuli because of a rapidly activating calcium-dependent K+ current which prematurely repolarizes the action potential.

  4. Implication of the calcium sensing receptor and the Phosphoinositide 3-kinase/Akt pathway in the extracellular calcium-mediated migration of RAW 264.7 osteoclast precursor cells.

    PubMed

    Boudot, Cédric; Saidak, Zuzana; Boulanouar, Abdel Krim; Petit, Laurent; Gouilleux, Fabrice; Massy, Ziad; Brazier, Michel; Mentaverri, Romuald; Kamel, Saïd

    2010-05-01

    While the processes involved in the formation, maturation and apoptosis of osteoclasts have been investigated extensively in previous studies, little is known about the mechanisms responsible for the localization and homing of osteoclast precursor cells to the bone environment in order to initiate the bone remodeling process. Recent studies have suggested that the extracellular Ca(2+) (Ca(2+)(o)) concentration gradient present near the bone environment may be one of the participating factors, producing a chemoattractant effect on osteoclast precursors. Using the murine osteoclast precursor cells of the monocyte-macrophage lineage, the RAW 264.7 cell line, we have shown that Ca(2+)(o) increases the migration of these cells in a directional manner. The participation of the calcium sensing receptor (CaR) in this effect was tested by knocking down its expression through RNA interference, which resulted in an abolition of the migratory response. By the use of specific pathway inhibitors and western blot analysis, the phosphoinositide 3-kinase (PI3K)/Akt and phospholipase Cbeta pathways were shown to be implicated in the migratory effect. The implication of the Akt pathway in the Ca(2+)(o)-induced chemoattraction of RAW 264.7 cells was also confirmed by transducing the cells with the fusion protein TAT-dominant negative-Akt, which decreased the migratory effect. In contrast, the MAPK pathways (ERK1/2, p38 and JNK) were not involved in the production of the migratory effect. We conclude that through the activation of the CaR and subsequent signaling via the PI3K/Akt pathway, Ca(2+)(o) produces a chemoattractant effect on the osteoclast precursor RAW 264.7 cells. These results suggest that the Ca(2+)(o) gradient present near the bone may be one of the initiating factors for the homing of osteoclast precursors to bone, thus possibly playing a role in the initiation of bone remodeling. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  5. Aquaporin-4 Deficient Mice have Increased Extracellular Space Without Tortuosity Change

    PubMed Central

    Yao, Xiaoming; Hrabětová, Sabina; Nicholson, Charles; Manley, Geoffrey T.

    2008-01-01

    Aquaporin-4 (AQP4) is the major water channel expressed at fluid-tissue barriers throughout the brain and plays a crucial role in cerebral water balance. To assess whether these channels influence brain extracellular space (ECS) under resting physiological conditions, we used the established real-time iontophoresis method with tetramethylammonium (TMA+) to measure three diffusion parameters: ECS volume fraction (α), tortuosity (λ), and TMA+ loss (k’). In vivo measurements were performed in the somatosensory cortex of AQP4 deficient (AQP4-/-) mice and wild-type controls with matched age. Mice lacking AQP4 showed a 28% increase in α (0.23 ± 0.007 vs. 0.18 ± 0.003) with no differences in λ (1.62 ± 0.04 vs. 1.61 ± 0.02) and k’ (0.0045 ± 0.0001 1/sec vs. 0.0031 ± 0.0009 1/sec). Additional recordings in brain slices showed similarly elevated α in AQP4-/- mice, and no differences in λ and k’ between the two genotypes. This is the first direct comparison of ECS properties in adult mice lacking AQP4 water channels with wild-type animals and demonstrates a significant enlargement of the volume fraction but no difference in hindrance to TMA+ diffusion, expressed as tortuosity. These findings provide direct evidence for involvement of AQP4 in modulation of the ECS volume fraction and provide a basis for future modeling of water and ion transport in the central nervous system. PMID:18495879

  6. Extracellular ATP Hydrolysis Inhibits Synaptic Transmission by Increasing pH Buffering in the Synaptic Cleft

    PubMed Central

    Vroman, Rozan; Klaassen, Lauw J.; Howlett, Marcus H.C.; Cenedese, Valentina; Klooster, Jan; Sjoerdsma, Trijntje; Kamermans, Maarten

    2014-01-01

    Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca2+ channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation

  7. Does Potassium Citrate Medical Therapy Increase the Risk of Calcium Phosphate Stone Formation?

    NASA Astrophysics Data System (ADS)

    Leitao, Victor; Haleblian, George E.; Robinson, Marnie R.; Pierre, Sean A.; Sur, Roger L.; Preminger, Glenn M.

    2007-04-01

    Potassium citrate has been extensively used in the treatment of recurrent nephrolithiasis. Recent evidence suggests that it may contribute to increasing urinary pH and, as such, increase the risk of calcium phosphate stone formation. We performed a retrospective review of our patients to further investigate this phenomenon.

  8. Congenital Heart Block Maternal Sera Autoantibodies Target an Extracellular Epitope on the α1G T-Type Calcium Channel in Human Fetal Hearts

    PubMed Central

    Rath, Arianna; Liu, Jie; Silverman, Earl D.; Liu, Xiaoru; Siragam, Vinayakumar; Ackerley, Cameron; Su, Brenda Bin; Yan, Jane Yuqing; Capecchi, Marco; Biavati, Luca; Accorroni, Alice; Yuen, William; Quattrone, Filippo; Lung, Kalvin; Jaeggi, Edgar T.; Backx, Peter H.; Deber, Charles M.; Hamilton, Robert M.

    2013-01-01

    Background Congenital heart block (CHB) is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV) block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit α1G may be a fetal target of maternal sera autoantibodies in CHB. Methodology/Principal Findings We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G (α1G gene) in the AV junction of human fetal hearts compared to the apex (18–22.6 weeks gestation). Using human fetal hearts (20–22 wks gestation), our immunoprecipitation (IP), Western blot analysis and immunofluorescence (IF) staining results, taken together, demonstrate accessibility of the α1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the α1G protein. By ELISA we demonstrated maternal sera reactivity to α1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305–319 of the extracellular loop linking transmembrane segments S5–S6 in α1G repeat I). Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved) of the α1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN) cells. Conclusions/Significance Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of α1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for α1H suggesting that autoantibodies can target multiple fetal targets. PMID:24039792

  9. Congenital heart block maternal sera autoantibodies target an extracellular epitope on the α1G T-type calcium channel in human fetal hearts.

    PubMed

    Strandberg, Linn S; Cui, Xuezhi; Rath, Arianna; Liu, Jie; Silverman, Earl D; Liu, Xiaoru; Siragam, Vinayakumar; Ackerley, Cameron; Su, Brenda Bin; Yan, Jane Yuqing; Capecchi, Marco; Biavati, Luca; Accorroni, Alice; Yuen, William; Quattrone, Filippo; Lung, Kalvin; Jaeggi, Edgar T; Backx, Peter H; Deber, Charles M; Hamilton, Robert M

    2013-01-01

    Congenital heart block (CHB) is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV) block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit α1G may be a fetal target of maternal sera autoantibodies in CHB. We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G (α1G gene) in the AV junction of human fetal hearts compared to the apex (18-22.6 weeks gestation). Using human fetal hearts (20-22 wks gestation), our immunoprecipitation (IP), Western blot analysis and immunofluorescence (IF) staining results, taken together, demonstrate accessibility of the α1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the α1G protein. By ELISA we demonstrated maternal sera reactivity to α1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305-319 of the extracellular loop linking transmembrane segments S5-S6 in α1G repeat I). Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved) of the α1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN) cells. Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of α1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for α1H suggesting that autoantibodies can target multiple fetal targets.

  10. A fungal homologue of neuronal calcium sensor-1, Bbcsa1, regulates extracellular acidification and contributes to virulence in the entomopathogenic fungus Beauveria bassiana.

    PubMed

    Fan, Yanhua; Ortiz-Urquiza, Almudena; Kudia, Ramsha A; Keyhani, Nemat O

    2012-07-01

    Neuronal calcium sensor proteins and their homologues participate in transducing extracellular signals that affect intracellular Ca(2+) levels, which in turn regulate enzyme activities, secretion, gene expression and other biological processes. The filamentous fungus Beauveria bassiana is a broad-host-range pathogen of insects that acidifies the extracellular milieu during growth and pathogenesis towards target hosts. A collection of B. bassiana random insertion mutants were screened on pH indicator plates and one mutant was isolated that displayed reduced acidification. The random insertion site was mapped to a gene that displayed homology to the neuronal calcium sensor/frequenin protein family and was designated Bbcsa1. To validate the role of Bbcsa1 in B. bassiana, a targeted gene-knockout was constructed. Data confirmed that Bbcsa1 was not an essential gene and the ΔBbcsa1 strain displayed delayed acidification of the medium when grown in Czapek-Dox medium, as compared with the wild-type parent. HPLC profiling of secreted metabolites did not detect any major changes in the production of organic acids, although downregulation of the membrane H(+) pump/ATPase was noted in the mutant. A slight growth-deficient phenotype was observed for the ΔBbcsa1 strain on Czapek-Dox and potato dextrose media, which was accentuated at high calcium concentrations (500 mM) and 1.5 M sorbitol, but was unaffected by EDTA or SDS. Perturbations in vacuole morphology were also noted for the mutant. Insect bioassays using Galleria mellonella as the target host revealed decreased virulence in the ΔBbcsa1 mutant when applied topically, representing the natural route of infection, but no significant effect was observed when fungal cells were directly injected into target hosts. These results suggest that Bbcsa1 participates in pre-penetration or early penetration events, but is dispensable once the insect cuticle has been breached.

  11. The hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) increases cortical extracellular glutamate levels in rats.

    PubMed

    Scruggs, Jennifer L; Schmidt, Dennis; Deutch, Ariel Y

    2003-08-07

    Activation of the cerebral cortex is seen during hallucinations. The 5-HT(2A/C) agonist 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) is a potent hallucinogen that has been proposed to act by targeting 5-HT(2A) heteroceptors on thalamocortical neurons and eliciting release of glutamate from these cells, which in turn drives cortical neurons. We used in vivo microdialysis to determine if DOI increases extracellular glutamate levels. Systemic administration of DOI significantly increased extracellular glutamate levels in the somatosensory cortex of the freely-moving rat. Similarly, intracortical administration of DOI by reverse dialysis increased cortical extracellular glutamate levels. No consistent changes in either extracellular GABA or glycine levels were observed in response to DOI. The increase in glutamate levels elicited by intracortical DOI was blocked by treatment with the selective 5-HT(2A) antagonist MDL 100,907. These data are consistent with the hypothesis that 5-HT(2A) receptor-mediated regulation of glutamate release is the mechanism through which hallucinogens activate the cerebral cortex.

  12. Oxytocin Inhibits the Membrane Depolarization-Induced Increase in Intracellular Calcium in Capsaicin Sensitive Sensory Neurons: A Peripheral Mechanism of Analgesic Action

    PubMed Central

    Hobo, Shotaro; Hayashida, Ken-ichiro; Eisenach, James C.

    2011-01-01

    Background Lumbar intrathecal injection of oxytocin produces antinociception in rats and analgesia in humans. Classically, oxytocin receptors couple to stimulatory G proteins, increase inositol-3-phosphate production, and result in neuronal excitation. Most work to date has focused on a spinal site of oxytocin to excite γ-aminobutyric acid interneurons to produce analgesia. Here we ask whether oxytocin might also affect primary sensory afferents by modulating high voltage-gated calcium channels, such as it does in the brain. Methods Dorsal root ganglion cells from adult rats were acutely dissociated and cultured, and changes in intracellular calcium determined by fluorescent microscopy using an indicator dye. The effects of oxytocin alone and in the presence of transient depolarization from increased extracellular KCl concentration were determined, then the pharmacology of these effects were studied. Cells from injured dorsal root ganglion cells after spinal nerve ligation were also studied. Results Oxytocin produced a concentration-dependent inhibition of the increase in intracellular calcium from membrane depolarization, an effect blocked more efficiently by oxytocin- than vasopressin-receptor selective antagonists. Oxytocin-induced inhibition was present in cells responding to capsaicin, and when internal stores of calcium were depleted with thapsigargin. Oxytocin produced similar inhibition in cells from animals with spinal nerve ligation. Conclusions These data suggest that oxytocin produces antinociception after intrathecal delivery in part by reducing excitatory neurotransmitter release from the central terminals of nociceptors. PMID:22104073

  13. Endogenous dopamine increases extracellular concentrations of glutamate and GABA in striatum of the freely moving rat: involvement of D1 and D2 dopamine receptors.

    PubMed

    Expósito, I; Del Arco, A; Segovia, G; Mora, F

    1999-07-01

    Interactions between endogenous dopamine, glutamate, GABA, and taurine were investigated in striatum of the freely moving rat by using microdialysis. Intrastriatal infusions of the selective dopamine uptake inhibitor nomifensine (NMF) were used to increase the endogenous extracellular dopamine. NMF produced a dose-related increase in extracellular dopamine and also increased extracellular concentrations of glutamate, GABA, and taurine. Extracellular increases of dopamine were significantly correlated with extracellular increases of glutamate and GABA, but not taurine. To investigate whether the increased extracellular dopamine produced by NMF was responsible for the concomitant increase of glutamate and GABA, D1, and D2 receptor antagonists were used. Dopamine receptor antagonists D1 (SCH23390) and D2 (sulpiride) significantly attenuated the increases of glutamate and GABA produced by NMF. These data suggest that endogenous dopamine, through both D1 and D2 dopamine receptors, plays a role in releasing glutamate and GABA in striatum of the freely moving rat.

  14. Increased Extracellular Glutamate In the Nucleus Accumbens Promotes Excessive Ethanol Drinking in Ethanol Dependent Mice

    PubMed Central

    Griffin III, William C; Haun, Harold L; Hazelbaker, Callan L; Ramachandra, Vorani S; Becker, Howard C

    2014-01-01

    Using a well-established model of ethanol dependence and relapse, this study examined adaptations in glutamatergic transmission in the nucleus accumbens (NAc) and their role in regulating voluntary ethanol drinking. Mice were first trained to drink ethanol in a free-choice, limited access (2 h/day) paradigm. One group (EtOH mice) received repeated weekly cycles of chronic intermittent ethanol (CIE) exposure with intervening weeks of test drinking sessions, whereas the remaining mice (CTL mice) were similarly treated but did not receive CIE treatment. Over repeated cycles of CIE exposure, EtOH mice exhibited significant escalation in drinking (up to ∼3.5 g/kg), whereas drinking remained relatively stable at baseline levels (2–2.5 g/kg) in CTL mice. Using in vivo microdialysis procedures, extracellular glutamate (GLUEX) levels in the NAc were increased approximately twofold in EtOH mice compared with CTL mice, and this difference was observed 7 days after final CIE exposure, indicating that this hyperglutamatergic state persisted beyond acute withdrawal. This finding prompted additional studies examining the effects of pharmacologically manipulating GLUEX in the NAc on ethanol drinking in the CIE model. The non-selective glutamate reuptake antagonist, threo-β-benzyloxyaspartate (TBOA), was bilaterally microinjected into the NAc and found to dose-dependently increase drinking in nondependent (CTL) mice to levels attained by dependent (EtOH) mice. TBOA also further increased drinking in EtOH mice. In contrast, reducing glutamatergic transmission in the NAc via bilateral injections of the metabotropic glutamate receptor-2/3 agonist LY379268 reduced drinking in dependent (EtOH) mice to nondependent (CTL) levels, whereas having a more modest effect in decreasing ethanol consumption in CTL mice. Taken together, these data support an important role of glutamatergic transmission in the NAc in regulating ethanol drinking. Additionally, these results indicate that

  15. Increasing serotonin concentrations alter calcium and energy metabolism in dairy cows.

    PubMed

    Laporta, Jimena; Moore, Spencer A E; Weaver, Samantha R; Cronick, Callyssa M; Olsen, Megan; Prichard, Austin P; Schnell, Brian P; Crenshaw, Thomas D; Peñagaricano, Francisco; Bruckmaier, Rupert M; Hernandez, Laura L

    2015-07-01

    A 4×4 Latin square design in which varied doses (0, 0.5, 1.0, and 1.5 mg/kg) of 5-hydroxy-l-tryptophan (5-HTP, a serotonin precursor) were intravenously infused into late-lactation, non-pregnant Holstein dairy cows was used to determine the effects of serotonin on calcium and energy metabolism. Infusion periods lasted 4 days, with a 5-day washout between periods. Cows were infused at a constant rate for 1 h each day. Blood was collected pre- and 5, 10, 30, 60, 90, and 120 min post-infusion, urine was collected pre- and post-infusion, and milk was collected daily. All of the 5-HTP doses increased systemic serotonin as compared to the 0 mg/kg dose, and the 1.0 and 1.5 mg/kg doses increased circulating glucose and non-esterified fatty acids (NEFA) and decreased beta-hydroxybutyrate (βHBA) concentrations. Treatment of cows with either 1.0 or 1.5 mg/kg 5-HTP doses decreased urine calcium elimination, and the 1.5 mg/kg dose increased milk calcium concentrations. No differences were detected in the heart rates, respiration rates, or body temperatures of the cows; however, manure scores and defecation frequency were affected. Indeed, cows that received 5-HTP defecated more, and the consistency of their manure was softer. Treatment of late-lactation dairy cows with 5-HTP improved energy metabolism, decreased loss of calcium into urine, and increased calcium secretion into milk. Further research should target the effects of increasing serotonin during the transition period to determine any benefits for post-parturient calcium and glucose metabolism.

  16. Plum and Soy Aglycon Extracts Superior at Increasing Bone Calcium Retention in Ovariectomized Sprague Dawley Rats

    PubMed Central

    2015-01-01

    Plant-derived polyphenols have been shown to influence bone turnover and bone properties in the estrogen-depleted state. We used a crossover design in ovariectomized rats (n = 16 rats for each diet) to investigate the effect of supplementation of two doses each of blueberry, plum, grape, grape seed extract, and resveratrol on bone. We tested the aglycon and glucoside forms of genistein to quantify differences in efficacy on bone calcium retention. Rats were given an intravenous dose of 45Ca to prelabel bone, and bone calcium retention was assessed by urinary excretion of 45Ca:Ca ratio during an intervention period compared with nonintervention. Genistein aglycon increased bone calcium retention significantly (p < 0.05) more than the glucoside (22% vs 13%, respectively). Plum extract (0.45% w/w total dietary polyphenols) and resveratrol (0.2% w/w total dietary polyphenols) were also effective, increasing bone calcium retention by 20% (p = 0.0153) and 14% (p = 0.0012), respectively. Several polyphenolic-rich diets improved bone calcium retention. PMID:24894797

  17. Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: potential role in regulation of a nonselective cation channel

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Ye, C.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D(3) (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in Ca

  18. Broiler chicks fed low-calcium diets. 2. Increased sensitivity to copper toxicity.

    PubMed

    Leach, R M; Rosenblum, C I; Amman, M J; Burdette, J

    1990-11-01

    Young broiler chicks were more sensitive to copper toxicity when they were fed diets deficient or marginal in calcium content. Growth rate was depressed and liver copper concentration was increased under these conditions. Chicks fed a casein-gelatin diet were more sensitive to copper toxicity than those fed a corn-soybean meal diet. Addition of phytic acid to the casein-gelatin basal diet enhanced copper toxicity as evidenced by effects on growth rate and liver copper content. Measurements of intestinal and biliary copper content suggested that the influence of calcium on copper toxicity was mediated via intestinal absorption rather than through influences on copper excretion.

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

    PubMed

    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.

  20. Increased phase synchronization of spontaneous calcium oscillations in epileptic human versus normal rat astrocyte cultures

    NASA Astrophysics Data System (ADS)

    Balázsi, Gábor; Cornell-Bell, Ann H.; Moss, Frank

    2003-06-01

    Stochastic synchronization analysis is applied to intracellular calcium oscillations in astrocyte cultures prepared from epileptic human temporal lobe. The same methods are applied to astrocyte cultures prepared from normal rat hippocampus. Our results indicate that phase-repulsive coupling in epileptic human astrocyte cultures is stronger, leading to an increased synchronization in epileptic human compared to normal rat astrocyte cultures.

  1. AHNAK enables mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility

    PubMed Central

    Dzik, Luciana M.; Iglesia, Rebeca P.; Cruz, Mário C.; Zelanis, André; de Siqueira, Adriane S.; Serrano, Solange M.T.; Goldberg, Gary S.; Jaeger, Ruy G.; Freitas, Vanessa M.

    2016-01-01

    Extracellular vesicles play important roles in tumor development. Many components of these structures, including microvesicles and exosomes, have been defined. However, mechanisms by which extracellular vesicles affect tumor progression are not fully understood. Here, we investigated vesicular communication between mammary carcinoma cells and neighboring nontransformed mammary fibroblasts. Nonbiased proteomic analysis found that over 1% of the entire proteome is represented in these vesicles, with the neuroblast differentiation associated protein AHNAK and annexin A2 being the most abundant. In particular, AHNAK was found to be the most prominent component of these vesicles based on peptide number, and appeared necessary for their formation. In addition, we report here that carcinoma cells produce vesicles that promote the migration of recipient fibroblasts. These data suggest that AHNAK enables mammary carcinoma cells to produce and release extracellular vesicles that cause disruption of the stroma by surrounding fibroblasts. This paradigm reveals fundamental mechanisms by which vesicular communication between carcinoma cells and stromal cells can promote cancer progression in the tumor microenvironment. PMID:27374178

  2. AHNAK enables mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility.

    PubMed

    Silva, Thaiomara A; Smuczek, Basílio; Valadão, Iuri C; Dzik, Luciana M; Iglesia, Rebeca P; Cruz, Mário C; Zelanis, André; de Siqueira, Adriane S; Serrano, Solange M T; Goldberg, Gary S; Jaeger, Ruy G; Freitas, Vanessa M

    2016-08-02

    Extracellular vesicles play important roles in tumor development. Many components of these structures, including microvesicles and exosomes, have been defined. However, mechanisms by which extracellular vesicles affect tumor progression are not fully understood. Here, we investigated vesicular communication between mammary carcinoma cells and neighboring nontransformed mammary fibroblasts. Nonbiased proteomic analysis found that over 1% of the entire proteome is represented in these vesicles, with the neuroblast differentiation associated protein AHNAK and annexin A2 being the most abundant. In particular, AHNAK was found to be the most prominent component of these vesicles based on peptide number, and appeared necessary for their formation. In addition, we report here that carcinoma cells produce vesicles that promote the migration of recipient fibroblasts. These data suggest that AHNAK enables mammary carcinoma cells to produce and release extracellular vesicles that cause disruption of the stroma by surrounding fibroblasts. This paradigm reveals fundamental mechanisms by which vesicular communication between carcinoma cells and stromal cells can promote cancer progression in the tumor microenvironment.

  3. Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media.

    PubMed Central

    Pierson, E S; Miller, D D; Callaham, D A; Shipley, A M; Rivers, B A; Cresti, M; Hepler, P K

    1994-01-01

    Lily pollen tubes possess a steep, tip-focused intracellular Ca2+ gradient and a tip-directed extracellular Ca2+ influx. Ratiometric ion imaging revealed that the gradient extends from above 3.0 microM at the apex to approximately 0.2 microM within 20 microns from the tip, while application of the Ca(2+)-specific vibrating electrode indicated that the extracellular influx measured between 1.4 and 14 pmol cm-2 sec-1. We examined the relationship between these phenomena and their role in tube growth by using different 1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA)-type buffers and hypertonic media. Injection of active BAPTA-type buffers or application of elevated levels of sucrose reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca2+ flux. Of the BAPTA-type buffers, 5,5'-dibromo-BAPTA (dissociation constant [Kd] is 1.5 microM) and 4,4'-difluoro-BAPTA (Kd of 1.7 microM) exhibited greater activity than those buffers with either a higher affinity (5,5'-dimethyl-BAPTA, Kd of 0.15 microM; BAPTA, Kd of 0.21 microM; 5,5'-difluoro-BAPTA, Kd of 0.25 microM) or lower affinity (5-methyl, 5'-nitro-BAPTA, Kd of 22 microM) for Ca2+. Our findings provide evidence that growing pollen tubes have open Ca2+ channels in their tip and that these channels become inactivated in nongrowing tubes. The studies with elevated sucrose support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca2+ signal. PMID:7866026

  4. Extracellular ATP signaling in plants

    PubMed Central

    Tanaka, Kiwamu; Gilroy, Simon; Jones, Alan M.; Stacey, Gary

    2016-01-01

    Extracellular adenosine-5′-triphosphate (ATP) induces a number of cellular responses in plants and animals. Some of the molecular components for purinergic signaling in animal cells appear to be lacking in plant cells, although some cellular responses are similar in both systems [e.g. increased levels of cytosolic free calcium, nitric oxide (NO), and reactive oxygen species (ROS)]. The purpose of this review is to compare and contrast purinergic signaling mechanisms in animal and plant cells. This comparison will aid our overall understanding of plant physiology and also provide details of the general fundamentals of extracellular ATP signaling in eukaryotes. PMID:20817461

  5. The Effects of Extracellular Calcium on Motility, Pseudopod and Uropod Formation, Chemotaxis and the Cortical Localization of Myosin II in Dictyostelium discoideum

    PubMed Central

    Lusche, Daniel F.; Wessels, Deborah; Soll, David R.

    2009-01-01

    Extracellular Ca++, a ubiquitous cation in the soluble environment of cells both free living and within the human body, regulates most aspects of amoeboid cell motility, including shape, uropod formation, pseudopod formation, velocity and turning in Dictyostelium discoideum. Hence it affects the efficiency of both basic motile behavior and chemotaxis. Extracellular Ca++ is optimal at 10 mM. A gradient of the chemoattractant cAMP generated in the absence of added Ca++ only affects turning, but in combination with extracellular Ca++, enhances the effects of extracellular Ca++. Potassium, at 40 mM, can substitute for Ca++. Mg++, Mn++, Zn++ and Na+ cannot. Extracellular Ca++, or K+, also induce the cortical localization of myosin II in a polar fashion. The effects of Ca++, K+ or a cAMP gradient do not appear to be similarly mediated by an increase in the general pool of free cytosolic Ca++. These results suggest a model, in which each agent functioning through different signaling systems, converge to affect the cortical localization of myosin II, which in turn effects the behavioral changes leading to efficient cell motility and chemotaxis. PMID:19363786

  6. Increased calcium absorption from synthetic stable amorphous calcium carbonate: double-blind randomized crossover clinical trial in postmenopausal women.

    PubMed

    Vaisman, Nachum; Shaltiel, Galit; Daniely, Michal; Meiron, Oren E; Shechter, Assaf; Abrams, Steven A; Niv, Eva; Shapira, Yami; Sagi, Amir

    2014-10-01

    Calcium supplementation is a widely recognized strategy for achieving adequate calcium intake. We designed this blinded, randomized, crossover interventional trial to compare the bioavailability of a new stable synthetic amorphous calcium carbonate (ACC) with that of crystalline calcium carbonate (CCC) using the dual stable isotope technique. The study was conducted in the Unit of Clinical Nutrition, Tel Aviv Sourasky Medical Center, Israel. The study population included 15 early postmenopausal women aged 54.9 ± 2.8 (mean ± SD) years with no history of major medical illness or metabolic bone disorder, excess calcium intake, or vitamin D deficiency. Standardized breakfast was followed by randomly provided CCC or ACC capsules containing 192 mg elemental calcium labeled with 44Ca at intervals of at least 3 weeks. After swallowing the capsules, intravenous CaCl2 labeled with 42Ca on was administered on each occasion. Fractional calcium absorption (FCA) of ACC and CCC was calculated from the 24-hour urine collection following calcium administration. The results indicated that FCA of ACC was doubled (± 0.96 SD) on average compared to that of CCC (p < 0.02). The higher absorption of the synthetic stable ACC may serve as a more efficacious way of calcium supplementation. © 2014 American Society for Bone and Mineral Research.

