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Sample records for reticulum calcium atpase

  1. Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate.

    Science.gov (United States)

    Fraqueza, Gil; Ohlin, C André; Casey, William H; Aureliano, Manuel

    2012-02-01

    Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca(2+)-ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca(2+)-ATPase, with the following IC(50) values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb(10)), is the strongest P-type enzyme inhibitor, after decavanadate (V(10)). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca(2+)-ATPase stoichiometry. Furthermore, V(10) binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H(2)VO(4)(2-); V(1)) to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate-Ca(2+)-ATPase, with a dissociation constant, k(d) of 1 μM(-1). The interaction of decavanadate V(10)O(28)(6-) (V(10)) with Ca(2+)-ATPase is prevented by the isostructural and isoelectronic decaniobate Nb(10)O(28)(6-) (Nb(10)), whereas no significant effects were

  2. Ameliorated stress related proteins are associated with improved cardiac function by sarcoplasmic reticulum calcium ATPase gene transfer in heart failure

    Institute of Scientific and Technical Information of China (English)

    Zhi-Qing Fu; Xiao-Ying Li; Xiao-Chun Lu; Ya-Fei Mi; Tao Liu; Wei-Hua Ye

    2012-01-01

    Background Previous studies showed that overexpression of sarco-endoplasmic reticulum calcium ATPase (SERCA2a) in a variety of heart failure (HF) models was associated with greatly enhanced cardiac performance. However, it still undefined the effect of SERCA2a overexpression on the systemic inflammatory response and neuro-hormonal factors. Methods A rapid right ventricular pacing model of experimental HF was used in beagles. Then the animals underwent recombinant adeno-associated virus 1 (rAAV1) mediated gene transfection by direct intra-myocardium injection. HF animals were randomized to receive the SERCA2a gene, enhanced green fluorescent protein (control) gene, or equivalent phosphate buffered saline. Thirty days after gene delivery, the cardiac function was evaluated by echocardiographic testing. The protein level of SERCA2a was measured by western blotting. The proteomic analysis of left ventricular (LV) sample was determined using two-dimensional (2-D) gel electrophoresis and MALDI-TOF-MS. The serum levels of the systemic inflammatory and neuro-hormonal factors were assayed using radioimmunoassay kits. Results The cardiac function improved after SERCA- 2a gene transfer due to the significantly increased SERCA2a protein level. Beagles treated with SERCA2a had significantly decreased serum levels of the inflammatory markers (interleukin-6 and tumor necrosis factor-α) and neuro-hormonal factors (brain natriuretic peptide, endothelin-1 and angiotensin Ⅱ) compared with HF animals. The myocardial proteomic analysis showed that haptoglobin heavy chain, heat shock protein (alpha-crystallin-related, B6) were down-regulated, and galectin-1 was up-regulated in SERCA2a group compared with HF group, companied by up-regulated contractile proteins and NADH dehydrogenase. Conclusions These findings demonstrate that regional intramyocardial injections of rAAV1-SERCA2a vectors may improve global LV function, correlating with reverse activation of the systemic inflammatory

  3. Sarcoplasmic reticulum calcium ATPase is inhibited by organic vanadium coordination compounds: pyridine-2,6-dicarboxylatodioxovanadium(V), BMOV, and an amavadine analogue.

    Science.gov (United States)

    Aureliano, Manuel; Henao, Fernando; Tiago, Teresa; Duarte, Rui O; Moura, J J G; Baruah, Bharat; Crans, Debbie C

    2008-07-07

    The general affinity of the sarcoplasmic reticulum (SR) Ca (2+)-ATPase was examined for three different classes of vanadium coordination complexes including a vanadium(V) compound, pyridine-2,6-dicarboxylatodioxovanadium(V) (PDC-V(V)), and two vanadium(IV) compounds, bis(maltolato)oxovanadium(IV) (BMOV), and an analogue of amavadine, bis( N-hydroxylamidoiminodiacetato)vanadium(IV) (HAIDA-V(IV)). The ability of vanadate to act either as a phosphate analogue or as a transition-state analogue with enzymes' catalysis phosphoryl group transfer suggests that vanadium coordination compounds may reveal mechanistic preferences in these classes of enzymes. Two of these compounds investigated, PDC-V(V) and BMOV, were hydrolytically and oxidatively reactive at neutral pH, and one, HAIDA-V(IV), does not hydrolyze, oxidize, or otherwise decompose to a measurable extent during the enzyme assay. The SR Ca (2+)-ATPase was inhibited by all three of these complexes. The relative order of inhibition was PDC-V(V) > BMOV > vanadate > HAIDA-V(IV), and the IC 50 values were 25, 40, 80, and 325 microM, respectively. Because the observed inhibition is more potent for PDC-V(V) and BMOV than that of oxovanadates, the inhibition cannot be explained by oxovanadate formation during enzyme assays. Furthermore, the hydrolytically and redox stable amavadine analogue HAIDA-V(IV) inhibited the Ca (2+)-ATPase less than oxovanadates. To gauge the importance of the lipid environment, studies of oxidized BMOV in microemulsions were performed and showed that this system remained in the aqueous pool even though PDC-V(V) is able to penetrate lipid interfaces. These findings suggest that the hydrolytic properties of these complexes may be important in the inhibition of the calcium pump. Our results show that two simple coordination complexes with known insulin enhancing effects can invoke a response in calcium homeostasis and the regulation of muscle contraction through the SR Ca (2+)-ATPase.

  4. Comparison of current docking tools for the simulation of inhibitor binding by the transmembrane domain of the sarco/endoplasmic reticulum calcium ATPase

    Science.gov (United States)

    Lape, Michael; Elam, Christopher; Paula, Stefan

    2010-01-01

    Inhibitors of the transmembrane protein sarco/endoplasmic reticulum calcium ATPase (SERCA) are invaluable tools for the study of the enzyme’s physiological functions and they have been recognized as a promising new class of anticancer agents. For the discovery of novel enzyme inhibitors, small molecule docking for virtual screens of large compound libraries has become increasingly important. Since the performance of various docking routines varies considerably, depending on the target and the chemical nature of the ligand, we critically evaluated the performance of four frequently used programs – GOLD, AutoDock, Surflex-Dock, and FRED – for the docking of SERCA inhibitors based on the structures of thapsigargin, di-tert-butylhydroquinone, and cyclopiazonic acid. Evaluation criteria were docking accuracy using crystal structures as references, docking reproducibility, and correlation between docking scores and known bioactivities. The best overall results were obtained by GOLD and FRED. Docking runs with conformationally flexible binding sites produced no significant improvement of the results. PMID:20167416

  5. Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

    Science.gov (United States)

    Tong, X; Kono, T; Evans-Molina, C

    2015-06-18

    The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

  6. Decavanadate interactions with sarcoplasmic reticulum calcium pump

    OpenAIRE

    2007-01-01

    Although not stable, once formed, decameric vanadate (V10) disintegration is in general slow enough to allow the study of its effects even in the micromolar range. Besides, it may become inaccessible to decomposition due to their specific interaction upon target proteins such as the Ca2+-ATPase from sarcoplasmic reticulum (SR). Characterization of the vanadate solutions and interactions with compounds containing phosphate as well as with the SR Ca2+-ATPase was analysed by 51V NMR spectr...

  7. Formalin evokes calcium transients from the endoplasmatic reticulum.

    Directory of Open Access Journals (Sweden)

    Michael J M Fischer

    Full Text Available The formalin test is the most widely used behavioral screening test for analgesic compounds. The cellular mechanism of action of formaldehyde, inducing a typically biphasic pain-related behavior in rodents is addressed in this study. The chemoreceptor channel TRPA1 was suggested as primary transducer, but the high concentrations used in the formalin test elicit a similar response in TRPA1 wildtype and knockout animals. Here we show that formaldehyde evokes a dose-dependent calcium release from intracellular stores in mouse sensory neurons and primary keratinocytes as well as in non-neuronal cell lines, and independent of TRPA1. The source of calcium is the endoplasmatic reticulum and inhibition of the sarco/endoplasmic reticulum calcium-ATPase has a major contribution. This TRPA1-independent mechanism may underlie formaldehyde-induced pan-neuronal excitation and subsequent inflammation.

  8. Identification of calcium-transporting ATPases of Entamoeba histolytica and cellular localization of the putative SERCA.

    Science.gov (United States)

    Martinez-Higuera, Aarón; Salas-Casas, Andrés; Calixto-Gálvez, Mercedes; Chávez-Munguía, Bibiana; Pérez-Ishiwara, D Guillermo; Ximénez, Cecilia; Rodríguez, Mario A

    2013-09-01

    Calcium has an important role on signaling of different cellular processes in the protozoa parasite Entamoeba histolytica, including development and pathogenesis. However, the systems that control calcium responses in this parasite are incompletely understood. Calcium-ATPases (Ca(2+)-ATPases) are proteins that play an important role in calcium homeostasis by catalyzing the active efflux of this ion from cytoplasm and are essential to the correct functioning of the cell machinery. Here, we reported the identification of five E. histolytica genes encoding putative Ca(2+)-ATPases, three related to PMCA, and two related to organellar ATPases. RT-PCR assays showed that all those genes are expressed in trophozoites and specific antibodies against the SERCA-like member located this protein in a continuous cytoplasmic network, supporting the hypothesis that it corresponds to the Ca(2+)-ATPase responsible to sequester calcium in the endoplasmic reticulum of this parasite.

  9. Ion pathways in the sarcoplasmic reticulum Ca2+-ATPase.

    Science.gov (United States)

    Bublitz, Maike; Musgaard, Maria; Poulsen, Hanne; Thøgersen, Lea; Olesen, Claus; Schiøtt, Birgit; Morth, J Preben; Møller, Jesper Vuust; Nissen, Poul

    2013-04-12

    The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a transmembrane ion transporter belonging to the P(II)-type ATPase family. It performs the vital task of re-sequestering cytoplasmic Ca(2+) to the sarco/endoplasmic reticulum store, thereby also terminating Ca(2+)-induced signaling such as in muscle contraction. This minireview focuses on the transport pathways of Ca(2+) and H(+) ions across the lipid bilayer through SERCA. The ion-binding sites of SERCA are accessible from either the cytoplasm or the sarco/endoplasmic reticulum lumen, and the Ca(2+) entry and exit channels are both formed mainly by rearrangements of four N-terminal transmembrane α-helices. Recent improvements in the resolution of the crystal structures of rabbit SERCA1a have revealed a hydrated pathway in the C-terminal transmembrane region leading from the ion-binding sites to the cytosol. A comparison of different SERCA conformations reveals that this C-terminal pathway is exclusive to Ca(2+)-free E2 states, suggesting that it may play a functional role in proton release from the ion-binding sites. This is in agreement with molecular dynamics simulations and mutational studies and is in striking analogy to a similar pathway recently described for the related sodium pump. We therefore suggest a model for the ion exchange mechanism in P(II)-ATPases including not one, but two cytoplasmic pathways working in concert.

  10. Green fluorescent protein-tagged sarco(endo)plasmic reticulum Ca2+-ATPase overexpression in Paramecium cells: isoforms, subcellular localization, biogenesis of cortical calcium stores and functional aspects.

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    Hauser, K; Pavlovic, N; Klauke, N; Geissinger, D; Plattner, H

    2000-08-01

    We have followed the time-dependent transfection of Paramecium cells with a vector containing the gene of green fluorescent protein (GFP) attached to the C-terminus of the PtSERCA1 gene. The outlines of alveolar sacs (ASs) are labelled, as is the endoplasmic reticulum (ER) throughout the cell. When GFP fluorescence is compared with previous anti-PtSERCA1 antibody labelling, the much wider distribution of GFP (ER+ASs) indicates that only a small amount of SERCA molecules is normally retained in the ER. A second isoform, PtSERCA2, also occurs and its C-terminal GFP-tagging results in the same distribution pattern. However, when GFP is inserted in the major cytoplasmic loop, PtSERCA1 and two fusion proteins are mostly retained in the ER, probably because of the presence of the overt C-terminal KKXX ER-retention signal and/or masking of a signal for transfer into ASs. On the overall cell surface, new SERCA molecules seem to be permanently delivered from the ER to ASs by vesicle transport, whereas in the fission zone of dividing cells ASs may form anew. In cells overexpressing PtSERCA1 (with C-terminal GFP) in ASs, [Ca2+]i regulation during exocytosis is not significantly different from controls, probably because their Ca2+ pump has to mediate only slow reuptake.

  11. Interactions of vanadate oligomers with sarcoplasmic reticulum Ca(2+)-ATPase.

    Science.gov (United States)

    Aureliano, M; Mdeira, V M

    1994-04-28

    Upon addition of sarcoplasmic reticulum (SR), the line width of tetrameric vanadate signal of 51V-NMR spectra narrowed in the presence of ATP and Ca2+, whereas monomeric vanadate line widths were broadened. Thus, ATP decreases the affinity of the enzyme for tetravanadate whereas it induces the interaction with monomeric vanadate. In the presence of Ca2+ it was observed that tetrameric and decameric vanadate bind to SR ATPase whereas monomeric vanadate only binds to SR when ATP is present. However, decameric vanadate clearly differs from vanadate oligomers present in monovanadate solutions in preventing the accumulation of Ca2+ by sarcoplasmic reticulum (SR) vesicles coupled to ATP hydrolysis. Mg2+ increased the inhibitory effect promoted by decavanadate whereas a slight enhancement of Ca2+ uptake was observed in the presence of monovanadate. For 5 mM Mg2+, a nominal 2 mM vanadium 'decavanadate' solution containing about 190 to 200 microM decameric and less than 100 microM monomeric species depressed the rate of Ca2+ uptake by 50% whereas a nominal 2 mM monovanadate solution containing about 662 microM monomeric, 143 microM dimeric and 252 microM tetrameric species had no effect on the rate of Ca2+ accumulation. However, 2 mM 'decavanadate' inhibits by 75% the SR Ca(2+)-ATPase activity whereas the presence of 2 mM 'monovanadate' produces an inhibitory effect below 50%. Therefore, the Ca:ATP stoichiometry of Ca2+ transport is enhanced by monovanadate. In the presence of oxalate, inhibition of SR Ca(2+)-ATPase activity by these solutions is enhanced to 97% and 86% whereas in the presence of the ionophore lasalocid, the inhibitory values were 87% and 19% for 2 mM decavanadate and 2 mM monovanadate solutions, respectively. Apparently, the increase of vesicular Ca2+ concentration counteracts monovanadate inhibition of SR Ca(2+)-ATPase activity but it does not significantly affect decavanadate inhibition.

