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Sample records for calcium sensitization induced

  1. Polyamines transduce the nongenomic, androgen-induced calcium sensitization in intestinal smooth muscle.

    PubMed

    González-Montelongo, María C; Marín, Raquel; Pérez, José A; Gómez, Tomás; Díaz, Mario

    2013-10-01

    Androgens regulate body development and differentiation through a variety of genotropic mechanisms, mostly in reproductive organs. In recent years a different scenario for sex hormone actions has emerged: the intestinal muscle. Thus, although estrogens relax intestinal muscle, androgens are powerful inducers of mechanical potentiation. This effect of androgens was intriguing because it is observed at physiological concentrations, is mediated by nongenomic mechanisms, and involves a phenomenon of calcium sensitization of contractile machinery by stimulating phosphorylation of 20 kDa myosin light chain by Rho-associated kinase. Here we have deciphered the molecular mechanisms underlying calcium sensitization and mechanical potentiation by androgens in male intestinal muscle as well as its tight relationship to polyamine metabolism. Thus, androgens stimulate polyamine synthesis, and the inhibition of polyamine synthesis abolishes androgen-induced calcium sensitization and 20 kDa myosin light chain phosphorylation. We demonstrate that the first molecular step in the induction of calcium sensitization is a nonconventional activation of the adaptor protein RhoA, triggered by a transglutaminase-catalyzed polyamination of RhoA, which is then targeted to the membrane to activate Rho-associated kinase. Altogether, these results demonstrate that the physiological levels of androgens, through the modulation of polyamine metabolism and posttanslational modification of RhoA, activate a new signal transduction pathway in the intestinal smooth muscle to induce calcium sensitization. Furthermore, apart from being one of the few physiologically relevant nongenomic effects of androgens, these results might underlie the well-known gender differences in intestinal transits, thus expanding the nature's inventory of sex hormones effects.

  2. Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

    While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixty-fold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling.

  3. Impact of hydroxyl radical-induced injury on calcium handling and myofilament sensitivity in isolated myocardium

    PubMed Central

    Haizlip, Kaylan M.; Hiranandani, Nitisha; Biesiadecki, Brandon J.

    2012-01-01

    Hydroxyl radicals (OH˙) are involved in the pathogenesis of reperfusion injury and are observed in acute heart failure, stroke, and myocardial infarction. Two different subcellular defects are involved in the pathogenesis of OH˙ injury, deranged calcium handling, and alterations of myofilament responsiveness, but their temporal impact on contractile function is not resolved. Initially, after brief OH˙ exposure, there is a corresponding marked increase in diastolic calcium and diastolic force. We followed these parameters until a new steady-state level was reached at ∼45 min post-OH˙ exposure. At this new baseline, diastolic calcium had returned to near-normal, pre-OH˙ levels, whereas diastolic force remained markedly elevated. An increased calcium sensitivity was observed at the new baseline after OH˙-induced injury compared with the pre-OH˙ state. The acute injury that occurs after OH˙ exposure is mainly due to calcium overload, while the later sustained myocardial dysfunction is mainly due to the altered/increased myofilament responsiveness. PMID:22773772

  4. Adenosine restores angiotensin II-induced contractions by receptor-independent enhancement of calcium sensitivity in renal arterioles.

    PubMed

    Lai, En Yin; Martinka, Peter; Fähling, Michael; Mrowka, Ralf; Steege, Andreas; Gericke, Adrian; Sendeski, Mauricio; Persson, P B; Persson, A Erik G; Patzak, Andreas

    2006-11-10

    Adenosine is coupled to energy metabolism and regulates tissue blood flow by modulating vascular resistance. In this study, we investigated isolated, perfused afferent arterioles of mice, which were subjected to desensitization during repeated applications of angiotensin II. Exogenously applied adenosine restores angiotensin II-induced contractions by increasing calcium sensitivity of the arterioles, along with augmented phosphorylation of the regulatory unit of the myosin light chain. Adenosine restores angiotensin II-induced contractions via intracellular action, because inhibition of adenosine receptors do not prevent restoration, but inhibition of NBTI sensitive adenosine transporters does. Restoration was prevented by inhibition of Rho-kinase, protein kinase C, and the p38 mitogen-activated protein kinase, which modulate myosin light chain phosphorylation and thus calcium sensitivity in the smooth muscle. Furthermore, adenosine application increased the intracellular ATP concentration in LuciHEK cells. The results of the study suggest that restoration of the angiotensin II-induced contraction by adenosine is attributable to the increase of the calcium sensitivity by phosphorylation of the myosin light chain. This can be an important component of vascular control during ischemic and hypoxic conditions. Additionally, this mechanism may contribute to the mediation of the tubuloglomerular feedback by adenosine in the juxtaglomerular apparatus of the kidney. PMID:17038642

  5. Serotonin-induced bistability of turtle motoneurones caused by a nifedipine-sensitive calcium plateau potential.

    PubMed Central

    Hounsgaard, J; Kiehn, O

    1989-01-01

    plateau response was induced by Bay K 8644 (1-15 microM), this L-channel agonist could not reproduce the pronounced effect of serotonin. 9. It is concluded that serotonin induces a Ca2+-dependent and nifedipine-sensitive plateau potential in turtle motoneurones primarily by reducing a K+-current responsible for the slow after-hyperpolarization. PMID:2607432

  6. Osteoblasts detect pericellular calcium concentration increase via neomycin-sensitive voltage gated calcium channels.

    PubMed

    Sun, Xuanhao; Kishore, Vipuil; Fites, Kateri; Akkus, Ozan

    2012-11-01

    The mechanisms underlying the detection of critically loaded or micro-damaged regions of bone by bone cells are still a matter of debate. Our previous studies showed that calcium efflux originates from pre-failure regions of bone matrix and MC3T3-E1 osteoblasts respond to such efflux by an increase in the intracellular calcium concentration. The mechanisms by which the intracellular calcium concentration increases in response to an increase in the pericellular calcium concentration are unknown. Elevation of the intracellular calcium may occur via release from the internal calcium stores of the cell and/or via the membrane bound channels. The current study applied a wide range of pharmaceutical inhibitors to identify the calcium entry pathways involved in the process: internal calcium release from endoplasmic reticulum (ER, inhibited by thapsigargin and TMB-8), calcium receptor (CaSR, inhibited by calhex), stretch-activated calcium channel (SACC, inhibited by gadolinium), voltage-gated calcium channels (VGCC, inhibited by nifedipine, verapamil, neomycin, and ω-conotoxin), and calcium-induced-calcium-release channel (CICRC, inhibited by ryanodine and dantrolene). These inhibitors were screened for their effectiveness to block intracellular calcium increase by using a concentration gradient induced calcium efflux model which mimics calcium diffusion from the basal aspect of cells. The inhibitor(s) which reduced the intracellular calcium response was further tested on osteoblasts seeded on mechanically loaded notched cortical bone wafers undergoing damage. The results showed that only neomycin reduced the intracellular calcium response in osteoblasts, by 27%, upon extracellular calcium stimulus induced by concentration gradient. The inhibitory effect of neomycin was more pronounced (75% reduction in maximum fluorescence) for osteoblasts seeded on notched cortical bone wafers loaded mechanically to damaging load levels. These results imply that the increase in

  7. Sensitivity to calcium intake in calcium stone forming patients.

    PubMed

    Heilberg, I P; Martini, L A; Draibe, S A; Ajzen, H; Ramos, O L; Schor, N

    1996-01-01

    The absorptive or renal origin of hypercalciuria can be discriminated using an acute oral calcium load test (ACLT). Of 86 patients with calcium oxalate kidney stones, 28 (23%) were found to be hypercalciuric (HCa) and 58 (67%) normocalciuric (NCa) on their customary free diet, containing 542 +/- 29 mg/day (mean +/- SE) of calcium. Since the apparently normal 24-hour calcium excretion of many calcium stone formers (CSF) may be due to a combination of high calcium absorption with moderately low calcium intake, all patients were investigated by ACLT. Of 28 HCa patients, 13 (46%) were classified as absorptive (AH) and 15 (54%) as renal hypercalciuria (RH). Of the 58 NCa patients, 38 (65%) presented features of intestinal hyperabsorption and were therefore designated as AH-like, and 20 (35%) as RH-like. To further elucidate the role of dietary calcium in these CSF, a chronic calcium load test (CCLT), consisting of 1 g/day of oral Ca for 7 days, was designed. A positive response to the CCLT was considered to occur when urinary calcium (uCa) was > or = 4 mg/ kg/24 h on the 7th day. Among NCa patients, 29% of AH-like subjects responded to the CCLT and 71% did not; 50% of RH-like subjects also responded and 50% did not. In HCa patients, 85% of AH and 67% of RH subjects maintained uCa > or = 4 mg/kg/24 h after the CCLT and 15% of AH and 23% of RH subjects did not. However, a significant additional increase in mean uCa was not observed among HCa patients. All patients were submitted to a second evaluation of fasting calciuria (Ca/Cr). A modification of this parameter was noticed in 89% of RH-like and 78% of RH patients. In conclusion, these data suggest the presence of subpopulations of patients sensitive or not to calcium intake, regardless of whether the acute response to a calcium overload test suggested AH or RH. The CCLT disclosed dietary hypercalciuria in 21/58 (36%) of previously NCa patients. In these NCa patients, the ACLT may be replaced by the CCLT. The distinction

  8. CALCIUM-INDUCED SUPRAMOLECULAR STRUCTURES IN THE CALCIUM CASEINATE SYSTEM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular details deciphering the spontaneous calcium-induced protein aggregation process in the calcium caseinate system remain obscure. Understanding this complex process could lead to potential new applications of this important food ingredient. In this work, we studied calcium-induced supra...

  9. [The dynamics of the domains of the IP3-binding site of the inositol-1,4,5-triphosphate-sensitive calcium channel, induced by inositol-1,4,5-triphosphate and calcium].

    PubMed

    Veresov, V G; Konev, S V

    2005-01-01

    The dynamics of the inositol-1,4,5-triphosphate-sensitive calcium channel after binding of inositol-1,4,5-triphosphate and Ca2+ was analyzed by the Monte Carlo minimization technique. It was shown that the binding of Ca2+ with the unliganded receptor (channel) leads to a turning of the beta-sheet domain relative to the alpha-helical domain with the formation of the receptor conformation that is open for the entry of ions into the cytoplasmic channel vestibule, sterically closed for their passage through the vestibule in the part adjacent to the alpha-helical domains, and unfavourable for subsequent binding of inositol-1,4,5-triphosphate with the receptor. When both co-agonists bind to the receptor, the structure rearrangements induced eliminate both these steric obstacles for the passage of ions through the IP3-binding domain: one at the entrance of the channel cytoplasmic vestibule and the other that is placed deeper in the vestibule near the alpha-domains. The role of the dynamics of the receptor binding core in the IP3-sensitive channel gating is discussed.

  10. Oxygen increases ductus arteriosus smooth muscle cytosolic calcium via release of calcium from inositol triphosphate-sensitive stores.

    PubMed

    Keck, Maggie; Resnik, Ernesto; Linden, Bradley; Anderson, Franklin; Sukovich, David J; Herron, Jean; Cornfield, David N

    2005-05-01

    In utero, blood shunts away from the lungs via the ductus arteriosus (DA) and the foramen ovale. After birth, the DA closes concomitant with increased oxygen tension. The present experimental series tests the hypothesis that oxygen directly increases DA smooth muscle cell (SMC) cytosolic calcium ([Ca(2+)](i)) through inactivation of a K(+) channel, membrane depolarization, and entry of extracellular calcium. To test the hypothesis, DA SMC were isolated from late-gestation fetal lambs and grown to subconfluence in primary culture in low oxygen tension (25 Torr). DA SMC were loaded with the calcium-sensitive fluorophore fura-2 under low oxygen tension conditions and studied using microfluorimetry while oxygen tension was acutely increased (120 Torr). An acute increase in oxygen tension progressively increased DA SMC [Ca(2+)](i) by 11.7 +/- 1.4% over 40 min. The effect of acute normoxia on DA SMC [Ca(2+)](i) was mimicked by pharmacological blockade of the voltage-sensitive K(+) channel. Neither removal of extracellular calcium nor voltage-operated calcium channel blockade prevented the initial increase in DA SMC [Ca(2+)](i). Manganese quenching experiments demonstrated that acute normoxia initially decreases the rate of extracellular calcium entry. Pharmacological blockade of inositol triphosphate-sensitive, but not ryanodine-sensitive, intracellular calcium stores prevented the oxygen-induced increase in [Ca(2+)](i). Endothelin increased [Ca(2+)](i) in acutely normoxic, but not hypoxic, DA SMC. Thus acute normoxia 1) increases DA SMC [Ca(2+)](i) via release of calcium from intracellular calcium stores, and subsequent entry of extracellular calcium, and 2) potentiates the effect of contractile agonists. Prolonged patency of the DA may result from disordered intracellular calcium homeostasis.

  11. Calcium wave propagation by calcium-induced calcium release: an unusual excitable system.

    PubMed

    Sneyd, J; Girard, S; Clapham, D

    1993-03-01

    We discuss in detail the behaviour of a model, proposed by Goldbeter et al. (1990. Proc. natn. Acad. Sci. 87, 1461-1465), for intracellular calcium wave propagation by calcium-induced calcium release, focusing our attention on excitability and the propagation of waves in one spatial dimension. The model with no diffusion behaves like a generic excitable system, and threshold behaviour, excitability and oscillations can be understood within this general framework. However, when diffusion is included, the model no longer behaves like a generic excitable system; the fast and slow variables are not distinct and previous results on excitable systems do not necessarily apply. We consider a piecewise linear simplification of the model, and construct travelling pulse and periodic plane wave solutions to the simplified model. The analogous behaviour in the full model is studied numerically. Goldbeter's model for calcium-induced calcium release is an excitable system of a type not previously studied in detail.

  12. 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.

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

    ERIC Educational Resources Information Center

    Liang, Willmann

    2008-01-01

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

  14. Calcium sensitivity of residual force enhancement in rabbit skinned fibers.

    PubMed

    Joumaa, V; Herzog, W

    2014-08-15

    Isometric force after active stretch of muscles is higher than the purely isometric force at the corresponding length. This property is termed residual force enhancement. Active force in skeletal muscle depends on calcium attachment characteristics to the regulatory proteins. Passive force has been shown to influence calcium attachment characteristics, specifically the sarcomere length dependence of calcium sensitivity. Since one of the mechanisms proposed to explain residual force enhancement is the increase in passive force that results from engagement of titin upon activation and stretch, our aim was to test if calcium sensitivity of residual force enhancement was different from that of its corresponding purely isometric contraction and if such a difference was related to the molecular spring titin. Force-pCa curves were established in rabbit psoas skinned fibers for reference and residual force-enhanced states at a sarcomere length of 3.0 μm 1) in a titin-intact condition, 2) after treatment with trypsin to partially eliminate titin, and 3) after treatment with trypsin and osmotic compression with dextran T-500 to decrease the lattice spacing in the absence of titin. The force-pCa curves of residual force enhancement were shifted to the left compared with their corresponding controls in titin-intact fibers, indicating increased calcium sensitivity. No difference in calcium sensitivity was observed between reference and residual force-enhanced contractions in trypsin-treated and osmotically compressed trypsin-treated fibers. Furthermore, calcium sensitivity after osmotic compression was lower than that observed for residual force enhancement in titin-intact skinned fibers. These results suggest that titin-based passive force regulates the increase in calcium sensitivity of residual force enhancement by a mechanism other than reduction of the myofilament lattice spacing.

  15. Simplification and analysis of models of calcium dynamics based on IP3-sensitive calcium channel kinetics.

    PubMed

    Tang, Y; Stephenson, J L; Othmer, H G

    1996-01-01

    We study the models for calcium (Ca) dynamics developed in earlier studies, in each of which the key component is the kinetics of intracellular inositol-1,4,5-trisphosphate-sensitive Ca channels. After rapidly equilibrating steps are eliminated, the channel kinetics in these models are represented by a single differential equation that is linear in the state of the channel. In the reduced kinetic model, the graph of the steady-state fraction of conducting channels as a function of log10(Ca) is a bell-shaped curve. Dynamically, a step increase in inositol-1,4,5-trisphosphate induces an incremental increase in the fraction of conducting channels, whereas a step increase in Ca can either potentiate or inhibit channel activation, depending on the Ca level before and after the increase. The relationships among these models are discussed, and experimental tests to distinguish between them are given. Under certain conditions the models for intracellular calcium dynamics are reduced to the singular perturbed form epsilon dx/d tau = f(x, y, p), dy/d tau = g(x, y, p). Phase-plane analysis is applied to a generic form of these simplified models to show how different types of Ca response, such as excitability, oscillations, and a sustained elevation of Ca, can arise. The generic model can also be used to study frequency encoding of hormonal stimuli, to determine the conditions for stable traveling Ca waves, and to understand the effect of channel properties on the wave speed.

  16. Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells

    PubMed Central

    1988-01-01

    The inactivation of calcium channels in mammalian pituitary tumor cells (GH3) was studied with patch electrodes under voltage clamp in cell- free membrane patches and in dialyzed cells. The calcium current elicited by depolarization from a holding potential of -40 mV passed predominantly through one class of channels previously shown to be modulated by dihydropyridines and cAMP-dependent phosphorylation (Armstrong and Eckert, 1987). When exogenous calcium buffers were omitted from the pipette solution, the macroscopic calcium current through those channels inactivated with a half time of approximately 10 ms to a steady state level 40-75% smaller than the peak. Inactivation was also measured as the reduction in peak current during a test pulse that closely followed a prepulse. Inactivation was largely reduced or eliminated by (a) buffering free calcium in the pipette solution to less than 10(-8) M; (b) replacing extracellular calcium with barium; (c) increasing the prepulse voltage from +10 to +60 mV; or (d) increasing the intracellular concentration of cAMP, either 'directly' with dibutyryl-cAMP or indirectly by activating adenylate cyclase with forskolin or vasoactive intestinal peptide. Thus, inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells only occurs when membrane depolarization leads to calcium ion entry and intracellular accumulation. PMID:2849631

  17. Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles

    PubMed Central

    Perrino, Brian A

    2016-01-01

    An increase in intracellular Ca2+ is the primary trigger of contraction of gastrointestinal (GI) smooth muscles. However, increasing the Ca2+ sensitivity of the myofilaments by elevating myosin light chain phosphorylation also plays an essential role. Inhibiting myosin light chain phosphatase activity with protein kinase C-potentiated phosphatase inhibitor protein-17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation is considered to be the primary mechanism underlying myofilament Ca2+ sensitization. The relative importance of Ca2+ sensitization mechanisms to the diverse patterns of GI motility is likely related to the varied functional roles of GI smooth muscles. Increases in CPI-17 and MYPT1 phosphorylation in response to agonist stimulation regulate myosin light chain phosphatase activity in phasic, tonic, and sphincteric GI smooth muscles. Recent evidence suggests that MYPT1 phosphorylation may also contribute to force generation by reorganization of the actin cytoskeleton. The mechanisms responsible for maintaining constitutive CPI-17 and MYPT1 phosphorylation in GI smooth muscles are still largely unknown. The characteristics of the cell-types comprising the neuroeffector junction lead to fundamental differences between the effects of exogenous agonists and endogenous neurotransmitters on Ca2+ sensitization mechanisms. The contribution of various cell-types within the tunica muscularis to the motor responses of GI organs to neurotransmission must be considered when determining the mechanisms by which Ca2+ sensitization pathways are activated. The signaling pathways regulating Ca2+ sensitization may provide novel therapeutic strategies for controlling GI motility. This article will provide an overview of the current understanding of the biochemical basis for the regulation of Ca2+ sensitization, while also discussing the functional importance to different smooth muscles of the GI tract. PMID:26701920

  18. Sulfhydryl oxidation modifies the calcium dependence of ryanodine-sensitive calcium channels of excitable cells.

    PubMed Central

    Marengo, J J; Hidalgo, C; Bull, R

    1998-01-01

    The calcium dependence of ryanodine-sensitive single calcium channels was studied after fusing with planar lipid bilayers sarcoendoplasmic reticulum vesicles isolated from excitable tissues. Native channels from mammalian or amphibian skeletal muscle displayed three different calcium dependencies, cardiac (C), mammalian skeletal (MS), and low fractional open times (low Po), as reported for channels from brain cortex. Native channels from cardiac muscle presented only the MS and C dependencies. Channels with the MS or low Po behaviors showed bell-shaped calcium dependencies, but the latter had fractional open times of <0.1 at all [Ca2+]. Channels with C calcium dependence were activated by [Ca2+] < 10 microM and were not inhibited by increasing cis [Ca2+] up to 0.5 mM. After oxidation with 2,2'-dithiodipyridine or thimerosal, channels with low Po or MS dependencies increased their activity. These channels modified their calcium dependencies sequentially, from low Po to MS and C, or from MS to C. Reduction with glutathione of channels with C dependence (native or oxidized) decreased their fractional open times in 0.5 mM cis [Ca2+], from near unity to 0.1-0.3. These results show that all native channels displayed at least two calcium dependencies regardless of their origin, and that these changed after treatment with redox reagents. PMID:9512024

  19. Calcium sensitive ring-like oligomers formed by synaptotagmin

    PubMed Central

    Wang, Jing; Bello, Oscar; Auclair, Sarah M.; Wang, Jing; Coleman, Jeff; Pincet, Frederic; Krishnakumar, Shyam S.; Sindelar, Charles V.; Rothman, James E.

    2014-01-01

    The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Here we report an unexpected circular arrangement (ring) of SYT’s cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18–43 nm, corresponding to 11–26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide–sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded. PMID:25201968

  20. Calcium-induced calcium release and gap junctions mediate large-scale calcium waves in olfactory ensheathing cells in situ.

    PubMed

    Stavermann, Maren; Meuth, Patrick; Doengi, Michael; Thyssen, Anne; Deitmer, Joachim W; Lohr, Christian

    2015-08-01

    Olfactory ensheathing cells (OECs) are a specialised type of glial cells, supporting axon growth and guidance during development and regeneration of the olfactory nerve and the nerve layer of the olfactory bulb. We measured calcium signalling in OECs in olfactory bulb in-toto preparations using confocal and epifluorescence microscopy and the calcium indicator Fluo-4. We identified two subpopulations of olfactory bulb OECs: OECs in the outer sublamina of the nerve layer responded to purinergic neurotransmitters such as adenosine triphosphate with calcium transients, while OECs in the inner sublamina of the nerve layer did not respond to neurotransmitters. However, the latter generated spontaneous calcium waves that covered hundreds of cells. These calcium waves persisted in the presence of tetrodotoxin and in calcium-free saline, but were abolished after calcium store depletion with cyclopiazonic acid or inositol trisphosphate receptor blockage with 2-APB. Calcium waves could be triggered by laser photolysis of caged inositol trisphosphate. Blocking purinoceptors with PPADS had no effect on calcium wave propagation, whereas blocking gap junctions with carbenoxolone or meclofenamic acid entirely suppressed calcium waves. Increasing calcium buffer capacity in OECs with NP-EGTA ("caged" Ca(2+)) prevented calcium wave generation, and laser photolysis of NP-EGTA in a small group of OECs resulted in a calcium increase in the irradiated cells followed by a calcium wave. We conclude that calcium waves in OECs can be initiated by calcium-induced calcium release via InsP3 receptors and propagate through gap junctions, while purinergic signalling is not involved.

  1. Intracellular calcium affects prestin's voltage operating point indirectly via turgor-induced membrane tension

    NASA Astrophysics Data System (ADS)

    Song, Lei; Santos-Sacchi, Joseph

    2015-12-01

    Recent identification of a calmodulin binding site within prestin's C-terminus indicates that calcium can significantly alter prestin's operating voltage range as gauged by the Boltzmann parameter Vh (Keller et al., J. Neuroscience, 2014). We reasoned that those experiments may have identified the molecular substrate for the protein's tension sensitivity. In an effort to understand how this may happen, we evaluated the effects of turgor pressure on such shifts produced by calcium. We find that the shifts are induced by calcium's ability to reduce turgor pressure during whole cell voltage clamp recording. Clamping turgor pressure to 1kPa, the cell's normal intracellular pressure, completely counters the calcium effect. Furthermore, following unrestrained shifts, collapsing the cells abolishes induced shifts. We conclude that calcium does not work by direct action on prestin's conformational state. The possibility remains that calcium interaction with prestin alters water movements within the cell, possibly via its anion transport function.

  2. Calcium signaling in UV-induced damage

    NASA Astrophysics Data System (ADS)

    Sun, Dan; Zhang, Su-juan; Li, Yuan-yuan; Qu, Ying; Ren, Zhao-Yu

    2007-05-01

    Hepa1-6 cells were irradiated with UV and incubated for varying periods of time. [Ca 2+] i (intracellular calcium concentration) of UV-irradiated cell was measured by ratio fluorescence imaging system. The comet assay was used to determine DNA damage. During the UVB-irradiation, [Ca 2+] i had an ascending tendency from 0.88 J/m2 to 92.4J/m2. Comet assay instant test indicated that when the irradiation dosage was above 0.88J/m2, DNA damage was observed. Even after approximate 2 h of incubation, DNA damage was still not detected by 0.88J/m2 of UVB irradiation. During UVA-irradiation, the elevation of [Ca 2+] i was not dose-dependent in a range of 1200 J/m2-6000J/m2 and DNA damage was not observed by comet assay. These results suggested that several intracellular UV receptors might induce [Ca 2+] i rising by absorption of the UV energy. Just [Ca 2+] i rising can't induce DNA damage certainly, it is very likely that the breakdown of calcium steady state induces DNA damage.u

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

    PubMed

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

    2007-10-01

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

  4. Detection of calcium activity in human monocytes by the fura-2 fluorescence method: in vitro differentiation sensitizes cells to dihydropyridine calcium channel modulators

    NASA Astrophysics Data System (ADS)

    Oraevsky, Alexander A.; Cabello, Olga A.; Shan, Qin; Tittel, Frank K.; Henry, Philip D.

    1994-07-01

    Dihydropyridine (DHP) calcium channel blockers have been shown to suppress atherogenesis in various species and controlled angiographic trials suggest that these drugs may retard the progression of occlusive coronary disease in humans. Because mononuclear leukocytes play a key role in the formation of early and advanced atheromatous lesions, we determined effects of DHP calcium channel modulators on calcium uptake by cells of the monocytic lineage. Human peripheral blood monocytes were evaluated before and after undergoing in vitro differentiation induced by two days of culture with fetal calf serum and FMLP. Changes in intracellular calcium activity were estimated with fura-2, a fluorescent calcium indicator. Freshly isolated (unactivated) monocytes were insensitive to DHP drugs both in the presence and absence of high potassium membrane depolarization. In contrast, nisoldipine, a DHP calcium channel blocker, and BAY K 8644, a DHP calcium channel activator, decreased and increased calcium uptake by KC1-depolarized differentiated monocytes. Results suggest that differentiation of monocytes to macrophages may involve a change in the expression and/or regulation of DHP- sensitive calcium channels.

  5. Influence of calcium sources on microbially induced calcium carbonate precipitation by Bacillus sp. CR2.

    PubMed

    Achal, Varenyam; Pan, Xiangliang

    2014-05-01

    Stimulation of microbially induced calcium carbonate precipitation (MICCP) is likely to be influenced by calcium sources. In order to study such influences, we performed MICCP using Bacillus sp. CR2 in nutrient broth containing urea, supplemented with different calcium sources (calcium chloride, calcium oxide, calcium acetate and calcium nitrate). The experiment lasted 7 days, during which bacterial growth, urease activity, calcite production and pH were measured. Our results showed that calcium chloride is the better calcium source for MICCP process, since it provides higher urease activity and more calcite production. The influences of calcium sources on MICCP were further studied using Fourier transform-infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses. These analyses confirmed that the precipitate formed was CaCO3 and composed of predominantly calcite crystals with a little amount of aragonite and vaterite crystals. The maximum yield of calcite precipitation was achievable with calcium chloride followed by calcium nitrate as a calcium source. The results of present study may be applicable to media preparation during efficient MICCP process.

  6. A Novel Phosphorylation Site, Serine 199, in the C-Terminus of Cardiac Troponin I Regulates Calcium Sensitivity and Susceptibility to Calpain-Induced Proteolysis

    PubMed Central

    Wijnker, Paul J.M.; Li, Yuejin; Zhang, Pingbo; Foster, D. Brian; dos Remedios, Cris; Van Eyk, Jennifer E.; Stienen, Ger J.M.; Murphy, Anne M.; van der Velden, Jolanda

    2015-01-01

    Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca2+] and at sarcomere lengths of 1.8 and 2.2 μm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca2+-sensitivity (pCa50) at 2.2 μm was significantly higher in 199D (pCa50=5.79±0.01) compared to 199A (pCa50=5.65±0.01) and Wt (pCa50=5.66±0.02) at ~63% cTn exchange. Myofilament Ca2+-sensitivity was significantly higher even with only 5.9±2.5% 199D exchange compared to 199A, and saturated at 12.3±2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~6%) increase myofilament Ca2+-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI. PMID:25771144

  7. Spreading depression induces expression of calcium-independent protein kinase C subspecies in ischaemia-sensitive cortical layers: regulation by N-methyl-D-aspartate receptors and glucocorticoids.

    PubMed

    Koponen, S; Keinänen, R; Roivainen, R; Hirvonen, T; Närhi, M; Chan, P H; Koistinaho, J

    1999-01-01

    Spreading depression is a wave of sustained depolarization challenging the energy metabolism of the cells without causing irreversible damage. In the ischaemic brain, sreading depression-like depolarization contributes to the evolution of ischaemia to infarction. The depolarization is propagated by activation of N-methyl-D-aspartate receptors, but changes in signal transduction downstream of the receptors are not known. Because protein phosphorylation is a general mechanism whereby most cellular processes are regulated, and inhibition of N-methyl-D-aspartate receptors or protein kinase C is neuroprotective, the expression of protein kinase C subspecies in spreading depression was examined. Cortical treatment with KCl induced an upregulation of protein kinase Cdelta and zeta messenger RNA at 4 and 8 h, whereas protein kinase Calpha, beta, gamma and epsilon did not show significant changes. The gene induction was the strongest in layers 2 and 3, and was followed by an increased number of protein kinase Cdelta-immunoreactive neurons. Protein kinase Cdelta and zeta inductions were inhibited by pretreatment with an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate, which also blocked spreading depression propagation, and with dexamethasone, which acted without blocking the propagation. Quinacrine, a phospholipase A2 inhibitor, reduced only protein kinase C5 induction. In addition, N(G)(-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, did not influence protein kinase Cdelta or zeta induction, whereas 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist, and the cyclo-oxygenase inhibitors indomethacin and diclophenac tended to increase gene expression. The data show that cortical spreading depression induces Ca2(+)-independent protein kinase C subspecies delta and zeta, but not Ca(2+)-dependent subspecies, through activation of N-methyl-D-aspartate receptors and

  8. Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress.

    PubMed

    Torres, Mauricio; Castillo, Karen; Armisén, Ricardo; Stutzin, Andrés; Soto, Claudio; Hetz, Claudio

    2010-12-29

    Prion-related disorders (PrDs) are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrP(RES). Altered endoplasmic reticulum (ER) homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrP(RES). Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrP(RES) and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models.

  9. Reversible loss of gravitropic sensitivity in maize roots after tip application of calcium chelators

    NASA Technical Reports Server (NTRS)

    Lee, J. S.; Mulkey, T. J.; Evans, M. L.

    1983-01-01

    The application of calcium chelating agents (EDTA or EGTA) to the tips of maize roots caused a loss of gravitropic sensitivity. When the chelator was replaced with calcium chloride, gravitropic sensitivity was restored. Asymmetric application of calcium chloride near the tip of a vertical root caused curvature toward the calcium source. When the calcium was applied to the upper surface of the tip of a root oriented horizontally, the root curved upward even though control roots exhibited strong downward curvature. Application of calcium chloride to the tips of decapped roots, which are known to be gravitropically insensitive, did not restore gravitropic sensitivity. However, asymmetric application of calcium chloride near the tips of decapped roots caused curvature toward the calcium source. Calcium may play a key role in linking gravity detection to gravitropic curvature in roots.

  10. Calcium channel blocker-induced gingival enlargement.

    PubMed

    Livada, R; Shiloah, J

    2014-01-01

    Despite the popularity and wide acceptance of the calcium channel blockers (CCBs) by the medical community, their oral impact is rarely recognized or discussed. CCBs, as a group, have been frequently implicated as an etiologic factor for a common oral condition seen among patients seeking dental care: drug-induced gingival enlargement or overgrowth. This enlargement can be localized or generalized, and can range from mild to extremely severe, affecting patient's appearance and function. Treatment options for these patients include cessation of the offending drug and substitution with another class of antihypertensive medication to prevent recurrence of the lesions. In addition, depending on the severity of the gingival overgrowth, nonsurgical and surgical periodontal therapy may be required. The overall objective of this article is to review the etiology and known risk factors of these lesions, their clinical manifestations and periodontal management.

  11. Acute Calcium Ingestion Attenuates Exercise-induced Disruption of Calcium Homeostasis

    PubMed Central

    Barry, Daniel W; Hansen, Kent C; Van Pelt, Rachael E; Witten, Michael; Wolfe, Pamela; Kohrt, Wendy M

    2011-01-01

    Purpose Exercise is associated with a decrease in bone mineral density under certain conditions. One potential mechanism is increased bone resorption due to an exercise-induced increase in parathyroid hormone (PTH), possibly triggered by dermal calcium loss. The purpose of this investigation was to determine whether calcium supplementation either before or during exercise attenuates exercise-induced increases in PTH and C-terminal telopeptide of type I collagen (CTX; a marker of bone resorption). Methods Male endurance athletes (n=20) completed three 35-km cycling time trials under differing calcium supplementation conditions: 1) 1000 mg calcium 20 minutes before exercise and placebo during, 2) placebo before and 250 mg calcium every 15 minutes during exercise (1000 mg total), or 3) placebo before and during exercise. Calcium was delivered in a 1000 mg/L solution. Supplementation was double-blinded and trials were performed in random order. PTH, CTX, bone-specific alkaline phosphatase (BAP; a marker of bone formation), and ionized calcium (iCa) were measured before and immediately after exercise. Results CTX increased and iCa decreased similarly in response to exercise under all test conditions. When compared to placebo, calcium supplementation before exercise attenuated the increase in PTH (55.8 ± 15.0 vs. 74.0 ± 14.2; mean ± SE; p=0.04); there was a similar trend (58.0 ± 17.4; p=0.07) for calcium supplementation during exercise. There were no effects of calcium on changes in CTX, BAP, and iCa. Conclusions Calcium supplementation before exercise attenuated the disruption of PTH. Further research is needed to determine the effects of repeated increases in PTH and CTX on bone (i.e., exercise training), and whether calcium supplementation can diminish any exercise-induced demineralization. PMID:20798655

  12. New inotropic concepts: rationale for and differences between calcium sensitizers and phosphodiesterase inhibitors.

    PubMed

    Holubarsch, C

    1997-01-01

    Positive inotropic compounds may be harmful in the long-term treatment of chronic congestive heart failure because they may induce a calcium overload, unwanted changes in cross-bridge kinetics and an acceleration in heart rate. As a result of all three alterations, energy consumption would be increased. Different pharmacological modes of action may have different effects on the molecular mechanisms underlying the positive inotropic effect, and hence on myocardial energy consumption. Therefore, we studied the effects of a variety of cardiotonic agents on the heat released from small guinea pig papillary muscles contracting isometrically at an experimental temperature of 21 degrees C and a stimulation frequency of 12 per minute using rapid antimony-bismuth thermopiles. We were able to define the economy of muscle contraction, which was lowest with phosphodiesterase inhibitors and highest with calcium sensitizers. Compared with an increase in extracellular calcium concentration, beta 1-adrenoceptor stimulators and phosphodiesterase inhibitors profoundly decrease the economy of myocardial contraction, and calcium-sensitizers (pimobendan and EMD-53998) slightly increase myocardial economy, whereas ouabain and the calcium channel agonist BAY K 8644 have no effect on this parameter. In addition, we provide evidence that acceleration of heart rate may be harmful not only from an energetic point of view: an increase in heart rate may also decrease the contractility of the failing human myocardium (inverse force-frequency relationship). Taking these observations into consideration, an "optimal' positive inotropic compound should have no, or even negative, chronotropic effects, should not be mediated by increases in calcium transients, and should decelerate, rather than accelerate, cross-bridge kinetics.

  13. Sensitive red protein calcium indicators for imaging neural activity

    PubMed Central

    Dana, Hod; Mohar, Boaz; Sun, Yi; Narayan, Sujatha; Gordus, Andrew; Hasseman, Jeremy P; Tsegaye, Getahun; Holt, Graham T; Hu, Amy; Walpita, Deepika; Patel, Ronak; Macklin, John J; Bargmann, Cornelia I; Ahrens, Misha B; Schreiter, Eric R; Jayaraman, Vivek; Looger, Loren L; Svoboda, Karel; Kim, Douglas S

    2016-01-01

    Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging. DOI: http://dx.doi.org/10.7554/eLife.12727.001 PMID:27011354

  14. PYK2: A Calcium-sensitive Protein Tyrosine Kinase Activated in Response to Fertilization of the Zebrafish Oocyte

    PubMed Central

    Sharma, Dipika; Kinsey, William H.

    2012-01-01

    Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone. PMID:23084926

  15. PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

    PubMed

    Sharma, Dipika; Kinsey, William H

    2013-01-01

    Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone.

  16. Mechanically Induced Intercellular Calcium Communication in Confined Endothelial Structures

    PubMed Central

    Junkin, Michael; Lu, Yi; Long, Juexuan; Deymier, Pierre A.; Hoying, James B.; Wong, Pak Kin

    2012-01-01

    Calcium signaling in the diverse vascular structures is regulated by a wide range of mechanical and biochemical factors to maintain essential physiological functions of the vasculature. To properly transmit information, the intercellular calcium communication mechanism must be robust against various conditions in the cellular microenvironment. Using plasma lithography geometric confinement, we investigate mechanically induced calcium wave propagation in networks of human umbilical vein endothelial cells organized. Endothelial cell networks with confined architectures were stimulated at the single cell level, including using capacitive force probes. Calcium wave propagation in the network was observed using fluorescence calcium imaging. We show that mechanically induced calcium signaling in the endothelial networks is dynamically regulated against a wide range of probing forces and repeated stimulations. The calcium wave is able to propagate consistently in various dimensions from monolayers to individual cell chains, and in different topologies from linear patterns to cell junctions. Our results reveal that calcium signaling provides a robust mechanism for cell-cell communication in networks of endothelial cells despite the diversity of the microenvironmental inputs and complexity of vascular structures. PMID:23267827

  17. 2-Chloroadenosine reduces the N calcium current of cultured mouse sensory neurones in a pertussis toxin-sensitive manner.

    PubMed Central

    Gross, R A; Macdonald, R L; Ryan-Jastrow, T

    1989-01-01

    1. The adenosine analogue 2-chloroadenosine (CADO) reduced the duration of calcium-dependent action potentials (CAPs) in mouse dorsal root ganglion (DRG) neurones in culture, by reducing voltage-activated calcium conductance (Macdonald, Skerritt & Werz, 1986). Using the single-electrode voltage clamp technique, we recorded three calcium current components in these neurones, the transient low-threshold (T), transient high-threshold (N) and slowly inactivating high-threshold (L) currents, as described previously (Nowycky, Fox & Tsien, 1985; Gross & Macdonald, 1987). CADO (100 microM) had no effect on the isolated T and L currents. In contrast, CADO reduced calcium currents evoked at clamp potentials positive to -20 mV from holding potentials (Vh) near the resting membrane potential; under these conditions, the calcium current consisted primarily of N and L calcium current components. 2. This effect of CADO was not voltage dependent. CADO reduced the magnitude of the calcium current without affecting the voltage dependence of the calcium current-voltage relation. In addition, similar reductions of calcium current were observed when currents were evoked from Vh of -60 or -80 mV. 3. In order to determine if a guanine nucleotide-binding (G) protein was involved in the CADO effect on calcium current, cultures were pre-treated with pertussis toxin (PT) for at least four hours. PT (100 ng/ml) reduced or abolished the CADO-induced reduction of CAP duration and calcium current. 4. Since CADO inhibits adenylate cyclase through the PT-sensitive G protein, Gi, we compared the effects of CADO and 8-Br-adenosine 3',5'-cyclic-monophosphate (8-Br-cyclic AMP) on calcium current. The effect of 8-Br-cyclic AMP was voltage dependent, unlike that of CADO. 8-Br-cyclic AMP reduced calcium currents evoked from Vh = -65 mV, but had no effect on currents evoked from Vh = -85 mV. 5. We conclude that the adenosine agonist CADO reduced CAP duration in mouse DRG neurones by selectively reducing

  18. Calcium-induced calcium release contributes to synaptic release from mouse rod photoreceptors

    PubMed Central

    Babai, N.; Morgans, C. W.; Thoreson, WB.

    2009-01-01

    We tested whether calcium-induced calcium release (CICR) contributes to synaptic release from rods in mammalian retina. Electron micrographs and immunofluorescent double labeling for the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) and synaptic ribbon protein, ribeye, showed a close association between ER and synaptic ribbons in mouse rod terminals. Stimulating CICR with 10 μM ryanodine evoked Ca2+ increases in rod terminals from mouse retinal slices visualized using confocal microscopy with the Ca2+-sensitive dye, Fluo-4. Ryanodine also stimulated membrane depolarization of individual mouse rods. Inhibiting CICR with a high concentration of ryanodine (100 μM) reduced the ERG b-wave but not a-wave consistent with inhibition of synaptic transmission from rods. Ryanodine (100 μM) also inhibited light-evoked voltage responses of individual rod bipolar cells (RBCs) and presumptive horizontal cells recorded with perforated patch recording techniques. A presynaptic site of action for ryanodine's effects is further indicated by the finding that ryanodine (100 μM) did not alter currents evoked in voltage-clamped RBCs by puffing the mGluR6 antagonist, (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG), onto bipolar cell dendrites in the presence of the mGluR6 agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4). Ryanodine (100 μM) also inhibited glutamatergic outward currents in RBCs evoked by electrical stimulation of rods using electrodes placed in the outer segment layer. Together, these results indicate that, like amphibian retina, CICR contributes to synaptic release from mammalian (mouse) rods. By boosting synaptic release in darkness, CICR may improve the detection of small luminance changes by post-synaptic neurons. PMID:19932743

  19. Neutron-Induced Cross Sections Measurements of Calcium

    SciTech Connect

    Guber, Klaus H; Kopecky, S.; Schillebeeckx, P.; Kauwenberghs, K.; Siegler, P.

    2013-01-01

    To support the US Department of Energy Nuclear Criticality Safety Program neutron induced cross section experiments were performed at the Geel Electron Linear Accelerator of the Institute for Reference Material and Measurements of the Joint Research Centers, European Union. Neutron capture and transmission measurements were carried out using a metallic calcium sample. The obtained data will be used for a new calcium evaluation, which will be submitted with its covariances to the ENDBF/B nuclear data base.

  20. Purification and reconstitution of the calcium antagonist receptor of the voltage-sensitive calcium channel

    SciTech Connect

    Curtis, B.M.

    1986-01-01

    Treatment with digitonin solubilized the calcium antagonist receptor as a stable complex with (/sup 3/H)nitrendipine from rat brain membranes. The solubilized complex retains allosteric coupling to binding sites for diltiazem, verapamil, and inorganic calcium antagonist sites. The calcium antagonist receptor from cardiac sarcolemma and the transverse-tubule membrane of skeletal muscle is also efficiently solubilized with digitonin and the receptor in all three tissues is a large glycoprotein with a sedimentation coefficient of 20 S. The T-tubule calcium antagonist receptor complex was extensively purified by a combination of chromatography on WGA-Sepharose, ion exchange chromatography, and sedimentation on sucrose gradients to yield preparations estimated to be 41% homogeneous by specific activity and 63% homogeneous by SDS gel electrophoresis. Analysis of SDS gels detect three polypeptides termed ..cap alpha..(Mr 135,000), ..beta..(Mr 50,000), and ..gamma..(Mr 32,000) as noncovalently associated subunits of the calcium antagonist receptor. The ..cap alpha.. and ..gamma.. subunits are glycosylated polypeptides, and the molecular weight of the core polypeptides are 108,000 and 24,000 respectively. The calcium antagonist receptor was reconstituted into a phospholipid bilayer by adding CHAPS and exogeneous lipid to the purified receptor followed by rapid detergent removal. This procedure resulted in the incorporation of 45% of the calcium antagonist receptor into closed phospholipid vesicles. Data suggests that the ..cap alpha.., ..beta.., and ..gamma.. subunits of the T-tubule calcium antagonist receptor are sufficient to form a functional calcium channel.

  1. Nefopam inhibits calcium influx, cGMP formation, and NMDA receptor-dependent neurotoxicity following activation of voltage sensitive calcium channels.

    PubMed

    Novelli, A; Díaz-Trelles, R; Groppetti, A; Fernández-Sánchez, M T

    2005-03-01

    Nefopam hydrochloride is a potent non sedative benzoxazocine analgesic that possesses a profile distinct from that of anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanism remains unclear. We have investigated the actions of nefopam on voltage sensitive calcium channels and calcium-mediated pathways. We found that nefopam prevented N-methyl-D-aspartate (NMDA)-mediated excitotoxicity following stimulation of L-type voltage sensitive calcium channels by the specific agonist BayK8644. Nefopam protection was concentration-dependent. 47 muM nefopam provided 50% protection while full neuroprotection was achieved at 100 muM nefopam. Neuroprotection was associated with a 73% reduction in the BayK8644-induced increase in intracellular calcium concentration. Nefopam also inhibited intracellular cGMP formation following BayK8644 in a concentration-dependent manner, 100 muM nefopam providing full inhibition of cGMP synthesis and 58 muM allowing 50% cGMP formation. Nefopam reduced NMDA receptor-mediated cGMP formation resulting from the release of glutamate following activation of channels by BayK8644. Finally, we also showed that nefopam effectively reduced cGMP formation following stimulation of cultures with domoic acid, while not providing neuroprotection against domoic acid. Thus, the novel action of nefopam we report here may be important both for its central analgesic effects and for its potential therapeutic use in neurological and neuropsychiatric disorders involving an excessive glutamate release.

  2. 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

  3. Calcium signal induced by mechanical perturbation of osteoclasts.

    PubMed

    Xia, S L; Ferrier, J

    1995-06-01

    Multinucleated osteoclasts from rabbit long bone, 1-6 days in culture, respond to mechanical perturbation with a transient increase of intracellular calcium concentration ([Ca2+]i), as measured with the fluorescent indicator fluo-3 on a confocal laser scanning microscope. In experiments with different extracellular calcium concentrations (from 11.8 mM to calcium-free), the incidence, the magnitude, and the duration of [Ca2+]i responses decreases with decreasing bathing [Ca2+]. Following mechanical perturbation, a thapsigargin-induced [Ca2+]i response has a lower magnitude than the thapsigargin-induced response without mechanical perturbation. In thapsigargin-pretreated osteoclasts the mechanical perturbation-induced rise in [Ca2+]i is larger and longer than in control cells. Ni2+ inhibits the incidence and decreases both the magnitude and the duration of the responses, while nifedipine, verapamil, and Gd3+ have no effect. These measurements show that rabbit osteoclasts transduce a mechanical perturbation of the cell membrane into a [Ca2+]i signal via both a calcium influx and an internal calcium release.

  4. Effectiveness of nano-calcium phosphate paste on sensitivity during and after bleaching: a randomized clinical trial.

    PubMed

    Loguercio, Alessandro Dourado; Tay, Lidia Yileng; Herrera, Daniel Rodrigo; Bauer, Jose; Reis, Alessandra

    2015-01-01

    The study aimed to evaluate the effectiveness of in-office bleaching and associated tooth sensitivity on application of nano-calcium phosphate paste as desensitizing agent. Bleaching was performed with 35% hydrogen peroxide gel in 40 patients who were randomly divided into placebo and nano-calcium phosphate paste groups. Bleaching efficacy (BE) was evaluated using a value-oriented Vita shade guide. Tooth sensitivity was recorded using a numeric rating scale (0-4) during bleaching and up to 48 h after each session. The primary outcome of absolute risk of tooth sensitivity was compared using the Fisher's exact test (α = 0.05). The intensity of tooth sensitivity and the efficacy of in-office bleaching were also statistically evaluated. No significant differences in absolute risk and intensity of tooth sensitivity were detected between the groups (p = 1.0 and p = 0.53, respectively). BE was also found to be similar between the groups (p = 0.67). Although the use of a nano-calcium phosphate paste associated with fluoride and potassium nitrate did not influence the whitening outcome, but it also did not reduce bleaching-induced tooth sensitivity. PMID:26313348

  5. TRICHLOROETHYLENE IHIBITS VOLTAGE-SENSITIVE CALCIUM CURRENTS IN DIFFERENTIATED PC 12 CELLS.

    EPA Science Inventory

    ABSTRACT BODY: It has been demonstrated recently that volatile organic compounds (VOCs)such as toluene, perchloroethylene and trichloroethylene inhibit function of voltage-sensitive calcium channels (VSSC). Such actions are hypothesized to contribute to the acute neurotoxicity of...

  6. Calcium regulation in aortic smooth muscle cells during the initial phase of tunicamycin-induced endo/sarcoplasmic reticulum stress.

    PubMed

    Ziomek, Gabriela; Cheraghi Zanjani, Parisa; Arman, Darian; van Breemen, Cornelis; Esfandiarei, Mitra

    2014-07-15

    Endo/sarcoplasmic reticulum stress and the unfolded protein response have been implicated as underlying mechanisms of cell death in many pathological conditions. We have confirmed that long-term exposure to 10µM tunicamycin induced the endo/sarcoplasmic reticulum stress in cultured vascular smooth muscle cells. Since tunicamycin is reported to induce the stress response by inhibiting protein glycosylation, we attempted to investigate a causal link between accumulation of unfolded proteins and dysregulation of cellular calcium transport. However, we found that tunicamycin caused an immediate release of calcium from the endo/sarcoplasmic reticulum, which was sensitive to thapsigargin, and an influx of calcium through the plasma membrane, resulting in a significant increase in cytoplasmic calcium and depletion of endo/sarcoplasmic reticulum calcium. Furthermore, we observed that tunicamycin also induced contraction in intact vascular smooth muscle. By applying established procedures and antagonists, we established that tunicamycin did not directly activate physiological calcium channels, such as store-operated channels, voltage gated calcium channels, ryanodine receptors or inositol trisphosphate receptors. Instead, we found that its effects on cellular calcium fluxes closely resembled those of the known calcium ionophore, ionomycin. We have concluded that tunicamycin directly permeabilizes the plasma membrane and endo/sarcoplasmic reticulum to calcium, and is, therefore, inappropriate for studying the relationship between accumulation of unfolded proteins and endo/sarcoplasmic reticulum calcium dysregulation during the endo/sarcoplasmic reticulum stress response. In contrast, we also report that two other well-known endo/sarcoplasmic reticulum stress inducers, brefeldin A and dithiothreitol, did not exhibit similar increases in calcium permeability.

  7. Light-induced ejection of calcium atoms from polymer surfaces

    NASA Astrophysics Data System (ADS)

    Mango, F.; Maccioni, E.

    2008-12-01

    Laser-induced fluorescence (LIF) of calcium atoms at room temperature has been observed in a polydimethylsiloxane (PDMS) coated cell when the walls are illuminated with non resonant visible light. Ca atomic density in the gas phase, monitored by the LIF, is much higher than normal room-temperature vapour pressure of calcium. In past years photon-stimulated desorption (PSD) was observed for several alkali metals that adsorbed to solid films of PDMS polymers. High yields of photo-desorbed atoms (and molecules in the case of sodium) can be induced, at room temperature and below, by weak intensity radiation. The desorption is characterised by a frequency threshold, whereas any power threshold is undetectable. The calcium photo-ejection is characterised both by a frequency threshold (about 18 500 cm-1) and by an observable power threshold (whose value becomes lower when the photo-ejecting light wavelength decreases).

  8. Verapamil induces calcium influx in the trachea.

    PubMed

    Mustafa, Seham; Abdulraheem, Abdulkareem; Alsughayer, Abdulhakeem

    2008-04-01

    Verapamil, a Ca(2+) entry blocker, can induce bronchorelaxation and bronchoconstriction. The mechanism of verapamil-induced bronchoconstriction is poorly understood. The present study determines the direct effect of verapamil on smooth muscle of isolated ovine airways and analyzes the mechanisms involved. Isolated tracheal strips were suspended in organ baths containing Krebs solution for isometric tension recording. Tissue responses to verapamil as assessed by basal tone were examined in the presence or absence of epithelium. The effects of verapamil on carbachol and cooling-induced contraction were also recorded. Measurement of unidirectional fluxes was carried out using (45)Ca(2+) in the absence or presence of verapamil. Verapamil induced contractions of basal tracheal smooth muscle that were proportional to its concentrations. Removal of epithelium did not affect the verapamil contractile effect. Verapamil-induced contractions were abolished in Ca(2+)-free Krebs solution containing 2 mM EGTA. Verapamil increased the (45)Ca(2+) influx into the tracheal smooth muscle. It caused relaxation of the muscle tone induced by carbachol or KCl, but it potentiated the effect of cooling-induced contraction. Verapamil induced Ca(2+) influx that may lead to bronchoconstriction. These results proved that verapamil may worsen bronchoconstriction; therefore verapamil should be used with caution in asthmatic individuals. PMID:18431040

  9. Oxidative stress-induced calcium signalling in Aspergillus nidulans.

    PubMed

    Greene, Vilma; Cao, Hong; Schanne, Francis A X; Bartelt, Diana C

    2002-05-01

    The effects of oxidative stress on levels of calcium ion (Ca(2+)) in Aspergillus nidulans were measured using strains expressing aequorin in the cytoplasm (Aeq(cyt)) and mitochondria (Aeq(mt)). When oxidative stress was induced by exposure to 10-mM H(2)O(2), the mitochondrial calcium response (Ca(mt)(2+)) was greater than the change in cytoplasmic calcium (Ca(c)(2+)). The Ca(mt)(2+) response to H(2)O(2) was dose dependent, while the increase in [Ca(c)(2+)] did not change with increasing H(2)O(2). The increase in both [Ca(c)(2+)] and [Ca(mt)(2+)] in response to oxidative stress was enhanced by exposure of cells to Ca(2+). The presence of chelator in the external medium only partially inhibited the Ca(mt)(2+) and Ca(c)(2+) responses to oxidative stress. Reagents that alter calcium fluxes had varied effects on the Ca(mt)(2+) response to peroxide. Ruthenium red blocked the increase in [Ca(mt)(2+)], while neomycin caused an even greater increase in [Ca(mt)(2+)]. Treatment with ruthenium red and neomycin had no effect on the Ca(c)(2+) response. Bafilomycin A and oligomycin had no effect on either the mitochondrial or cytoplasmic response. Inhibitors of both voltage-regulated calcium channels and intracellular calcium release channels inhibited the Ca(2+)-dependent component of the Ca(mt)(2+) response to oxidative stress. We conclude that the more significant Ca(2+) response to oxidative stress occurs in the mitochondria and that both intracellular and extracellular calcium pools can contribute to the increases in [Ca(c)(2+)] and [Ca(mt)(2+)] induced by oxidative stress.

  10. Suppression of store overload-induced calcium release by hydroxylated metabolites of carvedilol.

    PubMed

    Malig, Thomas; Xiao, Zhichao; Chen, S R Wayne; Back, Thomas G

    2016-01-01

    Carvedilol is a drug widely used in the treatment of heart failure and associated cardiac arrhythmias. A unique action of carvedilol is its suppression of store overload-induced calcium release (SOICR) through the cardiac ryanodine receptor (RyR2), which can trigger ventricular arrhythmias. Since the effects of carvedilol metabolites on SOICR have not yet been investigated, three carvedilol metabolites hydroxylated at the 3-, 4' and 5'-positions were synthesized and assayed for SOICR inhibition in mutant HEK 293 cells expressing the RyR2 mutant R4496C. This cell line is especially prone to SOICR and calcium release through the defective RyR2 channel was measured with a calcium-sensitive fluorescent dye. These results revealed that the 3- and 4'-hydroxy derivatives are slightly more effective than carvedilol in suppressing SOICR, while the 5'-analog proved slightly less active. Metabolic deactivation of carvedilol via these hydroxylation pathways is therefore insignificant.

  11. Deuterium oxide normalizes blood pressure and vascular calcium uptake in Dahl salt-sensitive hypertensive rats

    SciTech Connect

    Vasdev, S.; Prabhakaran, V.; Sampson, C.A. )

    1990-02-01

    This study examined the effect of 25% deuterium oxide in drinking water on systolic blood pressure, uptakes of calcium, and rubidium 86 by aortas of Dahl salt-sensitive rats on 0.4% (low) and 8% (high) sodium chloride (salt) diet. Twenty-four rats were divided into four groups. Groups I and II were on the low salt diet and groups III and IV on the high salt diet from 6 weeks of age. Additionally, at 10 weeks of age groups I and III were placed on 100% water and groups II and IV on 25% deuterium oxide. At 14 weeks, systolic blood pressure, uptakes of calcium, and rubidium 86 by aortas were significantly higher (p less than 0.01) in rats on the high salt diet as compared with those on the low salt diet. Deuterium oxide intake normalized systolic blood pressure and aortic calcium uptake but not aortic rubidium 86 uptake in hypertensive rats on the high salt diet. Deuterium oxide had no effect on blood pressure or aortic calcium uptake in rats on the low salt diet. The parallel increase in systolic blood pressure and vascular calcium uptake suggests that increased calcium uptake mechanisms are associated with hypertension in salt-sensitive Dahl rats. Furthermore, deuterium oxide appears to normalize elevated blood pressure in salt-sensitive hypertensive rats by normalizing elevated vascular (aortic) calcium uptake.

  12. Abscisic acid induces a cytosolic calcium decrease in barley aleurone protoplasts.

    PubMed

    Wang, M; Van Duijn, B; Schram, A W

    1991-01-14

    Cytosolic calcium concentrations (Cai) of barley aleurone protoplasts after stimulation with the plant hormone abscisic acid (ABA) were measured by using the calcium-sensitive fluorescent dye Indo-1. The measured basal Cai is about 200 nM. Stimulation with ABA induces a strong dose-dependent decrease in Cai to a minimal value of about 50 nM. This decrease occurs within 5 s. The Ca2+ antagonists La3+ and Cd2+ inhibit the ABA-induced Cai decrease in a dose-dependent manner, while the Ca2+ channel blockers verapamil and nifedipine give no inhibition. The induction of Cai decrease by ABA is consistent with activation of the plasma membrane Ca2(+)-ATPase by ABA. The possible role of this ABA-induced Cai decrease in ABA signal transduction and in counteracting the effects of gibberellic acid are discussed.

  13. Involvement of the Sodium-Calcium exchanger 3 (NCX3) in ziram-induced calcium dysregulation and toxicity

    PubMed Central

    Jin, J.; Lao, A.J.; Katsura, M.; Caputo, A.; Schweizer, F. E.; Sokolow, S.

    2014-01-01

    Ziram is a dimethyldithiocarbamate fungicide which can cause intraneuronal calcium (Ca2+) dysregulation and subsequently neuronal death. The signaling mechanisms underlying ziram-induced Ca2+ dyshomeostasis and neurotoxicity are not fully understood. NCX3 is the third isoform of the sodium-calcium exchanger (NCX) family and plays an important role in regulating Ca2+ homeostasis in excitable cells. We previously generated a mouse model deficient for the sodium-calcium exchanger 3 and showed that NCX3 is protective against ischemic damage. In the present study, we aim to examine whether NCX3 exerts a similar role against toxicological injury caused by the pesticide ziram. Our data show baby hamster kidney (BHK) cells stably transfected with NCX3 (BHK-NCX3) are more susceptible to ziram toxicity than cells transfected with the empty vector (BHK-WT). Increased toxicity in BHK-NCX3 was associated with a rapid rise in cytosolic Ca2+ concentration [Ca2+i] induced by ziram. Profound mitochondrial dysfunction and ATP depletion were also observed in BHK-NCX3 cells following treatment with ziram. Lastly, primary dopaminergic neurons lacking NCX3 (NCX3−/−) were less sensitive to ziram neurotoxicity than wildtype control dopaminergic neurons. These results demonstrate that NCX3 genetic deletion protects against ziram-induced neurotoxicity and suggest NCX3 and its downstream molecular pathways as key factors involved in ziram toxicity. Our study identifies new molecular events through which pesticides (e.g. ziram) can lead to pathological features of degenerative diseases such as Parkinson’s disease and indicates new targets to slow down neuronal degeneration. PMID:25284465

  14. Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells.

    PubMed

    Freitas, Hercules R; Ferraz, Gabriel; Ferreira, Gustavo C; Ribeiro-Resende, Victor T; Chiarini, Luciana B; do Nascimento, José Luiz M; Matos Oliveira, Karen Renata H; Pereira, Tiago de Lima; Ferreira, Leonardo G B; Kubrusly, Regina C; Faria, Robson X; Herculano, Anderson Manoel; Reis, Ricardo A de Melo

    2016-01-01

    Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1-10 mM) showed that 5-10 mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50 mM KCl (labeled as βIII tubulin positive cells). BBG 100 nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70 μM and MK-801 20 μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5 mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit. PMID:27078878

  15. Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells

    PubMed Central

    Freitas, Hercules R.; Ferraz, Gabriel; Ferreira, Gustavo C.; Ribeiro-Resende, Victor T.; Chiarini, Luciana B.; do Nascimento, José Luiz M.; Matos Oliveira, Karen Renata H.; Pereira, Tiago de Lima; Ferreira, Leonardo G. B.; Kubrusly, Regina C.; Faria, Robson X.