  7. Acamprosate blocks the increase in dopamine extracellular levels in nucleus accumbens evoked by chemical stimulation of the ventral hippocampus.

    PubMed

    Cano-Cebrián, M J; Zornoza-Sabina, T; Guerri, C; Polache, A; Granero, L

    2003-10-01

    Recently, we have shown that acamprosate is able to modulate extracellular dopamine (DA) levels in the nucleus accumbens (NAc) and may act as an antagonist of N-methyl-D-aspartate (NMDA) receptors. Neurochemical studies show that chemical stimulation (using NMDA) of the ventral subiculum (vSub) of the hippocampus produces robust and sustained increases in extracellular DA levels in the NAc, an effect mediated through ionotropic glutamate (iGlu) receptors. The present study examines whether acamprosate locally infused in the NAc of rats could block or attenuate the increase in NAc extracellular DA elicited by chemical stimulation (with 5 mM NMDA) of the ventral subiculum of the hippocampus. The stimulation of the vSub during perfusion of artificial cerebrospinal fluid in NAc induced a significant and persistent increase in NAc DA levels. Reverse dialysis of 0.05 mM acamprosate in NAc blocked the increase in DA evoked by the chemical stimulation of the vSub. These data support the possibility that the antagonism at the NMDA receptors in NAc can explain, at least in part, the mechanism of action of this drug.

  8. Increasing urinary calcium excretion after ceftriaxone and cephalothin therapy in adults: possible association with urolithiasis.

    PubMed

    Otunctemur, Alper; Ozbek, Emin; Polat, Emre Can; Cekmen, Mustafa; Dursun, Murat; Cakir, Suleyman Sami

    2014-04-01

    In children, stone formation after ceftriaxone (CTRX) therapy by increasing calcium excretion was showed in the literature. In this study, we investigated the effect of CTRX, cephalothin (CP) and ampicillin (AS) therapy on urinary calcium excretion in adults. 180 participants included in the study who divided into six equal groups. The groups were; (1) CTRX therapy in stone free patients, (2) CTRX therapy in patients who have urinary stone; (3) CP therapy in stone free patients, (4) CP therapy in patients with urinary stone, (5) AS therapy in stone free patients, (6) AS therapy in patients with urinary stone. The patients received 2 g/day intravenous CTRX, CP and AS for 5 days in all groups respectively. There were no significant differences in demographic characteristics and blood biochemistry between the groups. Before and 5 days after the antibiotic therapies, the participants were evaluated by 24-h urinary calcium to creatinine ratio. Results were compared between the groups statistically by ANOVA and Tukey test. After drug therapies in group 2 and 4, the excretion of calcium to creatinine ratio in 24-h urine was more than the other groups. We found that both groups of two drugs therapy with or without stones (groups 1, 2, 3, 4), have significantly increased calcium to creatinine ratio in 24-h urine (p < 0.05). We did not find statistically difference in groups 5 and 6, after AS therapy. As a result of the study, we suggest that the patients who have taken antibiotic therapy with CTRX or CP, have an increased risk for the urolithiasis. In addition, we think that these drugs should be used carefully especially in patients with urolithiasis.

  9. Genetically modified Medicago truncatula lacking calcium oxalate has increased calcium bioavailability and partially rescues vitamin D receptor knockout mice phenotypes

    USDA-ARS?s Scientific Manuscript database

    How the distribution and sequestered form of plant macro/micro-nutrients influence their bioavailability, and ultimately impact human health, is poorly understood. The legume Medicago truncatula has a portion of its tissue calcium sequestered in the form of the calcium oxalate crystal, which reduces...

  10. Deletion of aquaporin-4 increases extracellular K(+) concentration during synaptic stimulation in mouse hippocampus.

    PubMed

    Haj-Yasein, Nadia Nabil; Bugge, Cecilie Elisabeth; Jensen, Vidar; Østby, Ivar; Ottersen, Ole Petter; Hvalby, Øivind; Nagelhus, Erlend Arnulf

    2015-07-01

    The coupling between the water channel aquaporin-4 (AQP4) and K(+) transport has attracted much interest. In this study, we assessed the effect of Aqp4 deletion on activity-induced [K(+)]o changes in acute slices from hippocampus and corpus callosum of adult mice. We show that Aqp4 deletion has a layer-specific effect on [K(+)]o that precisely mirrors the known effect on extracellular volume dynamics. In CA1, the peak [K(+)]o in stratum radiatum during 20 Hz stimulation of Schaffer collateral/commissural fibers was significantly higher in Aqp4 (-/-) mice than in wild types, whereas no differences were observed throughout the [K(+)]o recovery phase. In stratum pyramidale and corpus callosum, neither peak [K(+)]o nor post-stimulus [K(+)]o recovery was affected by Aqp4 deletion. Our data suggest that AQP4 modulates [K(+)]o during synaptic stimulation through its effect on extracellular space volume.

  11. Increase of glycosaminoglycans and metalloproteinases 2 and 9 in liver extracellular matrix on early stages of extrahepatic cholestasis.

    PubMed

    Guedes, Pedro Luiz Rodrigues; Castañon, Maria Christina Marques Nogueira; Nagaoka, Márcia Regina; Aguiar, Jair Adriano Kopke de

    2014-01-01

    Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs) activities. Animals (6-8 weeks; n = 40) were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT), alkaline phosphatase (Alk-P), alanine and aspartate aminotransferases (ALT and AST), tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064) and 14 (P = 0.0002) groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667), MMP-2 (P = 0.0003) and MMP-9 (P<0.0001) activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. Cholestasis led to many changes on rats' liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content.

  12. Expression of extracellular calcium (Ca2 + o)-sensing receptor in the clonal osteoblast-like cell lines, UMR-106 and SAOS-2

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Kifor, O.; Chattopadhyay, N.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2 + o) homeostasis in parathyroid gland and kidney. More recent data have suggested the presence of this receptor in additional tissues, such as brain, intestine and skin. In this study, we examined the expression of the CaR in the rat and human osteosarcoma cell lines, UMR-106 and SAOS-2, respectively, which possess osteoblast-like characteristics. Both immunocytochemistry and Western blot analysis, using a polyclonal antiserum specific for the CaR, detected CaR protein in UMR-106 and SAOS-2 cells. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in each cell line. Therefore, taken together, our data strongly suggest that the osteoblast-like cell lines, UMR-106 and SAOS-2, possess both CaR protein and mRNA very similar if not identical to those in parathyroid and kidney.

  13. Impact of Increasing Dietary Calcium Levels on Calcium Excretion and Vitamin D Metabolites in the Blood of Healthy Adult Cats

    PubMed Central

    Paßlack, Nadine; Schmiedchen, Bettina; Raila, Jens; Schweigert, Florian J.; Stumpff, Friederike; Kohn, Barbara; Neumann, Konrad; Zentek, Jürgen

    2016-01-01

    Background Dietary calcium (Ca) concentrations might affect regulatory pathways within the Ca and vitamin D metabolism and consequently excretory mechanisms. Considering large variations in Ca concentrations of feline diets, the physiological impact on Ca homeostasis has not been evaluated to date. In the present study, diets with increasing concentrations of dicalcium phosphate were offered to ten healthy adult cats (Ca/phosphorus (P): 6.23/6.02, 7.77/7.56, 15.0/12.7, 19.0/17.3, 22.2/19.9, 24.3/21.6 g/kg dry matter). Each feeding period was divided into a 10-day adaptation and an 8-day sampling period in order to collect urine and faeces. On the last day of each feeding period, blood samples were taken. Results Urinary Ca concentrations remained unaffected, but faecal Ca concentrations increased (P < 0.001) with increasing dietary Ca levels. No effect on whole and intact parathyroid hormone levels, fibroblast growth factor 23 and calcitriol concentrations in the blood of the cats were observed. However, the calcitriol precursors 25(OH)D2 and 25(OH)D3, which are considered the most useful indicators for the vitamin D status, decreased with higher dietary Ca levels (P = 0.013 and P = 0.033). Increasing dietary levels of dicalcium phosphate revealed an acidifying effect on urinary fasting pH (6.02) and postprandial pH (6.01) (P < 0.001), possibly mediated by an increase of urinary phosphorus (P) concentrations (P < 0.001). Conclusions In conclusion, calcitriol precursors were linearly affected by increasing dietary Ca concentrations. The increase in faecal Ca excretion indicates that Ca homeostasis of cats is mainly regulated in the intestine and not by the kidneys. Long-term studies should investigate the physiological relevance of the acidifying effect observed when feeding diets high in Ca and P. PMID:26870965

  14. High calcium enhances calcium oxalate crystal binding capacity of renal tubular cells via increased surface annexin A1 but impairs their proliferation and healing.

    PubMed

    Chutipongtanate, Somchai; Fong-ngern, Kedsarin; Peerapen, Paleerath; Thongboonkerd, Visith

    2012-07-06

    Hypercalciuria is associated with kidney stone formation and impaired renal function. However, responses of renal tubular cells upon exposure to high-calcium environment remain largely unknown. We thus performed a proteomic analysis of altered proteins in renal tubular cells induced by high-calcium and evaluated functional significance of these changes. MDCK cells were maintained with or without 20 mM CaCl(2) for 72 h. Cellular proteins were then analyzed by two-dimensional electrophoresis (2-DE) (n = 5 gels derived from 5 independent culture flasks per group). Spot matching and quantitative intensity analysis revealed 20 protein spots (from a total of 700) that were differentially expressed between the two groups. These altered proteins were then identified by Q-TOF-MS and MS/MS analyses, including those involved in calcium binding, protein synthesis, carbohydrate metabolism, lipid metabolism, cell proliferation, mitosis regulation, apoptosis, cell migration, oxidative stress, and ion transport. Protein network analysis and functional validation revealed that high-calcium-exposed cells had 36.5% increase in calcium oxalate monohydrate (COM) crystal-binding capacity. This functional change was consistent to the expression data in which annexin A1 (ANXA1), a membrane-associated calcium-binding protein, was markedly increased on the apical surface of high-calcium-exposed cells. Pretreatment with anti-ANXA1 antibody could neutralize this increasing crystal-binding capacity. Moreover, high-calcium exposure caused defects in cell proliferation and wound healing. These expression and functional data demonstrate the enhanced crystal-binding capacity but impaired cell proliferation and wound healing in renal tubular cells induced by high-calcium. Taken together, these phenomena may contribute, at least in part, to the pathogenic mechanisms of hypercalciuria-induced nephrolithiasis and impaired renal function. Our in vitro study offers several candidates for further

  15. Extracellular calcium is involved in egg yolk-induced head-to-head agglutination of bull sperm.

    PubMed

    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 <5% at 72 h (P < 0.001), but addition of 5 mM CaCl(2) to BD failed to induce 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 < 0.05), partially restored by addition of 5 mM CaCl2 (70%; P < 0.05), but the calcium effect was neutralized by addition of 5 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid (1.7%; P < 0.05), again implicating calcium. Addition of 30 μM of a protein kinase A inhibitor (H-89) to an agglutinating diluent failed to inhibit 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

  16. Parent/Child training to increase preteens' calcium, physical activity, and bone density: a controlled trial.

    PubMed

    Hovell, Melbourne F; Nichols, Jeanne F; Irvin, Veronica L; Schmitz, Katharine E; Rock, Cheryl L; Hofstetter, C Richard; Keating, Kristen; Stark, Lori J

    2009-01-01

    To test effects of parent/child training designed to increase calcium intake, bone-loading physical activity (PA), and bone density. Two-group randomized controlled trial. Family-based intervention delivered at research center. 117 healthy children aged 10-13 years (58.1% female, 42.7% Hispanic, 40.2% White). Ninety-seven percent of participants had at least one parent graduate from high school and 37.2 % had at least one parent graduate from a 4-year university. Children and parents were randomly assigned to diet and exercise (experimental) or injury prevention (control) interventions. Children were taught in eight weekly classes how to engage in bone-loading PA and eat calcium-rich foods or avoid injuries. Parents were taught behavior management techniques to modify children's behaviors. Measures at baseline and at 3, 9, and 12 months included 24-hour diet and PA recalls, and bone mineral density (BMD) by dual-energy x-ray absorptiometry. Analysis of variance and generalized estimating equations (GEE) assessed group by time differences. Comparisons were conducted separately for boys and girls. For boys, cross-sectional differences between experimental and control groups were achieved for 3- and 9-month calcium intake (1352 vs. 1052 mg/day, 1298 vs. 970 mg/day, p < .05). For girls, marginal cross-sectional differences were achieved for high-impact PA at 12 months (p < .10). For calcium intake, a significant group by time interaction was observed from pretest to posttest for the full sample (p = .008) and for girls (p = .006) but not for boys. No significant group by time differences in calcium were observed across the follow-up period. No group by time differences were observed for high-impact PA. Among boys, longitudinal group by time differences reached significance for total hip BMD (p = .045) and femoral neck BMD (p = .033), even after adjusting for skeletal growth. Similar differential increases were observed among boys for bone mineral content (BMC) at the

  17. Parent/child training to increase preteens’ calcium, physical activity and bone density: A controlled trial

    PubMed Central

    Hovell, Melbourne F.; Nichols, Jeanne F.; Irvin, Veronica L.; Schmitz, Katharine E.; Rock, Cheryl L.; Hofstetter, C. Richard; Keating, Kristen; Stark, Lori

    2012-01-01

    PURPOSE To test effects of parent/child training designed to increase calcium intake, bone-loading physical activity (PA), and bone density. DESIGN Two-group randomized controlled trial. SETTING Family-based intervention delivered at research center. SUBJECTS 117 healthy children aged 10-13 years (58.1% female, 42.7% Hispanic, 40.2% White). Ninety-seven percent of participants had at least one parent graduate from high school and 37.2% had at least one parent graduate from a 4-year university. INTERVENTION Children and parents were randomly assigned to diet and exercise (experimental) or injury prevention (control) interventions. Children were taught in eight weekly classes how to engage in bone-loading PA and eat calcium-rich foods or avoid injuries. Parents were taught behavior management techniques to modify children’s behaviors. MEASURES Measures at baseline, three, nine and twelve months included 24-hour diet and PA recalls, and bone mineral density (BMD) by DXA. ANALYSIS ANOVA and Generalized Estimating Equations assessed group by time differences. Comparisons were conducted separately for boys and girls. RESULTS For boys, cross-sectional differences between experimental versus control group were achieved for 3 and 9-month calcium intake (1352 vs. 1052mg/day, 1298 vs. 970mg/day, p<0.05). For girls, marginal cross-sectional differences were achieved for high-impact PA at 12 months (p<0.10). For calcium intake, a significant group by time interaction was observed from pre to post test for the full sample (p=.008) and for girls (p=.006) but not for boys. No significant group by time differences in calcium were observed across the follow-up period. No group by time differences were observed for high impact physical activity. Among boys, longitudinal group by time differences reached significance for total hip BMD (p=.045) and femoral neck BMD (p=.033), even after adjusting for skeletal growth. Similar differential increases were observed among boys for BMC at

  18. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation.

    PubMed

    González-Ramos, Marta; Calleros, Laura; López-Ongil, Susana; Raoch, Viviana; Griera, Mercedes; Rodríguez-Puyol, Manuel; de Frutos, Sergio; Rodríguez-Puyol, Diego

    2013-02-01

    The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.

  19. Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5

    PubMed Central

    Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad

    2010-01-01

    Background Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. Methods/Results We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. Conclusion In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline. PMID:20927353

  20. NG2 proteoglycan increases mesangial cell proliferation and extracellular matrix production

    SciTech Connect

    Xiong Jing; Wang Yang; Zhu, Zhonghua; Liu Jianshe; Wang Yumei; Zhang Chun; Hammes, Hans-Peter; Lang, Florian; Feng Yuxi

    2007-10-05

    As a membrane-spanning protein, NG2 chondroitin sulfate proteoglycan interacts with molecules on both sides of plasma membrane. The present study explored the role of NG2 in the pathogenesis of diabetic nephropathy. In the normal kidneys, NG2 was observed predominantly in glomerular mesangium, Bowman's capsule and interstitial vessels. Both mRNA and protein expression in kidneys was significantly higher in strepozotocin-induced diabetic rats than that in normal rats. In the cultured rat mesangial cell line HBZY-1, overexpression of NG2 promoted mesangial cell proliferation and extracellular matrix (ECM) production, such as type VI collagen and laminin. Furthermore, target knockdown of NG2 resulted in decreased cell proliferation and ECM formation. The observations suggest that NG2 is up-regulated in diabetic nephropathy. It actively participates in the development and progression of glomerulosclerosis by stimulating proliferation of mesangial cells and deposition of ECM.

  1. Lifetime high calcium intake increases osteoporotic fracture risk in old age.

    PubMed

    Klompmaker, Thijs R

    2005-01-01

    Caloric restriction prolongs life span. Calcium restriction may preserve bone health. In osteoporosis, bone mineral density (BMD) has significantly decreased, due to a lack of osteoblast bone formation. Traditional osteoporosis prevention is aimed at maximizing BMD, but the lifetime effects of continuously maintaining a high BMD on eventual bone health in old age, have not been studied. Strikingly, in countries with a high mean BMD, fracture rates in the elderly are significantly higher than in countries with a low mean BMD. Studies show that this is not based on genetic differences. Also, in primary hyperparathyroidism, on the brink of osteoporosis, BMD levels may be significantly higher than normal. Maybe, BMD does not represent long term bone health, but merely momentary bone strength. And maybe, maintaining a high BMD might actually wear out bone health. Since osteoporosis particularly occurs in the elderly, and because in osteoporotic bone less osteoblasts are available, the underlying process may have to do with ageing of osteoblastic cells. In healthy subjects, osteoblastic bone cells respond to the influx of calcium by composing a matrix upon which calcium precipitates. In the process of creating this matrix, 50-70% of the involved osteoblasts die. The greater the influx of calcium, the greater osteoblast activity, and the greater osteoblast apoptosis rate. An increased osteoblast apoptosis rate leads to a decrease in the age-related osteoblast replicative capacity (ARORC). In comparison to healthy bone, in osteoporotic bone the decrease in the replicative capacity of osteoblastic cells is greater. Due to the eventual resulting lack of osteoblast activity, micro-fractures cannot be repaired. Continuously maintaining a high BMD comes with continuously high bone remodeling rates, which regionally exhaust the ARORC, eventually leading to irreparable microfractures. Regarding long time influences on bone health, adequate estrogen levels are known to be

  2. Evidence for increased renal tubule and parathyroid gland sensitivity to serum calcium in human idiopathic hypercalciuria.

    PubMed

    Worcester, Elaine M; Bergsland, Kristin J; Gillen, Daniel L; Coe, Fredric L

    2013-09-15

    Patients with idiopathic hypercalciuria (IH) have decreased renal calcium reabsorption, most marked in the postprandial state, but the mechanisms are unknown. We compared 29 subjects with IH and 17 normal subjects (N) each fed meals providing identical amounts of calcium. Urine and blood samples were collected fasting and after meals. Levels of three candidate signalers, serum calcium (SCa), insulin (I), and plasma parathyroid hormone (PTH), did not differ between IH and N either fasting or fed, but all changed with feeding, and the change in SCa was greater in IH than in N. Regression analysis of fractional excretion of calcium (FECa) was significant for PTH and SCa in IH but not N. With the use of multivariable analysis, Sca entered the model while PTH and I did not. To avoid internal correlation we decomposed FECa into its independent terms: adjusted urine calcium (UCa) and UFCa molarity. Analyses using adjusted Uca and unadjusted Uca parallel those using FECa, showing a dominant effect of SCa with no effect of PTH or I. The effect of SCa may be mediated via vitamin D receptor-stimulated increased abundance of basolateral Ca receptor, which is supported by the fact PTH levels also seem more responsive to serum Ca in IH than in N. Although our data support an effect of SCa on FECa and UCa, which is more marked in IH than in N, it can account for only a modest fraction of the meal effect, perhaps 10-20%, suggesting additional mediators are also responsible for the exaggerated postprandial hypercalciuria seen in IH.

  3. Sucralose, an activator of the glucose-sensing receptor, increases ATP by calcium-dependent and -independent mechanisms.

    PubMed

    Li, Longfei; Ohtsu, Yoshiaki; Nakagawa, Yuko; Masuda, Katsuyoshi; Kojima, Itaru

    2016-08-31

    Sucralose is an artificial sweetener and activates the glucose-sensing receptor expressed in pancreatic β-cells. Although sucralose does not enter β-cells nor acts as a substrate for glucokinase, it induces a marked elevation of intracellular ATP ([ATP]c). The present study was conducted to identify the signaling pathway responsible for the elevation of [ATP]c induced by sucralose. Previous studies have shown that sucralose elevates cyclic AMP (cAMP), activates phospholipase C (PLC) and stimulates Ca(2+) entry by a Na(+)-dependent mechanism in MIN6 cells. The addition of forskolin induced a marked elevation of cAMP, whereas it did not affect [ATP]c. Carbachol, an activator of PLC, did not increase [ATP]c. In addition, activation of protein kinase C by dioctanoylglycerol did not affect [ATP]c. In contrast, nifedipine, an inhibitor of the voltage-dependent Ca(2+) channel, significantly reduced [ATP]c response to sucralose. Removal of extracellular Na(+) nearly completely blocked sucralose-induced elevation of [ATP]c. Stimulation of Na(+) entry by adding a Na(+) ionophore monensin elevated [ATP]c. The monensin-induced elevation of [ATP]c was only partially inhibited by nifedipine and loading of BAPTA, both of which completely abolished elevation of [Ca(2+)]c. These results suggest that Na(+) entry is critical for the sucralose-induced elevation of [ATP]c. Both calcium-dependent and -independent mechanisms are involved in the action of sucralose.

  4. Depression of serum calcium by increased plasma free fatty acids in the rat: a mechanism for hypocalcemia in acute pancreatitis.

    PubMed

    Warshaw, A L; Lee, K H; Napier, T W; Fournier, P O; Duchainey, D; Axelrod, L

    1985-10-01

    Some patients with hypertriglyceridemia and acute pancreatitis have marked hypocalcemia and high levels of plasma free fatty acids (FFAs). This study tests the hypothesis that increased plasma FFAs can significantly reduce the calcium level in vivo, a phenomenon which is different from local formation of calcium soaps due to lipolysis of adipose tissue lipids. Free fatty acid elevation was induced in rats by the administration of heparin and by the infusion of triglycerides. The results show that, compared with controls, induction of elevated FFA (from 1.57 +/- 0.08 mEq/L to 5.64 +/- 0.35, mean +/- SEM) causes the concentration of calcium to fall rapidly (from 9.04 +/- 0.06 mg/dl to 8.42 +/- 0.10, p less than 0.001). There is a significant (p less than 0.001) positive correlation between spontaneous baseline concentration of FFA and the responsiveness of calcium concentration to FFA challenge. At near-normal levels of FFA there is a significant (p less than 0.001) correlation between the magnitude of increased FFA concentration and decreased calcium concentration. Additional studies in vivo and in vitro show that elevated plasma triglycerides per se did not interfere with measurement of calcium concentration; however, FFA-albumin complexes bind calcium and lower its measured value. These findings suggest that (a) changes in the concentration of FFA occurring spontaneously may affect measured serum calcium concentration; (b) the observed depression of serum calcium concentration may be due in part to intravascular sequestration of calcium by FFA, but increased flux of circulating calcium-FFA complexes into extravascular and intracellular sites may also be important; (c) the markedly increased FFA concentration in some patients with acute pancreatitis may contribute significantly to hypocalcemia and calcium flux in these patients. As parathyroid hormone secretion, function, or integrity may be impaired in pancreatitis, the depressant effect of FFA could be even

  5. Emerging roles of the extracellular calcium-sensing receptor in nutrient sensing: control of taste modulation and intestinal hormone secretion.

    PubMed

    Brennan, Sarah C; Davies, Thomas S; Schepelmann, Martin; Riccardi, Daniela

    2014-06-01

    The extracellular Ca-sensing receptor (CaSR) is a sensor for a number of key nutrients within the body, including Ca ions (Ca²⁺) and L-amino acids. The CaSR is expressed in a number of specialised cells within the gastrointestinal (GI) tract, and much work has been done to examine CaSR's role as a nutrient sensor in this system. This review article examines two emerging roles for the CaSR within the GI tract--as a mediator of kokumi taste modulation in taste cells and as a regulator of dietary hormone release in response to L-amino acids in the intestine.

  6. Increase of extracellular glutamate concentration increases its oxidation and diminishes glucose oxidation in isolated mouse hippocampus: reversible by TFB-TBOA.

    PubMed

    Torres, Felipe Vasconcelos; Hansen, Fernanda; Locks-Coelho, Lucas Doridio

    2013-08-01

    Glutamate concentration at the synaptic level must be kept low in order to prevent excitotoxicity. Astrocytes play a key role in brain energetics, and also astrocytic glutamate transporters are responsible for the vast majority of glutamate uptake in CNS. Experiments with primary astrocytic cultures suggest that increased influx of glutamate cotransported with sodium at astrocytes favors its flux to the tricarboxylic acid cycle instead of the glutamate-glutamine cycle. Although metabolic coupling can be considered an emergent field of research with important recent discoveries, some basic aspects of glutamate metabolism still have not been characterized in brain tissue. Therefore, the aim of this study was to investigate whether the presence of extracellular glutamate is able to modulate the use of glutamate and glucose as energetic substrates. For this purpose, isolated hippocampi of mice were incubated with radiolabeled substrates, and CO2 radioactivity and extracellular lactate were measured. Our results point to a diminished oxidation of glucose with increasing extracellular glutamate concentration, glutamate presumably being the fuel, and might suggest that oxidation of glutamate could buffer excitotoxic conditions by high glutamate concentrations. In addition, these findings were reversed when glutamate uptake by astrocytes was impaired by the presence of (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA). Taken together, our findings argue against the lactate shuttle theory, because glutamate did not cause any detectable increase in extracellular lactate content (or, presumably, in glycolysis), because the glutamate is being used as fuel instead of going to glutamine and back to neurons. Copyright © 2013 Wiley Periodicals, Inc.

  7. Morphine sensitization increases the extracellular level of glutamate in CA1 of rat hippocampus via μ-opioid receptor.

    PubMed

    Farahmandfar, Maryam; Karimian, Seyed Morteza; Zarrindast, Mohammad-Reza; Kadivar, Mehdi; Afrouzi, Hossein; Naghdi, Nasser

    2011-04-25

    Repeated administration of abuse drugs such as morphine elicits a progressive enhancement of drug-induced behavioral responses, a phenomenon termed behavioral sensitization. These changes in behavior may reflect plastic changes requiring regulation of glutamatergic system in the brain. In this study, we investigated the effect of morphine sensitization on extracellular glutamate concentration in the hippocampus, a brain region rich in glutamatergic neurons. Sensitization was induced by subcutaneous (s.c.) injection of morphine, once daily for 3 days followed by 5 days free of the opioid treatment. The results showed that extracellular glutamate concentration in the CA1 was decreased following administration of morphine in non-sensitized rats. However, morphine-induced behavioral sensitization significantly increased the extracellular glutamate concentration in this area. The enhancement of glutamate in morphine sensitized rats was prevented by administration of naloxone 30 min before each of three daily doses of morphine. These results suggest an adaptation of the glutamatergic neuronal transmission in the hippocampus after morphine sensitization and it is postulated that opioid receptors may play an important role in this effect.