  12. Water-mediated interactions influence the binding of thapsigargin to sarco/endoplasmic reticulum calcium adenosinetriphosphatase

    DEFF Research Database (Denmark)

    Paulsen, Eleonora S.; Villadsen, Jesper; Tenori, Eleonora;

    2013-01-01

    A crystal structure suggests four water molecules are present in the binding cavity of thapsigargin in sarco/endoplasmic reticulum calcium ATPase (SERCA). Computational chemistry indicates that three of these water molecules mediate an extensive hydrogen-bonding network between thapsigargin...

  13. Drug action of benzocaine on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle.

    Science.gov (United States)

    Di Croce, D; Trinks, P W; Grifo, M B; Takara, D; Sánchez, G A

    2015-11-01

    The effect of the local anesthetic benzocaine on sarcoplasmic reticulum membranes isolated from fast-twitch muscles was tested. The effects on Ca-ATPase activity, calcium binding and uptake, phosphoenzyme accumulation and decomposition were assessed using radioisotopic methods. The calcium binding to the Ca-ATPase was noncompetitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner (IC50 47.1 mM). The inhibition of the activity depended on the presence of the calcium ionophore calcimycin and the membrane protein concentration. The pre-exposure of the membranes to benzocaine enhanced the enzymatic activity in the absence of calcimycin, supporting the benzocaine permeabilizing effect, which was prevented by calcium. Benzocaine also interfered with the calcium transport capability by decreasing the maximal uptake (IC50 40.3 mM) without modification of the calcium affinity for the ATPase. It inhibited the phosphorylation of the enzyme, and at high benzocaine concentration, the dephosphorylation step became rate-limiting as suggested by the biphasic profile of phosphoenzyme accumulation at different benzocaine concentrations. The data reported in this paper revealed a complex pattern of inhibition involving two sites for interaction with low and high benzocaine concentrations. It is concluded that benzocaine not only exerts an indirect action on the membrane permeability to calcium but also affects key steps of the Ca-ATPase enzymatic cycle.

  14. Conformational changes in the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum detected using phosphorescence polarization.

    Science.gov (United States)

    Restall, C J; Coke, M; Murray, E K; Chapman, D

    1985-02-28

    The technique of time-averaged phosphorescence has been used to study the interaction of calcium ions and ATP with the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum vesicles. The presence of excess calcium ions was found to cause a 20% decrease in the phosphorescence emission anisotropy. This is interpreted as being due to a conformational change in the protein and is supported by data from time-resolved phosphorescence measurements which also show a lowering of the anisotropy. This change in the decay of the emission anisotropy is associated with only minor changes in the rotational relaxation time of the protein and is again suggestive of a conformational change in the protein. In some cases ATP was also observed to lower the time-averaged phosphorescence anisotropy possibly via an interaction with the low-affinity regulatory site of the protein.

  15. Effects of tetrandrine on calcium transport, protein fluorescences and membrane fluidity of sarcoplasmic reticulum.

    Science.gov (United States)

    Chen, L Y; Chen, X; Tian, X L; Yu, X H

    2000-10-01

    To understand whether the molecular mechanism of Tetrandrine (Tet)'s pharmacological effects is concerned with sarcoplasmic reticulum calcium transport so as to be involved in myocardial contractility, we observed the effects of Tet on calcium transport and membrane structure of rabbit skeletal muscle sarcoplasmic reticulum vesicles (SR) and rat cardiac sarcoplasmic reticulum vesicles (CSR). Calcium uptake was monitored with a dual-wavelength spectrophotometer. Protein conformation and fluorescence polarization were measured by fluospectrophotometric method and membrane lipids labelled with fluorescence probes for SR, respectively. 128 micromol l(-1) Tet reduced the initial rate of calcium uptake to 59% of control 6 min after reaction. Tet un-competitively inhibited SR Ca(2+), Mg(2+)-ATPase activity, causing the stoichiometric ratio of SR Ca(2+)/ATP to decrease to 1.43 from 2.0 of control. Inhibitory rates on SR Ca(2+),Mg(2+)-ATPase by Tet were reduced from 60% in the absence of phosphate to 50% in the presence of phosphate and reduced from 92% in 1 mmol l(-1) ATP to 60% in 5 mmol l(-1) ATP. Tet markedly reduced SR intrinsic protein fluorescence, while it slightly decreased the thiol(SH)-modified protein fluorescence of SR labelled with N-(3-pyrene)-maleimide. Tet slightly increased fluorescence polarization in the middle and deep layers of SR membrane lipids labelled with 7- or 12-(9-anthroyloxy) stearic acid (AS) probes, whereas it did not change that of SR labelled with 1, 6-diphenyl-1,3,5-hexatrine (DPH). These results revealed that prevention of SR calcium uptake by Tet was due to inhibition of the SR calcium pump Ca(2+),Mg(2+)-ATPase, changes in spatial conformation of the pumps protein molecules and a decrease in the extent of motion of membrane lipid molecules, thus altering the regulation of [Ca(2+)](i) and myocardial contractility.

  16. Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study.

    Science.gov (United States)

    Picard, Martin; Dahmane, Tassadite; Garrigos, Manuel; Gauron, Carole; Giusti, Fabrice; le Maire, Marc; Popot, Jean-Luc; Champeil, Philippe

    2006-02-14

    Amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. Previous work has suggested both advantages and disadvantages to the use of a polyacrylate-based amphipol, A8-35, for studying the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). We investigated this issue further using a set of four amphipols with different chemical structures. Previous size exclusion chromatography experiments had shown that A8-35 and SERCA1a/A8-35 complexes aggregate under certain conditions. We show here that aggregation can be prevented by omitting calcium from buffers or by using a sulfonated version of A8-35. A8-35 had previously been shown to protect Ca2+-ATPase from irreversible denaturation, while inhibiting its activity in a reversible manner. We show here that the other three amphipols tested also display these properties and that all four amphipols slow down backward calcium dissociation from the nonphosphorylated solubilized enzyme, a priori an unrelated step. As this calcium dissociation involves the opening up of the bundle of transmembrane ATPase segments, the slowing of this process may indicate that multipoint attachment of the polymers to the hydrophobic transmembrane surface damps protein dynamics ("Gulliver" effect). Damping might be the reason why amphipols also simultaneously protect membrane proteins against irreversible denaturation and may inhibit the activity of those of them that display large rearrangements of their transmembrane surface during their catalytic cycle.

  17. 3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

    Science.gov (United States)

    Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

    2012-05-01

    3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA.

  18. Ca2+ and endoplasmic reticulum Ca2+-ATPase regulate the formation of silk fibers with favorable mechanical properties.

    Science.gov (United States)

    Wang, Xin; Li, Yi; Xie, Kang; Yi, Qiying; Chen, Quanmei; Wang, Xiaohuan; Shen, Hong; Xia, Qingyou; Zhao, Ping

    2015-02-01

    Calcium ions (Ca(2+)) are crucial for the conformational transition of silk fibroin in vitro, and silk fibroin conformations correlate with the mechanical properties of silk fibers. To investigate the relationship between Ca(2+) and mechanical properties of silk fibers, CaCl2 was injected into silkworms (Bombyx mori). Fourier-transform infrared spectroscopy (FTIR) analysis and mechanical testing revealed that injection of CaCl2 solution (7.5mg/g body weight) significantly increased the levels of α-helix and random coil structures of silk proteins. In addition, extension of silk fibers increased after CaCl2 injection. In mammals, sarcoplasmic reticulum Ca(2+)-ATPase in muscle and endoplasmic reticulum Ca(2+)-ATPase in other tissues (together denoted by SERCA) are responsible for calcium balance. Therefore, we analyzed the expression pattern of silkworm SERCA (BmSERCA) in silk glands and found that BmSERCA was abundant in the anterior silk gland (ASG). After injection of thapsigargin (TG) to block SERCA activity, silkworms showed a silk-spinning deficiency and their cocoons had higher calcium content compared to that of controls. Moreover, FTIR analysis revealed that the levels of α-helix and β-sheet structures increased in silk fibers from TG-injected silkworms compared to controls. The results provide evidence that BmSERCA has a key function in calcium transportation in ASG that is related to maintaining a suitable ionic environment. This ionic environment with a proper Ca(2+) concentration is crucial for the formation of silk fibers with favorable mechanical performances.

  19. Decavanadate, decaniobate, tungstate and molybdate interactions with sarcoplasmic reticulum Ca(2+)-ATPase: quercetin prevents cysteine oxidation by vanadate but does not reverse ATPase inhibition.

    Science.gov (United States)

    Fraqueza, Gil; Batista de Carvalho, Luís A E; Marques, M Paula M; Maia, Luisa; Ohlin, C André; Casey, William H; Aureliano, Manuel

    2012-11-07

    Recently we demonstrated that the decavanadate (V(10)) ion is a stronger Ca(2+)-ATPase inhibitor than other oxometalates, such as the isoelectronic and isostructural decaniobate ion, and the tungstate and molybdate monomer ions, and that it binds to this protein with a 1 : 1 stoichiometry. The V(10) interaction is not affected by any of the protein conformations that occur during the process of calcium translocation (i.e. E1, E1P, E2 and E2P) (Fraqueza et al., J. Inorg. Biochem., 2012). In the present study, we further explore this subject, and we can now show that the decaniobate ion, [Nb(10) = Nb(10)O(28)](6-), is a useful tool in deducing the interaction and the non-competitive Ca(2+)-ATPase inhibition by the decavanadate ion [V(10) = V(10)O(28)](6-). Moreover, decavanadate and vanadate induce protein cysteine oxidation whereas no effects were detected for the decaniobate, tungstate or molybdate ions. The presence of the antioxidant quercetin prevents cysteine oxidation, but not ATPase inhibition, by vanadate or decavanadate. Definitive V(IV) EPR spectra were observed for decavanadate in the presence of sarcoplasmic reticulum Ca(2+)-ATPase, indicating a vanadate reduction at some stage of the protein interaction. Raman spectroscopy clearly shows that the protein conformation changes that are induced by V(10), Nb(10) and vanadate are different from the ones induced by molybdate and tungstate monomer ions. Here, Mo and W cause changes similar to those by phosphate, yielding changes similar to the E1P protein conformation. The putative reduction of vanadium(V) to vanadium(IV) and the non-competitive binding of the V(10) and Nb(10) decametalates may explain the differences in the Raman spectra compared to those seen in the presence of molybdate or tungstate. Putting it all together, we suggest that the ability of V(10) to inhibit the Ca(2+)-ATPase may be at least in part due to the process of vanadate reduction and associated protein cysteine oxidation. These

  20. Calcium handling by the sarcoplasmic reticulum during oscillatory contractions of skinned skeletal muscle fibres.

    Science.gov (United States)

    Szentesi, P; Zaremba, R; Stienen, G J

    1998-08-01

    Isometric ATP consumption and force were investigated in mechanically skinned fibres from iliofibularis muscle of Xenopus laevis. Measurements were performed at different [Ca2+], in the presence and absence of caffeine (5 nM). In weakly Ca2+-buffered solutions without caffeine, spontaneous oscillations in force and ATPase activity occurred. The repetition frequency was [Ca2+]-and temperature-dependent. The Ca2+ threshold (+/- SEM) for the oscillations corresponded to a pCa of 6.5 +/- 0.1. The maximum ATP consumption associated with calcium uptake by the sarcoplasmic reticulum (SR) reached during the oscillations was similar to the activity under steady-state conditions at saturating calcium concentrations in the presence of caffeine. Maximum activity was reached when the force relaxation was almost complete. The calculated amount of Ca2+ taken up by the SR during a complete cycle corresponded to 5.4 +/ 0.4 mmol per litre cell volume. In strongly Ca2+-buffered solutions, caffeine enhanced the calcium sensitivity of the contractile apparatus and, at low calcium concentrations, SR Ca uptake. These results suggest that when the SR is heavily loaded by net Ca uptake, there is a massive calcium-induced calcium release. Subsequent net Ca uptake by the SR then gives rise to the periodic nature of the calcium transient.

  1. Stable inhibition of brain synaptic plasma membrane calcium ATPase in rats anesthetized with halothane.

    Science.gov (United States)

    Franks, J J; Horn, J L; Janicki, P K; Singh, G

    1995-01-01

    The authors recently showed that plasma membrane Ca(2+)-ATPase (PMCA) activity in cerebral synaptic plasma membrane (SPM) is diminished in a dose-related fashion during exposure in vitro to halothane, isoflurane, xenon, and nitrous oxide at clinically relevant partial pressures. They have now extended their work to in vivo studies, examining PMCA pumping in SPM obtained from control rats decapitated without anesthetic exposure, from rats decapitated during halothane anesthesia, and from rats decapitated after recovery from halothane anesthesia. Three treatment groups were studied: 1) C, control rats that were decapitated without anesthetic exposure, 2) A, anesthetized rats exposed to 1 minimum effective dose (MED) for 20 min and then decapitated, and 3) R, rats exposed to 1 MED for 20 min and then decapitated after recovery from anesthesia, defined as beginning to groom. Plasma membrane Ca(2+)-ATPase pumping and Ca(2+)-dependent ATPase hydrolytic activity, as well as sodium-calcium exchanger activity and Na+-K+-ATPase hydrolytic activity, were assessed in cerebral SPM. In addition, halothane effect on smooth endoplasmic reticulum Ca(2+)-ATPase (SERCA) was examined. Plasma membrane Ca(2+)-ATPase transport of Ca2+ into SPM vesicles from anesthetized rats was reduced to 71% of control (P SPM from rats killed while anesthetized with halothane, compared with rats killed without anesthesia or after recovery from anesthesia. The studies described in this report, in conjunction with previously reported inhibition of PMCA activity in vitro by a wide range of anesthetic agents, indicate a relationship between inhibition of PMCA and action of inhalational anesthetics.

  2. Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression.

    Science.gov (United States)

    Zatyka, Malgorzata; Da Silva Xavier, Gabriela; Bellomo, Elisa A; Leadbeater, Wendy; Astuti, Dewi; Smith, Joel; Michelangeli, Frank; Rutter, Guy A; Barrett, Timothy G

    2015-02-01

    Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca(2+) imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca(2+) concentration ([Ca(2+)]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis.

  3. The calcium uptake of the rat heart sarcoplasmic reticulum is altered by dietary lipid.

    Science.gov (United States)

    Taffet, G E; Pham, T T; Bick, D L; Entman, M L; Pownall, H J; Bick, R J

    1993-01-01

    Small amounts of dietary n-3 fatty acids can have dramatic physiological effects, including the reduction of plasma triglycerides and an elevation of cellular eicosapentanoic (EPA) and docosahexanoic acids (DHA) at the expense of arachidonic acid (AA). We investigated the effects of alterations in the fatty acid compositions of cardiac sarcoplasmic reticulum (CSR) produced by dietary manipulation on the calcium pump protein that is required for energy dependent calcium transport. CSR was isolated from rats fed menhaden oil, which is rich in n-3 fatty acids, and from control animals that were given corn oil. Relative to control membranes, those isolated from rats fed menhaden oil, had a lower content of saturated phospholipids, an increased DHA/AA ratio, and an increased ratio of n-3 to n-6 fatty acids. These changes were associated with a 30% decrease in oxalate-facilitated, ATP-dependent calcium uptake and concomitant decreased Ca-ATPase activity in the membranes from the animals fed menhaden oil. In contrast, there was no alteration in active pump sites as measured by phosphoenzyme formation. Thus, the CSR Ca-ATPase function can be altered by dietary interventions that change the composition, and possibly structure, of the phospholipid membranes thereby affecting enzyme turnover.