    2016-01-01

    Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1–10mM) showed that 5–10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as βIII tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit. PMID:27078878

  16. Calcium-induced conidiation in Penicillium cyclopium: calcium triggers cytosolic alkalinization at the hyphal tip.

    PubMed Central

    Roncal, T; Ugalde, U O; Irastorza, A

    1993-01-01

    Addition of Ca2+ (1 to 10 mM) to submerged cultures of Penicillium cyclopium induces conidiation. Ca2+ induced an increase in cytosolic pH from approximately 7.00 to > 7.60 in less than 10 min, as determined with the fluorescent pH probe fluorescein. Measurement of the H(+)-ATPase activity in total membrane fractions did not show any stable activation in vivo as a result of Ca2+ treatment. By fluorescence ratio imaging microscopy, it was observed that vegetative hyphae exhibit a tip-to-base pH gradient, with the tip being more acidic. Ca2+ caused this gradient to dissipate within 10 min. The effect of several agents that are supposed to cause internal acidification, by different means, on conidiation was tested. Concentrations of these agents that did not significantly affect growth but inhibited Ca(2+)-induced conidiation also prevented the intracellular alkalinization observed after exposure to the cation. Calcium channel blockers (lanthanum, cobalt, verapamil, and nifedipine) were not able to inhibit Ca(2+)-induced conidiation, although their effect on calcium uptake was not evaluated. However, the combined results point towards externally bound Ca2+ as the primary agent of conidiation induction, causing changes in plasma membrane function which disrupt the pH gradient observed during apical growth. Images PMID:8380805

  17. Calcium sensitizer agents: a new class of inotropic agents in the treatment of decompensated heart failure.

    PubMed

    Perrone, Sergio V; Kaplinsky, Edgardo J

    2005-09-01

    The clinician's primary objective in treating a patient with decompensated heart failure is rapid and effective stabilization. This goal often is achieved through the use of inotropic support. Classic inotropic agents (beta-adrenergic agonists and phosphodiesterase III inhibitors) can provide short-term hemodynamic benefits, but their long-term use has been correlated with poor survival rates. Calcium sensitizers comprise a new drug class that offers hemodynamic and symptomatic improvements without increasing cAMP and intracellular calcium concentrations. These agents enhance contractility without a concurrent increase in the risk of cardiac events and thus represent a significant improvement over classic positive inotropic agents. Levosimendan is the most potent calcium sensitizer to date, exhibiting a unique dual mechanism of action that combines a positive inotropic action mediated via calcium sensitization and a vasodilator property via ATP-dependent potassium channels. Available clinical data suggest that calcium sensitizer agents represent a promising class of inotropic agents in a field that has seen few advances in recent decades.

  18. Design and Synthesis of a Calcium-Sensitive Photocage.

    PubMed

    Heckman, Laurel M; Grimm, Jonathan B; Schreiter, Eric R; Kim, Charles; Verdecia, Mark A; Shields, Brenda C; Lavis, Luke D

    2016-07-11

    Photolabile protecting groups (or "photocages") enable precise spatiotemporal control of chemical functionality and facilitate advanced biological experiments. Extant photocages exhibit a simple input-output relationship, however, where application of light elicits a photochemical reaction irrespective of the environment. Herein, we refine and extend the concept of photolabile groups, synthesizing the first Ca(2+) -sensitive photocage. This system functions as a chemical coincidence detector, releasing small molecules only in the presence of both light and elevated [Ca(2+) ]. Caging a fluorophore with this ion-sensitive moiety yields an "ion integrator" that permanently marks cells undergoing high Ca(2+) flux during an illumination-defined time period. Our general design concept demonstrates a new class of light-sensitive material for cellular imaging, sensing, and targeted molecular delivery. PMID:27218487

  19. Steroid hormone 20-hydroxyecdysone promotes higher calcium mobilization to induce apoptosis.

    PubMed

    Wang, Di; Pei, Xu-Yang; Zhao, Wen-Li; Zhao, Xiao-Fan

    2016-07-01

    Calcium ions are essential secondary messengers that regulate diverse cellular processes including gene transcription, cell proliferation, and apoptosis. The steroid hormone 20-hydroxyecdysone (20E) promotes programmed cell death during insect metamorphosis, whereas juvenile hormone (JH) counteracts 20E activity to prevent metamorphosis. Both 20E and JH can induce cellular calcium increase; however, the mechanisms and physiological consequences resulting from calcium increase caused by the two counteracting hormones are unclear. Here, using Helicoverpa armigera epidermal cell line, we show that 20E via a G-protein-coupled receptor induced a major calcium rise in the cells, whereas JH via receptor tyrosine kinase induced a minor calcium increase. The calcium release-activated calcium modulator 1 (Orai1) and transient receptor potential (TRP) channels were necessary for 20E-induced rapid calcium influx. A higher calcium level was maintained in a long time and more genes including Orai1 and TRP channels showed elevated expression after the treatment of 20E than did after JH treatment. Caspase3/7 activation, cell death and pro-apoptotic gene expression were elicited by 20E induction, but not by JH. JH could repress 20E-induced calcium influx, caspase3/7 activation and gene expression. Higher calcium levels induced apoptosis. These results suggest that 20E and JH via different pathways regulate calcium mobilization and homeostasis at different levels, thus inform different gene expression and cellular responses. PMID:27209368

  20. Caffeine-induced myocardial injury in calcium-free perfused rat hearts.

    PubMed

    Vander Heide, R S; Ganote, C E

    1985-01-01

    Hearts depleted of extracellular calcium become susceptible to injury caused by repletion of extracellular calcium (calcium paradox). It has been suggested that calcium-free perfusion causes weakening of intercalated disks and that the physical stress of contracture may cause sarcolemmal membrane rupture and creatine kinase (CK) release. To further investigate this hypothesis, the effects of caffeine on contracture, cellular morphology, and CK release were studied in control and calcium-free perfused isolated rat hearts. Control hearts perfused with 2.5 mM calcium retained normal ultrastructure for long periods of perfusion. Calcium-free hearts perfused for 12 minutes developed separations of fascia adherens portions of intercalated disks but retained intact nexus junctions. Hearts subjected to 5-minute calcium-free perfusion, followed by calcium repletion, developed a massive CK release and extensive contraction band necrosis (calcium paradox). Ten millimolar caffeine, which causes rapid calcium release from sarcoplasmic reticulum (SR), produced contracture, but not CK release, from control hearts perfused with medium containing 2.5 mM calcium. In calcium-free perfused hearts, caffeine caused sudden CK release accompanied by contracture, development of contraction bands, wide separations of cells at intercalated disks, and sarcolemmal membrane injury. Caffeine-induced injury occurred despite 3 mM amobarbital inhibition of mitochondrial respiration. Hearts perfused with caffeine in the presence of calcium relaxed when made calcium-free and did not release CK. Addition of caffeine following calcium-free perfusion at 22 C, which protects the heart from the calcium paradox, produced a rapid, transient contracture. These results are compatible with the hypothesis that myocardial cell injury in calcium-free hearts is not dependent on repletion of extracellular calcium or mitochondrial function, but can result from contracture following caffeine-induced release of

  1. A novel method using Autographa californica multiple nucleopolyhedrovirus for increasing the sensitivity of insecticide through calcium influx in insect cell line.

    PubMed

    Licznar, Patricia; List, Olivier; Goven, Delphine; Nna, Rolande Ndong; Lapied, Bruno; Apaire-Marchais, Véronique

    2014-01-01

    Due to an intensive use of chemical insecticides, resistance mechanisms to insecticides together with adverse effects on non-target organisms have been largely reported. Improvement in pest control strategy represents an urgent need to optimize efficiency in the control of pest insects. In this context, a novel method based on the use of insect specific virus applied in combination with chemical insecticide, which could lead to sensitization of the insect target to insecticides is described. Insect virus, the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), applied onto Sf9 cells induces an increase of intracellular calcium concentration via extracellular calcium influx. Co-application of AcMNPV with chlorpyrifos-ethyl onto Sf9 cells expressing the key enzyme acetylcholinesterase (AChE), known to be targeted by organophosphate insecticides, increases 1.5-fold the sensitivity of AChE to the insecticide. This effect is correlated with intracellular calcium concentration rise since AcMNPV-induced potentiating insecticide effect is counteracted by pretreatment with the calcium channel blocker, cadmium chloride. Increasing insecticide target sensitivity through intracellular calcium modulation by using insect virus co-applied with a chemical insecticide is a very promising strategy allowing optimization of insecticide treatment while reducing the concentration of insecticides used.

  2. Mammalian homologue of the calcium-sensitive phosphoglycoprotein, parafusin.

    PubMed

    Wyroba, E; Widding Høyer, A; Storgaard, P; Satir, B H

    1995-12-01

    Three specific antipeptide antibodies and oligonucleotide probes synthesized to internal sequences of parafusin have been used to search for mammalian counterpart(s) of this protein. Parafusin is an exocytic-sensitive phosphoglycoprotein from a unicellular eukaryote Paramecium that was recently cloned and sequenced (Subramanian et al., Proc. Natl. Acad. Sci. USA 91, 9832-9836 (1994)). Western and Southern blot analyses, polymerase chain reaction (PCR) and reverse transcriptase coupled PCR (RT-PCR) techniques have been used to examine rat liver and pancreas, human pancreas and a murine pancreatic beta-cell line (beta TC3) arising in transgenic mice. The parafusin-specific antibodies showed cross-reaction with a protein at approximately 63 kDa in 4 tissues, whereas a phosphoglucomutase-specific antibody also detected a second band of similar molecular weight in the beta TC3 cells. The presence of two bands shows that parafusin homologue(s) and phosphoglucomutase are separate entities. beta TC3 cells were shown to incorporate [beta 35]UDPGlc into the parafusin homologue in a Ca(++)-sensitive manner characteristic of parafusin. Southern blot analysis revealed that the parafusin-specific probe hybridized with restriction enzyme digests of rat DNA in distinct patterns different from those observed with a phosphoglucomutase-specific probe. Rat genomic DNA and mRNA from the beta TC3 cells were used as the templates for PCR and RT-PCR using internal parafusin primers. In both cases similarly sized products were obtained which hybridized in Southern analysis with a specific parafusion probe located within the amplified region. These results indicate that a parafusin homologue exists in mammalian cells.

  3. Dynamics and calcium sensitivity of the Ca2+/myristoyl switch protein hippocalcin in living cells

    PubMed Central

    O'Callaghan, Dermott W.; Tepikin, Alexei V.; Burgoyne, Robert D.

    2003-01-01

    Hippocalcin is a neuronal calcium sensor protein that possesses a Ca2+/myristoyl switch allowing it to translocate to membranes. Translocation of hippocalcin in response to increased cytosolic [Ca2+] was examined in HeLa cells expressing hippocalcin–enhanced yellow fluorescent protein (EYFP) to determine the dynamics and Ca2+ affinity of the Ca2+/myristoyl switch in living cells. Ca2+-free hippocalcin was freely diffusible, as shown by photobleaching and use of a photoactivable GFP construct. The translocation was dependent on binding of Ca2+ by EF-hands 2 and 3. Using photolysis of NP-EGTA, the maximal kinetics of translocation was determined (t1/2 = 0.9 s), and this was consistent with a diffusion driven process. Low intensity photolysis of NP-EGTA produced a slow [Ca2+] ramp and revealed that translocation of hippocalcin–EYFP initiated at around 180 nM and was half maximal at 290 nM. Histamine induced a reversible translocation of hippocalcin–EYFP. The data show that hippocalcin is a sensitive Ca2+ sensor capable of responding to increases in intracellular Ca2+ concentration over the narrow dynamic range of 200–800 nM free Ca2+. PMID:14638856

  4. N-METHYL-d-ASPARTATE RECEPTORS AND LARGE CONDUCTANCE CALCIUM-SENSITIVE POTASSIUM CHANNELS INHIBIT THE RELEASE OF OPIOID PEPTIDES THAT INDUCE μ-OPIOID RECEPTOR INTERNALIZATION IN THE RAT SPINAL CORD

    PubMed Central

    SONG, B.; MARVIZÓN, J. C. G.

    2006-01-01

    Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the μ-opioid receptor, we measured μ-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced μ-opioid receptor internalization in half of the μ-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-d-aspartate (IC50=2 μM), and N-methyl-d-aspartate antagonists prevented this effect. μ-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-d-aspartate receptor activation. N-methyl-d-aspartate did not affect μ-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-d-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-d-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase μ-opioid receptor internalization in the absence of N-methyl-d-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked μ-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-d-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since μ-opioid receptors in the dorsal horn

  5. N-methyl-D-aspartate receptors and large conductance calcium-sensitive potassium channels inhibit the release of opioid peptides that induce mu-opioid receptor internalization in the rat spinal cord.

    PubMed

    Song, B; Marvizón, J C G

    2005-01-01

    Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the mu-opioid receptor, we measured mu-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced mu-opioid receptor internalization in half of the mu-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-D-aspartate (IC50=2 microM), and N-methyl-D-aspartate antagonists prevented this effect. mu-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-D-aspartate receptor activation. N-methyl-D-aspartate did not affect mu-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-D-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-D-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase mu-opioid receptor internalization in the absence of N-methyl-D-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked mu-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-D-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since mu-opioid receptors in the dorsal horn

  6. Calcium oxalate calculi-induced clusterin expression in kidney.

    PubMed

    Li, Jin-Yi; Liu, Junjiang; Jiang, Junyi; Pumill, Chris; Elaiho, Cordelia; Zhang, Yunxia; Li, Shoubin; Zhou, Tie

    2015-10-01

    The aim of the study was to investigate clusterin expression in the kidney and evaluate the urine clusterin level in the kidney stone formers. (1) In vitro, we treated the Madin-Darby canine kidney (MDCK) cell line with different concentrations of calcium oxalate (CaOx), and then the clusterin protein expression in the cells was evaluated by Western blotting. (2) Kidney stone patients who received percutaneous nephrolithotomy were enrolled in our study. Urine samples were collected before surgery, the kidney punctured to obtain kidney tissue guided by ultrasound intraoperatively. Clusterin expression in the human kidney tissue was evaluated by immunochemistry. The urine clusterin level was determined by enzyme-linked immunosorbent assay. Non-kidney disease subjects were chosen as controls. In vitro, the clusterin expression was up-regulated in the MDCK cells induced by CaOx. The study included 49 patients and 41 non-kidney disease subjects. All calculi were composed of calcium oxalate monohydrate or calcium oxalate dihydrate and a few also contained protein or uric acid. Mean ± SD urine clusterin level was 17.47 ± 18.61 μg/ml in patients, and 3.31 ± 5.42 μg/ml in non-kidney disease subjects, respectively (p < 0.001). Immunohistochemistry revealed the clusterin was located in the cytoplasm of the renal distal and collecting tubular epithelial cells. Also the tissue clusterin expression increased significantly in the kidney stone formers compared to the control groups (p = 0.001). CaOx could induce clusterin expression in renal tubular cells, and increase clusterin levels in the kidney and urine from the kidney stone formers.

  7. Contribution of potential EF hand motifs to the calcium-dependent gating of a mouse brain large conductance, calcium-sensitive K(+) channel.

    PubMed

    Braun, A P; Sy, L

    2001-06-15

    1. The large conductance, calcium-sensitive K(+) channel (BK(Ca) channel) is a unique member of the K(+)-selective ion channel family in that activation is dependent upon both direct calcium binding and membrane depolarization. Calcium binding acts to dynamically shift voltage-dependent gating in a negative or left-ward direction, thereby adjusting channel opening to changes in cellular membrane potential. 2. We hypothesized that the intrinsic calcium-binding site within the BK(Ca) channel alpha subunit may contain an EF hand motif, the most common, naturally occurring calcium binding structure. Following identification of six potential sites, we introduced a single amino acid substitution (D/E to N/Q or A) at the equivalent of the -z position of a bona fide EF hand that would be predicted to lower calcium binding affinity at each of the six sites. 3. Using macroscopic current recordings of wild-type and mutant BK(Ca) channels in excised inside-out membrane patches from HEK 293 cells, we observed that a single point mutation in the C-terminus (Site 6, FLD(923)QD to N), adjacent to the 'calcium bowl' described by Salkoff and colleagues, shifted calcium-sensitive gating right-ward by 50--65 mV over the range of 2--12 microM free calcium, but had little effect on voltage-dependent gating in the absence of calcium. Combining this mutation at Site 6 with a similar mutation at Site 1 (PVD(81)EK to N) in the N-terminus produced a greater shift (70--90 mV) in calcium-sensitive gating over the same range of calcium. We calculated that these combined mutations decreased the apparent calcium binding affinity approximately 11-fold (129.5 microM vs. 11.3 microm) compared to the wild-type channel. 4. We further observed that a bacterially expressed protein encompassing Site 6 of the BK(Ca) channel C-terminus and bovine brain calmodulin were both able to directly bind (45)Ca(2+) following denaturation and polyacrylamide gel electrophoresis (e.g. SDS-PAGE). 5. Our results suggest

  8. Calcium feedback and sensitivity regulation in primate rods

    PubMed Central

    1991-01-01

    Membrane current was recorded from a single primate rod with a suction pipette while the cell was bath perfused with solutions maintained at a temperature of approximately 38 degrees C. A transient inward current was observed at the onset of bright illumination after briefly exposing the outer segment in darkness to Ringer's (Locke) solution containing 3- isobutyl-1-methylxanthine (IBMX), an inhibitor of cGMP phosphodiesterase. After briefly removing external Na+ from around the outer segment in darkness, a similar current was observed upon Na+ restoration in bright light. By analogy to amphibian rods, this inward current was interpreted to represent the activity of an electrogenic Na(+)-dependent Ca2+ efflux, which under physiological conditions in the light is expected to reduce the free Ca2+ in the outer segment and provide negative feedback (the "Ca2+ feedback") to the phototransduction process. The exchange current had a saturated amplitude of up to approximately 5 pA and a decline time course that appeared to have more than one exponential component. In the absence of the Ca2+ feedback, made possible by removing the Ca2+ influx and efflux at the outer segment using a 0 Na(+)-0 Ca2+ external solution, the response of a rod to a dim flash was two to three times larger and had a longer time to peak than in physiological solution. These changes can be approximately accounted for by a simple model describing the Ca2+ feedback in primate rods. The dark hydrolytic rate for cGMP was estimated to be 1.2 s-1. The incremental hydrolytic rate, beta*(t), activated by one photoisomerization was approximately 0.09 s-1 at its peak, with a time-integrated activity, integral of beta*(t)dt, of approximately 0.033, both numbers being derived assuming spatial homogeneity in the outer segment. Finally, we have found that primate rods adapt to light in much the same way as amphibian and other mammalian rods, such as showing a Weber-Fechner relation between flash sensitivity and

  9. PYRETHROID INDUCED ALTERATIONS IN TRANSCRIPTION OF CALCIUM RESPONSIVE AND IMMEDIATE EARLY GENES IN VIVO.

    EPA Science Inventory

    Multiple molecular targets for pyrethroid insecticides have been evaluated in in vitro preparations, including but not limited to voltage-sensitive sodium channels (VSSCs), voltage-sensitive calcium channels (VSCCs), GABAergic receptors, ATPases and mitochondrial respiratory chai...

  10. Calcium influx and release mechanism(s) in histamine-induced myometrial contraction in buffaloes.

    PubMed

    Sharma, Abhishek; Choudhury, Soumen; Nakade, Udayraj P; Yadav, Rajkumar Singh; Garg, Satish Kumar

    2014-05-01

    The present study was undertaken to characterize the presence of histamine H1R using molecular biology tools and unravel the influx and release mechanism(s) involved in calcium signalling cascades in histamine-induced myometrial contraction in buffaloes. The presence of H1R mRNA transcript and immunoreactive membrane protein in buffalo myometrium was confirmed by RT-PCR and Western blot analysis. Further, histamine produced concentration-dependent (1nM-10μM) contraction in buffalo myometrium with a potency of 7.13±0.11. When myometrial strips were pre-incubated either with Ca(2+) free solution or with nifedipine, a L-type Ca(2+) channel blocker, dose response curve (DRC) of histamine was significantly (P<0.05) shifted towards right with decline in maximal contraction (Emax). Reduction in Emax of histamine in the presence of nifedipine (55.75±3.10%) was significantly (P<0.001) greater than that in the presence of ruthenium red (93.61±3.43%), a blocker of IP3-gated and RyR-sensitive Ca(2+) channels. Moreover, histamine produced only 26.87±1.99% of the maximum contraction in the presence of both nifedipine and CPA (blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase). Interestingly, following concurrent exposure to U-73122 (a PL-C inhibitor) and nifedipine, the DRC of histamine was significantly (P<0.05) shifted towards left with increase in maximal contraction (126.30±3.36%). Our findings in buffalo uterus thus suggest that influx of extracellular calcium plays a major role in histamine-induced myometrial contraction, while release of intracellular calcium through calcium-release channels of sarcoplasmic reticulum has a minor role. A possible involvement of non-selective cation channels in histamine-induced myometrial contraction cannot be ruled out, and therefore requires further investigations. PMID:24631173

  11. Calcium influx and release mechanism(s) in histamine-induced myometrial contraction in buffaloes.

    PubMed

    Sharma, Abhishek; Choudhury, Soumen; Nakade, Udayraj P; Yadav, Rajkumar Singh; Garg, Satish Kumar

    2014-05-01

    The present study was undertaken to characterize the presence of histamine H1R using molecular biology tools and unravel the influx and release mechanism(s) involved in calcium signalling cascades in histamine-induced myometrial contraction in buffaloes. The presence of H1R mRNA transcript and immunoreactive membrane protein in buffalo myometrium was confirmed by RT-PCR and Western blot analysis. Further, histamine produced concentration-dependent (1nM-10μM) contraction in buffalo myometrium with a potency of 7.13±0.11. When myometrial strips were pre-incubated either with Ca(2+) free solution or with nifedipine, a L-type Ca(2+) channel blocker, dose response curve (DRC) of histamine was significantly (P<0.05) shifted towards right with decline in maximal contraction (Emax). Reduction in Emax of histamine in the presence of nifedipine (55.75±3.10%) was significantly (P<0.001) greater than that in the presence of ruthenium red (93.61±3.43%), a blocker of IP3-gated and RyR-sensitive Ca(2+) channels. Moreover, histamine produced only 26.87±1.99% of the maximum contraction in the presence of both nifedipine and CPA (blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase). Interestingly, following concurrent exposure to U-73122 (a PL-C inhibitor) and nifedipine, the DRC of histamine was significantly (P<0.05) shifted towards left with increase in maximal contraction (126.30±3.36%). Our findings in buffalo uterus thus suggest that influx of extracellular calcium plays a major role in histamine-induced myometrial contraction, while release of intracellular calcium through calcium-release channels of sarcoplasmic reticulum has a minor role. A possible involvement of non-selective cation channels in histamine-induced myometrial contraction cannot be ruled out, and therefore requires further investigations.

  12. Calcium

    MedlinePlus

    ... milligrams) of calcium each day. Get it from: Dairy products. Low-fat milk, yogurt, cheese, and cottage ... lactase that helps digest the sugar (lactose) in dairy products, and may have gas, bloating, cramps, or ...

  13. Calcium

    MedlinePlus

    ... supplements and fortified foods include gluconate, lactate, and phosphate. Calcium absorption is best when a person consumes ... also interfere with the body's ability to absorb iron and zinc, but this effect is not well ...

  14. Mapping Calcium-Sensitive Regions in the Neuronal Calcium Sensor GCAP2 by Site-Specific Fluorescence Labeling.

    PubMed

    Sulmann, Stefan; Wallisch, Melanie; Scholten, Alexander; Christoffers, Jens; Koch, Karl-Wilhelm

    2016-05-10

    Myristoylation of most neuronal calcium sensor proteins, a group of EF-hand calcium-binding proteins mainly expressed in neuronal tissue, can have a strong impact on protein dynamics and functional properties. Intracellular oscillations of the free Ca(2+) concentration can trigger conformational changes in Ca(2+) sensors. The position and possible movements of the myristoyl group in the photoreceptor cell-specific Ca(2+) sensor GCAP2 are not well-defined but appear to be different from those of the highly homologous cognate GCAP1. We designed and applied a new group of diaminoterephthalate-derived fluorescent probes to label GCAP2 at a covalently attached 12-azido-dodecanoic acid (a myristoyl substitute) and at cysteine residues in critical positions. Fluorescence emission of dye-labeled GCAP2 decreased when going from low (10(-9) M) to high [Ca(2+)] (10(-3) M), reaching a half-maximal effect of fluorescence emission at 0.44 ± 0.07 μM. The modified acyl group can therefore monitor changes in the protein conformation during binding and dissociation of Ca(2+) in the physiological range of free [Ca(2+)]. However, fluorescence quenching studies showed that the dye-acyl chain was shielded from the quencher by an adjacent polypeptide region. Further probing three cysteine positions (C35, C111, and C131) by dye labeling revealed that all positions were also sensitive to a change in [Ca(2+)], but only one (C131) was sensitive to a change in [Mg(2+)]. We suggest a scenario during illumination of the photoreceptor cell in which Ca(2+) dissociates first from low and medium affinity binding sites. These steps are sensed by dyes in cysteines at positions 35 and 111. Release of Ca(2+) from high affinity sites is sensed by regions adjacent to the dye-labeled fatty acid and involves the critical conformational change leading to activating guanylate cyclase.

  15. Store-operated calcium entry in human oocytes and sensitivity to oxidative stress.

    PubMed

    Martín-Romero, Francisco Javier; Ortíz-de-Galisteo, Jose Ramón; Lara-Laranjeira, Javier; Domínguez-Arroyo, Jose Antonio; González-Carrera, Ernesto; Alvarez, Ignacio S

    2008-02-01

    Calcium signaling is a cellular event that plays a key role at many steps of fertilization and early development. However, little is known regarding the contribution of extracellular Ca(2+) influx into the cell to this signaling in gametes and early embryos. To better know the significance of calcium entry on oocyte physiology, we have evaluated the mechanism of store-operated calcium entry (SOCE) in human metaphase II (MII) oocytes and its sensitivity to oxidative stress, one of the major factors implicated in the outcome of in vitro fertilization (IVF) techniques. We show that depletion of intracellular Ca(2+) stores through inhibition of sarco(endo)plasmic Ca(2+)-ATPase with thapsigargin triggers Ca(2+) entry in resting human oocytes. Ba(2+) and Mn(2+) influx was also stimulated following inhibition, and Ca(2+) entry was sensitive to pharmacological inhibition because the SOCE blocker 2-aminoethoxydiphenylborate (2-APB) reduced calcium and barium entry. These results support the conclusion that there is a plasma membrane mechanism responsible for the capacitative divalent cation entry in human oocytes. Moreover, the Ca(2+) entry mechanism described in MII oocytes was found to be highly sensitive to oxidative stress. Hydrogen peroxide, at micromolar concentrations that could mimic culture conditions in IVF, elicited an increase of [Ca(2+)](i) that was dependent on the presence of extracellular Ca(2+). This rise was preventable by 2-APB, indicating that it was mainly due to the enhanced influx through store-operated calcium channels. In sum, our results demonstrate the occurrence of SOCE in human MII oocytes and the modification of this pathway due to oxidative stress, with possible consequences in IVF. PMID:18003943

  16. Pseudopterosin A inhibits phagocytosis and alters intracellular calcium turnover in a pertussis toxin sensitive site in Tetrahymena thermophila.

    PubMed

    Moya, Claudia E; Jacobs, Robert S

    2006-08-01

    The free living ciliate Tetrahymena thermophila was chosen as a cellular model in order to investigate the mode of action of the anti-inflammatory marine natural product Pseudopterosin A (PsA). In this paper we present evidence that PsA inhibits phagosome formation (KD=10.5 microM) and triggers a discrete intracellular calcium release (depletion) from a site in T. thermophila cells (KD=6.4 microM). Pre-treatment with the Gi/o protein inhibitor, pertussis toxin (PTX), inhibits PsA activity of both responses providing pharmacological evidence that the site of action for PsA is at a PTX sensitive G protein or a G protein coupled receptor (GPCR). Addition of extracellular calcium induced a concentration dependent increase in the incidence of phagosome formation (KD=30.3 microM) and was blocked by PsA pre-treatment. This particular effect of PsA on extracellular calcium was not blocked by PTX pre-treatment.

  17. Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation

    PubMed Central

    Colinet, Anne-Sophie; Sengottaiyan, Palanivelu; Deschamps, Antoine; Colsoul, Marie-Lise; Thines, Louise; Demaegd, Didier; Duchêne, Marie-Clémence; Foulquier, François; Hols, Pascal; Morsomme, Pierre

    2016-01-01

    Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca2+ exchangers. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca2+ uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca2+/H+ antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn2+ to the cells. PMID:27075443

  18. The atrazine metabolite diaminochlorotriazine suppresses LH release from murine LβT2 cells by suppressing GnRH-induced intracellular calcium transients.

    PubMed

    Dooley, Gregory P; Tjalkens, Ronald B; Hanneman, William H

    2013-05-01

    The primary metabolite of the herbicide atrazine (ATRA), diaminochlorotriazine (DACT), has been suggested to cause disruption in the hypothalamic-pituitary-gonadal axis leading to inhibition of luteinizing hormone (LH) release. DACT is a reactive electrophile known to form covalent protein adducts both in vitro and in vivo following ATRA exposure and maybe targeting proteins involved in GnRH-induced calcium signaling and subsequent LH release. To test this hypothesis, LβT2 pituitary cells were exposed to 300 μM DACT for 24 hrs and examined by fluorescence microscopy for GnRH-induced changes in intracellular calcium and LH release. LβT2 cells exposed to DACT had markedly diminished GnRH-induced intracellular calcium transients and a significant decreased LH release in response to GnRH. DACT appeared to cause a selective decrease in caffeine-sensitive ryanodine receptor-operated calcium stores in LβT2 cells, rather than in thapsigargin-sensitive ER calcium stores. This sensitivity correlated with the formation of covalent protein adducts by DACT, as determined by mass spectrometry. ERp57 was identified by mass spectrometry as a target of DACT adduction in the ER that could potentially mediate the effects of DACT on inhibition of GnRH-induced calcium signaling and inhibition of LH release. Intracellular calcium responses to GnRH and release of LH were restored in DACT-treated cells with the addition of a calcium ionophore (A23187). These data suggest that DACT forms adducts on proteins involved in calcium handling within the ER and that dysfunction in this critical signaling system is associated with loss of normal sensitivity to GnRH and subsequent decreased release of LH.

  19. The atrazine metabolite diaminochlorotriazine suppresses LH release from murine LβT2 cells by suppressing GnRH-induced intracellular calcium transients

    PubMed Central

    Dooley, Gregory P.; Tjalkens, Ronald B.; Hanneman, William H.

    2013-01-01

    The primary metabolite of the herbicide atrazine (ATRA), diaminochlorotriazine (DACT), has been suggested to cause disruption in the hypothalamic-pituitary-gonadal axis leading to inhibition of luteinizing hormone (LH) release. DACT is a reactive electrophile known to form covalent protein adducts both in vitro and in vivo following ATRA exposure and maybe targeting proteins involved in GnRH-induced calcium signaling and subsequent LH release. To test this hypothesis, LβT2 pituitary cells were exposed to 300 μM DACT for 24 hrs and examined by fluorescence microscopy for GnRH-induced changes in intracellular calcium and LH release. LβT2 cells exposed to DACT had markedly diminished GnRH-induced intracellular calcium transients and a significant decreased LH release in response to GnRH. DACT appeared to cause a selective decrease in caffeine-sensitive ryanodine receptor-operated calcium stores in LβT2 cells, rather than in thapsigargin-sensitive ER calcium stores. This sensitivity correlated with the formation of covalent protein adducts by DACT, as determined by mass spectrometry. ERp57 was identified by mass spectrometry as a target of DACT adduction in the ER that could potentially mediate the effects of DACT on inhibition of GnRH-induced calcium signaling and inhibition of LH release. Intracellular calcium responses to GnRH and release of LH were restored in DACT-treated cells with the addition of a calcium ionophore (A23187). These data suggest that DACT forms adducts on proteins involved in calcium handling within the ER and that dysfunction in this critical signaling system is associated with loss of normal sensitivity to GnRH and subsequent decreased release of LH. PMID:24052811

  20. Enzymatic modification of pectin to increase its calcium sensitivity while preserving its molecular weight.

    PubMed

    Hotchkiss, Arland T; Savary, Brett J; Cameron, Randall G; Chau, Hoa K; Brouillette, Janine; Luzio, Gary A; Fishman, Marshall L

    2002-05-01

    A commercial high-methoxy citrus pectin was treated with a purified salt-independent pectin methylesterase (PME) isozyme isolated from Valencia orange peel to prepare a series of deesterified pectins. A series of alkali-deesterified pectins was also prepared at pH 10 under conditions permitting beta-elimination. Analysis of these pectins using high-performance size exclusion chromatography (HPSEC) with on-line multiangle laser light-scattering, differential viscometer, and refractive index (RI) detectors revealed no reduction in weight-average molecular weight (M(w); 150000) in the PME-treated pectin series, whereas a 16% reduction in intrinsic viscosity (IV) occurred below a degree of esterification (DE) of 47%. In contrast, alkali deesterification rapidly reduced both M(w) and IV to less than half of that observed for untreated pectin. PME treatment of a non-calcium-sensitive citrus pectin introduced calcium sensitivity with only a 6% reduction in the DE. Triad blocks of unesterified galacturonic acid were observed in (1)H nuclear magnetic resonance spectra of this calcium-sensitive pectin (CSP). These results demonstrate that the orange salt-independent PME isozyme utilizes a blockwise mode of action. This is the first report of the preparation of a CSP by PME treatment without significant loss of the pectin's M(w) due to depolymerization.

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

    SciTech Connect

    Kile, J.P.

    1986-01-01

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

  2. Acute biochemical variations induced by two different calcium salts in healthy perimenopausal women.

    PubMed

    Gonnelli, S; Cepollaro, C; Camporeale, A; Nardi, P; Rossi, S; Gennari, C

    1995-09-01

    Despite the potential utility of calcium supplementation and the availability of many calcium supplements in the market, there are few data concerning the absorbability of different calcium salts in different conditions. We have compared the acute metabolic responses following oral administration of calcium citrate (CC) or calcium gluconolactate and carbonate (CGC) given to 20 healthy perimenopausal women (aged 48-55 years). Ten women received two effervescent tablets of CC (each containing 500 mg of calcium) and 10 women received two effervescent tablets of CGC (each containing 500 mg of calcium). Before and on an hourly basis for 6 hours, serum total and ionized calcium, phosphate, and immunoreactive parathyroid hormone (iPTH) were measured. Urinary calcium and creatinine were also measured. Both calcium salts induced significant increase in serum total and ionized calcium and in urinary calcium excretion; they also significantly reduced circulating levels of iPTH. The analysis of ionized calcium and iPTH response curves to CC and CGC administration revealed a significantly greater bioavailability of CC compared with CGC. Our data suggest that CC could be prefered to CGC for its characteristics of absorbability and bioavailability. PMID:8574932

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

    PubMed

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

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

  4. 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

  5. A model of calcium dynamics in cardiac myocytes based on the kinetics of ryanodine-sensitive calcium channels.

    PubMed Central

    Tang, Y; Othmer, H G

    1994-01-01

    The ryanodine-sensitive calcium channels are pivotal to signal transduction and cell function in many cell types, including cardiac myocytes. In this paper a kinetic model is proposed for these channels. In the model there are two Ca regulatory sites on the channel protein, one positive and the other negative. Cytoplasmic Ca binds to these regulatory sites independently It is assumed that the binding of Ca to the positive site is a much faster process than binding to the negative site. At steady state, the channel opening as a function of the Ca concentration is a bell-shaped curve. The model predicts the adaptation of channels to constant Ca stimulus. When this model is applied to cardiac myocytes, it predicts excitability with respect to Ca perturbations, smoothly graded responses, and Ca oscillations in certain pathological circumstances. In a spatially distributed system, traveling Ca waves in individual myocytes exist under certain conditions. This model can also be applied to other systems where the ryanodine-sensitive channels have been identified. Images FIGURE 1 FIGURE 15 PMID:7696464

  6. Asbestos-induced disruption of calcium homeostasis induces endoplasmic reticulum stress in macrophages.

    PubMed

    Ryan, Alan J; Larson-Casey, Jennifer L; He, Chao; Murthy, Shuhba; Carter, A Brent

    2014-11-28

    Although the mechanisms for fibrosis development remain largely unknown, recent evidence indicates that endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) may act as an important fibrotic stimulus in diseased lungs. ER stress is observed in lungs of patients with idiopathic pulmonary fibrosis. In this study we evaluated if ER stress and the UPR was present in macrophages exposed to chrysotile asbestos and if ER stress in macrophages was associated with asbestos-induced pulmonary fibrosis. Macrophages exposed to chrysotile had elevated transcript levels of several ER stress genes. Macrophages loaded with the Ca(2+)-sensitive dye Fura2-AM showed that cytosolic Ca(2+) increased significantly within minutes after chrysotile exposure and remained elevated for a prolonged time. Chrysotile-induced increases in cytosolic Ca(2+) were partially inhibited by either anisomycin, an inhibitor of passive Ca(2+) leak from the ER, or 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator known to deplete ER Ca(2+) stores. Anisomycin inhibited X-box-binding protein 1 (XBP1) mRNA splicing and reduced immunoglobulin-binding protein (BiP) levels, whereas BAPTA-AM increased XBP1 splicing and BiP expression, suggesting that ER calcium depletion may be one factor contributing to ER stress in cells exposed to chrysotile. To evaluate ER stress in vivo, asbestos-exposed mice showed fibrosis development, and alveolar macrophages from fibrotic mice showed increased expression of BiP. Bronchoalveolar macrophages from asbestosis patients showed increased expression of several ER stress genes compared with normal subjects. These findings suggest that alveolar macrophages undergo ER stress, which is associated with fibrosis development.

  7. Action potential-induced dendritic calcium dynamics correlated with synaptic plasticity in developing hippocampal pyramidal cells.

    PubMed

    Isomura, Y; Kato, N

    1999-10-01

    In hippocampal CA1 pyramidal cells, intracellular calcium increases are required for induction of long-term potentiation (LTP), an activity-dependent synaptic plasticity. LTP is known to develop in magnitude during the second and third postnatal weeks in the rats. Little is known, however, about development of intracellular calcium dynamics during the same postnatal weeks. We investigated postnatal development of intracellular calcium dynamics in the proximal apical dendrites of CA1 pyramidal cells by whole cell patch-clamp recordings and calcium imaging with the Ca(2+) indicator fura-2. Dendritic calcium increases induced by intrasomatically evoked action potentials were slight during the first postnatal week but gradually became robust 3 to 6-fold during the second and third postnatal weeks. These calcium increases were blocked by application of 250 microM CdCl(2), a nonspecific blocker for high-threshold voltage-dependent calcium channels (VDCCs). Under the voltage-clamp condition, both calcium currents and dendritic calcium accumulations induced by depolarization were larger at the late developmental stage (P15-18) than the early stage (P4-7), indicating developmental enhancement of calcium influx mediated by high-threshold VDCCs. Moreover, theta-burst stimulation (TBS), a protocol for LTP induction, induced large intracellular calcium increases at the late developmental stage, in synchrony with maturation of TBS-induced LTP. These results suggest that developmental enhancement of intracellular calcium increases induced by action potentials may underlie maturation of calcium-dependent functions such as synaptic plasticity in hippocampal neurons.

  8. Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations

    PubMed Central

    Boudreault, Francis; Grygorczyk, Ryszard

    2004-01-01

    Mechanical stresses release ATP from a variety of cells by a poorly defined mechanism(s). Using custom-designed flow-through chambers, we investigated the kinetics of cell swelling-induced ATP secretion, cell volume and intracellular calcium changes in epithelial A549 and 16HBE14o− cells, and NIH/3T3 fibroblasts. Fifty per cent hypotonic shock triggered transient ATP release from cell confluent monolayers, which consistently peaked at around 1 min 45 s for A549 and NIH/3T3, and at 3 min for 16HBE14o− cells, then declined to baseline within the next 15 min. Whereas the release time course had a similar pattern for the three cell types, the peak rates differed significantly (294 ± 67, 70 ± 22 and 17 ± 2.8 pmol min−1 (106 cells)−1, for A549, 16HBE14o− and NIH/3T3, respectively). The concomitant volume changes of substrate-attached cells were analysed by a 3-dimensional cell shape reconstruction method based on images acquired from two perpendicular directions. The three cell types swelled at a similar rate, reaching maximal expansion in 1 min 45 s, but differed in the duration of the volume plateau and regulatory volume decrease (RVD). These experiments revealed that ATP release does not correlate with either cell volume expansion and the expected activation of stretch-sensitive channels, or with the activation of volume-sensitive, 5-nitro-2-(3-phenylpropylamino) benzoic acid-inhibitable anion channels during RVD. By contrast, ATP release was tightly synchronized, in all three cell types, with cytosolic calcium elevations. Furthermore, loading A549 cells with the calcium chelator BAPTA significantly diminished ATP release (71% inhibition of the peak rate), while the calcium ionophore ionomycin triggered ATP release in the absence of cell swelling. Lowering the temperature to 10°C almost completely abolished A549 cell swelling-induced ATP release (95% inhibition of the peak rate). These results strongly suggest that calcium-dependent exocytosis plays a

  9. Annexin A4 induces platinum resistance in a chloride-and calcium-dependent manner.

    PubMed

    Morimoto, Akiko; Serada, Satoshi; Enomoto, Takayuki; Kim, Ayako; Matsuzaki, Shinya; Takahashi, Tsuyoshi; Ueda, Yutaka; Yoshino, Kiyoshi; Fujita, Masami; Fujimoto, Minoru; Kimura, Tadashi; Naka, Tetsuji

    2014-09-15

    Platinum resistance has long been a major issue in the treatment of various cancers. We previously reported that enhanced annexin A4 (ANXA4) expression, a Ca2+-regulated phospholipid-binding protein, induces chemoresistance to platinum-based drugs. In this study, we investigated the role of annexin repeats, a conserved structure of all the annexin family, responsible for platinum-resistance as well as the effect of knockdown of ANXA4. ANXA4 knockdown increased sensitivity to platinum-based drugs both in vitro and in vivo. To identify the domain responsible for chemoresistance, ANXA4 deletion mutants were constructed by deleting annexin repeats one by one from the C terminus. Platinum resistance was induced both in vitro and in vivo in cells expressing either full-length ANXA4 or the deletion mutants, containing at least one intact annexin repeat. However, cells expressing the mutant without any calcium-binding sites in the annexin repeated sequence, which is essential for ANXA4 translocation from the cytosol to plasma membrane, failed to acquire platinum resistance. After cisplatin treatment, the intracellular chloride ion concentration, whose channel is partly regulated by ANXA4, significantly increased in the platinum-resistant cells. These findings indicate that the calcium-binding site in the annexin repeat induces chemoresistance to the platinum-based drug by elevating the intracellular chloride concentration.

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

    PubMed

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

    2014-10-01

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

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

    PubMed Central

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

    2014-01-01

    Calcium signaling has a versatile role in many important cellular functions. Despite its importance, regulation of calcium signaling in bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) has not been explored extensively. Our previous study revealed that cyclic adenosine monophosphate (cAMP) enabled BMSCs to generate calcium signal upon stimulation by dopamine, KCl and glutamate. Concurrently, cAMP transiently activated the transcription factor cAMP response element binding protein (CREB) in BMSCs. Activity of CREB can be modulated by the calcium/calmodulin-dependent kinase signaling pathway, however, whether the calcium signaling observed in cAMP-induced BMSCs requires CREB has not been investigated. In an effort to uncover the role of CREB in the generation of calcium signaling in response to modulators such as dopamine and KCl, we knocked down CREB activity in BMSCs. Our study indicated that BMSCs, but not its close relative fibroblasts, are responsive to dopamine and KCl after cAMP treatment. Calcium signal elicited by dopamine depends, in part, on calcium influx whereas that elicited by KCl depends completely on calcium influx. Knock-down of CREB activity significantly reduced or abolished the cAMP-induced calcium response, and reintroducing a constitutively active CREB partially restored the calcium response. PMID:25154887

  12. On the localization of voltage-sensitive calcium channels in the flagella of Chlamydomonas reinhardtii

    PubMed Central

    1994-01-01

    This study was undertaken to prove that voltage-sensitive calcium channels controlling the photophobic stop response of the unicellular green alga Chlamydomonas reinhardtii are exclusively found in the flagellar region of the cell and to answer the question as to their exact localization within the flagellar membrane. The strategy used was to amputate flagella to a variable degree without perturbing the electrical properties of the cell and measure flagellar currents shortly after amputation and during the subsequent regeneration process. Under all conditions, a close correlation was found between current size and flagellar length, strongly suggesting that the channels that mediate increases in intraflagellar calcium concentration are confined to and distributed over the total flagellar length. Bald mutants yielded tiny flagellar currents, in agreement with the existence of residual flagellar stubs. In the presence of the protein synthesis inhibitor cycloheximide, flagellar length and flagellar currents also recovered in parallel. Recovery came to an earlier end, however, leveling off at a time when in the absence of cycloheximide only half maximal values were achieved. This suggests the existence of a pool of precursors, which permits the maintenance of a constant ratio between voltage-sensitive calcium channels and other intraflagellar proteins. PMID:8195293

  13. Effects of ethanol on RhoA/Rho-kinase-mediated calcium sensitization in mouse lung parenchymal tissue.

    PubMed

    Aydinoglu, Fatma; Ergurhan Kiroglu, Olcay; Astarci, Erhan; Balli, Ebru; Ogulener, Nuran

    2015-10-01

    Calcium sensitization by the RhoA/Rho-kinase (ROCK) pathway contributes to the contraction in smooth muscle. Contractile stimuli can sensitize myosin to Ca(2+) by activating RhoA/Rho-kinase that inhibits myosin light chain phosphatase activity. The present study was aimed at investigating the possible involvement of RhoA/Rho-kinase pathway in contractile responses to agonist (phenylephrine) and depolarizing (KCl) of mouse lung parenchymal tissues. Also, we investigated the effect of ethanol on RhoA/Rho-kinase pathway. Phenylephrine (10(-8)-10(-4) M) and KCl (10-80 mM) induced sustained contractions in parenchymal strips. Ethanol significantly attenuated the contractions to phenylephrine and KCl. The Rho-kinase inhibitors fasudil (5×10(-5) M) and Y-27632 (5×10(-5) M) inhibited contractions to in both control and ethanol-treated parenchymal strips. In addition, the relaxations induced by fasudil (10(-4) M) and Y-27632 (5×10(-4) M) on parenchymal strips contracted by phenylephrine but not KCl was decreased in ethanol-treatment group. Also, RhoA, ROCK1 and ROCK2 expressions were detected in mouse lung parenchymal tissue. In ethanol-treated group, expression of RhoA and ROCK1 but not ROCK2 decreased compared to control. Furthermore, ethanol causes apoptotic changes in alveolar type I epithelial cells of parenchymal tissue. These results suggest that RhoA/Rho-kinase signaling pathway plays an important role in phenylephrine- and KCl-induced Ca(2)(+) sensitization in mouse lung parenchymal tissue. Also, ethanol may be decrease phenylephrine- and KCl-induced contraction due to lowering the RhoA/Rho-kinase-mediated Ca(2+)-sensitizing by inhibiting RhoA/Rho-kinase pathway in parenchymal tissue. These results may be lead to important insights into the mechanisms of lung diseases due to alcohol consumption.

  14. Noise-induced sensitization of human brain

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yoshiharu; Hidaka, Ichiro; Nozaki, Daichi; Iso-o, Noriko; Soma, Rika; Kwak, Shin

    2002-11-01

    In the past decade, it has been recognized that noise can enhance the response of nonlinear systems to weak signals, via a mechanism known as stochastic resonance (SR). Particularly, the concept of SR has generated considerable interest in sensory biology, because it has been shown in several experimental studies that noise can assist neural systems in detecting weak signals which could not be detected in its absence. Recently, we have shown a similar type of noise-induced sensitization of human brain; externally added noise to the brain stem baroreflex centers sensitized their responses in maintaining adequate blood perfusion to the brain itself. Furthermore, the addition of noise has also shown to be useful in compensating for dysfunctions of the baroreflex centers in certain neurological diseases. It is concluded that the statistical physics concept of SR could be useful in sensitizing human brain in health and disease.

  15. CXCL12 induces hepatic stellate cell contraction through a calcium-independent pathway.

    PubMed

    Saiman, Yedidya; Agarwal, Ritu; Hickman, DaShawn A; Fausther, Michel; El-Shamy, Ahmed; Dranoff, Jonathan A; Friedman, Scott L; Bansal, Meena B

    2013-09-01

    Liver fibrosis, with subsequent development of cirrhosis and ultimately portal hypertension, results in the death of patients with end-stage liver disease if liver transplantation is not performed. Hepatic stellate cells (HSCs), central mediators of liver fibrosis, resemble tissue pericytes and regulate intrahepatic blood flow by modulating pericapillary resistance. Therefore, HSCs can contribute to portal hypertension in patients with chronic liver disease (CLD). We have previously demonstrated that activated HSCs express functional chemokine receptor, CXCR4, and that receptor engagement by its ligand, CXCL12, which is increased in patients with CLD, leads to further stellate cell activation in a CXCR4-specific manner. We therefore hypothesized that CXCL12 promotes HSC contraction in a CXCR4-dependent manner. Stimulation of HSCs on collagen gel lattices with CXCL12 led to gel contraction and myosin light chain (MLC) phosphorylation, which was blocked by addition of AMD3100, a CXCR4 small molecule inhibitor. These effects were further mediated by the Rho kinase pathway since both Rho kinase knockdown or Y-27632, a Rho kinase inhibitor, blocked CXCL12 induced phosphorylation of MLC and gel contraction. BAPTA-AM, a calcium chelator, had no effect, indicating that this pathway is calcium sensitive but not calcium dependent. In conclusion, CXCL12 promotes stellate cell contractility in a predominantly calcium-independent fashion. Our data demonstrates a novel role of CXCL12 in stellate cell contraction and the availability of small molecule inhibitors of the CXCL12/CXCR4 axis justifies further investigation into its potential as therapeutic target for portal hypertension. PMID:23812037

  16. The modulation of action potential generation by calcium-induced calcium release is enhanced by mitochondrial inhibitors in mudpuppy parasympathetic neurons.

    PubMed

    Barstow, K L; Locknar, S A; Merriam, L A; Parsons, R L

    2004-01-01

    Previously, we demonstrated that outward currents activated by calcium-induced calcium release (CICR) opposed depolarization-induced action potential (AP) generation in dissociated mudpuppy parasympathetic neurons [J Neurophysiol 88 (2002) 1119]. In the present study, we tested whether AP generation by depolarizing current ramps could be altered by dissipating the mitochondrial membrane potential and thus interrupting mitochondrial Ca2+ buffering. Exposure to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP; 2 microM) alone or in combination with the mitochondrial ATP synthase inhibitor oligomycin (8 microg/ml), increased the latency to AP generation. Exposure to the electron transport chain inhibitor rotenone (10 microM) alone or in combination with oligomycin (8 microg/ml) similarly increased the latency to AP generation. CCCP and oligomycin or rotenone and oligomycin treatment caused rhodamine 123 loss from mitochondria within a few minutes, confirming that the mitochondrial membrane potential was dissipated during drug exposure. Oligomycin alone had no effect on the latency to AP generation and did not cause loss of rhodamine 123 from mitochondria. The increase in latency induced by CCCP and oligomycin was similar when recordings were made with either the perforated patch or standard whole cell patch recording configuration. Exposure to the endoplasmic reticulum Ca-ATPase inhibitor thapsigargin (1 microM), decreased the latency to AP generation. In cells pretreated with thapsigargin to eliminate CICR, CCCP and oligomycin had no effect on AP latency. Pretreatment with iberiotoxin (IBX; 100 nM), an inhibitor of large conductance, calcium- and voltage-activated potassium channels, reduced the extent of the CCCP- and oligomycin-induced increase in latency to AP generation. These results indicate that treatment with CCCP or rotenone to dissipate the mitochondrial membrane potential, a condition which should minimize sequestration of Ca2+ by

  17. Extracellular calcium increases bisphosphonate-induced growth inhibition of breast cancer cells

    PubMed Central

    Journé, Fabrice; Kheddoumi, Naïma; Chaboteaux, Carole; Duvillier, Hugues; Laurent, Guy; Body, Jean-Jacques

    2008-01-01

    Introduction Bisphosphonates have become standard therapy for the treatment of skeletal complications related to breast cancer. Although their therapeutic effects mainly result from an inhibition of osteoclastic bone resorption, in vitro data indicate that they also act directly on breast cancer cells, inhibiting proliferation and inducing apoptosis. Methods The present study examined the effects of calcium (from 0.6 to 2.0 mmol/l) on the antitumour activity of the bisphosphonate ibandronate (1 to 1,000 nmol/l) on MDA-MB-231 and MCF-7 breast cancer cells. Cell culture densities were determined using crystal violet staining assay. Apoptotic cell death was assessed by annexin V-phycoerythrin and 7-amino-actinomycin double staining. Results At low calcium concentration, 30 μmol/l ibandronate had no effect on MDA-MB-231 cells growth and only slightly inhibited MCF-7 cells growth. Higher calcium levels significantly increased growth inhibition as well as cell apoptosis induced by ibandronate. We observed similar effects with zoledronic acid. Of note, enhancement of ibandronate-induced growth inhibition was also observed in other breast cancer cell lines (T-47D, ZR-75, Hs-578T and BT-549 cells). The growth inhibitory effect of ibandronate in the presence of high concentrations of calcium was partly suppressed by the calcium chelator EGTA (ethylene glycol tetra-acetic acid). In addition, in the presence of calcium at high concentrations, cells accumulated more [14C]ibandronate than at low calcium concentrations. We obtained further evidence of enhancement of cellular ibandronate accumulation by calcium by demonstrating that high calcium levels increased the inhibition of protein prenylation induced by the bisphosphonate. Conclusion Altogether, our data suggest that extracellular calcium, probably through its binding to ibandronate, markedly increased its cellular accumulation and its inhibitory activity on breast tumour cells. Thus, calcium released during the process of

  18. Phosphoprotein Phosphatase 1 Is Required for Extracellular Calcium-Induced Keratinocyte Differentiation

    PubMed Central

    Fan, Hong; Zeng, Qin; Pennypacker, Sally D.; Xie, Zhongjian

    2016-01-01

    Extracellular calcium is a major regulator of keratinocyte differentiation in vitro and appears to play that role in vivo, but the mechanism is unclear. We have previously demonstrated that, following calcium stimulation, PIP5K1α is recruited by the E-cadherin-β-catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-γ1. This signaling pathway is critical for calcium-induced generation of second messengers including IP3 and intracellular calcium and keratinocyte differentiation. In this study, we explored the upstream regulatory mechanism by which calcium activates PIP5K1α and the role of this activation in calcium-induced keratinocyte differentiation. We found that treatment of human keratinocytes in culture with calcium resulted in an increase in serine dephosphorylation and PIP5K1α activation. PP1 knockdown blocked extracellular calcium-induced increase in serine dephosphorylation and activity of PIP5K1α and induction of keratinocyte differentiation markers. Knockdown of PLC-γ1, the downstream effector of PIP5K1α, blocked upstream dephosphorylation and PIP5K1α activation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1α complex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1α complex in the plasma membrane to activate PIP5K1α, which is required for PLC-γ1 activation leading to keratinocyte differentiation. PMID:27340655

  19. Antagonism of T-type calcium channels inhibits high-fat diet–induced weight gain in mice

    PubMed Central

    Uebele, Victor N.; Gotter, Anthony L.; Nuss, Cindy E.; Kraus, Richard L.; Doran, Scott M.; Garson, Susan L.; Reiss, Duane R.; Li, Yuxing; Barrow, James C.; Reger, Thomas S.; Yang, Zhi-Qiang; Ballard, Jeanine E.; Tang, Cuyue; Metzger, Joseph M.; Wang, Sheng-Ping; Koblan, Kenneth S.; Renger, John J.

    2009-01-01

    The epidemics of obesity and metabolic disorders have well-recognized health and economic burdens. Pharmacologic treatments for these diseases remain unsatisfactory with respect to both efficacy and side-effect profiles. Here, we have identified a potential central role for T-type calcium channels in regulating body weight maintenance and sleep. Previously, it was shown that mice lacking CaV3.1 T-type calcium channels have altered sleep/wake activity. We found that these mice were also resistant to high-fat diet–induced weight gain, without changes in food intake or sensitivity to high-fat diet–induced disruptions of diurnal rhythm. Administration of a potent and selective antagonist of T-type calcium channels, TTA-A2, to normal-weight animals prior to the inactive phase acutely increased sleep, decreased body core temperature, and prevented high-fat diet–induced weight gain. Administration of TTA-A2 to obese rodents reduced body weight and fat mass while concurrently increasing lean muscle mass. These effects likely result from better alignment of diurnal feeding patterns with daily changes in circadian physiology and potentially an increased metabolic rate during the active phase. Together, these studies reveal what we believe to be a previously unknown role for T-type calcium channels in the regulation of sleep and weight maintenance and suggest the potential for a novel therapeutic approach to treating obesity. PMID:19451696

  20. Reductions in calcium uptake induced in rat brain synaptosomes by ionizing radiation

    SciTech Connect

    Kandasamy, S.B.; Howerton, T.C.; Hunt, W.A. )

    1991-02-01

    Gamma irradiation (60Co) reduced KCl-stimulated voltage-dependent 45Ca2+ uptake in whole-brain, cortical, and striatal synaptosomes. The time course (3, 10, 30, and 60 s) of calcium uptake by irradiated (3 Gy) and nonirradiated synaptosomes, as well as the effect of KCl (15-65 mM), was measured in whole-brain synaptosomes. The fastest and highest rate of depolarization-dependent calcium uptake occurred at 3 s with 65 mM KCl. Irradiation reduced calcium uptake at all incubation times and KCl concentrations. Bay K 8644 enhancement of KCl-stimulated calcium influx was also reduced by radiation exposure. Nimodipine binding to dihydropyridine (DHP) L-type calcium channel receptors was not altered following radiation exposure. These results demonstrate an inhibitory effect of ionizing radiation on the voltage-sensitive calcium channels in rat brain synaptosomes that are not mediated by DHP receptors.

  1. Pressure induced reactions amongst calcium aluminate hydrate phases

    SciTech Connect

    Moon, Ju-hyuk; Oh, Jae Eun; Balonis, Magdalena; Glasser, Fredrik P.; Clark, Simon M.; Monteiro, Paulo J.M.

    2011-06-15

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

  2. Block of L-type calcium channels by charged dihydropyridines. Sensitivity to side of application and calcium

    PubMed Central

    1991-01-01

    We have studied block of L-type calcium channels by intracellular and extracellular application of the ionized dihydropyridine derivatives amlodipine and SDZ 207-180. We find that extracellular application of either drug causes voltage-dependent block of calcium channels. However, neither drug is effective when applied intracellularly. The insensitivity of calcium channels to intracellular drug is not due to the low concentrations of cytosolic calcium, because voltage-dependent block by ionized amlodipine, SDZ 207-180, and the neutral drug nisoldipine persists under conditions in which Ca0 is buffered by EGTA. In fact, the time course of the development of block by the ionized but not neutral drug molecules studied, is slower in the presence than in the absence of calcium. Our results indicate that the DHP binding site of the L-type calcium channel is close to the extracellular surface of the cell membrane and that ionized DHP molecules may interact with the receptor in a manner that is uniquely affected by calcium. PMID:1658191

  3. Nuclear membrane R-type calcium channels mediate cytosolic ET-1-induced increase of nuclear calcium in human vascular smooth muscle cells.

    PubMed

    Bkaily, Ghassan; Avedanian, Levon; Al-Khoury, Johny; Chamoun, Marc; Semaan, Rana; Jubinville-Leblanc, Cynthia; D'Orléans-Juste, Pedro; Jacques, Danielle

    2015-04-01

    The objective of this work was to verify whether, as in the case of the plasma membrane of human vascular smooth muscle cells (hVSMCs), cytosolic ET-1-induced increase of nuclear calcium is mediated via the activation of calcium influx through the steady-state R-type calcium channel. Pharmacological tools to identify the R-type calcium channels, as well as real 3-D confocal microscopy imaging techniques coupled to calcium fluorescent probes, were used to study the effect of cytosolic ET-1 on nuclear calcium in isolated nuclei of human hepatocytes and plasma membrane perforated hVSMCs. Our results showed that pre-treatment with pertussis toxin (PTX) or cholera toxin (CTX) prevented cytosolic ET-1 (10(-9) mol/L) from inducing a sustained increase in nuclear calcium. Furthermore, the L-type calcium channel blocker nifedipine did not prevent cytosolic ET-1 from inducing an increase in nuclear calcium, as opposed to the dual L- and R-type calcium channel blocker isradipine (PN200-110) (in the presence of nifedipine). In conclusion, the preventative effect with PTX and CTX, and the absence of an effect with nifedipine, as well as the blockade by isradipine on cytosolic ET-1-induced increase in nuclear calcium, suggest that this nuclear calcium influx in hVSMCs is due to activation of the steady-state R-type calcium channel. The sarcolemmal and nuclear membrane R-type calcium channels in hVSMCs are involved in ET-1 modulation of vascular tone in physiology and pathology.

  4. Effects of calcium buffering on glucose-induced insulin release in mouse pancreatic islets: an approximation to the calcium sensor

    PubMed Central

    Pertusa, José A G; Sanchez-Andrés, Juan V; Martín, Franz; Soria, Bernat

    1999-01-01

    The properties of the calcium sensor for glucose-induced insulin secretion have been studied using cell-permeant Ca2+ buffers with distinct kinetics and affinities. In addition, submembrane cytosolic Ca2+ distribution has been modelled after trains of glucose-induced action potential-like depolarizations. Slow Ca2+ buffers (around 1 mmol l−1 intracellular concentration) with different affinities (EGTA and Calcium Orange-5N) did not significantly affect glucose-induced insulin release. Modelling showed no effect on cytosolic Ca2+ concentrations at the outermost shell (0.05 μm), their effects being observed in the innermost shells dependent on Ca2+ affinity. In contrast, fast Ca2+ buffers (around 1 mmol l−1 intracellular concentration) with different affinities (BAPTA and Calcium Green-5N) caused a 50% inhibition of early insulin response and completely blocked the late phase of glucose-induced insulin response, their simulations showing a decrease of [Ca2+]i at both the inner and outermost shells. These data are consistent with the existence in pancreatic β-cells of a higher affinity Ca2+ sensor than that proposed for neurons. Moreover, these data are consistent with the proposed existence of two distinct pools of granules: (i) ‘primed’ vesicles, colocalized with Ca2+ channels and responsible of the first phase of insulin release; and (ii) ‘reserved pool’ vesicles, not colocalized and responsible for the second phase. PMID:10523416

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

    PubMed

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

    2016-06-16

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

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

    PubMed Central

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

    2016-01-01

    Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and metabolism. Accumulation of unfolded proteins within the calcium-depleted lumen of the endoplasmic reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections linking disturbance of ER calcium equilibrium to ER stress and UPR signaling are evolutionary conserved and highlight the crucial role of metabolism in modulating the effects induced by calcium shortage. PMID:27305947

  7. Artemisinin Induces Calcium-Dependent Protein Secretion in the Protozoan Parasite Toxoplasma gondii▿ †

    PubMed Central

    Nagamune, Kisaburo; Beatty, Wandy L.; Sibley, L. David

    2007-01-01

    Intracellular calcium controls several crucial cellular events in apicomplexan parasites, including protein secretion, motility, and invasion into and egress from host cells. The plant compound thapsigargin inhibits the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), resulting in elevated calcium and induction of protein secretion in Toxoplasma gondii. Artemisinins are natural products that show potent and selective activity against parasites, making them useful for the treatment of malaria. While the mechanism of action is uncertain, previous studies have suggested that artemisinin may inhibit SERCA, thus disrupting calcium homeostasis. We cloned the single-copy gene encoding SERCA in T. gondii (TgSERCA) and demonstrate that the protein localizes to the endoplasmic reticulum in the parasite. In extracellular parasites, TgSERCA partially relocalized to the apical pole, a highly active site for regulated secretion of micronemes. TgSERCA complemented a calcium ATPase-defective yeast mutant, and this activity was inhibited by either thapsigargin or artemisinin. Treatment of T. gondii with artemisinin triggered calcium-dependent secretion of microneme proteins, similar to the SERCA inhibitor thapsigargin. Artemisinin treatment also altered intracellular calcium in parasites by increasing the periodicity of calcium oscillations and inducing recurrent, strong calcium spikes, as imaged using Fluo-4 labeling. Collectively, these results demonstrate that artemisinin perturbs calcium homeostasis in T. gondii, supporting the idea that Ca2+-ATPases are potential drug targets in parasites. PMID:17766463

  8. Low-calcium-induced enhancement of chemical synaptic transmission from photoreceptors to horizontal cells in the vertebrate retina.