  8. Cerebral extracellular lactate increase is predominantly nonischemic in patients with severe traumatic brain injury

    PubMed Central

    Sala, Nathalie; Suys, Tamarah; Zerlauth, Jean-Baptiste; Bouzat, Pierre; Messerer, Mahmoud; Bloch, Jocelyne; Levivier, Marc; Magistretti, Pierre J; Meuli, Reto; Oddo, Mauro

    2013-01-01

    Growing evidence suggests that endogenous lactate is an important substrate for neurons. This study aimed to examine cerebral lactate metabolism and its relationship with brain perfusion in patients with severe traumatic brain injury (TBI). A prospective cohort of 24 patients with severe TBI monitored with cerebral microdialysis (CMD) and brain tissue oxygen tension (PbtO2) was studied. Brain lactate metabolism was assessed by quantification of elevated CMD lactate samples (>4 mmol/L); these were matched to CMD pyruvate and PbtO2 values and dichotomized as glycolytic (CMD pyruvate >119 μmol/L vs. low pyruvate) and hypoxic (PbtO2 <20 mm Hg vs. nonhypoxic). Using perfusion computed tomography (CT), brain perfusion was categorized as oligemic, normal, or hyperemic, and was compared with CMD and PbtO2 data. Samples with elevated CMD lactate were frequently observed (41±8%), and we found that brain lactate elevations were predominantly associated with glycolysis and normal PbtO2 (73±8%) rather than brain hypoxia (14±6%). Furthermore, glycolytic lactate was always associated with normal or hyperemic brain perfusion, whereas all episodes with hypoxic lactate were associated with diffuse oligemia. Our findings suggest predominant nonischemic cerebral extracellular lactate release after TBI and support the concept that lactate may be used as an energy substrate by the injured human brain. PMID:23963367

  9. Increased red cell calcium, decreased calcium adenosine triphosphatase, and altered membrane proteins during fava bean hemolysis in glucose-6-phosphate dehydrogenase-deficient (Mediterranean variant) individuals.

    PubMed

    Turrini, F; Naitana, A; Mannuzzu, L; Pescarmona, G; Arese, P

    1985-08-01

    RBCs from four glucose-6-phosphate dehydrogenase (G6PD)-deficient (Mediterranean variant) subjects were studied during fava bean hemolysis. In the density-fractionated RBC calcium level, Ca2+-ATPase activity, reduced glutathione level, and ghost protein pattern were studied. In the bottom fraction, containing most heavily damaged RBCs, calcium level ranged from 143 to 244 mumol/L RBCs (healthy G6PD-deficient controls: 17 +/- 5 mumol/L RBCs). The Ca2+-ATPase activity ranged from 0.87 to 1.84 mumol ATP consumed/g Hb/min (healthy G6PD-deficient controls: 2.27 +/- 0.4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of ghosts showed: (1) the presence of high mol wt aggregates (in three cases they were reduced by dithioerythritol; in one case, only partial reduction was possible); (2) the presence of multiple, scattered new bands; and (3) the reduction of band 3. Oxidant-mediated damage to active calcium extrusion, hypothetically associated with increased calcium permeability, may explain the large increase in calcium levels. They, in turn, could activate calcium-dependent protease activity, giving rise to the profound changes in the ghost protein pattern.

  10. Increased 10-year cardiovascular disease and mortality risk scores in asymptomatic patients with calcium oxalate urolithiasis.

    PubMed

    Aydin, Hasan; Yencilek, Faruk; Erihan, Ismet Bilger; Okan, Binnur; Sarica, Kemal

    2011-12-01

    Both the prevalence of cardiovascular risk factors and event rate are increased in patients with urolithiasis. Screening is recommended to all patients who have high cardiovascular risk. The aim of this study was to document 10-year risk of cardiovascular disease and mortality in asymptomatic patients with urolithiasis. Consecutive 200 patients with calcium oxalate urolithiasis were compared with 200 age- and sex-matched healthy controls. Ten-year cardiovascular disease risk was calculated with the Framingham Risk Score and mortality risk with SCORE risk score. Calcium, oxalate, and citrate excretion were studied as urinary stone risk factors. The results indicate that patients with urolithiasis had higher total cholesterol (p < 0.0001), lower HDL-cholesterol (p < 0.0001), and higher systolic blood pressure (p < 0.0001) and hsCRP (p < 0.0001) compared with controls. Patients with urolithiasis had a higher Framingham Risk Scores [OR 8.36 (95% CI 3.81-18.65), p = 0.0001] and SCORE risk score [OR 3.02 (95% CI 1.30-7.02), p = 0.0006] compared with controls. The Framingham and SCORE risk score were significantly correlated with urinary calcium (p = 0.0001, r = 0.460, and p = 0.005, r = 0.223, respectively) and oxalate excretion (p = 0.0001, r = 0.516, p = 0.001, r = 0.290, respectively). In multiple linear regression analysis, urinary calcium and oxalate excretion, age, sex, total cholesterol, HDL-cholesterol, hsCRP and smoking were the independent predictors of 10-year cardiovascular disease risk and urinary calcium and oxalate excretion, age, sex, total cholesterol, fasting blood glucose for 10-year cardiovascular mortality. In conclusion, patients with calcium oxalate urolithiasis carry high risk of cardiovascular disease and mortality. All patients should be screened at the initial diagnosis of urolithiasis for the risk factors.

  11. A grape-enriched diet increases bone calcium retention and cortical bone properties in ovariectomized rats.

    PubMed

    Hohman, Emily E; Weaver, Connie M

    2015-02-01

    Grapes and their associated phytochemicals have been investigated for beneficial effects on cardiovascular health, cancer prevention, and other chronic diseases, but the effect of grape consumption on bone health has not been fully determined. We previously found short-term benefits of grape products on reducing bone turnover in ovariectomized rats. The objective of this study was to determine the long-term benefits of a grape-enriched diet on bone in ovariectomized rats. Rats were ovariectomized at 3 mo of age and were administered a single dose of (45)Ca to prelabel bones at 4 mo of age. After a 1-mo equilibration period, baseline urinary (45)Ca excretion was determined. Rats (n = 22/group) were then randomly assigned to a modified AIN93M diet containing 25% freeze-dried grape powder or to a control diet for 8 wk. Urinary (45)Ca excretion was monitored throughout the study to determine changes in bone (45)Ca retention. Calcium balance was assessed after 1 and 8 wk of consuming the experimental diets, and a calcium kinetic study was performed at 8 wk. After 8 wk, femurs were collected for micro-computed tomographic imaging, 3-point bending, and reference point indentation. Rats fed the grape-enriched diet had 44% greater net bone calcium retention than did rats fed the control diet. There were no differences in calcium balance due to diet at either week 1 or week 8, but there was a significant increase in net calcium absorption (10.6%) and retention (5.7%) from week 1 to week 8 in the grape-enriched diet group only. Grape-enriched diet-fed rats had 3% greater cortical thickness and 11% greater breaking strength. There were no differences in femur bone mineral density, trabecular microarchitecture, or reference point indentation variables due to diet. This study of ovariectomized rats indicates that the consumption of grape products may improve calcium utilization and suppress bone turnover, resulting in improvements in bone quality. © 2015 American Society for

  12. Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells.

    PubMed

    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.

  13. Androgens induce increases in intracellular calcium via a G protein-coupled receptor in LNCaP prostate cancer cells.

    PubMed

    Sun, Ying-Hao; Gao, Xu; Tang, Yuan-Jie; Xu, Chuan-Liang; Wang, Lin-Hui

    2006-01-01

    The receptor mechanism of testosterone-induced nongenomic Ca2+ signaling in prostate cancer cells is poorly understood. In this study we investigated androgen-induced intracellular Ca2+ increases in LNCaP human prostate cancer cells with Fura-2 as a Ca2+ probe. 5alpha-dihydrotestosterone (DHT) produced fast and transient increases in intracellular Ca2+ in LNCaP cells in a concentration-dependent manner. These effects were abolished by extracellular Ca2+ removal or pretreatment with L-type Ca2+ channel inhibitors (nifedipine, verapamil, and diltiazem). Pretreatment with endoplasmic reticulum ryanodine receptor blocker (procaine) or phospholipase C inhibitor (neomycin sulfate) did not alter DHT-induced Ca2+ influx. The concentration of Ca2+ was also increased by impermeable testosterone conjugated to bovine serum albumin. Neither an antagonist of intracellular androgen receptors (cyproterone acetate) nor a protein synthesis inhibitor (cycloheximide) affected this fast Ca2+ influx. Furthermore, the effect of DHT was abolished in cells incubated with a G protein inhibitor (pertussis toxin) and a nonhydrolyzable analog of guanosine triphosphate (guanosine 5-[beta-thio]disphosphate) but not in cells incubated with the tyrosine kinase inhibitor genistein. These results indicate that androgens induced an L-type calcium channel-dependent intracellular Ca2+ increase in LNCaP prostate cancer cells. The rapid responses triggered by DHT did not appear to be mediated through classic intracellular androgen receptors, c-Src kinase-androgen receptor complex, or sex hormone-binding globulin but through a G protein-coupled receptor in LNCaP prostate cancer cells. These results may provide a new explanation for progression of prostate cancer.

  14. Endogenous glutamate increases extracellular concentrations of dopamine, GABA, and taurine through NMDA and AMPA/kainate receptors in striatum of the freely moving rat: a microdialysis study.

    PubMed

    Segovia, G; Del Arco, A; Mora, F

    1997-10-01

    Interactions between glutamate (Glu), dopamine (DA), GABA, and taurine (Tau) were investigated in striatum of the freely moving rat by using microdialysis. Intrastriatal infusions of the selective Glu uptake inhibitor L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC) were used to increase the endogenous extracellular [Glu]. Correlations between extracellular [Glu] and extracellular [DA], [GABA], and [Tau], and the effects of a selective blockade of ionotropic Glu receptors, were studied. PDC (1, 2, and 4 mM) produced a dose-related increase in extracellular [Glu]. At the highest dose of PDC, [Glu] increased from 1.55 +/- 0.35 to 6.11 +/- 0.88 microM. PDC also increased extracellular [DA], [GABA], and [Tau]. The increasing [Glu] was correlated significantly with increasing [DA], [GABA], and [Tau]. PDC also decreased extracellular concentrations of DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (HVA). Perfusion with the NMDA-receptor antagonist 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (1 mM) or the AMPA/kainate-receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) (1 mM) attenuated the increases produced by PDC (4 mM) on [DA], [GABA], and [Tau], and decreases in [DOPAC] and [HVA]. DNQX also attenuated the increases in [Glu] induced by PDC. These data show that endogenous Glu plays a role in modulating the extracellular concentrations of DA, GABA, and Tau in striatum of the freely moving rat.

  15. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    PubMed

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

  16. Alcohol-induced apoptosis of canine cerebral vascular smooth muscle cells: role of extracellular and intracellular calcium ions.

    PubMed

    Li, Wenyan; Li, Jianfeng; Liu, Weiming; Altura, Bella T; Altura, Burton M

    2004-01-16

    Exposure of canine cerebral vascular smooth muscle cells (VSMCs) to ethanol (10, 25 and 100 mM) for 1, 3 and 5 days induced apoptosis with its typical characteristics of nuclear shrinkage, condensation, and DNA breakage as well as formation of apoptotic bodies observed by fluorescence staining, terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling and comet assays. Such effects of alcohol on cerebral VSMCs were time- and concentration-dependent. The threshold ethanol concentration for induction of the apoptotic process was found to be 10 mM. Extracellular and intracellular Ca2+ chelators, i.e. ethylglycol-bisbeta-aminoethylether-N,N,N'N'-tetraacetic acid (EGTA, 5 mM) and 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid AM (BAPTA, 10(-6) M), respectively, ameliorated greatly the number of cerebral VSMCs which underwent apoptosis. Verapamil, however, failed to inhibit apoptosis of cerebral VSMCs. From these new findings, we suggest that alcohol-induced apoptosis may contribute to alcohol-induced brain-vascular damage and stroke. In addition, our findings point to potential caution for humans who imbibe two or more standard drinks per day or who undergo 'binge drinking'.

  17. Effect of increased calcium concentration in sea water on calcification and photosynthesis in the scleractinian coral Galaxea fascicularis.

    PubMed

    Marshall, Alan T; Clode, Peta L

    2002-07-01

    The relationship between calcification and photosynthesis in coral was investigated using standard sea water with enhanced calcium concentration. In standard sea water at 23 degrees C with the calcium concentration increased by 2.5 mmol l(-1), incorporation of calcium into the skeleton increased by 30-61 %, depending on the method of data normalisation, and photosynthesis, measured as (14)C incorporation into the tissues, also increased by 87 %. At 29 degrees C, calcium incorporation into the skeleton increased by 54-84 % and (14)C incorporation increased by 32 % when sea water calcium concentration was increased by 5 mmol l(-1). However, photosynthesis measured as net photosynthetic oxygen production did not increase. Similarly there was no change in respiration rate when coral polyps were incubated in high-calcium sea water. It is conjectured that an increase in photorespiration may be responsible for the latter observations. Bisphosphonate has been considered to inhibit calcification but not photosynthesis in corals. We show that bisphosphonate may not inhibit formation of amorphous calcium carbonate and that the inhibition of calcification is possibly illusory. The data are consistent with the trans-calcification model, which suggests that calcification is a source of CO(2) for photosynthesis in corals.

  18. Theophylline and adenosine modulate the inflammatory functions of the human neutrophil by exerting an opposing influence on the stimulus-induced increase in intracellular calcium

    SciTech Connect

    Schmeichel Morley, C.J.

    1988-01-01

    Based on evidence that endogenously-produced adenosine inhibited neutrophil responses, the influence of methylxanthine bronchodilators on neutrophil responses stimulated in vitro by n-formyl-methionyl-leucyl-phenylalanine (fMLP) was examined. At concentrations between 10/sup /minus/5/ M and 10/sup /minus/4/ M, theophylline potentiated lysosomal enzyme release by 30 to 50%, superoxide anion formation by 30 to 60%, and neutrophil aggregation. Theophylline at concentrations >10/sup /minus/4/ M inhibited the same responses by >90%. Adenosine deaminase mimicked, whereas adenosine reversed the theophylline potentiation. A potential role for calcium in the modulation of the neutrophil responses by theophylline and adenosine was explored. Theophylline enhanced by >150% the fMLP-stimulated increase in cytoplasmic calcium concentration ((Ca/sup 2 +/)/sub i/) at time points between 5 and 90 sec as measured by Fura-2. Adenosine deaminase induced a comparable enhancement, whereas 3 /times/ 10/sup /minus/7/ M adenosine and 10/sup /minus/7/ M N-ethylcarboxamideadenosine decreased the (Ca/sup 2 +/)/sub i/ in fMLP-stimulated neutrophils. Extracellular calcium was not required for the opposing influences of theophylline and adenosine and neither compound altered fMLP-stimulated /sup 45/Ca uptake at the early time points.

  19. Increasing the intestinal resistance of rats to the invasive pathogen Salmonella enteritidis: additive effects of dietary lactulose and calcium.

    PubMed Central

    Bovee-Oudenhoven, I M; Termont, D S; Heidt, P J; Van der Meer, R

    1997-01-01

    BACKGROUND AND AIMS: Lactulose fermentation by the intestinal microflora acidifies the gut contents, resulting in an increased resistance to colonisation by acid sensitive pathogens. The extent of fermentation should be controlled to prevent acid induced epithelial cell damage. Considering the buffering capacity of calcium phosphate and its intestinal cytoprotective effects, whether supplemental calcium phosphate adds to the increased resistance to intestinal infections by lactulose fermentations was studied. METHODS: In a strictly controlled experiment, rats were fed a purified low calcium control diet, a low calcium/lactulose diet, or a high calcium/lactulose diet, and subsequently infected orally with Salmonella enteritidis. RESULTS: Lactulose fermentation lowered the pH and increased the lactic acid concentration of the intestinal contents, which significantly reduced excretion of this pathogen in faeces; thus it improved the resistance to colonisation. This agreed with the high sensitivity of S enteritidis to lactic acid (main metabolite of lactulose fermentation) in vitro. Calcium phosphate decreased translocation of S enteritidis to the systemic circulation, an effect independent of lactulose. The unfavourable increased cytotoxicity of faecal water caused by lactulose fermentation was more than counteracted by supplemental calcium phosphate. Moreover, calcium phosphate stimulated lactulose fermentation, as judged by the reduced lactulose excretion in faeces and increased lactic acid, ammonia, and faecal nitrogen excretion. CONCLUSION: Extra calcium phosphate added to a lactulose diet improves the resistance to colonisation and translocation of S enteritidis. This is probably mediated by a calcium induced stimulation of lactulose fermentation by the intestinal microflora and reversion of the lactulose mediated increased luminal cytotoxicity, which reduces damage inflicted on the intestinal mucosa. PMID:9176078

  20. Milk calcium taken with cheese increases bone mineral density and bone strength in growing rats.

    PubMed

    Kato, Ken; Takada, Yukihiro; Matsuyama, Hiroaki; Kawasaki, Yoshihiro; Aoe, Seiichiro; Yano, Hideo; Toba, Yasuhiro

    2002-11-01

    We investigated the calcium bioavailability of milk calcium, taken with or without cheese. Twenty-four 6-week-old male rats for a meal-feeding experiment were trained to consume an AIN-76 diet within 2 h (2 times per day) for 2 weeks. The rats were then divided into three experimental groups, each fed 2 types of experimental diets: Control group, Cheese group, and Ca-Cheese group. The rats were each alternately given 2 types of experimental diets at 2-h meal-feeding for 31 days. The breaking force and energy of the femur in the Ca-Cheese group were significantly higher than in the control group. The bone mineral density (BMD) of the lumbar spine and the femur in the Ca-Cheese group was also significantly higher than in the other two groups. These results indicate that milk calcium taken with cheese increases bone strength and BMD efficiently, results that may be useful for the prevention of osteoporosis.

  1. Increased Calcium Supplementation Postpartum Is Associated with Breastfeeding among Chinese Mothers: Finding from Two Prospective Cohort Studies.

    PubMed

    Zhao, Jian; Zhao, Yun; Binns, Colin W; Lee, Andy H

    2016-10-09

    The calcium supplementation status during the postpartum period among Chinese lactating women is still unclear. The objective of this study is to utilize data from two population-based prospective cohort studies to examine the calcium supplementation status and to identify whether breastfeeding is associated with increased calcium supplementation among Chinese mothers after child birth. Information from 1540 mothers on breastfeeding and calcium supplementation measured at discharge, 1, 3, and 6 months postpartum were extracted to evaluate the association between breastfeeding and calcium supplementation postpartum. A generalized linear mixed model was applied to each study initially to account for the inherent correlation among repeated measurements, adjusting for socio-demographic, obstetric factors and calcium supplementation during pregnancy. In addition, breastfeeding status measured at different follow-up time points was treated as a time dependent variable in the longitudinal analysis. Furthermore, the effect sizes of the two cohort studies were pooled using fixed effect model. Based on the two cohort studies, the pooled likelihood of taking calcium supplementation postpartum among breastfeeding mothers was 4.02 times (95% confidence interval (2.30, 7.03)) higher than that of their non-breastfeeding counterparts. Dietary supplementation intervention programs targeting different subgroups should be promoted in Chinese women, given currently a wide shortage of dietary calcium intake and calcium supplementation postpartum.

  2. Increased Calcium Supplementation Postpartum Is Associated with Breastfeeding among Chinese Mothers: Finding from Two Prospective Cohort Studies

    PubMed Central

    Zhao, Jian; Zhao, Yun; Binns, Colin W.; Lee, Andy H.

    2016-01-01

    The calcium supplementation status during the postpartum period among Chinese lactating women is still unclear. The objective of this study is to utilize data from two population-based prospective cohort studies to examine the calcium supplementation status and to identify whether breastfeeding is associated with increased calcium supplementation among Chinese mothers after child birth. Information from 1540 mothers on breastfeeding and calcium supplementation measured at discharge, 1, 3, and 6 months postpartum were extracted to evaluate the association between breastfeeding and calcium supplementation postpartum. A generalized linear mixed model was applied to each study initially to account for the inherent correlation among repeated measurements, adjusting for socio-demographic, obstetric factors and calcium supplementation during pregnancy. In addition, breastfeeding status measured at different follow-up time points was treated as a time dependent variable in the longitudinal analysis. Furthermore, the effect sizes of the two cohort studies were pooled using fixed effect model. Based on the two cohort studies, the pooled likelihood of taking calcium supplementation postpartum among breastfeeding mothers was 4.02 times (95% confidence interval (2.30, 7.03)) higher than that of their non-breastfeeding counterparts. Dietary supplementation intervention programs targeting different subgroups should be promoted in Chinese women, given currently a wide shortage of dietary calcium intake and calcium supplementation postpartum. PMID:27735835

  3. Acute stress-mediated increases in extracellular glutamate levels in the rat amygdala: differential effects of antidepressant treatment.

    PubMed

    Reznikov, Leah R; Grillo, Claudia A; Piroli, Gerardo G; Pasumarthi, Ravi K; Reagan, Lawrence P; Fadel, Jim

    2007-05-01

    Depressive illness is associated with changes in amygdalar volume, and stressful life events are known to precipitate depressive episodes in this patient population. Stress affects amygdalar synaptic plasticity and several neurotransmitter systems have been implicated in stress-mediated changes in the brain, including the glutamatergic system. However, the role of the glutamatergic system in stress-mediated plasticity in the amygdala remains to be determined. Accordingly the current study examined the stress modulation of extracellular glutamate levels in the basolateral nucleus (BLA) and the central nucleus (CeA) of the amygdala by in vivo microdialysis. Acute stress increased extracellular glutamate levels in the BLA and CeA, although the dynamics of these stress-mediated changes were dramatically different in these amygdalar nuclei. Tetrodotoxin administration reduced basal, and completely eliminated stress-mediated increases in glutamate efflux in the amygdala, demonstrating that stress effects are dependent on local axonal depolarization. Moreover, stress-mediated increases in glutamate efflux in the BLA were inhibited by the antidepressant tianeptine but not by the selective serotonin-reuptake inhibitor fluoxetine. Collectively, these data demonstrate that stress-induced modulation of glutamate neurochemistry reflects a fundamental pathological change that may contribute to the aetiology and progression of depressive illness, and suggest that some antidepressants such as tianeptine may elicit their clinical effects by modulation of glutamatergic neurotransmission.

  4. Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis.

    PubMed

    Jokela, Tiina; Kärnä, Riikka; Rauhala, Leena; Bart, Genevieve; Pasonen-Seppänen, Sanna; Oikari, Sanna; Tammi, Markku I; Tammi, Raija H

    2017-03-24

    The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 μm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

  5. Increased extracellular dopamine and 5-hydroxytryptamine levels contribute to enhanced subthalamic nucleus neural activity during exhausting exercise

    PubMed Central

    Hu, Y; Liu, X

    2015-01-01

    The purpose of the study was to explore the mechanism underlying the enhanced subthalamic nucleus (STN) neural activity during exhausting exercise from the perspective of monoamine neurotransmitters and changes of their corresponding receptors. Rats were randomly divided into microdialysis and immunohistochemistry study groups. For microdialysis study, extracellular fluid of the STN was continuously collected with a microdialysis probe before, during and 90 min after one bout of exhausting exercise. Dopamine (DA) and 5-hydroxytryptamine (5-HT) levels were subsequently detected with high-performance liquid chromatography (HPLC). For immunohistochemistry study, the expression of DRD2 and HT2C receptors in the STN, before, immediately after and 90 min after exhaustion was detected through immunohistochemistry technique. Microdialysis study results showed that the extracellular DA and 5-HT neurotransmitters increased significantly throughout the procedure of exhausting exercise and the recovery period (P<0.05 or P<0.01). Immunohistochemistry study results showed that the expression levels of DRD2 and HT2C in the rat STN immediately after exhausting exercise and at the time point of 90 min after exhaustion were both higher than those of the rest condition, but the difference was not significant (P>0.05). Our results suggest that the increased extracellular DA and 5-HT in the STN might be one important factor leading to the enhanced STN neural activity and the development of fatigue during exhausting exercise. This study may essentially offer useful evidence for better understanding of the mechanism of the central type of exercise-induced fatigue. PMID:26424920

  6. Calcium Pre-Rinse Greatly Increases Overnight Salivary Fluoride after a 228 ppm Fluoride Rinse

    PubMed Central

    Vogel, G.L.; Chow, L.C.; Carey, C.M.

    2008-01-01

    Background Large increases in salivary fluoride were reported 1 h after a calcium pre-rinse/NaF rinse. Aims This study examined the persistence of these increases. Methods 12 subjects rinsed in the evening with water, with a 228 μg/g (ppm) F rinse or with 150 mmol/l calcium lactate followed by a 228 μg/g F rinse. In a second experiment these same patients rinsed with a 912 μg/g F rinse. Saliva samples were obtained the morning after rinsing, centrifuged and the supernatants analyzed. Results The Ca pre-rinse/228 μg/g F rinse induced an increase in overnight salivary F over the 912 μg/g F rinse (≈2.5 times) and a statistically significant increase over the 228 μg/g F rinse (≈5.5 times). Conclusions The results suggest that a Ca pretreatment may increase the cariostatic effect of topical F agents. PMID:18781069

  7. Triiodothyronine increases calcium loss in a bed rest antigravity model for space flight.

    PubMed

    Smith, Steven R; Lovejoy, Jennifer C; Bray, George A; Rood, Jennifer; Most, Marlene M; Ryan, Donna H

    2008-12-01

    Bed rest has been used as a model to simulate the effects of space flight on bone metabolism. Thyroid hormones accelerate bone metabolism. Thus, supraphysiologic doses of this hormone might be used as a model to accelerate bone metabolism during bed rest and potentially simulate space flight. The objective of the study was to quantitate the changes in bone turnover after low doses of triiodothyronine (T(3)) added to short-term bed rest. Nine men and 5 women were restricted to bed rest for 28 days with their heads positioned 6 degrees below their feet. Subjects were randomly assigned to receive either placebo or oral T(3) at doses of 50 to 75 microg/d in a single-blind fashion. Calcium balance was measured over 5-day periods; and T(3), thyroxine, thyroid-stimulating hormone, immunoreactive parathyroid hormone, osteocalcin, bone alkaline phosphatase, and urinary deoxypyridinoline were measured weekly. Triiodothyronine increased 2-fold in the men and 5-fold in the women during treatment, suppressing both thyroxine and thyroid-stimulating hormone. Calcium balance was negative by 300 to 400 mg/d in the T(3)-treated volunteers, primarily because of the increased fecal loss that was not present in the placebo group. Urinary deoxypyridinoline to creatinine ratio, a marker of bone resorption, increased 60% in the placebo group during bed rest, but more than doubled in the T(3)-treated subjects (P < .01), suggesting that bone resorption was enhanced by treatment with T(3). Changes in serum osteocalcin and bone-specific alkaline phosphatase, markers of bone formation, were similar in T(3)- and placebo-treated subjects. Triiodothyronine increases bone resorption and fecal calcium loss in subjects at bed rest.

  8. Increased intracellular calcium activates serum and glucocorticoid-inducible kinase 1 (SGK1) through a calmodulin-calcium calmodulin dependent kinase kinase pathway in Chinese hamster ovary cells.

    PubMed

    Imai, Seiji; Okayama, Naotsuka; Shimizu, Manabu; Itoh, Makoto

    2003-04-04

    SGK1 is one of the protein-serine/threonine kinases that is activated by insulin in a PI3K-dependent manner. Although SGK1 mediates a variety of biological activities, the mechanisms regulating its activity remain unclear. In this study, we examined the potential roles of calcium signaling in the activation of SGK1. Treatment of CHO-IR cells with a cell-permeable calcium chelator, BAPTA-AM, abolished the insulin-induced activation of SGK1. Increasing intracellular calcium concentration by treating cells with thapsigargin or ionomycin induced a 6-8 fold increase in SGK1 activation. This was not affected by a PI3K inhibitor, wortmannin, but was completely inhibited by the calmodulin inhibitors, W 7 and W 5. Co-transfection of CHO cells with FLAG-SGK1 and CaMKK revealed the direct association of CaMKK with SGK1. These results suggest a calcium-triggered signaling cascade in which an increase in intracellular calcium concentration directly stimulates SGK1 through CaMKK.