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

    Science.gov (United States)

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

    2016-04-29

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-04-29

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

  6. Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis.

    Science.gov (United States)

    Strosova, Miriam K; Karlovska, Janka; Zizkova, Petronela; Kwolek-Mirek, Magdalena; Ponist, Silvester; Spickett, Corinne M; Horakova, Lubica

    2011-07-01

    Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression.

  7. Geographic structuring of the Plasmodium falciparum sarco(endoplasmic reticulum Ca2+ ATPase (PfSERCA gene diversity.

    Directory of Open Access Journals (Sweden)

    Ronan Jambou

    Full Text Available Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp, of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC(50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates. This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte.

  8. Molecular characterisation of the smooth endoplasmic reticulum Ca(2+)-ATPase of Porcellio scaber and its expression in sternal epithelia during the moult cycle.

    Science.gov (United States)

    Hagedorn, Monica; Weihrauch, Dirk; Towle, David W; Ziegler, Andreas

    2003-07-01

    The anterior sternal epithelial cells of the terrestrial isopod Porcellio scaber transport large amounts of calcium during the formation and resorption of intermittent calcium carbonate deposits. Recent investigations on epithelia involved in mineralisation processes suggest a role of the smooth endoplasmic reticulum Ca(2+)-ATPase (SERCA) in transcellular calcium transport. We present the first molecular characterisation of a SERCA within a crustacean mineralising epithelium. We cloned the SERCA from a cDNA library of the anterior sternal epithelium and used in situ hybridisation to compare the expression of the SERCA mRNA between three different moulting stages. The full-length SERCA cDNA has an open reading frame of 3006 nucleotides. The deduced 1002 amino-acid polypeptide has a predicted molecular mass of 109.7 kDa and 87% identity to the SERCA of Procambarus clarkii axial muscle isoform. In situ hybridisation confirmed expression within the anterior sternal epithelium and revealed an increase in SERCA mRNA abundance from the non-transporting, early premoult stage to the calcium transporting, late premoult and intramoult stage. The results support previous indications of a contribution by the smooth endoplasmic reticulum to transcellular calcium transport and suggest a transcriptional regulation of SERCA activity.

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

    Science.gov (United States)

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

    2016-09-01

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

  10. Lipogenesis mitigates dysregulated sarcoplasmic reticulum calcium uptake in muscular dystrophy.

    Science.gov (United States)

    Paran, Christopher W; Zou, Kai; Ferrara, Patrick J; Song, Haowei; Turk, John; Funai, Katsuhiko

    2015-12-01

    Muscular dystrophy is accompanied by a reduction in activity of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) that contributes to abnormal Ca(2+) homeostasis in sarco/endoplasmic reticulum (SR/ER). Recent findings suggest that skeletal muscle fatty acid synthase (FAS) modulates SERCA activity and muscle function via its effects on SR membrane phospholipids. In this study, we examined muscle's lipid metabolism in mdx mice, a mouse model for Duchenne muscular dystrophy (DMD). De novo lipogenesis was ~50% reduced in mdx muscles compared to wildtype (WT) muscles. Gene expressions of lipogenic and other ER lipid-modifying enzymes were found to be differentially expressed between wildtype (WT) and mdx muscles. A comprehensive examination of muscles' SR phospholipidome revealed elevated phosphatidylcholine (PC) and PC/phosphatidylethanolamine (PE) ratio in mdx compared to WT mice. Studies in primary myocytes suggested that defects in key lipogenic enzymes including FAS, stearoyl-CoA desaturase-1 (SCD1), and Lipin1 are likely contributing to reduced SERCA activity in mdx mice. Triple transgenic expression of FAS, SCD1, and Lipin1 (3TG) in mdx myocytes partly rescued SERCA activity, which coincided with an increase in SR PE that normalized PC/PE ratio. These findings implicate a defect in lipogenesis to be a contributing factor for SERCA dysfunction in muscular dystrophy. Restoration of muscle's lipogenic pathway appears to mitigate SERCA function through its effects on SR membrane composition.

  11. Cloning, Characterization, and Expression Patterns of Three Sarco/Endoplasmic Reticulum Ca2+-ATPase Isoforms from Pearl Oyster (Pinctada fucata)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A large amount of calcium is required for mollusk biomineralization. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a well-known protein with the function of sustaining the calcium homeostasis. How does it possibly function in the process of pearl oyster biomineralization? Three SERCA isoforms, namely PSERA, PSERB, and PSERC were cloned from the pearl oyster, Pinctada fucata. The cDNAs of the three isoforms were isolated by reverse transcription-polymerase chain reaction (RT-PCR)and rapid amplification of cDNA ends. PSERA consisted of 3568 bp encoding 1007 amino acids, PSERB included 3953 bp encoding 1024 amino acids, and PSERC comprised of 3450 bp encoding 1000 amino acids.The three isoforms showed high homology (65%-87%) with SERCAs from other species. Consistent with the results from other invertebrates, Southern blot analysis revealed that the three isoforms originated from a single gene that was also related to SERCA1, SERCA2, and SERCA3 of vertebrates. The splicing mechanism of the three isoforms was similar to that of isoforms of vertebrate SERCA3. Semiquantitative RT-PCR was carried out to study the expression patterns of the three isoforms. The results showed that PSERB was ubiquitously expressed in all tested tissues and was a potential "housekeeping" SERCA isoform; PSERA was expressed in the adductor muscle and foot and was likely to be a muscle-specific isoform, and PSERC was expressed in the other tissues except the adductor muscle or foot with the highest expression levels in the gill and mantle, indicating that it was a non-muscle-specific isoform and might be involved in calcium homeostasis during pearl oyster biomineralization.

  12. Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition.

    Science.gov (United States)

    Dalton, K A; East, J M; Mall, S; Oliver, S; Starling, A P; Lee, A G

    1998-02-01

    The sarcoplasmic reticulum of skeletal muscle contains anionic phospholipids as well as the zwitterionic phosphatidylcholine and phosphatidylethanolamine. Here we study the effects of anionic phospholipids on the activity of the Ca2+-ATPase purified from the membrane. Reconstitution of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C18:1)PS] or dioleoylphosphatidic acid [di(C18:1)PA] leads to a decrease in ATPase activity. Measurements of the quenching of the tryptophan fluorescence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcholine close to 1 and suggest that phosphatidic acid only binds to the ATPase at the bulk lipid sites around the ATPase. Addition of di(C18:1)PS or di(C18:1)PA to the ATPase in the short-chain dimyristoleoylphosphatidylcholine [di(C14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the response of tryptophan fluorescence intensity to Ca2+ binding. It is concluded that the lipid headgroup and lipid fatty acyl chains have separate effects on the function of the ATPase. The anionic phospholipids have no significant effect on Ca2+ binding to the ATPase; the level of Ca2+ binding to the ATPase, the affinity of binding and the rate of dissociation of Ca2+ are unchanged by reconstitution into di(C18:1)PA. The major effect of the anionic lipids is a reduction in the maximal level of binding of MgATP. This is attributed to the formation of oligomers of the Ca2+-ATPase, in which only one molecule of the ATPase can bind MgATP dimers in di(C18:1)PS and trimers or tetramers in di(C18:1)PA. The rates of phosphorylation and dephosphorylation for the proportion of the ATPase still able to bind ATP are unaffected by reconstitution. Larger changes were observed in the level of phosphorylation of the ATPase by Pi, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase

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

    DEFF Research Database (Denmark)

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

    1990-01-01

    Thapsigargin, a tumor-promoting sesquiterpene lactone, discharges intracellular Ca2+ in rat hepatocytes, as it does in many vertebrate cell types. It appears to act intracellularly, as incubation of isolated rat liver microsomes with thapsigargin induces a rapid, dose-dependent release of stored Ca...... isoform of the Ca2(+)-ATPase is highly selective, as thapsigargin has little or no effect on the Ca2(+)-ATPases of hepatocyte or erythrocyte plasma membrane or of cardiac or skeletal muscle sarcoplasmic reticulum. These results suggest that thapsigargin increases the concentration of cytosolic free Ca2...

  14. Glucose-6-phosphate reduces calcium accumulation in rat brain endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Jeffrey Thomas Cole

    2012-04-01

    Full Text Available Brain cells expend large amounts of energy sequestering calcium (Ca2+, while loss of Ca2+ compartmentalization leads to cell damage or death. Upon cell entry, glucose is converted to glucose-6-phosphate (G6P, a parent substrate to several metabolic major pathways, including glycolysis. In several tissues, G6P alters the ability of the endoplasmic reticulum to sequester Ca2+. This led to the hypothesis that G6P regulates Ca2+ accumulation by acting as an endogenous ligand for sarco-endoplasmic reticulum calcium ATPase (SERCA. Whole brain ER microsomes were pooled from adult male Sprague-Dawley rats. Using radio-isotopic assays, 45Ca2+ accumulation was quantified following incubation with increasing amounts of G6P, in the presence or absence of thapsigargin, a potent SERCA inhibitor. To qualitatively assess SERCA activity, the simultaneous release of inorganic phosphate (Pi coupled with Ca2+ accumulation was quantified. Addition of G6P significantly and decreased Ca2+ accumulation in a dose-dependent fashion (1-10 mM. The reduction in Ca2+ accumulation was not significantly different that seen with addition of thapsigargin. Addition of glucose-1-phosphate or fructose-6-phosphate, or other glucose metabolic pathway intermediates, had no effect on Ca2+ accumulation. Further, the release of Pi was markedly decreased, indicating G6P-mediated SERCA inhibition as the responsible mechanism for reduced Ca2+ uptake. Simultaneous addition of thapsigargin and G6P did decrease inorganic phosphate in comparison to either treatment alone, which suggests that the two treatments have different mechanisms of action. Therefore, G6P may be a novel, endogenous regulator of SERCA activity. Additionally, pathological conditions observed during disease states that disrupt glucose homeostasis, may be attributable to Ca2+ dystasis caused by altered G6P regulation of SERCA activity

  15. The yeast p5 type ATPase, spf1, regulates manganese transport into the endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Yifat Cohen

    Full Text Available The endoplasmic reticulum (ER is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn(2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn(2+ in ∆spf1 cells and an increase following it's overexpression. In agreement with the observed loss of luminal Mn(2+ we could observe concurrent reduction in many Mn(2+-related process in the ER lumen. Conversely, cytosolic Mn(2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn(2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn(2+-dependent neurological disorders.

  16. Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

    DEFF Research Database (Denmark)

    Laursen, Mette; Bublitz, Maike; Moncoq, Karine;

    2009-01-01

    Abstract: We have determined the structure of the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) in an E2.P-i-like form stabilized as a complex with MgF42-, an ATP analog, adenosine 5'-(beta,gamma-methylene) triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.......5 angstrom resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which...... is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca2+-ATPases, e. g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing...

  17. The Ca(2+)-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum.

    Science.gov (United States)

    Espinoza-Fonseca, L Michel

    2017-03-28

    Ca(2+) transport across the sarco/endoplasmic reticulum (SR) plays an essential role in intracellular Ca(2+) homeostasis, signalling, cell differentiation and muscle contractility. During SR Ca(2+) uptake and release, proton fluxes are required to balance the charge deficit generated by the exchange of Ca(2+) and other ions across the SR. During Ca(2+) uptake by the SR Ca(2+)-ATPase (SERCA), two protons are countertransported from the SR lumen to the cytosol, thus partially compensating for the charge moved by Ca(2+) transport. Studies have shown that protons are also transported from the cytosol to the lumen during Ca(2+) release, but a transporter that facilitates proton transport into the SR lumen has not been described. In this article we propose that SERCA forms pores that facilitate bidirectional proton transport across the SR. We describe the location and structure of water-filled pores in SERCA that form cytosolic and luminal pathways for protons to cross the SR membrane. Based on this structural information, we suggest mechanistic models for proton translocation to the cytosol during active Ca(2+) transport, and into the SR lumen during SERCA inhibition by endogenous regulatory proteins. Finally, we discuss the physiological consequences of SERCA-mediated bidirectional proton transport across the SR membrane of muscle and non-muscle cells.

  18. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Directory of Open Access Journals (Sweden)

    Narendranath Reddy Chintagari

    Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

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

    DEFF Research Database (Denmark)

    Busti, Stefano; Mapelli, Valeria; Tripodi, Farida

    2016-01-01

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

  20. Short and long range functions of amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase. A mutational study.

    Science.gov (United States)

    Chen, L; Sumbilla, C; Lewis, D; Zhong, L; Strock, C; Kirtley, M E; Inesi, G

    1996-05-01

    Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.