    PubMed Central

    Piccolino, M; Byzov, A L; Kurennyi, D E; Pignatelli, A; Sappia, F; Wilkinson, M; Barnes, S

    1996-01-01

    According to the classical calcium hypothesis of synaptic transmission, the release of neurotransmitter from presynaptic terminals occurs through an exocytotic process triggered by depolarization-induced presynaptic calcium influx. However, evidence has been accumulating in the last two decades indicating that, in many preparations, synaptic transmitter release can persist or even increase when calcium is omitted from the perfusing saline, leading to the notion of a "calcium-independent release" mechanism. Our study shows that the enhancement of synaptic transmission between photoreceptors and horizontal cells of the vertebrate retina induced by low-calcium media is caused by an increase of calcium influx into presynaptic terminals. This paradoxical effect is accounted for by modifications of surface potential on the photoreceptor membrane. Since lowering extracellular calcium concentration may likewise enhance calcium influx into other nerve cells, other experimental observations of "calcium-independent" release may be reaccommodated within the framework of the classical calcium hypothesis without invoking unconventional processes. PMID:8637867

  9. Sources of activator calcium for potassium- and serotonin-induced constriction of isolated bovine cerebral arteries

    SciTech Connect

    Not Available

    1986-03-01

    Previous in vitro studies with the calcium channel blockers (CCB) indirectly suggest that K/sup +/ and serotonin (5HT) constrict bovine middle cerebral arteries (BMCA) by promoting the influx of extracellular calcium (Ca) through CCB-sensitive channels. In this study, the authors directly determined the sources of activator Ca for K/sup +/- and 5HT-induced constriction of BMCA, using radiolabelled /sup 4/)2%Ca and /sup 3/H-sorbitol. EGTA-resistant Ca uptake, an estimate of Ca influx into vascular smooth muscle, was determined by exposure to Ca-deficient 2 mM EGTA solutions at 1/sup 0/C. The total Ca content of BMCA was 4.4 nmole/mg (wet wt.) after equilibration at 37/sup 0/C. The total exchangeable Ca content was 1.64 nmole/mg after 1 hr of /sup 45/Ca loading; the Ca content of the extracellular water was 0.30 nmole/mg, as estimated from the /sup 3/H-sorbitol space (0.25 ul/mg). The EGTA-resistant Ca uptake at 1 hr was 134 pmole/mg. K/sup +/ and 5HT significantly increased EGTA-resistant Ca uptake during 5 min of /sup 45/Ca loading; for K/sup +/, Ca uptake increased from 71 to 202 pmole/mg, and for 5HT, from 65 to 102 pmole/mg. Verapamil (10/sup -5/ M) or nifedipine (3.3 x 10/sup -7/ M) significantly blocked the increase in EGTA-resistant Ca uptake induced by K/sup +/ or 5HT. These results provide direct evidence that K/sup +/ or 5HT may constrict BMCA by promoting the influx of extracellular Ca through CCB-sensitive channels.

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2014-06-21

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

  12. Acute changes in serum calcium and parathyroid hormone circulating levels induced by the oral intake of five currently available calcium salts in healthy male volunteers.

    PubMed

    Deroisy, R; Zartarian, M; Meurmans, L; Nelissenne, N; Micheletti, M C; Albert, A; Reginster, J Y

    1997-05-01

    Several calcium supplements are currently available and many of them are marketed without proper comparison of the bioavailability of the actual preparations. The aim of the present trial was to evaluate and compare the acute changes in serum calcium (Ca) and parathyroid hormone (PTH) levels following the oral administration of a vehicle and of five calcium salts currently prescribed in Western Europe. No significant changes in serum Ca or PTH levels were observed after administration of the vehicle. All calcium salts induced significant increases in serum Ca and decreases in serum PTH compared to baseline values. Comparison of the six response curves revealed a significantly greater increase in serum Ca and a greater decrease in serum PTH after each of the calcium salts than observed after the vehicle. However, no statistically significant differences were observed between the different calcium salts for serum Ca increments. The decrease in serum PTH observed after administration of an ossein-hydroxyapatite complex was significantly less important than after the four other calcium salts, even if statistically different than after vehicle. When assessing the area under the curve (AUC) of PTH values, we observed that calcium carbonate and citrate induce a significantly greater decrease in serum PTH than the other calcium salts which are, however, statistically more active than the vehicle. Serum PTH is decreased under the lower limit of the normal range (10 pg/ml), between t60 and t120 for calcium carbonate and citrate and between t60 and t90 for calcium gluconolactate while the mean PTH values remain within the normal range throughout the study with calcium pidolate, the ossein-hydroxyapatite complex and the vehicle. In conclusion, all calcium preparations significantly increase serum calcium and decrease serum parathormone, compared to what is observed after oral intake of a vehicle. However, significant differences in suppression of parathormone are observed

  13. Hormone-induced calcium oscillations in liver cells can be explained by a simple one pool model.

    PubMed

    Somogyi, R; Stucki, J W

    1991-06-15

    Hormone-induced oscillations of the free intracellular calcium concentration are thought to be relevant for frequency encoding of hormone signals. In liver cells, such Ca2+ oscillations occur in response to stimulation by hormones acting via phosphoinositide breakdown. This observation may be explained by cooperative, positive feedback of Ca2+ on its own release from one inositol 1,4,5-trisphosphate-sensitive pool, obviating oscillations of inositol 1,4,5-trisphosphate. The kinetic rate laws of the associated model have a mathematical structure reminiscent of the Brusselator, a hypothetical chemical model involving a rather improbable trimolecular reaction step, thus giving a realistic biological interpretation to this hallmark of dissipative structures. We propose that calmodulin is involved in mediating this cooperativity and positive feedback, as suggested by the presented experiments. For one, hormone-induced calcium oscillations can be inhibited by the (nonphenothiazine) calmodulin antagonists calmidazolium or CGS 9343 B. Alternatively, in cells overstimulated by hormone, as characterized by a non-oscillatory elevated Ca2+ concentration, these antagonists could again restore sustained calcium oscillations. The experimental observations, including modulation of the oscillations by extracellular calcium, were in qualitative agreement with the predictions of our mathematical model. PMID:1904060

  14. Interaction between calcium channel blockers and sweetening agents on morphine-induced analgesia in mice by formalin test.

    PubMed

    Nikfar, S; Abdollahi, M; Sarkarati, F; Etemad, F

    1998-09-01

    1. Calcium is known to be an important ion in the modulation of nociception and inflammation. Previous research has shown that mice drinking sweet-tasting solutions such as sucrose, saccharin and aspartame exhibit significant changes in morphine-induced analgesia in both phases of the formalin test. 2. In this study, the role of calcium channel blockers on the effectivity of a 12-day regimen of different sweetening agents (sucrose 32%, saccharin 0.08% and aspartame 0.16%) on the alteration of the morphine response has been investigated. 3. Male albino mice weighing 20-27 g were used for experiments. Animals were given 12 days to adapt to dietary conditions. Animals were given morphine (1.5, 3, 6, 9 mg/kg) subcutaneously 30 min before observation. Nifedipine (5 mg/kg), verapamil (5 mg/kg) and diltiazem (10 mg/kg) were administered intraperitoneally 20 min before morphine injection. 4. Recording of the early phase started immediately and lasted for 10 min after formalin injection. Recording of the late response started 20 min after formalin injection and lasted for 10 min. 5. Calcium channel blockers potentiated the antinociceptive effects of sweetening agents and diminished the antagonistic effects of these compounds on morphine-induced analgesia in the early and late phases of the formalin test. 6. It is proposed that calcium has a role for the interactive effects of sweetening agents and morphine on pain sensitivity.

  15. [Dynamics of calcium metabolism and calcium-regulating hormones in pregnancy-induced hypertension].

    PubMed

    Ohara, N; Yamasaki, M; Morikawa, H; Ueda, Y; Mochizuki, M

    1986-08-20

    Serum concentrations of total calcium, ionized calcium and inorganic phosphorus in severe PIH were significantly lower than those in normal pregnancy during the 3rd trimester of pregnancy and continued to be low even at puerperium. On the other hand, serum concentrations of parathyroid hormone in severe PIH were significantly higher during the 3rd trimester of pregnancy and decreased at puerperium. Any remarkable differences in serum calcitonin levels were not found between severe PIH and normal pregnancy through the last trimester of pregnancy and puerperium. Serum concentrations of 1 alpha, 25-(OH)2 vitamin D3 increased significantly in the 3rd trimester of normal pregnancy, but in severe PIH, their increase was not observed, remaining at the normal levels of non-pregnant women. The kidney functions in the both groups were within the normal limits of non-pregnant women, but placental dysfunction was observed in severe PIH. These results suggest that the decrease in serum calcium and phosphorus levels might have occurred as a result of the decrease in the absorption of calcium and phosphorus from the intestine due to the decrease in serum 1 alpha, 25-(OH)2 vitamin D3 levels and that low serum 1 alpha, 25-(OH)2 vitamin D3 concentrations might be caused by the disturbance of the synthesis in the placenta rather than in the kidney. PMID:3781066

  16. Redox Nanodomains Are Induced by and Control Calcium Signaling at the ER-Mitochondrial Interface.

    PubMed

    Booth, David M; Enyedi, Balázs; Geiszt, Miklós; Várnai, Péter; Hajnóczky, György

    2016-07-21

    The ER-mitochondrial interface is central to calcium signaling, organellar dynamics, and lipid biosynthesis. The ER and mitochondrial membranes also host sources and targets of reactive oxygen species (ROS), but their local dynamics and relevance remained elusive since measurement and perturbation of ROS at the organellar interface has proven difficult. Employing drug-inducible synthetic ER-mitochondrial linkers, we overcame this problem and demonstrate that the ER-mitochondrial interface hosts a nanodomain of H2O2, which is induced by cytoplasmic [Ca(2+)] spikes and exerts a positive feedback on calcium oscillations. H2O2 nanodomains originate from the mitochondrial cristae, which are compressed upon calcium signal propagation to the mitochondria, likely due to Ca(2+)-induced K(+) and concomitant water influx to the matrix. Thus, ER-mitochondrial H2O2 nanodomains represent a component of inter-organelle communication, regulating calcium signaling and mitochondrial activities.

  17. The endocannabinoid 2-arachidonoylglicerol decreases calcium induced cytochrome c release from liver mitochondria.

    PubMed

    Zaccagnino, Patrizia; D'Oria, Susanna; Romano, Luigi Luciano; Di Venere, Almerinda; Sardanelli, Anna Maria; Lorusso, Michele

    2012-04-01

    2-Arachidonoylglicerol (2-AG) is an endocannabinoid that mimics the pharmacological effects of Δ⁹ tetrahydrocannabinol, the psychoactive component of the plant Cannabis sativa. It is present in many mammalian tissues, such as brain, liver, spleen, heart and kidney, where it exerts different biological effects either receptor mediated or independently of receptor activation. This work analyzes the effects of 2-AG on liver mitochondrial functions. It is shown that 2-AG causes a relevant decrease of calcium induced cyclosporine A sensitive cytochrome c release from mitochondria, a process representing an early event of the apoptotic program. Cyclosporin sensitive matrix swelling and ROS production measured under the same conditions are, on the contrary, almost unaffected or even enhanced, respectively, by 2-AG. Furthemore, 2-AG is found to stimulate resting state succinate oxidase activity and to inhibit oligomycin sensitive F₀F₁ ATP synthase activity. All these effects are apparently associated with 2-AG dependent alteration in the fluidity of the mitochondrial membranes, which was measured as generalized polarization of laurdan fluorescence. PMID:22437740

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

    PubMed Central

    Isokawa, Masako

    2016-01-01

    GABAergic inhibition plays a critical role in the regulation of neuron excitability; thus, it is subject to modulations by many factors. Recent evidence suggests the elevation of intracellular calcium ([Ca2+]i) and calcium-dependent signaling molecules underlie the modulations. Caffeine induces a release of calcium from intracellular stores. We tested whether caffeine modulated GABAergic transmission by increasing [Ca2+]i. A brief local puff-application of caffeine to hippocampal CA1 pyramidal cells transiently suppressed GABAergic inhibitory postsynaptic currents (IPSCs) by 73.2 ± 6.98%. Time course of suppression and the subsequent recovery of IPSCs resembled DSI (depolarization-induced suppression of inhibition), mediated by endogenous cannabinoids that require a [Ca2+]i rise. However, unlike DSI, caffeine-induced suppression of IPSCs (CSI) persisted in the absence of a [Ca2+]i rise. Intracellular applications of BAPTA and ryanodine (which blocks caffeine-induced calcium release from intracellular stores) failed to prevent the generation of CSI. Surprisingly, ruthenium red, an inhibitor of multiple calcium permeable/release channels including those of stores, induced metaplasticity by amplifying the magnitude of CSI independently of calcium. This metaplasticity was accompanied with the generation of a large inward current. Although ionic basis of this inward current is undetermined, the present result demonstrates that caffeine has a robust Ca2+-independent inhibitory action on GABAergic inhibition and causes metaplasticity by opening plasma membrane channels. PMID:26998364

  19. Houttuyniae Herba Attenuates Kainic Acid-Induced Neurotoxicity via Calcium Response Modulation in the Mouse Hippocampus.

    PubMed

    Kim, Hyo Geun; Jeong, Hyun Uk; Hong, Sung In; Oh, Myung Sook

    2015-12-01

    Epilepsy is a complex neurological disorder characterized by the repeated occurrence of electrical activity known as seizures. This activity induces increased intracellular calcium, which ultimately leads to neuronal damage. Houttuyniae Herba, the aerial part of Houttuynia cordata, has various pharmacological effects and is widely used as a traditional herb. In the present study, we evaluated the protective effects of Houttuyniae Herba water extract on kainic acid-induced neurotoxicity. Kainic acid directly acts on calcium release, resulting in seizure behavior, neuronal damage, and cognitive impairment. In a rat primary hippocampal culture system, Houttuyniae Herba water extract significantly protected neuronal cells from kainic acid toxicity. In a seizure model where mice received intracerebellar kainic acid injections, Houttuyniae Herba water extract treatment resulted in a lower seizure stage score, ameliorated cognitive impairment, protected neuronal cells against kainic acid-induced toxicity, and suppressed neuronal degeneration in the hippocampus. In addition, Houttuyniae Herba water extract regulated increases in the intracellular calcium level, its related downstream pathways (reactive oxygen species production and mitochondrial dysfunction), and calcium/calmodulin complex kinase type II immunoreactivity in the mouse hippocampus, which resulted from calcium influx stimulation induced by kainic acid. These results demonstrate the neuroprotective effects of Houttuyniae Herba water extract through inhibition of calcium generation in a kainic acid-induced epileptic model. PMID:26366753

  20. Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity.

    PubMed Central

    Hirschi, K D

    1999-01-01

    Calcium (Ca(2)+) efflux from the cytosol modulates Ca(2+) concentrations in the cytosol, loads Ca(2+) into intracellular compartments, and supplies Ca(2+) to organelles to support biochemical functions. The Ca(2+)/H(+) antiporter CAX1 (for CALCIUM EXCHANGER 1) of Arabidopsis is thought to be a key mediator of these processes. To clarify the regulation of CAX1, we examined CAX1 RNA expression in response to various stimuli. CAX1 was highly expressed in response to exogenous Ca(2+). Transgenic tobacco plants expressing CAX1 displayed symptoms of Ca(2+) deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium concentrations, and to cold shock, but increasing the Ca(2+) in the media abrogated these sensitivities. Tobacco plants expressing CAX1 also demonstrated increased Ca(2+) accumulation and altered activity of the tonoplast-enriched Ca(2+)/H(+) antiporter. These results emphasize that regulated expression of Ca(2+)/H(+) antiport activity is critical for normal growth and adaptation to certain stresses. PMID:10559438

  1. A pH-sensitive, biobased calcium carbonate aragonite nanocrystal as a novel anticancer delivery system.

    PubMed

    Shafiu Kamba, Abdullahi; Ismail, Maznah; Tengku Ibrahim, Tengku Azmi; Zakaria, Zuki Abu Bakar

    2013-01-01

    The synthesised biobased calcium carbonate nanocrystals had demonstrated to be an effective carrier for delivery of anticancer drug doxorubicin (DOX). The use of these nanocrystals displayed high levels of selectivity and specificity in achieving effective cancer cell death without nonspecific toxicity. These results confirmed that DOX was intercalated into calcium carbonate nanocrystals at high loading and encapsulation efficiency (4.8 and 96%, resp.). The CaCO₃/DOX nanocrystals are relatively stable at neutral pH (7.4), resulting in slow release, but the nanocrystals progressively dissociated in acidic pH (4.8) regimes, triggering faster release of DOX. The CaCO₃/DOX nanocrystals exhibited high uptake by MDA MB231 breast cancer cells and a promising potential delivery of DOX to target cells. In vitro chemosensitivity using MTT, modified neutral red/trypan blue assay, and LDH on MDA MB231 breast cancer cells revealed that CaCO₃/DOX nanocrystals are more sensitive and gave a greater reduction in cell growth than free DOX. Our findings suggest that CaCO₃ nanocrystals hold tremendous promise in the areas of controlled drug delivery and targeted cancer therapy.

  2. Sensitivity of calcium binding in cerebral tissue to weak environmental electric fields oscillating at low frequency.

    PubMed Central

    Bawin, S M; Adey, W R

    1976-01-01

    Weak sinusoidal electric fields modify the calcium efflux from freshly isolated chick and cat cerebral tissues bathed in Ringer's solution, at 36 degrees. Following incubation (30 min) with radioactive calcium (45Ca2+), each sample, immersed in fresh solution, was exposed for 20 min to fields at 1, 6, 16, 32, or 75 Hz, with electric gradients of 5, 10, 56, and 100 V/m in air. 45Ca2+ efflux in the solution was then measured in 0.2 ml aliquots and compared with efflux from unexposed control samples. Field exposures resulted in a general trend toward a reduction in the release of the preincubated 45Ca2+. Both frequency and amplitude sensitivities were observed. Maximum decreases occurred at 6 and 16 Hz (12-15%). Thresholds were around 10 and 56 V/m for chick and cat tissues, respectively. Similar but nonsignificant trends occurred during other field exposures. All results were statistically compared with matched samples of controls. Tissue gradients could not be measured, but estimates were of the order of 0.1 muV/cm. The susceptibility of the electrochemical equilibrium in the neuronal membrane to small extracellular perturbations is discussed and a possible role for weak intrinsic cerebral fields in neuronal excitability is suggested. Images PMID:1064869

  3. Protein kinase A changes calcium sensitivity but not crossbridge kinetics in human cardiac myofibrils

    PubMed Central

    Walker, Lori A.; Margulies, Ken; Buttrick, Peter; de Tombe, Pieter

    2011-01-01

    We investigated the effect of PKA treatment (1 U/ml) on the mechanical properties of isolated human cardiac myofibrils. PKA treatment was associated with significant incorporation of radiolabeled phosphate into several sarcomeric proteins including troponin I and myosin binding protein C and was also associated with a right shift in the tension-pCa relation (ΔpCa50 = 0.2 ± 0.1). PKA treatment also caused right shifts in the pCa dependence of the rate of tension development, tension redevelopment, and the linear and exponential phases of myofibril relaxation. However, there was no change in the same measures of crossbridge turnover when expressed as a function of tension. We conclude that the changes in crossbridge kinetics as a function of calcium concentration reflect a reduced tension due to a lower calcium sensitivity and that the relationship between crossbridge kinetics and tension was unchanged, indicating no direct effect of PKA treatment on crossbridge cycling. PMID:21498779

  4. KCNJ2 Mutation Causes an Adrenergic-Dependent Rectification Abnormality with Calcium Sensitivity and Ventricular Arrhythmia

    PubMed Central

    Kalscheur, Matthew M.; Vaidyanathan, Ravi; Orland, Kate M.; Abozeid, Sara; Fabry, Nicholas; Maginot, Kathleen R.; January, Craig T.; Makielski, Jonathan C.; Eckhardt, Lee L.

    2014-01-01

    Background KCNJ2 mutations are associated with a variety of inherited arrhythmia syndromes including CPVT3. Objective Detailed cellular and mechanistic characterization of the clinically recognized KCNJ2 mutation R67Q. Methods Kir2.1 current density was measured using the whole-cell voltage clamp technique from COS-1 cells transiently transfected with WT-Kir2.1 and/or R67Q-Kir2.1. Catecholamine activity was simulated with PKA stimulating cocktail exposure. Phosphorylation deficient mutants, S425N-Kir2.1 and S425N-Kir2.1/R67Q-S425N-Kir2.1, were used in a separate set of experiments. HA- or Myc-Tag-WT-Kir2.1 or HA-Tag-R67Q-Kir2.1 were used for confocal imaging. Results A 33 year old presented with a CPVT-like clinical phenotype and was found to have KCNJ2 missense mutation R67Q. Treatment with nadolol and flecainide resulted in complete suppression of arrhythmias and symptom resolution. Under baseline conditions, R67Q-Kir2.1 expressed alone did not produce IK1 while cells co-expressing WT-Kir2.1 and R67Q-Kir2.1 showed rectification index (RI) similar to WT-Kir2.1. After PKA stimulation, R67Q-Kir2.1/WT-Kir2.1 failed to increase peak outward current density; WT-Kir2.1 increased 46% (n=5) while R67Q-Kir2.1/WT-Kir2.1 decreased 6% (n=6), p=0.002. Rectification properties in R67Q-Kir2.1/WT-Kir2.1 demonstrated sensitivity to calcium with decreased RI in high-calcium pipette solution (RI 20.3 ± 4.1%) compared to low-calcium (RI 36.5 ± 5.7%) (p< 0.05). Immunostaining of WT-Kir2.1 and R67Q-Kir2.1 individually and together showed a normal membrane expression pattern and co-localization by Pearson’s correlation coefficient. Conclusion R67Q-Kir2.1 is associated with an adrenergic-dependent clinical and cellular phenotype with rectification abnormality enhanced by increased calcium. These findings are a significant advancement of our knowledge and understanding of phenotype-genotype relationship of arrhythmia syndromes related to KCNJ2 mutations. PMID:24561538

  5. 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.

  6. Calcium-induced assembly of adherens junctions in keratinocytes

    PubMed Central

    1987-01-01

    Extracellular calcium concentration has been shown to control the stratification of cultured keratinocytes, presumably by regulation of formation of desmosomes. Previous studies have shown that keratinocytes cultured in medium containing 0.1 mM Ca++ form loose colonies without desmosomes. If the Ca++ is raised to 1 mM, desmosomes are assembled and the distribution of keratin filaments is altered. We have examined the disposition of vinculin and actin in keratinocytes under similar conditions. Using immunofluorescence microscopy we show that raising [Ca++] in the medium dramatically alters the distribution of vinculin and actin and results in the formation of adherens-type junctions within 15 min after switching to high calcium medium. Borders of cells at the edge of colonies, which are not proximal to other cells, are not affected, while cells in the interior of the colony form junctions around their periphery. Attachment plaques in keratinocytes grown in low calcium medium are located at the ventral plane of the cell, but junctions formed after switching to high calcium are not, as demonstrated by interference reflection microscopy. In cells colabeled with antibodies against vinculin and desmoplakin, vinculin-containing adherens junctions were visible before desmosomal junctions when cells were switched to high calcium. Although newly formed vinculin- containing structures in high calcium cells, like desmosomes, colocalize with phase-dense structures, superimposition of video fluorescence images using digitized fluorescence microscopy indicates that adherens junctions and desmosomes are discrete structures. Adherens junctions, like desmosomes, may play an essential role in controlling stratification of keratinocytes. PMID:2442175

  7. [Calcium-induced changes in bilayer membranes from oxidized cholesterol].

    PubMed

    Hianik, T; Miklovichova, J; Foltinova, O; Bajchi, A

    1985-01-01

    Elastic properties of oxidized cholesterol bilayers in n-octane and membrane solvent free were studied by measuring Young modulus E perpendicular in the direction perpendicular to the membrane plane as a function of concentration of calcium ions. Interaction between calcium ions and solvent free bilayers resulted in a significant increases of Young modulus E perpendicular in the concentration range 20-40 mmol/l Ca2+. It is suggested that the hardening of the membrane is caused by some structural changes in the hydrophobic region of the membrane.

  8. Beta-adrenergic modulation of cardiac ion channels. Differential temperature sensitivity of potassium and calcium currents

    PubMed Central

    1989-01-01

    beta-Adrenergic stimulation of ventricular heart cells results in the enhancement of two important ion currents that regulate the plateau phase of the action potential: the delayed rectifier potassium channel current (IK) and L-type calcium channel current (ICa). The temperature dependence of beta-adrenergic modulation of these two currents was examined in patch-clamped guinea pig ventricular myocytes at various steps in the beta-receptor/cyclic AMP-dependent protein kinase pathway. External applications of isoproterenol and forskolin were used to activate the beta-receptor and the enzyme adenylate cyclase, respectively. Internal dialysis of cyclic 3',5'-adenosine monophosphate (cAMP) or the catalytic subunit of cAMP-dependent protein kinase (CS), as well as the external addition of 8-chlorphenylthio cAMP (CPT-cAMP) was applied to increase intracellular levels of cAMP and CS. Isoproterenol-mediated increases in IK, but not ICa, were found to be very temperature dependent over the range of 20-37 degrees C. At room temperature (20-22 degrees C) isoproterenol produced a large (threefold) enhancement of ICa but had no effect on IK. In contrast, at warmer temperatures (30-37 degrees C) both currents increased in the presence of this agonist and the kinetics of IK were slowed at -30 mV. A similar temperature sensitivity also existed after exposure to forskolin, CPT-cAMP, cAMP, and CS, suggesting that this temperature sensitivity of IK may arise at the channel protein level. Modulation of IK during each of these interventions was accompanied by a slowing in IK kinetics. Thus, regulation of cardiac potassium channels but not calcium channels involves a temperature-dependent step that occurs after activation of the catalytic subunit of cAMP-dependent protein kinase. PMID:2472462

  9. Omega-conotoxin GVIA binding to a high-affinity receptor in brain: characterization, calcium sensitivity, and solubilization

    SciTech Connect

    Wagner, J.A.; Snowman, A.M.; Biswas, A.; Olivera, B.M.; Snyder, S.H.

    1988-09-01

    We describe unique, high-affinity binding sites for omega(/sup 125/I)conotoxin GVIA in membranes from rat brain and rabbit sympathetic ganglia which appear to be primarily associated with N-type voltage-dependent calcium channels. The dissociation constant (KD) for the toxin in rat brain membranes is 60 pM. Physiologic extracellular concentrations of calcium inhibit toxin binding noncompetitively (IC50 = 0.2 mM). The regional distribution of the binding sites in rat brain differs markedly from that of dihydropyridine calcium antagonist receptors associated with L-type calcium channels. In detergent-solubilized brain membranes, toxin binding retains the same affinity, specificity, and ionic sensitivity as in particulate preparations.

  10. Loss of β2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction

    PubMed Central

    Chand, Kirat K; Lee, Kah Meng; Schenning, Mitja P; Lavidis, Nickolas A; Noakes, Peter G

    2015-01-01

    β2-laminin is a key mediator in the differentiation and formation of the skeletal neuromuscular junction. Loss of β2-laminin results in significant structural and functional aberrations such as decreased number of active zones and reduced spontaneous release of transmitter. In vitro β2-laminin has been shown to bind directly to the pore forming subunit of P/Q-type voltage-gated calcium channels (VGCCs). Neurotransmission is initially mediated by N-type VGCCs, but by postnatal day 18 switches to P/Q-type VGCC dominance. The present study investigated the changes in neurotransmission during the switch from N- to P/Q-type VGCC-mediated transmitter release at β2-laminin-deficient junctions. Analysis of the relationship between quantal content and extracellular calcium concentrations demonstrated a decrease in the calcium sensitivity, but no change in calcium dependence at β2-laminin-deficient junctions. Electrophysiological studies on VGCC sub-types involved in transmitter release indicate N-type VGCCs remain the primary mediator of transmitter release at matured β2-laminin-deficient junctions. Immunohistochemical analyses displayed irregularly shaped and immature β2-laminin-deficient neuromuscular junctions when compared to matured wild-type junctions. β2-laminin-deficient junctions also maintained the presence of N-type VGCC clustering within the presynaptic membrane, which supported the functional findings of the present study. We conclude that β2-laminin is a key regulator in development of the NMJ, with its loss resulting in reduced transmitter release due to decreased calcium sensitivity stemming from a failure to switch from N- to P/Q-type VGCC-mediated synaptic transmission. PMID:25556799

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

    PubMed

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

    2012-01-10

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

  12. Calcium-induced changes in cytoskeleton and motility of cultured human keratinocytes.

    PubMed

    Magee, A I; Lytton, N A; Watt, F M

    1987-09-01

    In normal epidermis keratinocytes migrate upward from the basal layer as they undergo terminal differentiation, yet they also have the capacity for lateral movement during wound healing. The purpose of our experiments was to investigate these two types of movement by manipulating the calcium ion concentration of the medium so that keratinocytes formed monolayers (0.1 mM calcium) or stratified sheets (2.0 mM calcium). Time-lapse video recording indicated that keratinocytes in low-calcium medium were laterally more motile than keratinocytes in normal medium. This was consistent with the ultrastructural appearance of the cells and the lack of desmosomal junctions, determined by scanning and transmission electron microscopy. During calcium-induced stratification keratinocytes moved upward from the basal layer by gliding over their neighbors and forming contacts with other suprabasal cells. Keratinocytes in low-calcium medium migrated into wounds made in the cultures, a process which was inhibited by monensin; however, stratified keratinocytes in normal medium did not enter wounds. Cytochalasin D caused rapid cell rounding and disruption of actin filaments in keratinocytes grown in low-calcium but not in normal medium, indicating more rapid treadmilling of actin and consistent with the greater motility of keratinocytes in low-calcium medium. Our results suggest that desmosome formation may place constraints on the movement of individual keratinocytes and that the actomyosin cytoskeleton is involved in lateral migration.

  13. Polymer surface interacts with calcium in aqueous media to induce stem cell assembly.

    PubMed

    Hung, Kun-Che; Hsu, Shan-Hui

    2015-10-28

    Bioinspired surface with functional group rearrangement abilities are highly desirable for designing functional materials. Calcium ion (Ca(2+) ) is a pivotal life element and the ion transport is tightly regulated through calcium channels. It is demonstrated here that Ca(2+) can be transported by polymer surface to induce cell assembly. A series of polyurethane materials is synthesized with different abilities to rearrange the surface functional groups in response to aqueous environment. It is observed that surface recruitment of carboxyl and amino groups from the bulk material can interact with Ca(2+) and facilitate its translocation from aqueous media into cells. The surface rearrangement of functional group triggers the calcium trafficking and turns on signals involving cell merging and assembly. This observation provides an insight on adjusting material-calcium interaction to design nature-inspired smart interfaces to induce cell organization and tissue regeneration.

  14. Calcium-induced contraction of sarcomeres changes the regulation of mitochondrial respiration in permeabilized cardiac cells.

    PubMed

    Anmann, Tiia; Eimre, Margus; Kuznetsov, Andrey V; Andrienko, Tatiana; Kaambre, Tuuli; Sikk, Peeter; Seppet, Evelin; Tiivel, Toomas; Vendelin, Marko; Seppet, Enn; Saks, Valdur A

    2005-06-01

    The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system. PMID:15955072

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

    PubMed

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

    1975-04-01

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

  16. Catechin prevents the calcium oxalate monohydrate induced renal calcium crystallization in NRK-52E cells and the ethylene glycol induced renal stone formation in rat

    PubMed Central

    2013-01-01

    Background Reactive oxygen species play important roles in renal calcium crystallization. In this study, we examined the effects of catechin, which have been shown to have antioxidant properties on the renal calcium crystallization. Methods In the vitro experiment, the changes of the mitochondrial membrane potential, expression of superoxide dismutase (SOD), 4-hydroxynonenal (4-HNE), cytochrome c, and cleaved caspase 3 were measured to show the effects of catechin treatment on the NRK-52E cells induced by calcium oxalate monohydrate (COM). In the vivo study, Sprague–Dawley rats were administered 1% ethylene glycol (EG) to generate a rat kidney stone model and then treated with catechin (2.5 and 10 mg/kg/day) for 14 days. The urine and serum variables were dected on 7 and 14 days after EG administration. The expression of cytochrome c, cleaved caspase 3, SOD, osteopontin (OPN), malondialdehyde (MDA), 8-hydroxy-2′-deoxyguanosine (8-OHdG) in kidney were measured. Furthermore, the mitochondrial microstructure in the kidney was also examined by transmission electron microscopy. Results Catechin treatment could prevent the changes in mitochondrial membrane potential and expression of SOD, 4-HNE, cytochrome c, and cleaved caspase 3 in NRK-52E cells induced by the COM. For the in vivo experiments, the EG administration induced renal calcium crystallization was also prevented by the catechin. The expression of SOD, OPN, MDA, OPN and 8-OHdG, were increased after EG administration and this increase was diminished by catechin. Moreover, catechin also prevented EG induced mitochondrial collapse in rat. Conclusions Catechin has preventive effects on renal calcium crystallization both in vivo and in vitro, and provide a potential therapeutic treatment for this disease. PMID:24044655

  17. Stereocontrolled synthesis of rosuvastatin calcium via iodine chloride-induced intramolecular cyclization.

    PubMed

    Xiong, Fangjun; Wang, Haifeng; Yan, Lingjie; Han, Sheng; Tao, Yuan; Wu, Yan; Chen, Fener

    2016-01-28

    A novel, stereoselective approach towards rosuvastatin calcium from the known (S)-homoallylic alcohol has been developed. The synthesis is highlighted by a regio- and stereocontrolled ICl-induced intramolecular cyclization of chiral homoallylic carbonate to deliver the C6-formyl statin side chain with a syn-1,3-diol moiety. An improved synthesis of the rosuvastatin pyrimidine core moiety is also included. Moreover, this methodology is useful in the asymmetric synthesis of structural variants of statins such as pitavastatin calcium and atorvastatin calcium and their related analogs.

  18. Calcium transients and the effect of a photolytically released calcium chelator during electrically induced contractions in rabbit rectococcygeus smooth muscle.

    PubMed Central

    Arner, A; Malmqvist, U; Rigler, R

    1998-01-01

    Intracellular Ca2+ was determined with the fura-2 technique during electrically induced contractions in the rabbit rectococcygeus smooth muscle at 22 degreesC. The muscles were electrically activated to give short, reproducible contractions. Intracellular [Ca2+] increased during activation; the increase in [Ca2+] preceded force development by approximately 2 s. After cessation of stimulation Ca2+ fell, preceding the fall in force by approximately 4 s. The fluorescence properties of fura-2 were determined with time-resolved spectroscopy using synchrotron light at the MAX-storage ring, Lund, Sweden. The fluorescence decay of free fura-2 was best described by two exponential decays (time constants approximately 0.5 and 1.5 ns) at low Ca2+ (pCa 9). At high Ca2+ (pCa 4.5), fluorescence decay became slower and could be fitted by one exponential decay (1.9 ns). Time-resolved anisotropy of free fura-2 was characteristic of free rotational motion (correlation time 0.3 ns). Motion of fura-2 could be markedly inhibited by high concentrations of creatine kinase. Time-resolved spectroscopy measurements of muscle fibers loaded with fura-2 showed that the fluorescence lifetime of the probe was longer, suggesting an influence of the chemical environment. Anisotropy measurements revealed, however, that the probe was mobile in the cells. The Ca2+-dependence of contraction and relaxation was studied using a photolabile calcium chelator, diazo-2, which could be loaded into the muscle cells in a similar manner as fura-2. Photolysis of diazo-2 leads to an increase in its Ca2+-affinity and a fall in free Ca2+. When muscles that had been loaded with diazo-2 were illuminated with UV light flashes during the rising phase of contraction, the rate of contraction became slower, suggesting a close relation between intracellular Ca2+ and the cross-bridge interaction. In contrast, photolysis during relaxation did not influence the rate of force decay, suggesting that relaxation of these

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

    PubMed

    Moheno, Phillip; Pfleiderer, Wolfgang; Fuchs, Dietmar

    2009-01-01

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

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

    PubMed

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

    2013-07-01

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

  1. Membrane perturbation: studies employing a calcium-sensitive dye, arsenazo III, in liposomes.

    PubMed Central

    Weissmann, G; Collins, T; Evers, A; Dunham, P

    1976-01-01

    A metallochromic dye, arsenazo III [2,7-bis-(2-arsonophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid], has been incorporated into the aquenous interspaces of multilamellar liposomes. multilamellar liposomes. Addition of Ca produced no shift in the absorbance spectrum of dye captured by liposomes, whereas disruption of liposomes by Triton X-100, followed by Ca, produced the spectrum chracteristic of the dye-Ca complex: evidence of latency. Addition of excess ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) reversed the spectal shift. Differences between spectra obtained in this sequence yielded dye efflus. To measure Ca efflux, difference spectra (+/-EGTA) were obtained from cationic liposomes containing Ca after detergent lysis (sensitivity less than 10 mmol/ml). Since liposomes were impermeable either to dye or Ca until perturbed, it was possible to test a variety of membrane-active steroids (diethylstilbesterol, deoxycorticosterone, etiocholanolone) for their capacity to provoke dye efflux from liposomes; preincorporation of cortisol stablized liposomes against dye leak. Immunoglobulin-coated liposomes containing dye were taken up by phagocytes of Mustelus canis, and phagocytic vacuoles stained red-purple after ingestions. Liposomes containing the calcium-sensitive dye constitute a simple, accurate means for determining membrane perturbation and Ca fluxes; their uptake by cells or organelles remains to be exploited further. PMID:1061152

  2. Aging, motor function, and sensitivity to calcium channel blockers: An investigation using chronic methylmercury exposure.

    PubMed

    Shen, Andrew Nathanael; Cummings, Craig; Hoffman, Daniel; Pope, Derek; Arnold, Megan; Newland, M Christopher

    2016-12-15

    Methylmercury (MeHg) neurotoxicity is thought to be mediated, in part, by dysregulation of calcium (Ca(2+)) homeostasis, a mechanism that may also slowly and progressively degrade neuronal function during normal aging. Longitudinal studies of MeHg exposure provide a powerful approach to studying neural and behavioral mechanisms by which both MeHg toxicity and aging affect motor function. Wheel-running and rotarod performance were assessed in two age groups of BALB/c mice chronically exposed to 0 or 1.2mg/kg/day MeHg and 0 or 20mg/kg/day nimodipine, a 1,4-dihyrdopyridine L-type calcium channel blocker (CCB), for approximately 8.5 months. Adults began exposure on postnatal day (PND) 72 and retired breeders on PND 296. A log-survivor bout analysis partitioned wheel-running into bouts that identified motor (within-bout rates) and motivational (bout-initiation rates) influences. Retired breeders ran farther, because of a higher bout-initiation rates, but performed more poorly on the rotarod than younger adults, a difference unaffected by nimodipine. MeHg produced relatively age-independent deficits in wheel-running and rotarod performance, whereas nimodipine afforded greater protection to adult mice than to retired breeders. Rotarod performance and within-bout response rate were more sensitive to and more reliable predictors of MeHg toxicity than bout-initiation rate, which was least affected by MeHg exposure. Thus the motivation to run was unimpaired as the ability to do so declined. While chronic MeHg exposure produced functionally similar behavior deficits between age groups, the age-dependent neuroprotection by nimodipine supports the notion that underlying neurobiological systems mediated by Ca(2+) signaling, are differentially affected in older adults. PMID:27481695

  3. Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation

    PubMed Central

    Peshenko, Igor V.; Olshevskaya, Elena V.; Dizhoor, Alexander M.

    2012-01-01

    Guanylyl cyclase activating proteins (GCAPs) are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS) of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala) on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca2+ binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity of RetGC reconstituted with the G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand proximal to the myristoyl residue in three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism. PMID:22371697

  4. Membrane depolarization induces calcium-dependent upregulation of Hsp70 and Hmox-1 in skeletal muscle cells.

    PubMed

    Jorquera, Gonzalo; Juretić, Nevenka; Jaimovich, Enrique; Riveros, Nora

    2009-09-01

    Heat shock proteins (HSPs) are a conserved family of cytoprotective polypeptides, synthesized by cells in response to stress. Hsp70 and heme oxygenase 1 (Hmox-1) are induced by a variety of cellular stressors in skeletal muscle, playing a role in long-term adaptations and muscle fibers regeneration. Though HSPs expression after exercise has been intensely investigated, the molecular mechanisms concerning Hsp70 and Hmox-1 induction are poorly understood. The aim of this work was to investigate the involvement of calcium in Hsp70 and Hmox-1 expression upon depolarization of skeletal muscle cells. We observed that depolarization of myotubes increased both mRNA levels and protein expression for Hsp70 and Hmox-1. Stimulation in the presence of intracellular calcium chelator BAPTA-AM resulted in a complete inhibition of Hsp70-induced expression. It is known that inositol-1,4,5-trisphophate (IP(3))-mediated slow Ca(2+) transients, evoked by membrane depolarization, are involved in the regulation of gene expression. Here we demonstrated that inhibition of IP(3)-dependent calcium signals decreased both Hsp70 mRNA induction and Hsp70 and Hmox-1 protein expression. Inhibitors of calcium-dependent protein kinase C also abolished Hsp70 mRNA induction. Our results provide evidence that membrane depolarization increases Hsp70 and Hmox-1 expression in cultured skeletal muscle cells, which the effect is critically dependent on Ca(2+) released from IP(3)-sensitive intracellular stores and that it involves PKC as an upstream effector in Hsp70 mRNA-induced expression. PMID:19570893

  5. Cardiac contraction, calcium transients, and myofilament calcium sensitivity fluctuate with the estrous cycle in young adult female mice.

    PubMed

    MacDonald, Jennifer K; Pyle, W Glen; Reitz, Cristine J; Howlett, Susan E

    2014-04-01

    This study established conditions to induce regular estrous cycles in female C57BL/6J mice and investigated the impact of the estrous cycle on contractions, Ca2+ transients, and underlying cardiac excitation-contraction (EC)-coupling mechanisms. Daily vaginal smears from group-housed virgin female mice were stained to distinguish estrous stage (proestrus, estrus, metestrus, diestrus). Ventricular myocytes were isolated from anesthetized mice. Contractions and Ca2+ transients were measured simultaneously (4 Hz, 37 °C). Interestingly, mice did not exhibit regular cycles unless they were exposed to male pheromones in bedding added to their cages. Field-stimulated myocytes from mice in estrus had larger contractions (∼2-fold increase), larger Ca2+ transients (∼1.11-fold increase), and longer action potentials (>2-fold increase) compared with other stages. Larger contractions and Ca2+ transients were not observed in estrus myocytes voltage-clamped with shorter action potentials. Voltage-clamp experiments also demonstrated that estrous stage had no effect on Ca2+ current, EC-coupling gain, diastolic Ca2+, sarcoplasmic reticulum (SR) Ca2+ content, or fractional release. Although contractions were largest in estrus, myofilament Ca2+ sensitivity was lowest (EC50 values ∼1.15-fold higher) in conjunction with increased phosphorylation of myosin binding protein C in estrus. Contractions were enhanced in ventricular myocytes from mice in estrus because action potential prolongation increased SR Ca2+ release. These findings demonstrate that cyclical changes in reproductive hormones associated with the estrous cycle can influence myocardial electrical and contractile function and modify Ca2+ homeostasis. However, such changes are unlikely to occur in female mice housed in groups under conventional conditions, since these mice do not exhibit regular estrous cycles.

  6. Polymerization of calcium caseinates solutions induced by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Lacroix, M.; Jobin, M.; Mezgheni, E.; Srour, M.; Boileau, S.

    1998-06-01

    Solutions of calcium caseinate (5%) combined with propylene glycol (PG) or triethylene glycol(TEG) (0, 2.5% and 5%) and used for the development of edible films and coatings, were irradiated at doses between 0 to 128 kGy. Solutions were chromatographed through toyopearl HW 55F resin to observe the effect of irradiation on cross-link reactions. In unirradiated calcium caseinate solutions, two peaks could be observed (fractions 30 and 37) while samples irradiated at 64 kGy and 128 kGy showed one shifted peak at fraction 32 and 29 respectively. No effect of the plasticizers was observed. According to proteins standards of knowed molecular weights, the molecular weight of calcium caseinate increased approximately 10 times when irradiated at 128 kGy and 5 times when irradiated at 64 kGy. The physico-chemical properties of bio-films prepared with the irradiated solutions, demonstrated that tensile strength at break increased with increase of irradiation dose. A maximum dose was obtained at 16 kGy.

  7. Effects of calcium antagonists on isolated bovine cerebral arteries: inhibition of constriction and calcium-45 uptake induced by potassium or serotonin

    SciTech Connect

    Wendling, W.W.; Harakal, C.

    1987-05-01

    The purpose of this study was to determine the mechanisms by which organic calcium channel blockers inhibit cerebral vasoconstriction. Isolated bovine middle cerebral arteries were cut into rings to measure contractility or into strips to measure radioactive calcium (/sup 45/Ca) influx and efflux. Calcium channel blockers (10(-5) M verapamil or 3.3 X 10(-7) M nifedipine) and calcium-deficient solutions all produced near-maximal inhibition of both potassium- and serotonin-induced constriction. In calcium-deficient solutions containing potassium or serotonin, verapamil and nifedipine each blocked subsequent calcium-induced constriction in a competitive manner. Potassium and serotonin significantly increased /sup 45/Ca uptake into cerebral artery strips during 5 minutes of /sup 45/Ca loading; for potassium /sup 45/Ca uptake increased from 62 to 188 nmol/g, and for serotonin from 65 to 102 nmol/g. Verapamil or nifedipine had no effect on basal /sup 45/Ca uptake but significantly blocked the increase in /sup 45/Ca uptake induced by potassium or serotonin. Potassium, and to a lesser extent serotonin, each induced a brief increase in the rate of /sup 45/Ca efflux into calcium-deficient solutions. Verapamil or nifedipine had no effect on basal or potassium-stimulated /sup 45/Ca efflux. The results demonstrate that verapamil and nifedipine block /sup 45/Ca uptake through both potential-operated (potassium) and receptor-operated (serotonin) channels in bovine middle cerebral arteries.

  8. Unsaturated fatty acids induce calcium influx into keratinocytes and cause abnormal differentiation of epidermis.

    PubMed

    Katsuta, Yuji; Iida, Toshii; Inomata, Shinji; Denda, Mitsuhiro

    2005-05-01

    Abnormal follicular keratinization is involved in comedogenesis in acne vulgaris. We recently demonstrated that calcium influx into epidermal keratinocytes is associated with impaired skin barrier function and epidermal proliferation. Based on these results, we hypothesized that sebum components affect calcium dynamics in the keratinocyte and consequently induce abnormal keratinization. To test this idea, we first observed the effects of topical application of sebum components, triglycerides (triolein), saturated fatty acids (palmitic acid and stearic acid), and unsaturated fatty acids (oleic acid and palmitoleic acid) on hairless mouse skin. Neither triglyceride nor saturated fatty acids affected the skin surface morphology or epidermal proliferation. On the other hand, application of unsaturated fatty acids, oleic acid, and palmitoleic acid induced scaly skin, abnormal keratinization, and epidermal hyperplasia. Application of triglycerides and saturated fatty acids on cultured human keratinocytes did not affect the intracellular calcium concentration ([Ca(2+)](i)), whereas unsaturated fatty acids increased the [Ca(2+)](i) of the keratinocytes. Moreover, application of oleic acid on hairless mouse skin induced an abnormal calcium distribution in the epidermis. These results suggest that unsaturated fatty acids in sebum alter the calcium dynamics in epidermal keratinocytes and induce abnormal follicular keratinization.

  9. Individual aggregates of amyloid beta induce temporary calcium influx through the cell membrane of neuronal cells

    PubMed Central

    Drews, Anna; Flint, Jennie; Shivji, Nadia; Jönsson, Peter; Wirthensohn, David; De Genst, Erwin; Vincke, Cécile; Muyldermans, Serge; Dobson, Chris; Klenerman, David

    2016-01-01

    Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations. PMID:27553885

  10. Individual aggregates of amyloid beta induce temporary calcium influx through the cell membrane of neuronal cells.

    PubMed

    Drews, Anna; Flint, Jennie; Shivji, Nadia; Jönsson, Peter; Wirthensohn, David; De Genst, Erwin; Vincke, Cécile; Muyldermans, Serge; Dobson, Chris; Klenerman, David

    2016-01-01

    Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations. PMID:27553885

  11. Different calcium sensitivity in osteoclasts on glass and on bone and maintenance of cytoskeletal structures on bone in the presence of high extracellular calcium.

    PubMed

    Lakkakorpi, P T; Lehenkari, P P; Rautiala, T J; Väänänen, H K

    1996-09-01

    The sensitivity of rat osteoclasts to increased extracellular calcium concentrations ([Ca2+]e) was investigated by single cell measurements of free cytosolic calcium concentrations ([Ca2+]i), by changes in microfilament organization of resorbing osteoclasts, and by in vitro bone resorption assays. Osteoclasts cultured on glass and on bone showed clear differences in their responses, as in 44% and 52% of osteoclasts on glass but in only 21% and 25% of osteoclasts on bone [Ca2+]i increased when [Ca2+]e was increased from 2 mM to 6 or 10 mM via perfusion, respectively. Bone resorption was inhibited without changes in the osteoclast numbers only by 10 mM [Ca2+]e in 2 day cultures. Furthermore, there were no changes in the organization of microfilament structures in resorbing osteoclasts after increased [Ca2+]e (up to 20 mM [Ca2+]e, 30 min incubation). These results suggest that the sensitivity of osteoclasts to increased [Ca2+]e is dependent on their activation phase (resting/migrating vs. resorbing) and that resorbing osteoclasts are not sensitive to increased [Ca2+]e or that the sensing system cannot be reached in polarized resorbing osteoclasts. In contrast, increasing [Ca2+]i through the use of calcium ionophores dispersed specific microfilament structures at the sealing zone transiently in a few minutes. This shows that [Ca2+]i is used as a signaling mechanism to inactivate osteoclasts, with a similar end result on microfilament structures at the sealing zone as caused by increased concentration of cAMP and activation of protein kinase C.

  12. Calcium-sensing receptor induces rat neonatal ventricular cardiomyocyte apoptosis

    SciTech Connect

    Sun Yihua; Liu Meina; Li Hong; Shi Sa; Zhao Yajun; Wang Rui; Xu Changqing . E-mail: syh200415@yahoo.com.cn

    2006-12-01

    The calcium-sensing receptor (CaSR) exists in many tissues, and its expression has been identified in rat cardiac tissue. However, Physiological importance and pathophysiological involvement of CaSR in homeostatic regulation of cardiac function are unclear. To investigate the relation of CaSR and apoptosis in cardiomyocytes, we examined the role of the CaSR activator gadolinium chloride (GdCl{sub 3}) in rat neonatal ventricular cardiomyocytes. Expression of the CaSR protein was observed by Western blot. The apoptotic ratio of rat neonatal ventricular cardiomyocytes was measured with flow cytometry and immunofluorescence techniques. A laser scan confocal microscope was used to detect the intracellular concentration of calcium ([Ca{sup 2+}]{sub i}) in rat neonatal ventricular cardiomyocytes using the acetoxymethyl ester of fluo-3 (fluo-3/(AM)) as a fluorescent dye. The results showed that GdCl{sub 3} increased the phosphorylation of extracellular signal-regulated protein kinase (ERK), c-Jun NH{sub 2}-terminal protein kinases (JNK), and p38. GdCl{sub 3} also activated caspase 9 and increased apoptosis in myocyte by increasing [Ca{sup 2+}]{sub i}. In conclusion, these results suggest that CaSR promotes cardiomyocyte apoptosis in rat neonatal ventricular cardiomyocytes through activation of mitogen-activated protein kinases and caspase 9 signaling pathways.

  13. Studies on psychomotoric effects and pharmacokinetic interactions of the new calcium sensitizing drug levosimendan and ethanol.

    PubMed

    Antila, S; Järvinen, A; Akkila, J; Honkanen, T; Karlsson, M; Lehtonen, L

    1997-07-01

    Levosimendan (CAS 141505-33-1) is a calcium sensitizing drug intended for the treatment of congestive heart failure. In animal experiments levosimendan has potentiated the sedative effects of ethanol. Due to poor water solubility of the compound, ethanol is used as a diluent in the intravenous formulation. In this study the possible interactions between levosimendan and ethanol in human have been studied. Twelve healthy male volunteers were included in this double-blind, randomized, cross-over study. The study consisted of three treatment periods: levosimendan 1 mg intravenously, levosimendan combined with ethanol orally and ethanol 0.8 g/kg alone. Blood samples for determination of levosimendan and ethanol concentrations were collected for 8 h after the dosing. To observe possible pharmacodynamic interactions psychomotoric tests were made before drug administration and 1h, 2h, 3h and 6h thereafter. These tests included Digit symbol substitution test, Maddox wing, Critical Flicker fusion and VAS-test for subjective assessment of performance status. Plasma levosimendan concentrations were not changed by the concomitant ethanol administration. Ethanol did not alter the pharmacokinetics of levosimendan except the volume of distribution of central compartment which was decreased. Levosimendan did neither affect elimination of ethanol. Levosimendan did not potentiate the psychomotoric effects of ethanol neither did it have any psychomotoric effects itself. In conclusion, levosimendan is not likely to have any psychomotoric adverse effects or any clinically significant interactions with ethanol.

  14. Trolox-Sensitive Reactive Oxygen Species Regulate Mitochondrial Morphology, Oxidative Phosphorylation and Cytosolic Calcium Handling in Healthy Cells

    PubMed Central

    Distelmaier, Felix; Valsecchi, Federica; Forkink, Marleen; van Emst-de Vries, Sjenet; Swarts, Herman G.; Rodenburg, Richard J.T.; Verwiel, Eugène T.P.; Smeitink, Jan A.M.; Willems, Peter H.G.M.

    2012-01-01

    Abstract Aims: Cell regulation by signaling reactive oxygen species (sROS) is often incorrectly studied through extracellular oxidant addition. Here, we used the membrane-permeable antioxidant Trolox to examine the role of sROS in mitochondrial morphology, oxidative phosphorylation (OXPHOS), and cytosolic calcium (Ca2+) handling in healthy human skin fibroblasts. Results and Innovation: Trolox treatment reduced the levels of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein (CM-H2DCF) oxidizing ROS, lowered cellular lipid peroxidation, and induced a less oxidized mitochondrial thiol redox state. This was paralleled by increased glutathione- and mitofusin-dependent mitochondrial filamentation, increased expression of fully assembled mitochondrial complex I, elevated activity of citrate synthase and OXPHOS enzymes, and a higher cellular O2 consumption. In contrast, Trolox did not alter hydroethidium oxidation, cytosolic thiol redox state, mitochondrial NAD(P)H levels, or mitochondrial membrane potential. Whole genome expression profiling revealed that Trolox did not trigger significant changes in gene expression, suggesting that Trolox acts downstream of this process. Cytosolic Ca2+ transients, induced by the hormone bradykinin, were of a higher amplitude and decayed faster in Trolox-treated cells. These effects were dose-dependently antagonized by hydrogen peroxide. Conclusions: Our findings suggest that Trolox-sensitive sROS are upstream regulators of mitochondrial mitofusin levels, morphology, and function in healthy human skin fibroblasts. This information not only facilitates the interpretation of antioxidant effects in cell models (of oxidative-stress), but also contributes to a better understanding of ROS-related human pathologies, including mitochondrial disorders. Antioxid. Redox Signal. 17, 1657–1669. PMID:22559215

  15. [Thapsigargin-sensitive and insensitive intracellular calcium stores in acinar cells of the submandibular salivary gland in rats].

    PubMed

    Kopach, O V; Kruhlykov, I A; Voĭtenko, N V; Fedirko, N V

    2005-01-01

    Acinar cells of rat submandibular salivary gland are characterized by heterogeneity of intracellular Ca2+ stores. In the present work we have studied this heterogeneity using Arsenazo III dye to measure a cellular total calcium content and Fura-2/AM, to determine free cytosolic calcium concentration ([Ca2+]i). We have found that the amount of Ca2+ released by inhibition of Ca2+ ATPase of the ER with thapsigargin comprises approximately 30% of total ER calcium. This result was obtained in experiments on both intact and permeabilized acinar cells. We have also shown that both Ca2+ ATPase inhibition with thapsigargin and emptying the stores with acetylcholine (ACh) led to activation of store-operated Ca2+ influx (an increase in total calcium content of approximately 14%). In permeabilized cells application of ACh after preincubation with thapsigargin led to a further decrease in total cellular calcium content (approximately 38%). At the same time in intact cells it resulted in generation of [Ca2+]i transients with gradually decreasing amplitudes. Thus, ACh is capable of producing an additional release of Ca2+ from thapsigargin-insensitive stores. This additional release is IP3-dependent since it was completely blocked by heparin. We conclude that in acinar cells of rat submandibular gland thapsigargin-sensitive and thapsigargin-insensitive Ca2+ stores could exist.

  16. Calcium induced ATP synthesis: Isotope effect, magnetic parameters and mechanism

    NASA Astrophysics Data System (ADS)

    Buchachenko, A. L.; Kuznetsov, D. A.; Breslavskaya, N. N.; Shchegoleva, L. N.; Arkhangelsky, S. E.

    2011-03-01

    ATP synthesis by creatine kinase with calcium ions is accompanied by 43Ca/ 40Ca isotope effect: the enzyme with 43Ca 2+ was found to be 2.0 ± 0.3 times more active than enzymes, in which Ca 2+ ions have nonmagnetic nuclei 40Ca. The effect demonstrates that primary reaction in ATP synthesis is electron transfer between reaction partners, Сa( HO)n2+ ( n ⩽ 3) and Ca 2+(ADP) 3-. It generates ion-radical pair, in which spin conversion results in the isotope effect. Magnetic parameters (g-factors and HFC constants a( 43Ca) and a( 31P)) confirm that namely terminal oxygen atom of the ADP ligand in the complex Ca 2+(ADP) 3- donates electron to the Ca( HO)n2+ ion.

  17. Electrically Induced Calcium Handling in Cardiac Progenitor Cells

    PubMed Central

    Wagner, Mary B.

    2016-01-01

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

  18. Healing and Preventive Effects of Calcium Alginate on Carbon Tetrachloride Induced Liver Injury in Rats

    PubMed Central

    Khotimchenko, Yuri S.; Khotimchenko, Maxim Y.

    2004-01-01

    The purpose of this study was to investigate the pharmacological effects of calcium alginate on carbon tetrachloride (CCL4)-induced hepatotoxicity in rats. The study included two experiments. In the first experiment the animals were given daily CCL4 through gavage for 7 days and then 10, 50, or 250 mg/kg b.w. of calcium alginate for 21 days. The increased bilirubin level, enhanced alanine and aspartate aminotransferase activity in plasma and reduced liver glycogen content induced by CCL4 were partly normalized by alginate administration in a dose-dependent manner. In addition, alginate significantly improved CCL4-induced alterations of pro-oxidant and antioxidant biochemical parameters in liver and plasma compared to those of rats administered CCL4. In the second experiment the animals were given daily 10, 50 or 250 mg/kg b.w. of calcium alginate for 21 days before 7-day administration of CCL4. Pretreatment with alginate before CCL4 administration resulted in significantly inhibited increase of the blood enzymatic activities of alanine and aspartate aminotransferases and bilirubin level in a dose-dependent manner. Also, preliminary administration of alginate prevented elevation of lipid peroxidation products and reduction of liver glutathione content in rats given CCL4. These results suggest that calcium alginate exerts healing and preventive effects on CCL4-induced hepatotoxicity in rats.

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

    ERIC Educational Resources Information Center

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

    2016-01-01

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

  20. Transient calcium release induced by successive increments of inositol 1,4,5-trisphosphate.

    PubMed Central

    Meyer, T; Stryer, L

    1990-01-01

    Many hormonal, neurotransmitter, and sensory stimuli trigger the formation of inositol 1,4,5-trisphosphate, which in turn releases calcium from intracellular stores. We report here that inositol 1,4,5-trisphosphate-induced calcium release from saponin-permeabilized rat basophilic leukemia cells at 37 degrees C is markedly biphasic, in contrast with nearly monophasic release kinetics at 11 degrees C. Hepatoma, PC-12 neuronal cells, and several other cell types exhibit similar biphasic release at 37 degrees C. The biphasic kinetics are not due to degradation of inositol 1,4,5-trisphosphate or to increased Ca2(+)-ATPase pump activity. Biphasic calcium release was also seen when ATP was quenched to less than 0.4 microM by adding hexokinase and glucose, suggesting that phosphorylation is not involved. External calcium (100 nM-600 nM) range had little influence on the biphasic kinetics. Rapid-mixing experiments revealed that rapid efflux of calcium is followed in approximately 0.5 s by a 30-fold slower efflux. Most striking, successive additions of the same amount of inositol 1,4,5-trisphosphate induced short bursts of calcium release of similar size. This retention of responsiveness, which we term increment detection, may be a distinct mode of signal transduction. Like inactivation and adaptation, increment detection gives rise to transient responses to sustained stimuli. Systems exhibiting inactivation, adaptation, and increment detection differ in their responsiveness (none, partial, and full, respectively) to stepwise increases in stimulus intensity. Increment detection could be advantageous in generating receptor-triggered calcium oscillations. Images PMID:2339124

  1. Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism

    PubMed Central

    Millay, Douglas P.; Goonasekera, Sanjeewa A.; Sargent, Michelle A.; Maillet, Marjorie; Aronow, Bruce J.; Molkentin, Jeffery D.