  9. Calcium modulation of the renin-aldosterone axis.

    PubMed

    Porter, L; Conlin, P R; Scott, J; Brown, E M; El-Hajj Fuleihan, G

    1999-02-01

    Changes in the extracellular calcium concentration have important effects on hormone secretion. In vitro, kidney juxtaglomerular cells have been shown to secrete renin in an inverse relationship to the extracellular calcium concentration. The effect of physiologic changes in calcium on renin secretion in humans is less clear. We therefore investigated the effects of physiologic changes in extracellular calcium levels on renin and aldosterone secretion in 7 healthy men. Serum ionized calcium, ACTH, plasma renin activity (PRA), and aldosterone levels were serially measured during 90-min infusions of dextrose, disodium edetate (EDTA) and calcium gluconate, and the latter two infusions were repeated with dexamethasone pre-treatment. ACTH levels decreased during the 5% dextrose in water and the EDTA infusions but increased during the calcium infusion. Similarly, PRA and aldosterone levels fell during the D5W and EDTA infusions but this decrease was absent with increments in calcium levels. Dexamethasone administration suppressed ACTH and the aldosterone response to calcium but did not affect the PRA response to the calcium infusion. Our data indicate that increases in calcium within the physiological range blunt the diurnal decline in both PRA and aldosterone. This appears to be a direct effect of calcium on PRA but mediated through ACTH in the case of aldosterone.

  10. In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase

    PubMed Central

    Nizar, Krystal; Uhlirova, Hana; Tian, Peifang; Saisan, Payam A.; Cheng, Qun; Reznichenko, Lidia; Weldy, Kimberly L.; Steed, Tyler C.; Sridhar, Vishnu B.; MacDonald, Christopher L.; Cui, Jianxia; Gratiy, Sergey L.; Sakadžić, Sava; Boas, David A.; Beka, Thomas I.; Einevoll, Gaute T.; Chen, Ju; Masliah, Eliezer; Dale, Anders M.; Silva, Gabriel A.; Devor, Anna

    2013-01-01

    Calcium-dependent release of vasoactive gliotransmitters is widely assumed to trigger vasodilation associated with rapid increases in neuronal activity. Inconsistent with this hypothesis, intact stimulus-induced vasodilation was observed in inositol 1,4,5-triphosphate (IP3) type-2 receptor (R2) knockout (KO) mice, in which the primary mechanism of astrocytic calcium increase – the release of calcium from intracellular stores following activation of an IP3-dependent pathway – is lacking. Further, our results in wild type (WT) mice indicate that in vivo onset of astrocytic calcium increase in response to sensory stimulus could be considerably delayed relative to the simultaneously measured onset of arteriolar dilation. Delayed calcium increases in WT mice were observed in both astrocytic cell bodies and perivascular endfeet. Thus, astrocytes may not play a role in the initiation of blood flow response, at least not via calcium-dependent mechanisms. Moreover, an increase in astrocytic intracellular calcium was not required for normal vasodilation in the IP3R2-KO animals. PMID:23658179

  11. Interaction between Calcium Ions and Bacillus thuringiensis Toxin Activity against Sf9 Cells (Spodoptera frugiperda, Lepidoptera)

    PubMed Central

    Monette, R.; Potvin, L.; Baines, D.; Laprade, R.; Schwartz, J. L.

    1997-01-01

    The effects of calcium ions and modulators of calcium movement on Bacillus thuringiensis insecticidal protein toxicity were investigated with Sf9 cells (Spodoptera frugiperda, fall armyworm) by a new B. thuringiensis toxicity assay based on measurement of fluorescence of ethidium homodimer, a high-affinity DNA stain. CryIC toxicity was substantially stimulated by extracellular calcium in a dose-dependent way (in the millimolar range), while toxicity enhancement could not be replicated when calcium was replaced by barium. This incremental toxicity was reduced by cobalt and lanthanum ions, two inorganic-calcium transport inhibitors. Methoxyverapamil, a voltage-dependent calcium channel blocker, and nifedipine, an inhibitor of dihydropyridine-sensitive L-type calcium channels, had no effect on CryIC toxin activity, but BAY K 8644, an L-type calcium channel activator, increased CryIC activity at high concentrations of extracellular calcium. While A23187, a calcium ionophore, and TMB-8, an inhibitor of intracellular-calcium mobilization, did not change CryIC-induced mortality, thapsigargin, an inhibitor of calcium uptake in intracellular stores, and more particularly trifluoperazine, which inhibits calcium-calmodulin-dependent processes, increased CryIC-mediated toxicity. The incremental effect of extracellular calcium on CryIC-induced toxicity was consistent with an increased concentration of intracellular calcium. PMID:16535509

  12. Modulation by extracellular ATP of L-type calcium channels in guinea-pig single sinoatrial nodal cell.

    PubMed Central

    Qi, A. D.; Kwan, Y. W.

    1996-01-01

    1. The effects of extracellular adenosine 5'-triphosphate ([ATP]zero) on the L-type Ca2+ channel currents in guinea-pig single sinoatrial nodal (SAN) cells, isolated by enzymatic dissociation, were investigated by use of whole-cell patch-clamp techniques. 2. The application of [ATP]zero (2 microM-1 mM) produced an inhibitory effect on the L-type Ca2+ channel current peak amplitude (10 mM Ba2+ as charge carrier) in a concentration-dependent and reversible manner with an IC50 of 100 microM and a Hill coefficient of 1.83. 3. The presence of the adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM) and 8-phenyltheophylline (10 microM) did not affect the [ATP]zero-induced inhibition of the Ca2+ channel currents. Adenosine (100 microM) had little effect on the basal Ca2+ channel currents. Adenosine 500 microM, caused 23% inhibition of the Ca2+ channel current, which was abolished by 0.1 microM DPCPX. 4. The presence of the P2-purinoceptor antagonists, suramin (1, 10 and 100 microM), reactive blue 2 (1 and 10 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 50 and 100 microM) failed to affect the inhibitory action of [ATP]zero on Ca2+ channel currents. 5. The relative rank order of potency of different nucleotides and nucleosides, at a concentration of 100 microM, on the inhibition of the Ca2+ channel currents is as follows: adenosine 5'-triphosphate (ATP) = alpha,beta-methylene-ATP (alpha,beta MeATP) > > 2-methylthioATP (2-MeSATP) > or = adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > > uridine 5'-triphosphate (UTP) = adenosine 5'-diphosphate (ADP) > adenosine 5'-monophosphate (AMP) > or = adenosine. 6. These results suggest that [ATP]zero may play an important role in the heart beat by inhibiting the L-type Ca2+ channel currents in single SAN cells. This inhibitory effect is not due to the formation of adenosine resulting from the enzymatic degradation of [ATP]zero. Based on the relative order of inhibitory

  13. Increased mitochondrial calcium coexists with decreased reperfusion injury in postconditioned (but not preconditioned) hearts.

    PubMed

    Argaud, Laurent; Gateau-Roesch, Odile; Augeul, Lionel; Couture-Lepetit, Elisabeth; Loufouat, Joseph; Gomez, Ludovic; Robert, Dominique; Ovize, Michel

    2008-01-01

    Ca(2+) is the main trigger for mitochondrial permeability transition pore opening, which plays a key role in cardiomyocyte death after ischemia-reperfusion. We investigated whether a reduced accumulation of mitochondrial Ca(2+) might explain the attenuation of lethal reperfusion injury by postconditioning. Anesthetized New Zealand White rabbits underwent 30 min of ischemia, followed by either 240 (infarct size protocol) or 60 (mitochondria protocol) min of reperfusion. They received either no intervention (control), preconditioning by 5-min ischemia and 5-min reperfusion, postconditioning by four cycles of 1-min reperfusion and 1-min ischemia at the onset of reflow, or pharmacological inhibition of the transition pore opening by N-methyl-4-isoleucine-cyclosporin (NIM811; 5 mg/kg iv) given at reperfusion. Area at risk and infarct size were assessed by blue dye injection and triphenyltetrazolium chloride staining. Mitochondria were isolated from the risk region for measurement of 1) Ca(2+) retention capacity (CRC), and 2) mitochondrial content of total (atomic absorption spectrometry) and ionized (potentiometric technique) calcium concentration. CRC averaged 0.73 +/- 0.16 in control vs. 4.23 +/- 0.17 mug Ca(2+)/mg proteins in shams (P < 0.05). Postconditioning, preconditioning, or NIM811 significantly increased CRC (P < 0.05 vs. control). In the control group, total and free mitochondrial calcium significantly increased to 2.39 +/- 0.43 and 0.61 +/- 0.10, respectively, vs. 1.42 +/- 0.09 and 0.16 +/- 0.01 mug Ca(2+)/mg in sham (P < 0.05). Surprisingly, whereas total and ionized mitochondrial Ca(2+) decreased in preconditioning, it significantly increased in postconditioning and NIM811 groups. These data suggest that retention of calcium within mitochondria may explain the decreased reperfusion injury in postconditioned (but not preconditioned) hearts.

  14. High concentration of insulin promotes apoptosis of primary cultured rat ovarian granulosa cells via its increase in extracellular HMGB1.

    PubMed

    Ni, Xiao-Rong; Sun, Zhou-Jun; Hu, Guo-Hua; Wang, Rong-Hui

    2015-03-01

    Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting women of reproductive age. Insulin resistance/hyperinsulinemia is a prevalent finding in women with PCOS, which indicates that insulin resistance/hyperinsulinemia may be an important player in the pathogenesis of the PCOS. However, the underlying mechanism of insulin resistance/hyperinsulinemia on the pathogenesis of the PCOS remains elusive. In this study, we found an increased high-mobility group box 1 (HMGB1) in the serum from women with PCOS having insulin resistance/hyperinsulinemia. Furthermore, we discovered that high concentration of insulin, which mimics insulin resistance model, promoted apoptosis in primary cultured rat ovarian granulosa cells (GCs) via its effect on the increase in extracellular HMGB1. Our data presented the first evidence that increased HMGB1 induced by insulin resistance/hyperinsulinemia promoted apoptosis of ovarian GCs, which provided new molecular basis for the PCOS pathogenesis.

  15. Contribution of pannexin 1 and connexin 43 hemichannels to extracellular calcium-dependent transport dynamics in human blood-brain barrier endothelial cells.

    PubMed

    Kaneko, Yosuke; Tachikawa, Masanori; Akaogi, Ryo; Fujimoto, Kazuhisa; Ishibashi, Megumi; Uchida, Yasuo; Couraud, Pierre-Olivier; Ohtsuki, Sumio; Hosoya, Ken-ichi; Terasaki, Tetsuya

    2015-04-01

    Dysregulation of blood-brain barrier (BBB) transport function is thought to exacerbate neuronal damage in acute ischemic stroke. The purpose of this study was to clarify the characteristics of pannexin (Px) and/or connexin (Cx) hemichannel(s)-mediated transport of organic anions and cations in human BBB endothelial cell line hCMEC/D3 and to identify inhibitors of hemichannel opening in hCMEC/D3 cells in the absence of extracellular Ca(2+), a condition mimicking acute ischemic stroke. In the absence of extracellular Ca(2+), the cells showed increased uptake and efflux transport of organic ionic fluorescent dyes. Classic hemichannel inhibitors markedly inhibited the enhanced uptake and efflux. Quantitative targeted absolute proteomics confirmed Px1 and Cx43 protein expression in plasma membrane of hCMEC/D3 cells. Knockdown of Px1 and Cx43 with the small interfering RNAs significantly inhibited the enhanced uptake and efflux of organic anionic and cationic fluorescent dyes. Clinically used cilnidipine and progesterone, which have neuroprotective effects in animal ischemia models, were identified as inhibitors of hemichannel opening. These findings suggest that altered transport dynamics at the human BBB in the absence of extracellular Ca(2+) is at least partly attributable to opening of Px1 and Cx43 hemichannels. Therefore, we speculate that Px1 and Cx43 may be potential drug targets to ameliorate BBB transport dysregulation during acute ischemia.

  16. Sucrose increases calcium-dependent protein kinase and phosphatase activities in potato plants.

    PubMed

    Raíces, M; MacIntosh, G C; Ulloa, R M; Gargantini, P R; Vozza, N F; Téllez-Inón, M T

    2003-09-01

    The effect of sucrose on tuber formation, calcium-dependent protein kinase (CDPK) and phosphatase activities was analysed using in vitro cultured potato plants. In short treatments, sucrose induced CDPK and phosphatase activities. In long treatments, sucrose induced tuber formation in the absence of other tuber inducing stimuli. Sorbitol caused a minor increase in CDPK activity and affected plant morphology but did not induce tuber development. The addition of the protein kinase inhibitor Staurosporine precluded sucrose-induced tuberization. Altogether, our results suggest that phosphorylation/dephosphorylation events are involved in sucrose-induced tuber development.

  17. Stimulation of high affinity gamma-aminobutyric acidB receptors potentiates the depolarization-induced increase of intraneuronal ionized calcium content in cerebellar granule neurons.

    PubMed

    De Erausquin, G; Brooker, G; Costa, E; Wojcik, W J

    1992-09-01

    In the treatment of spasticity, the therapeutic cerebrospinal fluid levels of (+/-)-baclofen, a gamma-aminobutyric acid (GABA)B receptor agonist, are below 1 microM. However, the mechanism of the therapeutic action of (+/-)-baclofen remains unknown, because, for the most part, the action of (+/-)-baclofen on GABAB receptors requires micromolar concentrations. Using fura-2 fluorescence microscopy, intracellular ionized calcium was measured in cerebellar granule neurons. Stimulation of a high affinity GABAB receptor potentiated by 2-3-fold the rise in intracellular calcium observed after depolarization of the cell with a Krebs Ringer's buffered solution containing 40 mM K+. Both GABA (100 nM) and (+/-)-baclofen (10-100 nM) stimulated this high affinity receptor. The potentiation of the depolarization-induced rise in intracellular calcium by (+/-)-baclofen (100 nM) was completely blocked by the GABAB receptor antagonist CGP 35348 (200 microM). Also, the intracellular calcium response induced by the activation of high affinity GABAB receptors was prevented by dantrolene (10 microM). The cerebellar granule neurons contained calcium-induced calcium release (CICR) stores. Caffeine (3 mM) and ryanodine (100 microM) potentiated the depolarization-induced rise in intracellular calcium, and this response to both drugs was blocked by dantrolene (10 microM). Because dantrolene does not prevent the rise in intracellular calcium after cell depolarization (this calcium originated from the influx of extracellular calcium), (+/-)-baclofen acting via the high affinity GABAB receptor indirectly activates the CICR stores, allowing the influx of extracellular calcium to trigger the release of calcium from these dantrolene-sensitive CICR stores. Thus, this high affinity GABAB receptor might become activated during persistent depolarization caused by pathological states and could be a mechanism to be studied for the therapeutic action of (+/-)-baclofen in spasticity.

  18. Increased calcium absorption from synthetic stable amorphous calcium carbonate: Double-blind randomized crossover clinical trial in post-menopausal women

    USDA-ARS?s Scientific Manuscript database

    Calcium supplementation is a widely recognized strategy for achieving adequate calcium intake. We designed this blinded, randomized, crossover interventional trial to compare the bioavailability of a new stable synthetic amorphous calcium carbonate (ACC) with that of crystalline calcium carbonate (C...

  19. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils.

    PubMed

    Hasan, Md Ashraful; Ahn, Won-Gyun; Song, Dong-Keun

    2016-09-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca(2+) signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca(2+)]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca(2+)]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca(2+)]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca(2+)]i in human neutrophils was observed. In Ca(2+)-free buffer, NAC- and cysteine-induced [Ca(2+)]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca(2+)]i in human neutrophils occur through Ca(2+) influx. NAC- and cysteine-induced [Ca(2+)]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na(+)-free HEPES, both NAC and cysteine induced a marked increase in [Ca(2+)]i in human neutrophils, arguing against the possibility that Na(+)-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca(2+)]i increasing activity. Our results show that NAC and cysteine induce [Ca(2+)]i increase through Ca(2+) influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

  20. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

    PubMed Central

    Hasan, Md. Ashraful; Ahn, Won-Gyun

    2016-01-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca2+]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca2+]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca2+]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca2+]i in human neutrophils was observed. In Ca2+-free buffer, NAC- and cysteine-induced [Ca2+]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca2+]i in human neutrophils occur through Ca2+ influx. NAC- and cysteine-induced [Ca2+]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na+-free HEPES, both NAC and cysteine induced a marked increase in [Ca2+]i in human neutrophils, arguing against the possibility that Na+-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca2+]i increasing activity. Our results show that NAC and cysteine induce [Ca2+]i increase through Ca2+ influx in human neutrophils via SKF96365- and ruthenium red-dependent way. PMID:27610031

  1. Serum Calcium Increase Correlates With Worsening of Lipid Profile: An Observational Study on a Large Cohort From South Italy.

    PubMed

    Gallo, Luigia; Faniello, Maria C; Canino, Giovanni; Tripolino, Cesare; Gnasso, Agostino; Cuda, Giovanni; Costanzo, Francesco S; Irace, Concetta

    2016-02-01

    Despite the well-documented role of calcium in cell metabolism, its role in the development of cardiovascular disease is still under heavy debate. Several studies suggest that calcium supplementation might be associated with an increased risk of coronary heart disease, whereas others underline a significant effect on lowering high blood pressure and hyperlipidemia. The purpose of this study was to investigate, in a large nonselected cohort from South Italy, if serum calcium levels correlate with lipid values and can therefore be linked to higher individual cardiovascular risk.Eight-thousand-six-hundred-ten outpatients addressed to the Laboratory of Clinical Biochemistry, University of Magna Græcia, Catanzaro, Italy from January 2012 to December 2013 for routine blood tests, were enrolled in the study. Total HDL-, LDL- and non-HDL colesterol, triglycerides, and calcium were determined with standard methods.We observed a significant association between total cholesterol, LDL-cholesterol, HDL-cholesterol, non-HDL cholesterol, triglycerides, and serum calcium in men and postmenopause women. Interestingly, in premenopause women, we only found a direct correlation between serum calcium, total cholesterol, and HDL-cholesterol. Calcium significantly increased while increasing total cholesterol and triglycerides in men and postmenopause women.Our results confirm that progressive increase of serum calcium level correlates with worsening of lipid profile in our study population. Therefore, we suggest that a greater caution should be used in calcium supplement prescription particularly in men and women undergoing menopause, in which an increase of serum lipids is already known to be associated with a higher cardiovascular risk.

  2. Calcium intake is not associated with increased coronary artery calcification: The Framingham Study

    USDA-ARS?s Scientific Manuscript database

    Adequate calcium intake is known to protect the skeleton. However, studies that have reported adverse effects of calcium supplementation on vascular events have raised widespread concern. We assessed the association between calcium intake (from diet and supplements) and coronary artery calcification...

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

    PubMed

    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 < 0.01), and VSM calcium was 200 ± 20 nmol/l in PAs vs. 104 ± 15 nmol/l in MCAs (P < 0.01). In vessels permeabilized with Staphylococcus aureus α-toxin, PAs were not more sensitive to calcium, suggesting calcium sensitization was not at the level of the contractile apparatus. PAs were 30-fold more sensitive to the voltage-dependent calcium channel (VDCC) inhibitor nifedipine than MCAs (EC50 for PAs was 3.5 ± 0.4 vs. 82.1 ± 2.1 nmol/l for MCAs;P < 0.01); however, electrophysiological properties of the VDCC were not different in VSM. PAs had little to no response to the calcium-activated potassium 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.

  4. Formulation and sensory evaluation of Prosopis alba (Algarrobo) pulp cookies with increased iron and calcium dialyzabilities.

    PubMed

    Bernardi, C; Drago, S; Sabbag, N; Sanchez, H; Freyre, M

    2006-03-01

    Prosopis alba (algarrobo) is an important indigenous specie, which fruits are used as food and feed since ancient times. Cookies containing algarrobo pulp (AP) with increased iron and calcium availabilities were formulated and sensory evaluated. AP is preferred as food ingredient because of its high sugar content and pleasant flavour. Formulated cookies mean proximal composition was 8.9 g/100 g protein, 7.2 g/100 g dietary fiber, 25 g/100 g total sugar, and 18.5 g/100 g crude fat with iron and calcium contents 30 ppm and 340 ppm, respectively. Ascorbic (AA) and citric (CA) acids at different mM acid: mM Fe were added in order to increase mineral availabilities being evaluated by an in vitro method. Those ratios were 5:1 and 10:1 for AA:Fe whereas for CA:Fe were 50:1 and 100:1 and combinations of them. After chosen the best AA:Fe and CA:Fe ratios (5:1 and 50:1, respectively), sensory evaluation with trained sensory panel and a consumer acceptability test with one hundred and seventy untrained judges were carried out. Acceptability test showed that 77.65% of the people (< 25 years old 41.76%, 25-50 years old 20.00% and > 50 years old 15.89%) tasting final formulated cookies indicated that they "like very much" or "moderately like" and there were not consumers rejecting them.

  5. Increased asynchronous release and aberrant calcium channel activation in amyloid precursor protein deficient neuromuscular synapses.

    PubMed

    Yang, L; Wang, B; Long, C; Wu, G; Zheng, H

    2007-11-23

    Despite the critical roles of the amyloid precursor protein (APP) in Alzheimer's disease pathogenesis, its physiological function remains poorly established. Our previous studies implicated a structural and functional activity of the APP family of proteins in the developing neuromuscular junction (NMJ). Here we performed comprehensive analyses of neurotransmission in mature neuromuscular synapse of APP deficient mice. We found that APP deletion led to reduced paired-pulse facilitation and increased depression of synaptic transmission with repetitive stimulation. Readily releasable pool size and total releasable vesicles were not affected, but probability of release was significantly increased. Strikingly, the amount of asynchronous release, a measure sensitive to presynaptic calcium concentration, was dramatically increased, and pharmacological studies revealed that it was attributed to aberrant activation of N- and L-type Ca(2+) channels. We propose that APP modulates synaptic transmission at the NMJ by ensuring proper Ca(2+) channel function.

  6. [Methods of increasing the activity of extracellular esterase, beta-fructofuranosidase and proteases of wine yeast].

    PubMed

    Abdurazakova, S Kh; Salomov, Kh T

    1975-01-01

    Upon regular fermentation changes in the activity of the enzymes esterase, beta-fructofuranosidase and protease of the yeast Saccharomyces mini of the Parkent I race were examined. The maximum activity of the enzymes occurred in the stationary phase of the yeast growth. An increase in the activity of the above enzymes was shown possible during a prolonged stabilization of the stationary conditions in the process of a continuous chemostat cultivation of wine yeast.

  7. Increased extracellular pressure provides a novel adjuvant stimulus for enhancement of conventional dendritic cell maturation strategies

    SciTech Connect

    Craig, David H.; Shiratsuchi, Hiroe; Basson, Marc D.

    2009-09-11

    Dendritic cell (DC)-based vaccine strategies have gained increasing popularity in recent years. Methods for ex vivo generation of immunocompetent mature DCs still require optimization. DCs have been shown to phenotypically mature under elevated pressure. We compared the effects of pressure on DC maturation with LPS- and cytokine-stimulation. Human monocyte-derived immature or LPS- and cytokine-matured DCs were exposed to ambient or 40 mmHg increased pressure for 12 h, then assessed for expression of CD80, CD86, CD40, MHC-I/II, and inflammatory cytokine production. DCs were also evaluated for capacity to stimulate T-cell proliferation by co-culture with allogeneic lymphocytes. Pressure significantly increased cytokine production and expression of all surface molecules on immature DC other than MHC-I and CD40. Pressure/LPS-treated DCs displayed further upregulation of MHC-I, CD40, and IL-12p70. Cytokine-matured DCs appeared less responsive to pressure. T-cell proliferation correlated with MHC expression. Results suggest mechanical stimulation of DCs may provide a useful adjuvant to TLR-agonist maturation strategies.

  8. Calcium and bone disease

    PubMed Central

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

    2013-01-01

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

  9. Regional increase in extracellular potassium can be arrhythmogenic due to nonuniform muscle contraction in rat ventricular muscle

    PubMed Central

    Hattori, Taiki; Murai, Naomi; Nagano, Tsuyoshi; Nishio, Taichi; Boyden, Penelope A.; Shindoh, Chiyohiko

    2012-01-01

    In the ischemic myocardium, extracellular potassium ([K+]o) increases to ≥20 mmol/l. To determine how lethal arrhythmias occur during ischemia, we investigated whether the increased spatial pattern of [K+]o, i.e., a regional or a global increase, affects the incidence of arrhythmias. Force, sarcomere length, membrane potential, and nonuniform intracellular Ca2+ ([Ca2+]i) were measured in rat ventricular trabeculae. A “regional” or “global” increase in [K+]o was produced by exposing a restricted region of muscle to a jet of 30 mmol/l KCl or by superfusing trabeculae with a solution containing 30 mmol/l KCl, respectively. The increase in [Ca2+]i (CaCW) during Ca2+ waves was measured (24°C, 3.0 mmol/l [Ca2+]o). A regional increase in [K+]o caused nonuniform [Ca2+]i and contraction. In the presence of isoproterenol, the regional increase in [K+]o induced sustained arrhythmias in 10 of 14 trabeculae, whereas the global increase did not induce such arrhythmias. During sustained arrhythmias, Ca2+ surged within the jet-exposed region. In the absence of isoproterenol, the regional increase in [K+]o increased CaCW, whereas the global increase decreased it. This increase in CaCW with the regional increase in [K+]o was not suppressed by 100 μmol/l streptomycin, whereas it was suppressed by 1) a combination of 10 μmol/l cilnidipine and 3 μmol/l SEA0400; 2) 20 mmol/l 2,3-butanedione monoxime; and 3) 10 μmol/l blebbistatin. A regional but not a global increase in [K+]o induces sustained arrhythmias, probably due to nonuniform excitation-contraction coupling. The same mechanism may underlie arrhythmias during ischemia. PMID:22447939

  10. Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity-dependent bulk endocytosis.

    PubMed

    Morton, Andrew; Marland, Jamie R K; Cousin, Michael A

    2015-08-01

    Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine-dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium-dependent events such as activity-dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity-dependent dynamin I dephosphorylation was also arrested in EGTA-treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. To determine the minimal requirements for ADBE triggering, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. We found that SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient to trigger ADBE.

  11. Alcohol consumption increases basal extracellular glutamate in the nucleus accumbens core of Sprague-Dawley rats without increasing spontaneous glutamate release.

    PubMed

    Pati, Dipanwita; Kelly, Kyle; Stennett, Bethany; Frazier, Charles J; Knackstedt, Lori A

    2016-07-01

    Glutamate neurotransmission in the nucleus accumbens core (NAc) mediates ethanol consumption. Previous studies using non-contingent and voluntary alcohol administration in inbred rodents have reported increased basal extracellular glutamate levels in the NAc. Here, we assessed basal glutamate levels in the NAc following intermittent alcohol consumption in male Sprague-Dawley rats that had access to ethanol for 7 weeks on alternating days. We found increased basal NAc glutamate at 24 h withdrawal from ethanol and thus sought to identify the source of this glutamate. To do so, we employed a combination of microdialysis, slice electrophysiology and western blotting. Reverse dialysis of the voltage-gated sodium channel blocker tetrodotoxin did not affect glutamate levels in either group. Electrophysiological recordings in slices made after 24 h withdrawal revealed a decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency relative to controls, with no change in sEPSC amplitude. No change in metabotropic glutamate receptor 2/3 (mGlu2/3) function was detected as bath application of the mGlu2/3 agonist LY379268 decreased spontaneous and miniature EPSC frequency in slices from both control and ethanol-consuming rats. The increase in basal glutamate was not associated with changes in the surface expression of GLT-1, however, a decrease in slope of the no-net-flux dialysis function was observed following ethanol consumption, indicating a potential decrease in glutamate reuptake. Taken together, these findings indicate that the increase in basal extracellular glutamate occurring after chronic ethanol consumption is not mediated by an increase in action potential-dependent glutamate release or a failure of mGlu2/3 autoreceptors to regulate such release. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  12. Does EDTA-infusion affect calcium homeostatis leading to increased resistance to challenge?