  1. Ca2+ uptake by the endoplasmic reticulum Ca2+-ATPase in rat microvascular endothelial cells.

    Science.gov (United States)

    Moccia, Francesco; Berra-Romani, Roberto; Baruffi, Silvana; Spaggiari, Santina; Signorelli, Silvia; Castelli, Loretta; Magistretti, Jacopo; Taglietti, Vanni; Tanzi, Franco

    2002-01-01

    In non-excitable cells, many agonists increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing an inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from the intracellular stores. Ca(2+) influx from the extracellular medium may then sustain the Ca(2+) signal. [Ca(2+)](i) recovers its resting level as a consequence of Ca(2+)-removing mechanisms, i.e. plasma-membrane Ca(2+)-ATPase (PMCA) pump, Na(+)/Ca(2+) exchanger (NCX) and sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump. In a study performed in pancreatic acinar cells, evidence has been provided suggesting that, during the decay phase of the agonist-evoked Ca(2+) transients, the Ca(2+) concentration within the intracellular stores remains essentially constant [Mogami, Tepikin and Petersen (1998) EMBO J. 17, 435-442]. It was therefore hypothesized that, in such a situation, intracellular Ca(2+) is not only picked up by the SERCA pump, but is also newly released through IP(3)-sensitive Ca(2+) channels, with the balance between these two processes being approximately null. The main aim of the present work was to test this hypothesis by a different experimental approach. Using cardiac microvascular endothelial cells, we found that inhibition of the SERCA pump has no effect on the time course of agonist-evoked Ca(2+) transients. This result was not due to a low capacity of the SERCA pump since, after agonist removal, this pump proved to be very powerful in clearing the excess of intracellular Ca(2+). We showed further that: (i) in order to avoid a rapid removal of Ca(2+) by the SERCA pump, continuous IP(3) production appears to be required throughout all of the decay phase of the Ca(2+) transient; and (ii) Ca(2+) picked up by the SERCA pump can be fully and immediately released by agonist application. All these results support the model of Mogami, Tepikin and Petersen [(1998) EMBO J. 17, 435-442]. Since the SERCA pump did not appear to be involved in shaping the decay phase of the

  2. Endoplasmic reticulum-mitochondria calcium signaling in hepatic metabolic diseases.

    Science.gov (United States)

    Rieusset, Jennifer

    2017-06-01

    The liver plays a central role in glucose homeostasis, and both metabolic inflexibility and insulin resistance predispose to the development of hepatic metabolic diseases. Mitochondria and endoplasmic reticulum (ER), which play a key role in the control of hepatic metabolism, also interact at contact points defined as mitochondria-associated membranes (MAM), in order to exchange metabolites and calcium (Ca(2+)) and regulate cellular homeostasis and signaling. Here, we overview the role of the liver in the control of glucose homeostasis, mainly focusing on the independent involvement of mitochondria, ER and Ca(2+) signaling in both healthy and pathological contexts. Then we focus on recent data highlighting MAM as important hubs for hormone and nutrient signaling in the liver, thus adapting mitochondria physiology and cellular metabolism to energy availability. Lastly, we discuss how chronic ER-mitochondria miscommunication could participate to hepatic metabolic diseases, pointing MAM interface as a potential therapeutic target for metabolic disorders. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. 肌浆网钙ATP酶基因转导对慢性心力衰竭犬心肌蛋白质组影响的初步研究%Overexpression of sarcoplasmic reticulum calcium ATPase induced hemodynamic and proteomic changes in a dog model of heart failure

    Institute of Scientific and Technical Information of China (English)

    付治卿; 李小鹰; 刘秀华; 孙胜; 刘涛; 米亚非; 周声安; 叶卫华; 王青松

    2008-01-01

    handling,myofibrils,and energy production in this dog model of chronic heart failure.%目的 分析心肌肌浆网Ca2+-ATP酶(sarcoplasmic reticulum Ca2+ ATPase 2a,SERCA2a)基因转导对慢性心力衰竭(HF)犬心肌蛋白质组的影响,探讨SERCA2a基因转导改善心功能的机制.方法 快速右心室起搏建立HF犬模型并随机分为HF组、HF+绿色荧光蛋白(enhanced green fluorescent pmtein,EGFP)组、HF+SERCA2a组.后两组分别向心肌内注射携带EGFP和SERCA2a基因的rAAV载体.于基因转导30 d时停止起搏后进行超声心动图和血流动力学检查并制备心室肌双向电泳蛋白样品和心肌双向电泳图谱,图像分析软件分析蛋白表达差异点,MALDI-TOF-MS数据库搜索鉴定蛋白质.结果 基因转导30 d时,HF+SERCA2a组犬的症状、超声心动图和血流动力学指标与HF+EGFP组相比有显著好转(P<0.05);与对照组相比差异无统计学意义(P>0.05).挑选SERCA2a基因转导后表达量发生明显改变的10个蛋白点进行分析,经质谱鉴定分别为心肌收缩相关蛋白、线粒体能量代谢酶类和应激相关蛋白.结论 以rAAV为载体介导SERCA2a基因转导能够改善HF犬心脏的收缩和舒张功能,其可能的机制是恢复了心肌收缩相关蛋白正常表型或正常表达量,增加了心肌能量的产生,改变了应激相关蛋白的表达.

  4. The pmr gene, encoding a Ca2+-ATPase, is required for calcium and manganese homeostasis and normal development of hyphae and conidia in Neurospora crassa.

    Science.gov (United States)

    Bowman, Barry J; Abreu, Stephen; Johl, Jessica K; Bowman, Emma Jean

    2012-11-01

    The pmr gene is predicted to encode a Ca(2+)-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmr(RIP), in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl(2) (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca(2+)-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca(2+)-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmr(RIP) Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.

  5. Temperature and Ca2+-dependence of the sarcoplasmic reticulum Ca2(+)-ATPase in haddock, salmon, rainbow trout and zebra cichlid

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming

    2002-01-01

    Temperature dependence of Ca2+-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degreesC. Whether the break point arises as a result of temperature dependent changes...... nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degreesC, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the. temperature area, 6-14 degreesC. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca2.......5) between 60 and 250 muM. Results indicated that interaction between SR Ca2+-ATPase and its lipid environment may play an important role for the different Arrhenius plot of the different types of fish species investigated. (C) 2002 Elsevier Science Inc. All rights reserved....

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

    DEFF Research Database (Denmark)

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

    1990-01-01

    Thapsigargin, a tumor-promoting sesquiterpene lactone, discharges intracellular Ca2+ in rat hepatocytes, as it does in many vertebrate cell types. It appears to act intracellularly, as incubation of isolated rat liver microsomes with thapsigargin induces a rapid, dose-dependent release of stored Ca....... This hypothesis is strongly supported by the demonstration that thapsigargin causes a rapid inhibition of the Ca2(+)-activated ATPase activity of rat liver microsomes, with an identical dose dependence to that seen in whole cell or isolated microsome Ca2+ discharge. The inhibition of the endoplasmic reticulum...

  7. Sarco(endoplasmic Reticulum Ca2+-ATPase-2 Gene: Structure and Transcriptional Regulation of the Human Gene

    Directory of Open Access Journals (Sweden)

    Angel Zarain-Herzberg

    2002-01-01

    Full Text Available The sarco(endoplasmic reticulum Ca2+-ATPases (SERCAs belong to a family of active calcium transport enzymes encoded by the SERCA1, 2, and 3 genes. In this study, we describe the complete structure of the human SERCA2 gene and its 5’ -regulatory region. The hSERCA2 gene is located in chromosome 12 position q24.1 in Contig NT_009770.8, spans 70 kb, and is organized in 21 exons intervened by 20 introns. The last two exons of the pre-mRNA produce by alternatively splicing the cardiac/slow-twitch muscle-specific SERCA2a isoform and the ubiquitous SERCA2b isoform. The sequence of the proximal 225-bp regulatory region of the SERCA2 genes is 80% G+C-rich and is conserved among human, rabbit, rat, and mouse species. It contains a TATA-like-box, an E-box/USF sequence, a CAAT-box, four Sp1 binding sites, and a thyroid hormone responsive element (TRE. There are two other conserved regulatory regions located between positions -410 to -661 bp and from -919 to -1410 bp. Among the DNA cis-elements present in these two regulatory regions there are potential binding sites for: GATA-4, -5, -6, Nkx-2.5/Csx, OTF-1, USF, MEF-2, SRF, PPAR/RXR, AP-2, and TREs. Upstream from position -1.5 kb, there is no significant homology among the SERCA2 genes cloned. In addition, the human gene has several repeated sequences mainly of the Alu and L2 type located upstream from position -1.7 kb, spanning in a continuous fashion for more than 40 kb. In this study, we report the cloning of 2.4 kb of 5’-regulatory region and demonstrate that the proximal promoter region is sufficient for expression in cardiac myocytes, and the region from -225 to -1232 bp contains regulatory DNA elements which down-regulate the expression of the SERCA2 gene in neonatal cardiomyocytes.

  8. Cardiac function improved by sarcoplasmic reticulum Ca2+-ATPase overexpression in a heart failure model induced by chronic myocardial ischemia

    Directory of Open Access Journals (Sweden)

    Wei XIN

    2011-04-01

    Full Text Available Objective Chronic myocardial ischemia(CMI has become an important cause of heart failure(HF.The aim of present study was to examine the effects of Sarco-endoplasmic reticulum calcium ATPase(SERCA2a gene transfer in HF model in large animal induced by CMI.Methods HF was reproduced in minipigs by ligating the initial segment of proximal left anterior descending(LAD coronary artery with an ameroid constrictor to produce progressive vessel occlusion and ischemia.After confirmation of myocardial perfusion defect and cardiac function impairment by SPECT and echocardiography in the model,animals were divided into 4 groups: HF group;HF+enhanced green fluorescent protein(EGFP group;HF+SERCA2a group;and sham operation group as control.rAAV1-EGFP and rAAV1-SERCA2a(1×1012 vg for each animal were directly and intramyocardially injected to the animals of HF+EGFP and HF+SERCA2a groups.Sixty days after the gene transfer,the expression of SERCA2a at the protein level was examined by Western blotting and immunohistochemistry,the changes in cardiac function were determined by echocardiographic and hemodynamic analysis,and the changes in serum inflammatory and neuro-hormonal factors(including BNP,TNF-a,IL-6,ET-1 and Ang II were determined by radioimmunoassay.Results Sixty days after gene transfer,LVEF,Ev/Av and ±dp/dtmax increased significantly(P < 0.05,along with an increase of SERCA2a protein expression in the ischemic myocardium(PP < 0.05,accompanied by a significant decrease of inflammatory and neural-hormonal factors(PP < 0.05 in HF+SERCA2a group as compared with HF/HF+EGFP group.Conclusions Overexpression of SERCA2a may significantly improve the cardiac function of the ischemic myocardium of HF model induced by CMI and reverse the activation of neural-hormonal factors,implying that it has a potential therapeutic significance in CMI related heart failure.

  9. Improvement in cardiac function after sarcoplasmic reticulum Ca2+-ATPase gene transfer in a beagle heart failure model

    Institute of Scientific and Technical Information of China (English)

    MI Ya-fei; LI Xiao-ying; TANG Li-jiang; LU Xiao-chun; FU Zhi-qing; YE Wei-hua

    2009-01-01

    Background Heart failure (HF) is a major cause of morbidity and mortality worldwide, but current treatment modalities cannot reverse the underlying pathological state of the heart. Gene-based therapies are emerging as promising therapeutic modalities in HF patients. Our previous studies have shown that recombinant adeno-associated viral (rAAV) gene transfer of Sarco-endoplasmic reticulum calcium ATPase (SERCA2a) can be effective in treating rats with chronic heart failure (CHF). The aim of this study was to examine the effects of SERCA2a gene transfer in a large HF animal model.Methods HF was induced in beagles by rapid right ventricular pacing (230 beats/min) for 30 days. A reduced rate ventricular pacing (180 beats/min) was continued for another 30 days. The beagles were assigned to four groups: (a) control group (n=4); (b) HF group (n=4); (c) enhanced green fluorescent protein group (n=4); and (d) SERCA2.a group (n=4). rAAVl-EGFP (lx1012 μg) and rAAVl-SERCA2a (lx1012 μg) were delivered intramyocardially. SERCA2.a expression was assessed by Western blotting and immunohistochemistry.Results Following 30 days of SERCA2a gene transfer in HF beagles its protein expression was significantly higher than in the HF group than in the control group (P <0.05). Heart function improved along with the increase in SERCA2a expression. Left ventricular systolic function significantly improved, including the ejection fraction, left ventricular systolic pressure, maximal rate of rise of left ventricular pressure (+dp/dtmax), and the maximal rate of decline of left ventricular pressure (-dp/dtmax) (P <0.05). Left ventricular end-diastole pressure significantly decreased (P <0.05). The expression of SERCA2a in the myocardial tissue was higher in the SERCA2a group than in the HF group (P<0.05). Conclusions Intramyocardial injection of rAAVl-SERCA2a can improve the cardiac function in beagles induced with HE We expect further studies on SERCA2a's long-term safety, efficacy, dosage

  10. Phospholamban Modulates the Functional Coupling between Nucleotide Domains in Ca-ATPase Oligomeric Complexes in Cardiac Sarcoplasmic Reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Chen, L.; Yao, Qing; Soares, Thereza A.; Squier, Thomas C.; Bigelow, Diana J.

    2009-03-24

    Oligomeric interactions between Ca-ATPase polypeptide chains and their modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic reticulum (SR) microsomes. Progressive modification of Lys514 with fluorescein-5-isothiocyanate (FITC), which physically blocks access to the nucleotide binding site by ATP, demonstrates that Ca-ATPase active sites function independently of one another prior to the phosphorylation of PLB. However, upon PKA-dependent phosphorylation of PLB, a second-order dependence between enzyme activity and the fraction of active sites is observed, consistent with a dimeric functional complex. Complementary distance measurements were made using FITC or 5-iodoacetamido-fluorescein (IAF) bound to Cys674 within the N- or P-domains respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 ± 2 Å decrease in the proximity between FITC sites within the N-domain and a 9 ± 3 Å increase in the proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB induces spatial rearrangements between the N- and P-domain elements of proximal Ca-ATPase polypeptide chains which restore functional interactions between neighboring polypeptide chains and, in turn, result in increased rates of catalytic turnover. These results are interpreted in terms of a structural model, calculated through optimization of shape complementarity, desolvation, and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase polypeptide chains through the proximal association of N-domains. We suggest that the phosphorylation of PLB acts to release constraints involving interdomain subunit interactions that enhance catalytically important N-domain motions.

  11. Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells

    DEFF Research Database (Denmark)

    Cardozo, Alessandra K; Ortis, Fernanda; Storling, Joachim

    2005-01-01

    , beta-cells showed marked sensitivity to apoptosis induced by SERCA blockers, as compared with fibroblasts. Cytokine-induced ER Ca(2+) depletion was paralleled by an NO-dependent induction of CHOP protein and activation of diverse components of the ER stress response, including activation of inositol......Cytokines and free radicals are mediators of beta-cell death in type 1 diabetes. Under in vitro conditions, interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma) induce nitric oxide (NO) production and apoptosis in rodent and human pancreatic beta-cells. We have previously shown......, by microarray analysis of primary beta-cells, that IL-1beta + IFN-gamma decrease expression of the mRNA encoding for the sarcoendoplasmic reticulum pump Ca(2+) ATPase 2b (SERCA2b) while inducing expression of the endoplasmic reticulum stress-related and proapoptotic gene CHOP (C/EBP [CCAAT/enhancer binding...

  12. Sulindac sulfide inhibits sarcoendoplasmic reticulum Ca2+ ATPase, induces endoplasmic reticulum stress response, and exerts toxicity in glioma cells: relevant similarities to and important differences from celecoxib.