    2009-01-01

    Muscular dystrophy is a general term encompassing muscle disorders that cause weakness and wasting, typically leading to premature death. Membrane instability, as a result of a genetic disruption within the dystrophin-glycoprotein complex (DGC), is thought to induce myofiber degeneration, although the downstream mechanism whereby membrane fragility leads to disease remains controversial. One potential mechanism that has yet to be definitively proven in vivo is that unregulated calcium influx initiates disease in dystrophic myofibers. Here we demonstrate that calcium itself is sufficient to cause a dystrophic phenotype in skeletal muscle independent of membrane fragility. For example, overexpression of transient receptor potential canonical 3 (TRPC3) and the associated increase in calcium influx resulted in a phenotype of muscular dystrophy nearly identical to that observed in DGC-lacking dystrophic disease models, including a highly similar molecular signature of gene expression changes. Furthermore, transgene-mediated inhibition of TRPC channels in mice dramatically reduced calcium influx and dystrophic disease manifestations associated with the mdx mutation (dystrophin gene) and deletion of the δ-sarcoglycan (Scgd) gene. These results demonstrate that calcium itself is sufficient to induce muscular dystrophy in vivo, and that TRPC channels are key disease initiators downstream of the unstable membrane that characterizes many types of muscular dystrophy. PMID:19864620

  2. Scrophularia orientalis extract induces calcium signaling and apoptosis in neuroblastoma cells

    PubMed Central

    LANGE, INGO; MOSCHNY, JULIA; TAMANYAN, KAMILLA; KHUTSISHVILI, MANANA; ATHA, DANIEL; BORRIS, ROBERT P.; KOOMOA, DANA-LYNN

    2016-01-01

    Effective neuroblastoma (NB) treatments are still limited despite treatment options available today. Therefore, this study attempted to identify novel plant extracts that have anticancer effects. Cytotoxicity and increased intracellular calcium levels were determined using the Sulforhodamine B (SRB) assay and Fluo4-AM (acetoxymethyl) staining and fluorescence microscopy in NB cells in order to screen a library of plant extracts. The current study examined the anticancer effects of a dichloromethane extract from Scrophularia orientalis L. (Scrophulariaceae), a plant that has been used in Traditional Chinese Medicine. This extract contained highly potent agents that significantly reduced cell survival and increased calcium levels in NB cells. Further analysis revealed that cell death induced by this extract was associated with intracellular calcium release, opening of the MPTP, caspase 3- and PARP-cleavage suggesting that this extract induced aberrant calcium signaling that resulted in apoptosis via the mitochondrial pathway. Therefore, agents from Scrophularia orientalis may have the potential to lead to new chemo therapeutic anticancer drugs. Furthermore, targeting intracellular calcium signaling may be a novel strategy to develop more effective treatments for NB. PMID:26848085

  3. Transmembrane potential polarization, calcium influx, and receptor conformational state modulate the sensitivity of the imidacloprid-insensitive neuronal insect nicotinic acetylcholine receptor to neonicotinoid insecticides.

    PubMed

    Bodereau-Dubois, Béatrice; List, Olivier; Calas-List, Delphine; Marques, Olivier; Communal, Pierre-Yves; Thany, Steeve H; Lapied, Bruno

    2012-05-01

    Neonicotinoid insecticides act selectively on insect nicotinic acetylcholine receptors (nAChRs). Recent studies revealed that their efficiency was altered by the phosphorylation/dephosphorylation process and the intracellular signaling pathway involved in the regulation of nAChRs. Using whole-cell patch-clamp electrophysiology adapted for dissociated cockroach dorsal unpaired median (DUM) neurons, we demonstrated that intracellular factors involved in the regulation of nAChR function modulated neonicotinoid sensitivity. DUM neurons were known to express two α-bungarotoxin-insensitive nAChR subtypes: nAChR1 and nAChR2. Whereas nAChR1 was sensitive to imidacloprid, nAChR2 was insensitive to this insecticide. Here, we demonstrated that, like nicotine, acetamiprid and clothianidin, other types of neonicotinoid insecticides, acted as agonists on the nAChR2 subtype. Using acetamiprid, we revealed that both steady-state depolarization and hyperpolarization affected nAChR2 sensitivity. The measurement of the input membrane resistance indicated that change in the acetamiprid-induced agonist activity was related to the receptor conformational state. Using cadmium chloride, ω-conotoxin GVIA, and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-acetamide (LOE 908), we found that inhibition of calcium influx through high voltage-activated calcium channels and transient receptor potential γ (TRPγ) activated by both depolarization and hyperpolarization increased nAChR2 sensitivity to acetamiprid. Finally, using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7), forskolin, and cAMP, we demonstrated that adenylyl cyclase sensitive to the calcium/calmodulin complex regulated internal cAMP concentration, which in turn modulated TRPγ function and nAChR2 sensitivity to acetamiprid. Similar TRPγ-induced modulatory effects were also obtained when clothianidin was tested. These findings bring insights into the signaling pathway modulating

  4. Transmembrane potential polarization, calcium influx, and receptor conformational state modulate the sensitivity of the imidacloprid-insensitive neuronal insect nicotinic acetylcholine receptor to neonicotinoid insecticides.

    PubMed

    Bodereau-Dubois, Béatrice; List, Olivier; Calas-List, Delphine; Marques, Olivier; Communal, Pierre-Yves; Thany, Steeve H; Lapied, Bruno

    2012-05-01

    Neonicotinoid insecticides act selectively on insect nicotinic acetylcholine receptors (nAChRs). Recent studies revealed that their efficiency was altered by the phosphorylation/dephosphorylation process and the intracellular signaling pathway involved in the regulation of nAChRs. Using whole-cell patch-clamp electrophysiology adapted for dissociated cockroach dorsal unpaired median (DUM) neurons, we demonstrated that intracellular factors involved in the regulation of nAChR function modulated neonicotinoid sensitivity. DUM neurons were known to express two α-bungarotoxin-insensitive nAChR subtypes: nAChR1 and nAChR2. Whereas nAChR1 was sensitive to imidacloprid, nAChR2 was insensitive to this insecticide. Here, we demonstrated that, like nicotine, acetamiprid and clothianidin, other types of neonicotinoid insecticides, acted as agonists on the nAChR2 subtype. Using acetamiprid, we revealed that both steady-state depolarization and hyperpolarization affected nAChR2 sensitivity. The measurement of the input membrane resistance indicated that change in the acetamiprid-induced agonist activity was related to the receptor conformational state. Using cadmium chloride, ω-conotoxin GVIA, and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-acetamide (LOE 908), we found that inhibition of calcium influx through high voltage-activated calcium channels and transient receptor potential γ (TRPγ) activated by both depolarization and hyperpolarization increased nAChR2 sensitivity to acetamiprid. Finally, using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7), forskolin, and cAMP, we demonstrated that adenylyl cyclase sensitive to the calcium/calmodulin complex regulated internal cAMP concentration, which in turn modulated TRPγ function and nAChR2 sensitivity to acetamiprid. Similar TRPγ-induced modulatory effects were also obtained when clothianidin was tested. These findings bring insights into the signaling pathway modulating

  5. Microscopic heat pulses induce contraction of cardiomyocytes without calcium transients

    SciTech Connect

    Oyama, Kotaro; Mizuno, Akari; Shintani, Seine A.; Itoh, Hideki; Serizawa, Takahiro; Fukuda, Norio; Suzuki, Madoka

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Infra-red laser beam generates microscopic heat pulses. Black-Right-Pointing-Pointer Heat pulses induce contraction of cardiomyocytes. Black-Right-Pointing-Pointer Ca{sup 2+} transients during the contraction were not detected. Black-Right-Pointing-Pointer Skinned cardiomyocytes in free Ca{sup 2+} solution also contracted. Black-Right-Pointing-Pointer Heat pulses regulated the contractions without Ca{sup 2+} dynamics. -- Abstract: It was recently demonstrated that laser irradiation can control the beating of cardiomyocytes and hearts, however, the precise mechanism remains to be clarified. Among the effects induced by laser irradiation on biological tissues, temperature change is one possible effect which can alter physiological functions. Therefore, we investigated the mechanism by which heat pulses, produced by infra-red laser light under an optical microscope, induce contractions of cardiomyocytes. Here we show that microscopic heat pulses induce contraction of rat adult cardiomyocytes. The temperature increase, {Delta}T, required for inducing contraction of cardiomyocytes was dependent upon the ambient temperature; that is, {Delta}T at physiological temperature was lower than that at room temperature. Ca{sup 2+} transients, which are usually coupled to contraction, were not detected. We confirmed that the contractions of skinned cardiomyocytes were induced by the heat pulses even in free Ca{sup 2+} solution. This heat pulse-induced Ca{sup 2+}-decoupled contraction technique has the potential to stimulate heart and skeletal muscles in a manner different from the conventional electrical stimulations.

  6. Positive lusitropic effect and diminished myofibrillar sensitivity to calcium produced by cAMP on toad (Bufo arenarum Hensel) ventricle.

    PubMed

    Vila Petroff, M; Vittone, L; Mundiña, C; Chiappe de Cingolani, G; Alicia, M

    1992-01-01

    In intact ventricular strips from toad heart, we studied the relaxant or positive lusitropic effect of different interventions known to increase intracellular cAMP levels. Isoproterenol increased developed tension (DT), maximal rate of contraction (+T), and maximal velocity of relaxation (-T). From 10(-8) to 10(-4)M isoproterenol, -T increased proportionally more than +T being the ratio +T/-T significantly decreased. A single dose of isoproterenol (3 x 10(-8)M) increased cAMP levels from 0.174 +/- 0.022 to 0.329 +/- 0.039 pmoles/mg ww (P < 0.05), increased contractility by 69 +/- 13% and decreased +T/-T by 18.5 +/- 4.55%. Administration of 10(-3)M of dibutyryl cyclic AMP (dcAMP) significantly increased DT and +T and decreased the ratio +T/-T. Similar effects were obtained with milrinone, a specific cAMP phosphodiesterase inhibitor. Papaverine, a non selective phosphodiesterase inhibitor, failed to increase +T but significantly increased -T. In chemically skinned ventricular trabeculae, calcium sensitivity of the myofibrils was significantly increased by 10(-5)M of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). 10(-3)M dcAMP failed to affect calcium sensitivity of chemically skinned ventricular trabeculae when given alone, but produced a decrease in calcium sensitivity of the myofibrils in the presence of 10(-5)M of either IBMX or papaverine. The results would indicate that the relaxant effect of isoproterenol is mediated in toad ventricle by an increase in intracellular cAMP levels. They furthermore suggest that a decrease in myofilament sensitivity to calcium may be a mechanism by which cAMP produces its relaxant effect.

  7. Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

    PubMed

    Zhang, Junjie; He, Shanping; Wang, Yi; Brulois, Kevin; Lan, Ke; Jung, Jae U; Feng, Pinghui

    2015-03-01

    G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

  8. Detection of light-induced changes of intracellular ionized calcium concentration in Limulus ventral photoreceptors using arsenazo III

    PubMed Central

    Brown, J. E.; Brown, P. K.; Pinto, L. H.

    1977-01-01

    1. The metallochromic indicator dye, arsenazo III, was injected intracellularly into Limulus ventral photoreceptor cells to concentrations greater than 1 mM. 2. The absorption spectrum (450-750 nm) of the dye in single dark-adapted cells was measured by a scanning microspectrophotometer. When a cell was light-adapted, the absorption of the dye changed; the difference spectrum had two maxima at about 610 and 660 nm, a broad minimum at about 540 nm and an isosbestic point at about 585 nm. 3. When intracellular calcium concentration was raised in dark-adapted cells previously injected with arsenazo III, the difference spectum had two maxima at about 610 and 660 nm, a broad minimum at about 530 nm and an isosbestic point at about 585 nm. The injection of Mg2+ into dark-adapted cells previously injected with the dye induced a difference spectrum that had a single maximum at about 620 nm. Also, decreasing the intracellular pH of cells previously injected with the dye induced a difference spectrum that had a minimum at about 620 nm. The evidence suggests that there is a rise of intracellular ionized calcium when a Limulus ventral photoreceptor is light-adapted. 4. The intracellular calcium concentration, [Ca2+]1, in light-adapted photoreceptors was estimated to reach at least 10-4 M by compaing the light-induced difference spectra measured in ventral photoreceptors with a standard curve determined in microcuvettes containing 2mM arsenazo III in 400 mM-KCl, 1 mM-MgCl2 and 25 mM MOPS at pH 7·0. 5. In cells injected to less than 3 mM arsenazo III, light induced a transient decrease in optical transmission at 660 nm (T660). This decrease in T660 indicates that illumination of a ventral photoreceptor normally causes a transient increase of [Ca2+]1. 6. Arsenazo III was found to be sensitive, selective and rapid enough to measure light-induced changes of intracellular ionized calcium in Limulus ventral photoreceptor cells. PMID:17732

  9. Visual contrast sensitivity in drug-induced Parkinsonism.

    PubMed Central

    Bulens, C; Meerwaldt, J D; van der Wildt, G J; Keemink, C J

    1989-01-01

    The influence of stimulus orientation on contrast sensitivity function was studied in 10 patients with drug-induced Parkinsonism. Nine of the 10 patients had at least one eye with contrast sensitivity deficit for vertical and/or horizontal stimuli. Only generalised contrast sensitivity loss, observed in two eyes, was stimulus orientation independent. All spatial frequency-selective contrast deficits in 15 eyes were orientation dependent. The striking similarity between the pattern of contrast sensitivity loss in drug-induced Parkinsonism and that in idiopathic Parkinson's disease, suggests that generalised dopaminergic deficiency, from whatever cause, affects visual function in an analogous way. PMID:2926418

  10. Visual contrast sensitivity in drug-induced Parkinsonism.

    PubMed

    Bulens, C; Meerwaldt, J D; van der Wildt, G J; Keemink, C J

    1989-03-01

    The influence of stimulus orientation on contrast sensitivity function was studied in 10 patients with drug-induced Parkinsonism. Nine of the 10 patients had at least one eye with contrast sensitivity deficit for vertical and/or horizontal stimuli. Only generalised contrast sensitivity loss, observed in two eyes, was stimulus orientation independent. All spatial frequency-selective contrast deficits in 15 eyes were orientation dependent. The striking similarity between the pattern of contrast sensitivity loss in drug-induced Parkinsonism and that in idiopathic Parkinson's disease, suggests that generalised dopaminergic deficiency, from whatever cause, affects visual function in an analogous way.

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

    PubMed Central

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

    2016-01-01

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

  12. Ketamine-inhibition of calcium-induced contractions in depolarized rat uterus: a comparison with other calcium antagonists.

    PubMed Central

    Calixto, J. B.; Loch, S.

    1985-01-01

    The inhibitory effect of the intravenous anaesthetic ketamine on CaCl2-induced contractions in the isolated K+-depolarized uterus of the rat in Ca2+-free medium was compared with that produced by papaverine, theophylline and the calcium entry blocker verapamil. Pre-incubation for 20 min with either ketamine (0.3 to 3 mM), papaverine (3 to 30 microM), theophylline (0.1 to 1 mM) or verapamil (3 to 30 nM) induced parallel, concentration-dependent rightward displacements of the dose-response curves to Ca2+ (0.04 to 22 mM). The antagonism was competitive, except that due to verapamil, the Schild plot for which yielded a slope which differed significantly from unity. The calculated pA2 values (+/- s.e.mean) were: ketamine 3.90 +/- 0.07; papaverine 5.55 +/- 0.05; theophylline 3.99 +/- 0.1 and verapamil 9.54 +/- 0.24. These drugs differed in their ability to relax the sustained contraction induced by Ca2+ (1 mM) in K+-depolarizing solution. Ketamine and verapamil relaxed the preparation in a concentration-dependent manner whereas theophylline and especially papaverine were less potent and induced only partial maximal relaxation. The t1/2 of the relaxant effect was significantly less for ketamine than for verapamil (5 and 22 min, respectively). Only ketamine produced a relaxation comparable to that obtained by washing the preparation with Ca2+-free solution (t1/2 = approx. 5 min). Prior exposure of the depolarized uterine strip to a low concentration of Ca2+ (0.22 mM) increased the potency of ketamine, but decreased that of papaverine and theophylline, in antagonizing Ca2+ induced contractions. In contrast, this procedure did not affect the potency of verapamil.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:4027464

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

    NASA Astrophysics Data System (ADS)

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

    2009-07-01

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

  14. ATP releasing connexin 30 hemichannels mediate flow-induced calcium signaling in the collecting duct.

    PubMed

    Svenningsen, Per; Burford, James L; Peti-Peterdi, János

    2013-01-01

    ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC, and AQP2. Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30(-/-) mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca(2+)]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30(-/-) mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca(2+)]i in wild type CCDs. This response was blunted in Cx30(-/-) CCDs ([Ca(2+)]i increased only 1.2-fold, p < 0.0001 vs. WT, n = 6 each). To further test our hypothesis we performed CD [Ca(2+)]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca(2+)]i oscillations in free-flowing CDs of wild type but not Cx30(-/-) mice. The [Ca(2+)]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption. PMID:24137132

  15. Trypsin-Sensitive, Rapid Inactivation of a Calcium-Activated Potassium Channel

    NASA Astrophysics Data System (ADS)

    Solaro, Christopher R.; Lingle, Christopher J.

    1992-09-01

    Most calcium-activated potassium channels couple changes in intracellular calcium to membrane excitability by conducting a current with a probability that depends directly on submembrane calcium concentration. In rat adrenal chromaffin cells, however, a large conductance, voltage- and calcium-activated potassium channel (BK) undergoes rapid inactivation, suggesting that this channel has a physiological role different than that of other BK channels. The inactivation of the BK channel, like that of the voltage-gated Shaker B potassium channel, is removed by trypsin digestion and channels are blocked by the Shaker B amino-terminal inactivating domain. Thus, this BK channel shares functional and possibly structural homologies with other inactivating voltage-gated potassium channels.

  16. Dose-sensitive steroid-induced hyperglycaemia.

    PubMed

    Gannon, Craig; Dando, Nicholas

    2010-10-01

    Steroids cause significant but under-appreciated and poorly managed glucose intolerance. In this case we describe a patient with steroid-induced hyperglycaemia who obtained a large positive impact on glycaemic control from a small reduction in her steroid dose, sufficient to alleviate the need for insulin. Developments in the treatment of steroid-induced hyperglycaemia may mean that a more active approach needs to be considered when treating steroid-related diabetes in patients whose management is palliative. We advise checking for steroid-induced hyperglycaemia by testing capillary blood glucose values 2 hours after the lunchtime meal and recommend a single morning dose of long-acting insulin to treat the condition.

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

    PubMed

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

    2011-06-01

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

  18. Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

    PubMed Central

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

    2013-01-01

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

  19. TRPV1 Activation in Primary Cortical Neurons Induces Calcium-Dependent Programmed Cell Death.

    PubMed

    Song, Juhyun; Lee, Jun Hong; Lee, Sung Ho; Park, Kyung Ah; Lee, Won Taek; Lee, Jong Eun

    2013-03-01

    Transient receptor potential cation channel, subfamily V, member 1 (TRPV1, also known as vanilloid receptor 1) is a receptor that detects capsaicin, a pungent component of chili peppers, and noxious heat. Although its function in the primary nociceptor as a pain receptor is well established, whether TRPV1 is expressed in the brain is still under debate. In this study, the responses of primary cortical neurons were investigated. Here, we report that 1) capsaicin induces caspase-3-dependent programmed cell death, which coincides with increased production of nitric oxide and peroxynitrite ; that 2) the prolonged capsaicin treatment induces a steady increase in the degree of capase-3 activation, which is prevented by the removal of capsaicin; 3) and that blocking calcium entry and calcium-mediated signaling prevents capsaicin-induced cell death. These results indicate that cortical neurons express TRPV1 whose prolonged activation causes cell death. PMID:23585723

  20. Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system.

    PubMed

    Kiehart, D P

    1981-03-01

    I microinjected calcium ions into echinoderm eggs during mitosis to determine the calcium sensitivity of microtubules (Mts) in vivo. Spindle birefringence (BR), a measure of the number of aligned Mts in the spindle, is locally, rapidly, and reversibly abolished by small volumes of microinjected CaCl2 (1 mM). Rapid return of BR is followed by anaphase, and subsequent divisions are normal. Similar doses of MgCl2, BaCl2, KCl, NaCl, pH buffers, distilled water, or vegetable oil have no effect on spindle BR, whereas large doses of such agents sometimes cause slow, uniform loss in BR over the course of a minute or more. Of the ions tested, only Sr++ causes effects comparable to Ca++. Ca-EGTA buffers, containing greater than micromolar free Ca++, abolishes BR in a manner similar to millimolar concentrations of injected CaCl2. Caffeine, a potent uncoupler of the Ca++-pump/ATPase of sarcoplasmic reticulum, causes a local, transient depression in spindle BR in the injected region. Finally, injection of potassium oxalate results in the formation of small, highly BR crystals, presumably CA-oxalate, in Triton-sensitive compartments in the cytoplasm. Taken together, these findings demonstrate that spindle Mts are sensitive to levels of free Ca++ in the physiological range, provide evidence for the existence of a strong cytoplasmic Ca++-sequestering system, and support the notion that Mt assembly and disassembly in local regions of the spindle may be orchestrated by local changes in the cytoplasmic free Ca++ concentration during mitosis. An appendix offers the design of a new chamber for immobilizing echinoderm eggs for injection, a new method for determining the volume of the injected solution, and a description of the microinjection technique, which was designed, but never fully described, by Hiramoto (Y. Hiramoto, Exp. Cell. Res., 1962, 27:416-426.).

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

    PubMed Central

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

    1996-01-01

    To examine the role of membrane dynamics in transmembrane signal transduction, we studied changes in membrane fluidity in mucociliary tissues from frog palate and esophagus epithelia stimulated by extracellular ATP. Micromolar concentrations of ATP induced strong changes in fluorescence polarization, possibly indicating membrane fluidization. This effect was dosage dependent, reaching a maximum at 10-microM ATP. It was dependent on the presence of extracellular Ca2+ (or Mg2+), though it was insensitive to inhibitors of voltage-gated calcium channels. It was inhibited by thapsigargin and by ionomycin (at low extracellular Ca2+ concentration), both of which deplete Ca2+ stores. It was inhibited by the calcium-activated potassium channel inhibitors quinidine, charybdotoxin, and apamine and was reduced considerably by replacement of extracellular Na+ with K+. Hyperpolarization, or depolarization, of the mucociliary membrane induced membrane fluidization. The degree of membrane fluidization depended on the degree of hyperpolarization or depolarization of the ciliary membrane potential and was considerably lower than the effect induced by extracellular ATP. These results indicate that appreciable membrane fluidization induced by extracellular ATP depends both on an increase in intracellular Ca2+, mainly from its internal stores, and on hyperpolarization of the membrane. Calcium-dependent potassium channels couple the two effects. In light of recent results on the enhancement of ciliary beat frequency, it would appear that extracellular ATP-induced changes both in ciliary beat frequency and in membrane fluidity are triggered by similar signal transduction pathways. PMID:8789123

  2. IP(3)-dependent, post-tetanic calcium transients induced by electrostimulation of adult skeletal muscle fibers.

    PubMed

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

    2010-10-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2016-03-15

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

  5. Coagulation of β-conglycinin, glycinin and isoflavones induced by calcium chloride in soymilk.

    PubMed

    Hsiao, Yu-Hsuan; Yu, Chia-Jung; Li, Wen-Tai; Hsieh, Jung-Feng

    2015-08-11

    The coagulation of β-conglycinin (7S), glycinin (11S) and isoflavones induced by calcium chloride was investigated. Approximately 92.6% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) after the addition of 5 mM calcium chloride. SDS-PAGE and two-dimensional electrophoresis analysis indicated that most of the 7S (α', α and β), 11S acidic (A1a, A1b, A2, A3 and A4) and 11S basic (B1a) proteins in the SSF were coagulated into the SPF after treatment with 5 mM calcium chloride. Isoflavones, including daidzein and genistein, were also coagulated into the SPF after the addition of 5 mM calcium chloride. The amounts of daidzein and genistein in the SSF decreased to 39.4 ± 1.6 and 11.8 ± 7.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound with 7S and 11S proteins and then were coprecipitated into the SPF by 5 mM calcium chloride.

  6. Coagulation of β-conglycinin, glycinin and isoflavones induced by calcium chloride in soymilk

    NASA Astrophysics Data System (ADS)

    Hsiao, Yu-Hsuan; Yu, Chia-Jung; Li, Wen-Tai; Hsieh, Jung-Feng

    2015-08-01

    The coagulation of β-conglycinin (7S), glycinin (11S) and isoflavones induced by calcium chloride was investigated. Approximately 92.6% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) after the addition of 5 mM calcium chloride. SDS-PAGE and two-dimensional electrophoresis analysis indicated that most of the 7S (α’, α and β), 11S acidic (A1a, A1b, A2, A3 and A4) and 11S basic (B1a) proteins in the SSF were coagulated into the SPF after treatment with 5 mM calcium chloride. Isoflavones, including daidzein and genistein, were also coagulated into the SPF after the addition of 5 mM calcium chloride. The amounts of daidzein and genistein in the SSF decreased to 39.4 ± 1.6 and 11.8 ± 7.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound with 7S and 11S proteins and then were coprecipitated into the SPF by 5 mM calcium chloride.

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

    PubMed Central

    Mellau, LSB; Jørgensen, RJ; Enemark, JMD

    2001-01-01

    The intravenous Na2EDTA infusion technique allows effective specific chelation of circulating Ca2+ leading to a progressive hypocalcaemia. Methods previously used were not described in detail and results obtained by monitoring total and free ionic calcium were not comparable due to differences in sampling and analysis. This paper describes a standardized EDTA infusion technique that allowed comparison of the response of calcium, phosphorus and magnesium between 2 groups of experimental cows. The concentration of the Na2EDTA solution was 0.134 mol/l and the flow rate was standardized at 1.2 ml/kg per hour. Involuntary recumbency occurred when ionised calcium dropped to 0.39 – 0.52 mmol/l due to chelation. An initial fast drop of ionized calcium was observed during the first 20 min of infusion followed by a fluctuation leading to a further drop until recumbency. Pre-infusion [Ca2+] between tests does not correlate with the amount of EDTA required to induce involuntary recumbence. Total calcium concentration measured by atomic absorption remained almost constant during the first 100 min of infusion but declined gradually when the infusion was prolonged. The concentration of inorganic phosphate declined gradually in a fluctuating manner until recumbency. Magnesium concentration remained constant during infusion. Such electrolyte responses during infusion were comparable to those in spontaneous milk fever. The standardized infusion technique might be useful in future experimental studies. PMID:11503370

  8. Molecular mechanism of the calcium-induced conformational change in the spectrin EF-hands.

    PubMed Central

    Travé, G; Lacombe, P J; Pfuhl, M; Saraste, M; Pastore, A

    1995-01-01

    Calcium is a universally employed cytosolic messenger in eukaryotic cells. Most of the proteins that bind signalling calcium are members of the calmodulin superfamily and share two or more helix-loop-helix motifs known as EF-hands. A model, based on structure comparison of different domains and supported by preliminary NMR data, has suggested that EF-hands involved in signal transduction undergo a major conformational change upon calcium binding from a 'closed' to an 'open' state allowing protein-protein interaction. We have determined the solution structures of the EF-hand pair from alpha-spectrin in the absence and in the presence of calcium. The structures are in the closed and open conformation respectively, providing a definite experimental proof for the closed-to-open model. Our results allow formulation of the rules which govern the movement induced by calcium. These rules may be generalized to other EF-hands since the key residues involved are conserved within the calmodulin family. Images PMID:7588621

  9. K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity.

    PubMed

    Mokbel, Nancy; Ilkovski, Biljana; Kreissl, Michaela; Memo, Massimiliano; Jeffries, Cy M; Marttila, Minttu; Lehtokari, Vilma-Lotta; Lemola, Elina; Grönholm, Mikaela; Yang, Nan; Menard, Dominique; Marcorelles, Pascale; Echaniz-Laguna, Andoni; Reimann, Jens; Vainzof, Mariz; Monnier, Nicole; Ravenscroft, Gianina; McNamara, Elyshia; Nowak, Kristen J; Laing, Nigel G; Wallgren-Pettersson, Carina; Trewhella, Jill; Marston, Steve; Ottenheijm, Coen; North, Kathryn N; Clarke, Nigel F

    2013-02-01

    Mutations in the TPM2 gene, which encodes β-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of β-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-β-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-β-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity. PMID:23378224

  10. Coronal Discoloration Induced by Calcium-Enriched Mixture, Mineral Trioxide Aggregate and Calcium Hydroxide: A Spectrophotometric Analysis

    PubMed Central

    Esmaeili, Behnaz; Alaghehmand, Homayoun; Kordafshari, Tavoos; Daryakenari, Ghazaleh; Ehsani, Maryam; Bijani, Ali

    2016-01-01

    Introduction: The aim of this study was to compare the discoloration potential of calcium-enriched mixture (CEM) cement, white mineral trioxide aggregate (WMTA) and calcium hydroxide (CH), after placement in pulp chamber. Methods and Materials: Access cavities were prepared in 40 intact maxillary central incisors. Then, a 2×2 mm box was prepared on the middle third of the inner surface on the buccal wall of the access cavity. The specimens were randomly assigned into four groups; the boxes in the control group were left empty, in groups 1 to 3, the boxes were filled with CH, WMTA and CEM cement, respectively. The access cavities and the apical openings were sealed using resin modified glass ionomer (RMGI). The color measurement was performed with a spectrophotometer at the following intervals: before (T0), immediately after placement of the filling material (T1), one week (T2), 1 month (T3), 3 months (T4) and 5 months (T5) after filling of the box and finally immediately after removing the material from the boxes (T6). Color change (ΔE) values were calculated using the sample Kolmogorov-Smirnov test to determine the normal distribution of data, followed by ANOVA, repeated measured ANOVA and post-hoc Tukey’s tests. Results: All materials led to clinically perceptible crown discoloration after 1 week. The highest ΔE value belonged to WMTA group. Discoloration induced by CEM cement was not significantly different from CH or the control group (P>0.05). Conclusion: CEM cement may be the material of choice in the esthetic region, specifically pertaining to its lower color changing potential compared to WMTA. PMID:26843873

  11. H sup + -dependent calcium uptake into an IP sub 3 -sensitive calcium pool from rat parotid gland

    SciTech Connect

    Thevenod, F.; Schulz, I. )

    1988-10-01

    In permeabilized parotid cells and in isolated membrane vesicles from parotid endoplasmic reticulum (ER), Mg-ATP-dependent Ca{sup 2+} uptake was measured using a Ca{sup 2+}-specific macroelectrode and {sup 45}Ca{sup 2+}, respectively. Mg-ATP-dependent Ca{sup 2+} uptake was inhibited by vanadate by {approximately}45% in permeabilized cells and by {approximately}70% in membrane vesicles from ER during the initial 10 min. After this lag phase, Ca{sup 2+} uptake increased. Subsequent addition of inositol 1,4,5-trisphosphate (IP{sub 3}) caused a similar Ca{sup 2+} release compared with control. This indicates that in presence of vanadate an IP{sub 3}-sensitive Ca{sup 2+} pool was filled. However, when protonophores, such as nigericin or carbonyl cyanide-m-chlorophenylhydrazone, were added in addition to vanadate, this low steady-state free (Ca{sup 2+}) was not reached. {sup 45}Ca{sup 2+} uptake was reduced by {approximately}70% within 60 min, and IP{sub 3} did not cause {sup 45}Ca{sup 2+} release when given subsequently, indicating that filling of an IP{sub 3}-sensitive Ca{sup 2+} pool was prevented. Mg-ATP-driven H{sup +} uptake into ER vesicles was abolished by protonophores and by the H{sup +}-ATPase blockers N-ethylmaleimide and Dio 9 but was unaltered by vanadate. Preincubation of ER vesicles in a medium without Ca{sup 2+}, but with vanadate and with Mg-ATP to generate an H{sup +} gradient, allowed demonstration of {sup 45}Ca{sup 2+} uptake from a medium that did not contain ATP. The data indicate that both a Ca{sup 2+} and a H{sup +} pump are located in a compartment of ER that is also sensitive to IP{sub 3}. Ca{sup 2+} uptake is coupled to an H{sup +} gradient that is generated by the H{sup +} pump and most likely occurs via Mg-ATP-driven Ca{sup 2+}-H{sup +} countertransport but to some extent can also operate in absence of ATP at the expense of the H{sup +} gradient.

  12. Dysferlin Binds SNAREs (Soluble N-Ethylmaleimide-sensitive Factor (NSF) Attachment Protein Receptors) and Stimulates Membrane Fusion in a Calcium-sensitive Manner.

    PubMed

    Codding, Sara J; Marty, Naomi; Abdullah, Nazish; Johnson, Colin P

    2016-07-01

    Resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. Recent work suggests a critical role for dysferlin in the membrane repair process and that mutations in dysferlin are responsible for limb girdle muscular dystrophy 2B and Miyoshi myopathy. Beyond membrane repair, dysferlin has been linked to SNARE-mediated exocytotic events including cytokine release and acid sphingomyelinase secretion. However, it is unclear whether dysferlin regulates SNARE-mediated membrane fusion. In this study we demonstrate a direct interaction between dysferlin and the SNARE proteins syntaxin 4 and SNAP-23. In addition, analysis of FRET and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates syntaxin 4/SNAP-23 heterodimer formation and SNARE-mediated lipid mixing in a calcium-sensitive manner. These results support a function for dysferlin as a calcium-sensing SNARE effector for membrane fusion events. PMID:27226605

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

    PubMed

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

    2013-08-01

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

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

    PubMed Central

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

    2014-01-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulatenumerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca2+ overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca2+ levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca2+ influx, mitochondrial network fragmentation and loss of the mitochondrial Ca2+ buffer capacity. These biochemical events increase cytosolic Ca2+ levels and trigger cardiomyocyte death via the activation of calpains. PMID:23602992

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

    PubMed

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

    2013-08-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. PMID:23602992

  16. Synthetic peptides corresponding to human follicle-stimulating hormone (hFSH)-beta-(1-15) and hFSH-beta-(51-65) induce uptake of 45Ca++ by liposomes: evidence for calcium-conducting transmembrane channel formation

    SciTech Connect

    Grasso, P.; Santa-Coloma, T.A.; Reichert, L.E. Jr. )

    1991-06-01

    We have previously described FSH receptor-mediated influx of 45Ca++ in cultured Sertoli cells from immature rats and receptor-enriched proteoliposomes via activation of voltage-sensitive and voltage-independent calcium channels. We have further shown that this effect of FSH does not require cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding protein or activation of adenylate cyclase. In the present study, we have identified regions of human FSH-beta-subunit which appear to be involved in mediating calcium influx. We screened 11 overlapping peptide amides representing the entire primary structure of hFSH-beta-subunit for their effects on 45Ca++ flux in FSH receptor-enriched proteoliposomes. hFSH-beta-(1-15) and hFSH-beta-(51-65) induced uptake of 45Ca++ in a concentration-related manner. This effect of hFSH-beta-(1-15) and hFSH-beta-(51-65) was also observed in liposomes lacking incorporated FSH receptor. Reducing membrane fluidity by incubating liposomes (containing no receptor) with hFSH-beta-(1-15) or hFSH-beta-(51-65) at temperatures lower than the transition temperatures of their constituent phospholipids resulted in no significant (P greater than 0.05) difference in 45Ca++ uptake. The effectiveness of the calcium ionophore A23187, however, was abolished. Ruthenium red, a voltage-independent calcium channel antagonist, was able to completely block uptake of 45Ca++ induced by hFSH-beta-(1-15) and hFSH-beta-(51-65) whereas nifedipine, a calcium channel blocker specific for L-type voltage-sensitive calcium channels, was without effect. These results suggest that in addition to its effect on voltage-sensitive calcium channel activity, interaction of FSH with its receptor may induce formation of transmembrane aqueous channels which also facilitate influx of extracellular calcium.

  17. Potential Interactions of Calcium-Sensitive Reagents with Zinc Ion in Different Cultured Cells

    PubMed Central

    Fujikawa, Koichi; Fukumori, Ryo; Nakamura, Saki; Kutsukake, Takaya; Takarada, Takeshi; Yoneda, Yukio

    2015-01-01

    Background Several chemicals have been widely used to evaluate the involvement of free Ca2+ in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca2+-sensitive reagents in diverse cultured cells. Methodology/Principal Findings In rat astrocytic C6 glioma cells loaded with the fluorescent Ca2+ dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca2+ chelator EGTA irrespective of added CaCl2. The addition of the Ca2+ ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn2+ ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters. Conclusions/Significance Taken together, comprehensive analysis is absolutely required for the demonstration of a

  18. Protective effect of a calcium channel blocker "diltiazem" on aluminum chloride-induced dementia in mice.

    PubMed

    Rani, Anu; Neha; Sodhi, Rupinder K; Kaur, Amanpreet

    2015-11-01

    Many studies report that heavy metals such as aluminum are involved in amyloid beta aggregation and neurotoxicity. Further, high concentration of aluminum in the brain deregulates calcium signaling which contributes to synaptic dysfunction and halts neuronal communication which ultimately leads to the development of Alzheimer's disease. Recently, diltiazem, a calcium channel blocker clinically used in angina, is reported to decrease amyloid beta production by inhibiting calcium influx, decreasing inflammation and oxidative stress. However, the probable role of this drug in aluminum chloride (AlCl3)-induced experimental dementia is yet to be explored. Therefore, the present study is designed to investigate the effect of AlCl3-induced dementia in mice. Morris water maze test and elevated plus maze were utilized to evaluate learning and memory. Various biochemical estimations including brain acetylcholinesterase activity (AChE), brain total protein, thiobarbituric acid-reactive species (TBARS) level, reduced glutathione (GSH) level, nitrate/nitrite, and superoxide dismutase (SOD) were measured. AlCl3 significantly impaired learning and memory and increased brain AChE, brain total protein, TBARS, and nitrate/nitrite and decreased brain GSH or SOD. On the other hand, treatment with diltiazem significantly reversed AlCl3-induced behavioral and biochemical deficits. The present study indicates the beneficial role of diltiazem in AlCl3-induced dementia.

  19. Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin.

    PubMed

    Atanasijevic, Tatjana; Shusteff, Maxim; Fam, Peter; Jasanoff, Alan

    2006-10-01

    We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 microM Ca(2+), suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 microM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens.

  20. Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin

    PubMed Central

    Atanasijevic, Tatjana; Shusteff, Maxim; Fam, Peter; Jasanoff, Alan

    2006-01-01

    We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein–protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 μM Ca2+, suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 μM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens. PMID:17003117

  1. Antigen Sensitization Influences Organophosphorus Pesticide–Induced Airway Hyperreactivity

    PubMed Central

    Proskocil, Becky J.; Bruun, Donald A.; Lorton, Jesse K.; Blensly, Kirsten C.; Jacoby, David B.; Lein, Pamela J.; Fryer, Allison D.

    2008-01-01

    Background Recent epidemiologic studies have identified organophosphorus pesticides (OPs) as environmental factors potentially contributing to the increase in asthma prevalence over the last 25 years. In support of this hypothesis, we have demonstrated that environmentally relevant concentrations of OPs induce airway hyperreactivity in guinea pigs. Objectives Sensitization to allergen is a significant contributing factor in asthma, and we have shown that sensitization changes virus-induced airway hyperreactivity from an eosinophil-independent mechanism to one mediated by eosinophils. Here, we determine whether sensitization similarly influences OP-induced airway hyperreactivity. Methods Nonsensitized and ovalbumin-sensitized guinea pigs were injected subcutaneously with the OP parathion (0.001–1.0 mg/kg). Twenty-four hours later, animals were anesthetized and ventilated, and bronchoconstriction was measured in response to either vagal stimulation or intravenous acetylcholine. Inflammatory cells and acetylcholinesterase activity were assessed in tissues collected immediately after physiologic measurements. Results Ovalbumin sensitization decreased the threshold dose for parathion-induced airway hyperreactivity and exacerbated parathion effects on vagally induced bronchoconstriction. Pretreatment with antibody to interleukin (IL)-5 prevented parathion-induced hyperreactivity in sensitized but not in nonsensitized guinea pigs. Parathion did not increase the number of eosinophils in airways or the number of eosinophils associated with airway nerves nor did it alter eosinophil activation as assessed by major basic protein deposition. Conclusions Antigen sensitization increases vulnerability to parathion-induced airway hyperreactivity and changes the mechanism to one that is dependent on IL-5. Because sensitization to allergens is characteristic of 50% of the general population and 80% of asthmatics (including children), these findings have significant implications for

  2. Cannabidiol induces intracellular calcium elevation and cytotoxicity in oligodendrocytes.

    PubMed

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

    2010-11-01

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

  3. Luteolin inhibits lysophosphatidylcholine-induced apoptosis in endothelial cells by a calcium/mitocondrion/caspases-dependent pathway.

    PubMed

    Song, Junna; Liu, Kang; Yi, Jiali; Zhu, Deqiu; Liu, Gaolin; Liu, Baolin

    2010-03-01

    Luteolin, a naturally occurring polyphenol flavonoid, has demonstrated some beneficial modulation toward the endothelium. This study aims to investigate the effects of luteolin on lysophosphatidylcholine (LPC)-induced apoptosis, a key event in the pathogenesis of atherosclerosis, in endothelial cells. Luteolin reduced not only LPC-induced cell death but also lactate dehydrogenase (LDH) leakage. Luteolin inhibition of LPC-induced apoptosis in endothelial cells demonstrated its protection against the cytotoxicity of LPC. LPC-induced apoptosis is characterized by a calcium-dependent mitochondrial pathway, involving calcium influx, activation of calpains, cytochrome C release and caspases activation. Luteolin reduced calcium influx. It also inhibited calpains activation and prevented the release of cytochrome C from mitochondrion. The inhibition of cytochrome C release by luteolin blocked the activation of caspase-3 and thus prevented subsequent endothelial cell apoptosis. These results suggested that luteolin inhibits LPC-induced apoptosis in endothelial cells through the blockage of the calcium-dependent mitochondrial pathway. PMID:19830654

  4. T-type calcium channel Cav3.2 deficient mice show elevated anxiety, impaired memory and reduced sensitivity to psychostimulants

    PubMed Central

    Gangarossa, Giuseppe; Laffray, Sophie; Bourinet, Emmanuel; Valjent, Emmanuel

    2014-01-01

    The fine-tuning of neuronal excitability relies on a tight control of Ca2+ homeostasis. The low voltage-activated (LVA) T-type calcium channels (Cav3.1, Cav3.2 and Cav3.3 isoforms) play a critical role in regulating these processes. Despite their wide expression throughout the central nervous system, the implication of T-type Cav3.2 isoform in brain functions is still poorly characterized. Here, we investigate the effect of genetic ablation of this isoform in affective disorders, including anxiety, cognitive functions as well as sensitivity to drugs of abuse. Using a wide range of behavioral assays we show that genetic ablation of the cacna1h gene results in an anxiety-like phenotype, whereas novelty-induced locomotor activity is unaffected. Deletion of the T-type channel Cav3.2 also triggers impairment of hippocampus-dependent recognition memories. Acute and sensitized hyperlocomotion induced by d-amphetamine and cocaine are dramatically reduced in T-type Cav3.2 deficient mice. In addition, the administration of the T-type blocker TTA-A2 prevented the expression of locomotor sensitization observed in wildtype mice. In conclusion, our data reveal that physiological activity of this specific Ca2+ channel is required for affective and cognitive behaviors. Moreover, our work highlights the interest of T-type channel blockers as therapeutic strategies to reverse drug-associated alterations. PMID:24672455

  5. Vorinostat protects against calcium oxalate-induced kidney injury in mice.

    PubMed

    Wang, Li; Chen, Wei; Peng, Zhongjiang; Liu, Changcheng; Zhang, Caihong; Guo, Zhiyong

    2015-09-01

    The present study aimed to examine the effect of the histone deacetylase inhibitor, vorinostat (SAHA), on renal function in a calcium oxalate crystal mouse model, and to investigate the mechanism underlying the renoprotective effect of SAHA. Calcium oxalate crystal formation was induced in 8 week‑old male C57BL/6 mice by administering 100 mg/kg glyoxylate for 7 days. A total of 24 male C57BL/6 mice were randomly divided into a control group and the following experimental groups: 50 mg/kg normal saline + 100 mg/kg glyoxylate; 50 mg/kg dimethyl sulfoxide (DMSO) + 100 mg/kg glyoxylate; and 50 mg/kg SAHA + 100 mg/kg glyoxylate. The mice in each of the experimental groups were injected with the saline, DMSO or SAHA into their abdominal cavities 6 h prior to the glyoxylate injection. The mice were sacrificed after 7 days, following which blood and urine samples were collected. The kidneys were harvested to analyze the levels of calcium concentrations and the levels of malondialdehyde (MDA), superoxide dismutase and glutathione reductase. Immunohistochemical staining and semi‑quantitative analyses were performed to detect the expression levels of osteopontin (OPN) and CD44. Renal tubular cell apoptosis was detected using a TUNEL assay. The concentrations of calcium and malondialdehyde were significantly decreased in the SAHA group, and calcium oxalate crystals in the kidney tissue and the expression levels of OPN and CD44 in the SAHA group were lower, compared with the other experimental groups. SAHA significantly reduced the urinary excretion of KIM‑1 and renal tubular cell apoptosis. In conclusion, SAHA reduced calcium oxalate crystal deposition and protected against kidney injury.

  6. Sodium-dependent calcium extrusion and sensitivity regulation in retinal cones of the salamander.

    PubMed Central

    Nakatani, K; Yau, K W

    1989-01-01

    1. Membrane current was recorded from an isolated, dark-adapted salamander cone by sucking its inner segment into a tight-fitting glass pipette containing Ringer solution. The outer segment of the cell was exposed to a bath solution that could be changed rapidly. 2. After removing Na+ from the bath Ringer solution for a short period of time in darkness (the 'loading period'), a transient inward current was observed upon restoring it in bright light. A similar but longer-lasting current was observed when Na+ was restored in the light after a large Ca2+ influx was induced through the light-sensitive conductance in darkness. 3. The above transient current was not observed if Li+ or guanidinium was substituted for Na+ in the light, or if Ba2+ was substituted for Ca2+ during the dark loading period. However, a current was observed if Sr2+ was the substituting ion for Ca2+ during loading. These observations suggested that the current was associated with an electrogenic Na+-dependent Ca2+ efflux at the cone outer segment. 4. The saturated amplitude of the exchange current was 12-25 pA with a mean around 16 pA. This is very comparable to that measured in the outer segment of a salamander rod under similar conditions. 5. By comparing a known Ca2+ load in a cone outer segment to the subsequent charge transfer through the exchange, we estimated that the stoichiometry of the exchange was near 3Na+:1Ca2+. 6. With a small Ca2+ load, or in the presence of Cs+ around the inner segment, the final temporal decline of the Na+-Ca2+ exchange current was roughly exponential, with a mean time constant of about 100 ms. This decline is about four times faster than that measured in rods. We interpret the shorter time constant in cones to reflect a faster rate of decline of intracellular free Ca2+ in their outer segments resulting from the exchange activity. 7. In the absence of external Na+, and hence any Na+-dependent Ca2+ efflux, the absolute sensitivity of a cone to a dim flash was

  7. Mechanisms underlying angiotensin II-induced calcium oscillations

    PubMed Central

    Edwards, Aurélie; Pallone, Thomas L.

    2008-01-01

    To gain insight into the mechanisms that underlie angiotensin II (ANG II)-induced cytoplasmic Ca2+ concentration ([Ca]cyt) oscillations in medullary pericytes, we expanded a prior model of ion fluxes. ANG II stimulation was simulated by doubling maximal inositol trisphosphate (IP3) production and imposing a 90% blockade of K+ channels. We investigated two configurations, one in which ryanodine receptors (RyR) and IP3 receptors (IP3R) occupy a common store and a second in which they reside on separate stores. Our results suggest that Ca2+ release from stores and import from the extracellular space are key determinants of oscillations because both raise [Ca] in subplasmalemmal spaces near RyR. When the Ca2+-induced Ca2+ release (CICR) threshold of RyR is exceeded, the ensuing Ca2+ release is limited by Ca2+ reuptake into stores and export across the plasmalemma. If sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps do not remain saturated and sarcoplasmic reticulum Ca2+ stores are replenished, that phase is followed by a resumption of leak from internal stores that leads either to [Ca]cyt elevation below the CICR threshold (no oscillations) or to elevation above it (oscillations). Our model predicts that oscillations are more prone to occur when IP3R and RyR stores are separate because, in that case, Ca2+ released by RyR during CICR can enhance filling of adjacent IP3 stores to favor a high subsequent leak that generates further CICR events. Moreover, the existence or absence of oscillations depends on the set points of several parameters, so that biological variation might well explain the presence or absence of oscillations in individual pericytes. PMID:18562632

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

    PubMed

    Singla, Neha; Dhawan, D K

    2014-10-01

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

  9. Modification of caffeine-induced injury in Ca2+-free perfused rat hearts. Relationship to the calcium paradox.

    PubMed Central

    Vander Heide, R. S.; Altschuld, R. A.; Lamka, K. G.; Ganote, C. E.

    1986-01-01

    The pathogenesis of the calcium paradox has not been established. In calcium-free perfused hearts, caffeine, which releases calcium from the sarcoplasmic reticulum, causes severe myocardial injury, with creatine kinase (CK) release and contraction band necrosis similar in many respects to the calcium paradox. It has been postulated that contracture, initiated by a small rise in intracellular calcium, may cause sarcolemmal injury in both the calcium paradox and caffeine-induced myocardial injury. The present study was initiated to determine whether interventions which modulate caffeine-induced contracture will also correspondingly alter cellular injury. The effects of caffeine dose, procaine, extended calcium-free perfusion, elevated potassium, temperature, and increasing intracellular sodium on caffeine-induced contracture were examined in Langendorff-perfused adult rat hearts. Caffeine-induced contracture at 22 C increased over a dose range of 5-40 mM caffeine. Procaine, which inhibits caffeine-induced calcium release at doses between 5 and 20 mM, progressively reduced contracture caused by addition of 20 mM caffeine at 22 C. Hearts perfused with calcium-free solution containing 16 mM K+ showed a reduction in caffeine-induced contracture. Extended calcium-free perfusion (20 minutes) at temperatures from 18 to 37 C resulted in a progressive reduction of caffeine-induced contracture. Each of these interventions was also found to inhibit caffeine-induced injury at 37 C. Low temperature was found to have complex effects. Hypothermia enhanced caffeine contractures but also protected hearts from cell separations and CK release. Increasing intracellular sodium was found to enhance caffeine-induced contracture at 37 C. There was a direct correlation between measured intracellular sodium levels and the magnitude and duration of caffeine-induced contracture. These results demonstrate a direct correlation between the magnitude of contracture and myocardial injury in calcium

  10. Study of the function of sarcoplasmic reticulum of vascular smooth muscle during activation due to depolarization-induced calcium influx

    SciTech Connect

    Hwang, K.S.

    1987-01-01

    The role of sarcoplasmic reticulum (SR) in vascular smooth muscle was evaluated with respect to regulation of myoplasmic Ca{sup 2+} during the Ca{sup 2+} entry induced by depolarization. Calcium agonist, Bay K8644, stimulated Ca{sup 2+} influx as well as tension in physiological salt solution, (PSS) in contrast to the priming effects due to the depolarization originally reported. Disparity, however, was found between the Ca{sup 2+} entered and tension developed. Correlation between the tension and {sup 45}Ca influx showed a typical threshold phenomenon; the basal Ca{sup 2+} influx can be raised to a certain level (25%) without tension induction, after which a minor increase in Ca{sup 2+} influx produced significant tension. This subthreshold Ca{sup 2+} influx was found accumulated in the caffeine-sensitive Ca stores, the SR. This confirmed the dependency of tension on the rate of Ca{sup 2+} entry demonstrated by a previous report.

  11. STX, a novel nonsteroidal estrogenic compound, induces rapid action in primate GnRH neuronal calcium dynamics and peptide release.

    PubMed

    Kenealy, B P; Keen, K L; Rønnekleiv, O K; Terasawa, E

    2011-08-01

    Previously, we reported that 1 nM 17ß-estradiol (E(2)) induces a rapid action, which is, in part, mediated through the G protein-coupled receptor GPR30 in primate GnRH neurons. Because it has been reported that the diphenylacrylamide compound, STX, causes estrogenic action in the mouse and guinea pig hypothalamus, the present study examined effects of STX in primate GnRH neurons and whether there is an action independent of GPR30. Results are summarized as follows. STX (10 nM) exposure increased 1) the oscillation frequency of intracellular calcium concentration ([Ca(2+)](i)), 2) the percentage of cells stimulated, and 3) the synchronization frequency of [Ca(2+)](i) oscillations. STX (10-100 nM) also stimulated GnRH release. The effects of STX on both [Ca(2+)](i) oscillations and GnRH release were similar to those caused by E(2) (1 nM), although with less magnitude. STX (10 nM)-induced changes in [Ca(2+)](i) oscillations were not altered by GPR30 small interfering RNA transfection, indicating that STX-sensitive receptors differ from GPR30. Finally, a higher dose of E(2) (10 nM) induced a larger change in [Ca(2+)](i) oscillations than that with a smaller dose of E(2) (1 nM), and the effects of 10 nM E(2) were reduced but not completely blocked by GPR30 small interfering RNA transfection, indicating that the effects of 10 nM E(2) in primate GnRH neurons are mediated by multiple membrane receptors, including GPR30 and STX-sensitive receptors. Collectively, the rapid action of E(2) mediated through GPR30 differs from that mediated through STX-sensitive receptors. The molecular structure of the STX-sensitive receptor remains to be identified.

  12. The role of prenucleation clusters in surface-induced calcium phosphate crystallization

    NASA Astrophysics Data System (ADS)

    Dey, Archan; Bomans, Paul H. H.; Müller, Frank A.; Will, Julia; Frederik, Peter M.; de With, Gijsbertus; Sommerdijk, Nico A. J. M.

    2010-12-01

    Unravelling the processes of calcium phosphate formation is important in our understanding of both bone and tooth formation, and also of pathological mineralization, for example in cardiovascular disease. Serum is a metastable solution from which calcium phosphate precipitates in the presence of calcifiable templates such as collagen, elastin and cell debris. A pathological deficiency of inhibitors leads to the uncontrolled deposition of calcium phosphate. In bone and teeth the formation of apatite crystals is preceded by an amorphous calcium phosphate (ACP) precursor phase. ACP formation is thought to proceed through prenucleation clusters-stable clusters that are present in solution already before nucleation-as was recently demonstrated for CaCO3 (refs 15,16). However, the role of such nanometre-sized clusters as building blocks for ACP has been debated for many years. Here we demonstrate that the surface-induced formation of apatite from simulated body fluid starts with the aggregation of prenucleation clusters leading to the nucleation of ACP before the development of oriented apatite crystals.

  13. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

    PubMed Central

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

    2016-01-01

    Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

  14. Dual pathways of calcium entry in spike and plateau phases of luteinizing hormone release from chicken pituitary cells: sequential activation of receptor-operated and voltage-sensitive calcium channels by gonadotropin-releasing hormone

    SciTech Connect

    Davidson, J.S.; Wakefield, I.K.; King, J.A.; Mulligan, G.P.; Millar, R.P.

    1988-04-01

    It has previously been shown that, in pituitary gonadotrope cells, the initial rise in cytosolic Ca2+ induced by GnRH is due to a Ca2+ mobilization from intracellular stores. This raises the possibility that the initial transient spike phase of LH release might be fully or partially independent of extracellular Ca2+. We have therefore characterized the extracellular Ca2+ requirements, and the sensitivity to Ca2+ channel blockers, of the spike and plateau phases of secretion separately. In the absence of extracellular Ca2+ the spike and plateau phases were inhibited by 65 +/- 4% and 106 +/- 3%, respectively. Both phases exhibited a similar dependence on concentration of extracellular Ca2+. However, voltage-sensitive Ca2+ channel blockers D600 and nifedipine had a negligible effect on the spike phase, while inhibiting the plateau phase by approximately 50%. In contrast, ruthenium red, Gd3+ ions, and Co2+ ions inhibited both spike and plateau phases to a similar extent as removal of extracellular Ca2+. A fraction (35 +/- 4%) of spike phase release was resistant to removal of extracellular Ca2+. This fraction was abolished after calcium depletion of the cells by preincubation with EGTA in the presence of calcium ionophore A23187, indicating that it depends on intracellular Ca2+ stores. Neither absence of extracellular Ca2+, nor the presence of ruthenium red or Gd3+ prevented mobilization of 45Ca2+ from intracellular stores by GnRH. We conclude that mobilization of intracellular stored Ca2+ is insufficient by itself to account for full spike phase LH release.

  15. Calcium channel blockade attenuates angiotensin II-induced drinking in rats.

    PubMed

    Calcagnetti, D J; Schechter, M D

    1993-01-01

    Lateral ventricular administration of angiotensin II (ANG II) produces potent dipsogenic effects in water-sated rats. ANG II seems to require functional voltage-gated calcium channels on neurons throughout circumventricular brain sites to exert its effects. Although there are at least three types of calcium channels, only L-type calcium channel-blocking drugs have been reported to decrease drinking. (4-(4-Benzofurazanyl)-1-4-dihydro-2,6-dimethyl-3,5-pyridine-dic arb oxylic acid methyl 1-methyl-ethyl ester) [PN 200-110; isradipine (ISR)], a selective L-type calcium channel blocker, has been shown to attenuate significantly the intake of sweetened water in water-sated rats following either peripheral or ICV administration, but ISR does not affect plain-water intake in water-deprived rats. The present experiment was designed to determine whether ISR would attenuate ANG II-induced drinking that is not either motivated by palatability or dependent on deprivation. Rats, each fitted with chronic indwelling ventricular cannulae, were pretreated with ISR (0.3, 3.0, and 30 micrograms/rat; ICV). ANG II (40 ng/rat; ICV) was administered 10 min later and rats were allowed free access to water for 15 min. Injections of ANG II plus saline and ANG II plus the ISR vehicle (dimethyl sulfoxide) did not attenuate ANG II-induced polydipsia, whereas ANG II+ISR (0.3 and 3.0 micrograms) attenuated ANG II-induced drinking to 62 and 22% of control, respectively. Results with the 30-micrograms dose were not different from the 3.0 dose.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Polychlorinated biphenyl quinone induces endoplasmic reticulum stress, unfolded protein response, and calcium release.

    PubMed

    Xu, Demei; Su, Chuanyang; Song, Xiufang; Shi, Qiong; Fu, Juanli; Hu, Lihua; Xia, Xiaomin; Song, Erqun; Song, Yang

    2015-06-15

    Organisms are able to respond to environmental insult to maintain cellular homeostasis, which include the activation of a wide range of cellular adaptive responses with tightly controlled mechanisms. The endoplasmic reticulum (ER) is an organelle responsible for protein folding and calcium storage. ER stress leads to the accumulation of unfolded proteins in the ER lumen. To be against or respond to this effect, cells have a comprehensive signaling system, called unfolded protein response (UPR), to restore homeostasis and normal ER function or activate the cell death program. Therefore, it is critical to understand how environmental insult regulates the ingredients of ER stress and UPR signalings. Previously, we have demonstrated that polychlorinated biphenyl (PCB) quinone caused oxidative stress, cytotoxicity, genotoxicity, and apoptosis in HepG2 cells. Here, we investigated the role of a PCB quinone, PCB29-pQ on ER stress, UPR, and calcium release. PCB29-pQ markedly increased the hallmark genes of ER stress, namely, glucose-regulated protein 78 (GRP78), GRP94, and C/EBP homologous protein (CHOP) on both protein and mRNA levels in HepG2 cells. We also confirmed PCB29-pQ induced ER morphological defects by using transmission electron microscopy. Moreover, PCB29-pQ induced intracellular calcium accumulation and calpain activity, which were significantly inhibited by the pretreatment of BAPTA-AM (Ca(2+) chelator). These results were correlated with the outcome that PCB29-pQ induces ER stress-related apoptosis through caspase family gene 12, while salubrinal and Z-ATAD-FMK (a specific inhibitor of caspase 12) partially ameliorated this effect, respectively. N-Acetyl-l-cysteine (NAC) scavenged ROS formation and consequently alleviated PCB29-pQ-induced expression of ER stress-related genes. In conclusion, our result demonstrated for the first time that PCB quinone leads to ROS-dependent induction of ER stress, and UPR and calcium release in HepG2 cells, and the

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

    NASA Astrophysics Data System (ADS)

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

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

  18. The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase-D toxin.

    PubMed

    Chaves-Moreira, Daniele; Souza, Fernanda N; Fogaça, Rosalvo T H; Mangili, Oldemir C; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Chaim, Olga M; Veiga, Silvio S

    2011-09-01

    Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying recombinant phospholipase-D, a potent hemolytic and phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase-D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium-sensitive fluorescent probe (Fluo-4). This calcium influx was shown to be channel

  19. Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation.

    PubMed

    Connolly, James; Kaufman, Megan; Rothman, Adam; Gupta, Rashmi; Redden, George; Schuster, Martin; Colwell, Frederick; Gerlach, Robin

    2013-09-01

    Two bacterial strains, Pseudomonas aeruginosa MJK1 and Escherichia coli MJK2, were constructed that both express green fluorescent protein (GFP) and carry out ureolysis. These two novel model organisms are useful for studying bacterial carbonate mineral precipitation processes and specifically ureolysis-driven microbially induced calcium carbonate precipitation (MICP). The strains were constructed by adding plasmid-borne urease genes (ureABC, ureD and ureFG) to the strains P. aeruginosa AH298 and E. coli AF504gfp, both of which already carried unstable GFP derivatives. The ureolytic activities of the two new strains were compared to the common, non-GFP expressing, model organism Sporosarcina pasteurii in planktonic culture under standard laboratory growth conditions. It was found that the engineered strains exhibited a lower ureolysis rate per cell but were able to grow faster and to a higher population density under the conditions of this study. Both engineered strains were successfully grown as biofilms in capillary flow cell reactors and ureolysis-induced calcium carbonate mineral precipitation was observed microscopically. The undisturbed spatiotemporal distribution of biomass and calcium carbonate minerals were successfully resolved in 3D using confocal laser scanning microscopy. Observations of this nature were not possible previously because no obligate urease producer that expresses GFP had been available. Future observations using these organisms will allow researchers to further improve engineered application of MICP as well as study natural mineralization processes in model systems.