    PubMed

    Mellau, L S; Jørgensen, R J

    2003-01-01

    Disodium EDTA is a chelating agent used in studies involving calcium. The sodium ion of the chelate is displaced by calcium to form soluble, non-toxic physiologically unavailable complexes that are excreted through urine. When EDTA binds the available calcium in the blood, an animal is forced into a state of negative calcium balance, a situation that mimics the principle of lowering dietary calcium in preventing milk fever. Although limited evidence is available, it is likely that calcium homeostatic mechanisms respond under such circumstances through the effect of parathyroid hormone and 1,25(OH)2 vit D3 on bone, gastrointestinal tract and the kidney. Investigations involving milk fever prevention by dietary manipulation using EDTA-induced hypocalcaemic or milk fever cow models should be interpreted with caution because EDTA is thought to be invasive to calcium homeostatic mechanisms.

  13. Extracellular calcium (Ca2+o)-sensing receptor in a mouse monocyte-macrophage cell line (J774): potential mediator of the actions of Ca2+o on the function of J774 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Kifor, O.; Chattopadhyay, N.; Bai, M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone remodeling and may play a role in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for bone marrow mononuclear cells in the vicinity, leading us to investigate whether such mononuclear cells express the CaR. In this study, we used the mouse J774 cell line, which exhibits a pure monocyte-macrophage phenotype. Both immunocytochemistry and Western blot analysis, using polyclonal antisera specific for the CaR, detected CaR protein in J774 cells. The use of reverse transcriptase-polymerase chain reaction with CaR-specific primers, including a set of intron-spanning primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in J774 cells. Exposure of J774 cells to high Ca2+o (2.8 mM or more) or the polycationic CaR agonist, neomycin (100 microM), stimulated both chemotaxis and DNA synthesis in J774 cells. Therefore, taken together, our data strongly suggest that the monocyte-macrophage cell line, J774, possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney.

  14. Extracellular calcium (Ca2+o)-sensing receptor in a mouse monocyte-macrophage cell line (J774): potential mediator of the actions of Ca2+o on the function of J774 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Kifor, O.; Chattopadhyay, N.; Bai, M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone remodeling and may play a role in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for bone marrow mononuclear cells in the vicinity, leading us to investigate whether such mononuclear cells express the CaR. In this study, we used the mouse J774 cell line, which exhibits a pure monocyte-macrophage phenotype. Both immunocytochemistry and Western blot analysis, using polyclonal antisera specific for the CaR, detected CaR protein in J774 cells. The use of reverse transcriptase-polymerase chain reaction with CaR-specific primers, including a set of intron-spanning primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in J774 cells. Exposure of J774 cells to high Ca2+o (2.8 mM or more) or the polycationic CaR agonist, neomycin (100 microM), stimulated both chemotaxis and DNA synthesis in J774 cells. Therefore, taken together, our data strongly suggest that the monocyte-macrophage cell line, J774, possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney.

  15. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Butters, R. R. Jr; Sugimoto, T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

  16. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Butters, R. R. Jr; Sugimoto, T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

  17. The Extracellular Domain of Notch2 Increases its Cell-Surface Abundance and Ligand Responsiveness during Kidney Development

    PubMed Central

    Liu, Zhenyi; Chen, Shuang; Boyle, Scott; Zhu, Yu; Zhang, Andrew; Piwnica-Worms, David R.; Ilagan, Ma. Xenia G.; Kopan, Raphael

    2013-01-01

    SUMMARY Notch2, but not Notch1, plays indispensable roles in kidney organogenesis and Notch2 haploinsufficiency is associated with Alagille syndrome. We proposed that proximal nephron fates are regulated by a threshold that requires nearly all available free Notch intracellular domains (ICDs), but we could not identify the mechanism explaining why Notch2 (N2) is more important than Notch1 (N1). By generating mice that swap their ICDs, we establish that overall protein concentration, expression domain, or ICD amino acid composition does not account for the differential requirement for these receptors. Instead, we find that the N2 extracellular domain (ECD) increases Notch protein localized to the cell surface during kidney development and is cleaved more efficiently upon ligand binding. This context-specific asymmetry in NICD release efficiency is further enhanced by Fringe. Our results indicate that elevating N1 surface level could compensate for the loss of N2 signal in specific cell contexts. PMID:23806616

  18. Barbiturates increase the rate of voltage-dependent inactivation of the calcium current in snail neurones.

    PubMed Central

    Nishi, K.; Oyama, Y.

    1983-01-01

    Effects of barbiturates (thiopentone, pentobarbitone, phenobarbitone and barbitone) on the calcium current (ICa) in identified Helix neurones were studied, using a conventional suction pipette technique. Barbiturates depressed the maximal peak amplitudes (MPA) of ICa in a dose-dependent manner without shifting the current-voltage relationships along the voltage axis. Barbiturates accelerated the decay phase of ICa at high concentrations (1 X 10(-4) to 3 X 10(-3) M), at which concentrations double-pulse experiments showed the increased rate of a voltage-dependent inactivation of ICa. It is concluded that the acceleration of the decay phase of ICa by barbiturates may be due to the increased rate of the voltage-dependent inactivation of ICa. PMID:6100847

  19. Increased Calcium Availability Leads to Greater Forest Floor Accumulation in an Adirondack Forest

    NASA Astrophysics Data System (ADS)

    Melvin, A.; Goodale, C. L.

    2010-12-01

    Nutrient availability in Northeastern US forests has been dramatically altered by anthropogenic activities. Acid deposition has not only increased nitrogen (N) availability, but has also been linked to soil acidification and a loss of base cations, largely calcium (Ca). We are studying the long-term effects of a Ca addition on carbon (C) and N cycling in a forested catchment in the Adirondack Park, New York. In 1989, calcium carbonate (lime) was added to two subcatchments within the Woods Lake Watershed to ameliorate the effects of soil Ca depletion. Two additional subcatchments were left as controls. Eighteen years after the Ca application, both soil pH and exchangeable Ca concentrations remain elevated in the organic horizons and upper mineral soils of the treated subcatchments. The forest floor mass in this watershed is very large and measurements show that the organic layer in the limed subcatchments is significantly larger than in the controls (212 t/ha vs. 116 t/ha), resulting in greater C and N stocks in the Ca-amended soils. This finding suggests that Ca may stabilize soil organic matter (SOM), resulting in greater C storage under high soil Ca conditions. We are investigating potential drivers of this SOM accumulation in the limed subcatchments, including rates of leaf litter production and the decomposition rate of forest floor material. This work will provide important insights into how long-term changes in soil Ca availability influence SOM stabilization, retention and nutrient cycling.

  20. TFEB-mediated increase in peripheral lysosomes regulates store-operated calcium entry

    PubMed Central

    Sbano, Luigi; Bonora, Massimo; Marchi, Saverio; Baldassari, Federica; Medina, Diego L.; Ballabio, Andrea; Giorgi, Carlotta; Pinton, Paolo

    2017-01-01

    Lysosomes are membrane-bound organelles mainly involved in catabolic processes. In addition, lysosomes can expel their contents outside of the cell via lysosomal exocytosis. Some of the key steps involved in these important cellular processes, such as vesicular fusion and trafficking, require calcium (Ca2+) signaling. Recent data show that lysosomal functions are transcriptionally regulated by transcription factor EB (TFEB) through the induction of genes involved in lysosomal biogenesis and exocytosis. Given these observations, we investigated the roles of TFEB and lysosomes in intracellular Ca2+ homeostasis. We studied the effect of transient modulation of TFEB expression in HeLa cells by measuring the cytosolic Ca2+ response after capacitative Ca2+ entry activation and Ca2+ dynamics in the endoplasmic reticulum (ER) and directly in lysosomes. Our observations show that transient TFEB overexpression significantly reduces cytosolic Ca2+ levels under a capacitative influx model and ER re-uptake of calcium, increasing the lysosomal Ca2+ buffering capacity. Moreover, lysosomal destruction or damage abolishes these TFEB-dependent effects in both the cytosol and ER. These results suggest a possible Ca2+ buffering role for lysosomes and shed new light on lysosomal functions during intracellular Ca2+ homeostasis. PMID:28084445

  1. Increased vitamin D and calcium intake associated with reduced mammographic breast density among premenopausal women.

    PubMed

    Fair, Alecia Malin; Lewis, Toni J; Sanderson, Maureen; Dupont, William D; Fletcher, Sarah; Egan, Kathleen M; Disher, Anthony C

    2015-10-01

    Vitamin D has been identified as a weak protective factor for postmenopausal breast cancer (relative risk, ~0.9), whereas high breast density has been identified as a strong risk factor (relative risk, ~4-6). To test the hypothesis that there is an association between vitamin D intake, but not circulating vitamin D levels, and mammographic breast density among women in our study, we conducted a cross-sectional study of 165 screening mammography patients at Nashville General Hospital's Breast Health Center, a public facility serving medically indigent and underserved women. Dietary and total (dietary plus supplements) vitamin D and calcium intakes were estimated by the Harvard African American Food Frequency Questionnaire, and blood samples were analyzed for 25-hydroxyvitamin D. Average percent breast density for the left and right breasts combined was estimated from digitized films using an interactive thresholding method available through Cumulus software. After statistical adjustment for age, race, and body mass index, the results revealed that there were significant trends of decreasing breast density with increasing vitamin D and calcium intake among premenopausal but not among postmenopausal women. There was no association between serum vitamin D and breast density in premenopausal or postmenopausal women. Confirmation of our findings in larger studies may assist in clarifying the role of vitamin D in breast density.

  2. Increased vitamin D and calcium intake associated with reduced mammographic breast density among premenopausal women

    PubMed Central

    Fair, Alecia Malin; Lewis, Toni J.; Sanderson, Maureen; Dupont, William D.; Fletcher, Sarah; Egan, Kathleen M.; Disher, Anthony C.

    2015-01-01

    Vitamin D has been identified as a weak protective factor for postmenopausal breast cancer (relative risk [RR]~0.9), while high breast density has been identified as a strong risk factor (RR~4–6). To test the hypothesis that there is an association between vitamin D intake, but not circulating vitamin D levels, and mammographic breast density among women in our study we conducted a cross-sectional study of 165 screening mammography patients at Nashville General Hospital’s Breast Health Center (NGH-BHC), a public facility serving medically indigent and underserved women. Dietary and total (dietary plus supplements) vitamin D, calcium intakes were estimated by the AAFQ and blood samples were analyzed for 25-Hydroxyvitamin D [25(OH)D3]. Average percent breast density for the left and right breasts combined was estimated from digitized films using an interactive-thresholding method available through Cumulus software. After statistical adjustment for age, race and body mass index, the results revealed there were significant trends of decreasing breast density with increasing vitamin D and calcium intake among premenopausal, but not among postmenopausal women. There was no association between serum vitamin D and breast density in pre- or postmenopausal women. Confirmation of our findings in larger studies may assist in clarifying the role of vitamin D in breast density. PMID:26321093

  3. Stem cell and extracellular matrix-related molecules increase following melatonin treatment in the skin of postmenopausal rats.

    PubMed

    Uslu, Serap; Oktem, Gulperi; Uysal, Aysegul; Soner, Burak Cem; Arbak, Serap; Ince, Umit

    2014-08-01

    The menopause has a negative effect in the skin. Melatonin affects skin functions and structures through actions mediated by cell-surface and putative-nuclear receptors expressed in skin cell. We have therefore determined the effects of melatonin treatment on stem cell in the epidermis and extracellular matrix related molecules in the dermis the skin of postmenopausal rats. A total of 45 female rats were divided into 5 groups: control group, group A [ovariectomy (OVX)], group B (OVX +10 mg/kg/day melatonin), group C (OVX +30 mg/kg/day melatonin), group S (sham operated + 10 mg/kg/day melatonin). Ventral skin samples were excised at 12th week after ovariectomy. Hematoxylin-eosin, periodic acid- methylamine silver, elastic van Gieson staining techniques were used to measure histomorphometrically the thickness of elastic fibers and basement membrane, depths of the epidermis, dermis, and subcutaneous fat layer. Immunohistochemical staining methods were used for fibroblast growth factor β (FGF β), collagen type I, fibronectin, β-catenin, c-kit, c-Myc evaluation. Epidermal thickness, subcutaneous fat layer, and elastic fibers were significantly decreased in group C, and there was a significant increase after melatonin treatment. Although there was no difference in dermal thickness of group C, melatonin also significantly increased the dermal thickness. High FGF β, type I collagen, fibronectin, β-catenin, c-Myc immunoreactivity developed following melatonin in all groups. Thus melatonin treatment of postmenopausal rats was mostly due to the decrease of stem cell and extracellular matrix-related molecules in the skin.

  4. Association Between Thoracic Aorta Calcium and Thoracic Aorta Geometry in a Cohort of Asymptomatic Participants at Increased Cardiovascular Risk.

    PubMed

    Craiem, Damian; Alsac, Jean-Marc; Casciaro, Mariano E; El Batti, Salma; Mousseaux, Elie; Sirieix, Marie-Emmanuelle; Simon, Alain

    2016-09-01

    Thoracic aorta calcium detection is known to improve cardiovascular risk prediction for cardiac and noncardiac events beyond traditional risk factors. We investigated the influence of thoracic aorta morphometry on the presence and extent of aortic calcifications. Nonenhanced computed tomography heart scans were performed in 970 asymptomatic participants at increased cardiovascular risk. An automated algorithm estimated the geometry of the entire thoracic aorta and quantified the aortic calcium Agatston score. A nonparametric model was used to analyze the percentiles of calcium score by age. Logistic regression models were calculated to identify anatomical associations with calcium levels. Calcifications were concentrated in the aortic arch and descending portions. Higher amounts of calcium were associated with an enlarged, unfolded, less tapered and more tortuous aorta. The size of the ascending aorta was not correlated with aortic calcium score, whereas enlargement of the descending aorta had the strongest association: the risk of having a global calcium score > 90th percentile was 3.62 times higher (confidence interval, 2.30-5.91; P < .001) for each 2.5-mm increase in descending aorta diameter. Vessel taper, tortuosity, unfolding and aortic arch and descending volumes were also correlated with higher amounts of calcium. Thoracic aorta calcium was predominantly found at the arch and descending aorta and was positively associated with the size of the descending aorta and the aortic arch, but not with the size of the ascending aorta. These findings suggest that aortic dilatation may have different mechanisms and may consequently require different preventive strategies according to the considered segments. Copyright © 2016 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

  5. 2,4,6-Trichlorophenol mediated increases in extracellular peroxidase activity in three species of Lemnaceae.

    PubMed

    Biswas, Dilip K; Scannell, Gillian; Akhmetov, Nurlan; Fitzpatrick, Dara; Jansen, Marcel A K

    2010-11-01

    Chlorinated phenols, or chlorophenols, are persistent priority pollutants that are widespread in the environment. Class III peroxidases are well-characterised plant enzymes that can catalyse the oxidative dechlorination of chlorophenols. Expression of these enzymes by plants is commonly associated with plant stress, therefore limiting scope for phytoremediation. In this study, we have quantitatively compared peroxidase activity and phytotoxicity as a function of 2,4,6-trichlorophenol (TCP) concentration in three species of Lemnaceae; Lemna minor, Lemna gibba and Landoltia punctata. Effects of TCP on the growth rates of the three species differed considerably with L. punctata being the most tolerant species. TCP also affected photosynthetic parameters, causing a decrease in open photosystem II reaction centres (qP) and, in L. punctata only, a decrease in non-photochemical quenching (qN). In parallel, TCP exposure resulted in increased peroxidase activity in all three species. Peroxidase activity in L. minor and L. gibba displayed an inverse relationship with biomass accumulation, i.e. the more growth reduction the more peroxidase activity. In contrast, induction of peroxidase activity in L. punctata was bi-phasic, with a TCP-induced activity peak at concentrations that had no major effect on growth, and further induction under phytotoxic concentrations. The mechanism by which L. punctata recognises and responds to low concentrations of an anthropogenic compound, in the absence of wide-ranging stress, remains enigmatic. However, we conclude that this "window" of peroxidase production in the absence of major growth inhibition offers potential for the development of sustainable, peroxidise-mediated phytoremediation systems. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Extremely Low Frequency Electromagnetic Fields Facilitate Vesicle Endocytosis by Increasing Presynaptic Calcium Channel Expression at a Central Synapse

    PubMed Central

    Sun, Zhi-cheng; Ge, Jian-long; Guo, Bin; Guo, Jun; Hao, Mei; Wu, Yi-chen; Lin, Yi-an; La, Ting; Yao, Pan-tong; Mei, Yan-ai; Feng, Yi; Xue, Lei

    2016-01-01

    Accumulating evidence suggests significant biological effects caused by extremely low frequency electromagnetic fields (ELF-EMF). Although exo-endocytosis plays crucial physical and biological roles in neuronal communication, studies on how ELF-EMF regulates this process are scarce. By directly measuring calcium currents and membrane capacitance at a large mammalian central nervous synapse, the calyx of Held, we report for the first time that ELF-EMF critically affects synaptic transmission and plasticity. Exposure to ELF-EMF for 8 to 10 days dramatically increases the calcium influx upon stimulation and facilitates all forms of vesicle endocytosis, including slow and rapid endocytosis, endocytosis overshoot and bulk endocytosis, but does not affect the RRP size and exocytosis. Exposure to ELF-EMF also potentiates PTP, a form of short-term plasticity, increasing its peak amplitude without impacting its time course. We further investigated the underlying mechanisms and found that calcium channel expression, including the P/Q, N, and R subtypes, at the presynaptic nerve terminal was enhanced, accounting for the increased calcium influx upon stimulation. Thus, we conclude that exposure to ELF-EMF facilitates vesicle endocytosis and synaptic plasticity in a calcium-dependent manner by increasing calcium channel expression at the nerve terminal. PMID:26887777

  7. Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding

    PubMed Central

    1993-01-01

    A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115- residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments. PMID:8245130

  8. Stable cavitation induces increased cytoplasmic calcium in L929 fibroblasts exposed to 1-MHz pulsed ultrasound.

    PubMed

    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.

  9. Lysergic acid diethylamide and [-]-2,5-dimethoxy-4-methylamphetamine increase extracellular glutamate in rat prefrontal cortex.

    PubMed

    Muschamp, John W; Regina, Meredith J; Hull, Elaine M; Winter, Jerrold C; Rabin, Richard A

    2004-10-08

    The ability of hallucinogens to increase extracellular glutamate in the prefrontal cortex (PFC) was assessed by in vivo microdialysis. The hallucinogen lysergic acid diethylamide (LSD; 0.1 mg/kg, i.p.) caused a time-dependent increase in PFC glutamate that was blocked by the 5-HT(2A) antagonist M100907 (0.05 mg/kg, i.p.). Similarly, the 5-HT(2A/C) agonist [-]-2,5-dimethoxy-4-methylamphetamine (DOM; 0.6 mg/kg, i.p.), which is a phenethylamine hallucinogen, increased glutamate to 206% above saline-treated controls. When LSD (10 microM) was directly applied to the PFC by reverse dialysis, a rapid increase in PFC glutamate levels was observed. Glutamate levels in the PFC remained elevated after the drug infusion was discontinued. These data provide direct evidence in vivo for the hypothesis that an enhanced release of glutamate is a common mechanism in the action of hallucinogens.

  10. Role of serotonin and/or norepinephrine in the MDMA-induced increase in extracellular glucose and glycogenolysis in the rat brain

    PubMed Central

    Pachmerhiwala, Rashida; Bhide, Nirmal; Straiko, Megan; Gudelsky, Gary A.

    2010-01-01

    The acute administration of MDMA has been shown to promote glycogenolysis and increase the extracellular concentration of glucose in the striatum. In the present study the role of serotonergic and/or noradrenergic mechanisms in the MDMA-induced increase in extracellular glucose and glycogenolysis was assessed. The relationship of these responses to the hyperthermia produced by MDMA also was examined. The administration of MDMA (10 mg/kg, i.p.) resulted in a significant and sustained increase of 65-100% in the extracellular concentration of glucose in the striatum, as well as in the prefrontal cortex and hippocampus, and a 35% decrease in brain glycogen content. Peripheral blood glucose was modestly increased by 32% after MDMA treatment. Treatment of rats with fluoxetine (10 mg/kg, i.p.) significantly attenuated the MDMA-induced increase in extracellular glucose in the striatum but had no effect on MDMA-induced glycogenolysis or hyperthermia. Treatment with prazosin (1 mg/kg, i.p.) did not alter the glucose or glycogen responses to MDMA but completely suppressed MDMA-induced hyperthermia. Finally, propranolol (3 mg/kg, i.p.) significantly attenuated the MDMA-induced increase in extracellular glucose and glycogenolysis but did not alter MDMA-induced hyperthermia. The present results suggest that MDMA increases extracellular glucose in multiple brain regions, and that this response involves both serotonergic and noradrenergic mechanisms. Furthermore, β-adrenergic and α-adrenergic receptors appear to contribute to MDMA-induced glycogenolysis and hyperthermia, respectively. Finally, hyperthermia, glycogenolysis and elevated extracellular glucose appear to be independent, unrelated responses to acute MDMA administration. PMID:20633550

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

    SciTech Connect

    Hashimoto, Ryota; Katoh, Youichi; Miyamoto, Yuki; Itoh, Seigo; Daida, Hiroyuki; Nakazato, Yuji; Okada, Takao

    2015-02-20

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

  12. Neutrophil extracellular trap formation is increased in psoriasis and induces human β-defensin-2 production in epidermal keratinocytes

    PubMed Central

    Hu, Stephen Chu-Sung; Yu, Hsin-Su; Yen, Feng-Lin; Lin, Chi-Ling; Chen, Gwo-Shing; Lan, Cheng-Che E.

    2016-01-01

    Neutrophil extracellular traps (NETs) have been implicated in the development of certain immune-mediated diseases, but their role in psoriasis has not been clearly defined. Human β-defensin-2 (HBD-2) is an important antimicrobial peptide overexpressed in psoriasis epidermis. We evaluated whether the amount of NETs is increased in psoriasis and determined the effect of NETs on HBD-2 production in epidermal keratinocytes. Using fluorescent microscopy, we found that patients with psoriasis (n = 48) had higher amount of NETotic cells in their peripheral blood compared to healthy controls (n = 48) and patients with eczema (n = 35). Psoriasis sera showed increased ability to induce NET formation in control neutrophils but normal NET degradation ability. The amount of NETs in the peripheral blood correlated with psoriasis disease severity. NETosis was also observed in the majority (18 of 20) of psoriasis skin specimens. Furthermore, NETs induced HBD-2 mRNA and protein production in keratinocytes, and immunohistochemical analysis confirmed strong expression of HBD-2 in psoriasis lesional skin. In summary, NET formation is increased in peripheral blood and lesional skin of psoriasis patients and correlates with disease severity. Additionally, NET-induced HBD-2 production may provide a novel mechanism for the decreased susceptibility of psoriasis plaques to microbial infections. PMID:27493143

  13. Functional expression of the extracellular calcium sensing receptor (CaSR) in equine umbilical cord matrix size-sieved stem cells.

    PubMed

    Martino, Nicola Antonio; Lange-Consiglio, Anna; Cremonesi, Fausto; Valentini, Luisa; Caira, Michele; Guaricci, Antonio Ciro; Ambruosi, Barbara; Sciorsci, Raffaele Luigi; Lacalandra, Giovanni Michele; Reshkin, Stephan Joel; Dell'Aquila, Maria Elena

    2011-03-17

    The present study investigates the effects of high external calcium concentration ([Ca(2+)](o)) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level. A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca(2+)](o) (0.37 mM); 2) high [Ca(2+)](o) (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca(2+)](o) and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca(2+)](o). Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca(2+)](o) was not effective in this cell line. In small cells, both higher [Ca(2+)](o) and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca(2+)](o) and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level. In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.

  14. Apelin Increases Cardiac Contractility via Protein Kinase Cε- and Extracellular Signal-Regulated Kinase-Dependent Mechanisms

    PubMed Central

    Perjés, Ábel; Skoumal, Réka; Tenhunen, Olli; Kónyi, Attila; Simon, Mihály; Horváth, Iván G.; Kerkelä, Risto; Ruskoaho, Heikki; Szokodi, István

    2014-01-01

    Background Apelin, the endogenous ligand for the G protein-coupled apelin receptor, is an important regulator of the cardiovascular homoeostasis. We previously demonstrated that apelin is one of the most potent endogenous stimulators of cardiac contractility; however, its underlying signaling mechanisms remain largely elusive. In this study we characterized the contribution of protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2) and myosin light chain kinase (MLCK) to the positive inotropic effect of apelin. Methods and Results In isolated perfused rat hearts, apelin increased contractility in association with activation of prosurvival kinases PKC and ERK1/2. Apelin induced a transient increase in the translocation of PKCε, but not PKCα, from the cytosol to the particulate fraction, and a sustained increase in the phosphorylation of ERK1/2 in the left ventricle. Suppression of ERK1/2 activation diminished the apelin-induced increase in contractility. Although pharmacological inhibition of PKC attenuated the inotropic response to apelin, it had no effect on ERK1/2 phosphorylation. Moreover, the apelin-induced positive inotropic effect was significantly decreased by inhibition of MLCK, a kinase that increases myofilament Ca2+ sensitivity. Conclusions Apelin increases cardiac contractility through parallel and independent activation of PKCε and ERK1/2 signaling in the adult rat heart. Additionally MLCK activation represents a downstream mechanism in apelin signaling. Our data suggest that, in addition to their role in cytoprotection, modest activation of PKCε and ERK1/2 signaling improve contractile function, therefore these pathways represent attractive possible targets in the treatment of heart failure. PMID:24695532

  15. Climate Change Increasing Calcium and Magnesium Leaching from Granitic Alpine Catchments.

    PubMed

    Kopáček, Jiří; Kaňa, Jiří; Bičárová, Svetlana; Fernandez, Ivan J; Hejzlar, Josef; Kahounová, Marie; Norton, Stephen A; Stuchlík, Evžen

    2017-01-03

    Climate change can reverse trends of decreasing calcium and magnesium [Ca + Mg] leaching to surface waters in granitic alpine regions recovering from acidification. Despite decreasing concentrations of strong acid anions (-1.4 μeq L(-1) yr(-1)) during 2004-2016 in nonacidic alpine lakes in the Tatra Mountains (Central Europe), the average [Ca + Mg] concentrations increased (2.5 μeq L(-1) yr(-1)), together with elevated terrestrial export of bicarbonate (HCO3(-); 3.6 μeq L(-1) yr(-1)). The percent increase in [Ca + Mg] concentrations in nonacidic lakes (0.3-3.2% yr(-1)) was significantly and positively correlated with scree proportion in the catchment area and negatively correlated with the extent of soil cover. Leaching experiments with freshly crushed granodiorite, the dominant bedrock, showed that accessory calcite and (to a lesser extent) apatite were important sources of Ca. We hypothesize that elevated terrestrial export of [Ca + Mg] and HCO3(-) resulted from increased weathering caused by accelerated physical erosion of rocks due to elevated climate-related mechanical forces (an increasing frequency of days with high precipitation amounts and air temperatures fluctuating around 0 °C) during the last 2-3 decades. These climatic effects on water chemistry are especially strong in catchments where fragmented rocks are more exposed to weathering, and their position is less stable than in soil.

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

    PubMed

    Bickler, P E; Fahlman, C S

    2004-01-01

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

  17. Marked increase of calcium uptake in the ATP-depleted red cells of patients with iron deficiency

    SciTech Connect

    Shimoda, M.; Yawata, Y.

    1985-05-01

    Calcium (Ca) uptake was markedly increased in ATP-depleted red cells of patients with iron deficiency anemia (IDA) compared to ATP- depleted normal red cells. The extent of increased Ca uptake was related to the severity of iron deficiency as judged by decreased mean cell volume. Moreover, the increased Ca uptake returned to normal levels after oral iron supplementation therapy. The net calcium content of fresh red cells from iron-deficient individuals was the same as in red cells from normal subjects. Sodium influx and ferric ion uptake appeared to be virtually unaffected in the iron deficient red cells.