    Science.gov (United States)

    White, M C; Johnson, G G; Zhang, W; Hobrath, J V; Piazza, G A; Grimaldi, M

    2013-03-01

    Malignant gliomas have low survival expectations regardless of current treatments. Nonsteroidal anti-inflammatory drugs (NSAIDs) prevent cell transformation and slow cancer cell growth by mechanisms independent of cyclooxygenase (COX) inhibition. Certain NSAIDs trigger the endoplasmic reticulum stress response (ERSR), as revealed by upregulation of molecular chaperones such as GRP78 and C/EBP homologous protein (CHOP). Although celecoxib (CELE) inhibits the sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA), an effect known to induce ERSR, sulindac sulfide (SS) has not been reported to affect SERCA. Here, we investigated these two drugs for their effects on Ca(2+) homeostasis, ERSR, and glioma cell survival. Our findings indicate that SS is a reversible inhibitor of SERCA and that both SS and CELE bind SERCA at its cyclopiazonic acid binding site. Furthermore, CELE releases additional Ca(2+) from the mitochondria. In glioma cells, both NSAIDS upregulate GRP78 and activate ER-associated caspase-4 and caspase-3. Although only CELE upregulates the expression of CHOP, it appears that CHOP induction could be associated with mitochondrial poisoning. In addition, CHOP induction appears to be uncorrelated with the gliotoxicity of these NSAIDS in our experiments. Our data suggest that activation of ERSR is primarily responsible for the gliotoxic effect of these NSAIDS. Because SS has good brain bioavailability, has lower COX-2 inhibition, and has no mitochondrial effects, it represents a more appealing molecular candidate than CELE to achieve gliotoxicity via activation of ERSR.

  13. Decavanadate, decaniobate, tungstate and molybdate interactions with sarcoplasmic reticulum Ca2+-ATPase: quercetin prevents cysteine oxidation by vanadate but does not reverse ATPase inhibition

    OpenAIRE

    Fraqueza, Gil; de Carvalho, Luís A. E. Batista; Marques, M. Paula M.; Maia, Luisa; Ohlin, C. André; Casey, William H.; Aureliano, M.

    2012-01-01

    Recently we demonstrated that the decavanadate (V10) ion is a stronger Ca2+-ATPase inhibitor than other oxometalates, such as the isoelectronic and isostructural decaniobate ion, and the tungstate and molybdate monomer ions, and that it binds to this protein with a 1 : 1 stoichiometry. The V10 interaction is not affected by any of the protein conformations that occur during the process of calcium translocation (i.e. E1, E1P, E2 and E2P) (Fraqueza et al., J. Inorg. Biochem., 2012). In the p...

  14. Endoplasmic reticulum stress in pathogenesis of diabetic retinopathy and effect of calcium dobesilate

    Institute of Scientific and Technical Information of China (English)

    Yu-Min Gui; Ming Zhao; Jie Ding

    2016-01-01

    Objective:To study the mechanism of endoplasmic reticulum stress in the pathogenesis of diabetic retinopathy and effect of calcium dobesilate.Methods:A total of 120 diabetic retinopathy patients treated in our hospital from January 2010 to September 2015 were enrolled in this article. The serum endoplasmic reticulum stress protein and interleukin protein expression levels were analyzed before and after calcium dobesilate treatment. A total of 55 cases of healthy subjects receiving physical examination in our hospital during the same period were taken as control group.Results:Serum endoplasmic reticulum stress proteins PERK, CHOP and IRE as well as interleukin proteins IL1, IL2, IL6 and IL10 expression significantly increased, serum MDA level significantly increased while SOD, CAT and GSHpx levels significantly decreased in diabetic retinopathy patients, and compared with control group (P<0.01); after calcium dobesilate treatment, above factors were significantly restored (P<0.01).Conclusions: Diabetic retinopathy is closely related to endoplasmic reticulum stress and calcium dobesilate treatment may improve diabetic retinopathy by inhibiting endoplasmic reticulum stress.

  15. The role of the calcium transporter protein plasma membrane calcium ATPase PMCA2 in cerebellar Purkinje neuron function.

    Science.gov (United States)

    Empson, R M; Akemann, W; Knöpfel, Thomas

    2010-01-01

    Genetic deletion of the plasma membrane calcium ATPase type 2 (PMCA2), a calcium transporter protein, is associated with an overtly ataxic phenotype in mice. PMCA2 is expressed at high levels in cerebellar Purkinje neurons (PNs) where functional integrity is essential for normal cerebellar function. Indeed, loss of PN function accompanies cerebellar ataxia in humans and mouse models. In the ataxic PMCA2 knockout (PMCA2-/-) mouse the ability of the PNs to control their cytosolic calcium levels was severely impaired; basal calcium levels were high and calcium recovery kinetics slow. Whole cell patch clamp recordings from PMCA2-/- PNs revealed that they possessed hyperpolarised membrane potentials, reduced frequency and increased irregularity of spontaneous action potential firing, curtailed complex spikes and sustained calcium-dependent outward K+ currents. We propose that these alterations limit pathological excursions in PN cytosolic calcium as an aid to survival but that they are insufficient to prevent loss of functional cerebellar output.

  16. The changes of cardioelectrical activity of rat with myocardial infarction receiving sarcoplasmic reticulum Ca2+-ATPase gene modified bone marrow stem cell transplantation by microelectrode array technology

    Institute of Scientific and Technical Information of China (English)

    范平

    2012-01-01

    Objective Therapy effects and cardiac electrical activity comparison of bone marrow stem cells (BMSCs) transplantation and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) gene modified BMSCs transplantation after acute myocardial infarction(AMI) in rats.Methods Rats with AMI were divided

  17. Calcium uptake by sarcoplasmic reticulum in the presence of organophosphorus insecticide methyl-parathion

    Energy Technology Data Exchange (ETDEWEB)

    Blasiak, J. [Lodz Univ. (Poland)

    1995-12-31

    Using an isotope labelling technique it has been shown that an organophosphorus insecticide methyl parathion (0,0-diethyl 0-4-nitrophenyl phosphorothionate) depressed calcium uptake by sarcoplasmic reticulum isolated from rabbit hind leg muscle. The effect was significant for insecticide concentrations of 50 and 100 {mu}M and was dose-dependent. The insecticide exerted a more pronounced effect on calcium uptake in the presence of ATP in the reticulum environment than in the absence of ATP. The inhibitory action of methyl parathion on Ca{sup 2+} accumulation by sarcoplasmic reticulum can cause a rise in myoplasmic free Ca{sup 2+}, the essential prerequisite for contracture activation. Because methyl parathion, as well as other organophosphorus insecticides, is primarily neurotoxic, evidence of non-specific effect could be important for assessing its environmental safety. (author). 20 refs, 2 figs.

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

    Science.gov (United States)

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

    2016-06-16

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

  19. Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

    Science.gov (United States)

    Kjelstrup, Signe; de Meis, Leopoldo; Bedeaux, Dick; Simon, Jean-Marc

    2008-11-01

    We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca(2+) in the Ca(2+)-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca(2+)-uptake, and heat up during ATP synthesis and Ca(2+)-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca(2+)-ATPases, not only act as Ca(2+)-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

  20. Decrease in sarcoplasmic reticulum calcium content, not myofilament function, contributes to muscle twitch force decline in isolated cardiac trabeculae

    Science.gov (United States)

    Milani-Nejad, Nima; Brunello, Lucia; Gyorke, Sándor; Janssen, Paul M.L.

    2014-01-01

    We set out to determine the factors responsible for twitch force decline in isolated intact rat cardiac trabeculae. The contractile force of trabeculae declined over extended periods of isometric twitch contractions. The force-frequency relationship within the frequency range of 4–8 Hz, at 37 °C, became more positive and the frequency optimum shifted to higher rates with this decline in baseline twitch tensions. The post-rest potentiation (37 °C), a phenomenon highly dependent on calcium handling mechanisms, became more pronounced with decrease in twitch tensions. We show that the main abnormality during muscle run-down was not due to a deficit in the myofilaments; maximal tension achieved using a K+ contracture protocol was either unaffected or only slightly decreased. Conversely, the sarcoplasmic reticulum (SR) calcium content, as assessed by rapid cooling contractures (from 27 °C to 0 °C), decreased, and had a close association with the declining twitch tensions (R2 ~ 0.76). SR Ca2+-ATPase, relative to Na+/Ca2+ exchanger activity, was not altered as there was no significant change in paired rapid cooling contracture ratios. Furthermore, confocal microscopy detected no abnormalities in the overall structure of the cardiomyocytes and t-tubules in the cardiac trabeculae (~23 °C). Overall, the data indicates that the primary mechanism responsible for force run-down in multi-cellular cardiac preparations is a decline in the SR calcium content and not the maximal tension generation capability of the myofilaments. PMID:25056841

  1. Comparative studies of cardiac and skeletal sarcoplasmic reticulum ATPases. Effect of a phospholamban antibody on enzyme activation by Ca2+.

    Science.gov (United States)

    Cantilina, T; Sagara, Y; Inesi, G; Jones, L R

    1993-08-15

    Vesicular fragments of skeletal (LSR) and cardiac (CSR) sarcoplasmic reticulum were compared with the aim of defining the effect of a monoclonal phospholamban (Pl) antibody (Ab). The Pl Ab has no effect on LSR, while enhancing the Ca2+ transport rates of CSR at Ca2+ concentrations below saturation. We found no direct effect of the Pl Ab on Ca2+ binding by the ATPase in the absence of ATP. Equilibrium measurements of Ca2+ binding yield positively cooperative isotherms which are best fit with a two-interacting sites equation. LSR and CSR display nearly identical affinities for Ca2+, and no effect of the Pl Ab is observed. Taking advantage of a stable CrATP-enzyme complex, we demonstrated that the stoichiometric ratio of occluded Ca2+ to catalytic sites is 2 in either LSR or CSR and that the addition of Pl Ab does not affect the Ca2+ concentration dependence of Ca2+ occluded after equilibration of the system. Interestingly, the cooperative interaction between the two Ca2+ sites is lost in the occluded state, with only one of the two sites acquiring lumenal exposure. The concentration dependence of Ca2+ inhibition of CSR ATPase phosphorylation with Pi is also unaffected by the Pl Ab. Contrary to the lack of Pl Ab effect on reactions measured at equilibrium, enhancement of phosphorylated intermediate formation by the Pl Ab is obtained in kinetic experiments in which nonsaturating Ca2+ and ATP are added to CSR preincubated with EGTA. Therefore, Ab binding to Pl reduces specifically the activation energy for a slow transition triggered by Ca2+ binding, with consequent enhancement of overall kinetics under conditions enhancing the rate-limiting contribution of this transition.

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

    Directory of Open Access Journals (Sweden)

    Junjie Zhang

    2015-03-01

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

  3. Luteolin improves cardiac dysfunction in heart failure rats by regulating sarcoplasmic reticulum Ca2+-ATPase 2a

    Science.gov (United States)

    Hu, Wenjing; Xu, Tongda; Wu, Pei; Pan, Defeng; Chen, Junhong; Chen, Jing; Zhang, Buchun; Zhu, Hong; Li, Dongye

    2017-01-01

    We previously found that luteolin (Lut) appeared to improve the contractility of cardiomyocytes during ischemia/reperfusion in rats. The enhancement was associated with the alteration in sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a). This finding prompted us to consider if the mechanism worked in heart failure (HF). We studied the regulation of SERCA2a by Lut in failing cardiomyocytes and intact heart of rats. Improvement of contractility and the mechanisms centered on SERCA2a were studied in isolated cardiomyocytes and intact heart. We found that Lut significantly improved contractility and Ca2+ transients, ameliorated expression, activity and stability of SERCA2a and upregulated expression of small ubiquitin-related modifier (SUMO) 1, which is a newfound SERCA2a regulator. Lut also increased phosphorylation of protein kinase B (Akt), phospholaban (PLB) and sumoylation of SERCA2a, specificity protein 1 (Sp1). Transcriptions of SUMO1 and SERCA2a were concurrently increased. Inhibition of posphatidylinositol 3 kinase/Akt (PI3K/Akt) pathway and SERCA2a activity both markedly abolished Lut-induced benefits in vitro and in vivo. Lut upregulated the expression ratio of Bcl-2/Bax, caspase-3/cleaved-Caspase3. Meanwhile, Lut ameliorated the myocardium fibrosis of HF. These discoveries provide an important potential therapeutic strategy that Lut targeted SERCA2a SUMOylation related to PI3K/Akt-mediated regulations on rescuing the dysfunction of HF.

  4. Luteolin Exerts Cardioprotective Effects through Improving Sarcoplasmic Reticulum Ca2+-ATPase Activity in Rats during Ischemia/Reperfusion In Vivo

    Directory of Open Access Journals (Sweden)

    Changsheng Nai

    2015-01-01

    Full Text Available The flavonoid luteolin exists in many types of fruits, vegetables, and medicinal herbs. Our previous studies have demonstrated that luteolin reduced ischemia/reperfusion (I/R injury in vitro, which was related with sarcoplasmic reticulum Ca2+-ATPase (SERCA2a activity. However, the effects of luteolin on SERCA2a activity during I/R in vivo remain unclear. To investigate whether luteolin exerts cardioprotective effects and to monitor changes in SERCA2a expression and activity levels in vivo during I/R, we created a myocardial I/R rat model by ligating the coronary artery. We demonstrated that luteolin could reduce the myocardial infarct size, lactate dehydrogenase release, and apoptosis during I/R injury in vivo. Furthermore, we found that luteolin inhibited the I/R-induced decrease in SERCA2a activity in vivo. However, neither I/R nor luteolin altered SERCA2a expression levels in myocardiocytes. Moreover, the PI3K/Akt signaling pathway played a vital role in this mechanism. In conclusion, the present study has confirmed for the first time that luteolin yields cardioprotective effects against I/R injury by inhibiting the I/R-induced decrease in SERCA2a activity partially via the PI3K/Akt signaling pathway in vivo, independent of SERCA2a protein level regulation. SERCA2a activity presents a novel biomarker to assess the progress of I/R injury in experimental research and clinical applications.

  5. Effects of Calcium on ATPase Activity and Lipid Composition of Plasma Membranes from Wheat Roots Under Aluminum Stress

    Institute of Scientific and Technical Information of China (English)

    HE Long-fei; SHEN Zhen-guo; LIU You-liang

    2003-01-01

    Effects of calcium on ATPase activities, lipid contents, and fatty acid compositions of plasma membrane from wheat roots were assayed under aluminum stress. The results showed that the increase of calcium concentration in the nutrient solution increased the activity of H+-ATPase and the phospholipid content, decreased the activity of Ca2+-ATPase and the galactolipid of plasma membrane. Owing to the decrease of linolenic acid content, the index of unsaturated fatty acid (IUFA) and index of double bond (DBI) decreased in Altas66. The IUFA and DBI of plasma membrane from Scout66 roots increased because its linolenic acid content increased obviously and its palmitic acid content decreased apparently.

  6. The contribution of the sodium-calcium exchanger (NCX) and plasma membrane Ca(2+) ATPase (PMCA) to cerebellar synapse function.