  20. Fracture Sealing with Microbially-Induced Calcium Carbonate Precipitation: A Field Study.

    PubMed

    Phillips, Adrienne J; Cunningham, Alfred B; Gerlach, Robin; Hiebert, Randy; Hwang, Chiachi; Lomans, Bartholomeus P; Westrich, Joseph; Mantilla, Cesar; Kirksey, Jim; Esposito, Richard; Spangler, Lee

    2016-04-01

    A primary environmental risk from unconventional oil and gas development or carbon sequestration is subsurface fluid leakage in the near wellbore environment. A potential solution to remediate leakage pathways is to promote microbially induced calcium carbonate precipitation (MICP) to plug fractures and reduce permeability in porous materials. The advantage of microbially induced calcium carbonate precipitation (MICP) over cement-based sealants is that the solutions used to promote MICP are aqueous. MICP solutions have low viscosities compared to cement, facilitating fluid transport into the formation. In this study, MICP was promoted in a fractured sandstone layer within the Fayette Sandstone Formation 340.8 m below ground surface using conventional oil field subsurface fluid delivery technologies (packer and bailer). After 24 urea/calcium solution and 6 microbial (Sporosarcina pasteurii) suspension injections, the injectivity was decreased (flow rate decreased from 1.9 to 0.47 L/min) and a reduction in the in-well pressure falloff (>30% before and 7% after treatment) was observed. In addition, during refracturing an increase in the fracture extension pressure was measured as compared to before MICP treatment. This study suggests MICP is a promising tool for sealing subsurface fractures in the near wellbore environment. PMID:26911511

  1. Fracture Sealing with Microbially-Induced Calcium Carbonate Precipitation: A Field Study.

    PubMed

    Phillips, Adrienne J; Cunningham, Alfred B; Gerlach, Robin; Hiebert, Randy; Hwang, Chiachi; Lomans, Bartholomeus P; Westrich, Joseph; Mantilla, Cesar; Kirksey, Jim; Esposito, Richard; Spangler, Lee

    2016-04-01

    A primary environmental risk from unconventional oil and gas development or carbon sequestration is subsurface fluid leakage in the near wellbore environment. A potential solution to remediate leakage pathways is to promote microbially induced calcium carbonate precipitation (MICP) to plug fractures and reduce permeability in porous materials. The advantage of microbially induced calcium carbonate precipitation (MICP) over cement-based sealants is that the solutions used to promote MICP are aqueous. MICP solutions have low viscosities compared to cement, facilitating fluid transport into the formation. In this study, MICP was promoted in a fractured sandstone layer within the Fayette Sandstone Formation 340.8 m below ground surface using conventional oil field subsurface fluid delivery technologies (packer and bailer). After 24 urea/calcium solution and 6 microbial (Sporosarcina pasteurii) suspension injections, the injectivity was decreased (flow rate decreased from 1.9 to 0.47 L/min) and a reduction in the in-well pressure falloff (>30% before and 7% after treatment) was observed. In addition, during refracturing an increase in the fracture extension pressure was measured as compared to before MICP treatment. This study suggests MICP is a promising tool for sealing subsurface fractures in the near wellbore environment.

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

    PubMed

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

    2015-06-01

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

  3. Calcium-regulated nuclear enzymes: potential mediators of phytochrome-induced changes in nuclear metabolism?

    NASA Technical Reports Server (NTRS)

    Roux, S. J.

    1992-01-01

    Calcium ions have been proposed to serve as important regulatory elements in stimulus-response coupling for phytochrome responses. An important test of this hypothesis will be to identify specific targets of calcium action that are required for some growth or development process induced by the photoactivated form of phytochrome (Pfr). Initial studies have revealed that there are at least two enzymes in pea nuclei that are stimulated by Pfr in a Ca(2+)-dependent fashion, a calmodulin-regulated nucleoside triphosphatase and a calmodulin-independent but Ca(2+)-dependent protein kinase. The nucleoside triphosphatase appears to be associated with the nuclear envelope, while the protein kinase co-purifies with a nuclear fraction highly enriched for chromatin. This short review summarizes the latest findings on these enzymes and relates them to what is known about Pfr-regulated nuclear metabolism.

  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. Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus

    PubMed Central

    Bettgenhaeuser, Jan; Iakobachvili, Nino; Bignell, Elaine M.; Read, Nick D.

    2015-01-01

    Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca2+-mediated signalling. Ca2+ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca2+ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuBKU80, were engineered to express the bioluminescent Ca2+-reporter aequorin. An aequorin-mediated method for routine Ca2+ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca2+]c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca2+, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The ‘signatures’ of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response. Concordant with the involvement of Ca2+-calmodulin complexes in these Ca2+-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca2+-signatures to all the challenges. The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses. PMID:26402916

  6. Acute biochemical variations induced by four different calcium salts in healthy male volunteers.

    PubMed

    Reginster, J Y; Denis, D; Bartsch, V; Deroisy, R; Zegels, B; Franchimont, P

    1993-09-01

    Calcium (Ca) supplements have positive effects in growing children, reduce bone loss in late-postmenopausal women with a low calcium diet and, in association with vitamin D3 supplements, may reduce non-vertebral fracture rates in elderly women. However, for many formulated pharmaceutical products their relative beneficial effects have not been conclusively established. We have compared the acute (6 h) metabolic responses following oral administration of two preparations of calcium gluconolactate and carbonate (CG and CG'), tricalcium phosphate (TCP) and calcium citrate (CC), given on separate occasions in each of 10 healthy young male volunteers. The subjects fasted overnight for 12 h and continued to fast during the experimental procedure. A 1000 mg dose of each Ca salt was ingested at weekly intervals. Blood was drawn after 30, 60, 90, 120, 180, 240, 300 and 360 min for measurement of serum Ca, phosphorus (P), parathyroid hormone (PTH) and whole plasma calcitonin (iCT). All Ca supplements induced significant (+6.4% to +8.1%; p < 0.01) increases in Ca and significant suppression of PTH (-37.4% to -57.4%; p < 0.01). Comparison of response curves revealed significantly (p < 0.01) more marked Ca increase and PTH suppression with CC than with the other three Ca salts. CG' and CC induced marginal decreases in serum P and the overall curve of P variations was different for TCP compared with CG, CG' and CC. No significant variation of iCT was recorded during the test.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Bacterially induced calcium carbonate precipitation and strontium coprecipitation in a porous media flow system.

    PubMed

    Lauchnor, Ellen G; Schultz, Logan N; Bugni, Steven; Mitchell, Andrew C; Cunningham, Alfred B; Gerlach, Robin

    2013-02-01

    Strontium-90 is a principal radionuclide contaminant in the subsurface at several Department of Energy sites in the Western U.S., causing a threat to groundwater quality in areas such as Hanford, WA. In this work, we used laboratory-scale porous media flow cells to examine a potential remediation strategy employing coprecipitation of strontium in carbonate minerals. CaCO(3) precipitation and strontium coprecipitation were induced via ureolysis by Sporosarcina pasteurii in two-dimensional porous media reactors. An injection strategy using pulsed injection of calcium mineralization medium was tested against a continuous injection strategy. The pulsed injection strategy involved periods of lowered calcite saturation index combined with short high fluid velocity flow periods of calcium mineralization medium followed by stagnation (no-flow) periods to promote homogeneous CaCO(3) precipitation. By alternating the addition of mineralization and growth media the pulsed strategy promoted CaCO(3) precipitation while sustaining the ureolytic culture over time. Both injection strategies achieved ureolysis with subsequent CaCO(3) precipitation and strontium coprecipitation. The pulsed injection strategy precipitated 71-85% of calcium and 59% of strontium, while the continuous injection was less efficient and precipitated 61% of calcium and 56% of strontium. Over the 60 day operation of the pulsed reactors, ureolysis was continually observed, suggesting that the balance between growth and precipitation phases allowed for continued cell viability. Our results support the pulsed injection strategy as a viable option for ureolysis-induced strontium coprecipitation because it may reduce the likelihood of injection well accumulation caused by localized mineral plugging while Sr coprecipitation efficiency is maintained in field-scale applications. PMID:23282003

  8. Acute biochemical variations induced by four different calcium salts in healthy male volunteers.

    PubMed

    Reginster, J Y; Denis, D; Bartsch, V; Deroisy, R; Zegels, B; Franchimont, P

    1993-09-01

    Calcium (Ca) supplements have positive effects in growing children, reduce bone loss in late-postmenopausal women with a low calcium diet and, in association with vitamin D3 supplements, may reduce non-vertebral fracture rates in elderly women. However, for many formulated pharmaceutical products their relative beneficial effects have not been conclusively established. We have compared the acute (6 h) metabolic responses following oral administration of two preparations of calcium gluconolactate and carbonate (CG and CG'), tricalcium phosphate (TCP) and calcium citrate (CC), given on separate occasions in each of 10 healthy young male volunteers. The subjects fasted overnight for 12 h and continued to fast during the experimental procedure. A 1000 mg dose of each Ca salt was ingested at weekly intervals. Blood was drawn after 30, 60, 90, 120, 180, 240, 300 and 360 min for measurement of serum Ca, phosphorus (P), parathyroid hormone (PTH) and whole plasma calcitonin (iCT). All Ca supplements induced significant (+6.4% to +8.1%; p < 0.01) increases in Ca and significant suppression of PTH (-37.4% to -57.4%; p < 0.01). Comparison of response curves revealed significantly (p < 0.01) more marked Ca increase and PTH suppression with CC than with the other three Ca salts. CG' and CC induced marginal decreases in serum P and the overall curve of P variations was different for TCP compared with CG, CG' and CC. No significant variation of iCT was recorded during the test.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8400610

  9. Bacterially induced calcium carbonate precipitation and strontium coprecipitation in a porous media flow system.

    PubMed

    Lauchnor, Ellen G; Schultz, Logan N; Bugni, Steven; Mitchell, Andrew C; Cunningham, Alfred B; Gerlach, Robin

    2013-02-01

    Strontium-90 is a principal radionuclide contaminant in the subsurface at several Department of Energy sites in the Western U.S., causing a threat to groundwater quality in areas such as Hanford, WA. In this work, we used laboratory-scale porous media flow cells to examine a potential remediation strategy employing coprecipitation of strontium in carbonate minerals. CaCO(3) precipitation and strontium coprecipitation were induced via ureolysis by Sporosarcina pasteurii in two-dimensional porous media reactors. An injection strategy using pulsed injection of calcium mineralization medium was tested against a continuous injection strategy. The pulsed injection strategy involved periods of lowered calcite saturation index combined with short high fluid velocity flow periods of calcium mineralization medium followed by stagnation (no-flow) periods to promote homogeneous CaCO(3) precipitation. By alternating the addition of mineralization and growth media the pulsed strategy promoted CaCO(3) precipitation while sustaining the ureolytic culture over time. Both injection strategies achieved ureolysis with subsequent CaCO(3) precipitation and strontium coprecipitation. The pulsed injection strategy precipitated 71-85% of calcium and 59% of strontium, while the continuous injection was less efficient and precipitated 61% of calcium and 56% of strontium. Over the 60 day operation of the pulsed reactors, ureolysis was continually observed, suggesting that the balance between growth and precipitation phases allowed for continued cell viability. Our results support the pulsed injection strategy as a viable option for ureolysis-induced strontium coprecipitation because it may reduce the likelihood of injection well accumulation caused by localized mineral plugging while Sr coprecipitation efficiency is maintained in field-scale applications.

  10. Cocaine-induced locomotor sensitization in rats correlates with nucleus accumbens activity on manganese-enhanced MRI.

    PubMed

    Perrine, Shane A; Ghoddoussi, Farhad; Desai, Kirtan; Kohler, Robert J; Eapen, Ajay T; Lisieski, Michael J; Angoa-Perez, Mariana; Kuhn, Donald M; Bosse, Kelly E; Conti, Alana C; Bissig, David; Berkowitz, Bruce A

    2015-11-01

    A long-standing goal of substance abuse research has been to link drug-induced behavioral outcomes with the activity of specific brain regions to understand the neurobiology of addiction behaviors and to search for drug-able targets. Here, we tested the hypothesis that cocaine produces locomotor (behavioral) sensitization that correlates with increased calcium channel-mediated neuroactivity in brain regions linked with drug addiction, such as the nucleus accumbens (NAC), anterior striatum (AST) and hippocampus, as measured using manganese-enhanced MRI (MEMRI). Rats were treated with cocaine for 5 days, followed by a 2-day drug-free period. The following day, locomotor sensitization was quantified as a metric of cocaine-induced neuroplasticity in the presence of manganese. Immediately following behavioral testing, rats were examined for changes in calcium channel-mediated neuronal activity in the NAC, AST, hippocampus and temporalis muscle, which was associated with behavioral sensitization using MEMRI. Cocaine significantly increased locomotor activity and produced behavioral sensitization compared with saline treatment of control rats. A significant increase in MEMRI signal intensity was determined in the NAC, but not AST or hippocampus, of cocaine-treated rats compared with saline-treated control rats. Cocaine did not increase signal intensity in the temporalis muscle. Notably, in support of our hypothesis, behavior was significantly and positively correlated with MEMRI signal intensity in the NAC. As neuronal uptake of manganese is regulated by calcium channels, these results indicate that MEMRI is a powerful research tool to study neuronal activity in freely behaving animals and to guide new calcium channel-based therapies for the treatment of cocaine abuse and dependence.

  11. Sarcolemmal ATP-sensitive potassium channel protects cardiac myocytes against lipopolysaccharide-induced apoptosis.

    PubMed

    Zhang, Xiaohui; Zhang, Xiaohua; Xiong, Yiqun; Xu, Chaoying; Liu, Xinliang; Lin, Jian; Mu, Guiping; Xu, Shaogang; Liu, Wenhe

    2016-09-01

    The sarcolemmal ATP-sensitive K+ (sarcKATP) channel plays a cardioprotective role during stress. However, the role of the sarcKATP channel in the apoptosis of cardiomyocytes and association with mitochondrial calcium remains unclear. For this purpose, we developed a model of LPS-induced sepsis in neonatal rat cardiomyocytes (NRCs). The TUNEL assay was performed in order to detect the apoptosis of cardiac myocytes and the MTT assay was performed to determine cellular viability. Exposure to LPS significantly decreased the viability of the NRCs as well as the expression of Bcl-2, whereas it enhanced the activity and expression of the apoptosis-related proteins caspase-3 and Bax, respectively. The sarcKATP channel blocker, HMR-1098, increased the apoptosis of NRCs, whereas the specific sarcKATP channel opener, P-1075, reduced the apoptosis of NRCs. The mitochondrial calcium uniporter inhibitor ruthenium red (RR) partially inhibited the pro-apoptotic effect of HMR-1098. In order to confirm the role of the sarcKATP channel, we constructed a recombinant adenovirus vector carrying the sarcKATP channel mutant subunit Kir6.2AAA to inhibit the channel activity. Kir6.2AAA adenovirus infection in NRCs significantly aggravated the apoptosis of myocytes induced by LPS. Elucidating the regulatory mechanisms of the sarcKATP channel in apoptosis may facilitate the development of novel therapeutic targets and strategies for the management of sepsis and cardiac dysfunction. PMID:27430376

  12. Sarcolemmal ATP-sensitive potassium channel protects cardiac myocytes against lipopolysaccharide-induced apoptosis

    PubMed Central

    Zhang, Xiaohui; Zhang, Xiaohua; Xiong, Yiqun; Xu, Chaoying; Liu, Xinliang; Lin, Jian; Mu, Guiping; Xu, Shaogang; Liu, Wenhe

    2016-01-01

    The sarcolemmal ATP-sensitive K+ (sarcKATP) channel plays a cardioprotective role during stress. However, the role of the sarcKATP channel in the apoptosis of cardiomyocytes and association with mitochondrial calcium remains unclear. For this purpose, we developed a model of LPS-induced sepsis in neonatal rat cardiomyocytes (NRCs). The TUNEL assay was performed in order to detect the apoptosis of cardiac myocytes and the MTT assay was performed to determine cellular viability. Exposure to LPS significantly decreased the viability of the NRCs as well as the expression of Bcl-2, whereas it enhanced the activity and expression of the apoptosis-related proteins caspase-3 and Bax, respectively. The sarcKATP channel blocker, HMR-1098, increased the apoptosis of NRCs, whereas the specific sarcKATP channel opener, P-1075, reduced the apoptosis of NRCs. The mitochondrial calcium uniporter inhibitor ruthenium red (RR) partially inhibited the pro-apoptotic effect of HMR-1098. In order to confirm the role of the sarcKATP channel, we constructed a recombinant adenovirus vector carrying the sarcKATP channel mutant subunit Kir6.2AAA to inhibit the channel activity. Kir6.2AAA adenovirus infection in NRCs significantly aggravated the apoptosis of myocytes induced by LPS. Elucidating the regulatory mechanisms of the sarcKATP channel in apoptosis may facilitate the development of novel therapeutic targets and strategies for the management of sepsis and cardiac dysfunction. PMID:27430376

  13. Iron Mediates N-Methyl-d-aspartate Receptor-dependent Stimulation of Calcium-induced Pathways and Hippocampal Synaptic Plasticity*

    PubMed Central

    Muñoz, Pablo; Humeres, Alexis; Elgueta, Claudio; Kirkwood, Alfredo; Hidalgo, Cecilia; Núñez, Marco T.

    2011-01-01

    Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-d-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP. PMID:21296883

  14. Combined use of UV-labile calcium chelators and calcium-sensitive dyes in a microscope with two light sources influencing different regions in a group of coordinated contracting cardiac myocytes

    NASA Astrophysics Data System (ADS)

    Pilarczyk, Goetz; Greulich, Karl-Otto

    1997-12-01

    The coordination of excitation in a biological system of cells such as cardiac myocytes in heart tissue has crucial influence on the function of the entire organ. This coordinated behavior can be visualized in a small group of embryonic cardiac myocytes derived from the hearts of unborn chicken. Loaded with a calcium sensitive dye the excitation can be imaged via the occurring transient rise in cytosolic calcium concentration. It can be shown that in regions with physiological or morphological restrictions the transient rise in cytosolic calcium occurs with a temporal delay compared to the ordinary array of coupled myocytes. The height of the transient rise of cytosolic calcium is related to the ability of the individual cell to participate in the coordinated contraction. The free cytosolic calcium concentration is decreased with the UV-labile calcium, chelator diazo-2. Our setup allows to decrease the free cytosolic calcium in a single cell of the contracting array of cells. This allows us to introduce mismatches in selected regions of the coordinated contraction and to visualize the effects simultaneously.

  15. Neuronal Expression of the Human Neuropeptide S Receptor NPSR1 Identifies NPS-Induced Calcium Signaling Pathways

    PubMed Central

    Erdmann, Frank; Kügler, Sebastian; Blaesse, Peter; Lange, Maren D.; Skryabin, Boris V.; Pape, Hans-Christian; Jüngling, Kay

    2015-01-01

    The neuropeptide S (NPS) system was discovered as a novel neurotransmitter system a decade ago and has since been identified as a key player in the modulation of fear and anxiety. Genetic variations of the human NPS receptor (NPSR1) have been associated with pathologies like panic disorders. However, details on the molecular fundamentals of NPSR1 activity in neurons remained elusive. We expressed NPSR1 in primary hippocampal cultures. Using single-cell calcium imaging we found that NPSR1 stimulation induced calcium mobilization from the endoplasmic reticulum via activation of IP3 and ryanodine receptors. Store-operated calcium channels were activated in a downstream process mediating entry of extracellular calcium. We provide the first detailed analysis of NPSR1 activity and the underlying intracellular pathways with respect to calcium mobilization in neurons. PMID:25714705

  16. Mammalian freeze-dried sperm can maintain their calcium oscillation-inducing ability when microinjected into mouse eggs.

    PubMed

    Liu, Qi-Cai; Chen, Tian-e; Huang, Xiu-Ying; Sun, Fang-Zhen

    2005-03-25

    Mammalian freeze-dried sperm can maintain their genetic integrity and event support full development to term when microinjected into mature oocytes. However, it is unknown whether freeze-dried sperm can still maintain their calcium oscillation-inducing capability. Here, we microinjected mouse and bovine freeze-dried sperm into mouse MII oocytes and examined their calcium oscillation-inducing ability following intracytoplasmic sperm injection (ICSI). Two pieces of information are revealed. First, nearly all oocytes injected with a freeze-dried mouse sperm head or a bovine sperm showed fertilization-like calcium oscillations, indicating that freeze-drying treatment does not affect the activity of the sperm factor responsible for calcium oscillations. Second, freeze-dried sperm exhibited high resistance to external temperature increase. This is shown by the finding that the freeze-dried sperm can maintain their calcium oscillation-inducing capacity even following exposure to 100 degrees C for 3 h. We therefore conclude that mammalian sperm can maintain their calcium oscillation-inducing capability following freeze-drying, rehydration, and ICSI treatments.

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

    PubMed

    Ma, Teng; Wu, Xiyan; Cai, Qiyan; Wang, Yun; Xiao, Lan; Tian, Yanping; Li, Hongli

    2015-08-13

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

  18. The role of calcium in hypoxia-induced signal transduction and gene expression.

    PubMed

    Seta, Karen A; Yuan, Yong; Spicer, Zachary; Lu, Gang; Bedard, James; Ferguson, Tsuneo K; Pathrose, Peterson; Cole-Strauss, Allyson; Kaufhold, Alexa; Millhorn, David E

    2004-01-01

    Mammalian cells require a constant supply of oxygen in order to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. Sophisticated mechanisms have therefore evolved which allow cells to respond and adapt to hypoxia. Specialized oxygen-sensing cells have the ability to detect changes in oxygen tension and transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in a wide variety of different organisms. An increase in intracellular calcium levels is a primary response of many cell types to hypoxia/ischemia. The response to hypoxia is complex and involves the regulation of multiple signaling pathways and coordinated expression of perhaps hundreds of genes. This review discusses the role of calcium in hypoxia-induced regulation of signal transduction pathways and gene expression. An understanding of the molecular events initiated by changes in intracellular calcium will lead to the development of therapeutic approaches toward the treatment of hypoxic/ischemic diseases and tumors. PMID:15261489

  19. Some Kinetic and Metabolic Characteristics of Calcium-Induced Potassium Transport in Human Red Cells

    PubMed Central

    Kregenow, Floyd M.; Hoffman, Joseph F.

    1972-01-01

    When fresh human erythrocytes or their ghosts are incubated with Ca + IAA (iodoacetic acid) + adenosine, K permeability increases; K permeability also increases when energy-depleted cells or their ghosts are incubated with Ca alone. Na transport decreases or remains unaltered in both situations. The Ca-induced increase in K permeability in the depleted cell system is qualitatively similar to that seen in the fresh cell system and furnishes a means for studying the metabolic dependence of calcium's action. Studies with the depleted system suggest that the normal refractiveness of the cell to calcium is provided by a metabolically dependent substrate. Removal of this substrate allows Ca to enter the cell and exert its effect. By using 47Ca, a maximum value was obtained (3–7 x 10-6 moles/liter of red blood cells) for the quantity of calcium that is taken up by the cell and responsible for the change in K permeability. Measurements of the unidirectional fluxes of K, obtained during the time Ca increases K permeability, appear to satisfy the flux ratio equation for passive diffusion through a membrane. PMID:5074809

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

    SciTech Connect

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

    2008-01-01

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

  1. Cadmium Induces Apoptosis in Freshwater Crab Sinopotamon henanense through Activating Calcium Signal Transduction Pathway

    PubMed Central

    Wang, Jinxiang; Zhang, Pingping; Liu, Na; Wang, Qian; Luo, Jixian; Wang, Lan

    2015-01-01

    Calcium ion (Ca2+) is one of the key intracellular signals, which is implicated in the regulation of cell functions such as impregnation, cell proliferation, differentiation and death. Cadmium (Cd) is a toxic environmental pollutant that can disturb cell functions and even lead to cell death. Recently, we have found that Cd induced apoptosis in gill cells of the freshwater crab Sinopotamon henanense via caspase activation. In the present study, we further investigated the role of calcium signaling in the Cd-induced apoptosis in the animals. Our data showed that Cd triggered gill cell apoptosis which is evidenced by apoptotic DNA fragmentation, activations of caspases-3, -8 and -9 and the presence of apoptotic morphological features. Moreover, Cd elevated the intracellular concentration of Ca2+, the protein concentration of calmodulin (CaM) and the activity of Ca2+-ATPase in the gill cells of the crabs. Pretreatment of the animals with ethylene glycol-bis-(b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), Ca2+ chelator, inhibited Cd-induced activation of caspases-3, -8 and -9 as well as blocked the Cd-triggered apoptotic DNA fragmentation. The apoptotic morphological features were no longer observed in gill cells pretreated with the Ca2+ signaling inhibitors before Cd treatment. Our results indicate that Cd evokes gill cell apoptosis through activating Ca2+-CaM signaling transduction pathway. PMID:26714174

  2. Calcium signaling and the novel anti-proliferative effect of the UTP-sensitive P2Y11 receptor in rat cardiac myofibroblasts.

    PubMed

    Certal, Mariana; Vinhas, Adriana; Pinheiro, Ana Rita; Ferreirinha, Fátima; Barros-Barbosa, Aurora Raquel; Silva, Isabel; Costa, Maria Adelina; Correia-de-Sá, Paulo

    2015-11-01

    During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and α-smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca(2+)]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca(2+)]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca(2+) in the extracellular fluid. The biphasic [Ca(2+)]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11 receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors. Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca(2+)]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.

  3. Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes

    SciTech Connect

    Zhang Weihua; Fu Songbin; Lu Fanghao . E-mail: lufanghao1973@yahoo.com.cn; Wu Bo; Gong Dongmei; Pan, Zhen-wei; Lv Yanjie; Zhao Yajun; Li Quanfeng; Wang Rui; Yang Baofeng; Xu Changqing . E-mail: xucq@163.com

    2006-09-08

    The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca{sup 2+}]{sub i}. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl{sub 3} was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl{sub 3} during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a collapsed {delta}{psi} {sub m}, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl{sub 3}. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl{sub 3} but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl{sub 3} in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.

  4. Endoplasmic reticulum stress and calcium imbalance are involved in cadmium-induced lipid aberrancy in Saccharomyces cerevisiae.

    PubMed

    Rajakumar, Selvaraj; Bhanupriya, Nagaraj; Ravi, Chidambaram; Nachiappan, Vasanthi

    2016-09-01

    The endoplasmic reticulum is the key organelle which controls protein folding, lipid biogenesis, and calcium (Ca(2+)) homeostasis. Cd exposure in Saccharomyces cerevisiae activated the unfolded protein response and was confirmed by the increased Kar2p expression. Cd exposure in wild-type (WT) cells increased PC levels and the PC biosynthetic genes. Deletion of the two phospholipid methyltransferases CHO2 and OPI3 modulated PC, TAG levels and the lipid droplets with cadmium exposure. Interestingly, we noticed an increase in the calcium levels upon Cd exposure in the mutant cells. This study concluded that Cd interrupted calcium homeostasis-induced lipid dysregulation leading to ER stress. PMID:27344570

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

    PubMed

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

    2015-04-01

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

  6. α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor activation protects against phencyclidine-induced caspase-3 activity by activating voltage-gated calcium channels.

    PubMed

    Timpe, Jennifer M; Wang, Cheng Z; Kim, Jisoo; Johnson, Kenneth M

    2014-12-01

    Phencyclidine (PCP) is a noncompetitive, open channel blocker of the N-methyl-D-aspartate (NMDA) receptor-ion channel complex. When administered to immature animals, it is known to cause apoptotic neurodegeneration in several regions, and this is followed by olanzapine-sensitive, schizophrenia-like behaviors in late adolescence and adulthood. Clarification of its mechanism of action could yield data that would help to inform the treatment of schizophrenia. In our initial experiments, we found that α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) inhibited PCP-induced apoptosis in organotypic neonatal rat brain slices in a concentration-dependent and 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive manner. Calcium signaling pathways are widely implicated in apoptosis, and PCP prevents calcium influx through NMDA receptor channels. We therefore hypothesized that AMPA could protect against this effect by activation of voltage-dependent calcium channels (VDCCs). In support of this hypothesis, pretreatment with the calcium channel blocker cadmium chloride eliminated AMPA-mediated protection against PCP. Furthermore, the L-type VDCC inhibitor nifedipine (10 µM) fully abrogated the effects of AMPA, suggesting that L-type VDCCs are required for AMPA-mediated protection against PCP-induced neurotoxicity. Whereas the P/Q-type inhibitor ω-agatoxin TK (200 nM) reduced AMPA protection by 51.7%, the N-type VDCC inhibitor ω-conotoxin (2 µM) had no effect. Decreased AMPA-mediated protection following cotreatment with K252a, a TrkB inhibitor, suggests that brain-derived neurotrophic factor signaling plays an important role. By analogy, these results suggest that activation of L-type, and to a lesser extent P/Q-type, VDCCs might be advantageous in treating conditions associated with diminished NMDAergic activity during early development. PMID:24995437

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

    PubMed

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

    2000-10-01

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

  8. Capsaicin sensitizes TRAIL-induced apoptosis through Sp1-mediated DR5 up-regulation: Involvement of Ca{sup 2+} influx

    SciTech Connect

    Moon, Dong-Oh; Kang, Chang-Hee; Kang, Sang-Hyuck; Choi, Yung-Hyun; Hyun, Jin-Won; Chang, Weon-Young; Kang, Hee-Kyoung; Koh, Young-Sang; Maeng, Young-Hee; Kim, Young-Ree; Kim, Gi-Young

    2012-02-15

    Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various malignant cells, several cancers including human hepatocellular carcinoma (HCC) exhibit potent resistance to TRAIL-induced cell death. The aim of this study is to evaluate the anti-cancer potential of capsaicin in TRAIL-induced cancer cell death. As indicated by assays that measure phosphatidylserine exposure, mitochondrial activity and activation of caspases, capsaicin potentiated TRAIL-resistant cells to lead to cell death. In addition, we found that capsaicin induces the cell surface expression of TRAIL receptor DR5, but not DR4 through the activation Sp1 on its promoter region. Furthermore, we investigated that capsaicin-induced DR5 expression and apoptosis are inhibited by calcium chelator or inhibitors for calmodulin-dependent protein kinase. Taken together, our data suggest that capsaicin sensitizes TRAIL-mediated HCC cell apoptosis by DR5 up-regulation via calcium influx-dependent Sp1 activation. Highlights: ► Capsaicin sensitizes TRAIL-induced apoptosis through activation of caspases. ► Capsaicin induces expression of DR5 through Sp1 activation. ► Capsaicin activates calcium signaling pathway.

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

  10. Effects of Troponin T Cardiomyopathy Mutations on the Calcium Sensitivity of the Regulated Thin Filament and the Actomyosin Cross-Bridge Kinetics of Human β-Cardiac Myosin

    PubMed Central

    Sutton, Shirley; Miller, Susan M.; Spudich, James A.; Ruppel, Kathleen M.

    2013-01-01

    Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) lead to significant cardiovascular morbidity and mortality worldwide. Mutations in the genes encoding the sarcomere, the force-generating unit in the cardiomyocyte, cause familial forms of both HCM and DCM. This study examines two HCM-causing (I79N, E163K) and two DCM-causing (R141W, R173W) mutations in the troponin T subunit of the troponin complex using human β-cardiac myosin. Unlike earlier reports using various myosin constructs, we found that none of these mutations affect the maximal sliding velocities or maximal Ca2+-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca2+ sensitivity using the human myosin isoform do, however, mimic changes seen previously with non-human myosin isoforms. Transient kinetic measurements show that these mutations alter the kinetics of Ca2+ induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin. PMID:24367593

  11. Physiological Levels of Virion-Associated Human Immunodeficiency Virus Type 1 Envelope Induce Coreceptor-Dependent Calcium Flux▿ †

    PubMed Central

    Melar, Marta; Ott, David E.; Hope, Thomas J.

    2007-01-01

    Human immunodeficiency virus (HIV) entry into target cells requires the engagement of receptor and coreceptor by envelope glycoprotein (Env). Coreceptors CCR5 and CXCR4 are chemokine receptors that generate signals manifested as calcium fluxes in response to binding of the appropriate ligand. It has previously been shown that engagement of the coreceptors by HIV Env can also generate Ca2+ fluxing. Since the sensitivity and therefore the physiological consequence of signaling activation in target cells is not well understood, we addressed it by using a microscopy-based approach to measure Ca2+ levels in individual CD4+ T cells in response to low Env concentrations. Monomeric Env subunit gp120 and virion-bound Env were able to activate a signaling cascade that is qualitatively different from the one induced by chemokines. Env-mediated Ca2+ fluxing was coreceptor mediated, coreceptor specific, and CD4 dependent. Comparison of the observed virion-mediated Ca2+ fluxing with the exact number of viral particles revealed that the viral threshold necessary for coreceptor activation of signaling in CD4+ T cells was quite low, as few as two virions. These results indicate that the physiological levels of virion binding can activate signaling in CD4+ T cells in vivo and therefore might contribute to HIV-induced pathogenesis. PMID:17121788

  12. Pharmacological blockade of the calcium plateau provides neuroprotection following organophosphate paraoxon induced status epilepticus in rats.

    PubMed

    Deshpande, Laxmikant S; Blair, Robert E; Huang, Beverly A; Phillips, Kristin F; DeLorenzo, Robert J

    2016-01-01

    Organophosphate (OP) compounds which include nerve agents and pesticides are considered chemical threat agents. Currently approved antidotes are crucial in limiting OP mediated acute mortality. However, survivors of lethal OP exposure exhibit delayed neuronal injury and chronic behavioral morbidities. In this study, we investigated neuroprotective capabilities of dantrolene and carisbamate in a rat survival model of paraoxon (POX) induced status epilepticus (SE). Significant elevations in hippocampal calcium levels were observed 48-h post POX SE survival, and treatment with dantrolene (10mg/kg, i.m.) and carisbamate (90mg/kg, i.m.) lowered these protracted calcium elevations. POX SE induced delayed neuronal injury as characterized by Fluoro Jade C labeling was observed in critical brain areas including the dentate gyrus, parietal cortex, amygdala, and thalamus. Dantrolene and carisbamate treatment provided significant neuroprotection against delayed neuronal damage in these brain regions when administered one-hour after POX-SE. These results indicate that dantrolene or carisbamate could be effective adjuvant therapies to the existing countermeasures to reduce neuronal injury and behavioral morbidities post OP SE survival. PMID:27224207

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

    SciTech Connect

    Han, T Y; Aizenberg, J

    2007-08-31

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

  14. Mitochondrial calcium uptake underlies ROS generation during aminoglycoside-induced hair cell death.

    PubMed

    Esterberg, Robert; Linbo, Tor; Pickett, Sarah B; Wu, Patricia; Ou, Henry C; Rubel, Edwin W; Raible, David W

    2016-09-01

    Exposure to aminoglycoside antibiotics can lead to the generation of toxic levels of reactive oxygen species (ROS) within mechanosensory hair cells of the inner ear that have been implicated in hearing and balance disorders. Better understanding of the origin of aminoglycoside-induced ROS could focus the development of therapies aimed at preventing this event. In this work, we used the zebrafish lateral line system to monitor the dynamic behavior of mitochondrial and cytoplasmic oxidation occurring within the same dying hair cell following exposure to aminoglycosides. The increased oxidation observed in both mitochondria and cytoplasm of dying hair cells was highly correlated with mitochondrial calcium uptake. Application of the mitochondrial uniporter inhibitor Ru360 reduced mitochondrial and cytoplasmic oxidation, suggesting that mitochondrial calcium drives ROS generation during aminoglycoside-induced hair cell death. Furthermore, targeting mitochondria with free radical scavengers conferred superior protection against aminoglycoside exposure compared with identical, untargeted scavengers. Our findings suggest that targeted therapies aimed at preventing mitochondrial oxidation have therapeutic potential to ameliorate the toxic effects of aminoglycoside exposure. PMID:27500493

  15. A MUTANT PRION PROTEIN SENSITIZES NEURONS TO GLUTAMATE-INDUCED EXCITOTOXICITY

    PubMed Central

    Biasini, Emiliano; Unterberger, Ursula; Solomon, Isaac H.; Massignan, Tania; Senatore, Assunta; Bian, Hejiao; Voigtlaender, Till; Bowman, Frederick P.; Bonetto, Valentina; Chiesa, Roberto; Luebke, Jennifer; Toselli, Paul; Harris, David A.

    2013-01-01

    Growing evidence suggests that a physiological activity of the cellular prion protein (PrPC) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer’s diseases. However, how the functional activity of PrPC is subverted to deliver neurotoxic signals remains uncertain. Transgenic mice expressing PrP with a deletion of residues 105–125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose-dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we have tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices, and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders due to toxic, β-rich oligomers that bind to PrPC. PMID:23392670

  16. Internal noise induced pattern formation and spatial coherence resonance for calcium signals of diffusively coupled cells

    NASA Astrophysics Data System (ADS)

    Wang, Maosheng; Sun, Runzhi; Huang, Wanxia; Tu, Yubing

    2014-01-01

    The effects of internal noise in a square-lattice Höfer calcium oscillation system have been studied numerically in the context of chemical Langevin equations. It was found that spatial pattern can be induced by internal noise and, interestingly, an optimal internal noise strength (or optimal cell size) exists which maximizes the spatial coherence of pattern, indicating the occurrence of spatial coherence resonance. The effects of control parameter and coupling strength on system’s spatial coherence have also been investigated. We found that larger internal noise strength is needed to induce spatial pattern for a small control parameter or a stronger coupling strength, and spatial coherence can be enhanced by coupling.

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

    NASA Astrophysics Data System (ADS)

    Gower, Laurie B.; Odom, Damian J.

    2000-03-01

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

  18. Presence of a thapsigargin-sensitive calcium pump in Trypanosoma evansi: Immunological, physiological, molecular and structural evidences.

    PubMed

    Pérez-Gordones, M C; Serrano, M L; Rojas, H; Martínez, J C; Uzcanga, G; Mendoza, M

    2015-12-01

    In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca(2+)-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 μM TG or 25 μM 2',5'-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca(2+) and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG.

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

    PubMed

    Kitchen, Carl-Christian; Nissen, Peter; Secher, Niels H; Nielsen, Henning B

    2014-01-01

    Vasopressor agents may affect cerebral oxygenation (rScO2) as determined by near-infrared spectroscopy on the forehead. This case series evaluated the effect of calcium chloride vs. α and β-adrenergic receptor agonists on rScO2 in patients (n = 47) undergoing surgery during i.v. anesthesia. Mean arterial pressure (MAP) and cardiac output (CO) were assessed by Model-flow(®) and ephedrine (55 ± 3 vs. 74 ± 9 mmHg; 10 mg, n = 9), phenylephrine (51 ± 5 vs. 78 ± 9 mmHg, 0.1 mg, n = 11), adrenaline (53 ± 3 vs. 72 ± 11 mmHg; 1-2 μg, n = 6), noradrenaline (53 ± 5 vs. 72 ± 12 mmHg; 2-4 μg, n = 11), and calcium chloride (49 ± 7 vs. 57 ± 16 mmHg; 5 mmol, n = 10) increased MAP (all P < 0.05). CO increased with ephedrine (4.3 ± 0.9 vs. 5.3 ± 1.2, P < 0.05) and adrenaline (4.7 ± 1.2 vs. 5.9 ± 1.1 l/min; P = 0.07) but was not significantly affected by phenylephrine (3.9 ± 0.7 vs. 3.6 ± 1.0 l/min), noradrenaline (3.8 ± 1.2 vs. 3.7 ± 0.7 l/min), or calcium chloride (4.0 ± 1.4 vs. 4.1 ± 1.5 l/min). Following administration of β-adrenergic agents and calcium chloride rScO2 was preserved while after administration of α-adrenergic drugs rScO2 was reduced by app. 2% (P < 0.05). Following α-adrenergic drugs to treat anesthesia-induced hypotension tissue oxygenation is reduced while the use of β-adrenergic agonists and calcium chloride preserve tissue oxygenation.

  20. Mcl-1 downregulation sensitizes glioma to bortezomib-induced apoptosis.

    PubMed

    Zhang, Yang; Zhu, Xiaobo; Hou, Kun; Zhao, Jinchuan; Han, Zhiguo; Zhang, Xiaona

    2015-05-01

    Glioma is the most aggressive form of primary brain tumor, with dismal patient outcome and no effective therapeutic approaches available. Targeting the ubiquitin-proteasome pathway has recently emerged as a potent rational anticancer strategy. Bortezomib, a specific proteasome inhibitor, has been approved for the treatment of relapsed or refractory multiple myeloma and other hematological malignancies as a single agent or as part of a combination therapy. However, bortezomib alone or in combination showed only minimal effects in the treatment of solid tumors. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein which protects tumor cells against spontaneous and chemotherapy-induced apoptosis. In multiple myeloma, specific downregulation of Mcl-1 induces apoptosis. Furthermore, previous studies demonstrated that proteasome inhibitors induce Mcl-1 accumulation that, in turn, slows down their pro-apoptotic effects, and the cell survival in multiple myeloma is highly dependent on Mcl-1. In the present study, we investigated the role of Mcl-1 downregulation in bortezomib-induced apoptosis in gliomas. We observed that bortezomib triggers caspase-3 and PARP activation, upregulates cytochrome c expression and induces apoptosis. Furthermore, we demonstrated that effective targeting of Mcl-1 in glioma cells by gene silencing technology augments the glioma cell sensitivity to bortezomib-induced apoptosis. In conclusion, the present study demonstrates that Mcl-1 plays a critical role in bortezomib-induced apoptosis. Mcl-1 inhibitor in combination with bortezomib present a promising novel strategy to trigger cell death pathways in the treatment of gliomas.

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

    PubMed Central

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

    2016-01-01

    Male infertility caused by testicular damage is one of the complications of diabetes mellitus. The calcium-sensing receptor (CaSR) is expressed in testicular tissues and plays a pivotal role in calcium homeostasis by activating cellular signaling pathways, but its role in testicular damage induced by diabetes remains unclear. A diabetic model was established by a single intraperitoneal injection of streptozotocin (STZ, 40 mg kg−1) in Wistar rats. Animals then received GdCl3 (an agonist of CaSR, 8.67 mg kg−1), NPS-2390 (an antagonist of CaSR, 0.20 g kg−1), or a combination of both 2 months after STZ injection. Diabetic rats had significantly lower testes weights and serum levels of testosterone compared to healthy rats, indicating testicular damage and dysfunction in STZ-induced diabetic rats. Compared with healthy controls, the testicular tissues of diabetic rats overexpressed the CaSR protein and had higher levels of malondialdehyde (MDA), lower superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, and higher numbers of apoptotic germ cells. The testicular tissues from diabetic rats also expressed lower levels of Bcl-2 and higher levels of Bax and cleaved caspase-3 in addition to higher phosphorylation rates of c-Jun NH2-terminal protein kinase (JNK), p38, and extracellular signaling-regulated kinase (ERK) 1/2. The above parameters could be further increased or aggravated by the administration of GdCl3, but could be attenuated by injection of NPS-2390. In conclusion, the present results indicate that CaSR activation participates in diabetes-induced testicular damage, implying CaSR may be a potential target for protective strategies against diabetes-induced testicular damage and could help to prevent infertility in diabetic men. PMID:26387585

  2. Targeting HSP70-induced thermotolerance for design of thermal sensitizers.

    PubMed

    Calderwood, S K; Asea, A

    2002-01-01

    Thermal therapy has been shown to be an extremely powerful anti-cancer agent and a potent radiation sensitizer. However, the full potential of thermal therapy is hindered by a number of considerations including highly conserved heat resistance pathways in tumour cells and inhomogeneous heating of deep-seated tumours due to energy deposition and perfusion issues. This report reviews recent progress in the development of hyperthermia sensitizing drugs designed to specifically amplify the effects of hyperthermia. Such agents might be particularly useful in situations where heating is not adequate for the full biological effect or is not homogeneously delivered to tumours. The particular pathway concentrated on is thermotolerance, a complex, inducible cellular response that leads to heat resistance. This paper will concentrate on the molecular pathways of thermotolerance induction for designing inhibitors of heat resistance/thermal sensitizers, which may allow the full potential of thermal therapy to be utilized.

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

    PubMed Central

    Jackson, Kia J.; Damaj, M. Imad

    2014-01-01

    Calcium activated second messengers such as calcium/calmodulin-dependent protein kinase II have been implicated in drug-induced antinociception. The less abundant calcium activated second messenger, calcium/calmodulin-dependent protein kinase IV (CaMKIV), mediates emotional responses to pain and tolerance to morphine analgesia; however its role in nicotine-mediated antinociception is currently unknown. The goal of this study was to evaluate the role of CaMKIV in the acute effects of nicotine, primarily acute nicotine- induced antinociception. CaMKIV knockout (−/−), heterozygote (+/−), and wild-type (+/+) mice were injected with various doses of nicotine and evaluated in a battery of tests, including the tail-flick and hot-plate tests for antinociception, body temperature, and locomotor activity. Our results show a genotype-dependent reduction in tail-flick and hot- plate latency in CaMKIV (+/−) and (−/−) mice after acute nicotine treatment, while no difference was observed between genotypes in the body temperature and locomotor activity assessments. The results of this study support a role for CaMKIV in acute nicotine-induced spinal and supraspinal pain mechanisms, and further implicate involvement of calcium-dependent mechanisms in drug-induced antinociception. PMID:24196027

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

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

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

    PubMed

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

    2013-12-01

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

  6. Calcium channels responsible for potassium-induced transmitter release at rat cerebellar synapses.

    PubMed

    Momiyama, A; Takahashi, T

    1994-04-15

    The effects of calcium channel blockers on potassium-induced transmitter release were studied in thin slices of cerebellum from neonatal rats using whole-cell patch clamp methods. Miniature inhibitory postsynaptic currents (mIPSCs) mediated by gamma-aminobutyric acid (GABA) were recorded from deep cerebellar nuclear neurones in the presence of tetrodotoxin. The frequency of mIPSCs was reproducibly increased by a brief application of high-potassium solution. In the presence of the L-type Ca2+ channel blocker nicardipine (10 microM), the potassium-induced increase in mIPSC frequency was suppressed by 49%. Neither the mean amplitude nor the time course of mIPSCs was affected by the blocker. The N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTX, 3 microM) had no effect on the frequency of potassium-induced mIPSCs. The P-type Ca2+ channel blocker omega-Aga-IVA (200 nM) suppressed the potassium-induced increase in mIPSC frequency by 83% without affecting the mean amplitude or time course of mIPSCs. Comparing these data with previous studies of neurally evoked transmission, it is concluded that the Ca2+ channel subtypes responsible for potassium-induced transmitter release may be different from those mediating fast synaptic transmission.

  7. Peptide induced crystallization of calcium carbonate on wrinkle patterned substrate: implications for chitin formation in molluscs.

    PubMed

    Ghatak, Anindita Sengupta; Koch, Marcus; Guth, Christina; Weiss, Ingrid M

    2013-06-04

    We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates.

  8. Peptide Induced Crystallization of Calcium Carbonate on Wrinkle Patterned Substrate: Implications for Chitin Formation in Molluscs

    PubMed Central

    Sengupta Ghatak, Anindita; Koch, Marcus; Guth, Christina; Weiss, Ingrid M.

    2013-01-01

    We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates. PMID:23736692

  9. Peptide induced crystallization of calcium carbonate on wrinkle patterned substrate: implications for chitin formation in molluscs.

    PubMed

    Ghatak, Anindita Sengupta; Koch, Marcus; Guth, Christina; Weiss, Ingrid M

    2013-01-01

    We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates. PMID:23736692

  10. Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

    PubMed Central

    Kludt, Eugen; Schild, Detlev

    2016-01-01

    The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the γ-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the γ-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb. PMID:27286501

  11. Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis.

    PubMed

    Brinkmann, Alexander; Okom, Camille; Kludt, Eugen; Schild, Detlev

    2016-01-01

    The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the γ-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the γ-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca(2+) traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb. PMID:27286501

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

    PubMed

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

    2006-12-01

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

  13. Development affects in vitro vascular tone and calcium sensitivity in ovine cerebral arteries

    PubMed Central

    Geary, Greg G; Osol, George J; Longo, Lawrence D

    2004-01-01

    We have shown recently that development from neonatal to adult life affects cerebrovascular tone of mouse cerebral arteries through endothelium-derived vasodilatory mechanisms. The current study tested the hypothesis that development from fetal to adult life affects cerebral artery vascular smooth muscle (VSM) [Ca2+]i sensitivity and tone through a mechanism partially dependent upon endothelium-dependent signalling. In pressurized resistance sized cerebral arteries (∼150 μm) from preterm (95 ± 2 days gestation (95 d)) and near-term (140 ± 2 days gestation (140 d)) fetuses, and non-pregnant adults, we measured vascular diameter (μm) and [Ca2+]i (nm) as a function of intravascular pressure. We repeated these studies in the presence of inhibition of nitric oxide synthase (NOS; with l-NAME), cyclo-oxygenase (COX; with indomethacin) and endothelium removal (E–). Cerebrovasculature tone (E+) was greater in arteries from 95 d fetuses and adults compared to 140 d sheep. Ca2+ sensitivity was similar in 95 d fetuses and adults, but much lower in 140 d fetuses. Removal of endothelium resulted in a reduction in lumen diameter as a function of pressure (greater tone) in all treatment groups. [Ca2+]i sensitivity differences among groups were magnified after E–. NOS inhibition decreased diameter as a function of pressure in each age group, with a significant increase in [Ca2+]i to pressure ratio only in the 140 d fetuses. Indomethacin increased tone and increased [Ca2+]i in the 140 d fetuses, but not the other age groups. Development from near-term to adulthood uncovered an interaction between NOS- and COX-sensitive substances that functioned to modulate artery diameter but not [Ca2+]i. This study suggests that development is associated with significant alterations in cerebral vascular smooth muscle (VSM), endothelium, NOS and COX responses to intravascular pressure. We speculate that these changes have important implications in the regulation of cerebral blood flow in

  14. Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

    NASA Astrophysics Data System (ADS)

    Taft, William C.; Delorenzo, Robert J.

    1984-05-01

    Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

  15. Vitamin D receptor is required for dietary calcium-induced repression of calbindin-D9k expression in mice.

    PubMed

    Bolt, Merry J G; Cao, Li-Ping; Kong, Juan; Sitrin, Michael D; Li, Yan Chun

    2005-05-01

    Calbindin (CaBP), the vitamin D-dependent calcium-binding protein, is believed to play an important role in intracellular calcium transport. The aim of this study was to investigate the effect of high dietary calcium on the expression of CaBP-D9k and CaBP-D28k in the presence and absence of a functional vitamin D receptor (VDR). Treatment with the HCa-Lac diet containing 2% calcium, 1.5% phosphorus and 20% lactose reversed the hypocalcemia seen in adult VDR-null mice in 3 weeks but did not significantly change the blood ionized calcium in wild-type mice. This dietary treatment dramatically suppressed both the duodenal and the renal CaBP-D9k expression in wild-type mice at both mRNA and protein levels but had little effect on the expression of the same gene in VDR-null mice. Removal of this diet gradually restored the expression of CaBP-D9k to the untreated level in wild-type mice. Only moderate or little change in CaBP-D28k expression was seen in wild-type and VDR-null mice fed with the HCa-Lac diet. The VDR content in the duodenum or kidney of wild-type mice was not altered by the dietary treatment. These results suggest that calcium regulates CaBP-D9k expression by modulating the circulating 1,25-dihydrxyvitamin D(3) level and that VDR is thus required for the dietary calcium-induced suppression of CaBP-D9k expression. Calcium regulation of the CaBP-D9k level may represent an important mechanism by which animals maintain their calcium balance. PMID:15866228

  16. Arachidonic acid-induced mobilization of calcium in human neutrophils: evidence for a multicomponent mechanism of action.

    PubMed Central

    Naccache, P. H.; McColl, S. R.; Caon, A. C.; Borgeat, P.

    1989-01-01

    1. The mechanism(s) involved in the mobilization of calcium induced by arachidonic acid in human neutrophils was investigated. 2. The addition of arachidonic acid to a suspension of human neutrophils led to a time- and concentration-dependent mobilization of calcium which was the result of two separate and experimentally differentiable processes. The latter consisted of a rapid and transient phase followed by a slower and more sustained response. 3. The initial phase of calcium mobilization elicited by arachidonic acid was decreased in the presence of EGTA, inhibited by pertussis toxin as well as by nordihydroguaiaretic acid (NDGA), and diminished following a pre-incubation with leukotriene B4, but not platelet-activating factor. 4. The characteristics of the first phase of the mobilization of calcium were consistent with an interaction of the fatty acid with the leukotriene B4 receptors, either directly or indirectly following the synthesis of leukotriene B4, as well as with a release of internal calcium. 5. The second, slower and more sustained phase of calcium mobilization was more apparent at high concentrations (greater than or equal to 8-16 microM) of arachidonic acid, and was relatively insensitive to pertussis toxin, EGTA or NDGA. 6. The characteristics of the 'slow' phase of calcium mobilization by arachidonic acid are consistent with its being associated primarily with a release of calcium from internal storage pools. 7. The data presented indicate that the mechanism of mobilization of calcium by arachidonic acid in human neutrophils is complex and involves specific activation pathways employed, in part at least, by other neutrophil agonists.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2547474

  17. Calcium-myristoyl Tug is a new mechanism for intramolecular tuning of calcium sensitivity and target enzyme interaction for guanylyl cyclase-activating protein 1: dynamic connection between N-fatty acyl group and EF-hand controls calcium sensitivity.

    PubMed

    Peshenko, Igor V; Olshevskaya, Elena V; Lim, Sunghyuk; Ames, James B; Dizhoor, Alexander M

    2012-04-20

    Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca(2+) sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca(2+) sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca(2+) binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca(2+)-bound and Mg(2+)-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca(2+) sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca(2+) binding to EF-hand 4 and Ca(2+) sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu(80) and Val(180)) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or "tug") between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca(2+) sensitivity.

  18. Osteoprotegerin induces podosome disassembly in osteoclasts through calcium, ERK, and p38 MAPK signaling pathways.

    PubMed

    Zhao, Hongyan; Liu, Xuezhong; Zou, Hui; Dai, Nannan; Yao, Lulian; Gao, Qian; Liu, Wei; Gu, Jianhong; Yuan, Yan; Bian, Jianchun; Liu, Zongping

    2015-02-01

    Osteoclasts are critical for bone resorption and use podosomes to attach to bone matrix. Osteoprotegerin (OPG) is a negative regulator of osteoclast function that can affect the formation and function of podosomes. However, the signaling pathways that link OPG to podosome function have not been well characterized. Therefore, this study examined the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts. We assessed the effects of the intracellular calcium chelator Bapta-AM, ERK inhibitor U0126, and p38 inhibitor SB202190 on OPG-treated osteoclast differentiation, adhesion structures, intracellular free Ca(2+) concentration and the phosphorylation state of podosome associated proteins (Pyk2 and Src). Mouse monocytic RAW 264.7 cells were differentiated to osteoclasts using RANKL (30ng/mL) and M-CSF (25ng/mL). The cells were pretreated with Bapta-AM (5μM), U0126 (5μM), or SB202190 (10μM) for 30min, followed by 40ng/mL OPG for 3h. Osteoclastogenesis, adhesion structure, viability and morphology, intracellular free Ca(2+) concentration and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, scanning electron microscopy, real-time cell analyzer, flow cytometry and western blotting, respectively. OPG significantly inhibited osteoclastogenesis, the formation of adhesion structures, and reduced the amount of phosphorylated Pyk2 and Src-pY527, but increased phosphorylation of Src-pY416. Bapta-AM, U0126, and SB202190 partially restored osteoclast differentiation and adhesion structures. Both Bapta-AM and U0126, but not SB202190, restored the levels of intracellular free Ca(2+) concentration, phosphorylated Pyk2 and Src-pY527. All three inhibitors blocked OPG-induced phosphorylation at Src-pY416. These results suggest OPG disrupts the attachment structures of osteoclasts and activates Src as an adaptor protein that competes for the reduced amount of phosphorylated Pyk2 through calcium- and ERK-dependent signaling

  19. Biotic and abiotic effects on CO2 sequestration during microbially-induced calcium carbonate precipitation.

    PubMed

    Okyay, Tugba Onal; Rodrigues, Debora F

    2015-03-01

    In this study, CO2 sequestration was investigated through the microbially-induced calcium carbonate precipitation (MICP) process with isolates obtained from a cave called 'Cave Without A Name' (Boerne, TX, USA) and the Pamukkale travertines (Denizli, Turkey). The majority of the bacterial isolates obtained from these habitats belonged to the genera Sporosarcina, Brevundimonas, Sphingobacterium and Acinetobacter. The isolates were investigated for their capability to precipitate calcium carbonate and sequester CO2. Biotic and abiotic effects of CO2 sequestration during MICP were also investigated. In the biotic effect, we observed that the rate and concentration of CO2 sequestered was dependent on the species or strains. The main abiotic factors affecting CO2 sequestration during MICP were the pH and medium components. The increase in pH led to enhanced CO2 sequestration by the growth medium. The growth medium components, on the other hand, were shown to affect both the urease activity and CO2 sequestration. Through the Plackett-Burman experimental design, the most important growth medium component involved in CO2 sequestration was determined to be urea. The optimized medium composition by the Plackett-Burman design for each isolate led to a statistically significant increase, of up to 148.9%, in CO2 uptake through calcification mechanisms.

  20. Gravity-Induced Polar Transport of Calcium across Root Tips of Maize 1

    PubMed Central

    Lee, June S.; Mulkey, Timothy J.; Evans, Michael L.

    1983-01-01

    Calcium movement across primary roots of maize (Zea mays, L.) was determined by application of 45Ca2+ to one side of the root and collection of radioactivity in an agar receiver block on the opposite side. Ca movement across the root tip was found to be at least 20 times greater than movement across the elongation zone. The rapid movement of Ca across the tip was severely inhibited in roots from which the root cap had been removed. Ca movement across the tip was also strongly retarded in roots pretreated with 2,4-dinitrophenol or potassium cyanide. Orientation of roots horizontally had no effect on Ca movement across the elongation zone but caused a strong asymmetry in the pattern of Ca movement across the tip. In gravistimulated roots, the movement of Ca from top to bottom increased while movement from bottom to top decreased. The data indicate that gravistimulation induces polar movement of Ca toward the lower side of the root cap. An earlier report (Lee, Mulkey, Evans 1983 Science 220: 1375-1376) from this laboratory showed that artificial establishment of calcium gradients at the root tip can cause gravitropic-like curvature. Together, the two studies indicate that Ca plays a key role in linking gravistimulation to the gravitropic growth response in roots. PMID:16663333

  1. Bacterially induced mineralization of calcium carbonate: the role of exopolysaccharides and capsular polysaccharides.

    PubMed

    Ercole, Claudia; Cacchio, Paola; Botta, Anna Lucia; Centi, Valeria; Lepidi, Aldo

    2007-02-01

    Bacterially induced carbonate mineralization has been proposed as a new method for the restoration of limestones in historic buildings and monuments. We describe here the formation of calcite crystals by extracellular polymeric substances isolated from Bacillus firmus and Bacillus sphaericus. We isolated bacterial outer structures (glycocalix and parietal polymers), such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) and checked for their influence on calcite precipitation. CPS and EPS extracted from both B. firmus and B. sphaericus were able to mediate CaCO3 precipitation in vitro. X-ray microanalysis showed that in all cases the formed crystals were calcite. Scanning electron microscopy showed that the shape of the crystals depended on the fractions utilized. These results suggest the possibility that biochemical composition of CPS or EPS influences the resulting morphology of CaCO3. There were no precipitates in the blank samples. CPS and EPS comprised of proteins and glycoproteins. Positive alcian blue staining also reveals acidic polysaccharides in CPS and EPS fractions. Proteins with molecular masses of 25-40 kDa and 70 kDa in the CPS fraction were highly expressed in the presence of calcium oxalate. This high level of synthesis could be related to the binding of calcium ions and carbonate deposition.

  2. Shock-induced devolatilization of calcium sulfate and implications for K-T extinctions

    NASA Technical Reports Server (NTRS)

    Chen, Guangqing; Tyburczy, James A.; Ahrens, Thomas J.

    1994-01-01

    The devolatilization of calcium sulfate, which is present in the target rock of the Chicxulub, Mexico impact structure, and dispersal in the stratosphere of the resultant sulfuric acid aerosol have been suggested as a possible mechanism for the Cretaceous-Tertiary extinctions. We measured the amount of SO2 produced from two shock-induced devolatilization reactions of calcium sulfate up to 42 GPa in the laboratory. We found both to proceed to a much lower extent than calculated by equilibrium thermodynamic calculations. Reaction products are found to be approx. 10(exp -2) times those calculated for equilibrium. Upon modeling the quantity of sulfur oxides degassed into the atmosphere from shock devolatilization of CaSO4 in the Chicxulub lithographic section, the resulting 9 x 10(exp 16) to 6 x 10(exp 17) g (in sulfur mass) is lower by a factor of 10-100 than previous upper limit estimates, the related environmental stress arising from the resultant global cooling and fallout of acid rain is insufficient to explain the widespread K-T extinctions.

  3. Shock-induced devolatization of calcium sulfate and implications for K-T extinctions

    NASA Technical Reports Server (NTRS)

    Chen, Guangqing; Tyburczy, James A.; Ahrens, Thomas J.

    1993-01-01

    Calcium sulfate devolatization during the impact at Chicxulub, Mexico and dispersal in the stratosphere of the resultant sulfuric acid aerosol have been suggested as a possible mechanism for the Cretaceous-Tertiary extinctions. In this paper, we investigated two shock-induced devolatization reactions of calcium sulfate up to 42 GPa in the laboratory: CaSO4 + SiO2 yields CaSiO3 + SO3(degassed) and CaSO4 yields CaO + SO2(degassed) + 1/2 O2(degassed). We found both to proceed to a much less extent than calculated by equilibrium thermodynamic calculations. Reaction products are found to be 10(exp -2) times those calculated for equilibrium. Consequently our estimate of the amount of sulfur oxides degassed into the atmosphere from shock devolatization of CaS04 in the Chicxulub lithographic section (6x10(exp 15)-2x10(exp 16)g in sulfur mass) is lower by a factor of 70 to 400 than previous estimates; the related environmental stress arising from the resultant global cooling of approximately 4 K and fallout of acid rain does not appear to suffice to explain the widespread K-T extinctions.