  18. Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury

    PubMed Central

    Cho, Young-Eun; Im, Eun-Ju; Moon, Pyong-Gon; Mezey, Esteban; Song, Byoung-Joon; Baek, Moon-Chang

    2017-01-01

    Drug- and alcohol-induced liver injury are a leading cause of liver failure and transplantation. Emerging evidence suggests that extracellular vesicles (EVs) are a source of biomarkers because they contain unique proteins reflecting the identity and tissue-specific origin of the EV proteins. This study aimed to determine whether potentially hepatotoxic agents, such as acetaminophen (APAP) and binge alcohol, can increase the amounts of circulating EVs and evaluate liver-specific EV proteins as potential biomarkers for liver injury. The circulating EVs, isolated from plasma of APAP-exposed, ethanol-fed mice, or alcoholic hepatitis patients versus normal control counterparts, were characterized by proteomics and biochemical methods. Liver specific EV proteins were analyzed by immunoblots and ELISA. The amounts of total and liver-specific proteins in circulating EVs from APAP-treated mice significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs from APAP-exposed mice revealed that the amounts of liver-specific and/or hepatotoxic proteins were increased compared to those of controls. Additionally, the increased protein amounts in EVs following APAP exposure returned to basal levels when mice were treated with N-acetylcysteine or glutathione. Similar results of increased amounts and liver-specific proteins in circulating EVs were also observed in mice exposed to hepatotoxic doses of thioacetamide or d-galactosamine but not by non-hepatotoxic penicillin or myotoxic bupivacaine. Additionally, binge ethanol exposure significantly elevated liver-specific proteins in circulating EVs from mice and alcoholics with alcoholic hepatitis, compared to control counterparts. These results indicate that circulating EVs in drug- and alcohol-mediated hepatic injury contain liver-specific proteins that could serve as specific biomarkers for hepatotoxicity. PMID:28225807

  19. Mepivacaine-induced contraction involves increased calcium sensitization mediated via Rho kinase and protein kinase C in endothelium-denuded rat aorta.

    PubMed

    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. Supplementing a low-protein diet with dibasic amino acids increases urinary calcium excretion in young women.

    PubMed

    Bihuniak, Jessica D; Sullivan, Rebecca R; Simpson, Christine A; Caseria, Donna M; Huedo-Medina, Tania B; O'Brien, Kimberly O; Kerstetter, Jane E; Insogna, Karl L

    2014-03-01

    Increasing dietary protein within a physiologic range stimulates intestinal calcium absorption, but it is not known if specific amino acids or dietary protein as a whole are responsible for this effect. Therefore, we selectively supplemented a low-protein (0.7 g/kg) diet with either the calcium-sensing receptor-activating amino acids (CaSR-AAAs) L-tryptophan, L-phenylalanine, and L-histidine, or the dibasic amino acids (DAAs) L-arginine and L-lysine, to achieve intakes comparable to the content of a high-protein diet (2.1 g/kg) and measured intestinal calcium absorption. Fourteen young women took part in a placebo-controlled, double-blind, crossover feeding trial in which each participant ingested a 6-d low-protein diet supplemented with CaSR-AAAs, DAAs, or methylcellulose capsules (control) after an 11-d adjustment period. All participants ingested all 3 diets in random order. Intestinal calcium absorption was measured between days 5 and 6 using dual-stable calcium isotopes ((42)Ca, (43)Ca, and (44)Ca). There was no difference in calcium absorption between the diet supplemented with CaSR-AAAs (22.9 ± 2.0%) and the control diet (22.3 ± 1.4%) (P = 0.64). However, calcium absorption tended to be greater during the DAA supplementation period (25.2 ± 1.4%) compared with the control diet period (22.3 ± 1.4%) (P < 0.10). Larger and longer clinical trials are needed to clarify the possible benefit of arginine and lysine on calcium absorption.

  1. Supplementing a Low-Protein Diet with Dibasic Amino Acids Increases Urinary Calcium Excretion in Young Women12

    PubMed Central

    Bihuniak, Jessica D.; Sullivan, Rebecca R.; Simpson, Christine A.; Caseria, Donna M.; Huedo-Medina, Tania B.; O’Brien, Kimberly O.; Kerstetter, Jane E.; Insogna, Karl L.

    2014-01-01

    Increasing dietary protein within a physiologic range stimulates intestinal calcium absorption, but it is not known if specific amino acids or dietary protein as a whole are responsible for this effect. Therefore, we selectively supplemented a low-protein (0.7 g/kg) diet with either the calcium-sensing receptor-activating amino acids (CaSR-AAAs) L-tryptophan, L-phenylalanine, and L-histidine, or the dibasic amino acids (DAAs) L-arginine and L-lysine, to achieve intakes comparable to the content of a high-protein diet (2.1 g/kg) and measured intestinal calcium absorption. Fourteen young women took part in a placebo-controlled, double-blind, crossover feeding trial in which each participant ingested a 6-d low-protein diet supplemented with CaSR-AAAs, DAAs, or methylcellulose capsules (control) after an 11-d adjustment period. All participants ingested all 3 diets in random order. Intestinal calcium absorption was measured between days 5 and 6 using dual-stable calcium isotopes (42Ca, 43Ca, and 44Ca). There was no difference in calcium absorption between the diet supplemented with CaSR-AAAs (22.9 ± 2.0%) and the control diet (22.3 ± 1.4%) (P = 0.64). However, calcium absorption tended to be greater during the DAA supplementation period (25.2 ± 1.4%) compared with the control diet period (22.3 ± 1.4%) (P < 0.10). Larger and longer clinical trials are needed to clarify the possible benefit of arginine and lysine on calcium absorption. PMID:24431325

  2. Acetylcholine induces voltage-independent increase of cytosolic calcium in mouse myotubes.

    PubMed Central

    Giovannelli, A; Grassi, F; Mattei, E; Mileo, A M; Eusebi, F; Giovanelli, A

    1991-01-01

    Electrophysiological, biochemical, and Ca2+ imaging studies of cultured mouse myotubes were used to investigate whether the neurotransmitter acetylcholine causes an increase in intracellular Ca2+ concentration ([Ca2+]i) through activation of a second messenger system. Bath applications of acetylcholine to myotubes (i) elicited a significant membrane current even in a Na(+)-free Ca2+ medium, when the current was carried mainly by calcium ions; (ii) caused a rapid and transient cytosolic accumulation of inositol 1,4,5-trisphosphate; (iii) evoked a conspicuous alpha-bungarotoxin-sensitive long-lasting [Ca2+]i enhancement even in the presence of Cd2+; and (iv) transiently increased [Ca2+]i when cells were equilibrated in a Ca(2+)-free atropine-containing medium. We propose that, in addition to opening ion channels, the nicotinic action of acetylcholine on the muscle cell membrane increases [Ca2+]i through activation of the inositol 1,4,5-trisphosphate second messenger system and mobilization of Ca2+ from intracellular stores. Images PMID:1946425

  3. Cardiac L-type calcium current is increased in a model of hyperaldosteronism in the rat.

    PubMed

    Martin-Fernandez, Beatriz; Miana, María; De Las Heras, Natalia; Ruiz-Hurtado, Gema; Fernandez-Velasco, María; Bas, Manuel; Ballesteros, Sandra; Lahera, Vicente; Cachofeiro, Victoria; Delgado, Carmen

    2009-06-01

    Accumulating evidence supports the importance of aldosterone as an independent risk factor in the pathophysiology of cardiovascular disease. It has been postulated that aldosterone could contribute to ventricular arrhythmogeneity by modulation of cardiac ionic channels. The aim of this study was to analyse ex vivo the electrophysiological characteristics of the L-type cardiac calcium current (I(CaL)) in a model of hyperaldosteronism in the rat. Aldosterone was administered for 3 weeks, and cardiac collagen deposition and haemodynamic parameters were analysed. In addition, RT-PCR and patch-clamp techniques were applied to study cardiac L-type Ca(2+) channels in isolated cardiomyocytes. Administration of aldosterone induced maladaptive cardiac remodelling that was related to increased collagen deposition, diastolic dysfunction and cardiac hypertrophy. In addition, ventricular myocytes isolated from the aldosterone-treated group showed increased I(CaL) density and conductance and prolongation of the action potential duration. No changes in kinetics or in voltage dependence of activation and inactivation of I(CaL) were observed, but relative expression of Ca(V)1.2 mRNA levels was higher in cardiomyocytes isolated from the aldosterone-treated group. The present study demonstrates that aldosterone treatment induces myocardial fibrosis, cardiac hypertrophy, increase of I(CaL) density, upregulation of L-type Ca(2+) channels and prolongation of action potential duration. It could be proposed that aldosterone, through these mechanisms, might exert pro-arrhythmic effects in the pathological heart.

  4. Spontaneous calcium waves in Bergman glia increase with age and hypoxia and may reduce tissue oxygen.

    PubMed

    Mathiesen, Claus; Brazhe, Alexey; Thomsen, Kirsten; Lauritzen, Martin

    2013-02-01

    Glial calcium (Ca(2+)) waves constitute a means to spread signals between glial cells and to neighboring neurons and blood vessels. These waves occur spontaneously in Bergmann glia (BG) of the mouse cerebellar cortex in vivo. Here, we tested three hypotheses: (1) aging and reduced blood oxygen saturation alters wave activity; (2) glial Ca(2+) waves change cerebral oxygen metabolism; and (3) neuronal and glial wave activity is correlated. We used two-photon microscopy in the cerebellar cortexes of adult (8- to 15-week-old) and aging (48- to 80-week-old) ketamine-anesthetized mice after bolus loading with OGB-1/AM and SR101. We report that the occurrence of spontaneous waves is 20 times more frequent in the cerebellar cortex of aging as compared with adult mice, which correlated with a reduction in resting brain oxygen tension. In adult mice, spontaneous glial wave activity increased on reducing resting brain oxygen tension, and ATP-evoked glial waves reduced the tissue O(2) tension. Finally, although spontaneous Purkinje cell (PC) activity was not associated with increased glia wave activity, spontaneous glial waves did affect intracellular Ca(2+) activity in PCs. The increased wave activity during aging, as well as low resting brain oxygen tension, suggests a relationship between glial waves, brain energy homeostasis, and pathology.

  5. Spontaneous calcium waves in Bergman glia increase with age and hypoxia and may reduce tissue oxygen

    PubMed Central

    Mathiesen, Claus; Brazhe, Alexey; Thomsen, Kirsten; Lauritzen, Martin

    2013-01-01

    Glial calcium (Ca2+) waves constitute a means to spread signals between glial cells and to neighboring neurons and blood vessels. These waves occur spontaneously in Bergmann glia (BG) of the mouse cerebellar cortex in vivo. Here, we tested three hypotheses: (1) aging and reduced blood oxygen saturation alters wave activity; (2) glial Ca2+ waves change cerebral oxygen metabolism; and (3) neuronal and glial wave activity is correlated. We used two-photon microscopy in the cerebellar cortexes of adult (8- to 15-week-old) and aging (48- to 80-week-old) ketamine-anesthetized mice after bolus loading with OGB-1/AM and SR101. We report that the occurrence of spontaneous waves is 20 times more frequent in the cerebellar cortex of aging as compared with adult mice, which correlated with a reduction in resting brain oxygen tension. In adult mice, spontaneous glial wave activity increased on reducing resting brain oxygen tension, and ATP-evoked glial waves reduced the tissue O2 tension. Finally, although spontaneous Purkinje cell (PC) activity was not associated with increased glia wave activity, spontaneous glial waves did affect intracellular Ca2+ activity in PCs. The increased wave activity during aging, as well as low resting brain oxygen tension, suggests a relationship between glial waves, brain energy homeostasis, and pathology. PMID:23211964

  6. Interaction between thapsigargin and ATP4- in the regulation of the intracellular calcium in rat submandibular glands.

    PubMed

    Métioui, M; Grosfils, K; Dehaye, J P

    1994-11-01

    Rat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura-2. In the absence of extracellular magnesium and calcium ([Ca2+]o), ATP had no effect; the response to ATP peaked at 1-2.5 mM [Ca2+]o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura-2; 10 microM Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca2+]in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca(2+)-free/Ca2+ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca2+]in and the calcium uptake but potentiated the response to ATP. These results suggest that, after binding to purinergic receptors, extracellular ATP4- increases [Ca2+]in. ATP4- does not mobilize thapsigargin-sensitive intracellular calcium pools (among which is the IP3-sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel.

  7. Effects of altered crystalline structure and increased initial compressive strength of calcium sulfate bone graft substitute pellets on new bone formation.

    PubMed

    Urban, Robert M; Turner, Thomas M; Hall, Deborah J; Infanger, Susan I; Cheema, Naveed; Lim, Tae-Hong; Moseley, Jon; Carroll, Michael; Roark, Michael

    2004-01-01

    A new, modified calcium sulfate has been developed with a different crystalline structure and a compressive strength similar to many calcium phosphate materials, but with a resorption profile only slightly slower than conventional surgical-grade calcium sulfate. A canine bilateral defect model was used to compare restoration of defects treated with the modified calcium sulfate compared to treatment using conventional calcium sulfate pellets after 6, 13, and 26 weeks. The modified calcium sulfate pellets were as effective as conventional calcium sulfate pellets with regard to the area fraction and compressive strength of newly formed bone in the treated bone defects. Mechanical testing demonstrated that the initial compressive strength of the modified material was increased nearly three-fold compared to that of conventional surgical-grade calcium sulfate. This increase potentially allows for its use in a broader range of clinical applications, such as vertebral and subchondral defects.

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

    NASA Technical Reports Server (NTRS)

    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.

  9. Ferric iron and extracellular electron shuttling increase xylose utilization and butanol production during fermentation with multiple solventogenic bacteria.

    PubMed

    Popovic, Jovan; Ye, Xiaofeng; Haluska, Anne; Finneran, Kevin T

    2017-09-29

    Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild-type cells are goals for biofuel production. Xylose consumption, butanol production, and hydrogen production increased in both Clostridium beijerinckii and a novel solventogenic bacterium (strain DC-1) when anthraquinone-2,6,-disulfonate (AQDS) or riboflavin were used as redox mediators to transfer electrons to poorly crystalline Fe(OH)3 as an extracellular electron sink. Strain DC-1 was most closely related to Rhizobiales bacterium Mfc52 based on 95% 16S rRNA gene sequence similarity, which demonstrates that this response is not limited to a single genus of xylose-fermenting bacteria. Xylose utilization and butanol production were negligible in control incubations containing cells plus 3% (w/v) xylose alone during a 10-day batch fermentation, for both strains tested (n-butanol titers of 0.05 g L(-1)). Micromolar concentrations of AQDS and riboflavin were added as electron shuttling compounds with poorly crystalline Fe(OH)3 as an insoluble electron acceptor, and respective n-butanol titers increased to 6.35 and 7.46 g L(-1). Increases in xylose consumption for the iron treatments were relatively high, from less than 0.49 g L(-1) (xylose alone, no iron or electron shuttling molecules) to 25.98 and 29.15 g L(-1) for the AQDS and riboflavin treatments, respectively. Hydrogen production was also 3.68 times greater for the AQDS treatment and 5.27 greater for the riboflavin treatment relative to controls. Strain DC-1 data were similar, again indicating that the effects are not specific to the genus Clostridium.

  10. Mitochondrial reactive oxygen species generation and calcium increase induced by visible light in astrocytes.

    PubMed

    Jou, Mei-Jie; Jou, Shuo-Bin; Guo, Mei-Jin; Wu, Hong-Yueh; Peng, Tsung-I

    2004-04-01

    Mitochondria contain photosensitive chromophores that can be activated or inhibited by light in the visible range. Rather than utilizing light energy, however, mitochondrial electron transport oxidation-reduction reaction and energy coupling could be stimulated or damaged by visible light. Our previous work demonstrated that reactive oxygen species (ROS) were generated in cultured astrocytes after visible laser irradiation. With confocal fluorescence microscopy, we found that ROS were generated mostly from mitochondria. This mitochondrial ROS (mROS) formation plays a critical role in photoirradiation-induced phototoxicity and apoptosis. In this study, we measured changes of mitochondrial calcium level ([Ca(2+)](m)) in cultured astrocytes (RBA-1 cell line) irradiated with blue light and examined the association between mROS formation and [Ca(2+)](m) level changes. Changes of intracellular ROS and [Ca(2+)](m) were visualized using fluorescent probes 2',7'-dichlorodihydrofluorescein (DCF), and rhod-2. After exposure to visible light irradiation, RBA-1 astrocytes showed a rapid increase in ROS accumulation particularly in the mitochondrial area. Increase in [Ca(2+)](m) was also induced by photoirradiation. The levels of increase in DCF fluorescence intensity varied among different astrocytes. Some of the cells generated much higher levels of ROS than others. For those cells that had high ROS levels, mitochondrial Ca(2+) levels were also high. In cells that had mild ROS levels, mitochondrial Ca(2+) levels were only slightly increased. The rate of increase in DCF fluorescence seemed to be close to the rate of rhod-2 fluorescence increase. There is a positive and close correlation between mitochondrial ROS levels and mitochondrial Ca(2+) levels in astrocytes irradiated by visible light.

  11. M1 muscarinic receptors increase calcium current and phosphoinositide turnover in guinea-pig ventricular cardiocytes.

    PubMed Central

    Gallo, M P; Alloatti, G; Eva, C; Oberto, A; Levi, R C

    1993-01-01

    1. Physiological and molecular evidence for the presence and functional role of M1 muscarinic cholinergic receptors (mAChRs) in adult guinea-pig ventricular cells is presented. 2. Whole-cell clamp measurements of the L-type calcium current (ICa) in isolated myocytes were performed. Caesium was used to suppress potassium currents. ICa was increased by the muscarinic agonist carbachol in cells pretreated with pertussis toxin which blocked the M2 mAChR-triggered cascade of intracellular signalling, while it was not changed in untreated cells. 3. If the M2-mediated regulation of ICa was blocked by directly saturating the cell with cyclic adenosine monophosphate (cAMP) through the patch pipette, application of carbachol induced a further small increase of the current above the level reached after cAMP perfusion. This increase was more pronounced in cells pretreated with pertussis toxin. 4. The carbachol-induced increase of ICa was blocked by the selective M1 mAChR antagonist pirenzepine. 5. The application of high concentrations of carbachol increased the accumulation of [3H]inositol monophosphate up to 240% above control levels. This increase was reduced by application of pirenzepine. 6. The expression of M1 receptor mRNA in ventricular cardiocytes was shown by reverse transcriptase-polymerase chain reaction. 7. These results suggest that M1 mAChR regulation of ICa can be a component of the paradoxical positive inotropism induced by high concentrations of muscarinic agonists. Images Fig. 11 PMID:8120813

  12. Extracellular magnesium regulates nuclear and perinuclear free ionized calcium in cerebral vascular smooth muscle cells: possible relation to alcohol and central nervous system injury.

    PubMed

    Altura, B M; Zhang, A; Cheng, T P; Altura, B T

    2001-02-01

    Quantitative digital imaging microscopy, confocal laser scanning microscopy (CLSM), and multiple molecular fluorescent probes were utilized to test the hypothesis that cerebral vascular muscle cell nuclear ([Ca(2+)](n)), perinuclear ([Ca(2+)](pn)), and cytoplasmic free calcium ([Ca(2+)](i)) levels are regulated by the concentration of extracellular free magnesium ions ([Mg(2+)](o)). Primary cultured canine cerebral vascular smooth muscle cells were loaded with either fura-2/AM, indo-1/AM, or fluo-3/AM, and the subcellular Ca(2+) responses to stepwise reduction in [Mg(2+)](o) (i.e., from 1.36 to 0.17 mM) were analyzed over time. With normal 1.36 mM [Mg(2+)](o)-containing incubation media, basal mean [Ca(2+)](i) was 89.6+/-15 nM. Lowering [Mg(2+)](o) to 1.07, 0.88, 0.48, and 0.17 mM resulted in rapid (<4 min) increments in [Ca(2+)](i) going to 213+/-43, 368+/-67, 471+/-77, and 642+/-98 nM, respectively; the longer the exposure time (up to 30 min) to lowered [Mg(2+)](o), the higher the [Ca(2+)](i). Restoration of [Mg(2+)](o) to normal caused decreases in [Ca(2+)](i) to 215.9+/-42.3 nM, but only complete removal of [Ca(2+)](o) returned [Ca(2+)](i) to basal levels. Results show that basal [Ca(2+)](pn) (282+/-92 nM) exceeds basal cytoplasmic Ca(2+) (61+/-27.8 nM) and [Ca(2+)](n) (20+/-7.6 nM). However, reduction of normal [Mg(2+)](o) to 0.48 mM resulted in dramatic, rapid rises in all subcellular compartments, where [Ca(2+)](pn) (1503+/-102 nM)>cytoplasmic Ca(2+) (688+/-49 nM) approximately equal to [Ca(2+)](n) (674+/-12 nM). Nuclear Ca(2+) rose dramatically (e.g., 35-40 times basal levels). Both verapamil (1 microM) and Ni(2+) (5 mM) prevented, completely, the rises in Ca(2+) in all compartments, suggesting that Mg(2+)-dependent Ca(2+) accumulation may be dependent on nuclear, endoplasmic reticulum-Golgi, and cytoplasmic L-type voltage membrane-regulated Ca(2+) channels. The normally low [Ca(2+)](n) suggests that Ca(2+) does not transport passively across the nuclear

  13. Royal Jelly Constituents Increase the Expression of Extracellular Superoxide Dismutase through Histone Acetylation in Monocytic THP-1 Cells.

    PubMed

    Makino, Junya; Ogasawara, Rie; Kamiya, Tetsuro; Hara, Hirokazu; Mitsugi, Yukari; Yamaguchi, Eiji; Itoh, Akichika; Adachi, Tetsuo

    2016-04-22

    Extracellular superoxide dismutase (EC-SOD) is one of the main SOD isozymes and plays an important role in the prevention of cardiovascular diseases by accelerating the dismutation reaction of superoxide. Royal jelly includes 10-hydroxy-2-decenoic acid (10H2DA, 2), which regulates the expression of various types of genes in epigenetics through the effects of histone deacetylase (HDAC) antagonism. The expression of EC-SOD was previously reported to be regulated epigenetically through histone acetylation in THP-1 cells. Therefore, we herein evaluated the effects of the royal jelly constituents 10-hydroxydecanoic acid (10HDA, 1), sebacic acid (SA, 3), and 4-hydroperoxy-2-decenoic acid ethyl ester (4-HPO-DAEE, 4), which is a derivative of 2, on the expression of EC-SOD in THP-1 cells. The treatment with 1 mM 1, 2, or 3 or 100 μM 4 increased EC-SOD expression and histone H3 and H4 acetylation levels. Moreover, the enrichment of acetylated histone H4 was observed in the proximal promoter region of EC-SOD and was caused by the partial promotion of ERK phosphorylation (only 4) and inhibition of HDAC activities, but not by the expression of HDACs. Overall, 4 exerted stronger effects than 1, 2, or 3 and has potential as a candidate or lead compound against atherosclerosis.

  14. Increased Expression of Extracellular Matrix Metalloproteinase Inducer (CD147) in Multiple Myeloma: Role in Regulation of Myeloma Cell Proliferation

    PubMed Central

    Arendt, Bonnie K.; Walters, Denise K.; Wu, Xiaosheng; Tschumper, Renee C.; Huddleston, Paul M.; Henderson, Kimberly J.; Dispenzieri, Angela; Jelinek, Diane F.

    2014-01-01

    Multiple myeloma (MM) is preceded by the asymptomatic premalignant state, monoclonal gammopathy of undetermined significance (MGUS). Although MGUS patients may remain stable for years, they are at increased risk of progressing to MM. A better understanding of the relevant molecular changes underlying the transition from an asymptomatic to symptomatic disease state is urgently needed. Our studies show for the first time that the CD147 molecule (extracellular matrix metalloproteinase inducer) may be playing an important biological role in MM. We first demonstrate that CD147 is over-expressed in MM plasma cells (PCs) vs. normal and premalignant PCs. Next, functional studies revealed that the natural CD147 ligand, cyclophilin B, stimulates MM cell growth. Moreover, when MM patient PCs displaying bimodal CD147 expression were separated into CD147bright and CD147dim populations and analyzed for proliferation potential, we discovered that CD147bright PCs displayed significantly higher levels of cell proliferation than did CD147dim PCs. Lastly, CD147 silencing significantly attenuated MM cell proliferation. Taken together, these data suggest that the CD147 molecule plays a key role in MM cell proliferation and may serve as an attractive target for reducing the proliferative compartment of this disease. PMID:22460757

  15. Increased Interleukin-32 Levels in Obesity Promote Adipose Tissue Inflammation and Extracellular Matrix Remodeling: Effect of Weight Loss.

    PubMed

    Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Valentí, Víctor; Moncada, Rafael; Landecho, Manuel F; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

    2016-12-01

    Interleukin (IL)-32 is a recently described cytokine involved in the regulation of inflammation. We aimed to explore whether IL-32 could function as an inflammatory and angiogenic factor in human obesity and obesity-associated type 2 diabetes. Samples obtained from 90 subjects were used in the study. Obese patients exhibited higher expression levels of IL-32 in visceral adipose tissue (AT) as well as in subcutaneous AT and peripheral blood mononuclear cells. IL32 was mainly expressed by stromovascular fraction cells, and its expression was significantly enhanced by inflammatory stimuli and hypoxia, whereas no changes were found after the incubation with anti-inflammatory cytokines. The addition of exogenous IL-32 induced the expression of inflammation and extracellular matrix-related genes in human adipocyte cultures, and IL32-silenced adipocytes showed a downregulation of inflammatory genes. Furthermore, adipocyte-conditioned media obtained from obese patients increased IL32 gene expression in human monocyte cultures, whereas the adipocyte-conditioned media from lean volunteers had no effect on IL32 mRNA levels. These findings provide evidence, for the first time, about the inflammatory and remodeling properties of IL-32 in AT, implicating this cytokine in obesity-associated comorbidities. © 2016 by the American Diabetes Association.

  16. Fatty Liver, Insulin Resistance, and Obesity: Relationships With Increase in Coronary Artery Calcium Over Time.

    PubMed

    Sung, Ki-Chul; Ryu, Seungho; Lee, Jong-Young; Lee, Sung Ho; Cheong, Eun Sun; Wild, Sarah H; Byrne, Christopher D

    2016-06-01

    Nonalcoholic fatty liver disease, insulin resistance (IR), and obesity frequently coexist with type 2 diabetes mellitus (DM), but it is uncertain whether these risk factors for vascular disease contribute to a change in atherosclerosis over time, independently of DM status. We hypothesized that the combination of fatty liver, IR, and obesity would be associated with an increase in coronary artery calcium (CAC) score over time, independently of DM status, other cardiovascular risk factors, and medications. Data were analyzed from a South Korean occupational cohort of 2175 people. The outcome was increase in cardiac computed tomography CAC score between baseline and follow-up. Insulin resistance was defined by homeostatic model assessment of insulin resistance (HOMA-IR) ≥75th percentile and fatty liver by ultrasound. In 592 (27.2%) participants, CAC score increased from baseline (mean ± SD; mean age at baseline, 44.8 ± 5.5 years); and in 1583 subjects, CAC did not change or improved during follow-up (mean age, 41.6 ± 5.6 years). Diabetes mellitus, HOMA-IR, fatty liver, and obesity prevalence were all higher (all P < 0.001) in participants whose CAC score increased from baseline. Adjusting for DM and potential confounders, the combination of IR, obesity, and fatty liver was independently associated with increase in CAC score over time (hazard ratio: 2.46, 95% confidence interval: 1.50-4.03). The combination of fatty liver, IR, and obesity is associated with progression of atherosclerosis over time independently of DM, cardiovascular risk factors, and all medications for cardiovascular disease and DM. © 2016 Wiley Periodicals, Inc.