    Science.gov (United States)

    Roome, Chris J; Empson, Ruth M

    2013-01-01

    The cerebellum, a part of the brain critically involved in motor learning and sensory adaptation, expresses high levels of the sodium-calcium exchanger (NCX) and the plasma membrane calcium ATPase (PMCA). Both these transporters control calcium dynamics at a variety of synapses, and here, we draw upon the available literature to discuss how NCX and PMCA work together to shape pre-synaptic calcium dynamics at cerebellar synapses.

  7. Plasma membrane calcium ATPase proteins as novel regulators of signal transduction pathways

    Institute of Scientific and Technical Information of China (English)

    Mary; Louisa; Holton; Michael; Emerson; Ludwig; Neyses; Angel; L; Armesilla

    2010-01-01

    Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular freecalcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulindependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.

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

    Science.gov (United States)

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

    2010-01-01

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

  9. Effects of type 1 diabetes, sprint training and sex on skeletal muscle sarcoplasmic reticulum Ca2+ uptake and Ca2+-ATPase activity.

    Science.gov (United States)

    Harmer, A R; Ruell, P A; Hunter, S K; McKenna, M J; Thom, J M; Chisholm, D J; Flack, J R

    2014-02-01

    Calcium cycling is integral to muscle performance during the rapid muscle contraction and relaxation of high-intensity exercise. Ca(2+) handling is altered by diabetes mellitus, but has not previously been investigated in human skeletal muscle. We investigated effects of high-intensity exercise and sprint training on skeletal muscle Ca(2+) regulation among men and women with type 1 diabetes (T1D, n = 8, 3F, 5M) and matched non-diabetic controls (CON, n = 8, 3F, 5M). Secondarily, we examined sex differences in Ca(2+) regulation. Subjects undertook 7 weeks of three times-weekly cycle sprint training. Before and after training, performance was measured, and blood and muscle were sampled at rest and after high-intensity exercise. In T1D, higher Ca(2+)-ATPase activity (+28%) and Ca(2+) uptake (+21%) than in CON were evident across both times and days (P women across both times and days. Intense exercise did not alter Ca(2+)-ATPase activity in T1D or CON. However, sex differences were evident: Ca(2+)-ATPase was reduced with exercise among men but increased among women across both days (time × sex interaction, P Sprint training reduced Ca(2+)-ATPase (-8%, P Sprint training reduced Ca(2+)-ATPase in T1D and CON. Sex differences in Ca(2+)-ATPase activity were evident and may be linked with fibre type proportion differences.

  10. Calcium signaling and endoplasmic reticulum dynamics during fertilization in marine protostome worms belonging to the phylum Nemertea.

    Science.gov (United States)

    Stricker, Stephen A

    2014-08-01

    Metaphase-I-arrested eggs of marine protostome worms in the phylum Nemertea generate a series of point-source calcium waves during fertilization. Such calcium oscillations depend on inositol-1,4,5-trisphosphate-mediated calcium release from endoplasmic reticulum (ER) stores that undergo structural reorganizations prior to and after fertilization. This article reviews fertilization-induced calcium transients and ER dynamics in nemertean eggs and compares these topics to what has been reported for other animals in order to identify unifying characteristics and distinguishing features of calcium responses during fertilization across the animal kingdom. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Inositol (1,4,5)-trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes.

    Science.gov (United States)

    Suárez-Isla, B. A.; Irribarra, V.; Oberhauser, A.; Larralde, L.; Bull, R.; Hidalgo, C.; Jaimovich, E.

    1988-01-01

    Sarcoplasmic reticulum membrane vesicles isolated from frog skeletal muscle display high conductance calcium channels when fused into phospholipid bilayers. The channels are selective for calcium and barium over Tris. The fractional open time was voltage-independent (-40 to +25 mV), but was steeply dependent on the free cis [Ca2+] (P0 = 0.02 at 10 microM cis Ca2+ and 0.77 at 150 microM Ca2+; estimated Hill coefficient: 1.6). Addition of ATP (1 mM; cis) further increased P0 from 0.77 to 0.94. Calcium activation was reversed by addition of EGTA to the cis compartment. Magnesium (2 mM) increased the frequency of rapid closures and 8 mM magnesium decreased the current amplitude from 3.4 to 1.2 pA at 0 mV, suggesting a reversible fast blockade. Addition of increasing concentrations of inositol (1, 4, 5)-triphosphate (cis), increased P0 from 0.10 +/- 0.01 (mean +/- SEM) in the control to 0.85 +/- 0.02 at 50 microM in an approximately sigmoidal fashion, with an apparent half-maximal activation at 15 microM inositol (1, 4, 5)-trisphosphate in the presence of 40 microM cis Ca2+. Lower concentrations of this agonist were required to produce a significant increase in P0 when 10 microM or less cis Ca2+ were used. The channel was blocked by the addition to the cis compartment of either 0.5 mM lanthanum, 0.5 microM ruthenium red, or 200 nM ryanodine, all known inhibitors of Ca2+ release from sarcoplasmic reticulum vesicles. These results demonstrate the presence of calcium channels in the sarcoplasmic reticulum from frog skeletal muscle with a pharmacological profile consistent with a role in excitation contraction coupling and with the hypothesis that inositol ( 1,4,5)-trisphosphate is a physiological agonist in this process. PMID:2852037

  12. Advances in the study of Plasmodium falciparum calcium ATPase 6 (PfATP6)%恶性疟原虫钙ATP蛋白6(PfATP6)研究进展

    Institute of Scientific and Technical Information of China (English)

    宋营改

    2012-01-01

    Plasmodium falciparum (Pf) calcium ATPase 6 (PfATP6) is an antimalaria target of artemisinin and its derivatives. PfATPG is a type of sarco/endoplasmic reticulum calcium ATPase (SERC'A). Its function is to maintain the calcium concentration within the cytoplasm of Pf through the consumption of ATP. Artemisinin and its derivatives suppress the function of PfATP6. increasing the concentration of calcium in the cytoplasm of Pf and killing it. Plasmodium develops drug resistance through mutations in PfATP6. Artemisinin and its derivatives can also suppress the SERCA of cancer cells, inducing changes in the calcium concentration within the cytoplasm of cancer cells and activating cancer cell apoptosis. Artemisinin and its derivatives can also he used to treat Toxoplastna gondii . Bahesia, and Pneu/nocystis ji-rovecii infections mainly through the mechanism of SERCA suppression. These phenomena have increased the significance of studies of PfATPG SERC'A and they have increased the clinic use of artemisinin and its derivatives.%恶性疟原虫钙ATP蛋白6(Plasmodium falciparum calcium ATPase 6,PfATP6)是青蒿素及其衍生物作用靶点之一,PfATP6是一类肌浆网/内质网钙ATP酶(sarco/endoplasmic reticulum calcium ATPase,SERCA),它通过消耗ATP来调节疟原虫胞浆内钙离子浓度,保持钙浓度的内稳状态.青蒿素及其衍生物通过抑制PfATP6,从而引发疟原虫胞浆内钙离子浓度上升,起到杀疟作用;疟原虫也通过PfATP6基因突变出现耐药现象.青蒿素及其衍生物通过对肿瘤细胞的SERCA抑制,引发肿瘤细胞内钙离子浓度的变化,激活凋亡程序,导致肿瘤细胞死亡;还能通过抑制SERCA治疗刚地弓形虫、巴贝斯虫和耶氏肺孢子菌感染,使PfATP6/SERCA基因研究更为重要,青蒿素及其衍生物临床应用更为广泛.

  13. Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [γ-(32)P]TNP-8N3-ATP Photolabeling.

    Science.gov (United States)

    Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter

    2016-01-01

    The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements.

  14. Endoplasmic reticulum calcium regulates the retrotranslocation of Trypanosoma cruzi calreticulin to the cytosol.

    Directory of Open Access Journals (Sweden)

    Carlos A Labriola

    Full Text Available For most secretory pathway proteins, crossing the endoplasmic reticulum (ER membrane is an irreversible process. However, in some cases this flow can be reversed. For instance, misfolded proteins retained in the ER are retrotranslocated to the cytosol to be degraded by the proteasome. This mechanism, known as ER associated degradation (ERAD, is exploited by several bacterial toxins to gain access to the cytosol. Interestingly, some ER resident proteins can also be detected in the cytosol or nucleus, calreticulin (CRT being the most studied. Here we show that in Trypanosoma cruzi a minor fraction of CRT localized to the cytosol. ER calcium depletion, but not increasing cytosolic calcium, triggered the retrotranslocation of CRT in a relatively short period of time. Cytosolic CRT was subsequently degraded by the proteasome. Interestingly, the single disulfide bridge of CRT is reduced when the protein is located in the cytosol. The effect exerted by ER calcium was strictly dependent on the C-terminal domain (CRT-C, since a CRT lacking it was totally retained in the ER, whereas the localization of an unrelated protein fused to CRT-C mirrored that of endogenous CRT. This finding expands the regulatory mechanisms of protein sorting and may represent a new crossroad between diverse physiological processes.

  15. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes.

    Science.gov (United States)

    Garrity, Abigail G; Wang, Wuyang; Collier, Crystal Md; Levey, Sara A; Gao, Qiong; Xu, Haoxing

    2016-05-23

    Impaired homeostasis of lysosomal Ca(2+) causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca(2+) are not known. We developed a physiological assay to monitor lysosomal Ca(2+) store refilling using specific activators of lysosomal Ca(2+) channels to repeatedly induce lysosomal Ca(2+) release. In contrast to the prevailing view that lysosomal acidification drives Ca(2+) into the lysosome, inhibiting the V-ATPase H(+) pump did not prevent Ca(2+) refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca(2+) prevented lysosomal Ca(2+) stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca(2+) refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca(2+) or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca(2+)for the lysosome.

  16. The Ca(2+)-ATPase of the sarcoplasmic reticulum in skeletal and cardiac muscle. An overview from the very beginning to more recent prospects.

    Science.gov (United States)

    Hasselbach, W

    1998-09-16

    The discovery of the ATP-driven calcium pump in the sarcoplasmic reticulum membranes reaches back to the postwar (World War II) years and would not be possible without the generous support by the American scientific community. It was this community that in pre- and postwar years gave shelter to many European scientists, which in return stimulated scientific development in the United States. These pre- and postwar relations helped to establish the calcium pump as a physiologically relevant mechanism in all kinds of cells. The pump and its counterpart, the calcium release channel, proved to be controlled by various intrinsic mechanisms. Rising hydrogen concentrations as occurring in ischemic muscles switch off pump activity and counteract allosterically caffeine-induced calcium release (CICR). Rising phosphate or the presence of other calcium-precipitating anions, on the other hand, prevents pump inhibition by intraluminal calcium precipitation, which, simultaneously, can increase the quantity of releasable calcium. The inactivation of CICR by removing medium chloride must be considered as a hint of additional mechanisms by which calcium-dependent activity regulation can be modified.

  17. The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy

    Science.gov (United States)

    Mohamed, Tamer M. A.; Abou-Leisa, Riham; Stafford, Nicholas; Maqsood, Arfa; Zi, Min; Prehar, Sukhpal; Baudoin-Stanley, Florence; Wang, Xin; Neyses, Ludwig; Cartwright, Elizabeth J.; Oceandy, Delvac

    2016-01-01

    The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition. PMID:27020607

  18. Ca2+ uptake, Ca2+-ATPase activity, phosphoprotein formation and phosphate turnover in a microsomal fraction of smooth muscle.

    Science.gov (United States)

    Raeymaekers, L; Hasselbach, W

    1981-05-15

    Vesicles capable of phosphate-stimulated calcium uptake were isolated from the microsomal fraction of the smooth muscle of the pig stomach according to a previously described procedure which consists in increasing the density of the vesicles by loading them with calcium phosphate and isolating them by centrifugation [Raeymaekers, L., Agostini, B., and Hasselbach, W. (1981) Histochemistry, 70, 139--150]. These vesicles, which contain calcium phosphate deposits, are able to accumulate an additional amount of calcium. This calcium uptake is accompanied by calcium-stimulated ATPase activity and by the formation of an acid-stable phosphoprotein. The acid-denatured phosphoprotein is dephosphorylated by hydroxylamine, which indicates that an acylphosphate is formed. This phosphoprotein probably represents a phosphorylated transport intermediate similar to that seen with the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle. As with the Ca2+-ATPase of sarcoplasmic reticulum vesicles, this vesicular fraction catalyses an exchange between inorganic phosphate and the gamma-phosphate of ATP (ATP-Pi exchange) which is dependent on the presence of intravesicular calcium, and an exchange of phosphate between ATP and ADP (ATP-ADP exchange). The results further indicate that the turnover rate of the calcium pump, calculated from the ratio of calcium-stimulated ATPase activity to the steady-state level of phosphoprotein, is similar to that of Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle.

  19. Role of endoplasmic reticulum calcium signaling in the pathogenesis of Alzheimer disease

    Directory of Open Access Journals (Sweden)

    Elena ePopugaeva

    2013-09-01

    Full Text Available Alzheimer disease (AD is a major threat of XXI century that is responsible for the majority of dementia in the elderly. Development of effective AD-preventing therapies are the top priority tasks for neuroscience research. Amyloid hypothesis of AD is a dominant idea in the field, but so far all amyloid-targeting therapies have failed in clinical trials. In addition to amyloid accumulation, there are consistent reports of abnormal calcium signaling in AD neurons. AD neurons exhibit enhanced intracellular calcium (Ca2+ liberation from the endoplasmic reticulum (ER and reduced store-operated Ca2+ entry (SOC. These changes occur primarily as a result of ER Ca2+ overload. We argue that normalization of intracellular Ca2+ homeostasis could be a strategy for development of effective disease-modifying therapies. The current review summarizes recent data about changes in ER Ca2+ signaling in AD. Ca2+ channels that are discussed in the current review include: inositol trisphosphate receptors (InsP3R, ryanodine receptors (RyanR, presenilins as ER Ca2+ leak channels and neuronal SOC channels. We discuss how function of these channels is altered in AD and how important are resulting Ca2+ signaling changes for AD pathogenesis.

  20. Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

    DEFF Research Database (Denmark)

    Laursen, Mette; Bublitz, Maike; Moncoq, Karine

    2009-01-01

    .5 angstrom resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which...... is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca2+-ATPases, e. g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing...