  4. Biotic and abiotic effects on CO2 sequestration during microbially-induced calcium carbonate precipitation.

    PubMed

    Okyay, Tugba Onal; Rodrigues, Debora F

    2015-03-01

    In this study, CO2 sequestration was investigated through the microbially-induced calcium carbonate precipitation (MICP) process with isolates obtained from a cave called 'Cave Without A Name' (Boerne, TX, USA) and the Pamukkale travertines (Denizli, Turkey). The majority of the bacterial isolates obtained from these habitats belonged to the genera Sporosarcina, Brevundimonas, Sphingobacterium and Acinetobacter. The isolates were investigated for their capability to precipitate calcium carbonate and sequester CO2. Biotic and abiotic effects of CO2 sequestration during MICP were also investigated. In the biotic effect, we observed that the rate and concentration of CO2 sequestered was dependent on the species or strains. The main abiotic factors affecting CO2 sequestration during MICP were the pH and medium components. The increase in pH led to enhanced CO2 sequestration by the growth medium. The growth medium components, on the other hand, were shown to affect both the urease activity and CO2 sequestration. Through the Plackett-Burman experimental design, the most important growth medium component involved in CO2 sequestration was determined to be urea. The optimized medium composition by the Plackett-Burman design for each isolate led to a statistically significant increase, of up to 148.9%, in CO2 uptake through calcification mechanisms. PMID:25764465

  5. Stereoselective inhibition of thromboxane-induced coronary vasoconstriction by 1,4-dihydropyridine calcium channel antagonists

    SciTech Connect

    Eltze, M.; Boer, R.; Sanders, K.H.; Boss, H.; Ulrich, W.R.; Flockerzi, D. )

    1990-01-01

    The biological activity of the (+)-S- and (-)-R-enantiomers of niguldipine, of the (-)-S- and (+)-R-enantiomers of felodipine and nitrendipine, and of rac-nisoldipine and rac-nimodipine was investigated in vitro and in vivo. Inhibition of coronary vasoconstriction due to the thromboxane A2 (TxA2)-mimetic U-46619 in guinea pig Langendorff hearts, displacement of (+)-({sup 3}H)isradipine from calcium channel binding sites of guinea pig skeletal muscle T-tubule membranes, and blood pressure reduction in spontaneously hypertensive rats were determined. The enantiomers were obtained by stereoselective synthesis. Cross-contamination was less than 0.5% for both S- and R-enantiomers of niguldipine and nitrendipine and less than 1% for those of felodipine. From the doses necessary for a 50% inhibition of coronary vasoconstriction, stereoselectivity ratios for (+)-(S)-/(-)-(R)-niguldipine, (-)-(S)-/(+)-(R)-felodipine, and (-)-(S)-/(+)-(R)-nitrendipine of 28, 13, and 7, respectively, were calculated. The potency ratio rac-nisoldipine/rac-nimodipine was 3.5. Ratios obtained from binding experiments and antihypertensive activity were (+)-(S)-/(-)-(R)-niguldipine = 45 and 35, (-)-(S)-/(+)-(R)-felodipine = 12 and 13, (-)-(S)-/(+)-(R)-nitrendipine = 8 and 8, and rac-nisoldipine/rac-nimodipine = 8 and 7, respectively. Highly significant correlations were found between the in vitro potency of the substances to prevent U-46619-induced coronary vasoconstriction and their affinity for calcium channel binding sites as well as their antihypertensive activity.

  6. High Sodium-Induced Oxidative Stress and Poor Anticrystallization Defense Aggravate Calcium Oxalate Crystal Formation in Rat Hyperoxaluric Kidneys

    PubMed Central

    Huang, Ho-Shiang; Ma, Ming-Chieh

    2015-01-01

    Enhanced sodium excretion is associated with intrarenal oxidative stress. The present study evaluated whether oxidative stress caused by high sodium (HS) may be involved in calcium oxalate crystal formation. Male rats were fed a sodium-depleted diet. Normal-sodium and HS diets were achieved by providing drinking water containing 0.3% and 3% NaCl, respectively. Rats were fed a sodium-depleted diet with 5% hydroxyl-L-proline (HP) for 7 and 42 days to induce hyperoxaluria and/or calcium oxalate deposition. Compared to normal sodium, HS slightly increased calcium excretion despite diuresis; however, the result did not reach statistical significance. HS did not affect the hyperoxaluria, hypocalciuria or supersaturation caused by HP; however, it increased calcium oxalate crystal deposition soon after 7 days of co-treatment. Massive calcium oxalate formation and calcium crystal excretion in HS+HP rats were seen after 42 days of treatment. HP-mediated hypocitraturia was further exacerbated by HS. Moreover, HS aggravated HP-induced renal injury and tubular damage via increased apoptosis and oxidative stress. Increased urinary malondialdehyde excretion, in situ superoxide production, NAD(P)H oxidase and xanthine oxidase expression and activity, and decreased antioxidant enzyme expression or activity in the HS+HP kidney indicated exaggerated oxidative stress. Interestingly, this redox imbalance was associated with reduced renal osteopontin and Tamm-Horsfall protein expression (via increased excretion) and sodium-dependent dicarboxylate cotransporter NaDC-1 upregulation. Collectively, our results demonstrate that a HS diet induces massive crystal formation in the hyperoxaluric kidney; this is not due to increased urinary calcium excretion but is related to oxidative injury and loss of anticrystallization defense. PMID:26241473

  7. High Sodium-Induced Oxidative Stress and Poor Anticrystallization Defense Aggravate Calcium Oxalate Crystal Formation in Rat Hyperoxaluric Kidneys.

    PubMed

    Huang, Ho-Shiang; Ma, Ming-Chieh

    2015-01-01

    Enhanced sodium excretion is associated with intrarenal oxidative stress. The present study evaluated whether oxidative stress caused by high sodium (HS) may be involved in calcium oxalate crystal formation. Male rats were fed a sodium-depleted diet. Normal-sodium and HS diets were achieved by providing drinking water containing 0.3% and 3% NaCl, respectively. Rats were fed a sodium-depleted diet with 5% hydroxyl-L-proline (HP) for 7 and 42 days to induce hyperoxaluria and/or calcium oxalate deposition. Compared to normal sodium, HS slightly increased calcium excretion despite diuresis; however, the result did not reach statistical significance. HS did not affect the hyperoxaluria, hypocalciuria or supersaturation caused by HP; however, it increased calcium oxalate crystal deposition soon after 7 days of co-treatment. Massive calcium oxalate formation and calcium crystal excretion in HS+HP rats were seen after 42 days of treatment. HP-mediated hypocitraturia was further exacerbated by HS. Moreover, HS aggravated HP-induced renal injury and tubular damage via increased apoptosis and oxidative stress. Increased urinary malondialdehyde excretion, in situ superoxide production, NAD(P)H oxidase and xanthine oxidase expression and activity, and decreased antioxidant enzyme expression or activity in the HS+HP kidney indicated exaggerated oxidative stress. Interestingly, this redox imbalance was associated with reduced renal osteopontin and Tamm-Horsfall protein expression (via increased excretion) and sodium-dependent dicarboxylate cotransporter NaDC-1 upregulation. Collectively, our results demonstrate that a HS diet induces massive crystal formation in the hyperoxaluric kidney; this is not due to increased urinary calcium excretion but is related to oxidative injury and loss of anticrystallization defense.

  8. Agonist-sensitive calcium pool in the pancreatic acinar cell. II. Characterization of reloading

    SciTech Connect

    Muallem, S.; Schoeffield, M.S.; Fimmel, C.J.; Pandol, S.J.

    1988-08-01

    45Ca2+ fluxes and free cytosolic Ca2+ measurements in guinea pig pancreatic acini indicated that after agonist stimulation and the release of Ca2+ from the agonist-sensitive pool at least part of the Ca2+ is extruded from the cell, resulting in 45Ca2+ efflux. In the continued presence of agonist, the pool remains permeable to Ca2+ but partially refills with Ca2+. This reloading is dependent on the concentration of extracellular Ca2+. In the absence of extracellular Ca2+, the pool is completely depleted of Ca2+. However, with increasing concentrations of CaCl2 in the incubation solution (from 0.5 to 2.0 mM) there is increasing repletion of the pool with Ca2+ during agonist stimulation. With termination of agonist stimulation, the Ca2+ permeability of the agonist-sensitive pool is rapidly reduced to that measured in the unstimulated cell. As a result, the Ca2+ incorporated into the pool during the stimulation period is rapidly trapped within the pool and exchanges poorly with medium Ca2+. Subsequently, the pool completely refills with Ca2+. The rate of Ca2+ reloading at the termination of agonist stimulation is slower than the conversion of the pool to the impermeable state. In incubation media containing 1.3 mM CaCl2, the half-time for reloading at the termination of stimulation is 5 min. These observations demonstrate the characteristics of Ca2+ reloading of the agonist-sensitive pool both during stimulation and at the termination of stimulation.

  9. Modulation of dihydropyridine-sensitive calcium channels in Drosophila by a cAMP-mediated pathway.

    PubMed

    Bhattacharya, A; Gu, G G; Singh, S

    1999-06-15

    Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduced the current and inhibited the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations. PMID:10380071

  10. Myofilament calcium sensitization delays decompensated hypertrophy differently between the sexes following myocardial infarction

    PubMed Central

    Shioura, Krystyna M.; Farjah, Mariam; Geenen, David L.; Solaro, R. John

    2011-01-01

    Contractile dysfunction is common to many forms of cardiovascular disease. Approaches directed at enhancing cardiac contractility at the level of the myofilaments during heart failure (HF) may provide a means to improve overall cardiovascular function. We are interested in gender-based differences in cardiac function and the effect of sarcomere activation agents that increase contractility. Thus, we studied the effect of gender and time on integrated arterial-ventricular function (A-V relationship) following myocardial infarction (MI). In addition, transgenic mice that overexpress the slow skeletal troponin I isoform were used to determine the impact of increased myofilament Ca2+ sensitivity following MI. Based on pressure-volume (P-V) loop measurements, we used derived parameters of cardiovascular function to reveal the effects of sex, time, and increased myofilament Ca2+ sensitivity among groups of post-MI mice. Analysis of the A-V relationship revealed that the initial increase was similar between the sexes, but the vascular unloading of the heart served to delay the decompensated stage in females. Conversely, the vascular response at 6 and 10 wk post-MI in males contributed to the continuous decline in cardiovascular function. Increasing the myofilament Ca2+ sensitivity appeared to provide sufficient contractile support to improve contractile function in both male and female transgenic mice. However, the improved contractile function was more beneficial in males as the concurrent vascular response contributed to a delayed decompensated stage in female transgenic mice post-MI. This study represents a quantitative approach to integrating the vascular-ventricular relationship to provide meaningful and diagnostic value following MI. Consequently, the data provide a basis for understanding how the A-V relationship is coupled between males and females and the enhanced ability of the cardiovascular system to tolerate pathophysiological stresses associated with HF in

  11. Inhibition of Store-Operated Calcium Entry Protects Endothelial Progenitor Cells from H2O2-Induced Apoptosis

    PubMed Central

    Wang, Yan-Wei; Zhang, Ji-Hang; Yu, Yang; Yu, Jie; Huang, Lan

    2016-01-01

    Store-operated calcium entry (SOCE), a major mode of extracellular calcium entry, plays roles in a variety of cell activities. Accumulating evidence indicates that the intracellular calcium ion concentration and calcium signaling are critical for the responses induced by oxidative stress. The present study was designed to investigate the potential effect of SOCE inhibition on H2O2-induced apoptosis in endothelial progenitor cells (EPCs), which are the predominant cells involved in endothelial repair. The results showed that H2O2-induced EPC apoptosis was reversed by SOCE inhibition induced either using the SOCE antagonist ML-9 or via silencing of stromal interaction molecule 1 (STIM1), a component of SOCE. Furthermore, SOCE inhibition repressed the increases in intracellular reactive oxygen species (ROS) levels and endoplasmic reticulum (ER) stress and ameliorated the mitochondrial dysfunction caused by H2O2. Our findings provide evidence that SOCE inhibition exerts a protective effect on EPCs in response to oxidative stress induced by H2O2 and may serve as a potential therapeutic strategy against vascular endothelial injury. PMID:27169819

  12. Calcium-dependent trichosanthin-induced generation of reactive oxygen species involved in apoptosis of human choriocarcinoma cells

    NASA Astrophysics Data System (ADS)

    Zhang, Chunyang; Ma, Hui; Chen, Die Yan

    2001-04-01

    The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumor and anti-HIV. We found for the first time that TCS induced production of reactive oxygen species (ROS) in JAR cells by using fluorescent probe 2',7'-dichlorofluorescin diacetate with confocal laser scanning microscopy. TCS-induced ROS showed dependence on the increase in intracellular calcium and on the presence of extracellular calcium. The production of ROS increased rapidly after the application of TCS, which paralleled TCS-indued increase in intracellular calcium monitored using fluo 3-AM, suggesting that TCS-induced ROS might mediate by the increase in intracellular Ca2PLU concentration. Simultaneous observation of the nuclear morphological changes and production of ROS in JAR cells with two-photon laser scanning microscopy and confocal laser scanning microscopy revealed that ROS involved in the apoptosis of JAR cells, which was confirmed by that antioxidant (alpha) -tocopherol prevented TCS-induced ROS formation and cell death. The finding that calcium-dependent TCS-induced ROS involved in the apoptosis of JAR cells might provide new insight into the anti-tumor and anti-HIV mechanism of TCS.

  13. Phosphorylation of a twitchin-related protein controls catch and calcium sensitivity of force production in invertebrate smooth muscle

    PubMed Central

    Siegman, Marion J.; Funabara, Daisuke; Kinoshita, Shigeharu; Watabe, Shugo; Hartshorne, David J.; Butler, Thomas M.

    1998-01-01

    “Catch” is a condition of prolonged, high-force maintenance at resting intracellular Ca2+ concentration ([Ca2+]) and very low energy usage, occurring in invertebrate smooth muscles, including the anterior byssus retractor muscle (ABRM) of Mytilus edulis. Relaxation from catch is rapid on serotonergic nerve stimulation in intact muscles and application of cAMP in permeabilized muscles. This release of catch occurs by protein kinase A-mediated phosphorylation of a high (≈600 kDa) molecular mass protein, the regulator of catch. Here, we identify the catch-regulating protein as a homologue of the mini-titin, twitchin, based on (i) a partial cDNA of the purified isolated protein showing 77% amino acid sequence identity to the kinase domain of Aplysia californica twitchin; (ii) a polyclonal antibody to a synthetic peptide in this sequence reacting with the phosphorylated catch-regulating protein band from permeabilized ABRM; and (iii) the similarity of the amino acid composition and molecular weight of the protein to twitchin. In permeabilized ABRM, at all but maximum [Ca2+], phosphorylation of twitchin results in a decreased calcium sensitivity of force production (half-maximum at 2.5 vs. 1.3 μM calcium). At a given submaximal force, with equal numbers of force generators, twitchin phosphorylation increased unloaded shortening velocity ≈2-fold. These data suggest that aspects of the catch state exist not only at resting [Ca2+], but also at higher submaximal [Ca2+]. The mechanism that gives rise to force maintenance in catch probably operates together, to some extent, with that of cycling myosin crossbridges. PMID:9560285

  14. Pressure-dependent contribution of Rho kinase-mediated calcium sensitization in serotonin-evoked vasoconstriction of rat cerebral arteries.

    PubMed

    El-Yazbi, Ahmed F; Johnson, Rosalyn P; Walsh, Emma J; Takeya, Kosuke; Walsh, Michael P; Cole, William C

    2010-05-15

    Our understanding of the cellular signalling mechanisms contributing to agonist-induced constriction is almost exclusively based on the study of conduit arteries. Resistance arteries/arterioles have received less attention as standard biochemical approaches lack the necessary sensitivity to permit quantification of phosphoprotein levels in these small vessels. Here, we have employed a novel, highly sensitive Western blotting method to assess: (1) the contribution of Ca(2+) sensitization mediated by phosphorylation of myosin light chain phosphatase targeting subunit 1 (MYPT1) and the 17 kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17) to serotonin (5-HT)-induced constriction of rat middle cerebral arteries, and (2) whether there is any interplay between pressure-induced myogenic and agonist-induced mechanisms of vasoconstriction. Arterial diameter and levels of MYPT1 (T697 and T855), CPI-17 and 20 kDa myosin light chain subunit (LC(20)) phosphorylation were determined following treatment with 5-HT (1 micromol l(1)) at 10 or 60 mmHg in the absence and presence of H1152 or GF109203X to suppress the activity of Rho-associated kinase (ROK) and protein kinase C (PKC), respectively. Although H1152 and GF109203X suppressed 5-HT-induced constriction and reduced phospho-LC(20) content at 10 mmHg, we failed to detect any increase in MYPT1 or CPI-17 phosphorylation. In contrast, an increase in MYPT1-T697 and MYPT1-T855 phosphorylation, but not phospho-CPI-17 content, was apparent at 60 mmHg following exposure to 5-HT, and the phosphorylation of both MYPT1 sites was sensitive to H1152 inhibition of ROK. The involvement of MYPT1 phosphorylation in the response to 5-HT at 60 mmHg was not dependent on force generation per se, as inhibition of cross-bridge cycling with blebbistatin (10 micromol l(1)) did not affect phosphoprotein content. Taken together, the data indicate that Ca(2+) sensitization owing to ROK-mediated phosphorylation of MYPT1 contributes to 5

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

    PubMed

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

    2005-12-01

    Prolactin, having been shown to stimulate transcellular active and solvent drag-induced calcium transport in the duodenum of female rats, was postulated to improve duodenal calcium transport in estrogen-deficient rats. The aim of the present study was, therefore, to demonstrate the effects of long-term prolactin exposure produced by anterior pituitary (AP) transplantation on the duodenal calcium transport in young (9-week-old) and adult (22-week-old) ovariectomized rats. We found that ovariectomy did not alter the transcellular active duodenal calcium transport in young and adult rats fed normal calcium diet (1.0% w/w Ca) but decreased the solvent drag-induced duodenal calcium transport from 75.50 +/- 10.12 to 55.75 +/- 4.77 nmol.hr(-1).cm(-2) (P < 0.05) only in adult rats. Long-term prolactin exposure stimulated the transcellular active calcium transport in young and adult AP-grafted ovariectomized rats fed with normal calcium diet by more than 2-fold from 7.56 +/- 0.79 to 16.54 +/- 2.05 (P < 0.001) and 9.78 +/- 0.72 to 15.99 +/- 1.75 (P < 0.001) nmol.hr(-1).cm(-2), respectively. However, only the solvent drag-induced duodenal calcium transport in young rats was enhanced by prolactin from 95.51 +/- 10.64 to 163.20 +/- 18.03 nmol.hr(-1).cm(-2) (P < 0.001) whereas that in adult rats still showed a decreased flux from 75.50 +/- 10.12 to 47.77 +/- 5.42 nmol.hr(-1).cm(-2) (P < 0.05). Because oral calcium supplement has been widely used to improve calcium balance in estrogen-deficient animals, the effect of a high-calcium diet (2.0% w/w Ca) was also investigated. The results showed that stimulatory action of long-term prolactin on the transcellular active duodenal calcium transport in both young and adult rats was diminished after being fed a high-calcium diet. The same diet also abolished prolactin-enhanced solvent drag-induced duodenal calcium transport in young and further decreased that in adult AP-grafted ovariectomized rats. We concluded that the solvent drag-induced

  16. The mechanism of injury-induced intracellular calcium concentration oscillations in the endothelium of excised rat aorta.

    PubMed

    Berra-Romani, Roberto; Raqeeb, Abdul; Torres-Jácome, Julián; Guzman-Silva, Alejandro; Guerra, Germano; Tanzi, Franco; Moccia, Francesco

    2012-01-01

    Endothelial injury is the primary event that leads to a variety of severe vascular disorders. Mechanical injury elicits a Ca(2+) response in the endothelium of excised rat aorta, which comprises an initial Ca(2+) release from inositol-1,4,5-trisphosphate (InsP(3))-sensitive stores followed by a long-lasting decay phase due to Ca(2+) entry through uncoupled connexons. The Ca(2+) signal may also adopt an oscillatory pattern, the molecular underpinnings of which are unclear. In the light of the role played by Ca(2+) spiking in tissue regeneration, this study aimed to unveil the mechanisms underlying injury-induced Ca(2+) oscillations. The latter reversibly ceased upon removal of extracellular Ca(2+) or addition of the gap junction blockers heptanol, 18 α,β-glycyrrhetinic acid, La(3+) and Ni(2+), but were insensitive to BTP-2 and SKF 96365. The spiking response was abolished by inhibiting the Ca(2+) entry mode of the Na(+)/Ca(2+) exchanger (NCX). The InsP(3)-producing agonist ATP resumed Ca(2+) oscillations in silent cells, while the phospholipase C inhibitor U73122 suppressed them. Injury-induced Ca(2+) transients were prevented by the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) blockers thapsigargin and cyclopiazonic acid, while they were unaffected by suramin and genistein. These data show for the first time that the coordinated interplay between NCX-mediated Ca(2+) entry and InsP(3)-dependent Ca(2+) release contributes to injury-induced intracellular Ca(2+) concentration oscillations.

  17. Extract of motorcycle exhaust particles induced macrophages apoptosis by calcium-dependent manner.

    PubMed

    Lee, Chen-Chen; Kang, Jaw-Jou

    2002-12-01

    Large survey and experiments have reported that environment pollutants from fossil fuel combustion would cause immune system deleterious by enhancement of allergic reaction and damage to respiratory tract. In this study, we reported that the extract of motorcycle exhaust particles (MEP) might affect the immune system by inducing cell apoptosis on macrophages. The motorcycle exhaust particles were collected from a two-stoke engine and their cytotoxic effect on macrophages was investigated. We found MEP is cytotoxic and induced apoptosis in RAW 264.7 cells, murine peritoneal macrophage, and rat alveolar macrophage. Pretreatment with mitochondria permeability transition inhibitor (cyclosporin A), intracellular (BAPTA-AM) and extracellular (EGTA) Ca(2+) chelator, and antioxidants (NAC, GSH, catalase, SOD) attenuated the MEP-induced cell apoptosis, and BAPTA-AM was the most effective one. Utilized Fura-2/AM loaded RAW 264.7 cells to directly detect the change of intracellular Ca(2+) concentration ([Ca(2+)](i)), we found that MEP could induce a sustained increase of [Ca(2+)](i). The raise of [Ca(2+)](i) induced by MEP could be completely blocked by the intracellular Ca(2+) chelator, BAPTA-AM, however, only partially inhibited by the extracellular Ca(2+) chelator, EGTA. These results suggested that both influx of extracellular Ca(2+) and release of Ca(2+) from the internal storage were involved. We also found that MEP caused a decrease of mitochondria membrane potential and an increase of oxidative stress in RAW 264.7 cells. In conclusion, we found that the particles, collected from the motorcycle exhaust, contain chemicals that will induce apoptosis of macrophage in calcium-dependent manner.

  18. Coupling of a transfected human brain A1 adenosine receptor in CHO-K1 cells to calcium mobilisation via a pertussis toxin-sensitive mechanism.

    PubMed Central

    Iredale, P. A.; Alexander, S. P.; Hill, S. J.

    1994-01-01

    1. The presence of A1 adenosine receptors in CHO-K1 cells transfected with the human brain A1 sequence was confirmed by ligand binding studies using 8-cyclopentyl-[3H] 1,3-dipropylxanthine ([3H]-DPCPX). 2. Alterations in intracellular calcium ([Ca2+]i) were measured with the calcium-sensitive dye, fura-2. 3. N6-cyclopentyladenosine (CPA), the selective A1 agonist, and 5'-N-ethylcarboxaminoadenosine (NECA), a relatively non-selective adenosine receptor agonist, elicited rapid, biphasic increases in [Ca2+]i which involved both mobilisation from intracellular stores and calcium entry. 4. The calcium response to CPA was significantly inhibited by the selective A1 antagonist DPCPX. The non-selective adenosine receptor, xanthine amino congener (XAC), was less potent. 5. The calcium response to CPA was completely prevented by pretreatment of the cells with pertussis toxin implicating the involvement of Gi in the receptor-mediated response. 6. In summary, we present evidence for the coupling of transfected human brain A1 adenosine receptors in CHO-K1 cells to mobilisation of [Ca2+]i via a pertussis toxin-sensitive G protein. PMID:8032613

  19. Intravital imaging reveals p53-dependent cancer cell death induced by phototherapy via calcium signaling

    PubMed Central

    Missiroli, Sonia; Poletti, Federica; Ramirez, Fabian Galindo; Morciano, Giampaolo; Morganti, Claudia; Pandolfi, Pier Paolo; Mammano, Fabio; Pinton, Paolo

    2015-01-01

    One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca2+). In the present study, we established conditions that allow the in vivo detection of Ca2+ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca2+ concentrations and, consequently, an increase in cell death in a p53-dependent pathway. PMID:25544762

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2016-02-01

    The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA - improvement of compressive strength of calcium phosphate scaffolds - is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts. PMID:26652403

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

    PubMed

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

    2016-07-01

    This study initially deals with the investigation of the induced strains during hardening stage of a self-setting calcium phosphate bone cement using fiber-Bragg grating (FBG) optical sensors. A complementary Scanning Electron Microscopy (SEM) investigation was also conducted at different time intervals of the hardening period and its findings were related to the FBG recordings. From the obtained results, it is demonstrated that the FBG response is affected by the microstructural changes taking place when the bone cement is immersed into the hardening liquid media. Subsequently, the FBG sensor was used to monitor the absorption process and hygroscopic response of the hardened and dried biocement when exposed to a liquid/humid environment. From the FBG-based calculated hygric strains as a function of moisture concentration, the coefficient of moisture expansion (CME) of the examined bone cement was obtained, exhibiting two distinct linear regions. PMID:26807773

  4. Intravital imaging reveals p53-dependent cancer cell death induced by phototherapy via calcium signaling.

    PubMed

    Giorgi, Carlotta; Bonora, Massimo; Missiroli, Sonia; Poletti, Federica; Ramirez, Fabian Galindo; Morciano, Giampaolo; Morganti, Claudia; Pandolfi, Pier Paolo; Mammano, Fabio; Pinton, Paolo

    2015-01-30

    One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca²⁺). In the present study, we established conditions that allow the in vivo detection of Ca²⁺ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca²⁺ concentrations and, consequently, an increase in cell death in a p53-dependent pathway.

  5. Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts

    SciTech Connect

    Zeng, Xiao R.; Sun Yubo; Wenger, Leonor; Cheung, Herman S. . E-mail: hcheung@med.miami.edu

    2005-05-13

    Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKC{alpha}-dependent p44/p42 MAPK pathway. Using a retrovirus gene expression system (Clontech) to overexpress Egr-1 in human fibroblast BJ-1 cells resulted in promotion of mitogenesis measured either by MTT cell proliferation analysis or by direct cell counting. The results demonstrate that Egr-1 may play a key role in mediating BCP crystal-induced synovial fibroblast mitogenesis.

  6. Calcium regulation by temperature-sensitive transient receptor potential channels in human uveal melanoma cells.

    PubMed

    Mergler, Stefan; Derckx, Raissa; Reinach, Peter S; Garreis, Fabian; Böhm, Arina; Schmelzer, Lisa; Skosyrski, Sergej; Ramesh, Niraja; Abdelmessih, Suzette; Polat, Onur Kerem; Khajavi, Noushafarin; Riechardt, Aline Isabel

    2014-01-01

    Uveal melanoma (UM) is both the most common and fatal intraocular cancer among adults worldwide. As with all types of neoplasia, changes in Ca(2+) channel regulation can contribute to the onset and progression of this pathological condition. Transient receptor potential channels (TRPs) and cannabinoid receptor type 1 (CB1) are two different types of Ca(2+) permeation pathways that can be dysregulated during neoplasia. We determined in malignant human UM and healthy uvea and four different UM cell lines whether there is gene and functional expression of TRP subtypes and CB1 since they could serve as drug targets to either prevent or inhibit initiation and progression of UM. RT-PCR, Ca(2+) transients, immunohistochemistry and planar patch-clamp analysis probed for their gene expression and functional activity, respectively. In UM cells, TRPV1 and TRPM8 gene expression was identified. Capsaicin (CAP), menthol or icilin induced Ca(2+) transients as well as changes in ion current behavior characteristic of TRPV1 and TRPM8 expression. Such effects were blocked with either La(3+), capsazepine (CPZ) or BCTC. TRPA1 and CB1 are highly expressed in human uvea, but TRPA1 is not expressed in all UM cell lines. In UM cells, the CB1 agonist, WIN 55,212-2, induced Ca(2+) transients, which were suppressed by La(3+) and CPZ whereas CAP-induced Ca(2+) transients could also be suppressed by CB1 activation. Identification of functional TRPV1, TRPM8, TRPA1 and CB1 expression in these tissues may provide novel drug targets for treatment of this aggressive neoplastic disease.

  7. On the effect of the injection of potassium phosphate in vivo inducing the precipitation of serum calcium with inorganic phosphate.

    PubMed

    Soares, Alcimar B; Ticianeli, José G; Soares, Letícia B M; Amaro, George

    2013-01-01

    High concentrations of inorganic phosphate (Pi) resulted from the hydrolysis of ATP is strongly associated to the weakness of the contractile mechanism of muscles due to its attractiveness to calcium. The majority of the experiments to study such effect are conducted in vitro. This work investigates the effects of different concentrations of Pi, induced by the injection of potassium phosphate in live animals, in the precipitation with serum calcium and the generation of calcium phosphate composites. The experiments were also designed to find out the ideal amount of potassium phosphate to induce an effective reaction. Potassium phosphate was injected in Wistar rats, randomly separated and distributed into seven groups. Group I was injected with 0.5 ml of saline solution (control) and groups II through VII were injected with 0.5, 1.5, 2.5, 5.0, 7.5 and 10.0 mg/kg of potassium phosphate, respectively. Blood collected from the inferior vena cava was submitted to biochemical analyses to measure the concentrations of calcium, Pi, urea and creatinine. The results showed that Pi, induced by the injection of potassium phosphate in live animals, causes precipitation with serum calcium, with statistically significant differences between the control and the treatment groups for doses up to 5.0 mg/kg. No statistically significant differences were found between the different doses and the concentration of urea and creatinine in the plasma. We conclude that potassium phosphate can be used to induce serum calcium precipitation in-vivo, with minor effects on other physiological variables, and the ideal dose to do so is 5.0 mg/kg.

  8. On the effect of the injection of potassium phosphate in vivo inducing the precipitation of serum calcium with inorganic phosphate

    PubMed Central

    Soares, Alcimar B; Ticianeli, José G; Soares, Letícia B M; Amaro, George

    2013-01-01

    High concentrations of inorganic phosphate (Pi) resulted from the hydrolysis of ATP is strongly associated to the weakness of the contractile mechanism of muscles due to its attractiveness to calcium. The majority of the experiments to study such effect are conducted in vitro. This work investigates the effects of different concentrations of Pi, induced by the injection of potassium phosphate in live animals, in the precipitation with serum calcium and the generation of calcium phosphate composites. The experiments were also designed to find out the ideal amount of potassium phosphate to induce an effective reaction. Potassium phosphate was injected in Wistar rats, randomly separated and distributed into seven groups. Group I was injected with 0.5 ml of saline solution (control) and groups II through VII were injected with 0.5, 1.5, 2.5, 5.0, 7.5 and 10.0 mg/kg of potassium phosphate, respectively. Blood collected from the inferior vena cava was submitted to biochemical analyses to measure the concentrations of calcium, Pi, urea and creatinine. The results showed that Pi, induced by the injection of potassium phosphate in live animals, causes precipitation with serum calcium, with statistically significant differences between the control and the treatment groups for doses up to 5.0 mg/kg. No statistically significant differences were found between the different doses and the concentration of urea and creatinine in the plasma. We conclude that potassium phosphate can be used to induce serum calcium precipitation in-vivo, with minor effects on other physiological variables, and the ideal dose to do so is 5.0 mg/kg. PMID:24379908

  9. 1H NMR and Rheological Studies of the Calcium Induced Gelation Process in Aqueous Low Methoxyl Pectin Solutions

    NASA Astrophysics Data System (ADS)

    Dobies, M.; Kuśmia, S.; Jurga, S.

    2006-07-01

    The 1H NMR relaxometry in combination with water proton spin-spin relaxation time measurements and rheometry have been applied to study the ionic gelation of 1% w/w aqueous low methoxyl pectin solution induced by divalent Ca2+ cations from a calcium chloride solution. The model-free approach to the analysis of 1H NMR relaxometry data has been used to separate the information on the static (β) and dynamic (<τ_c>) behaviour of the systems tested. The 1H NMR results confirm that the average mobility of both water and the pectin molecules is largely dependent on the concentration of the cross-linking agent. The character of this dependency (β,<τc> and T2 vs. CaCl2 concentration) is consistent with the two-stage gelation process of low methoxyl pectin, in which the formation of strongly linked dimer associations (in the range of 0-2.5 mM CaCl2) is followed by the appearance of weak inter-dimer aggregations (for CaCl2≥ 3.5 mM). The presence of the weak gel structure for the sample with 3.5 mM CaCl2 has been confirmed by rheological measurements. Apart from that, the T1 and T2 relaxation times have been found to be highly sensitive to the syneresis phenomenon, which can be useful to monitor the low methoxyl pectin gel network stability.

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

    PubMed Central

    Corral, María J.; Benito-Peña, Elena; Jiménez-Antón, M. Dolores; Cuevas, Laureano; Moreno-Bondi, María C.; Alunda, José M.

    2016-01-01

    Background Allicin has shown antileishmanial activity in vitro and in vivo. However the mechanism of action underlying its antiproliferative effect against Leishmania has been virtually unexplored. In this paper, we present the results obtained in L.infantum and a mechanistic basis is proposed. Methodology/Principal Finding Exposure of the parasites to allicin led to high Ca2+ levels and mitochondrial reactive oxygen species (ROS), collapse of the mitochondrial membrane potential, reduced production of ATP and elevation of cytosolic ROS. The incubation of the promastigotes with SYTOX Green revealed that decrease of ATP was not associated with plasma membrane permeabilization. Annexin V and propidium iodide (PI) staining indicated that allicin did not induce phospholipids exposure on the plasma membrane. Moreover, DNA agarose gel electrophoresis and TUNEL analysis demonstrated that allicin did not provoke DNA fragmentation. Analysis of the cell cycle with PI staining showed that allicin induced cell cycle arrest in the G2/M phase. Conclusions/Significance We conclude that allicin induces dysregulation of calcium homeostasis and oxidative stress, uncontrolled by the antioxidant defense of the cell, which leads to mitochondrial dysfunction and a bioenergetic catastrophe leading to cell necrosis and cell cycle arrest in the premitotic phase. PMID:27023069

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

  12. Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase-AIF pathway.

    PubMed

    U, Kin Pong; Subramanian, Venkataraman; Nicholas, Antony P; Thompson, Paul R; Ferretti, Patrizia

    2014-06-01

    PADs (peptidylarginine deiminases) are calcium-dependent enzymes that change protein-bound arginine to citrulline (citrullination/deimination) affecting protein conformation and function. PAD up-regulation following chick spinal cord injury has been linked to extensive tissue damage and loss of regenerative capability. Having found that human neural stem cells (hNSCs) expressed PAD2 and PAD3, we studied PAD function in these cells and investigated PAD3 as a potential target for neuroprotection by mimicking calcium-induced secondary injury responses. We show that PAD3, rather than PAD2 is a modulator of cell growth/death and that PAD activity is not associated with caspase-3-dependent cell death, but is required for AIF (apoptosis inducing factor)-mediated apoptosis. PAD inhibition prevents association of PAD3 with AIF and AIF cleavage required for its translocation to the nucleus. Finally, PAD inhibition also hinders calcium-induced cytoskeleton disassembly and association of PAD3 with vimentin, that we show to be associated also with AIF; together this suggests that PAD-dependent cytoskeleton disassembly may play a role in AIF translocation to the nucleus. This is the first study highlighting a role of PAD activity in balancing hNSC survival/death, identifying PAD3 as an important upstream regulator of calcium-induced apoptosis, which could be targeted to reduce neural loss, and shedding light on the mechanisms involved.

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

    SciTech Connect

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

    1989-02-28

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

  14. Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability

    PubMed Central

    Wang, Hsiu-Jung; Hsiao, Yu-Yuan; Chen, Yu-Pei; Ma, Tien-Yang

    2016-01-01

    Structural calcium sites control protein thermostability and activity by stabilizing native folds and changing local conformations. Alicyclobacillus acidocaldarius survives in thermal-acidic conditions and produces an endoglucanase Cel9A (AaCel9A) which contains a calcium-binding site (Ser465 to Val470) near the catalytic cleft. By superimposing the Ca2+-free and Ca2+-bounded conformations of the calcium site, we found that Ca2+ induces hydrophobic interactions between the calcium site and its nearby region by driving a conformational change. The hydrophobic interactions at the high-B-factor region could be enhanced further by replacing the surrounding polar residues with hydrophobic residues to affect enzyme thermostability and activity. Therefore, the calcium-binding residue Asp468 (whose side chain directly ligates Ca2+), Asp469, and Asp471 of AaCel9A were separately replaced by alanine and valine. Mutants D468A and D468V showed increased activity compared with those of the wild type with 0 mM or 10 mM Ca2+ added, whereas the Asp469 or Asp471 substitution resulted in decreased activity. The D468A crystal structure revealed that mutation D468A triggered a conformational change similar to that induced by Ca2+ in the wild type and developed a hydrophobic interaction network between the calcium site and the neighboring hydrophobic region (Ala113 to Ala117). Mutations D468V and D468A increased 4.5°C and 5.9°C, respectively, in melting temperature, and enzyme half-life at 75°C increased approximately 13 times. Structural comparisons between AaCel9A and other endoglucanases of the GH9 family suggested that the stability of the regions corresponding to the AaCel9A calcium site plays an important role in GH9 endoglucanase catalysis at high temperature. PMID:26729722

  15. Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability.

    PubMed

    Wang, Hsiu-Jung; Hsiao, Yu-Yuan; Chen, Yu-Pei; Ma, Tien-Yang; Tseng, Ching-Ping

    2016-03-01

    Structural calcium sites control protein thermostability and activity by stabilizing native folds and changing local conformations. Alicyclobacillus acidocaldarius survives in thermal-acidic conditions and produces an endoglucanase Cel9A (AaCel9A) which contains a calcium-binding site (Ser465 to Val470) near the catalytic cleft. By superimposing the Ca(2+)-free and Ca(2+)-bounded conformations of the calcium site, we found that Ca(2+) induces hydrophobic interactions between the calcium site and its nearby region by driving a conformational change. The hydrophobic interactions at the high-B-factor region could be enhanced further by replacing the surrounding polar residues with hydrophobic residues to affect enzyme thermostability and activity. Therefore, the calcium-binding residue Asp468 (whose side chain directly ligates Ca(2+)), Asp469, and Asp471 of AaCel9A were separately replaced by alanine and valine. Mutants D468A and D468V showed increased activity compared with those of the wild type with 0 mM or 10 mM Ca(2+) added, whereas the Asp469 or Asp471 substitution resulted in decreased activity. The D468A crystal structure revealed that mutation D468A triggered a conformational change similar to that induced by Ca(2+) in the wild type and developed a hydrophobic interaction network between the calcium site and the neighboring hydrophobic region (Ala113 to Ala117). Mutations D468V and D468A increased 4.5°C and 5.9°C, respectively, in melting temperature, and enzyme half-life at 75°C increased approximately 13 times. Structural comparisons between AaCel9A and other endoglucanases of the GH9 family suggested that the stability of the regions corresponding to the AaCel9A calcium site plays an important role in GH9 endoglucanase catalysis at high temperature. PMID:26729722

  16. Calcium phosphate nanoparticles are associated with inorganic phosphate-induced osteogenic differentiation of rat bone marrow stromal cells.

    PubMed

    Chen, Xiao-rong; Bai, Jing; Yuan, Shuai-jun; Yu, Cai-xia; Huang, Jian; Zhang, Tian-lan; Wang, Kui

    2015-08-01

    In the present study, we demonstrated that calcium phosphate (CaP) nanoparticles formed in cell culture media were implicated in the process of high inorganic phosphate (Pi) mediated osteogenic differentiation of rat bone marrow stromal cells (BMSCs). Exposure of BMSCs in vitro to high Pi-containing media reduced alkaline phosphatase (ALP) activity and the expressions of osteoblast-specific genes. The sediments of CaP nanoparticles were observed at the cell surface and some of them were concomitantly found inside cells at high Pi concentration. In addition, treatment the cells with pyrophosphate (PPi), an inhibitor of calcium crystal formation, abrogated the ALP activity induced by high Pi, suggesting the contribution of CaP nanoparticles. Moreover, for isolated CaP nanoparticles, there was a trend of conversion from amorphous calcium phosphate to hydroxyapatite with elevated Pi. The particle size of CaP increased and the surface morphology changed from spherical to irregular due to increased concentrations of serum proteins incorporated into CaP nanoparticles. The study demonstrated that those physicochemical properties of CaP nanoparticles played an important role in modulating BMSCs differentiation. Furthermore, the addition of Pi in the osteogenic media resulted in a dose-dependent increase in matrix mineralization, while treatment of the cells with PPi suppressed Pi-induced calcium deposition. The findings indicated that calcium deposition in the matrix partly came from the spontaneous precipitation of CaP nanoparticles. PMID:26111760

  17. Evidence for inositol triphosphate as a second messenger for glucose-induced calcium signalling in budding yeast.

    PubMed

    Tisi, Renata; Belotti, Fiorella; Wera, Stefaan; Winderickx, Joris; Thevelein, Johan M; Martegani, Enzo

    2004-02-01

    The Saccharomyces cerevisiae phospholipase C Plc1 is involved in cytosolic transient glucose-induced calcium increase, which also requires the Gpr1/Gpa2 receptor/G protein complex and glucose hexokinases. Differing from mammalian cells, this increase in cytosolic calcium concentration is mainly due to an influx from the external medium. No inositol triphosphate receptor homologue has been identified in the S. cerevisiae genome; and, therefore, the transduction mechanism from Plc1 activation to calcium flux generation still has to be identified. Inositol triphosphate (IP(3)) in yeast is rapidly transformed into IP(4) and IP(5) by a dual kinase, Arg82. Then another kinase, Ipk1, phosphorylates the IP(5) into IP(6). In mutant cells that do not express either of these kinases, the glucose-induced calcium signal was not only detectable but was even wider than in the wild-type strain. IP(3) accumulation upon glucose addition was completely absent in the plc1Delta strain and was amplified both by deletion of either ARG82 or IPK1 genes and by overexpression of PLC1. These results taken together suggest that Plc1p activation by glucose, leading to cleavage of PIP(2) and generation of IP(3), seems to be sufficient for raising the calcium level in the cytosol. This is the first indication for a physiological role of IP(3) signalling in S. cerevisiae. Many aspects about the signal transduction mechanism and the final effectors require further study.

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

    PubMed

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

    2013-12-15

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

  19. Portuguese Man-of-war (Physalia physalis) venom induces calcium influx into cells by permeabilizing plasma membranes.

    PubMed

    Edwards, L; Hessinger, D A

    2000-08-01

    Portuguese Man-of-war (Physalia physalis) nematocyst venom dose-dependently stimulates calcium (45Ca(2+)) influx into L-929, GH(4)C(1), FRL, and embryonic chick heart cells. Venom-induced calcium influx is not blocked by ouabain, vanadate, nor organic calcium channel blockers, but is blocked by transition metal cations, such as lanthanum and zinc. Venom-induced calcium influx is accompanied in a dose-dependent manner by the release of intracellular lactate dehydrogenase, indicating a loss in plasma membrane integrity and cytolysis. Concentrations of zinc that block 45Ca(2+) influx also block lactate dehydrogenase release. Lanthanum, which also blocks 45Ca(2+) uptake, does not neutralize the cytolytic activity of the venom, but rather inhibits the venom's cytolytic action at the level of the target cell plasma membrane. Our findings indicate that Man-of-war venom causes an influx of calcium into several different cells types, not just those of the cardiovascular system, and this influx likely occurs by permeabilizing the plasma membranes of cells. PMID:10708794

  20. Calcium phosphate nanoparticles are associated with inorganic phosphate-induced osteogenic differentiation of rat bone marrow stromal cells.

    PubMed

    Chen, Xiao-rong; Bai, Jing; Yuan, Shuai-jun; Yu, Cai-xia; Huang, Jian; Zhang, Tian-lan; Wang, Kui

    2015-08-01

    In the present study, we demonstrated that calcium phosphate (CaP) nanoparticles formed in cell culture media were implicated in the process of high inorganic phosphate (Pi) mediated osteogenic differentiation of rat bone marrow stromal cells (BMSCs). Exposure of BMSCs in vitro to high Pi-containing media reduced alkaline phosphatase (ALP) activity and the expressions of osteoblast-specific genes. The sediments of CaP nanoparticles were observed at the cell surface and some of them were concomitantly found inside cells at high Pi concentration. In addition, treatment the cells with pyrophosphate (PPi), an inhibitor of calcium crystal formation, abrogated the ALP activity induced by high Pi, suggesting the contribution of CaP nanoparticles. Moreover, for isolated CaP nanoparticles, there was a trend of conversion from amorphous calcium phosphate to hydroxyapatite with elevated Pi. The particle size of CaP increased and the surface morphology changed from spherical to irregular due to increased concentrations of serum proteins incorporated into CaP nanoparticles. The study demonstrated that those physicochemical properties of CaP nanoparticles played an important role in modulating BMSCs differentiation. Furthermore, the addition of Pi in the osteogenic media resulted in a dose-dependent increase in matrix mineralization, while treatment of the cells with PPi suppressed Pi-induced calcium deposition. The findings indicated that calcium deposition in the matrix partly came from the spontaneous precipitation of CaP nanoparticles.

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

    PubMed

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

    2016-07-01

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

  2. The calcium chloride-induced rodent model of abdominal aortic aneurysm.

    PubMed

    Wang, Yutang; Krishna, Smriti; Golledge, Jonathan

    2013-01-01

    Abdominal aortic aneurysm (AAA) affects ∼5% men aged over 65 years and is an important cause of death in this population. Research into AAA pathogenesis has been fuelled by the need to identify new diagnostic biomarkers and therapeutic targets for this disease. One animal model of AAA involves peri-vascular application of calcium chloride (CaCl(2)) onto the infra-renal aorta of mice and rats to induce extracellular matrix remodelling. Twenty-three studies assessing CaCl(2)-induced AAA and six studies assessing AAA induced by a modified CaCl(2) method were identified. In the current report the preparation and pathological features of this AAA model are discussed. We also compared this animal model to human AAA. CaCl(2)-induced AAA shows the following pathological characteristics typically found in human AAA: calcification, inflammatory cell infiltration, oxidative stress, neovascularisation, elastin degradation and vascular smooth muscle cell apoptosis. A number of mechanisms involved in CaCl(2)-induced AAA have been identified which may be relevant to the pathogenesis of human AAA. Key molecules include c-Jun N-terminal kinase, peroxisome proliferator-activated receptor-γ, chemokine (C-C motif) receptor 2, group x secretory phospholipase A2 and plasminogen. CaCl(2)-induced AAA does not display aortic thrombus, atherosclerosis and rupture which are classical features of human AAA. Advantages of the CaCl(2)-induced AAA technique include (1) it can be applied to wild type mice making assessment of transgenic rodent models more straight forward and rapid; and (2) CaCl(2)-induced AAAs are usually developed in the infra-renal abdominal aorta, which is the most common location of human AAA. Currently findings obtained from the CaCl(2)-induced AAA model or other animal models of AAA have not been translated into the human situation. It is hoped that this deficiency will be corrected over the next decade with a number of clinical trials currently examining novel

  3. Molecular cloning of the alpha-1 subunit of an omega-conotoxin-sensitive calcium channel.

    PubMed Central

    Dubel, S J; Starr, T V; Hell, J; Ahlijanian, M K; Enyeart, J J; Catterall, W A; Snutch, T P

    1992-01-01

    Of the four major types of Ca channel described in vertebrate cells (designated T, L, N, and P), N-type Ca channels are unique in that they are found specifically in neurons, have been correlated with control of neurotransmitter release, and are blocked by omega-conotoxin, a neuropeptide toxin isolated from the marine snail Conus geographus. A set of overlapping cDNA clones were isolated and found to encode a Ca channel alpha-1 subunit, designated rbB-I. Polyclonal antiserum generated against a peptide from the rbB-I sequence selectively immunoprecipitates high-affinity 125I-labeled omega-conotoxin-binding sites from labeled rat forebrain membranes. PCR analysis shows that, like N-type Ca channels, expression of rbB-I is limited to the nervous system and neuronally derived cell lines. This brain Ca channel may mediate the omega-conotoxin-sensitive Ca influx required for neurotransmitter release at many synapses. Images PMID:1317580

  4. Nitric oxide lowers the calcium sensitivity of tension in the rat tail artery

    PubMed Central

    Tran, Nguyen N P; Spitzbarth, Esther; Robert, Alain; Giummelly, Philippe; Atkinson, Jeffrey; Capdeville-Atkinson, Christine

    1998-01-01

    Controversy exists as to whether a fall in the intracellular Ca2+ concentration ([Ca2+]i) is a requisite element of the vasodilatory response to nitric oxide (NO). We studied the effect of NO on the coupling between [Ca2+]i and vasoconstriction in arterial segments loaded with the [Ca2+]i-sensitive, intracellular dye fura-2. As data interpretation is equivocal when fura-2 is loaded into both endothelial and smooth muscle cells, we compared results from in vitro experiments on segments of the rat tail artery in which fura-2 and noradrenaline were applied on the luminal or adventitial side, and endothelium was removed ‘physically’ (rubbing or air) or ‘functionally’ (Nω-nitro-l-arginine methyl ester). The use of air perfusion to remove endothelium is of considerable benefit since it allows paired observations in a single tissue. Fura-2 loaded into endothelial cells but endothelial ‘contamination’ of the smooth muscle cell [Ca2+]i signal was minimal. Endogenous NO decreased vasoconstrictor responses to noradrenaline but had no effect on [Ca2+]i. Nitroglycerine decreased vasoconstrictor responses in a concentration-dependent fashion but had no effect on [Ca2+]i. In conclusion, NO causes vasodilatation via a mechanism which is downstream of [Ca2+]i mobilization. PMID:9490833

  5. Biphasic modulation by mGlu5 receptors of TRPV1-mediated intracellular calcium elevation in sensory neurons contributes to heat sensitivity

    PubMed Central

    Masuoka, T; Nakamura, T; Kudo, M; Yoshida, J; Takaoka, Y; Kato, N; Ishibashi, T; Imaizumi, N; Nishio, M

    2015-01-01

    Background and Purpose Elevation of glutamate, an excitatory amino acid, during inflammation and injury plays a crucial role in the reception and transmission of sensory information via ionotropic and metabotropic receptors. This study aimed to investigate the mechanisms underlying the biphasic effects of metabotropic glutamate mGlu5 receptor activation on responses to noxious heat. Experimental Approach We assessed the effects of intraplantar quisqualate, a non-selective glutamate receptor agonist, on heat and mechanical pain behaviours in mice. In addition, the effects of quisqualate on the intracellular calcium response and on membrane currents mediated by TRPV1 channels, were examined in cultured dorsal root ganglion neurons from mice. Key Results Activation of mGlu5 receptors in hind paw transiently increased, then decreased, the response to noxious heat. In sensory neurons, activation of mGlu5 receptors potentiated TRPV1-mediated intracellular calcium elevation, while terminating activation of mGlu5 receptors depressed it. TRPV1-induced currents were potentiated by activation of mGlu5 receptors under voltage clamp conditions and these disappeared after washout. However, voltage-gated calcium currents were inhibited by the mGlu5 receptor agonist, even after washout. Conclusions and Implications These results suggest that, in sensory neurons, mGlu5 receptors biphasically modulate TRPV1-mediated intracellular calcium response via transient potentiation of TRPV1 channel-induced currents and persistent inhibition of voltage-gated calcium currents, contributing to heat hyper- and hypoalgesia. PMID:25297838

  6. Agonist-sensitive calcium pool in the pancreatic acinar cell. I. Permeability properties

    SciTech Connect

    Muallem, S.; Schoeffield, M.S.; Fimmel, C.J.; Pandol, S.J.

    1988-08-01

    45Ca2+ fluxes and free cytosolic Ca2+ (( Ca2+)i) were used to describe the Ca2+ permeability and Ca2+ reloading of the agonist-sensitive pool at rest, during stimulation, and at termination of stimulation. A sequence of stimulation with carbachol, inhibition with atropine (cycling), and restimulation with cholecystokinin octapeptide (CCK-8) was used to follow Ca2+ reloading. Reloading of the pool required extracellular Ca2+ and was measured as an increased rate and extent of 45Ca2+ uptake into the acini. The 45Ca2+ incorporated into cycled acini could be completely released with CCK-8. The dose-response curves for 45Ca uptake and release were identical to those of the hormonally evoked (Ca2+)i increase. The increased 45Ca2+ uptake during reloading was not due to an expansion of any intracellular pool size but reflects the labeling of the pool to isotopic equilibrium in cycled acini. The rate constant of Ca2+ efflux from the pool of resting cells was approximately 0.67 +/- 0.01/h. With stimulation, the Ca2+ permeability of the pool membrane rapidly increased, resulting in Ca2+ release into the cytosol and an increase in (Ca2+)i. With termination of stimulation, the Ca2+ permeability of the pool membrane rapidly decreased while the pool continued to reload with extracellular Ca2+. Labeling of the pool to isotopic equilibrium allowed determination of the amount of Ca2+ released from the pool, which was 2.94 +/- 0.06 nmol/mg protein. This indicates that total Ca2+ concentration in the pool is in the millimolar range.

  7. Targeting SLUG sensitizes leukemia cells to ADR-induced apoptosis

    PubMed Central

    Wei, Chang-Rong; Liu, Jun; Yu, Xiao-Jun

    2015-01-01

    Background and Aims: Slug is an E-cadherin repressor and a suppressor of PUMA (p53 upregulated modulator of apoptosis) and it has recently been demonstrated that Slug plays an important role in controlling apoptosis. In this study, we examined whether Slug’s ability to silence expression suppresses the growth of leukemia HL-60 cells and/or sensitizes leukemia HL-60 cells to adriamycin (ADR) through induction of apoptosis. Methods: SLUG siRNA was transfected into the HL-60 and HL-60ADR cell lines (an adriamycin resistant cell line). The stably SLUG siRNA transfected HL-60 and HL-60ADR cells was transiently transfected with PUMA siRNA. The mRNA and protein expression of SLUG and PUMA were determined by Quantitative real-time RT-PCR and Western blot assay. The effects of SLUG siRNA alone or combined with ADR or PUMA siRNA on growth and apoptosis in HL-60 and HL-60ADR cells was detected by MTT, ELISA and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Results: The results showed that SLUG was less expressed in the HL-60 cells, and high expressed in the HL-60ADR cells. Obvious down-regulation of SLUG mRNA and protein levels and up-regulation of PUMA mRNA and protein levels after SLUG siRNA transfection was showed in the HL-60ADR cells. Treatment with ADR induced SLUG mRNA and protein in the HL-60 cells. Significant positive correlation was observed between basal SLUG mRNA and protein and ADR sensitivity. SLUG gene silencing by SLUG siRNA transfection inhibited growth and induced apoptosis, and increased ADR killing of the HL-60 and HL-60ADR cell lines. After the SLUG siRNA transfected HL-60 and HL-60ADR cells was transiently transfected with PUMA siRNA, did not increase ADR killing of the HL-60 and HL-60ADR cell lines. Conclusion: SLUG level positively correlated with sensitivity to ADR. SLUG siRNA could effectively reduce SLUG expression and induce PUMA expression and restore the drug sensitivity of resistant leukemic cells to

  8. Prostaglandin F2 alpha-induced calcium transient in ovine large luteal cells: II. Modulation of the transient and resting cytosolic free calcium alters progesterone secretion.

    PubMed

    Wegner, J A; Martinez-Zaguilan, R; Gillies, R J; Hoyer, P B

    1991-02-01

    A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in

  9. The CYP3A4 inhibitor intraconazole does not affect the pharmacokinetics of a new calcium-sensitizing drug levosimendan.

    PubMed

    Antila, S; Honkanen, T; Lehtonen, L; Neuvonen, P J

    1998-08-01

    Itraconazole is a potent inhibitor of CYP3A4 isoenzyme and it can cause clinically significant interactions with some other drugs. Levosimendan is a new calcium-sensitizing drug intended for congestive heart failure. We aimed to study possible interactions of itraconazole with levosimendan in healthy volunteers. Twelve healthy male volunteers were included into a randomized, double-blind, two-phase crossover study. A wash-out period of 4 weeks was held between the phases. The subjects were given orally itraconazole 200 mg or placebo daily for 5 days. On the fifth day, they received a single oral dose of 2 mg of levosimendan. Levosimendan plasma concentrations were determined up to 12 hours and ECG, heart rate, and blood pressure followed-up to 8 hours after intake of levosimendan. Itraconazole had no significant effects on the pharmacokinetic parameters of levosimendan. Neither were there any differences in heart rate, PQ-, QTc- or QRS intervals between the placebo and itraconazole phases. The systolic blood pressure was decreased slightly more (p < 0.05) during the itraconazole phase than during the placebo phase. In conclusion, because the potent CYP3A4 inhibitor itraconazole had no significant pharmacokinetic interaction with levosimendan, interactions with CYP3A4 inhibitor, and oral levosimendan are unlikely.

  10. The calcium pump plasma membrane Ca2+-ATPase 2 (PMCA2) regulates breast cancer cell proliferation and sensitivity to doxorubicin

    PubMed Central

    Peters, Amelia A.; Milevskiy, Michael J. G.; Lee, Wei C.; Curry, Merril C.; Smart, Chanel E.; Saunus, Jodi M.; Reid, Lynne; da Silva, Leonard; Marcial, Daneth L.; Dray, Eloise; Brown, Melissa A.; Lakhani, Sunil R.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.

    2016-01-01

    Regulation of Ca2+ transport is vital in physiological processes, including lactation, proliferation and apoptosis. The plasmalemmal Ca2+ pump isoform 2 (PMCA2) a calcium ion efflux pump, was the first protein identified to be crucial in the transport of Ca2+ ions into milk during lactation in mice. In these studies we show that PMCA2 is also expressed in human epithelia undergoing lactational remodeling and also report strong PMCA2 staining on apical membranes of luminal epithelia in approximately 9% of human breast cancers we assessed. Membrane protein expression was not significantly associated with grade or hormone receptor status. However, PMCA2 mRNA levels were enriched in Basal breast cancers where it was positively correlated with survival. Silencing of PMCA2 reduced MDA-MB-231 breast cancer cell proliferation, whereas silencing of the related isoforms PMCA1 and PMCA4 had no effect. PMCA2 silencing also sensitized MDA-MB-231 cells to the cytotoxic agent doxorubicin. Targeting PMCA2 alone or in combination with cytotoxic therapy may be worthy of investigation as a therapeutic strategy in breast cancer. PMCA2 mRNA levels are also a potential tool in identifying poor responders to therapy in women with Basal breast cancer. PMID:27148852

  11. Involvements of calcium channel and potassium channel in Danshen and Gegen decoction induced vasodilation in porcine coronary LAD artery.

    PubMed

    Hu, Fan; Koon, Chi Man; Chan, Judy Yuet Wa; Lau, Kit Man; Kwan, Y W; Fung, Kwok Pui

    2012-09-15

    Danshen (Salviae Miltiorrhizae Radix) and Gegen (Puerariae Lobatae Radix) have been widely used in treating cardiovascular diseases for thousands of years in China. The present study was carried out to evaluate the effects of a Danshen and Gegen decoction (DG) on the vascular reactivity of a porcine isolated coronary artery and the underlying mechanisms involved. Porcine coronary rings were precontracted with 15 nM U46619. The involvement of endothelium-dependent mechanisms was explored by removing the endothelium; the involvement of potassium channels was investigated by the pretreatment of the artery rings with various blockers, and the involvement of the calcium channels was investigated by incubating the artery rings with Ca²⁺-free buffer and priming them with high [K⁺] prior to adding CaCl₂ to elicit contraction. The involvement of Ca²⁺ sensitization was explored by evaluating the Rho-activity expression. The results revealed that DG elicited a concentration-dependent relaxation on a U46619-precontracted coronary artery ring. These relaxation responses were not altered by the pretreatment of inhibitors of endothelium-related dilator synthases, cGMP and cAMP pathway inhibitors, potassium channel (BK(Ca), SK(Ca), K(V) and K(ATP)) blockers and endothelium removal. The K(IR) channel blocker BaCl₂ only slightly attenuated the DG-induced relaxation. However, the Ca²⁺-induced artery contraction was inhibited by DG. Additionally, the expression of the phosphorylated myosin light chain was inhibited by DG whereas the activity of RhoA was not affected. Therefore, DG could be a useful cardioprotective agent for vasodilation in patients who have hypertension.

  12. Ammonium-induced calcium mobilization in 1321N1 astrocytoma cells

    SciTech Connect

    Hillmann, Petra; Koese, Meryem; Soehl, Kristina; Mueller, Christa E.

    2008-02-15

    High blood levels of ammonium/ammonia (NH{sub 4}{sup +}/NH{sub 3}) are associated with severe neurotoxicity as observed in hepatic encephalopathy (HE). Astrocytes are the main targets of ammonium toxicity, while neuronal cells are less vulnerable. In the present study, an astrocytoma cell line 1321N1 and a neuroblastoma glioma hybrid cell line NG108-15 were used as model systems for astrocytes and neuronal cells, respectively. Ammonium salts evoked a transient increase in intracellular calcium concentrations ([Ca{sup 2+}]{sub i}) in astrocytoma (EC{sub 50} = 6.38 mM), but not in NG108-15 cells. The ammonium-induced increase in [Ca{sup 2+}]{sub i} was due to an intracellular effect of NH{sub 4}{sup +}/NH{sub 3} and was independent of extracellular calcium. Acetate completely inhibited the ammonium effect. Ammonium potently reduced calcium signaling by G{sub q} protein-coupled receptors (H{sub 1} and M3) expressed on the cells. Ammonium (5 mM) also significantly inhibited the proliferation of 1321N1 astrocytoma cells. While mRNA for the mammalian ammonium transporters RhBG and RhCG could not be detected in 1321N1 astrocytoma cells, both transporters were expressed in NG108-15 cells. RhBG and RhBC in brain may promote the excretion of NH{sub 3}/NH{sub 4}{sup +} from neuronal cells. Cellular uptake of NH{sub 4}{sup +}/NH{sub 3} was mainly by passive diffusion of NH{sub 3}. Human 1321N1 astrocytoma cells appear to be an excellent, easily accessible human model for studying HE, which can substitute animal studies, while NG108-15 cells may be useful for investigating the role of the recently discovered Rhesus family type ammonium transporters in neuronal cells. Our findings may contribute to the understanding of pathologic ammonium effects in different brain cells, and to the treatment of hyperammonemia.