  17. Secular decline of seawater calcium increases seawater buffering and pH

    NASA Astrophysics Data System (ADS)

    Hain, M.; Sigman, D. M.; Higgins, J. A.; Haug, G. H.

    2015-12-01

    Reconstructed changes in seawater calcium and magnesium concentration ([Ca2+], [Mg2+]) predictably affect the ocean's acid/base and carbon chemistry. Yet inaccurate formulations of chemical equilibrium "constants" are currently in use to account for these changes. Here we develop an efficient implementation of the MIAMI Ionic Interaction Model (Millero and Pierrot, 1998) to predict all chemical equilibrium constants required for carbon chemistry calculations under variable [Ca2+] and [Mg2+] (Hain et al., 2015). We investigate the impact of [Ca2+] and [Mg2+] on the relationships among the ocean's pH, CO2, dissolved inorganic carbon (DIC), saturation state of CaCO3 (Ω), and buffer capacity. Increasing [Ca2+] and/or [Mg2+] enhances "ion pairing," which increases seawater buffering by increasing the concentration ratio of total to "free" (uncomplexed) carbonate ion. An increase in [Ca2+], however, also causes a decline in carbonate ion to maintain a given Ω, thereby overwhelming the ion pairing effect and decreasing seawater buffering. Given the reconstructions of Eocene [Ca2+] and [Mg2+] ([Ca2+]~20mM; [Mg2+]~30 mM), Eocene seawater would have required essentially the same DIC as today to simultaneously explain a similar-to-modern Ω and the estimated Eocene atmospheric CO2 of ~1000 ppm. During the Cretaceous, at ~4 times modern [Ca2+], ocean buffering would have been at a minimum. Overall, during times of high seawater [Ca2+], CaCO3 saturation, pH, and atmospheric CO2 were more susceptible to perturbations of the global carbon cycle. For example, given both Eocene and Cretaceous seawater [Ca2+] and [Mg2+], a doubling of atmospheric CO2 would require less carbon addition to the ocean/atmosphere system than under modern seawater composition. Moreover, increase in seawater buffering since the Cretaceous may have been a driver of evolution by raising energetic demands of biologically controlled calcification and CO2 concentration mechanisms that aid photosynthesis.

  18. Yeast respond to hypotonic shock with a calcium pulse

    NASA Technical Reports Server (NTRS)

    Batiza, A. F.; Schulz, T.; Masson, P. H.

    1996-01-01

    We have used the transgenic AEQUORIN calcium reporter system to monitor the cytosolic calcium ([Ca2+]cyt) response of Saccharomyces cerevisiae to hypotonic shock. Such a shock generates an almost immediate and transient rise in [Ca2+]cyt which is eliminated by gadolinium, a blocker of stretch-activated channels. In addition, this transient rise in [Ca2+]cyt is initially insensitive to 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular calcium chelator. However, BAPTA abruptly attenuates the maintenance of that transient rise. These data show that hypotonic shock generates a stretch-activated channel-dependent calcium pulse in yeast. They also suggest that the immediate calcium influx is primarily generated from intracellular stores, and that a sustained increase in [Ca2+]cyt depends upon extracellular calcium.

  19. Yeast respond to hypotonic shock with a calcium pulse

    NASA Technical Reports Server (NTRS)

    Batiza, A. F.; Schulz, T.; Masson, P. H.

    1996-01-01

    We have used the transgenic AEQUORIN calcium reporter system to monitor the cytosolic calcium ([Ca2+]cyt) response of Saccharomyces cerevisiae to hypotonic shock. Such a shock generates an almost immediate and transient rise in [Ca2+]cyt which is eliminated by gadolinium, a blocker of stretch-activated channels. In addition, this transient rise in [Ca2+]cyt is initially insensitive to 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular calcium chelator. However, BAPTA abruptly attenuates the maintenance of that transient rise. These data show that hypotonic shock generates a stretch-activated channel-dependent calcium pulse in yeast. They also suggest that the immediate calcium influx is primarily generated from intracellular stores, and that a sustained increase in [Ca2+]cyt depends upon extracellular calcium.

  20. Soluble corn fiber increases bone calcium retention in postmenopausal women in a dose-dependent manner: a randomized crossover trial.

    PubMed

    Jakeman, Steven A; Henry, Courtney N; Martin, Berdine R; McCabe, George P; McCabe, Linda D; Jackson, George S; Peacock, Munro; Weaver, Connie M

    2016-09-01

    Dietary soluble corn fiber (SCF) significantly improves calcium absorption in adolescents and the bone strength and architecture in rodent models. In this study, we aimed to determine the skeletal benefits of SCF in postmenopausal women. We used our novel technology of determining bone calcium retention by following the urinary appearance of (41)Ca, a rare long-lived radioisotope, from prelabeled bone to rapidly and sensitively evaluate the effectiveness of SCF in reducing bone loss. A randomized-order, crossover, double-blinded trial was performed in 14 healthy postmenopausal women to compare doses of 0, 10, and 20 g fiber from SCF/d for 50 d. A dose-response effect was shown with 10 and 20 g fiber from SCF/d, whereby bone calcium retention was improved by 4.8% (P < 0.05) and 7% (P < 0.04), respectively. The bone turnover biomarkers N-terminal telopeptide and osteocalcin were not changed by the interventions; however, a significant increase in bone-specific alkaline phosphatase, which is a bone-formation marker, was detected between 0 and 20 g fiber from SCF/d (8%; P = 0.035). Daily SCF consumption significantly increased bone calcium retention in postmenopausal women, which improved the bone calcium balance by an estimated 50 mg/d. This study was registered at clinicaltrials.gov as NCT02416947. © 2016 American Society for Nutrition.

  1. Sulfur mustard-induced increase in intracellular calcium: A mechanism of mustard toxicity

    SciTech Connect

    Ray, R.; Majerus, B.J.; Munavalli, G.S.; Petrali, J.P.

    1993-05-13

    The effect of sulfur mustard SM, bis-(2-chloroethyl) sulfide on intracellular free Ca2+ concentration (Ca2+)i was studied in vitro using the clonal mouse neuroblastoma-rat glioma hybrid NG108-15 and primary normal human epidermal keratinocyte (NHEK) cell culture models. SM depletes cellular glutathione (GSH) and thus may inhibit GSH-dependent Ca2+-ATPase (Ca2+ pump), leading to a high (Ca2+) and consequent cellular toxicity. Following 0.3 mM SM exposure, GSH levels decreased 20-34% between 1-6 hr in NG108-15 cells. SM increased (Ca2+)i, measured using the Ca2+-specific fluorescent probe Fluo-3 AM, in both NG108-15 cells (1030% between 2-6 hr) and NHEK (23-30% between 0.5-3 hr) . Depletion of cellular GSH by buthionine sulfoximine (1 mM), a specific GSH biosynthesis inhibitor, also increased Ca2+, (88% at 1 hr) in NHEK, suggesting that GSH depletion may lead to increased (Ca2+)i. Calcium, localized cytochemically with antimony, accumulated in increased amounts around mitochondria and endoplasmic reticula, in the cytosol, and in particular in the euchromatin regions of the nucleus beginning at 6 hr after 0.3 mM SM exposure of NG108-15 cells. Cell membrane integrity examined with the fluorescent membrane probe calcein AM was unaffected through 6 hr following 1 mM SM exposure; and cell viability (NG108-15 cells) measured by trypan blue exclusion was >80% of control through 9 hr following 0.3 mM SM exposure.

  2. Involvement of two-pore channels in hydrogen peroxide-induced increase in the level of calcium ions in the cytoplasm of human umbilical vein endothelial cells.

    PubMed

    Avdonin, P V; Nadeev, A D; Tsitrin, E B; Tsitrina, A A; Avdonin, P P; Mironova, G Yu; Zharkikh, I L; Goncharov, N V

    2017-05-01

    Hydrogen peroxide at concentrations below cytotoxic ones causes an increase in the cytoplasmic calcium concentration in human umbilical vein endothelial cells as a result of calcium release from intracellular stores. Two-pore calcium channel blocker trans-NED19 partially suppresses the increase in the level of calcium ions in the cells in response to the addition of hydrogen peroxide. The staining of endothelial cells with the fluorescent stereoisomer cis-NED19 and LysoTracker confirmed the localization of two-pore calcium channels in lysosomes and endolysosomal vesicles.

  3. Hunting increases phosphorylation of calcium/calmodulin-dependent protein kinase type II in adult barn owls.

    PubMed

    Nichols, Grant S; DeBello, William M

    2015-01-01

    Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB) provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII) in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX), the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is "off" in adults.

  4. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

    PubMed Central

    Nichols, Grant S.; DeBello, William M.

    2015-01-01

    Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB) provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII) in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX), the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is “off” in adults. PMID:25789177

  5. Does a higher ratio of serum calcium to magnesium increase the risk for postmenopausal breast cancer?

    PubMed Central

    Sahmoun, Abe E.; Singh, Brij B.

    2011-01-01

    SUMMARY Breast cancer is the most commonly diagnosed cancer among United States (US) women. Established risk factors explain only about 13% of breast cancer incidence among women in the US. Thus, the cause of most cases of breast cancer remains unknown. In postmenopausal women, serum calcium (Ca) and serum magnesium (Mg) play an important role in skeletal health, cell proliferation and cancer. Mg is essential for DNA duplication and repair and Mg deficiency favors DNA mutations leading to carcinogenesis. Dietary intake of Mg in the US is less than the recommended amount, and the deficit is more pronounced in older individuals where gastrointestinal and renal mechanisms for Mg conservation are not as efficient. Furthermore, healthy postmenopausal women are frequently recommended to take supplemental Ca, but not Mg and vitamin D to maintain bone and overall health. Most women with hormone sensitive breast cancer are recommended to take aromatase inhibitors, which causes bone loss and thus are generally prescribed Ca and vitamin D, but not Mg. Although the association between serum Ca and breast cancer risk remains controversial, we hypothesize that this may be because Mg levels have not been accounted for. Mg level directly influences transient receptor potential melastatin 7 (TRPM7) related Ca influx, calcium–adenosine triphosphatase (Ca–ATP) levels, and cell proliferation, and thereby could lead to cancer. Thus a high serum Ca/Mg ratio is more appropriate and alterations in this ratio could lead to increased development of new and recurrent breast cancer. PMID:20371155

  6. Arginine vasopressin increases cellular free calcium concentration and adenosine 3',5'-monophosphate production in rat renal papillary collecting tubule cells in culture

    SciTech Connect

    Ishikawa, S.; Okada, K.; Saito, T.

    1988-09-01

    The role of calcium (Ca) in the cellular action of arginine vasopressin (AVP) was examined in rat renal papillary collecting tubule cells in culture. AVP increased both the cellular free Ca concentration ((Ca2+)i) using fura-2, and cAMP production in a dose-dependent manner. AVP-induced cellular Ca mobilization was totally blocked by the antagonist to the antidiuretic action of AVP, and somewhat weakened by the antagonist to the vascular action of AVP. 1-Deamino-8-D-AVP (dDAVP). an antidiuretic analog of AVP, also increased (Ca2+) significantly. Cellular Ca mobilization was not obtained with cAMP, forskolin (a diterpene activator of adenylate cyclase), or phorbol-12-myristate-13-acetate. The early phase of (Ca2+)i depended on the intracellular Ca pool, since an AVP-induced rise in (Ca2+)i was obtained in cells pretreated with Ca-free medium containing 1 mM EGTA, verapamil, or cobalt, which blocked cellular Ca uptake. Also, AVP increased /sup 45/Ca2+ influx during the initial 10 min, which initiated the sustained phase of cellular Ca mobilization. However, cellular cAMP production induced by AVP during the 10-min observation period was diminished in the cells pretreated with Ca-free medium, verapamil, or cobalt, but was still significantly higher than the basal level. This was also diminished by a high Ca concentration in medium. These results indicate that 1) AVP concomitantly regulates cellular free Ca as well as its second messenger cAMP production; 2) AVP-induced elevation of cellular free Ca is dependent on both the cellular Ca pool and extracellular Ca; and 3) there is an optimal level of extracellular Ca to modulate the AVP action in renal papillary collecting tubule cells.

  7. Increased calcium/calmodulin-dependent protein kinase II activity by morphine-sensitization in rat hippocampus.

    PubMed

    Kadivar, Mehdi; Farahmandfar, Maryam; Ranjbar, Faezeh Esmaeli; Zarrindast, Mohammad-Reza

    2014-07-01

    Repeated exposure to drugs of abuse, such as morphine, elicits a progressive enhancement of drug-induced behavioral responses, a phenomenon termed behavioral sensitization. These changes in behavior may reflect long-lasting changes in some of the important molecules involved in memory processing such as calcium/calmodulin-dependent protein kinase II (CaMKII). In the present study, we investigated the effect of morphine sensitization on mRNA expression of α and β isoforms and activity of CaMKII in the hippocampus of male rats. Animals were treated for 3 days with saline or morphine (20mg/kg) and following a washout period of 5 days, a challenge dose of morphine (5mg/kg) were administered. The results indicate that morphine administration in pre-treated animals produces behavioral sensitization, as determined by significant increase in locomotion and oral stereotypy behavior. In addition, repeated morphine treatment increased mRNA expression of both α and β isoforms of CaMKII in the hippocampus. The present study also showed that induction of morphine sensitization significantly increased both Ca2+/calmodulin-independent and Ca2+/calmodulin-dependent activities of CaMK II in the rat hippocampus. However, acute administration of morphine (5mg/kg) did not alter either α and β CaMKII mRNA expression or CaMKII activity in the hippocampus. The stimulation effects of morphine sensitization on mRNA expression and activity of CaMKII were completely abolished by administration of naloxone, 30min prior to s.c. injections of morphine (20mg/kg/day×3 days). Our data demonstrated that induction of morphine sensitization could effectively modulate the activity and the mRNA expression of CaMKII in the hippocampus and this effect of morphine was exerted by the activation of opioid receptors.

  8. Resistance training over 2 years increases bone mass in calcium-replete postmenopausal women.

    PubMed

    Kerr, D; Ackland, T; Maslen, B; Morton, A; Prince, R

    2001-01-01

    Understanding the stress/strain relationship between exercise and bone is critical to understanding the potential benefit of exercise in preventing postmenopausal bone loss. This study examined the effect of a 2-year exercise intervention and calcium supplementation (600 mg) on bone mineral density (BMD) in 126 postmenopausal women (mean age, 60 +/- 5 years). Assignment was by block randomization to one of three groups: strength (S), fitness (F), or nonexercise control (C). The two exercise groups completed three sets of the same nine exercises, three times a week. The S group increased the loading, while the F group had additional stationary bicycle riding with minimal increase in loading. Retention at 2 years was 71% (59% in the S group, 69% in the F group, and 83% in the C group), while the exercise compliance did not differ between the exercise groups (S group, 74 +/- 13%; F group, 77 +/- 14%). BMD was measured at the hip, lumbar spine, and forearm sites every 6 months using a Hologic 4500. Whole body BMD also was measured every 6 months on a Hologic 2000. There was no difference between the groups at the forearm, lumbar spine, or whole body sites. There was a significant effect of the strength program at the total (0.9 +/- 2.6%; p < 0.05) and intertrochanter hip site (1.1 +/- 3.0%; p < 0.01). There was a significant time and group interaction (p < 0.05) at the intertrochanter site by repeated measures. This study shows the effectiveness of a progressive strength program in increasing bone density at the clinically important hip site. We concluded that a strength program could be recommended as an adjunct lifestyle approach to osteoporosis treatment or used in combination with other therapies.

  9. Increased osteoblast density in the presence of novel calcium phosphate coated magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Pareta, Rajesh A.; Taylor, Erik; Webster, Thomas J.

    2008-07-01

    Bone diseases (including osteoporosis, osteoarthritis and bone cancer) are of great concern to the medical world. Drugs are available to treat such diseases, but often these drugs are not specifically targeted to the site of the disease and, thus, lack an immediate directed therapeutic effect. The optimal drug delivery system should enhance healthy bone growth with high specificity to the site of bone disease. It has been previously shown that magnetic nanoparticles can be directed in the presence of a magnetic field to any part of the body, allowing for site-specific drug delivery and possibly an immediate increase in bone density. The objective of the present study was to build off of this evidence and determine the density of osteoblasts (bone forming cells) in the presence of various uncoated and coated magnetic nanoparticles that could eventually be used in drug delivery applications. Results showed that some magnetic nanoparticles (specifically, γ-Fe2O3) significantly promoted osteoblast density (that is, cells per well) after 5 and 8 days of culture compared to controls (no particles). These magnetic nanoparticles were further coated with calcium phosphate (CaP; the main inorganic component of bone) to tailor them for treating various bone diseases. The coatings were conducted in the presence of either bovine serum albumin (BSA) or citric acid (CA) to reduce magnetic nanoparticle agglomeration, a common problem resulting from the use of nanoparticles which decreases their effectiveness. Results with these coatings showed that magnetic nanoparticles, specifically (γ-Fe2O3), coated in the presence of BSA significantly increased osteoblast density compared to controls after 1 day. In this manner, this study provided unexpected evidence that CaP-coated γ-Fe2O3 magnetic nanoparticles increased osteoblast density (compared to no particles) and, thus, should be further studied to treat numerous bone diseases.

  10. Increasing Superoxide Production and the Labile Iron Pool in Tumor Cells may Sensitize Them to Extracellular Ascorbate.

    PubMed

    McCarty, Mark Frederick; Contreras, Francisco

    2014-01-01

    Low millimolar concentrations of ascorbate are capable of inflicting lethal damage on a high proportion of cancer cells lines, yet leave non-transformed cell lines unscathed. Extracellular generation of hydrogen peroxide, reflecting reduction of molecular oxygen by ascorbate, has been shown to mediate this effect. Although some cancer cell lines express low catalase activity, this cannot fully explain the selective sensitivity of cancer cells to hydrogen peroxide. Ranzato and colleagues have presented evidence for a plausible new explanation of this sensitivity - a high proportion of cancers, via NADPH oxidase complexes or dysfunctional mitochondria, produce elevated amounts of superoxide. This superoxide, via a transition metal-catalyzed transfer of an electron to the hydrogen peroxide produced by ascorbate, can generate deadly hydroxyl radical (Haber-Weiss reaction). It thus can be predicted that concurrent measures which somewhat selectively boost superoxide production in cancers will enhance their sensitivity to i.v. ascorbate therapy. One way to achieve this is to increase the provision of substrate to cancer mitochondria. Measures which inhibit the constitutive hypoxia-inducible factor-1 (HIF-1) activity in cancers (such as salsalate and mTORC1 inhibitors, or an improvement of tumor oxygenation), or that inhibit the HIF-1-inducible pyruvate dehydrogenase kinase (such as dichloroacetate), can be expected to increase pyruvate oxidation. A ketogenic diet should provide more lipid substrate for tumor mitochondria. The cancer-killing activity of 42°C hyperthermia is to some degree contingent on an increase in oxidative stress, likely of mitochondrial origin; reports that hydrogen peroxide synergizes with hyperthermia in killing cancer cells suggest that hyperthermia and i.v. ascorbate could potentiate each other's efficacy. A concurrent enhancement of tumor oxygenation might improve results by decreasing HIF-1 activity while increasing the interaction of ascorbic

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

    PubMed

    Lipin, Mikhail Y; Vigh, Jozsef

    2015-01-15

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

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

    PubMed Central

    Lipin, Mikhail Y.

    2014-01-01

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

  13. Induction of extracellular ATP mediates increase in intracellular thioredoxin in RAW264.7 cells exposed to low-dose γ-rays.

    PubMed

    Ohshima, Yasuhiro; Kitami, Akihiro; Kawano, Ayumi; Tsukimoto, Mitsutoshi; Kojima, Shuji

    2011-09-15

    We previously showed that low doses (0.25-0.5 Gy) of γ-rays elevated thioredoxin (Trx-1) in various organs of mice after whole-body irradiation. Also, it is reported that extracellular ATP, which is released in response to various stresses, regulates the expression of intracellular antioxidants through activation of P2 receptors. We have recently found that low-dose γ-rays induce ATP release from the exposed cells. However, it is not yet clear whether the radiation-induced extracellular ATP modulates the cellular redox balance. Here, we investigated whether γ-ray irradiation-induced release of extracellular ATP contributes to the induction of the cellular antioxidant Trx-1, using mouse macrophage-like RAW264.7 cells. Irradiation with γ-rays or exogenously added ATP increased the expression of Trx-1, and in both cases the increase was blocked by pretreatment with an ectonucleotidase, apyrase. Then, the involvement of ATP-dependent reactive oxygen species (ROS) generation in the increase in antioxidant capacity was examined. ATP stimulation promoted the generation of intracellular ROS and also increased Trx-1 expression. The increase in Trx-1 expression was significantly suppressed by pretreatment of the cells with antioxidants. In conclusion, the γ-ray irradiation-induced release of extracellular ATP may, at least in part, contribute to the production of ROS via purinergic signaling, leading to promotion of intracellular antioxidants as an adaptive response to an oxidative stress.

  14. Sphingolipid metabolites selectively elicit increases in nuclear calcium concentration in cell suspension cultures and in isolated nuclei of tobacco.

    PubMed

    Xiong, Tou Cheu; Coursol, Sylvie; Grat, Sabine; Ranjeva, Raoul; Mazars, Christian

    2008-01-01

    Sphingolipids are known to interfere with calcium-based signalling pathways. Here we report that these compounds modulate nuclear calcium signalling in tobacco BY-2 cells. Nuclear protein kinase activity phosphorylated endogenous sphingoid long-chain bases (LCBs), suggesting that LCBs are actively metabolized in the nucleus of tobacco BY-2 cells. The Delta4-unsaturated LCB D-erythro-sphingosine and the saturated LCB D-ribo-phytosphingosine elicited increases in free calcium in the nucleus in a dose-dependent and structure-related manner. However, neither sphingosine-1-phosphate nor C2-ceramide was able to stimulate nuclear calcium changes. N-,N-Dimethyl-D-erythro-sphingosine, a structural analogue of D-erythro-sphingosine, was the most efficient LCB so far tested in eliciting nuclear calcium changes both in intact tobacco BY-2 cells and in isolated nuclei. TRP channel inhibitors prevent the effect of DMS, suggesting that LCBs may activate TRP-like channels located on the inner nuclear membrane Collectively, the obtained data show that nuclei respond to LCBs on their own independently of the cytosolic compartment.

  15. Abundance and co-occurrence of extracellular capsules increase environmental breadth: Implications for the emergence of pathogens

    PubMed Central

    Néron, Bertrand; Touchon, Marie; Rocha, Eduardo P. C.

    2017-01-01

    Extracellular capsules constitute the outermost layer of many bacteria, are major virulence factors, and affect antimicrobial therapies. They have been used as epidemiological markers and recently became vaccination targets. Despite the efforts to biochemically serotype capsules in a few model pathogens, little is known of their taxonomic and environmental distribution. We developed, validated, and made available a computational tool, CapsuleFinder, to identify capsules in genomes. The analysis of over 2500 prokaryotic genomes, accessible in a database, revealed that ca. 50% of them—including Archaea—encode a capsule. The Wzx/Wzy-dependent capsular group was by far the most abundant. Surprisingly, a fifth of the genomes encode more than one capsule system—often from different groups—and their non-random co-occurrence suggests the existence of negative and positive epistatic interactions. To understand the role of multiple capsules, we queried more than 6700 metagenomes for the presence of species encoding capsules and showed that their distribution varied between environmental categories and, within the human microbiome, between body locations. Species encoding capsules, and especially those encoding multiple capsules, had larger environmental breadths than the other species. Accordingly, capsules were more frequent in environmental bacteria than in pathogens and, within the latter, they were more frequent among facultative pathogens. Nevertheless, capsules were frequent in clinical samples, and were usually associated with fast-growing bacteria with high infectious doses. Our results suggest that capsules increase the environmental range of bacteria and make them more resilient to environmental perturbations. Capsules might allow opportunistic pathogens to profit from empty ecological niches or environmental perturbations, such as those resulting from antibiotic therapy, to colonize the host. Capsule-associated virulence might thus be a by-product of

  16. Regional increase of extracellular potassium leads to electrical instability and reentry occurrence through the spatial heterogeneity of APD restitution.

    PubMed

    Sidorov, Veniamin Y; Uzelac, Ilija; Wikswo, John P

    2011-07-01

    The heterogeneities of electrophysiological properties of cardiac tissue are the main factors that control both arrhythmia induction and maintenance. Although the local increase of extracellular potassium ([K(+)](o)) due to coronary occlusion is a well-established metabolic response to acute ischemia, the role of local [K(+)](o) heterogeneity in phase 1a arrhythmias has yet to be determined. In this work, we created local [K(+)](o) heterogeneity and investigated its role in fast pacing response and arrhythmia induction. The left marginal vein of a Langendorff-perfused rabbit heart was cannulated and perfused separately with solutions containing 4, 6, 8, 10, and 12 mM of K(+). The fluorescence dye was utilized to map the voltage distribution. We tested stimulation rates, starting from 400 ms down to 120 ms, with steps of 5-50 ms. We found that local [K(+)](o) heterogeneity causes action potential (AP) alternans, 2:1 conduction block, and wave breaks. The effect of [K(+)](o) heterogeneity on electrical stability and vulnerability to arrhythmia induction was largest during regional perfusion with 10 mM of K(+). We detected three concurrent dynamics: normally propagating activation when excitation waves spread over tissue perfused with normal K(+), alternating 2:2 rhythm near the border of [K(+)](o) heterogeneity, and 2:1 aperiodicity when propagation was within the high [K(+)](o) area. [K(+)](o) elevation changed the AP duration (APD) restitution and shifted the restitution curve toward longer diastolic intervals and shorter APD. We conclude that spatial heterogeneity of the APD restitution, created with regional elevation of [K(+)](o), can lead to AP instability, 2:1 block, and reentry induction.

  17. Myocardial extracellular volume fraction quantified by cardiovascular magnetic resonance is increased in hypertension and associated with left ventricular remodeling.

    PubMed

    Wang, Shuli; Hu, Hongjie; Lu, Minjie; Sirajuddin, Arlene; Li, Jinghui; An, Jing; Chen, Xiuyu; Yin, Gang; Lan, Tian; Dai, Linlin; Zhang, Yan; Yin, Xiaorong; Song, Lei; Dang, Aimin; Kellman, Peter; Arai, Andrew E; Zhao, Shihua

    2017-04-24

    To determine whether extracellular volume fraction (ECV) quantification by cardiac magnetic resonance (CMR) can demonstrate left ventricle (LV) abnormalities and relationship between ECV and LV remodeling in hypertension (HTN) patients METHODS: ECV quantification was prospectively performed in 134 consecutive HTN patients and 97 healthy subjects. Individual and regional ECV were compared to the regions on late gadolinium enhancement (LGE) images. Statistical analysis of the relationship between LV global functional parameters and ECV was carried out using Pearson's correlation, Student's t test and multiple regressions. In the HTN group, 70.1% (94/134) were LGE negative and 29.9% (40/134) LGE positive. The mean ECV after adjusting for age, sex, BMI, diabetes, smoking and dyslipidaemia in healthy controls and LGE-negative patients were 26.9 ± 2.67% and 28.5 ± 2.9% (p < 0.001), respectively. The differences in ECV reached statistical significance among the regions of LGE, LGE-Peri, LGE remote and the normal area between the control and LGE-positive subgroup (all p < 0.05). Global ECV significantly correlated with LVEF (r = -0.466, p < 0 .001) and LV hypertrophy (r = 0.667, p < 0.001). ECV can identify LV abnormalities at an early stage in HTN patients without LGE. These abnormalities may reflect an increase in diffuse myocardial fibrosis and are associated with LV remodeling. • Diffuse myocardial fibrosis may develop in hypertensive cardiomyopathy before conventional MRI detectable LGE. • ECV can identify myocardial fibrosis at an early stage in hypertensive patients. • Elevated ECV is associated with decreased LV global function and LV remodeling in hypertension.