  1. Sarcoplasmic reticulum calcium release in frog skeletal muscle fibres estimated from Arsenazo III calcium transients.

    Science.gov (United States)

    Baylor, S M; Chandler, W K; Marshall, M W

    1983-01-01

    Single twitch fibres, dissected from frog muscle, were injected with the metallochromic dye Arsenazo III. Changes in dye-related absorbance measured at 650 or 660 nm were used to estimate the time course of myoplasmic free [Ca2+] following either action potential stimulation or voltage-clamp depolarization (temperature, 15-17 degrees C). The amplitude of the Ca2+ transient decreased when fibres were stretched to sarcomere spacings approaching 4 microns. The effect appeared to be less marked in H2O Ringer than in D2O Ringer, where a reduction of about 40% was observed in going from 3.0 microns to 3.7-3.9 microns. In fibres heavily injected with dye (1.5-2.2 mM-dye) at least 0.1 mM-Ca2+ was complexed with Arsenazo III following a single action potential, implying that at least 0.1 mM-Ca2+ was released from the sarcoplasmic reticulum (s.r.) into the myoplasm. Computer simulations were carried out to estimate the flux of Ca2+ between the s.r. and myoplasm (in fibres containing no more that 0.8 mM-dye). The amounts and time courses of Ca2+ bound to the Ca2+-regulatory sites on troponin and to the Ca2+, Mg2+ sites on parvalbumin were estimated from the free [Ca2+] wave form and the law of mass action. In the computations the total myoplasmic [Ca2+] was taken as the total amount of Ca2+ existing either as free ion or as ion complexed with dye, troponin or parvalbumin. The time derivative of total myoplasmic [Ca2+] was used as an estimate of net Ca2+ flux (release minus uptake) from the s.r. into myoplasm. Rate constants for formation of cation: receptor complex were taken from published values. For the Ca2+-regulatory sites on troponin, three sets of rate constants, corresponding to two values of dissociation constant (0.2 and 2 microM) were used. Each set of three simulations was carried out both with and without parvalbumin. The simulations show that following action potential stimulation, 0.2-0.3 mM-Ca2+ enters the myoplasm from the s.r. The wave form of s.r. Ca2

  2. Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes.

    Science.gov (United States)

    Pierce, D H; Scarpa, A; Trentham, D R; Topp, M R; Blasie, J K

    1983-12-01

    Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane is x-ray diffraction. Therefore, the kinetics of ATP-induced calcium uptake by isolated SR vesicles in dispersions and hydrated, oriented multilayers were compared. Ca2+ uptake was necessarily initiated by the addition of ATP through flash photolysis of caged ATP, P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate, with either a frequency-doubled ruby laser or a 200 W Hg arc lamp, and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III, which is sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 degrees to 26 degrees C. The Ca2+ uptake kinetics of SR membranes in both the vesicular dispersions and oriented multilayers consist of at least two phases, an initial fast phase and a subsequent slow phase. The fast phase, generally believed to be associated with the formation of the phosphorylated enzyme, E approximately P, is kinetically comparable in both SR dispersions and multilayers. The slow phase mathematically follows first-order kinetics with specific rate constants of approximately 0.6 s-1 and approximately 1.2 s-1 for the dispersions at 26 degrees C and multilayers at 21 degrees C, respectively, with the given experimental conditions. The slow phase, generally believed to be associated with the translocation of Ca+2, across the membrane profile, appears to be the same process in SR dispersions and multilayers through their virtually identical rate constants

  3. Interactions of vanadium(V)-citrate complexes with the sarcoplasmic reticulum calcium pump.

    Science.gov (United States)

    Aureliano, Manuel; Tiago, Teresa; Gândara, Ricardo M C; Sousa, Andrea; Moderno, A; Kaliva, M; Salifoglou, A; Duarte, Rui O; Moura, José J G

    2005-12-01

    Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR). To this end, initial research efforts were launched with two vanadium(V)-citrate complexes, namely (NH(4))(6)[V(2)O(4)(C(6)H(4)O(7))(2)].6H(2)O and (NH(4))(6)[V(2)O(2)(O(2))(2)(C(6)H(4)O(7))(2)].4H(2)O, potentially capable of interacting with the SR calcium pump by combining kinetic studies with (51)V NMR spectroscopy. Upon dissolution in the reaction medium (concentration range: 4-0.5mM), both vanadium(V):citrate (VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A 1mM solution of the PVC complex, containing 184microM of the PVC complex, 94microM oxoperoxovanadium(V) (PV) species, 222microM monomeric (V1), 43microM dimeric (V2) and 53microM tetrameric (V4) species, inhibits Ca(2+) accumulation by 75 %, whereas a solution of the VC complex of the same vanadium concentration, containing 98microM of the VC complex, 263microM monomeric (V1), 64microM dimeric (V2) and 92microM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a 1 mM metavanadate solution, containing 460microM monomeric (V1), 90.2microM dimeric (V2) and 80microM tetrameric (V4) species, has no effect on Ca(2+) accumulation. The NMR signals from the VC complex (-548.0ppm), PVC complex (-551.5ppm) and PV (-611.1ppm) are broadened upon SR vesicle addition (2.5mg/ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the interaction with the protein, was found to be V4>PVC>VC>PV>V2=V1=1, with no effect observed for the V1 and V2 signals. Putting it all together the effects of two vanadium(V)-citrate complexes on the modulation of calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity into the nature of key species forming upon dissolution of the title complexes in the reaction media.

  4. Direct demonstration of Ca2+ binding defects in sarco-endoplasmic reticulum Ca2+ ATPase mutants overexpressed in COS-1 cells transfected with adenovirus vectors.

    Science.gov (United States)

    Strock, C; Cavagna, M; Peiffer, W E; Sumbilla, C; Lewis, D; Inesi, G

    1998-06-12

    Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+ inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 microM Ca2+ and in the absence of ATP, the Glu771 --> Gln, Thr799 --> Ala, Asp800 --> Asn, and Glu908 --> Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 --> Gln and the Asn796 --> Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 --> Gln mutant does not bind any Ca2+, and its phosphorylation by Pi is not inhibited by Ca2+. On the contrary, the Asn796 --> Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 --> Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 --> Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+ can be overcome by raising

  5. Cloning of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) gene from white shrimp, Litopenaeus vannamei and its expression level analysis under salinity stress.

    Science.gov (United States)

    Wang, Yanhong; Luo, Peng; Zhang, Lvping; Hu, Chaoqu; Ren, Chunhua; Xia, Jianjun

    2013-11-01

    Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is an intracellular membrane bound enzyme that utilizes the free energy of ATP to transport Ca(2+) against a concentration gradient. In the present study, a new SERCA gene (LvSERCA) from white shrimp (Litopenaeus vannamei) was cloned using suppression subtractive hybridization and rapid amplification of cDNA ends. The full-length cDNA of LvSERCA contained an open reading frame of 3,009 bp coding for 1,002 amino acids with a calculated molecular weight of approximately 109.8 kDa. The identity analysis of the amino acid sequence of LvSERCA showed that it is highly conserved with 10 transmembrane α-helices, one P-domain, one A-domain and one N-domain. The phylogenetic analysis revealed that LvSERCA is similar to other Arthropoda SERCA proteins. The mRNA levels of LvSERCA under salinity stress (3 and 40 g L(-1)) were analyzed by reverse transcription PCR and quantitative real-time PCR. The results showed that LvSERCA was expressed in all tissues detected. LvSERCA mRNA levels were significantly higher under hyper-salinity than hypo-salinity. These results highlight that Ga(2+)-ATPase plays an essential role in adjustment salinity stress, which may be useful for selective breeding of L. vannamei.

  6. Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Ren-Jie Tang; Hua Liu; Yang Yang; Lei Yang; Xiao-Shu Gao; Veder J Garcia; Sheng Luan; Hong-Xia Zhang

    2012-01-01

    Plant responses to developmental and environmental cues are often mediated by calcium(Ca2+)signals that are transmitted by diverse calcium sensors.The calcineurin B-like(CBL)protein family represents calcium sensors that decode calcium signals through specific interactions with a group of CBL-interacting protein kinases.We report functional analysis of Arabidopsis CBL2 and CBL3,two closely related CBL members that are localized to the vacuolar membrane through the N-terminal tonoplast-targeting sequence.While cbl2 or cbl3 single mutant did not show any phenotypic difference from the wild type,the cbl2 cbl3 double mutant was stunted with leaf tip necrosis,underdeveloped roots,shorter siliques and fewerseeds.These defects were reminiscent of those in the vha-a2 vha-a3 double mutant deficient in vacuolar H+-ATPase(V-ATPase).Indeed,the V-ATPase activity was reduced in the cbl2 cbl3 double mutant,connecting tonoplast CBL-type calcium sensors to the regulation of V-ATPase.Furthermore,cbl2 cbl3 double mutant was compromised in ionic tolerance and micronutrient accumulation,consistent with the defect in V-ATPase activity that has been shown to function in ion compartmentalization.Our results suggest that calcium sensors CBL2 and CBL3 serve as molecular links between calcium signaling and V-ATPase,a central regulator of intracellular ion homeostasis.

  7. The Plasma Membrane Ca2+ ATPase and the Plasma Membrane Sodium Calcium Exchanger Cooperate in the Regulation of Cell Calcium

    Science.gov (United States)

    Brini, Marisa; Carafoli, Ernesto

    2011-01-01

    Calcium is an ambivalent signal: it is essential for the correct functioning of cell life, but may also become dangerous to it. The plasma membrane Ca2+ ATPase (PMCA) and the plasma membrane Na+/Ca2+ exchanger (NCX) are the two mechanisms responsible for Ca2+ extrusion. The NCX has low Ca2+ affinity but high capacity for Ca2+ transport, whereas the PMCA has a high Ca2+ affinity but low transport capacity for it. Thus, traditionally, the PMCA pump has been attributed a housekeeping role in maintaining cytosolic Ca2+, and the NCX the dynamic role of counteracting large cytosolic Ca2+ variations (especially in excitable cells). This view of the roles of the two Ca2+ extrusion systems has been recently revised, as the specific functional properties of the numerous PMCA isoforms and splicing variants suggests that they may have evolved to cover both the basal Ca2+ regulation (in the 100 nM range) and the Ca2+ transients generated by cell stimulation (in the μM range). PMID:21421919

  8. Role of plasma membrane calcium ATPase 2 in spinal cord pathology

    Institute of Scientific and Technical Information of China (English)

    Amanda; Kathleen; Fakira; Stella; Elkabes

    2010-01-01

    A number of studies have indicated that plasma membrane calcium ATPases(PMCAs) are expressed in the brain and spinal cord and could play important roles not only in the maintenance of cellular calcium homeostasis but also in the survival and function of central nervous system cells under pathological conditions.The different regional and cellular distributions of the various PMCA isoforms and splice variants in the nervous system and the diverse phenotypes of PMCA knockout mice support the notion that each isoform might play a distinct role. Especially in the spinal cord,the survival of neurons and,in particular,motor neurons could be dependent on PMCA2.This is indicated by the knockdown of PMCA2 in pure spinal cord neuronal cultures that leads to cell death via a decrease in collapsing response mediator protein 1 levels.Moreover,the progressive decline in the number of motor neurons in PMCA2-null mice andheterozygous mice further supports this notion.Therefore,the reported reduction in PMCA2 mRNA and protein levels in the inflamed spinal cord of mice affected by experimental autoimmune encephalomyelitis(EAE) ,an animal model of multiple sclerosis,and after spinal cord contusion injury,suggests that changes in PMCA2 expression could be a cause of neuronal pathology and death during inflammation and injury.Glutamate excitotoxicity mediated via kainate receptors has been implicated in the neuropathology of both EAE and spinal cord injury,and has been identified as a trigger that reduces PMCA2 levels in pure spinal cord neuronal cultures through degradation of the pump by calpain without affecting PMCA2 transcript levels.It remains to be determined which other stimuli modulate PMCA2 mRNA expression in the aforementioned pathological conditions of the spinal cord.

  9. Islet Oxygen Consumption and Insulin Secretion Tightly Coupled to Calcium Derived from L-type Calcium Channels but Not from the Endoplasmic Reticulum*

    OpenAIRE

    Gilbert, Merle; Jung, Seung-Ryoung; Reed, Benjamin J.; Sweet, Ian R.

    2008-01-01

    The aim of the study was to test whether the source of intracellular calcium (Ca2+) is a determinant of beta cell function. We hypothesized that elevations in cytosolic Ca2+ caused by the release of Ca2+ from the endoplasmic reticulum (ER) have little physiologic impact on oxygen consumption and insulin secretion. Ca2+ release from the ER was induced in isolated rat islets by acetylcholine and response of oxygen consumption rate (OCR), NAD(P)H, cytosolic Ca2+, and ...

  10. Temperature and Ca2+-dependence of the sarcoplasmic reticulum Ca2(+)-ATPase in haddock, salmon, rainbow trout and zebra cichlid

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming

    2002-01-01

    in the enzyme or its membrane lipid environment is still a matter of discussion. In this study we compared the temperature dependence and Ca2+-dependence of SR Ca2+-ATPase in haddock (Melanogrammus aeglefinus), salmon (Salmo, salar), rainbow trout (Oncorhynchus mykiss) and zebra cichlid (Cichlasoma...... nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degreesC, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the. temperature area, 6-14 degreesC. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca2...

  11. Concerted but Noncooperative Activation of Nucleotide and Actuator Domains of the Ca-ATPase Upon Calcium Binding

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baowei; Mahaney, James E.; Mayer, M. Uljana; Bigelow, Diana J.; Squier, Thomas C.

    2008-11-25

    Calcium-dependent domain movements of the nucleotide (N) and actuator (A) domains of the SERCA2a isoform of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4’,5’-bis(1,3,2-dithoarsolan-2-yl)fluorescein (FlAsH-EDT2). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT2. Distance measurements using the nucleotide analog TNP-ATP, which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 Å increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures. No significant distance changes occur across the N-domain between FlAsH and TNP-ATP, indicating that calcium activation induces rigid body domain movements rather than intradomain conformational changes. Calcium-dependent decreases in the fluorescence of FlAsH bound respectively to either the N- or A-domains indicate coordinated and noncooperative domain movements, where both N- and A-domains domains display virtually identical calcium dependencies (i.e., Kd = 4.8 ± 0.4 μM). We suggest that occupancy of a single high-affinity calcium binding site induces the rearrangement of the A- and N-domains of the Ca-ATPase to form an intermediate state, which facilitates ATP utilization upon occupancy of the second high-affinity calcium site to enhance transport efficiency.

  12. A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.

    Science.gov (United States)

    Henderson, Mark J; Baldwin, Heather A; Werley, Christopher A; Boccardo, Stefano; Whitaker, Leslie R; Yan, Xiaokang; Holt, Graham T; Schreiter, Eric R; Looger, Loren L; Cohen, Adam E; Kim, Douglas S; Harvey, Brandon K

    2015-01-01

    Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

  13. A Low Affinity GCaMP3 Variant (GCaMPer for Imaging the Endoplasmic Reticulum Calcium Store.

    Directory of Open Access Journals (Sweden)

    Mark J Henderson

    Full Text Available Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19. A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19 fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19 has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

  14. The Ubiquitin Regulatory X (UBX) Domain-containing Protein TUG Regulates the p97 ATPase and Resides at the Endoplasmic Reticulum-Golgi Intermediate Compartment*

    Science.gov (United States)

    Orme, Charisse M.; Bogan, Jonathan S.