  13. (−)-Epicatechin induces calcium and translocation independent eNOS activation in arterial endothelial cells

    PubMed Central

    Ramirez-Sanchez, Israel; Maya, Lisandro; Ceballos, Guillermo

    2011-01-01

    The consumption of cacao-derived (i.e., cocoa) products provides beneficial cardiovascular effects in healthy subjects as well as individuals with endothelial dysfunction such as smokers, diabetics, and postmenopausal women. The vascular actions of cocoa are related to enhanced nitric oxide (NO) production. These actions can be reproduced by the administration of the cacao flavanol (−)-epicatechin (EPI). To further understand the mechanisms behind the vascular action of EPI, we investigated the effects of Ca2+ depletion on endothelial nitric oxide (NO) synthase (eNOS) activation/phosphorylation and translocation. Human coronary artery endothelial cells were treated with EPI or with bradykinin (BK), a well-known Ca2+-dependent eNOS activator. Results demonstrate that both EPI and BK induce increases in intracellular calcium and NO levels. However, under Ca2+-free conditions, EPI (but not BK) is still capable of inducing NO production through eNOS phosphorylation at serine 615, 633, and 1177. Interestingly, EPI-induced translocation of eNOS from the plasmalemma was abolished upon Ca2+ depletion. Thus, under Ca2+-free conditions, EPI can stimulate NO synthesis independent of calmodulin binding to eNOS and of its translocation into the cytoplasm. We also examined the effect of EPI on the NO/cGMP/vasodilator-stimulated phosphoprotein (VASP) pathway activation in isolated Ca2+-deprived canine mesenteric arteries. Results demonstrate that under these conditions, EPI induces the activation of this vasorelaxation-related pathway and that this effect is inhibited by pretreatment with nitro-l-arginine methyl ester, suggesting a functional relevance for this phenomenon. PMID:21209365

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

    SciTech Connect

    Yoneda, Akihiro; Watanabe, Tomomasa

    2015-05-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  16. Simultaneous formation of calcium oxalate (mono-, di-, and trihydrate) induced by potassium tartrate in gelatinous system

    NASA Astrophysics Data System (ADS)

    Ouyang, Jian-Ming; Zheng, Hui; Deng, Sui-Ping

    2006-07-01

    Calcium oxalate (CaOx) is the major crystalline component of kidney stone. Calcium oxalate mono-(COM), di-(COD), and trihydrate (COT) crystals were simultaneously induced by potassium tartrate (K 2tart) in a gelatinous system by a double diffusion technique. The phase composition and morphology of the crystals were determined by scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy. In the presence of low concentration of K 2tart (0.01 mol/l), the COM crystals with sharp tips are the dominant crystal phase. The percentage of COD is less than 5% and no COT crystals are observed. When the concentration of K 2tart is increased to 0.10 and 0.50 mol/l, the percentage of COT increases to 56% and 82%, respectively. The morphology of COM crystals becomes blunter. The crystallization temperature affects the phase composition and morphology of CaOx crystals. In the presence of 0.01 mol/l K 2tart, COM and COT are the main phases at 37 °C. However, COD is dominant at 7 °C. The result that K 2tart can increase the content of COT and inhibit the aggregation and decrease the surface area of COM crystals indicates K 2tart can inhibit the formation of CaOx stones. This result also supports the clinical use of K 2tart and may be helpful in elucidating the mechanisms by which the formation of CaOx calculi can be inhibited.

  17. Regression of Glomerular and Tubulointerstitial Injuries by Dietary Salt Reduction with Combination Therapy of Angiotensin II Receptor Blocker and Calcium Channel Blocker in Dahl Salt-Sensitive Rats

    PubMed Central

    Rafiq, Kazi; Nishiyama, Akira; Konishi, Yoshio; Morikawa, Takashi; Kitabayashi, Chizuko; Kohno, Masakazu; Masaki, Tsutomu; Mori, Hirohito; Kobori, Hiroyuki; Imanishi, Masahito

    2014-01-01

    A growing body of evidence indicates that renal tissue injuries are reversible. We investigated whether dietary salt reduction with the combination therapy of angiotensin II type 1 receptor blocker (ARB) plus calcium channel blocker (CCB) reverses renal tissue injury in Dahl salt-sensitive (DSS) hypertensive rats. DSS rats were fed a high-salt diet (HS; 4% NaCl) for 4 weeks. Then, DSS rats were given one of the following for 10 weeks: HS diet; normal-salt diet (NS; 0.5% NaCl), NS + an ARB (olmesartan, 10 mg/kg/day), NS + a CCB (azelnidipine, 3 mg/kg/day), NS + olmesartan + azelnidipine or NS + hydralazine (50 mg/kg/day). Four weeks of treatment with HS diet induced hypertension, proteinuria, glomerular sclerosis and hypertrophy, glomerular podocyte injury, and tubulointerstitial fibrosis in DSS rats. A continued HS diet progressed hypertension, proteinuria and renal tissue injury, which was associated with inflammatory cell infiltration and increased proinflammatory cytokine mRNA levels, NADPH oxidase activity and NADPH oxidase-dependent superoxide production in the kidney. In contrast, switching to NS halted the progression of hypertension, renal glomerular and tubular injuries. Dietary salt reduction with ARB or with CCB treatment further reduced blood pressure and partially reversed renal tissues injury. Furthermore, dietary salt reduction with the combination of ARB plus CCB elicited a strong recovery from HS-induced renal tissue injury including the attenuation of inflammation and oxidative stress. These data support the hypothesis that dietary salt reduction with combination therapy of an ARB plus CCB restores glomerular and tubulointerstitial injury in DSS rats. PMID:25233358

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

    PubMed

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

    2012-04-01

    An increased intestinal permeability is associated with several diseases. Previously, we have shown that dietary Ca decreases colonic permeability in rats. This might be explained by a calcium-phosphate-induced increase in luminal buffering capacity, which protects against an acidic pH due to microbial fermentation. Therefore, we investigated whether dietary phosphate is a co-player in the effect of Ca on permeability. Rats were fed a humanised low-Ca diet, or a similar diet supplemented with Ca and containing either high, medium or low phosphate concentrations. Chromium-EDTA was added as an inert dietary intestinal permeability marker. After dietary adaptation, short-chain fructo-oligosaccharides (scFOS) were added to all diets to stimulate fermentation, acidify the colonic contents and induce an increase in permeability. Dietary Ca prevented the scFOS-induced increase in intestinal permeability in rats fed medium- and high-phosphate diets but not in those fed the low-phosphate diet. This was associated with higher faecal water cytotoxicity and higher caecal lactate levels in the latter group. Moreover, food intake and body weight during scFOS supplementation were adversely affected by the low-phosphate diet. Importantly, luminal buffering capacity was higher in rats fed the medium- and high-phosphate diets compared with those fed the low-phosphate diet. The protective effect of dietary Ca on intestinal permeability is impaired if dietary phosphate is low. This is associated with a calcium phosphate-induced increase in luminal buffering capacity. Dragging phosphate into the colon and thereby increasing the colonic phosphate concentration is at least part of the mechanism behind the protective effect of Ca on intestinal permeability. PMID:21851756

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

    NASA Astrophysics Data System (ADS)

    Kaufman, I.; Luchinsky, D. G.; Tindjong, R.; McClintock, P. V. E.; Eisenberg, R. S.

    2013-11-01

    We use Brownian dynamics (BD) simulations to study the ionic conduction and valence selectivity of a generic electrostatic model of a biological ion channel as functions of the fixed charge Qf at its selectivity filter. We are thus able to reconcile the discrete calcium conduction bands recently revealed in our BD simulations, M0 (Qf=1e), M1 (3e), M2 (5e), with a set of sodium conduction bands L0 (0.5e), L1 (1.5e), thereby obtaining a completed pattern of conduction and selectivity bands vs Qf for the sodium-calcium channels family. An increase of Qf leads to an increase of calcium selectivity: L0 (sodium-selective, nonblocking channel) → M0 (nonselective channel) → L1 (sodium-selective channel with divalent block) → M1 (calcium-selective channel exhibiting the anomalous mole fraction effect). We create a consistent identification scheme where the L0 band is putatively identified with the eukaryotic sodium channel The scheme created is able to account for the experimentally observed mutation-induced transformations between nonselective channels, sodium-selective channels, and calcium-selective channels, which we interpret as transitions between different rows of the identification table. By considering the potential energy changes during permeation, we show explicitly that the multi-ion conduction bands of calcium and sodium channels arise as the result of resonant barrierless conduction. The pattern of periodic conduction bands is explained on the basis of sequential neutralization taking account of self-energy, as Qf(z,i)=ze(1/2+i), where i is the order of the band and z is the valence of the ion. Our results confirm the crucial influence of electrostatic interactions on conduction and on the Ca2+/Na+ valence selectivity of calcium and sodium ion channels. The model and results could be also applicable to biomimetic nanopores with charged walls.

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

    PubMed

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

    2013-11-01

    We use Brownian dynamics (BD) simulations to study the ionic conduction and valence selectivity of a generic electrostatic model of a biological ion channel as functions of the fixed charge Q(f) at its selectivity filter. We are thus able to reconcile the discrete calcium conduction bands recently revealed in our BD simulations, M0 (Q(f)=1e), M1 (3e), M2 (5e), with a set of sodium conduction bands L0 (0.5e), L1 (1.5e), thereby obtaining a completed pattern of conduction and selectivity bands vs Q(f) for the sodium-calcium channels family. An increase of Q(f) leads to an increase of calcium selectivity: L0 (sodium-selective, nonblocking channel) → M0 (nonselective channel) → L1 (sodium-selective channel with divalent block) → M1 (calcium-selective channel exhibiting the anomalous mole fraction effect). We create a consistent identification scheme where the L0 band is putatively identified with the eukaryotic sodium channel The scheme created is able to account for the experimentally observed mutation-induced transformations between nonselective channels, sodium-selective channels, and calcium-selective channels, which we interpret as transitions between different rows of the identification table. By considering the potential energy changes during permeation, we show explicitly that the multi-ion conduction bands of calcium and sodium channels arise as the result of resonant barrierless conduction. The pattern of periodic conduction bands is explained on the basis of sequential neutralization taking account of self-energy, as Q(f)(z,i)=ze(1/2+i), where i is the order of the band and z is the valence of the ion. Our results confirm the crucial influence of electrostatic interactions on conduction and on the Ca(2+)/Na(+) valence selectivity of calcium and sodium ion channels. The model and results could be also applicable to biomimetic nanopores with charged walls. PMID:24329301

  1. Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway.

    PubMed

    Lee, Eunkyung; Choi, So-Young; Yang, Jae-Ho; Lee, Youn Ju

    2016-07-01

    Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway. PMID:27382356

  2. Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway

    PubMed Central

    Lee, Eunkyung; Choi, So-Young; Yang, Jae-Ho

    2016-01-01

    Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway. PMID:27382356

  3. L-type calcium channels and MAP kinase contribute to thyrotropin-releasing hormone-induced depolarization in thalamic paraventricular nucleus neurons

    PubMed Central

    Kolaj, Miloslav; Zhang, Li

    2016-01-01

    In rat paraventricular thalamic nucleus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances neuronal excitability via concurrent decrease in a G protein-coupled inwardly rectifying K (GIRK)-like conductance and opening of a cannabinoid receptor-sensitive transient receptor potential canonical (TRPC)-like conductance. Here, we investigated the calcium (Ca2+) contribution to the components of this TRH-induced response. TRH-induced membrane depolarization was reduced in the presence of intracellular BAPTA, also in media containing nominally zero [Ca2+]o, suggesting a critical role for both intracellular Ca2+ release and Ca2+ influx. TRH-induced inward current was unchanged by T-type Ca2+ channel blockade, but was decreased by blockade of high-voltage-activated Ca2+ channels (HVACCs). Both the pharmacologically isolated GIRK-like and the TRPC-like components of the TRH-induced response were decreased by nifedipine and increased by BayK8644, implying Ca2+ influx via L-type Ca2+ channels. Only the TRPC-like conductance was reduced by either thapsigargin or dantrolene, suggesting a role for ryanodine receptors and Ca2+-induced Ca2+ release in this component of the TRH-induced response. In pituitary and other cell lines, TRH stimulates MAPK. In PVT neurons, only the GIRK-like component of the TRH-induced current was selectively decreased in the presence of PD98059, a MAPK inhibitor. Collectively, the data imply that TRH-induced depolarization and inward current in PVT neurons involve both a dependency on extracellular Ca2+ influx via opening of L-type Ca2+ channels, a sensitivity of a TRPC-like component to intracellular Ca2+ release via ryanodine channels, and a modulation by MAPK of a GIRK-like conductance component. PMID:27009047

  4. Effects of calcium phosphate/chitosan composite on bone healing in rats: calcium phosphate induces osteon formation.

    PubMed

    Fernández, Tulio; Olave, Gilberto; Valencia, Carlos H; Arce, Sandra; Quinn, Julian M W; Thouas, George A; Chen, Qi-Zhi

    2014-07-01

    Vascularization of an artificial graft represents one of the most significant challenges facing the field of bone tissue engineering. Over the past decade, strategies to vascularize artificial scaffolds have been intensively evaluated using osteoinductive calcium phosphate (CaP) biomaterials in animal models. In this work, we observed that CaP-based biomaterials implanted into rat calvarial defects showed remarkably accelerated formation and mineralization of new woven bone in defects in the initial stages, at a rate of ∼60 μm/day (0.8 mg/day), which was considerably higher than normal bone growth rates (several μm/day, 0.1 mg/day) in implant-free controls of the same age. Surprisingly, we also observed histological evidence of primary osteon formation, indicated by blood vessels in early-region fibrous tissue, which was encapsulated by lamellar osteocyte structures. These were later fully replaced by compact bone, indicating complete regeneration of calvarial bone. Thus, the CaP biomaterial used here is not only osteoinductive, but vasculogenic, and it may have contributed to the bone regeneration, despite an absence of osteons in normal rat calvaria. Further investigation will involve how this strategy can regulate formation of vascularized cortical bone such as by control of degradation rate, and use of models of long, dense bones, to more closely approximate repair of human cortical bone. PMID:24460696

  5. Effects of calcium phosphate/chitosan composite on bone healing in rats: calcium phosphate induces osteon formation.

    PubMed

    Fernández, Tulio; Olave, Gilberto; Valencia, Carlos H; Arce, Sandra; Quinn, Julian M W; Thouas, George A; Chen, Qi-Zhi

    2014-07-01

    Vascularization of an artificial graft represents one of the most significant challenges facing the field of bone tissue engineering. Over the past decade, strategies to vascularize artificial scaffolds have been intensively evaluated using osteoinductive calcium phosphate (CaP) biomaterials in animal models. In this work, we observed that CaP-based biomaterials implanted into rat calvarial defects showed remarkably accelerated formation and mineralization of new woven bone in defects in the initial stages, at a rate of ∼60 μm/day (0.8 mg/day), which was considerably higher than normal bone growth rates (several μm/day, 0.1 mg/day) in implant-free controls of the same age. Surprisingly, we also observed histological evidence of primary osteon formation, indicated by blood vessels in early-region fibrous tissue, which was encapsulated by lamellar osteocyte structures. These were later fully replaced by compact bone, indicating complete regeneration of calvarial bone. Thus, the CaP biomaterial used here is not only osteoinductive, but vasculogenic, and it may have contributed to the bone regeneration, despite an absence of osteons in normal rat calvaria. Further investigation will involve how this strategy can regulate formation of vascularized cortical bone such as by control of degradation rate, and use of models of long, dense bones, to more closely approximate repair of human cortical bone.

  6. Transglutaminase-induced crosslinking of gelatin-calcium carbonate composite films.

    PubMed

    Wang, Yuemeng; Liu, Anjun; Ye, Ran; Wang, Wenhang; Li, Xin

    2015-01-01

    The effects of transglutaminase (TGase) on the rheological profiles and interactions of gelatin-calcium carbonate solutions were studied. In addition, mechanical properties, water vapour permeability and microstructures of gelatin-calcium carbonate films were also investigated and compared. Fluorescence data suggested that the interaction of TGase and gelation-calcium carbonate belonged to a static quenching mechanism, and merely one binding site between TGase and gelatin-calcium carbonate was identified. Moreover, differential scanning calorimetry (DSC), the mechanical properties and the water vapour permeability studies revealed that TGase favoured the strong intramolecular polymerisation of the peptides in gelatin. The microstructures of the surfaces and cross sections in gelatin-calcium carbonate films were shown by scanning electron microscope (SEM) micrographs. The results of the fourier transform infrared spectroscopy (FTIR) indicated that TGase caused conformational changes in the proteins films. Therefore, TGase successfully facilitated the formation of gelatin-calcium carbonate composite films.

  7. Mean Field Strategies Induce Unrealistic Non-Linearities in Calcium Puffs

    PubMed Central

    Solovey, Guillermo; Fraiman, Daniel; Dawson, Silvina Ponce

    2011-01-01

    Mean field models are often useful approximations to biological systems, but sometimes, they can yield misleading results. In this work, we compare mean field approaches with stochastic models of intracellular calcium release. In particular, we concentrate on calcium signals generated by the concerted opening of several clustered channels (calcium puffs). To this end we simulate calcium puffs numerically and then try to reproduce features of the resulting calcium distribution using mean field models were all the channels open and close simultaneously. We show that an unrealistic non-linear relationship between the current and the number of open channels is needed to reproduce the simulated puffs. Furthermore, a single channel current which is five times smaller than the one of the stochastic simulations is also needed. Our study sheds light on the importance of the stochastic kinetics of the calcium release channel activity to estimate the release fluxes. PMID:21869877

  8. Surface Sensitivity in Cluster-Ion-Induced Sputtering

    SciTech Connect

    Szakal, Christopher; Kozole, Joseph; Russo, Michael F. Jr.; Garrison, Barbara J.; Winograd, Nicholas

    2006-06-02

    The ion beam-induced removal of thin water ice films condensed onto Ag and bombarded by energetic Au, Au{sub 2}, Au{sub 3}, and C{sub 60} projectiles is examined both experimentally and with molecular dynamics computer simulations. For water overlayers of thicknesses greater than 10 A, the yields of sputtered Ag{sup +} secondary ions decay exponentially with increasing ice thickness, revealing characteristic decay lengths of 24, 20, 18, and 7.0 A ring , respectively. It is shown that these values manifest the characteristic depths of projectile energy loss, rather than escape depths of the sputtered Ag atoms through the water ice overlayer. Computer simulations show that the mechanism of ejection involves the sweeping away of overlayer water molecules, allowing for an unimpeded escape of ejected Ag atoms. The relevance of these data with respect to surface sensitivity in secondary ion mass spectrometry is discussed.

  9. Zinc and calcium reduce lead induced perturbations in the aminergic system of developing brain.

    PubMed

    Jaya Prasanthi, R P; Hariprasad Reddy, G; Bhuvaneswari Devi, C; Rajarami Reddy, G

    2005-12-01

    Since alterations in monoamines and monoamine oxidase (MAO) have been postulated to play a role in toxic effects of lead (Pb) on the central nervous system, we have examined the protective effects of calcium (Ca2+) and zinc (Zn2+) supplementation on Pb-induced perturbations in the levels of monoamines and the activity of MAO. Swiss albino mice were lactationally exposed to low (0.2%) and high (1%) levels of Pb-acetate via drinking water of the mother. Pb-exposure commenced on postnatal day (PND) 1, continued up to PND 21 and stopped at weaning. Ca2+ or Zn2+ (0.02% in 0.2% Pb-water or 0.1% in 1% Pb-water) was supplemented separately to the mother up to PND 21. The levels of monoamines (epinephrine, norepinephrine, dopamine and serotonin) and the activity of MAO in the brain regions such as hippocampus, cortex, cerebellum and medulla of young (1 month old) and adult (3 month old) mice were determined in the synaptosomal fractions. The synaptosomal monoamines though increased with low level (0.2%) Pb-exposure, significantly decreased with high level (1%) Pb-exposure in all the brain regions in both the age groups. In general, the young mice seem to be more vulnerable to Pb-neurotoxicity. Ca2+ or Zn2+ supplementation significantly reversed the Pb-induced perturbations both in the levels of monoamines and in the activity of MAO. However, the recovery in monoamine levels and MAO activity was more pronounced with Ca2+ supplementation as compared to Zn2+. These results provide evidence that dietary Ca2+ and/or Zn2+ provide protection against Pb-induced neurotoxic effects.

  10. Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration

    PubMed Central

    2010-01-01

    Background Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine. Methods Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted. Results Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium. Conclusions Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential. PMID:20920278

  11. 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. PMID:24233561

  12. Light-induced basilar membrane vibrations in the sensitive cochlea

    NASA Astrophysics Data System (ADS)

    Grosh, Karl; Ren, Tianying; He, Wenxuan; Fridberger, Anders; Li, Yizeng; Nankali, Amir

    2015-12-01

    The exceptional sensitivity of mammalian hearing organ is attributed to an outer hair cell-mediated active process, where forces produced by sensory cells boost sound-induced vibrations, making soft sounds audible. This process is thought to be local, with each section of the hearing organ capable of amplifying sound-evoked movement, and nearly instantaneous, since amplification can work for sounds at frequencies up to 100 kHz in some species. To test these precepts, we developed a method for focally stimulating the living hearing organ with light. Light pulses caused intense and highly damped mechanical responses followed by traveling waves that developed with considerable delay. The delayed response was identical to movements evoked by click-like sounds. A physiologically based mathematical model shows that such waves engage the active process, enhancing hearing sensitivity. The experiments and the theoretical analysis show that the active process is neither local nor instantaneous, but requires mechanical waves traveling from the cochlear base toward its apex.

  13. Arsenic induced neuronal apoptosis in guinea pigs is Ca2+ dependent and abrogated by chelation therapy: role of voltage gated calcium channels.

    PubMed

    Pachauri, Vidhu; Mehta, Ashish; Mishra, Deepshikha; Flora, Swaran J S

    2013-03-01

    Arsenic contaminated drinking water has affected more than 200 million people globally. Chronic arsenicism has also been associated with numerous neurological diseases. One of the prime mechanisms postulated for arsenic toxicity is reactive oxygen species (ROS) mediated oxidative stress. In this study, we explored the kinetic relationship of ROS with calcium and attempted to dissect the calcium ion channels responsible for calcium imbalance after arsenic exposure. We also explored if mono- or combinational chelation therapy prevents arsenic-induced (25ppm in drinking water for 4 months) neuronal apoptosis in a guinea pig animal model. Results indicate that chronic arsenic exposure caused a significant increase in ROS followed by NO and calcium influx. This calcium influx is mainly dependent on L-type voltage gated channels that disrupt mitochondrial membrane potential, increase bax/bcl2 levels and caspase 3 activity leading to apoptosis. Interestingly, blocking of ROS could completely reduce calcium influx whereas calcium blockage partially reduced ROS increase. While in general mono- and combinational chelation therapies were effective in reversing arsenic induced alteration, combinational therapy of DMSA and MiADMSA was most effective. Our results provide evidence for the role of L-type calcium channels in regulating arsenic-induced calcium influx and DMSA+MiADMSA combinational therapy may be a better protocol than monotherapy in mitigating chronic arsenicosis.

  14. Arsenic induced neuronal apoptosis in guinea pigs is Ca2+ dependent and abrogated by chelation therapy: role of voltage gated calcium channels.

    PubMed

    Pachauri, Vidhu; Mehta, Ashish; Mishra, Deepshikha; Flora, Swaran J S

    2013-03-01

    Arsenic contaminated drinking water has affected more than 200 million people globally. Chronic arsenicism has also been associated with numerous neurological diseases. One of the prime mechanisms postulated for arsenic toxicity is reactive oxygen species (ROS) mediated oxidative stress. In this study, we explored the kinetic relationship of ROS with calcium and attempted to dissect the calcium ion channels responsible for calcium imbalance after arsenic exposure. We also explored if mono- or combinational chelation therapy prevents arsenic-induced (25ppm in drinking water for 4 months) neuronal apoptosis in a guinea pig animal model. Results indicate that chronic arsenic exposure caused a significant increase in ROS followed by NO and calcium influx. This calcium influx is mainly dependent on L-type voltage gated channels that disrupt mitochondrial membrane potential, increase bax/bcl2 levels and caspase 3 activity leading to apoptosis. Interestingly, blocking of ROS could completely reduce calcium influx whereas calcium blockage partially reduced ROS increase. While in general mono- and combinational chelation therapies were effective in reversing arsenic induced alteration, combinational therapy of DMSA and MiADMSA was most effective. Our results provide evidence for the role of L-type calcium channels in regulating arsenic-induced calcium influx and DMSA+MiADMSA combinational therapy may be a better protocol than monotherapy in mitigating chronic arsenicosis. PMID:23376091

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

    PubMed Central

    Madias, Christopher; Garlitski, Ann C.; Kalin, John; Link, Mark S.

    2016-01-01

    Background. In a commotio cordis swine model, ventricular fibrillation (VF) can be induced by a ball blow to the chest believed secondary to activation of mechanosensitive ion channels. The purpose of the current study is to evaluate whether stretch induced activation of the L-type calcium channel may cause intracellular calcium overload and underlie the VF in commotio cordis. Method and Results. Anesthetized juvenile swine received 6 chest wall strikes with a 17.9 m/s lacrosse ball timed to the vulnerable period for VF induction. Animals were randomized to IV verapamil (n = 6) or placebo (n = 6). There was no difference in the observed frequency of VF between verapamil (19/26: 73%) and placebo (20/36: 56%) treated animals (p = 0.16). There was also no significant difference in the combined endpoint of VF or nonsustained VF (21/26: 81% in verapamil versus 24/36: 67% in controls, p = 0.22). Conclusions. In this experimental model of commotio cordis, verapamil did not prevent VF induction. Thus, in commotio cordis it is unlikely that stretch activation of the L-type calcium channel with resultant intracellular calcium overload plays a prominent role. PMID:26925288

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

    PubMed

    Madias, Christopher; Garlitski, Ann C; Kalin, John; Link, Mark S

    2016-01-01

    Background. In a commotio cordis swine model, ventricular fibrillation (VF) can be induced by a ball blow to the chest believed secondary to activation of mechanosensitive ion channels. The purpose of the current study is to evaluate whether stretch induced activation of the L-type calcium channel may cause intracellular calcium overload and underlie the VF in commotio cordis. Method and Results. Anesthetized juvenile swine received 6 chest wall strikes with a 17.9 m/s lacrosse ball timed to the vulnerable period for VF induction. Animals were randomized to IV verapamil (n = 6) or placebo (n = 6). There was no difference in the observed frequency of VF between verapamil (19/26: 73%) and placebo (20/36: 56%) treated animals (p = 0.16). There was also no significant difference in the combined endpoint of VF or nonsustained VF (21/26: 81% in verapamil versus 24/36: 67% in controls, p = 0.22). Conclusions. In this experimental model of commotio cordis, verapamil did not prevent VF induction. Thus, in commotio cordis it is unlikely that stretch activation of the L-type calcium channel with resultant intracellular calcium overload plays a prominent role. PMID:26925288

  17. Characterization of L-type calcium channel activity in atrioventricular nodal myocytes from rats with streptozotocin-induced Diabetes mellitus.

    PubMed

    Yuill, Kathryn H; Al Kury, Lina T; Howarth, Frank Christopher

    2015-11-01

    Cardiovascular complications are common in patients with Diabetes mellitus (DM). In addition to changes in cardiac muscle inotropy, electrical abnormalities are also commonly observed in these patients. We have previously shown that spontaneous cellular electrical activity is altered in atrioventricular nodal (AVN) myocytes, isolated from the streptozotocin (STZ) rat model of type-1 DM. In this study, utilizing the same model, we have characterized the changes in L-type calcium channel activity in single AVN myocytes. Ionic currents were recorded from AVN myocytes isolated from the hearts of control rats and from those with STZ-induced diabetes. Patch-clamp recordings were used to assess the changes in cellular electrical activity in individual myocytes. Type-1 DM significantly altered the cellular characteristics of L-type calcium current. A reduction in peak ICaL density was observed, with no corresponding changes in the activation parameters of the current. L-type calcium channel current also exhibited faster time-dependent inactivation in AVN myocytes from diabetic rats. A negative shift in the voltage dependence of inactivation was also evident, and a slowing of restitution parameters. These findings demonstrate that experimentally induced type-1 DM significantly alters AVN L-type calcium channel cellular electrophysiology. These changes in ion channel activity may contribute to the abnormalities in cardiac electrical function that are associated with high mortality levels in patients with DM. PMID:26603460

  18. Sustained release of vascular endothelial growth factor from calcium-induced alginate hydrogels reinforced by heparin and chitosan.

    PubMed

    Lee, K W; Yoon, J J; Lee, J H; Kim, S Y; Jung, H J; Kim, S J; Joh, J W; Lee, H H; Lee, D S; Lee, S K

    2004-10-01

    A possible alternative for immunosuppression is a microencapsulation technique using hydrogels, which have been utilized for cell immobilization and drug delivery systems. Angiogenesis is crucial for delivery of the metabolic products to the host tissues as well as to supply oxygen and nutrients to cells. The local delivery of angiogenic growth factors, such as VEGF and basic FGF, has been recently studied to enhance angiogenesis on peripheral tissue of graft. In this study, we evaluated sustained VEGF release with a model using hydrogels coated with chitosan and heparin in vitro. We fabricated calcium alginate gels and chitosan-coated calcium alginate gels. Heparinized chitosan-coated calcium-induced alginate hydrogel beads were prepared by soaking chitosan-coated calcium alginate gels in heparin solution. We compared the stability and VEGF release manner between three kinds of hydrogels. To compare the stability, 5 mL of each hydrogel was incubated with 20 mL PBS under the rotational culture. Compression forces were measured using a rheometer. The amount of VEGF released from the gels was measured by ELISA. The heparin-coated chitosan alginate hydrogels showed the highest surface stability among the three hydrogels. VEGF from the heparinized gel was released in sustained manner up to 10 days in vitro. Chitosan-coated alginate gels released 90% of loaded VEGF within 5 days. These results suggest that local delivery of VEGF using a heparinized hydrogel may provide a long-term supply of angiogenic growth factor that might induce new vessel formation in vivo.

  19. [Thrombocytopenia induced by rifampicin not previously sensitized: a case presentation].

    PubMed

    Neino Mourtala Mohamed, A; Tummino, C; Gouitaa, M; Chanez, P

    2013-11-01

    Thrombocytopenia induced by rifampicin in the absence of prior sensitization is exceptional, especially when it occurs in a patient without risk factors. We report the case of a patient aged 25 years with no past history of medical, surgical or knowledge of having taken rifampicin previously, who was hospitalized for treatment of thrombocytopenic purpura occurring after the initiation of fixed combination quadruple therapy (isoniazid, rifampicin, pyrazinamide and ethambutol) for pulmonary tuberculosis. The biological pretreatment and therapeutic education had not been made. The patient presented with thrombocytopenic purpura 30000/mm(3) on day 9 after the initiation of treatment. The platelet count returned to normal 10 days after discontinuation of treatment. We elected not to reintroduce rifampicin given the strong likelihood that it was responsible for this complication. We conducted a phased reintroduction of isoniazid, ethambutol and pyrazinamide. No recurrence of the thrombocytopenia occurred. Thus, the diagnosis of rifampicin-induced thrombocytopenia appears to have been confirmed and the patient tolerated the remainder of their treatment well.

  20. Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis.

    PubMed

    Rachlin, Kenneth; Moore, Dan H; Yount, Garret

    2013-11-01

    The development of nontoxic agents that can selectively enhance the cytotoxicity of chemotherapy is an important aim in oncology. This study evaluates the ability of infrasound exposure to sensitize glioblastoma cells to cisplatin-induced apoptosis. The infrasound was delivered using a device designed to replicate the unique infrasound emissions measured during external Qigong treatments. Human glioblastoma cell lines harboring wild-type p53 (U87) or mutant p53 (U251, SF210, and SF188) were treated in culture with cisplatin, infrasound emissions, or the combination of the 2 agents. Induction of apoptosis was quantified after 24 hours by flow cytometry following annexin V/propidium iodide staining. Infrasound emissions alone, delivered at moderate levels (~10 mPa) with dynamic frequency content (7-13 Hz), did not induce apoptosis, yet combining infrasound with cisplatin augmented the induction of apoptosis by cisplatin in all the 4 cell lines (P < .05). Increased cellular uptake of the fluorophore calcein associated with infrasound exposure was quantified by fluorescence microscopy as well as flow cytometry, demonstrating increased cell membrane permeability. The 4 cell lines differed in the degree to which infrasound exposure increased calcein uptake, and these differences were predictive of the extent to which infrasound enhanced cisplatin-induced apoptosis. When exposed to specific frequencies, membrane permeabilization also appeared to be differentially responsive for each cell line, suggesting the potential for selective targeting of tissue types using isolated infrasonic frequencies. Additionally, the pressure amplitudes used in this study were several orders of magnitude less than those used in similar studies involving ultrasound and shock waves. The results of this study provide support for using infrasound to enhance the chemotherapeutic effects of cisplatin in a clinical setting. PMID:23165942

  1. Tubifex: a sensitive model for UV-B-induced phototoxicity.

    PubMed

    Misra, R B; Babu, G Suresh; Ray, R S; Hans, R K

    2002-07-01

    The natural increase of UV-B radiation levels due to depletion of the ozone layer in the atmosphere may impose additional stress for the survival of zooplanktons which serve as a major constituent of the aquatic food chain. To study the adverse effects of UV-B radiation on the aquatic biomass, studies were conducted using the aquatic organism Tubifex as a model, as UV-B radiation is known to penetrate into the natural waters. UV-B radiation induced mortality in tubifex and the production of activated oxygen species by these organisms. Alterations in DNA, RNA, protein, glutathione (GSH), hydrogen peroxide H(2)O(2), thiobarbituric acid-reactive substance (TBA-RS), ATPase, AChE, GST, and LDH activities in Tubifex at various doses (0-2.0 J) of UV-B radiation were found. LC(50) value for UV-B-induced mortality of Tubifex was 0.80+/-0.15 J and the threshold dose was 0.35+/-0.05 J; mortality began within 3h postirradiation. UV-B dose-dependent production of singlet oxygen, superoxide anion, and hydroxyl radicals by Tubifex was observed. DNA, RNA, protein, and GSH contents were found to decrease significantly (P<0.001) while H(2)O(2) and TBA-RS increased (P<0.01) under the influence of UV-B radiation. The activities of ATpase, AChE, and GST enzymes were inhibited (P<0.01) and LDH activity was significantly increased (P<0.001) in Tubifex exposed to UV-B radiation. The results suggest that an increase in UV-B radiation alters several biochemical processes, leading to the mortality of the organism. Tubifex could be useful as a sensitive alternate model for studying UV-B-induced phototoxicity and possible mechanisms of action.

  2. Role of heterotrimeric G protein and calcium in cardiomyocyte hypertrophy induced by IGF-1.

    PubMed

    Carrasco, Loreto; Cea, Paola; Rocco, Paola; Peña-Oyarzún, Daniel; Rivera-Mejias, Pablo; Sotomayor-Flores, Cristian; Quiroga, Clara; Criollo, Alfredo; Ibarra, Cristian; Chiong, Mario; Lavandero, Sergio

    2014-04-01

    In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and β-myosin heavy chain (β-MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin.

  3. Role of heterotrimeric G protein and calcium in cardiomyocyte hypertrophy induced by IGF-1.

    PubMed

    Carrasco, Loreto; Cea, Paola; Rocco, Paola; Peña-Oyarzún, Daniel; Rivera-Mejias, Pablo; Sotomayor-Flores, Cristian; Quiroga, Clara; Criollo, Alfredo; Ibarra, Cristian; Chiong, Mario; Lavandero, Sergio

    2014-04-01

    In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and β-myosin heavy chain (β-MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin. PMID:24243530

  4. PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death.

    PubMed

    Wang, Yufeng; Kuramitsu, Yasuhiro; Baron, Byron; Kitagawa, Takao; Akada, Junko; Tokuda, Kazuhiro; Cui, Dan; Nakamura, Kazuyuki

    2015-09-22

    Calcium ions (Ca(2+)) are indispensable for the physiology of organisms and the molecular regulation of cells. We observed that CGK733, a synthetic chemical substance, induced non-apoptotic cell death and stimulated reversible calcium sequestration by vesicles in pancreatic cancer cells. The endoplasmic reticulum (ER) stress eukaryotic translation initiation factor 2-alpha kinase 3/C/EBP homologous protein (PERK/CHOP) signaling pathway was shown to be activated by treatment with CGK733. Ionomycin, an ER stress drug and calcium ionophore, can activate PERK/CHOP signaling and accelerate CGK733-induced calcium sequestration. Knockdown of CHOP diminished CGK733-induced vesicular calcium sequestration, but had no effects on the cell death. Proteomic analysis demonstrated that the ER-located calcium-binding proteins, calumenin and protein S100-A11, were altered in CGK733-treated cells compared to non-treated controls. Our study reveals that CGK733-induced intracellular calcium sequestration is correlated with the PERK/CHOP signaling pathway and may also be involved in the dysregulations of calcium-binding proteins.

  5. Tooth discoloration induced by calcium-silicate-based pulp-capping materials

    PubMed Central

    Yun, Da-A; Park, Su-Jung; Lee, Seok-Ryun; Min, Kyung-San

    2015-01-01

    Objectives: The aim of this study was to evaluate tooth discoloration induced by contact with various calcium silicate-based pulp capping materials in the presence or absence of blood in vitro. Materials and Methods: Eighty bovine samples were divided into six experimental groups and two control groups according to the type of material used (ProRoot [PR], Endocem [EC], or EndocemZr [ECZ]) and the presence or absence of contamination with blood. A spectrophotometer was used to calculate the color difference (ΔE) between the baseline measurement (after placement of materials) and measurements taken 1, 2, 4, and 8 weeks. The results were analyzed with repeated measures analysis of variance, Tukey's post-hoc tests and independent t-tests (P = 0.05). Results: The PR group and EC group showed significantly higher mean values of ΔE than the negative control group after 2 weeks (P < 0.05), whereas ECZ did not. There were larger ΔE values when there was contact with blood, especially in PR and EC group (P < 0.05). Conclusions: ECZ which contains zirconium oxide as a radiopacifier showed less discoloration irrespective of blood contamination compared to PR and EC. PMID:26038644

  6. Methotrexate inhibits osteoclastogenesis by decreasing RANKL-induced calcium influx into osteoclast progenitors.

    PubMed

    Kanagawa, Hiroya; Masuyama, Ritsuko; Morita, Mayu; Sato, Yuiko; Niki, Yasuo; Kobayashi, Tami; Katsuyama, Eri; Fujie, Atsuhiro; Hao, Wu; Tando, Toshimi; Watanabe, Ryuichi; Miyamoto, Kana; Morioka, Hideo; Matsumoto, Morio; Toyama, Yoshiaki; Saya, Hideyuki; Miyamoto, Takeshi

    2016-09-01

    The increasing number of osteoporosis patients is a pressing issue worldwide. Osteoporosis frequently causes fragility fractures, limiting activities of daily life and increasing mortality. Many osteoporosis patients take numerous medicines due to other health issues; thus, it would be preferable if a single medicine could ameliorate osteoporosis and other conditions. Here, we screened 96 randomly selected drugs targeting various diseases for their ability to inhibit differentiation of osteoclasts, which play a pivotal role in development of osteoporosis, and identified methotrexate (MTX), as a potential inhibitor. MTX is currently used to treat sarcomas or leukemic malignancies or auto-inflammatory diseases such as rheumatoid arthritis (RA) through its anti-proliferative and immunosuppressive activities; however, a direct effect on osteoclast differentiation has not been shown. Here, we report that osteoclast formation and expression of osteoclastic genes such as NFATc1 and DC-STAMP, which are induced by the cytokine RANKL, are significantly inhibited by MTX. We found that RANKL-dependent calcium (Ca) influx into osteoclast progenitors was significantly inhibited by MTX. RA patients often develop osteoporosis, and osteoclasts are reportedly required for joint destruction; thus, MTX treatment could have a beneficial effect on RA patients exhibiting high osteoclast activity by preventing both osteoporosis and joint destruction.

  7. Troponin I binds polycystin-L and inhibits its calcium-induced channel activation.

    PubMed

    Li, Qiang; Liu, Yan; Shen, Patrick Y; Dai, Xiao-Qing; Wang, Shaohua; Smillie, Lawrence B; Sandford, Richard; Chen, Xing-Zhen

    2003-06-24

    Polycystin-L (PCL) is an isoform of polycystin-2, the product of the second gene associated with autosomal dominant polycystic kidney disease, and functions as a Ca(2+)-regulated nonselective cation channel. We recently demonstrated that polycystin-2 interacts with troponin I, an important regulatory component of the actin microfilament complex in striated muscle cells and an angiogenesis inhibitor. In this study, using the two-microelectrode voltage-clamp technique and Xenopus oocyte expression system, we showed that the calcium-induced PCL channel activation is substantially inhibited by the skeletal and cardiac troponin I (60% and 31% reduction, respectively). Reciprocal co-immunoprecipitation experiments demonstrated that PCL physically associates with the skeletal and cardiac troponin I isoforms in overexpressed Xenopus oocytes and mouse fibroblast NIH 3T3 cells. Furthermore, both native PCL and cardiac troponin I were present in human heart tissues where they indeed associate with each other. GST pull-down and microtiter binding assays showed that the C-terminus of PCL interacts with the troponin I proteins. The yeast two-hybrid assay further verified this interaction and defined the corresponding interacting domains of the PCL C-terminus and troponin I. Taken together, this study suggests that troponin I acts as a regulatory subunit of the PCL channel complex and provides the first direct evidence that PCL is associated with the actin cytoskeleton through troponin I. PMID:12809519

  8. Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling

    PubMed Central

    Shih, Yu-Ru V.; Hwang, YongSung; Phadke, Ameya; Kang, Heemin; Hwang, Nathaniel S.; Caro, Eduardo J.; Nguyen, Steven; Siu, Michael; Theodorakis, Emmanuel A.; Gianneschi, Nathan C.; Vecchio, Kenneth S.; Chien, Shu; Lee, Oscar K.; Varghese, Shyni

    2014-01-01

    Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases. PMID:24395775

  9. Transient receptor potential ankyrin 1 activation enhances hapten sensitization in a T-helper type 2-driven fluorescein isothiocyanate-induced contact hypersensitivity mouse model

    SciTech Connect

    Shiba, Takahiro; Tamai, Takuma; Sahara, Yurina; Kurohane, Kohta; Watanabe, Tatsuo; Imai, Yasuyuki

    2012-11-01

    Some chemicals contribute to the development of allergies by increasing the immunogenicity of other allergens. We have demonstrated that several phthalate esters, including dibutyl phthalate (DBP), enhance skin sensitization to fluorescein isothiocyanate (FITC) in a mouse contact hypersensitivity model, in which the T-helper type 2 (Th2) response is essential. On the other hand, some phthalate esters were found to activate transient receptor potential ankyrin 1 (TRPA1) cation channels on sensory neurons. We then found a positive correlation between the enhancing effects of several types of phthalate esters on skin sensitization to FITC and their ability to activate TRPA1. Here we examined the involvement of TRPA1 in sensitization to FITC by using TRPA1 agonists other than phthalate esters. During skin sensitization to FITC, the TRPA1 agonists (menthol, carvacrol, cinnamaldehyde and DBP) augmented the ear-swelling response as well as trafficking of FITC-presenting dendritic cells to draining lymph nodes. We confirmed that these TRPA1 agonists induced calcium influx into TRPA1-expressing Chinese hamster ovary (CHO) cells. We also found that TRPA1 antagonist HC-030031 inhibited DBP-induced calcium influx into TRPA1-expressing CHO cells. After pretreatment with this antagonist upon skin sensitization to FITC, the enhancing effect of DBP on sensitization was suppressed. These results suggest that TRPA1 activation will become a useful marker to find chemicals that facilitate sensitization in combination with other immunogenic haptens. -- Highlights: ► Role of TRPA1 activation was revealed in a mouse model of skin sensitization to FITC. ► TRPA1 agonists enhanced skin sensitization as well as dendritic cell trafficking. ► Dibutyl phthalate (DBP) has been shown to enhance skin sensitization to FITC. ► TRPA1 activation by DBP was inhibited by a selective antagonist, HC-030031. ► HC-030031 inhibited the enhancing effect of DBP on skin sensitization to FITC.

  10. Calcium-sensing receptor inhibits secretagogue-induced electrolyte secretion by intestine via the enteric nervous system.

    PubMed

    Cheng, Sam X

    2012-07-01

    Bacterial toxins such as cholera toxin induce diarrhea by both direct epithelial cell generation of cyclic nucleotides as well as stimulation of the enteric nervous system (ENS). Agonists of the extracellular calcium-sensing receptor (CaSR) can reduce toxin-stimulated fluid secretion in ENS-absent colonic epithelial crypts by increasing phosphodiesterase-dependent cyclic-nucleotide degradation. Here we show that the CaSR is also highly expressed in tetrodotoxin (TTX)-sensitive neurons comprising the ENS, suggesting that CaSR agonists might also function through neuronal pathways. To test this hypothesis, rat colon segments containing intact ENS were isolated and mounted on Ussing chambers. Basal and cyclic nucleotide-stimulated electrolyte secretions were monitored by measuring changes in short-circuit current (I(sc)). CaSR was activated by R-568 and its effects were compared in the presence and absence of TTX. Consistent with active regulation of anion secretion by the ENS, a significant proportion of I(sc) in the proximal and distal colon was inhibited by serosal TTX, both at basal and under cyclic AMP-stimulated conditions. In the absence of TTX, activation of CaSR with R-568 significantly reduced basal I(sc) and cyclic AMP-stimulated I(sc); it also completely reversed the cAMP-stimulated secretory responses if the drug was applied after the forskolin stimulation. Such inhibitory effects of R-568 were either absent or significantly reduced when serosal TTX was present, suggesting that this agonist exerts its antisecretory effect on the intestine by inhibiting ENS. The present results suggest a new model for regulating intestinal fluid transport in which neuronal and nonneuronal secretagogue actions are modulated by the inhibitory effects of CaSR on the ENS. The ability of a CaSR agonist to reduce secretagogue-stimulated Cl(-) secretion might provide a new therapeutic approach for secretory and other ENS-mediated diarrheal conditions.

  11. Involvement of Potassium Channels and Calcium-Independent Mechanisms in Hydrogen Sulfide-Induced Relaxation of Rat Mesenteric Small Arteries.

    PubMed

    Hedegaard, Elise R; Gouliaev, Anja; Winther, Anna K; Arcanjo, Daniel D R; Aalling, Mathilde; Renaltan, Nirthika S; Wood, Mark E; Whiteman, Matthew; Skovgaard, Nini; Simonsen, Ulf

    2016-01-01

    Endogenous hydrogen sulfide (H2S) is involved in the regulation of vascular tone. We hypothesized that the lowering of calcium and opening of potassium (K) channels as well as calcium-independent mechanisms are involved in H2S-induced relaxation in rat mesenteric small arteries. Amperometric recordings revealed that free [H2S] after addition to closed tubes of sodium hydrosulfide (NaHS), Na2S, and GYY4137 [P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid] were, respectively, 14%, 17%, and 1% of added amount. The compounds caused equipotent relaxations in isometric myographs, but based on the measured free [H2S], GYY4137 caused more relaxation in relation to released free H2S than NaHS and Na2S in rat mesenteric small arteries. Simultaneous measurements of [H2S] and tension showed that 15 µM of free H2S caused 61% relaxation in superior mesenteric arteries. Simultaneous measurements of smooth muscle calcium and tension revealed that NaHS lowered calcium and caused relaxation of NE-contracted arteries, while high extracellular potassium reduced NaHS relaxation without corresponding calcium changes. In NE-contracted arteries, NaHS (1 mM) lowered the phosphorylation of myosin light chain, while phosphorylation of myosin phosphatase target subunit 1 remained unchanged. Protein kinase A and G, inhibitors of guanylate cyclase, failed to reduce NaHS relaxation, whereas blockers of voltage-gated KV7 channels inhibited NaHS relaxation, and blockers of mitochondrial complex I and III abolished NaHS relaxation. Our findings suggest that low micromolar concentrations of free H2S open K channels followed by lowering of smooth muscle calcium, and by another mechanism involving mitochondrial complex I and III leads to uncoupling of force, and hence vasodilation. PMID:26493746

  12. Involvement of Potassium Channels and Calcium-Independent Mechanisms in Hydrogen Sulfide-Induced Relaxation of Rat Mesenteric Small Arteries.

    PubMed

    Hedegaard, Elise R; Gouliaev, Anja; Winther, Anna K; Arcanjo, Daniel D R; Aalling, Mathilde; Renaltan, Nirthika S; Wood, Mark E; Whiteman, Matthew; Skovgaard, Nini; Simonsen, Ulf

    2016-01-01

    Endogenous hydrogen sulfide (H2S) is involved in the regulation of vascular tone. We hypothesized that the lowering of calcium and opening of potassium (K) channels as well as calcium-independent mechanisms are involved in H2S-induced relaxation in rat mesenteric small arteries. Amperometric recordings revealed that free [H2S] after addition to closed tubes of sodium hydrosulfide (NaHS), Na2S, and GYY4137 [P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid] were, respectively, 14%, 17%, and 1% of added amount. The compounds caused equipotent relaxations in isometric myographs, but based on the measured free [H2S], GYY4137 caused more relaxation in relation to released free H2S than NaHS and Na2S in rat mesenteric small arteries. Simultaneous measurements of [H2S] and tension showed that 15 µM of free H2S caused 61% relaxation in superior mesenteric arteries. Simultaneous measurements of smooth muscle calcium and tension revealed that NaHS lowered calcium and caused relaxation of NE-contracted arteries, while high extracellular potassium reduced NaHS relaxation without corresponding calcium changes. In NE-contracted arteries, NaHS (1 mM) lowered the phosphorylation of myosin light chain, while phosphorylation of myosin phosphatase target subunit 1 remained unchanged. Protein kinase A and G, inhibitors of guanylate cyclase, failed to reduce NaHS relaxation, whereas blockers of voltage-gated KV7 channels inhibited NaHS relaxation, and blockers of mitochondrial complex I and III abolished NaHS relaxation. Our findings suggest that low micromolar concentrations of free H2S open K channels followed by lowering of smooth muscle calcium, and by another mechanism involving mitochondrial complex I and III leads to uncoupling of force, and hence vasodilation.

  13. Simple and sensitive method for monitoring drug-induced cell injury in cultured cells

    SciTech Connect

    Shirhatti, V.; Krishna, G.

    1985-06-01

    A simple, sensitive method has been developed for evaluating cell injury noninvasively in monolayer cells in culture. The cell ATP pool was radiolabeled by incubating the cells with (/sup 14/C)adenine. The uptake and incorporation of (/sup 14/C)adenine was shown to proportional to the number of cells. As determined by HPLC, about 65-70% of the incorporated /sup 14/C label was in the ATP pool, 15-20% was in the ADP pool, and the rest was in the 5'-AMP pool. When prelabeled cells were exposed to toxic drugs (acetaminophen, calcium ionophore A-23187, or daunomycin) there was a marked decrease in cell ATP with a concomitant increase in leakage of labeled nucleotides, mainly 5'-AMP and 5'IMP. The authors have shown that leakage of /sup 14/C label into the medium from the prelabeled cells may be employed for quantitation of cell injury. This new measure of toxicity was shown to correlate very well with LDH leakage from the cells, which is a well accepted measure of cell injury. The leakage of 5'-(/sup 14/C)AMP also correlated very well with the reduction of cell ATP in cardiac myocytes. This method has been used for monitoring drug-induced toxicity in liver cells, cardiac myocytes, and LB cells.

  14. Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

    SciTech Connect

    Ostrow, Lyle W.; Suchyna, Thomas M.; Sachs, Frederick

    2011-06-24

    Highlights: {yields} Endothelin-1 expression by adult rat astrocytes correlates with cell proliferation. {yields} Stretch-induced ET-1 is inhibited by GsMtx-4, a specific inhibitor of Ca{sup 2+} permeant SACs. {yields} The less specific SAC inhibitor streptomycin also inhibits ET-1 secretion. {yields} Stretch-induced ET-1 production depends on a calcium influx. {yields} SAC pharmacology may provide a new class of therapeutic agents for CNS pathology. -- Abstract: The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch (<20%) of a rubber substrate increased ET-1 secretion, and 4 {mu}M GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca{sup 2+} threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

  15. Pharmacodynamics and safety of a new calcium sensitizer, levosimendan, and its metabolites during an extended infusion in patients with severe heart failure.

    PubMed

    Kivikko, Matti; Antila, Saila; Eha, Jaan; Lehtonen, Lasse; Pentikäinen, Pertti J

    2002-01-01

    Levosimendan is a new calcium sensitizer developed for the short-term intravenous treatment of congestive heart failure. The aims of the present open-label, nonrandomized study were to determine the tolerability, hemodynamic effects, and the basic pharmacokinetics of levosimendan and its metabolites during an extended continuous infusion of levosimendan. Twenty-four patients with New York Heart Association (NYHA) III-IV heart failure in two groups of 12 patients were exposed to either 0.05 microg/kg/min or 0.1 microg/kg/min of levosimendan for 7 days. Heart rate and blood pressure were measured, and blood samples for the determination of plasma concentrations of the parent drug and its metabolites were drawn daily during the infusion and the 10 to 15 days' follow-up. The 7-day infusion was well tolerated and no premature discontinuations occurred. Both systolic and diastolic blood pressure decreased maximally by 6 mmHg in the lower and by 11 mmHg in the higher levosimendan dose groups during the infusion period (p < 0.05 for both groups). The mean heart rate values increased maximally by 18 and 26 beats/min in the lower and higher levosimendan dose groups, respectively (p < 0.001 for both groups). The hemodynamic effects peaked at the end of the infusion period and thereafter slowly declined during the follow-up. After the recommended infusion period of 24 hours, the mean heart rate increase was only 2 and 6 beats/min in the lower and higher levosimendan dose groups, respectively. The elimination half-life of levosimendan was approximately 1 hour and of the metabolites 70 to 80 hours. It can be concluded that levosimendan, even administered considerably longer than the recommended 24 hours, was well tolerated. The 7-day infusion induced a prolonged increase in heart rate and a minor decrease in blood pressure. The long-lasting effects are probably explained by the active metabolite.

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

    PubMed Central

    Li, Shangfu; Yang, Bu; Teguh, Dian; Zhou, Lin; Xu, Jiake; Rong, Limin

    2016-01-01

    Osteoporosis and Alzheimer’s disease (AD) are common chronic degenerative disorders which are strongly associated with advanced age. We have previously demonstrated that amyloid beta peptide (Aβ), one of the pathological hallmarks of AD, accumulated abnormally in osteoporotic bone specimens in addition to having an activation effect on osteoclast (Bone 2014,61:164-75). However, the underlying molecular mechanisms remain unclear. Activation of NF-κB, extracellular signal-regulated kinase (ERK) phosphorylates, and calcium oscillation signaling pathways by receptor activator NF-κB ligand (RANKL) plays a pivotal role in osteoclast activation. Targeting this signaling to modulate osteoclast function has been a promising strategy for osteoclast-related diseases. In this study, we investigated the effects of Aβ on RANKL-induced osteoclast signaling pathways in vitro. In mouse bone marrow monocytes (BMMs), Aβ exerted no effect on RANKL-induced osteoclastogenesis but promoted osteoclastic bone resorption. In molecular levels, Aβ enhanced NF-κB activity and IκB-α degradation, activated ERK phosphorylation and stimulated calcium oscillation, thus leading to upregulation of NFAT-c1 expression during osteoclast activation. Taken together, our data demonstrate that Aβ enhances RANKL-induced osteoclast activation through IκB-α degradation, ERK phosphorylation, and calcium oscillation signaling pathways and that Aβ may be a promising agent in the treatment of osteoclast-related disease such as osteoporosis. PMID:27735865

  17. Aluminum Chloride Induces Osteoblasts Apoptosis via Disrupting Calcium Homeostasis and Activating Ca(2+)/CaMKII Signal Pathway.

    PubMed

    Cao, Zheng; Liu, Dawei; Zhang, Qiuyue; Sun, Xudong; Li, Yanfei

    2016-02-01

    Aluminum promotes osteoblast (OB) apoptosis. Apoptosis is induced by the disordered calcium homeostasis. Therefore, to investigate the relationship between Al-induced OB apoptosis and calcium homeostasis, calvarium OBs from neonatal rats (3-4 days) were cultured and exposed to 0.048-mg/mL Al(3+) or 0.048-mg/mL Al(3+) combined with 5 μM BAPTA-AM (OBs were pretreated with 5 μM BAPTA-AM for 1 h, then added 0.048 mg/mL Al(3+)), respectively. Then OB apoptosis rate, intracellular calcium ions concentration ([Ca(2+)]i), mRNA expression level of calmodulin (CaM), and protein expression levels of CaM and p-CaMKII in OBs were examined. The result showed that AlCl3 increased OB apoptosis rate, and [Ca(2+)]i and p-CaMKII expression levels and decreased CaM expression levels, whereas BAPTA-AM relieved the effects. These results proved that AlCl3 induced OB apoptosis by disrupting the intracellular Ca(2+) homeostasis and activating the Ca(2+)/CaMKII signal pathway. Our findings can provide new insights for revealing the apoptosis mechanism of OBs exposed to AlCl3.

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

    PubMed

    Hayer, Stefanie N; Bading, Hilmar

    2015-02-27

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

  19. Dihydropyridine-sensitive calcium channels in cardiac and skeletal muscle membranes: studies with antibodies against the. cap alpha. subunits

    SciTech Connect

    Takahashi, M.; Catterall, W.A.

    1987-08-25

    Polyclonal antibodies (PAC-2) against the purified skeletal muscle calcium channel were prepared and shown to be directed against ..cap alpha.. subunits of this protein by immunoblotting and immunoprecipitation. These polypeptides have an apparent molecular weight of 162,000 without reduction of disulfide bonds. Under conditions where the functional properties of the purified skeletal muscle calcium channel are retained, ..beta.. subunits (M/sub r/ 50,000) and lambda subunits (M/sub r/ 33,000) are coprecipitated, demonstrating specific noncovalent association of these three polypeptides in the purified skeletal muscle channel. PAC-2 immunoprecipitated cardiac calcium channels labeled with (/sup 3/H)isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-(methoxycarbonyl)pyridine-3-carboxylate ((/sup 3/H)PN200-110) at a 3-fold higher concentration than skeletal muscle channels. Preincubation with cardiac calcium channels blocked only 49% of the immunoreactivity of PAC-2 toward skeletal muscle channels, indicating that these two proteins have both homologous and distinct epitopes. The immunoreactive component of the cardiac calcium channel was identified by immunoprecipitation and polyacrylamide gel electrophoresis as a polypeptide with an apparent molecular weight of 170,000 before reduction of disulfide bonds and 141,000 after reduction, in close analogy with the properties of the ..cap alpha../sub 2/ subunits of the skeletal muscle channel. The calcium channels were radiolabeled with /sup 32/P and /sup 125/I. It is concluded that these two calcium channels have a homologous, but distinct, ..cap alpha.. subunit as a major polypeptide component.

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

    PubMed

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

    2015-10-01

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

  1. Noise-Induced Increase of Sensitivity in Bacterial Chemotaxis.

    PubMed

    He, Rui; Zhang, Rongjing; Yuan, Junhua

    2016-07-26

    Flagellated bacteria, like Escherichia coli, can swim toward beneficial environments by modulating the rotational direction of their flagellar motors through a chemotaxis signal transduction network. The noise of this network, the random fluctuation of the intracellular concentration of the signal protein CheY-P with time, has been identified in studies of single cell behavioral variability, and found to be important in coordination of multiple motors in a bacterium and in enhancement of bacterial drift velocity in chemical gradients. Here, by comparing the behavioral difference between motors of wild-type E. coli and mutants without signal noise, we measured the magnitude of this noise in wild-type cells, and found that the noise increases the sensitivity of the bacterial chemotaxis network downstream at the level of the flagellar motor. This provided a simple mechanism for the noise-induced enhancement of chemotactic drift, which we confirmed by simulating the E. coli chemotactic motion in various spatial profiles of chemo-attractant concentration. PMID:27463144

  2. Radiation-induced Genomic Instability and Radiation Sensitivity

    SciTech Connect

    Varnum, Susan M.; Sowa, Marianne B.; Kim, Grace J.; Morgan, William F.

    2013-01-19

    The obvious relationships between reactive oxygen and nitrogen species, mitochondrial dysfunction, inflammatory type responses and reactive chemokines and cytokines suggests a general stress response induced by ionizing radiation most likely leads to the non-targeted effects described after radiation exposure. We argue that true bystander effects do not occur in the radiation therapy clinic. But there is no question that effects outside the target volume do occur. These “out of field effects” are considered very low dose effects in the context of therapy. So what are the implications of non-targeted effects on radiation sensitivity? The primary goal of therapy is to eradicate the tumor. Given the genetic diversity of the human population, lifestyle and environment factors it is likely some combination of these will influence patient outcome. Non-targeted effects may contribute to a greater or lesser extent. But consider the potential situation involving a partial body exposure due to a radiation accident or radiological terrorism. Non-targeted effects suggest that the tissue at risk for demonstrating possible detrimental effects of radiation exposure might be greater than the volume actually irradiated.

  3. Differential sensitivity of mouse oocytes to colchicine-induced aneuploidy

    SciTech Connect

    Mailhes, J.B.; Yuan, Z.P.

    1987-01-01

    Unpublished results from our laboratory showed that colchicine increased the incidence of hyperploid mouse metaphase II (MII) oocytes when injected at the same time as human chorionic gonadotrophin (HCG). The objective of the present study was to determine whether the time of administering colchicine influenced the incidence of aneuploidy in MII oocytes. CD-1 mice were given pregnant mare's serum (PMS) and, 48 hr later, HCG. An intraperitoneal injection of 0.2 mg/kg colchicine was given at +4, +2, 0, -2, or -4 hr relative to HCG. Oocytes were collected 17 hr post-HCG and processed, and chromosomes were subsequently C-banded. The percentage of hyperploid oocytes was 0.77, 2.56, 5.71, 7.79, 3.54, and 2.70 for control, +4, +2, 0, -2, or -4 hr pre/post-HCG, respectively. Chi-square analyses of these data demonstrated that colchicine significantly increases the proportion of aneuploid oocytes, and that the relative sensitivity of colchicine-induced aneuploidy depends upon the time that this drug is administered relative to HCG.

  4. Calcium supplements

    MedlinePlus

    ... TYPES OF CALCIUM SUPPLEMENTS Forms of calcium include: Calcium carbonate: Over-the-counter (OTC) antacid products, such as Tums and Rolaids, contain calcium carbonate. These sources of calcium do not cost much. ...