  18. Human resistin promotes neutrophil pro-inflammatory activation, neutrophil extracellular trap formation, and increases severity of acute lung injury

    PubMed Central

    Jiang, Shaoning; Park, Dae Won; Tadie, Jean-Marc; Gregoire, Murielle; Deshane, Jessy; Pittet, Jean Francois; Abraham, Edward; Zmijewski, Jaroslaw W.

    2014-01-01

    Although resistin was recently found to modulate insulin resistance in preclinical models of type II diabetes and obesity, recent studies have also suggested that resistin has proinflammatory properties. In these studies, we examined if the human specific variant of resistin affects neutrophil activation as well as the severity of LPS-induced acute lung injury (ALI). Because human and mouse resistin have distinct patterns of tissue distribution, experiments were performed using resistin humanized mice that exclusively express human resistin (hRTN+/−/−), but are deficient in mouse resistin. Enhanced production of TNF-α or MIP-2 was found in LPS-treated hRtn+/−/−, compared to control Rtn−/−/− neutrophils. Expression of human resistin inhibited the activation of AMP-activated protein kinase (AMPK), a major sensor and regulator of cellular bioenergetics that is also implicated in inhibiting inflammatory activity of neutrophils and macrophages. In addition to the ability of resistin to sensitize neutrophils to LPS stimulation, human resistin also enhanced neutrophil extracellular trap formation. In LPS-induced ALI, humanized resistin mice demonstrated enhanced production of pro-inflammatory cytokines, more severe pulmonary edema, increased NET formation, and elevated concentration of the alarmins HMGB1 and histone 3 in the lungs. Our results suggest that human resistin may play an important contributory role in enhancing TLR4 induced inflammatory responses, and may be a target for future therapies aimed at diminishing the severity of acute lung injury and other inflammatory situations where neutrophils play a major role. PMID:24719460

  19. Mesenchymal Stem/Stromal Cells under Stress Increase Osteosarcoma Migration and Apoptosis Resistance via Extracellular Vesicle Mediated Communication

    PubMed Central

    Vallabhaneni, Krishna C.; Hassler, Meeves-Yoni; Abraham, Anu; Whitt, Jason; Mo, Yin-Yuan; Atfi, Azeddine; Pochampally, Radhika

    2016-01-01

    Studies have shown that mesenchymal stem/stromal cells (MSCs) from bone marrow are involved in the growth and metastasis of solid tumors but the mechanism remains unclear in osteosarcoma (OS). Previous studies have raised the possibility that OS cells may receive support from associated MSCs in the nutrient deprived core of the tumors through the release of supportive macromolecules and growth factors either in vesicular or non-vesicular forms. In the present study, we used stressed mesenchymal stem cells (SD-MSCs), control MSCs and OS cells to examine the hypothesis that tumor-associated MSCs in nutrient deprived core provide pro-proliferative, anti-apoptotic, and metastatic support to nearby tumor cells. Assays to study of the effects of SD-MSC conditioned media revealed that OS cells maintained proliferation when compared to OS cells grown under serum-starved conditions alone. Furthermore, OS cells in MSCs and SD-MSC conditioned media were significantly resistant to apoptosis and an increased wound healing rate was observed in cells exposed to either conditioned media or EVs from MSCs and SD-MSCs. RT-PCR assays of OS cells incubated with extracellular vesicles (EVs) from SD-MSCs revealed microRNAs that could potentially target metabolism and metastasis associated genes as predicted by in silico algorithms, including monocarboxylate transporters, bone morphogenic receptor type 2, fibroblast growth factor 7, matrix metalloproteinase-1, and focal adhesion kinase-1. Changes in the expression levels of focal adhesion kinase, STK11 were confirmed by quantitative PCR assays. Together, these data indicate a tumor supportive role of MSCs in osteosarcoma growth that is strongly associated with the miRNA content of the EVs released from MSCs under conditions that mimic the nutrient deprived core of solid tumors. PMID:27812189

  20. Increased maternofetal calcium flux in parathyroid hormone-related protein-null mice

    PubMed Central

    Bond, H; Dilworth, M R; Baker, B; Cowley, E; Requena Jimenez, A; Boyd, R D H; Husain, S M; Ward, B S; Sibley, C P; Glazier, J D

    2008-01-01

    The role of parathyroid hormone-related protein (PTHrP) in fetal calcium homeostasis and placental calcium transport was examined in mice homozygous for the deletion of the PTHrP gene (PTHrP−/− null; NL) compared to PTHrP+/+ (wild-type; WT) and PTHrP+/− (heterozygous; HZ) littermates. Fetal blood ionized calcium was significantly reduced in NL fetuses compared to WT and HZ groups at 18 days of pregnancy (dp) with abolition of the fetomaternal calcium gradient. In situ placental perfusion of the umbilical circulation at 18 dp was used to measure unidirectional clearance of 45Ca across the placenta in maternofetal (CaKmf) and fetoplacental (CaKfp) directions; CaKfp was < 5% of CaKmf for all genotypes. At 18 dp, CaKmf across perfused placenta and intact placenta (CaKmf(intact)) were similar and concordant with net calcium accretion rates in vivo. CaKmf was significantly raised in NL fetuses compared to WT and HZ littermates. Calcium accretion was significantly elevated in NL fetuses by 19 dp. Placental calbindin-D9K expression in NL fetuses was marginally enhanced (P < 0.07) but expression of TRPV6/ECaC2 and plasma membrane Ca2+-ATPase (PMCA) isoforms 1 and 4 were unaltered. We conclude that PTHrP is an important regulator of fetal calcium homeostasis with its predominant effect being on unidirectional maternofetal transfer, probably mediated by modifying placental calbindin-D9K expression. In situ perfusion of mouse placenta is a robust methodology for allowing detailed dissection of placental transfer mechanisms in genetically modified mice. PMID:18258656

  1. Testosterone induces an intracellular calcium increase by a nongenomic mechanism in cultured rat cardiac myocytes.

    PubMed

    Vicencio, Jose Miguel; Ibarra, Cristian; Estrada, Manuel; Chiong, Mario; Soto, Dagoberto; Parra, Valentina; Diaz-Araya, Guillermo; Jaimovich, Enrique; Lavandero, Sergio

    2006-03-01

    Androgens are associated with important effects on the heart, such as hypertrophy or apoptosis. These responses involve the intracellular androgen receptor. However, the mechanisms of how androgens activate several membrane signaling pathways are not fully elucidated. We have investigated the effect of testosterone on intracellular calcium in cultured rat cardiac myocytes. Using fluo3-AM and epifluorescence microscopy, we found that exposure to testosterone rapidly (1-7 min) led to an increase of intracellular Ca2+, an effect that persisted in the absence of external Ca2+. Immunocytochemical analysis showed that these effects occurred before translocation of the intracellular androgen receptor to the perinuclear zone. Pretreatment of the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester and thapsigargin blocked this response, suggesting the involvement of internal Ca2+ stores. U-73122, an inhibitor of phospholipase C, and xestospongin C, an inhibitor of inositol 1,4,5-trisphosphate receptor, abolished the Ca2+ signal. The rise in intracellular Ca2+ was not inhibited by cyproterone, an antagonist of intracellular androgen receptor. Moreover, the cell impermeant testosterone-BSA complex also produced the Ca2+ signal, indicating its origin in the plasma membrane. This effect was observed in cultured neonatal and adult rat cardiac myocytes. Pertussis toxin and the adenoviral transduction of beta- adrenergic receptor kinase carboxy terminal peptide, a peptide inhibitor of betagamma-subunits of G protein, abolished the testosterone-induced Ca2+ release. In summary, this is the first study of rapid, nongenomic intracellular Ca2+ signaling of testosterone in cardiac myocytes. Using various inhibitors and testosterone-BSA complex, the mechanism for the rapid, testosterone-induced increase in intracellular Ca2+ is through activation of a plasma membrane receptor associated with a Pertussis toxin-sensitive G protein-phospholipase C

  2. Extracellular calcium (Ca2+(o))-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+(o) on the function of ST2 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+(o)) homeostasis by mediating the actions of Ca2+(o) on parathyroid gland and kidney. Bone marrow stromal cells support the formation of osteoclasts from their progenitors as well as the growth of hematopoietic stem cells by secreting humoral factors and through cell to cell contact. Stromal cells also have the capacity to differentiate into bone-forming osteoblasts. Bone resorption by osteoclasts probably produces substantial local increases in Ca2+(o) that could provide a signal for stromal cells in the immediate vicinity, leading us to determine whether such stromal cells express the CaR. In this study, we used the murine bone marrow-derived, stromal cell line, ST2. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in ST2 cells. We also identified CaR transcripts in ST2 cells by Northern analysis using a CaR-specific probe and by RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of ST2 cells to high Ca2+(o) (4.8 mM) or to the polycationic CaR agonists, neomycin (300 microM) or gadolinium (100 microM), stimulated both chemotaxis and DNA synthesis in ST2 cells. Therefore, taken together, our data strongly suggest that the bone marrow-derived stromal cell line, ST2, possesses both CaR protein and messenger RNA that are very similar if not identical to those in parathyroid and kidney. Furthermore, as ST2 cells have the potential to differentiate into osteoblasts, the CaR in stromal cells could participate in bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local, osteoclast-mediated release of Ca2+(o) and, thereafter, initiating bone formation after their differentiation into osteoblasts.

  3. Extracellular calcium (Ca2+(o))-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+(o) on the function of ST2 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+(o)) homeostasis by mediating the actions of Ca2+(o) on parathyroid gland and kidney. Bone marrow stromal cells support the formation of osteoclasts from their progenitors as well as the growth of hematopoietic stem cells by secreting humoral factors and through cell to cell contact. Stromal cells also have the capacity to differentiate into bone-forming osteoblasts. Bone resorption by osteoclasts probably produces substantial local increases in Ca2+(o) that could provide a signal for stromal cells in the immediate vicinity, leading us to determine whether such stromal cells express the CaR. In this study, we used the murine bone marrow-derived, stromal cell line, ST2. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in ST2 cells. We also identified CaR transcripts in ST2 cells by Northern analysis using a CaR-specific probe and by RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of ST2 cells to high Ca2+(o) (4.8 mM) or to the polycationic CaR agonists, neomycin (300 microM) or gadolinium (100 microM), stimulated both chemotaxis and DNA synthesis in ST2 cells. Therefore, taken together, our data strongly suggest that the bone marrow-derived stromal cell line, ST2, possesses both CaR protein and messenger RNA that are very similar if not identical to those in parathyroid and kidney. Furthermore, as ST2 cells have the potential to differentiate into osteoblasts, the CaR in stromal cells could participate in bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local, osteoclast-mediated release of Ca2+(o) and, thereafter, initiating bone formation after their differentiation into osteoblasts.

  4. Increased Expression of Fatty-Acid and Calcium Metabolism Genes in Failing Human Heart

    PubMed Central

    Rodríguez-Penas, Diego; Feijóo-Bandín, Sandra; Noguera-Moreno, Teresa; Calaza, Manuel; Álvarez-Barredo, María; Mosquera-Leal, Ana; Parrington, John; Brugada, Josep; Portolés, Manuel; Rivera, Miguel; González-Juanatey, José Ramón; Lago, Francisca

    2012-01-01

    Background Heart failure (HF) involves alterations in metabolism, but little is known about cardiomyopathy-(CM)-specific or diabetes-independent alterations in gene expression of proteins involved in fatty-acid (FA) uptake and oxidation or in calcium-(Ca2+)-handling in the human heart. Methods RT-qPCR was used to quantify mRNA expression and immunoblotting to confirm protein expression in left-ventricular myocardium from patients with HF (n = 36) without diabetes mellitus of ischaemic (ICM, n = 16) or dilated (DCM, n = 20) cardiomyopathy aetiology, and non-diseased donors (CTL, n = 6). Results Significant increases in mRNA of genes regulating FA uptake (CD36) and intracellular transport (Heart-FA-Binding Protein (HFABP)) were observed in HF patients vs CTL. Significance was maintained in DCM and confirmed at protein level, but not in ICM. mRNA was higher in DCM than ICM for peroxisome-proliferator-activated-receptor-alpha (PPARA), PPAR-gamma coactivator-1-alpha (PGC1A) and CD36, and confirmed at the protein level for PPARA and CD36. Transcript and protein expression of Ca2+-handling genes (Two-Pore-Channel 1 (TPCN1), Two-Pore-Channel 2 (TPCN2), and Inositol 1,4,5-triphosphate Receptor type-1 (IP3R1)) increased in HF patients relative to CTL. Increases remained significant for TPCN2 in all groups but for TPCN1 only in DCM. There were correlations between FA metabolism and Ca2+-handling genes expression. In ICM there were six correlations, all distinct from those found in CTL. In DCM there were also six (all also different from those found in CTL): three were common to and three distinct from ICM. Conclusion DCM-specific increases were found in expression of several genes that regulate FA metabolism, which might help in the design of aetiology-specific metabolic therapies in HF. Ca2+-handling genes TPCN1 and TPCN2 also showed increased expression in HF, while HF- and CM-specific positive correlations were found among several FA and Ca2+-handling genes

  5. Angiotensin effects on calcium and steroidogenesis in adrenal glomerulosa cells

    SciTech Connect

    Elliott, M.E.; Siegel, F.L.; Hadjokas, N.E.; Goodfriend, T.L.

    1985-03-01

    We investigated the role of cellular calcium pools in angiotensin II-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. Angiotensin II decreased the size of the exchangeable cell calcium pool by 34%, consistent with previous observations that angiotensin II causes decreased uptake of /sup 45/Ca+2 into cells and increased efflux of /sup 45/Ca+2 from preloaded cells. Atomic absorption spectroscopy showed that angiotension II caused a decrease of 21% in total cellular calcium. Angiotensin II caused efflux of /sup 45/Ca+2 in the presence of EGTA and retarded uptake of /sup 45/Ca+2 when choline was substituted for sodium, suggesting that hormone effects on calcium pools do not involve influx of trigger calcium or sodium. Cells incubated in calcium-free buffer and 0.1 mM or 0.5 mM EGTA synthesized reduced (but still significant) amounts of the steroid in response to hormone. Cells incubated in increasing concentrations of extracellular calcium contained increasing amounts of intracellular calcium and synthesized increasing amounts of aldosterone in response to angiotensin II. These results point to the participation of intracellular calcium pools in angiotensin II-stimulated steroidogenesis and the importance of extracellular calcium in maintaining these pools.

  6. Increases in cellular calcium concentration stimulate pepsinogen secretion from dispersed chief cells

    SciTech Connect

    Raufman, J.P.; Berger, S.; Cosowsky, L.; Straus, E.

    1986-05-29

    Intracellular calcium concentration ((Ca)i) and pepsinogen secretion from dispersed chief cells from guinea pig stomach were determined before and after stimulation with calcium ionophores. (Ca)i was measured using the fluorescent probe quin2. Basal (Ca)i was 105 +/- 4 nM. Pepsinogen secretion was measured with a new assay using /sup 125/I-albumin substrate. This assay is 1000-fold more sensitive than the widely-used spectrophotometric assay, technically easy to perform, rapid, and relatively inexpensive. The kinetics and stoichiometry of ionophore-induced changes in (Ca)i and pepsinogen secretion were similar. These data support a role for calcium as a cellular mediator of pepsinogen secretion.

  7. Increased calcium deposits and decreased Ca2+ -ATPase in erythrocytes of ascitic broiler chickens.

    PubMed

    Li, Kai; Zhao, Lihong; Geng, Guangrui; Ma, Liqin; Dong, Shishan; Xu, Tong; Wang, Jianlin; Wang, Huiyu; Tian, Yong; Qiao, Jian

    2011-06-01

    The decrease of erythrocyte deformability may be one of the predisposing factors for pulmonary hypertension and ascites in broiler chickens. In mammals, the cytoplasmic calcium is a major regulator of erythrocyte deformability. In this study, the erythrocyte deformability was measured, and the precise locations of Ca2+ and Ca2+ -ATPase in the erythrocytes were investigated in chickens with ascites syndrome induced by low ambient temperature. The results showed that ascitic broilers had higher filtration index of erythrocyte compared with control groups, indicating a decrease in erythrocyte deformability in ascitic broilers. The more calcium deposits were observed in the erythrocytes of ascitic broilers compared with those of the age-matched control birds. The Ca2+ -ATPase reactive grains were significantly decreased on the erythrocyte membranes of ascitic broilers. Our data suggest that accumulation of intracellular calcium and inhibition of Ca2+ -ATPase might be important factors for the reduced deformability of the erythrocytes of ascitic broilers.

  8. Effect of increasing the colloidal calcium phosphate of milk on the texture and microstructure of yogurt.

    PubMed

    Ozcan, T; Horne, D; Lucey, J A

    2011-11-01

    The effect of increasing the colloidal calcium phosphate (CCP) content on the physical, rheological, and microstructural properties of yogurt was investigated. The CCP content of heated (85°C for 30 min) milk was increased by increasing the pH by the addition of alkali (NaOH). Alkalized milk was dialyzed against pasteurized skim milk at approximately 4°C for 72 h to attempt to restore the original pH and soluble Ca content. By adjustment of the milk to pH values 7.45, 8.84, 10.06, and 10.73, the CCP content was increased to approximately 107, 116, 123, and 128%, respectively, relative to the concentration in heated milk. During fermentation of milk, the storage modulus (G') and loss tangent values of yogurts were measured using dynamic oscillatory rheology. Large deformation rheological properties were also measured. The microstructure of yogurt was observed using fluorescence microscopy, and whey separation was determined. Acid-base titration was used to evaluate changes in the CCP content in milk. Total Ca and casein-bound Ca increased with an increase in the pH value of alkalization. During acidification, elevated buffering occurred in milk between pH values 6.7 to 5.2 with an increase in the pH of alkalization. When acidified milk was titrated with alkali, elevated buffering occurred in milk between pH values 5.6 to 6.4 with an increase in the pH of alkalization. The high residual pH of milk after dialysis could be responsible for the decreased contents of soluble Ca in these milks. The pH of gelation was higher in all dialyzed samples compared with the heated control milk, and the gelation pH was higher with an increase in CCP content. The sample with highest CCP content (128%) exhibited gelation at very high pH (6.3), which could be due to alkali-induced CN micellar disruption. The G' values at pH 4.6 were similar in gels with CCP levels up to 116%; at higher CCP levels, the G' values at pH 4.6 greatly decreased. Loss tangent values at pH 5.1 were similar

  9. Localized Apical Increases of Cytosolic Free Calcium Control Pollen Tube Orientation.

    PubMed Central

    Malho, R.; Trewavas, A. J.

    1996-01-01

    To reach the ovule, pollen tubes must undergo many changes in growth direction. We have shown in previous work that elevation of cytosolic free calcium ([Ca2+]c) can manipulate orientation in growing pollen tubes, but our results suggested that [Ca2+]c changes either in the tip or in more distal regions might regulate the critical orienting mechanism. To identify the spatial location of the orienting motor, we combined the techniques of ion imaging with confocal microscopy and localized photoactivation of loaded caged Ca2+ (nitr-5) and diazo-2 (a caged Ca2+ chelator) to manipulate [Ca2+]c in different pollen tube domains. We found that increasing [Ca2+]c on one side of the pollen tube apex induced reorientation of the growth axis toward that side. Similarly, a decrease in [Ca2+]c promoted bending toward the opposite side. These effects could be mimicked by imposing localized external gradients of an ionophore (A23187) or a Ca2+ channel blocker (GdCl3); the pollen tubes bend toward the highest concentration of A23187 and away from GdCl3. Manipulation of [Ca2+]c in regions farther back from the apical zone also induced changes in growth direction, but the new orientation was at random. We observed communication of these distal events to the tip through a slow-moving [Ca2+]c wave. These data show that localized changes of [Ca2+]c in the tip, which could result from asymmetric channel activity, control the direction of pollen tube growth. PMID:12239370

  10. Brief report: Does PTH increase with age, independent of 25-hydroxyvitamin D, phosphate, renal function, and ionized calcium?

    PubMed

    Carrivick, Simon J; Walsh, John P; Brown, Suzanne J; Wardrop, Robert; Hadlow, Narelle C

    2015-05-01

    Circulating PTH concentrations increase with age. It is uncertain whether an age-related PTH increase occurs independent of changes in circulating 25-hydroxyvitamin D, phosphate, renal function, and ionized calcium. The purpose of this article was to analyze the relationship between PTH and age, controlling for 25-hydroxyvitamin D, phosphate, renal function, and ionized calcium. This was a retrospective, cross-sectional study analyzing the relationship between PTH and age in 2 independent datasets (laboratory 1, n = 17 275 and laboratory 2, n = 4878). We further analyzed subgroups after excluding participants with estimated glomerular filtration rate of <60 mL/min/1.73 m(2) or 25-hydroxyvitamin D of <50 nmol/L (for subgroups, n = 12 051 for laboratory 1 and 3473 for laboratory 2). After adjustment for sex, ionized calcium, 25-hydroxyvitamin D, phosphate, and estimated glomerular filtration rate, each 10-year increase in age was associated with a 5.0% increase in PTH (95% confidence interval [CI], 4.4%-5.6%; P < .001) in laboratory 1 and a 4.2% increase in laboratory 2 (95% CI, 3.0%-5.4%; P < .001). In the subgroups, each 10-year increase in age was associated with a 6.1% increase in PTH (95% CI, 5.5%-6.8%; P < .001) in laboratory 1 and a 4.9% increase (95% CI 3.5%-6.2%; P < .001) in laboratory 2. PTH concentrations increase with age, independent of 25-hydroxyvitamin D, ionized calcium, phosphate, and renal function. Further research is required to explore the underlying mechanisms and clinical relevance and to determine whether the use of age-related PTH reference ranges improves diagnostic accuracy, particularly in elderly individuals.

  11. The influx of calcium ions into human erythrocytes during cold storage. The influences of extracellular pH, intracellular adenosine triphosphate and efflux of univalent cations.

    PubMed

    Mouat, B; Long, C

    1974-09-01

    1. When human erythrocytes are stored at 3 degrees C for several days as a suspension in iso-osmotic sucrose or KCl, containing CaCl(2), the rates of cellular ATP degradation are similar. 2. During cold storage of erythrocytes in sucrose-CaCl(2) medium, Ca(2+) influx and univalent-cation efflux occur, the pH value of the suspending medium rises and the intracellular pH falls. These pH changes correlate reasonably well with alterations in the membrane potential calculated from Cl(-) distribution. 3. The presence of Ca(2+) in the medium does not increase the rate of univalent-cation efflux from the cells. 4. When the pH of the medium is raised by addition of buffers, the rates of both Ca(2+) influx and univalent-cation efflux increase. 5. Replacement of sucrose by KCl as the main osmotic component of the medium completely suppresses Ca(2+) influx and univalent-cation efflux, although the pH of the KCl medium is higher than that of the sucrose medium. 6. When sucrose is replaced by choline chloride, Ca(2+) influx and univalent-cation efflux still occur, and the pH of the medium is similar to that found in iso-osmotic KCl. 7. When valinomycin, Pb(2+) or Cd(2+) are added to the iso-osmotic sucrose medium, the rate of efflux of univalent cations increases as also does the influx of Ca(2+). 8. From these and other observations, it was concluded that it is univalent-cation efflux rather than ATP depletion or elevated extracellular pH which is the prerequisite for Ca(2+) influx during cold storage.

  12. Extracellular superoxide dismutase in cultured astrocytes: decrease in cell-surface activity and increase in medium activity by lipopolysaccharide-stimulation.

    PubMed

    Iitsuka, Ichiro; Motoyoshi-Yamashiro, Akiko; Moriyama, Mitsuaki; Kannan-Hayashi, Yukiko; Fujimoto, Yuka; Takano, Katsura; Murakami, Koji; Yoneda, Yukio; Nakamura, Yoichi

    2012-10-01

    Under pathological conditions such as ischemia/reperfusion, a large amount of superoxide anion (O(2) (-)) is produced and released in brain. Among three isozymes of superoxide dismutase (SOD), extracellular (EC)-SOD, known to be excreted outside cells and bound to extracellular matrix, should play a role to detoxify O(2) (-) in extracellular space; however, a little is known about EC-SOD in brain. In order to evaluate the SOD activity in extracellular space of CNS as direct as possible, we attempted to measure the cell-surface SOD activity on primary cultured rat brain cells by the inhibition of color development of a water-soluble tetrazolium due to O(2) (-) generation by xanthine oxidase/hypoxanthine added into extracellular medium of intact cells. The cell-surface SOD activity on cultured neuron and microglia was below the detection limit; however, that on cultured astrocyte was high enough to measure. By means of RT-PCR, all mRNA of three isozymes of SOD could be detected in the three types of the cells examined; however, the semi-quantitative analysis revealed that the level of EC-SOD mRNA in astrocytes was significantly higher than that in neurons and microglia. When astrocytes were stimulated with lipopolysaccharide (LPS) for 12-24 h, the cell-surface SOD activity decreased to a half, whereas the activity recovered after 36-48 h. The decrease in the activity was dependent on the LPS concentration. On the other hand, the SOD activity in the medium increased by the LPS-stimulation in a dose dependent manner; suggesting that the SOD protein localized on cell-surface, probably EC-SOD, was released into the medium. These results suggest that EC-SOD of astrocyte play a role for detoxification of extracellular O(2) (-) and the regulation of EC-SOD in astrocytes may contribute to the defensive mechanism against oxidative stress in brain.

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

    PubMed

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

    2014-08-01

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

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

    PubMed

    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.

  15. Increased central nervous system production of extracellular matrix components and development of hydrocephalus in transgenic mice overexpressing transforming growth factor-beta 1.

    PubMed Central

    Wyss-Coray, T.; Feng, L.; Masliah, E.; Ruppe, M. D.; Lee, H. S.; Toggas, S. M.; Rockenstein, E. M.; Mucke, L.

    1995-01-01

    A number of important neurological diseases, including HIV-1 encephalitis, Alzheimer's disease, and brain trauma, are associated with increased cerebral expression of the multifunctional cytokine transforming growth factor-beta 1 (TGF-beta 1). To determine whether overexpression of TGF-beta 1 within the central nervous system (CNS) can contribute to the development of neuropathological alterations, a bioactive form of TGF-beta 1 was expressed in astrocytes of transgenic mice. Transgenic mice with high levels of cerebral TGF-beta 1 expression developed a severe communicating hydrocephalus, seizures, motor incoordination, and early runting. While unmanipulated heterozygous transgenic mice from a low expressor line showed no such alterations, increasing TGF-beta 1 expression in this line by injury-induced astroglial activation or generation of homozygous offspring did result in the abnormal phenotype. Notably, astroglial overexpression of TGF-beta 1 consistently induced a strong upmodulation of the extracellular matrix proteins laminin and fibronectin in the CNS, particularly in the vicinity of TGF-beta 1-expressing perivascular astrocytes, but was not associated with obvious CNS infiltration by hematogenous cells. While low levels of extracellular matrix protein expression may assist in CNS wound repair and regeneration, excessive extracellular matrix deposition could result in the development of hydrocephalus. As an effective inducer of extracellular matrix components, TGF-beta 1 may also contribute to the development of other neuropathological alterations, eg, the formation of amyloid plaques in Alzheimer's disease. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7604885

  16. Eplerenone inhibits the intracrine and extracellular actions of angiotensin II on the inward calcium current in the failing heart. On the presence of an intracrine renin angiotensin aldosterone system

    PubMed Central

    De Mello, Walmor C.; Gerena, Yamil

    2009-01-01

    The influence of chronic administration of eplerenone on the intracrine as well as on the extracellular action of angiotensin II (Ang II) on L-type inward calcium current was investigated in the failing heart of cardiomyopathic hamsters (TO-2).For this, eplerenone (200 mg/kg/day) was administered orally to 2 month-old cardiomyopathic hamsters for a period of 3 mon