  5. Vascularization of plastic calcium phosphate cement in vivo induced by in-situ-generated hollow channels.

    PubMed

    Yu, Tao; Dong, Chao; Shen, Zhonghua; Chen, Yan; Yu, Bo; Shi, Haishan; Zhou, Changren; Ye, Jiandong

    2016-11-01

    Despite calcium phosphate cement (CPC) is promising for bone repair therapy, slow biodegradation and insufficient vascularization in constructs negatively impacts its clinical application. A self-setting CPC composited with gelatin fiber is investigated to test the utility of this tissue engineering strategy to support rapid and extensive vascularization process. The interconnected hollow channels in CPC are formed after dissolution of gelatin fibers in vivo. The CPC-gelatin samples exhibit relatively decent/enhanced mechanical property, compared to the control. When implanted in vivo, the pre-established vascular networks in material anastomose with host vessels and accelerate vascular infiltration throughout the whole tissue construct. Different channel sizes induce different vascularization behaviors in vivo. Results indicate that the channel with the size of 250μm increases the expression of the representative angiogenic factors HIF1α, PLGF and migration factor CXCR4, which benefit the formation of small vessels. On the other hand, the channel with the size of 500μm enhances VEGF-A expression, which benefit the development of large vessels. Notably, the intersection area of channels has high invasive, sprouting and vasculogenesis potential under hypoxic condition, because more HIF1α-positive cells are observed there. Observation of the CD31-positive lumen in the border of scaffold indicates the ingrowth of blood vessels from its host into material through channel, benefited from gradually increased HIF1α expression. This kind of material was suggested to promote the effective application of bone regeneration through the combination of in situ self-setting, plasticity, angiogenesis, and osteoconductivity. PMID:27524007

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

    PubMed Central

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

    2015-01-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell–cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell–cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett’s, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin–catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells. PMID:26603452

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

    PubMed

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

    2015-11-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell-cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell-cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett's, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin-catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells. PMID:26603452

  8. Molecular mechanisms of corticotropin-releasing factor receptor-induced calcium signaling.

    PubMed

    Gutknecht, Eric; Van der Linden, Ilse; Van Kolen, Kristof; Verhoeven, Kim F C; Vauquelin, Georges; Dautzenberg, Frank M

    2009-03-01

    The molecular mechanisms governing calcium signal transduction of corticotropin-releasing factor (CRF) receptors CRF(1) and CRF(2(a)) stably expressed in human embryonic kidney (HEK) 293 cells were investigated. Calcium signaling strictly depended on intracellular calcium sources, and this is the first study to establish a prominent contribution of the three major G-protein families to CRF receptor-mediated calcium signaling. Overexpression of Galpha(q/11) and Galpha(16) led to leftward shifts of the agonist concentration-response curves. Blockade of Galpha(q/11) proteins by the small interfering RNA (siRNA) technology partially reduced agonist-mediated calcium responses in CRF(1)- and CRF(2(a))-expressing HEK293 cells, thereby proving a contribution of the G(q) protein family. A small but significant inhibition of calcium signaling was recorded by pharmacological inhibition of G(i/o) proteins with pertussis toxin treatment. This effect was mediated by direct binding of Gbetagamma subunits to phospholipase C. G(i/o) inhibition also elevated cAMP responses in CRF receptor-overexpressing HEK293 cells and in Y79 retinoblastoma cells endogenously expressing human CRF(1) and CRF(2(a)) receptors, thereby demonstrating natural coupling of G(i) proteins to both CRF receptors. The strongest reduction of CRF receptor-mediated calcium mobilization was noted when blocking the G(s) signaling protein either by cholera toxin or by siRNA. It is noteworthy that simultaneous inhibition of two G-proteins shed light on the additive effects of G(s) and G(q) on the calcium signaling and, hence, that they act in parallel. On the other hand, G(i) coupling required prior G(s) activation. PMID:19098121

  9. PhTx3-4, a Spider Toxin Calcium Channel Blocker, Reduces NMDA-Induced Injury of the Retina

    PubMed Central

    Binda, Nancy Scardua; Porto Petruceli Carayon, Charles; Agostini, Rafael Mourão; do Nascimento Pinheiro, Ana Cristina; Nascimento Cordeiro, Marta; Romano Silva, Marco Aurélio; Figueira Silva, Juliana; Rita Pereira, Elizete Maria; da Silva Junior, Claudio Antonio; de Castro Junior, Célio José; Sena Guimarães, Andre Luiz; Gomez, Marcus Vinicius

    2016-01-01

    The in vivo neuroprotective effect of PhTx3-4, a spider toxin N-P/Q calcium channel blocker, was studied in a rat model of NMDA-induced injury of the retina. NMDA (N-Methyl-d-Aspartate)-induced retinal injury in rats reduced the b-wave amplitude by 62% ± 3.6%, indicating the severity of the insult. PhTx3-4 treatment increased the amplitude of the b-wave, which was almost equivalent to the control retinas that were not submitted to injury. The PhTx3-4 functional protection of the retinas recorded on the ERG also was observed in the neuroprotection of retinal cells. NMDA-induced injury reduced live cells in the retina layers and the highest reduction, 84%, was in the ganglion cell layer. Notably, PhTx3-4 treatment caused a remarkable reduction of dead cells in the retina layers, and the highest neuroprotective effect was in the ganglion cells layer. NMDA-induced cytotoxicity of the retina increased the release of glutamate, reactive oxygen species (ROS) production and oxidative stress. PhTx3-4 treatment reduced glutamate release, ROS production and oxidative stress measured by malondialdehyde. Thus, we presented for the first time evidence of in vivo neuroprotection from NMDA-induced retinal injury by PhTx3-4 (-ctenitoxin-Pn3a), a spider toxin that blocks N-P/Q calcium channels. PMID:26978403

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

    PubMed

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

    2016-03-01

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

  11. Coral calcium hydride prevents hepatic steatosis in high fat diet-induced obese rats: A potent mitochondrial nutrient and phase II enzyme inducer.

    PubMed

    Hou, Chen; Wang, Yongyao; Zhu, Erkang; Yan, Chunhong; Zhao, Lin; Wang, Xiaojie; Qiu, Yingfeng; Shen, Hui; Sun, Xuejun; Feng, Zhihui; Liu, Jiankang; Long, Jiangang

    2016-03-01

    Diet-induced nonalcoholic fatty liver disease (NAFLD) is characterized by profound lipid accumulation and associated with an inflammatory response, oxidative stress and hepatic mitochondrial dysfunction. We previously demonstrated that some mitochondrial nutrients effectively ameliorated high fat diet (HFD)-induced hepatic steatosis and metabolic disorders. Molecular hydrogen in hydrogen-rich liquid or inhaling gas, which has been confirmed in scavenging reactive oxygen species and preventing mitochondrial decay, improved metabolic syndrome in patients and animal models. Coral calcium hydride (CCH) is a new solid molecular hydrogen carrier made of coral calcium. However, whether and how CCH impacts HFD-induced hepatic steatosis remains uninvestigated. In the present study, we applied CCH to a HFD-induced NAFLD rat model for 13 weeks. We found that CCH durably generated hydrogen in vivo and in vitro. CCH treatment significantly reduced body weight gain, improved glucose and lipid metabolism and attenuated hepatic steatosis in HFD-induced obese rats with no influence on food and water intake. Moreover, CCH effectively improved HFD-induced hepatic mitochondrial dysfunction, reduced oxidative stress, and activated phase II enzymes. Our results suggest that CCH is an efficient hydrogen-rich agent, which could prevent HFD-induced NAFLD via activating phase II enzymes and improving mitochondrial function. PMID:26774456

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  14. Cardioprotective activity of alcoholic extract of Tinospora cordifolia (Willd.) Miers in calcium chloride-induced cardiac arrhythmia in rats

    PubMed Central

    Sharma, Ashish Kumar; Kishore, Kunal; Sharma, Divya; Srinivasan, B.P; Agarwal, Shyam Sunder; Sharma, Ashok; Singh, Santosh Kumar; Gaur, Samir; Jatav, Vijay Singh

    2011-01-01

    The present study investigated the antiarrhythmic activity of alcoholic extract of Tinospora cordifolia (T. cordifolia) in CaCl2 induced arrhythmia. CaCl2 (25 mg/kg) was administered by intravenous infusion (iv) to produce arrhythmia in rats. The animals were then treated with T. cordifolia extract (150, 250, and 450 mg/kg) and verapamil (5 mg/kg,iv). Lead II electrocardiogram was monitored. Plasma calcium, sodium and potassium levels were measured. In CaCl2 induced arrhythmia, heart rate was decreased by 41.10%, T. cordifolia at 150, 300, and 450 mg/kg decreased the heart rate by 26.30%, 29.16%, and 38.29%, respectively, and verapamil reduced the heart rate by 9.70% compared to the normal group. The PQRST waves were normalized and atrial and ventricular fibrillation was controlled in rats treated with verapamil and T. cordifolia. CaCl2 increased calcium and sodium levels and decreased potassium levels in blood. T. cordifolia dose-dependently decreased calcium and sodium levels and increased potassium levels. Hence, T. cordifolia can be used in antiarrhythmic clinical settings and beneficial in atrial and ventricular fibrillation and flutter and may be indicated in ventricular tachyarrhythmia. PMID:23554702

  15. Opiates selectively increase intracellular calcium in developing type-1 astrocytes: role of calcium in morphine-induced morphologic differentiation.

    PubMed

    Stiene-Martin, A; Mattson, M P; Hauser, K F

    1993-12-17

    Endogenous opioids and opiate drugs inhibit nervous system maturation, in part, by affecting the growth of astrocytes. Opiates inhibit astrocyte proliferation and cause premature differentiation. The emerging importance of Ca2+ in astrocyte function prompted us to explore whether opiates might affect astrocyte development by altering Ca2+ homeostasis. Astrocyte-enriched cultures were derived from newborn ICR mouse cerebra. Quantitative fluorescent measurements of intracellular free Ca2+ ([Ca2+]i) using Fura-2 as well as fluo-3 and computer-aided image analysis showed that 1 microM morphine significantly increased [Ca2+]i in flat, polyhedral, glial fibrillary acidic protein (GFAP) immunoreactive astrocytes at 2 and 6 min, and at 72 h. Co-administration of 3 microM naloxone blocked morphine-dependent increases in [Ca2+]i. Treatment with 1 microM concentrations of the kappa-opioid receptor agonist, U69,593, but not equimolar amounts of mu ([D-Ala2,MePhe4,Gly(ol)5]enkephalin)- or delta ([D-Pen2,D-Pen5]enkephalin)-opioid receptor agonists, significantly increased [Ca2+]i in astrocytes. To assess the role of Ca2+ in morphine-induced astrocyte differentiation, untreated and 1 microM morphine-treated astrocyte cultures were incubated for 5 days in < 0.01, 0.3, 1.0, or 3.0 mM extracellular Ca2+ ([Ca2+]o), or incubated with 1.0 mM [Ca2+]o in the presence of 1 microM of the Ca2+ ionophore, A23187. The areas of single astrocytes were measured and there was a positive correlation between astrocyte area and [Ca2+]o. Morphine had an additive effect on area and form factor measures when [Ca2+]o was 1.0 mM. High [Ca2+]o (3.0 mM) alone mimicked the action of morphine. Morphine alone had no effect on astrocyte area in the presence of 3.0 mM Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. 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

  17. The hypoxia-inducible factor renders cancer cells more sensitive to vitamin C-induced toxicity.

    PubMed

    Tian, Weihua; Wang, Yu; Xu, Yan; Guo, Xiangpeng; Wang, Bo; Sun, Li; Liu, Longqi; Cui, Fenggong; Zhuang, Qiang; Bao, Xichen; Schley, Gunnar; Chung, Tung-Liang; Laslett, Andrew L; Willam, Carsten; Qin, Baoming; Maxwell, Patrick H; Esteban, Miguel A

    2014-02-01

    Megadose vitamin C (Vc) is one of the most enduring alternative treatments for diverse human diseases and is deeply engrafted in popular culture. Preliminary studies in the 1970s described potent effects of Vc on prolonging the survival of patients with terminal cancer, but these claims were later criticized. An improved knowledge of the pharmacokinetics of Vc and recent reports using cancer cell lines have renewed the interest in this subject. Despite these findings, using Vc as an adjuvant for anticancer therapy remains questionable, among other things because there is no proper mechanistic understanding. Here, we show that a Warburg effect triggered by activation of the hypoxia-inducible factor (HIF) pathway greatly enhances Vc-induced toxicity in multiple cancer cell lines, including von Hippel-Lindau (VHL)-defective renal cancer cells. HIF increases the intracellular uptake of oxidized Vc through its transcriptional target glucose transporter 1 (GLUT1), synergizing with the uptake of its reduced form through sodium-dependent Vc transporters. The resulting high levels of intracellular Vc induce oxidative stress and massive DNA damage, which then causes metabolic exhaustion by depleting cellular ATP reserves. HIF-positive cells are particularly sensitive to Vc-induced ATP reduction because they mostly rely on the rather inefficient glycolytic pathway for energy production. Thus, our experiments link Vc-induced toxicity and cancer metabolism, providing a new explanation for the preferential effect of Vc on cancer cells. PMID:24371136

  18. Light-induced changes of sensitivity in Limulus ventral photoreceptors

    PubMed Central

    1975-01-01

    The responses of Limulus ventral photoreceptors to brief test flashes and to longer adapting lights were measured under voltage clamp conditions. When the cell was dark adapted, there was a range of energy of the test flashes over which the peak amplitude of the responses (light-induced currents) was directly proportional to the flash energy. This was also true when test flashes were superposed on adapting stimuli but the proportionality constant (termed peak currently/photon) was reduced. The peak current/photon was attenuated more by brighter adapting stimuli than by less bright adapting stimuli. The peak current/photon is a measure of the sensitivity of the conductance- increase mechanism underlying the light response of the photo-receptor. The response elicited by an adapting stimulus had a large initial transient which declined to a smaller plateau. The peak current/photon decreased sharply during the declining phase of the transient and was relatively stable during the plateau. This indicates that the onset of light adaptation is delayed with respect to the onset of the response to the adapting stimulus. If the adaptational state just before the onset of each of a series of adapting stimuli was constant, the amplitude of the transient was a nearly linear function of intensity. When the total intensity was rapidly doubled (or halved) during a plateau response, the total current approximately doubled (or halved). We argue that the transition from transient to plateau, light-elicited changes of threshold, and the nonlinear function relating the plateau response to stimulus intensity all reflect changes of the responsiveness of the conductance-increase mechanism. PMID:1181378

  19. Three-photon induced fluorescence of the calcium probe Indo-1.

    PubMed Central

    Szmacinski, H; Gryczynski, I; Lakowicz, J R

    1996-01-01

    We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process. PMID:8770232

  20. Calcium-43 chemical shift and electric field gradient tensor interplay: a sensitive probe of structure, polymorphism, and hydration.

    PubMed

    Widdifield, Cory M; Moudrakovski, Igor; Bryce, David L

    2014-07-14

    Calcium is the 5th most abundant element on earth, and is found in numerous biological tissues, proteins, materials, and increasingly in catalysts. However, due to a number of unfavourable nuclear properties, such as a low magnetogyric ratio, very low natural abundance, and its nuclear electric quadrupole moment, development of solid-state (43)Ca NMR has been constrained relative to similar nuclides. In this study, 12 commonly-available calcium compounds are analyzed via(43)Ca solid-state NMR and the information which may be obtained by the measurement of both the (43)Ca electric field gradient (EFG) and chemical shift tensors (the latter of which are extremely rare with only a handful of literature examples) is discussed. Combined with density functional theory (DFT) computations, this 'tensor interplay' is, for the first time for (43)Ca, illustrated to be diagnostic in distinguishing polymorphs (e.g., calcium formate), and the degree of hydration (e.g., CaCl2·2H2O and calcium tartrate tetrahydrate). For Ca(OH)2, we outline the first example of (1)H to (43)Ca cross-polarization on a sample at natural abundance in (43)Ca. Using prior knowledge of the relationship between the isotropic calcium chemical shift and the calcium quadrupolar coupling constant (CQ) with coordination number, we postulate the coordination number in a sample of calcium levulinate dihydrate, which does not have a known crystal structure. Natural samples of CaCO3 (aragonite polymorph) are used to show that the synthetic structure is present in nature. Gauge-including projector augmented-wave (GIPAW) DFT computations using accepted crystal structures for many of these systems generally result in calculated NMR tensor parameters which are in very good agreement with the experimental observations. This combination of (43)Ca NMR measurements with GIPAW DFT ultimately allows us to establish clear correlations between various solid-state (43)Ca NMR observables and selected structural parameters

  1. Potentiation by nitric oxide synthase inhibitor and calcium channel blocker of aspartame-induced antinociception in the mouse formalin test.

    PubMed

    Abdollahi, M; Nikfar, S; Abdoli, N

    2001-04-01

    By applying a 12 day regimen of the non-calorific sweetener, aspartame, in combination with representative compounds of the calcium channel blocker and nitric oxide synthase inhibitor, we tried to investigate using a formalin-test in mice the relative role of aspartame on pain and its mechanism of action. Verapamil (2, 3.5, 5, 7.5 mg/kg) induced significant (P < 0.01) antinociception in both phases of the formalin test. L-Nitro-arginine-methyl-ester (L-NAME) at the doses used, induced significant (P < 0.01) antinociception in early phase (1, 2, 5, 10 mg/kg) and late phase (5, 10 mg/kg). Twelve days of treatment in animals by aspartame (0.16% w/v) significantly induced antinociception in both phases of the formalin test. Both verapamil (5 mg/kg) and L-NAME (10 mg/kg) significantly (P < 0.01) potentiated aspartame-induced antinociception in both phases of formalin test. The present findings support the hypothesis that the activation of NMDA receptors by aspartame modulates pain-related behaviour via a nitric oxide/cGMP/glutamate release cascade. It is concluded that aspartame would be a good analgesic agent if it would be used in combination with a calcium channel blocker or NOS inhibitor.

  2. Calcium-Myristoyl Tug Is a New Mechanism for Intramolecular Tuning of Calcium Sensitivity and Target Enzyme Interaction for Guanylyl Cyclase-activating Protein 1

    PubMed Central

    Peshenko, Igor V.; Olshevskaya, Elena V.; Lim, Sunghyuk; Ames, James B.; Dizhoor, Alexander M.

    2012-01-01

    Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca2+ sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca2+ sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca2+ binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca2+-bound and Mg2+-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca2+ sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca2+ binding to EF-hand 4 and Ca2+ sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu80 and Val180) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or “tug”) between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca2+ sensitivity. PMID:22383530

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

    PubMed

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

    2014-12-10

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

  4. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    SciTech Connect

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-08-26

    Highlights: {yields} Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. {yields} The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. {yields} Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  5. Recovery of bone strength in young pigs from an induced short-term dietary calcium deficit followed by a calcium replete diet.

    PubMed

    Aiyangar, Ameet K; Au, Anthony G; Crenshaw, Thomas D; Ploeg, Heidi-Lynn

    2010-12-01

    This study investigated whether the deficits in bone strength of pre-pubertal pigs, induced by short-term deficits in dietary calcium can be recovered if followed by a calcium-fortified diet. Young pigs were divided into two groups based on diet: a marginal Ca diet (70% of established Ca requirements) or an excess Ca diet (150% of established Ca requirements) for 4 weeks. Each group was then randomly sub-divided into two groups and fed diets with either marginal or excess dietary Ca for 6 weeks in a cross-over design, resulting in four treatment groups: H150-H150, H150-L70, L70-H150, and L70-L70. Animals were DXA scanned at 2-week intervals during the 10-week period to obtain whole body bone mineral content (BMC) and density (BMD). After animals were euthanized, right femurs were collected for this study. Traits such as bone mineral density, mass, volume, area moment of inertia (MI) and the section modulus (SM) were computed from computed tomography (CT) data and failure load was measured from four-point bending tests. DXA results showed significant reduction in BMC (61.6%) and BMD (37.5%) in the (L70-L70) group compared to the (H150-H150) group. DXA results additionally showed that deficiencies induced by the 4-week marginal Ca diet in the (L70-H150) group were not recovered with a subsequent excess Ca diet. While mechanical test results also showed significant reduction (75%) in strength in the L70-L70 group, compared to the H150-H150 group, they revealed no differences between the failure loads of the (L70-H150) group and the (H150-H150) group. Similar results were also found for bone mineral mass and volume, indicating that recovery from a short-term dietary Ca deficiency is possible at the pre-pubertal stage. Furthermore, bone mineral content and bone volume calculated from CT data correlated highly with failure load (R(2)=0.78 and 0.84, respectively), while density, MI and SM only showed weak-to-moderate correlations (R(2)=0.40-0.56), implying that bone

  6. Calcium-mediated responses and glutamine synthetase expression in greater duckweed (Spirodela polyrhiza L.) under diethyl phthalate-induced stress.

    PubMed

    Cheng, Lee-Ju; Hung, Meng-Ju; Cheng, Yen-I; Cheng, Tai-Sheng

    2013-11-15

    This study was carried out to assess the influence of diethyl phthalate (DEP) alone or associated with calcium chloride (CaCl2) on greater duckweed plants, emphasizing the implications of calcium in amelioration of DEP-induced stress on plant growth. Greater duckweed were treated with DEP in variable concentrations, as 0, 0.25, 0.5, 1.0 and 2.0mM for 7 days, or treated with the same concentration either 2mM DEP or 2mM DEP plus 10mM CaCl2·2H2O in different duration 0-7 days. Treatment with 2mM DEP resulted in increasing proline content, protease activity, and ammonia accumulation in duckweed tissues. NADH-glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) and Δ(1)-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2), two key enzymes in the glutamate pathway of proline synthesis, showed increase in activity with DEP treatment and positively correlated with proline accumulation. No further increase in proline accumulation was observed with addition of calcium chloride to the DEP-treated cultures. However, supplementation of Ca(2+) can mitigate the adverse effect of DEP, at least in part to decrease the DEP-induced superoxide accumulation and increase in GDH activity for ammonia assimilation in duckweed fronds. In addition, effects of calcium on mitigation of DEP injury were also observed in glutamine synthetase (GS; EC 6.3.1.2) expression. Both GS1 and GS2 polypeptide accumulation and the level of total GS activity were nearly equivalent to the control. Exogenous proline protects GS2 from DEP-modulated redox damage in the chloroplast lysates but there is no remarkable protection effects on D1 (the 32kDa protein in photosystem II reaction center) degradation. In conclusion, the glutamate pathway of proline synthesis might be involved in mitigation of DEP-induced injury, and calcium plays an important role in increasing GDH, P5CR, and GS expression.

  7. Rapid and Persistent Suppression of Feeding Behavior Induced by Sensitization Training in "Aplysia"

    ERIC Educational Resources Information Center

    Acheampong, Ama; Kelly, Kathleen; Shields-Johnson, Maria; Hajovsky, Julie; Wainwright, Marcy; Mozzachiodi, Riccardo

    2012-01-01

    In "Aplysia," noxious stimuli induce sensitization of defensive responses. However, it remains largely unknown whether such stimuli also alter nondefensive behaviors. In this study, we examined the effects of noxious stimuli on feeding. Strong electric shocks, capable of inducing sensitization, also led to the suppression of feeding. The use of…

  8. Calcium Induces Long-Term Legacy Effects in a Subalpine Ecosystem

    PubMed Central

    Schaffner, Urs; Alewell, Christine; Eschen, René; Matthies, Diethart; Spiegelberger, Thomas; Hegg, Otto

    2012-01-01

    Human activities have transformed a significant proportion of the world’s land surface, with profound effects on ecosystem processes. Soil applications of macronutrients such as nitrate, phosphorus, potassium or calcium are routinely used in the management of croplands, grasslands and forests to improve plant health or increase productivity. However, while the effects of continuous fertilization and liming on terrestrial ecosystems are well documented, remarkably little is known about the legacy effect of historical fertilization and liming events in terrestrial ecosystems and of the mechanisms involved. Here, we show that more than 70 years after the last application of lime on a subalpine grassland, all major soil and plant calcium pools were still significantly larger in limed than in unlimed plots, and that the resulting shift in the soil calcium/aluminium ratio continues to affect ecosystem services such as primary production. The difference in the calcium content of the vegetation and the topmost 10 cm of the soil in limed vs. unlimed plots amounts to approximately 19.5 g m−2, equivalent to 16.3% of the amount that was added to the plots some 70 years ago. In contrast, plots that were treated with nitrogen-phosphorus-potassium fertilizer in the 1930s did not differ from unfertilized plots in any of the soil and vegetation characteristics measured. Our findings suggest that the long-term legacy effect of historical liming is due to long-term storage of added calcium in stable soil pools, rather than a general increase in nutrient availability. Our results demonstrate that single applications of calcium in its carbonated form can profoundly and persistently alter ecosystem processes and services in mountain ecosystems. PMID:23284779

  9. Ischemia induces partial loss of surface membrane polarity and accumulation of putative calcium ionophores.

    PubMed Central

    Molitoris, B A; Wilson, P D; Schrier, R W; Simon, F R

    1985-01-01

    To determine if ischemia induces alterations in renal proximal tubule surface membranes, brush border (BBM) and basolateral membranes (BLM) were isolated simultaneously from the same cortical homogenate after 50 min of renal pedicle clamping. Ischemia caused a selective decrease in the specific activity of BBM marker enzymes leucine aminopeptidase and alkaline phosphatase, but did not effect enrichment (15 times). Neither specific activity nor enrichment (10 times) of BLM NaK-ATPase was altered by ischemia. Contamination of BBM by intracellular organelles was also unchanged, but there was an increase in the specific activity (41.1 vs. 60.0, P less than 0.01) and enrichment (2.3 vs. 4.3, P less than 0.01) of NaK-ATPase in the ischemic BBM fraction. Ischemia increased BLM lysophosphatidylcholine (1.3 vs. 2.5%, P less than 0.05) and phosphatidic acid (0.4 vs. 1.3%, P less than 0.05). Ischemia also decreased BBM sphingomyelin (38.5 vs. 29.6%, P less than 0.01) and phosphatidylserine (16.1 vs. 11.4%, P less than 0.01), and increased phosphatidylcholine (17.2 vs. 29.7%, P less than 0.01), phosphatidylinositol (1.8 vs. 4.6%, P less than 0.01), and lysophosphatidylcholine (1.0 vs. 1.8%, P less than 0.05). The large changes in BBM phospholipids did not result from new phospholipid synthesis, since the specific activity (32P dpm/nmol Pi) of prelabeled individual and total phospholipids was unaltered by ischemia. We next evaluated if these changes were due to inability of ischemic cells to maintain surface membrane polarity. Cytochemical evaluation showed that while NaK-ATPase could be detected only in control BLM, specific deposits of reaction product were present in the BBM of ischemic kidneys. Furthermore, using continuous sucrose gradients, the enzymatic profile of ischemic BBM NaK-ATPase shifted away from ischemic BLM NaK-ATPase and toward the BBM enzymatic marker leucine aminopeptidase. Taken together, these data suggest that NaK-ATPase activity determined enzymatically

  10. Exendin-4 antagonizes Aβ1-42-induced suppression of long-term potentiation by regulating intracellular calcium homeostasis in rat hippocampal neurons.

    PubMed

    Wang, Xiaohui; Wang, Li; Jiang, Ruirui; Yuan, Yuan; Yu, Qianqian; Li, Yameng

    2015-11-19

    An imbalance of intracellular calcium homeostasis induced by amyloid β-protein (Aβ) contributes to the pathogenesis of Alzheimer's disease (AD), such as deficits in learning and memory. Therefore, regulation of calcium homeostasis may represent a new strategy for treatment of AD. Growing evidence suggests that type 2 diabetes mellitus (T2DM) and AD are closely related in pathogenesis. Thus, drugs used in treatment of T2DM may modify the pathogenesis of AD. This study demonstrated that Exendin-4, which is a glucagon-like peptide-1 (GLP-1) analog used as a therapeutic drug for T2DM, significantly antagonized suppression of long-term potentiation (LTP) induced by Aβ1-42 in the rat hippocampal CA1 region in vivo. This neuroprotection may be mediated by regulation of calcium homeostasis. Pretreatment with Exendin-4 suppressed Aβ1-42-induced elevation in intracellular calcium concentration ([Ca(2+)]i) through L-type voltage-dependent calcium channels (L-VDCCs) and N-methyl-D-aspartate receptors (NMDARs). Furthermore, Exendin-4 antagonized the decrease in p-Ca(2+)/calmodulin-dependent protein kinase IIα (p-CaMKIIα) induced by Aβ1-42 in the rat hippocampal CA1 region. Thus, the neuroprotective effects of Exendin-4, which likely involve regulation of calcium homeostasis, provide theoretical support for using Exendin-4 to treat and prevent AD in the future.

  11. Exendin-4 antagonizes Aβ1-42-induced suppression of long-term potentiation by regulating intracellular calcium homeostasis in rat hippocampal neurons.

    PubMed

    Wang, Xiaohui; Wang, Li; Jiang, Ruirui; Yuan, Yuan; Yu, Qianqian; Li, Yameng

    2015-11-19

    An imbalance of intracellular calcium homeostasis induced by amyloid β-protein (Aβ) contributes to the pathogenesis of Alzheimer's disease (AD), such as deficits in learning and memory. Therefore, regulation of calcium homeostasis may represent a new strategy for treatment of AD. Growing evidence suggests that type 2 diabetes mellitus (T2DM) and AD are closely related in pathogenesis. Thus, drugs used in treatment of T2DM may modify the pathogenesis of AD. This study demonstrated that Exendin-4, which is a glucagon-like peptide-1 (GLP-1) analog used as a therapeutic drug for T2DM, significantly antagonized suppression of long-term potentiation (LTP) induced by Aβ1-42 in the rat hippocampal CA1 region in vivo. This neuroprotection may be mediated by regulation of calcium homeostasis. Pretreatment with Exendin-4 suppressed Aβ1-42-induced elevation in intracellular calcium concentration ([Ca(2+)]i) through L-type voltage-dependent calcium channels (L-VDCCs) and N-methyl-D-aspartate receptors (NMDARs). Furthermore, Exendin-4 antagonized the decrease in p-Ca(2+)/calmodulin-dependent protein kinase IIα (p-CaMKIIα) induced by Aβ1-42 in the rat hippocampal CA1 region. Thus, the neuroprotective effects of Exendin-4, which likely involve regulation of calcium homeostasis, provide theoretical support for using Exendin-4 to treat and prevent AD in the future. PMID:26390937

  12. Beneficial effect of the insulin sensitizer (HSP inducer) BGP-15 on olanzapine-induced metabolic disorders.

    PubMed

    Literáti-Nagy, B; Péterfai, E; Kulcsár, E; Literáti-Nagy, Zs; Buday, B; Tory, K; Mandl, J; Sümegi, B; Fleming, A; Roth, J; Korányi, L

    2010-11-20

    Olanzapine is a widely used atypical antipsychotic, with well known metabolic side effects such as weight gain, insulin resistance and blood glucose abnormalities. It has been previously shown in a phase II clinical trial that BGP-15, an amidoxim derivative has insulin-sensitizing effects. The aim of this study was to investigate the efficacy of BGP-15 for the treatment of olanzapine-induced metabolic side effects, in healthy volunteers. Thirty-seven (37) subjects (ages 18-55 years) with normal glucose metabolism were randomly assigned to 17 days of once-daily treatment with 400mg of BGP-15 or placebo and 5mg of olanzapine for 3 days followed by 10mg for 14 days. Total body and muscle tissue glucose utilization was determined by hyperinsulinemic-euglycemic clamp technique. As expected the 17-day olanzapine treatment provoked insulin resistance and body weight gain (p<0.05) in both groups. Administration of BGP-15 significantly reduced olanzapine-induced insulin resistance. The protective effect of BGP-15 on insulin stimulated glucose utilization had the highest magnitude in the values calculated for the muscle tissue (p=0.002). In healthy individuals BGP-15 was safe and well tolerated during the whole study period. It is suggested that BGP-15 can be a successful insulin sensitizer agent to prevent side effects of olanzapine treatment.

  13. Effect of neurotrophin-3 precursor on glutamate-induced calcium homeostasis deregulation in rat cerebellum granule cells.

    PubMed

    Safina, Dina R; Surin, Alexander M; Pinelis, Vsevolod G; Kostrov, Sergey V

    2015-12-01

    Neurotrophin-3 (NT-3) belongs to the family of highly conserved dimeric growth factors that controls the differentiation and activity of various neuronal populations. Mammals contain both the mature (NT-3) and the precursor (pro-NT-3) forms of neurotrophin. Members of the neurotrophin family are involved in the regulation of calcium homeostasis in neurons; however, the role of NT-3 and pro-NT-3 in this process remains unclear. The current study explores the effects of NT-3 and pro-NT-3 on disturbed calcium homeostasis and decline of mitochondrial potential induced by a neurotoxic concentration of glutamate (Glu; 100 µM) in the primary culture of rat cerebellar granule cells. In this Glu excitotoxicity model, mature NT-3 had no effect on the induced changes in Ca²⁺ homeostasis. In contrast, pro-NT-3 decreased the period of delayed calcium deregulation (DCD) and concurrent strong mitochondrial depolarization. According to the amplitude of the increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]i ) and Fura-2 fluorescence quenching by Mn²⁺ within the first 20 sec of exposure to Glu, pro-NT-3 had no effect on the initial rate of Ca²⁺ entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca²⁺]i rise was 1.2-fold greater in the presence of pro-NT-3 than in the presence of Glu alone (1.67 ±  0.07 and 1.39 ± 0.04, respectively, P < 0.05). The Glu-induced changes in Са²⁺ homeostasis in the presence of pro-NT-3 likely are due to the decreased rate of Са²⁺ removal from the cytosol during the DCD latency period.

  14. Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters

    PubMed Central

    Shinnawi, Rami; Huber, Irit; Maizels, Leonid; Shaheen, Naim; Gepstein, Amira; Arbel, Gil; Tijsen, Anke J.; Gepstein, Lior

    2015-01-01

    Summary The advent of the human-induced pluripotent stem cell (hiPSC) technology has transformed biomedical research, providing new tools for human disease modeling, drug development, and regenerative medicine. To fulfill its unique potential in the cardiovascular field, efficient methods should be developed for high-resolution, large-scale, long-term, and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs). To achieve this goal, we combined the hiPSC technology with genetically encoded voltage (ArcLight) and calcium (GCaMP5G) fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels, respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders, developmental biology, and drug development and testing. PMID:26372632

  15. Continuous angiotensin-(1-7) infusion improves myocardial calcium transient and calcium transient alternans in ischemia-induced cardiac dysfunction rats.

    PubMed

    Luo, Duan; Zhuang, Xiaodong; Luo, Chufan; Long, Ming; Deng, Chunyu; Liao, Xinxue; Wang, Lichun

    2015-11-27

    The aim of this study was to evaluate the impact of Ang-(1-7) on calcium transient (CaT) in cardiomyocytes during the pathogenesis of heart failure. Cardiac dysfunction was induced by ligation of left anterior descending coronary artery in adult SD rats. Randomly selected rats were ligated and continuously infused with Ang-(1-7) [HF + Ang-(1-7) group] or saline (HF + saline group) via osmotic minipumps. After 28 days, hemodynamic parameters, the CaT, and the heart rate threshold of CaT alternans (CaT-Alt) were measured. Continuous Ang-(1-7) treatment could attenuate the impairment of cardiac function following LAD ligation. The amplitudes (F/F0) and 50%/90% recovery time of CaT were significantly different among HF + saline, HF + Ang-(1-7) and Sham-operated group. Compared to the Sham-operated group, the HF + saline group showed decreased CaT amplitude, and a prolonged 50%/90% CaT recovery time; Ang-(1-7) significantly improved these abnormalities. Compared with Sham-operated group, heart rate thresholds of CaT-Alt significantly reduced in HF + saline group, and Ang-(1-7) partly restored it. These findings indicate that Ang-(1-7) attenuates the CaT disturbance and increases the heart rate threshold of CaT-Alt during the pathogenesis of ischemic heart failure.

  16. Nuclear Membranes ETB Receptors Mediate ET-1-induced Increase of Nuclear Calcium in Human Left Ventricular Endocardial Endothelial Cells.

    PubMed

    Jules, Farah; Avedanian, Levon; Al-Khoury, Johny; Keita, Ramatoulaye; Normand, Alexandre; Bkaily, Ghassan; Jacques, Danielle

    2015-07-01

    In fetal human left ventricular endocardial endothelial cells (EECLs), both plasma membrane (PM) ET(A)R and ET(B)R were reported to mediate ET-1-induced increase of intracellular calcium [Ca](i); however, this effect was mediated by ET(A)R in right EECs (EECRs). In this study, we verified whether, as for the PM, nuclear membranes (NMs) ET-1 receptors activation in EECLs and EECRs induce an increase of nuclear calcium ([Ca](n)) and if this effect is mediated through the same receptor type as in PM. Using a plasmalemma-perforated technique and 3D confocal microscopy, our results showed that, as in PM intact cells, superfusion of nuclei of both cell types with cytosolic ET-1 induced a concentration-dependent sustained increase of [Ca](n). In EECRs, the ET(A)R antagonist prevented the effect of ET-1 on [Ca](n) without affecting EECLs. However, in both cell types, the effect of cytosolic ET-1 on [Ca](n) was prevented by the ETBR antagonist. In conclusion, both NMs' ET(A)R and ET(B)R mediated the effect of cytosolic ET-1 on [Ca](n) in EECRs. In contrast, only NMs' ET(B)R activation mediated the effect of cytosolic ET-1 in EECLs. Hence, the type of NMs' receptors mediating the effect of ET-1 on [Ca](n) are different from those of PM mediating the increase in [Ca](i).

  17. Honeycomb chain-channel (HCC) signature in the calcium-induced Si(111)-(3 × 2) surface

    NASA Astrophysics Data System (ADS)

    Gallus, O.; Pillo, Th.; Starowicz, P.; Baer, Y.

    2002-12-01

    The electronic and structural properties of a calcium-induced chain system on Si(111) have been studied. Low-energy electron diffraction measurements clearly reveal a (3 × 2) periodicity and the Ca coverage is determined to be 1/6 monolayer. Angle-resolved photoemission measurements have been performed with two different light polarizations in order to study the symmetries of the surface states. In both polarizations no band crossing the Fermi level EF is found. The three detected surface state bands are in good agreement with theoretical calculations in the honeycomb chain-channel (HCC) model for an insulating case.

  18. Rapid changes in skeletal muscle calcium uptake induced in vitro by 1,25-dihydroxyvitamin D/sub 3/ are suppressed by calcium channel blockers

    SciTech Connect

    de Boland, A.R.; Boland, R.L.

    1987-05-01

    Previous investigations have shown that 1,25-dihydroxyvitamin D/sub 3/ (1,25-(OH)/sub 2/D/sub 3/) stimulates muscle Ca uptake through a nuclear mechanism. The possibility that 1,25-(OH)/sub 2/D/sub 3/ would induce rapid changes in muscle Ca fluxes independent of de novo protein synthesis was investigated in the present work. In vitro preparations of soleus muscles obtained from vitamin D-deficient chicks were used. A significant increase in /sup 45/Ca labeling of the tissue was already observed after 3-min treatment with 2.4 X 10(-10) M 1,25-(OH)/sub 2/D/sub 3/. This early stimulation in muscle Ca uptake became maximal at 10-15 min. Cycloheximide (50 microM) did not block the effect of the metabolite at 15 and 30 min. However, the antibiotic effectively blocked the increase in Ca uptake induced by 1,25-(OH)/sub 2/D/sub 3/ after 1-h treatment. The rapid 1,25-(OH)/sub 2/D/sub 3/-dependent stimulation of /sup 45/Ca labeling of soleus muscle was not associated to changes in lipid synthesis as assessed by measurements of /sup 3/H-glycerol incorporation into the tissue lipids. However, the calcium antagonists verapamil and nifedipine (50 microM) abolished the stimulation in Ca uptake produced by 1,25-(OH)/sub 2/D/sub 3/ in 5 min. These results suggest that 1,25-(OH)/sub 2/D/sub 3/ can act directly at the muscle membrane level affecting Ca fluxes through Ca channels.

  19. Mechanisms of pyrethroid insecticide-induced stimulation of calcium influx in neocortical neurons

    EPA Science Inventory

    Pyrethroid insecticides bind to voltage-gated sodium channels (VGSCs) and modify their gating kinetics, thereby disrupting neuronal function. Pyrethroids have also been reported to alter the function of other channel types, including activation of voltage-gated Ca2+ calcium chann...

  20. Effects of angiotensin II blockade on inflammation-induced alterations of pharmacokinetics and pharmacodynamics of calcium channel blockers

    PubMed Central

    Hanafy, S; Dagenais, N J; Dryden, W F; Jamali, F

    2007-01-01

    Background and purpose: Inflammation elevates plasma verapamil concentrations but diminishes pharmacological response. Angiotensin II is a pro-inflammatory mediator. We examined the effect of angiotensin II receptor blockade on the pharmacokinetics and pharmacodynamics of verapamil, as well as the binding properties and amounts of its target protein in calcium channels, in a rat model of inflammation. Experimental approach: We used 4 groups of male Sprague–Dawley rats (220–280 g): inflamed-placebo, inflamed-treated, control-placebo and control-treated. Inflammation as pre-adjuvant arthritis was induced by injecting Mycobacterium butyricum on day 0. From day 6 to 12, 30 mg kg−1 oral valsartan or placebo was administered twice daily. On day 12, a single oral dose of 25 mg kg−1 verapamil was administered and prolongation of the PR interval measured and plasma samples collected for verapamil and nor-verapamil analysis. The amounts of the target protein Cav1.2 subunit of L-type calcium channels in heart was measured by Western blotting and ligand binding with 3H-nitrendipine. Key results: Inflammation reduced effects of verapamil, although plasma drug concentrations were increased. This was associated with a reduction in ligand binding capacity and amount of the calcium channel target protein in heart extracts. Valsartan significantly reversed the down-regulating effect of inflammation on verapamil's effects on the PR interval, and the lower level of protein binding and the decreased target protein. Conclusions and implications: Reduced responses to calcium channel blockers in inflammatory conditions appeared to be due to a reduced amount of target protein that was reversed by the angiotensin II antagonist, valsartan. PMID:17965735

  1. Calcium channel blockade with verapamil. Effects on blood pressure, renal, and myocardial adrenergic, cholinergic, and calcium channel receptors in inbred Dahl hypertension-sensitive (S/JR) and hypertension-resistant (R/JR) rats.

    PubMed

    McCaughran, J A; Juno, C J

    1988-07-01

    Verapamil HCl was chronically administered to inbred Dahl S/JR and R/JR rats maintained on a diet containing 8.0% NaCl (w/w) and the effects on blood pressure (BP) and heart rate (HR) were investigated. Treatment over a 4-week period via implanted miniosmotic pumps attenuated but did not prevent the development of salt-induced hypertension (HT) in the S/JR rat. Elevated HR, possibly reflexive in origin, was observed in S/JR rats that received verapamil but not in similarly treated R/JR rats. Although verapamil retarded the development of HT in S/JR rats, BP rose to moderately hypertensive levels, and the ventricle/body weight ratio was elevated by the termination of the study. The effect of verapamil on the density and affinity of alpha 1-, alpha 2-, and beta-adrenergic, muscarinic cholinergic, and calcium channel receptors in renal and ventricular membranes was also assessed. The density of renal and ventricular alpha 1- and beta-adrenoceptors was not affected by chronic drug treatment. The density of renal alpha 2- and beta-adrenoceptors was greater in the S/JR strain than in the R/JR strain, regardless of the treatment. The density of muscarinic cholinergic and calcium channel receptors in the ventricle was not affected by the treatment. The results of this study suggest that the long-term antihypertensive effects of verapamil in the S/JR rat do not involve an alteration in the binding characteristics of adrenergic, cholinergic, or calcium channel receptor sites in ventricular and renal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Cold sensitivity of TRPA1 is unveiled by the prolyl hydroxylation blockade-induced sensitization to ROS.

    PubMed

    Miyake, Takahito; Nakamura, Saki; Zhao, Meng; So, Kanako; Inoue, Keisuke; Numata, Tomohiro; Takahashi, Nobuaki; Shirakawa, Hisashi; Mori, Yasuo; Nakagawa, Takayuki; Kaneko, Shuji

    2016-01-01

    Mammalian transient receptor potential ankyrin 1 (TRPA1) is a polymodal nociceptor that plays an important role in pain generation, but its role as a cold nociceptor is still controversial. Here, we propose that TRPA1 can sense noxious cold via transduction of reactive oxygen species (ROS) signalling. We show that inhibiting hydroxylation of a proline residue within the N-terminal ankyrin repeat of human TRPA1 by mutation or using a prolyl hydroxylase (PHD) inhibitor potentiates the cold sensitivity of TRPA1 in the presence of hydrogen peroxide. Inhibiting PHD in mice triggers mouse TRPA1 sensitization sufficiently to sense cold-evoked ROS, which causes cold hypersensitivity. Furthermore, this phenomenon underlies the acute cold hypersensitivity induced by the chemotherapeutic agent oxaliplatin or its metabolite oxalate. Thus, our findings provide evidence that blocking prolyl hydroxylation reveals TRPA1 sensitization to ROS, which enables TRPA1 to convert ROS signalling into cold sensitivity. PMID:27628562

  3. Cold sensitivity of TRPA1 is unveiled by the prolyl hydroxylation blockade-induced sensitization to ROS

    PubMed Central

    Miyake, Takahito; Nakamura, Saki; Zhao, Meng; So, Kanako; Inoue, Keisuke; Numata, Tomohiro; Takahashi, Nobuaki; Shirakawa, Hisashi; Mori, Yasuo; Nakagawa, Takayuki; Kaneko, Shuji

    2016-01-01

    Mammalian transient receptor potential ankyrin 1 (TRPA1) is a polymodal nociceptor that plays an important role in pain generation, but its role as a cold nociceptor is still controversial. Here, we propose that TRPA1 can sense noxious cold via transduction of reactive oxygen species (ROS) signalling. We show that inhibiting hydroxylation of a proline residue within the N-terminal ankyrin repeat of human TRPA1 by mutation or using a prolyl hydroxylase (PHD) inhibitor potentiates the cold sensitivity of TRPA1 in the presence of hydrogen peroxide. Inhibiting PHD in mice triggers mouse TRPA1 sensitization sufficiently to sense cold-evoked ROS, which causes cold hypersensitivity. Furthermore, this phenomenon underlies the acute cold hypersensitivity induced by the chemotherapeutic agent oxaliplatin or its metabolite oxalate. Thus, our findings provide evidence that blocking prolyl hydroxylation reveals TRPA1 sensitization to ROS, which enables TRPA1 to convert ROS signalling into cold sensitivity. PMID:27628562

  4. Vasorelaxation, Induced by Dictyota pulchella (Dictyotaceae), a Brown Alga, Is Mediated via Inhibition of Calcium Influx in Rats

    PubMed Central

    Queiroz, Thyago M.; Machado, Natália T.; Furtado, Fabíola F.; Oliveira-Filho, Abrahão A.; Alustau, Maria C.; Figueiredo, Camila S.; Miranda, George E. C.; Barbosa-Filho, José M.; Braga, Valdir A.; Medeiros, Isac A.

    2011-01-01

    This study aimed to investigate the cardiovascular effects elicited by Dictyota pulchella, a brown alga, using in vivo and in vitro approaches. In normotensive conscious rats, CH2Cl2/MeOH Extract (CME, 5, 10, 20 and 40 mg/kg) from Dictyota pulchella produced dose-dependent hypotension (−4 ± 1; −8 ± 2; −53 ± 8 and −63 ± 3 mmHg) and bradycardia (−8 ± 6; −17 ± 11; −257 ± 36 and −285 ± 27 b.p.m.). In addition, CME and Hexane/EtOAc Phase (HEP) (0.01–300 μg/mL) from Dictyota pulchella induced a concentration-dependent relaxation in phenylephrine (Phe, 1 μM)-pre-contracted mesenteric artery rings. The vasorelaxant effect was not modified by the removal of the vascular endothelium or pre-incubation with KCl (20 mM), tetraethylammonium (TEA, 3 mM) or tromboxane A2 agonist U-46619 (100 nM). Furthermore, CME and HEP reversed CaCl2-induced vascular contractions. These results suggest that both CME and HEP act on the voltage-operated calcium channel in order to produce vasorelaxation. In addition, CME induced vasodilatation after the vessels have been pre-contracted with L-type Ca2+ channel agonist (Bay K 8644, 200 nM). Taken together, our data show that CME induces hypotension and bradycardia in vivo and that both CME and HEP induce endothelium-independent vasodilatation in vitro that seems to involve the inhibition of the Ca2+ influx through blockade of voltage-operated calcium channels. PMID:22073010

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

    PubMed Central

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

    2016-01-01

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

  6. Effect of antispastic agents (calcium antagonists and/or isosorbide dinitrate) on high-sensitivity C-reactive protein in patients with coronary vasospastic angina pectoris and no hemodynamically significant coronary artery disease.

    PubMed

    Hung, Ming-Jui; Cherng, Wen-Jin; Cheng, Chi-Wen; Yang, Ning-I

    2005-01-01

    Levels of high-sensitivity C-reactive protein were measured before and after 3 months of treatment with antispastic agents (calcium antagonists and/or isosorbide dinitrate) in 27 patients who had coronary vasospastic angina pectoris and no hemodynamically significant coronary artery disease. Levels of high-sensitivity C-reactive protein decreased after treatment with antispastic agents.

  7. Ryanodine receptor gating controls generation of diastolic calcium waves in cardiac myocytes.

    PubMed

    Petrovič, Pavol; Valent, Ivan; Cocherová, Elena; Pavelková, Jana; Zahradníková, Alexandra

    2015-06-01

    The role of cardiac ryanodine receptor (RyR) gating in the initiation and propagation of calcium waves was investigated using a mathematical model comprising a stochastic description of RyR gating and a deterministic description of calcium diffusion and sequestration. We used a one-dimensional array of equidistantly spaced RyR clusters, representing the confocal scanning line, to simulate the formation of calcium sparks. Our model provided an excellent description of the calcium dependence of the frequency of diastolic calcium sparks and of the increased tendency for the production of calcium waves after a decrease in cytosolic calcium buffering. We developed a hypothesis relating changes in the propensity to form calcium waves to changes of RyR gating and tested it by simulation. With a realistic RyR gating model, increased ability of RyR to be activated by Ca2+ strongly increased the propensity for generation of calcium waves at low (0.05-0.1-µM) calcium concentrations but only slightly at high (0.2-0.4-µM) calcium concentrations. Changes in RyR gating altered calcium wave formation by changing the calcium sensitivity of spontaneous calcium spark activation and/or the average number of open RyRs in spontaneous calcium sparks. Gating changes that did not affect RyR activation by Ca2+ had only a weak effect on the propensity to form calcium waves, even if they strongly increased calcium spark frequency. Calcium waves induced by modulating the properties of the RyR activation site could be suppressed by inhibiting the spontaneous opening of the RyR. These data can explain the increased tendency for production of calcium waves under conditions when RyR gating is altered in cardiac diseases.

  8. Butyrate-induced changes in nuclease sensitivity of chromatin cannot be correlated with transcriptional activation

    SciTech Connect

    Birren, B.W.; Taplitz, S.J.; Herschman, H.R.

    1987-11-01

    The authors examined in the H4IIE rat heptoma cell line the relationship between butyrate-induced changes in the nuclease sensitivity of chromatin and changes in transcriptional activity of specific genes. The butyrate-inducible metallothionein I (MT-I) gene underwent a dramatic increase in DNase I sensitivity after 3 h of butyrate treatment. However, genes not transcribed in H4IIE cells underwent the same changes in DNase I sensitivity. Thus, butyrate-induced increases in DNase I sensitivity are not sufficient for the transcriptional activation of a gene. Butyrate treatment has also been reported to alter the sensitivity of sequence to micrococcal nuclease (MNase) in a manner reflecting their tissue-specific expression. Butyrate exposure caused increased digestion of the MT-I gene by MNase. However, butyrate-induced MNase sensitivity also occurred for genes which are neither transcribed in untreated cells nor butyrate inducible. Moreover, cadmium, a potent transcriptional activator of the MT-I gene, does not alter the sensitivity of the MT-I gene to MNase. Thus, the butyrate-induced alterations in MNase sensitivity are neither sufficient for, necessary for, nor indicative of transcriptional activation.

  9. Comparison of single versus repeated methamphetamine injection induced behavioral sensitization in mice.

    PubMed

    Jing, Li; Zhang, Min; Li, Jun-Xu; Huang, Ping; Liu, Qing; Li, Yu-Ling; Liang, Hui; Liang, Jian-Hui

    2014-02-01

    Repeated exposure to drugs of abuse produces a persistent behavioral sensitization to stimulants, which is often used to study drug-associated behavioral plasticity. Interestingly, even a single exposure to some drugs of abuse is sufficient to elicit long-lasting behavioral sensitization. However, few studies have directly compared the magnitude of sensitization between single versus repeated drug treatments. This study examined the magnitude and duration of single methamphetamine (METH) injection-induced behavioral sensitization and compared it to the more typical repeated drug injection-induced sensitization in mice. Different groups of mice were injected with METH (0.5, 1.0, 2.0mg/kg, i.p.) only once or daily for 7 consecutive days. A challenge dose of METH (1.0mg/kg, i.p.) was tested 7 days later. The time-course of a single METH injection-induced behavioral sensitization was assessed where METH (2.0mg/kg, i.p.) was injected and a challenge dose of METH (1.0mg/kg, i.p.) was tested after different drug-free periods. Single METH injection produced similar magnitude of behavioral sensitization as compared to repeated injection. Such a sensitized locomotor response peaked 8 days after METH injection and lasted for at least 21 days. This long lasting behavioral alteration induced by single METH injection suggests the value of future studies to explore the underlying neural mechanisms, particularly in comparison to those underlying repeated METH-induced sensitization.

  10. Capsaicin mimics mechanical load-induced intracellular signaling events: involvement of TRPV1-mediated calcium signaling in induction of skeletal muscle hypertrophy.

    PubMed

    Ito, Naoki; Ruegg, Urs T; Kudo, Akira; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi

    2013-01-01

    Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca ( 2+) ]i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca ( 2+) ]i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.

  11. Curcumin induces crosstalk between autophagy and apoptosis mediated by calcium release from the endoplasmic reticulum, lysosomal destabilization and mitochondrial events

    PubMed Central

    Moustapha, A; Pérétout, PA; Rainey, NE; Sureau, F; Geze, M; Petit, J-M; Dewailly, E; Slomianny, C; Petit, PX

    2015-01-01

    Curcumin, a major active component of turmeric (Curcuma longa, L.), has anticancer effects. In vitro studies suggest that curcumin inhibits cancer cell growth by activating apoptosis, but the mechanism underlying these effects is still unclear. Here, we investigated the mechanisms leading to apoptosis in curcumin-treated cells. Curcumin induced endoplasmic reticulum stress causing calcium release, with a destabilization of the mitochondrial compartment resulting in apoptosis. These events were also associated with lysosomal membrane permeabilization and of caspase-8 activation, mediated by cathepsins and calpains, leading to Bid cleavage. Truncated tBid disrupts mitochondrial homeostasis and enhance apoptosis. We followed the induction of autophagy, marked by the formation of autophagosomes, by staining with acridine orange in cells exposed curcumin. At this concentration, only the early events of apoptosis (initial mitochondrial destabilization with any other manifestations) were detectable. Western blotting demonstrated the conversion of LC3-I to LC3-II (light chain 3), a marker of active autophagosome formation. We also found that the production of reactive oxygen species and formation of autophagosomes following curcumin treatment was almost completely blocked by N-acetylcystein, the mitochondrial specific antioxidants MitoQ10 and SKQ1, the calcium chelators, EGTA-AM or BAPTA-AM, and the mitochondrial calcium uniporter inhibitor, ruthenium red. Curcumin-induced autophagy failed to rescue all cells and most cells underwent type II cell death following the initial autophagic processes. All together, these data imply a fail-secure mechanism regulated by autophagy in the action of curcumin, suggesting a therapeutic potential for curcumin. Offering a novel and effective strategy for the treatment of malignant cells. PMID:27551451

  12. Abscisic acid and CO2 signalling via calcium sensitivity priming in guard cells, new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus–response analyses

    PubMed Central

    Hubbard, Katharine E.; Siegel, Robert S.; Valerio, Gabriel; Brandt, Benjamin; Schroeder, Julian I.

    2012-01-01

    Background Stomatal guard cells are the regulators of gas exchange between plants and the atmosphere. Ca2+-dependent and Ca2+-independent mechanisms function in these responses. Key stomatal regulation mechanisms, including plasma membrane and vacuolar ion channels have been identified and are regulated by the free cytosolic Ca2+ concentration ([Ca2+]cyt). Scope Here we show that CO2-induced stomatal closing is strongly impaired under conditions that prevent intracellular Ca2+ elevations. Moreover, Ca2+ oscillation-induced stomatal closing is partially impaired in knock-out mutations in several guard cell-expressed Ca2+-dependent protein kinases (CDPKs) here, including the cpk4cpk11 double and cpk10 mutants; however, abscisic acid-regulated stomatal movements remain relatively intact in the cpk4cpk11 and cpk10 mutants. We further discuss diverse studies of Ca2+ signalling in guard cells, discuss apparent peculiarities, and pose novel open questions. The recently proposed Ca2+ sensitivity priming model could account for many of the findings in the field. Recent research shows that the stomatal closing stimuli abscisic acid and CO2 enhance the sensitivity of stomatal closing mechanisms to intracellular Ca2+, which has been termed ‘calcium sensitivity priming’. The genome of the reference plant Arabidopsis thaliana encodes for over 250 Ca2+-sensing proteins, giving rise to the question, how can specificity in Ca2+ responses be achieved? Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca2+ signal transduction, a topic of central interest in cell signalling research. In this article we further propose an individual stomatal tracking method for improved analyses of stimulus-regulated stomatal movements in Arabidopsis guard cells that reduces noise and increases fidelity in stimulus-regulated stomatal aperture responses ( Box 1). This method is recommended for stomatal response research, in parallel to previously

  13. Resonance-induced sensitivity enhancement method for conductivity sensors

    NASA Technical Reports Server (NTRS)

    Tai, Yu-Chong (Inventor); Shih, Chi-yuan (Inventor); Li, Wei (Inventor); Zheng, Siyang (Inventor)

    2009-01-01

    Methods and systems for improving the sensitivity of a variety of conductivity sensing devices, in particular capacitively-coupled contactless conductivity detectors. A parallel inductor is added to the conductivity sensor. The sensor with the parallel inductor is operated at a resonant frequency of the equivalent circuit model. At the resonant frequency, parasitic capacitances that are either in series or in parallel with the conductance (and possibly a series resistance) is substantially removed from the equivalent circuit, leaving a purely resistive impedance. An appreciably higher sensor sensitivity results. Experimental verification shows that sensitivity improvements of the order of 10,000-fold are possible. Examples of detecting particulates with high precision by application of the apparatus and methods of operation are described.

  14. Gender differences in ethanol-induced behavioral sensitivity in zebrafish.

    PubMed

    Dlugos, Cynthia A; Brown, Shereene J; Rabin, Richard A

    2011-02-01

    Gender-related differential sensitivity to ethanol has long been recognized. Our previous studies have demonstrated that the zebrafish, an animal model used currently to study genetics and development related to a variety of human diseases, is also sensitive to pharmacologically relevant concentrations of ethanol. Sensitivity to ethanol in the zebrafish can be easily gauged with a simple nonintrusive behavioral test that measures ethanol-related alterations in schooling by determining the distance between each fish and its nearest neighbor. The purpose of this study was to determine the influence of gender on the strain-specific ethanol sensitivity that we had observed previously. One hundred and sixty zebrafish of the wild-type (WT) and the long fin striped (LFS) strains were equally divided by gender for use in this study. For acute ethanol treatment, the fish were separated by gender and strain and exposed to 0.0, 0.125, 0.25 0.50, or 1.0% (vol/vol) ethanol. In the chronic study, eight fish of each strain and gender were exposed to 0.5% (vol/vol) ethanol for a period of 10 weeks and the swimming behavior tested before treatment and after each week of treatment. Results showed that female WT zebrafish displayed enhanced sensitivity to the effects of chronic ethanol exposure of increased nearest neighbor distances, whereas male and female LFS fish were not significantly affected by chronic ethanol exposure. Results of the acute ethanol study showed a dose-dependent effect in both strains and a gender effect that needs to be further investigated before enhanced female sensitivity to acute ethanol can be verified. PMID:20880661

  15. Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes.

    PubMed

    Granqvist, Emma; Sun, Jongho; Op den Camp, Rik; Pujic, Petar; Hill, Lionel; Normand, Philippe; Morris, Richard J; Downie, J Allan; Geurts, Rene; Oldroyd, Giles E D

    2015-08-01

    Plants that form root-nodule symbioses are within a monophyletic 'nitrogen-fixing' clade and associated signalling processes are shared with the arbuscular mycorrhizal symbiosis. Central to symbiotic signalling are nuclear-associated oscillations in calcium ions (Ca(2+) ), occurring in the root hairs of several legume species in response to the rhizobial Nod factor signal. In this study we expanded the species analysed for activation of Ca(2+) oscillations, including nonleguminous species within the nitrogen-fixing clade. We showed that Ca(2+) oscillations are a common feature of legumes in their association with rhizobia, while Cercis, a non-nodulating legume, does not show Ca(2+) oscillations in response to Nod factors from Sinorhizobium fredii NGR234. Parasponia andersonii, a nonlegume that can associate with rhizobia, showed Nod factor-induced calcium oscillations to S. fredii NGR234 Nod factors, but its non-nodulating sister species, Trema tomentosa, did not. Also within the nitrogen-fixing clade are actinorhizal species that associate with Frankia bacteria and we showed that Alnus glutinosa induces Ca(2+) oscillations in root hairs in response to exudates from Frankia alni, but not to S. fredii NGR234 Nod factors. We conclude that the ability to mount Ca(2+) oscillations in response to symbiotic bacteria is a common feature of nodulating species within the nitrogen-fixing clade. PMID:26010117

  16. A new system for profiling drug-induced calcium signal perturbation in human embryonic stem cell-derived cardiomyocytes.

    PubMed

    Lewis, Kimberley J; Silvester, Nicole C; Barberini-Jammaers, Steven; Mason, Sammy A; Marsh, Sarah A; Lipka, Magdalena; George, Christopher H

    2015-03-01

    The emergence of human stem cell-derived cardiomyocyte (hSCCM)-based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling "fingerprint" in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ≈ control. This rank order of drug-induced Ca(2+) signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework. PMID:25367900

  17. A New System for Profiling Drug-Induced Calcium Signal Perturbation in Human Embryonic Stem Cell–Derived Cardiomyocytes

    PubMed Central

    Lewis, Kimberley J.; Silvester, Nicole C.; Barberini-Jammaers, Steven; Mason, Sammy A.; Marsh, Sarah A.; Lipka, Magdalena

    2015-01-01

    The emergence of human stem cell–derived cardiomyocyte (hSCCM)–based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling “fingerprint” in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ≈ control. This rank order of drug-induced Ca2+ signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework. PMID:25367900

  18. Role for the magnetic field in the radiation-induced efflux of calcium ions from brain tissue in vitro

    SciTech Connect

    Blackman, C.F.; Benane, S.G.; Rabinowitz, J.R.; House, D.E.; Joines, W.T.

    1985-01-01

    Two independent laboratories have demonstrated that specific frequencies of electromagnetic radiation can cause a change in the efflux of calcium ions from brain tissue in vitro. Under a static magnetic field intensity of 38 microTesla (microT) due to the earth's magnetic field, 15- and 45-Hz electromagnetic signals (40 Vp-p/m in air) have been shown to induce a change in the efflux of calcium ions from the exposed tissues, while 1- and 30-Hz signals do not. The authors now show that the effective 15-Hz signal can be rendered ineffective when the net static magnetic field is reduced to 19 microT with Helmholtz coils. In addition, the ineffective 30-Hz signal becomes effective when the static magnetic field is changed to + or - 25.3 microT or to + or - 76 microT. These results demonstrate that the net intensity of the static magnetic field is an important variable. The results appear to describe a resonance-like relationship in which the extremely-low-frequency