    2012-01-01

    p97/VCP is a hexameric ATPase that is coupled to diverse cellular processes, such as membrane fusion and proteolysis. How p97 activity is regulated is not fully understood. Here we studied the potential role of TUG, a widely expressed protein containing a UBX domain, to control mammalian p97. In HEK293 cells, the vast majority of TUG was bound to p97. Surprisingly, the TUG UBX domain was neither necessary nor sufficient for this interaction. Rather, an extended sequence, comprising three regions of TUG, bound to the p97 N-terminal domain. The TUG C terminus resembled the Arabidopsis protein PUX1. Similar to the previously described action of PUX1 on AtCDC48, TUG caused the conversion of p97 hexamers into monomers. Hexamer disassembly was stoichiometric rather than catalytic and was not greatly affected by the p97 ATP-binding state or by TUG N-terminal regions in vitro. In HeLa cells, TUG localized to the endoplasmic reticulum-to-Golgi intermediate compartment and endoplasmic reticulum exit sites. Although siRNA-mediated TUG depletion had no marked effect on total ubiquitylated proteins or p97 localization, TUG overexpression caused an accumulation of ubiquitylated substrates and targeted both TUG and p97 to the nucleus. A physiologic role of TUG was revealed by siRNA-mediated depletion, which showed that TUG is required for efficient reassembly of the Golgi complex after brefeldin A removal. Together, these data support a model in which TUG controls p97 oligomeric status at a particular location in the early secretory pathway and in which this process regulates membrane trafficking in various cell types. PMID:22207755

  15. Structural dynamics and topology of phosphorylated phospholamban homopentamer reveal its role in the regulation of calcium transport in sarcoplasmic reticulum

    Science.gov (United States)

    Vostrikov, Vitaly V.; Mote, Kaustubh R.; Verardi, Raffaello; Veglia, Gianluigi

    2013-01-01

    Phospholamban (PLN) inhibits the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), thereby regulating cardiac diastole. In membranes, PLN assembles into homopentamers that in both the phosphorylated and non-phosphorylated states have been proposed to form ion-selective channels. Here, we determined the structure of the phosphorylated pentamer using a combination of solution and solid-state nuclear magnetic resonance methods. We found that the pinwheel architecture of the homopentamer is preserved upon phosphorylation, with each monomer having an L-shaped conformation of each monomer. The TM domains form a hydrophobic pore of approximately 24 Å long, and 2 Å in diameter, which is inconsistent with canonical Ca2+ selective channels. Phosphorylation, however, enhances the conformational dynamics of the cytoplasmic region of PLN, causing the partial unwinding of the amphipathic helix. We propose that PLN oligomers act as storage for active monomers, keeping SERCA function within a physiological window. PMID:24207128

  16. Lack of effect of flurothyl, a non-anesthetic fluorinated ether, on rat brain synaptic plasma membrane calcium-ATPase.

    Science.gov (United States)

    Horn, J L; Janicki, P K; Franks, J J

    1999-01-01

    Plasma membrane Ca2+-ATPase (PMCA), a regulator of intracellular calcium, is inhibited by volatile anesthetics and by xenon and nitrous oxide. Response of a cellular system to anesthetics, particularly to volatile agents, raises the question of non-specific, even toxic, side effects unrelated to anesthetic action. Compounds with chemical and physical properties similar to halogenated anesthetics, but which lack anesthetic effect, have been used to address this question. We have compared the effects of halothane and flurothyl, a non-anesthetic fluorinated ether, on PMCA Ca2+ transport across isolated brain synaptic plasma membranes (SPM). Flurothyl, at concentrations predicted by the Meyer-Overton curve to range from 0.4 to 2.6 MAC (minimum alveolar concentration), had no significant on PMCA activity. In contrast halothane, 1.3 MAC, reduced Ca2+ transport 30 to 40%. These findings provide further evidence for a specific effect of inhalation anesthetics on neuronal plasma membrane Ca2+-ATPase.

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

    Science.gov (United States)

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

    2017-05-01

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

  18. The secretory pathway calcium ATPase PMR-1/SPCA1 has essential roles in cell migration during Caenorhabditis elegans embryonic development.

    Directory of Open Access Journals (Sweden)

    Vida Praitis

    2013-05-01

    Full Text Available Maintaining levels of calcium in the cytosol is important for many cellular events, including cell migration, where localized regions of high calcium are required to regulate cytoskeletal dynamics, contractility, and adhesion. Studies show inositol-trisphosphate receptors (IP3R and ryanodine receptors (RyR, which release calcium into the cytosol, are important regulators of cell migration. Similarly, proteins that return calcium to secretory stores are likely to be important for cell migration. The secretory protein calcium ATPase (SPCA is a Golgi-localized protein that transports calcium from the cytosol into secretory stores. SPCA has established roles in protein processing, metal homeostasis, and inositol-trisphosphate signaling. Defects in the human SPCA1/ATP2C1 gene cause Hailey-Hailey disease (MIM# 169600, a genodermatosis characterized by cutaneous blisters and fissures as well as keratinocyte cell adhesion defects. We have determined that PMR-1, the Caenorhabditis elegans ortholog of SPCA1, plays an essential role in embryogenesis. Pmr-1 strains isolated from genetic screens show terminal phenotypes, such as ventral and anterior enclosure failures, body morphogenesis defects, and an unattached pharynx, which are caused by earlier defects during gastrulation. In Pmr-1 embryos, migration rates are significantly reduced for cells moving along the embryo surface, such as ventral neuroblasts, C-derived, and anterior-most blastomeres. Gene interaction experiments show changing the activity of itr-1/IP3R and unc-68/RyR modulates levels of embryonic lethality in Pmr-1 strains, indicating pmr-1 acts with these calcium channels to regulate cell migration. This analysis reveals novel genes involved in C. elegans cell migration, as well as a new role in cell migration for the highly conserved SPCA gene family.

  19. The secretory pathway calcium ATPase PMR-1/SPCA1 has essential roles in cell migration during Caenorhabditis elegans embryonic development.

    Science.gov (United States)

    Praitis, Vida; Simske, Jeffrey; Kniss, Sarah; Mandt, Rebecca; Imlay, Leah; Feddersen, Charlotte; Miller, Michael B; Mushi, Juliet; Liszewski, Walter; Weinstein, Rachel; Chakravorty, Adityarup; Ha, Dae-Gon; Schacht Farrell, Angela; Sullivan-Wilson, Alexander; Stock, Tyson

    2013-05-01

    Maintaining levels of calcium in the cytosol is important for many cellular events, including cell migration, where localized regions of high calcium are required to regulate cytoskeletal dynamics, contractility, and adhesion. Studies show inositol-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), which release calcium into the cytosol, are important regulators of cell migration. Similarly, proteins that return calcium to secretory stores are likely to be important for cell migration. The secretory protein calcium ATPase (SPCA) is a Golgi-localized protein that transports calcium from the cytosol into secretory stores. SPCA has established roles in protein processing, metal homeostasis, and inositol-trisphosphate signaling. Defects in the human SPCA1/ATP2C1 gene cause Hailey-Hailey disease (MIM# 169600), a genodermatosis characterized by cutaneous blisters and fissures as well as keratinocyte cell adhesion defects. We have determined that PMR-1, the Caenorhabditis elegans ortholog of SPCA1, plays an essential role in embryogenesis. Pmr-1 strains isolated from genetic screens show terminal phenotypes, such as ventral and anterior enclosure failures, body morphogenesis defects, and an unattached pharynx, which are caused by earlier defects during gastrulation. In Pmr-1 embryos, migration rates are significantly reduced for cells moving along the embryo surface, such as ventral neuroblasts, C-derived, and anterior-most blastomeres. Gene interaction experiments show changing the activity of itr-1/IP3R and unc-68/RyR modulates levels of embryonic lethality in Pmr-1 strains, indicating pmr-1 acts with these calcium channels to regulate cell migration. This analysis reveals novel genes involved in C. elegans cell migration, as well as a new role in cell migration for the highly conserved SPCA gene family.

  20. NO donors-relaxation is impaired in aorta from hypertensive rats due to a reduced involvement of K(+) channels and sarcoplasmic reticulum Ca(2+)-ATPase.

    Science.gov (United States)

    Bonaventura, Daniella; de Lima, Renata Galvão; da Silva, Roberto Santana; Bendhack, Lusiane Maria

    2011-10-24

    To examine the vasodilatation induce by the NO donors, [Ru(terpy)(bdq)NO](3+) (TERPY) and sodium nitroprusside (SNP), and to compare their effects in aortic rings from hypertensive 2K-1C and normotensive 2K rats. Vascular reactivity was performed in aortic rings pre-contracted with phenylephrine (Phe 100nM). We have analyzed the maximal relaxation (Emax) and potency (pD(2)) of NO donors. Potency of SNP was greater than TERPY in both arterial groups. The vasodilatation induced by TERPY was greater in 2K than in 2K-1C, and it was inhibited by sGC inhibitor ODQ in 2K and in 2K-1C aortic rings. ODQ did not alter the efficacy to SNP, but it reduced its potency in 2K and 2K-1C. The blockade of K(+) channels reduced the potency of TERPY only in aortic rings of 2K. On the other hand, the potency of SNP was reduced in both 2K and 2K-1C. The combination of ODQ and TEA reduced the relaxation induced by TERPY and SNP in 2K and reduced the efficacy to SNP in 2K-1C aortic rings but it had no additional effect on the TERPY relaxation in 2K-1C aortas. The production of cGMP induced by TERPY was greater than that produced by SNP, which was similarly increased in 2K and 2K-1C. Sarcoplasmic reticulum Ca-ATPase inhibition only impaired the relaxation induced by SNP in 2K aortic rings. Taken together, our results provide evidences that in this model of hypertension, impaired K(+) channels activation by TERPY and SERCA activation by SNP may contribute to decreased vasodilatation. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Inhibition of Calcium Influx Reduces Dysfunction and Apoptosis in Lipotoxic Pancreatic β-Cells via Regulation of Endoplasmic Reticulum Stress.

    Directory of Open Access Journals (Sweden)

    Yuren Zhou

    Full Text Available Lipotoxicity plays an important role in pancreatic β-cell failure during the development of type 2 diabetes. Prolonged exposure of β-cells to elevated free fatty acids level could cause deterioration of β-cell function and induce cell apoptosis. Therefore, inhibition of fatty acids-induced β-cell dysfunction and apoptosis might provide benefit for the therapy of type 2 diabetes. The present study examined whether regulation of fatty acids-triggered calcium influx could protect pancreatic β-cells from lipotoxicity. Two small molecule compounds, L-type calcium channel blocker nifedipine and potassium channel activator diazoxide were used to inhibit palmitic acid-induced calcium influx. And whether the compounds could reduce palmitic acid-induced β-cell failure and the underlying mechanism were also investigated. It was found that both nifedipine and diazoxide protected MIN6 pancreatic β-cells and primary cultured murine islets from palmitic acid-induced apoptosis. Meanwhile, the impaired insulin secretion was also recovered to varying degrees by these two compounds. Our results verified that nifedipine and diazoxide could reduce palmitic acid-induced endoplasmic reticulum stress to generate protective effects on pancreatic β-cells. More importantly, it suggested that regulation of calcium influx by small molecule compounds might provide benefits for the prevention and therapy of type 2 diabetes.

  2. Transient kinetics of Ca2+ transport of sarcoplasmic reticulum. A comparison of cardiac and skeletal muscle.

    Science.gov (United States)

    Sumida, M; Wang, T; Mandel, F; Froehlich, J P; Schwartz, A

    1978-12-25

    Current evidence supports similar functions and mechanisms for cardiac sarcoplasmic reticulum (CSR) as for skeletal sarcoplasmic reticulum (SSR). It is thought that the slower relaxation rate of cardiac muscle compared to fast skeletal muscle reflects the lower ATPase activity and calcium transport of CSR. Possible quantitative differences is phosphorylation, dephosphorylation, and calcium transport of the isolated preparations are studied using a quench-flow apparatus. The results show that both CSR and SSR bind calcium tightly in the absence of ATP, and coupling of E approximately P formation and calcium transport occurs in the transient phase of ATP hydrolysis. The rate of phosphorylation (t-1/2 - 10 ms) of sarcoplasmic reticulum (SR) preloaded with calcium is the same for cardiac and skeletal preparations. However, the rates of dissociation of extra vesicular calcium (10 s-1 versus 15 s-1), phosphorylation of calcium-free SR, and dephosphorylation of E approximately P (8 s-1 versus 12 s-1) are lower for CSR than for SSR. By computer simulation, the apparent rate constants associated with the reduced rates of phosphorylation of calcium-free SR were: 12 s-1 for CSR and 63 s-1 for SSR in the presence of high Mg2+. The difference in the rates may be partly responsible for the lower levels of ATPase and calcium transport activity with characterize cardiac muscle preparations.

  3. Cellular and subcellular localization of the neuron-specific plasma membrane calcium ATPase PMCA1a in the rat brain.

    Science.gov (United States)

    Kenyon, Katharine A; Bushong, Eric A; Mauer, Amy S; Strehler, Emanuel E; Weinberg, Richard J; Burette, Alain C

    2010-08-15

    Regulation of intracellular calcium is crucial both for proper neuronal function and survival. By coupling ATP hydrolysis with Ca(2+) extrusion from the cell, the plasma membrane calcium-dependent ATPases (PMCAs) play an essential role in controlling intracellular calcium levels in neurons. In contrast to PMCA2 and PMCA3, which are expressed in significant levels only in the brain and a few other tissues, PMCA1 is ubiquitously distributed, and is thus widely believed to play a "housekeeping" function in mammalian cells. Whereas the PMCA1b splice variant is predominant in most tissues, an alternative variant, PMCA1a, is the major form of PMCA1 in the adult brain. Here, we use immunohistochemistry to analyze the cellular and subcellular distribution of PMCA1a in the brain. We show that PMCA1a is not ubiquitously expressed, but rather is confined to neurons, where it concentrates in the plasma membrane of somata, dendrites, and spines. Thus, rather than serving a general housekeeping function, our data suggest that PMCA1a is a calcium pump specialized for neurons, where it may contribute to the modulation of somatic and dendritic Ca(2+) transients.

  4. Effects of fructose-1,6-diphosphate on concentration of calcium and activities of sarcoplosnic Ca2+-ATPase in cardiomyocytes of Adriamycin-treated rats

    Institute of Scientific and Technical Information of China (English)

    CAI Wei; CHEN Jun-zhu; RUAN Li-ming; WANG Yi-na

    2005-01-01

    Objective: To observe the effects of fructose-1,6-diphosphate (FDP) on serum levels of cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB), as well as the concentration of calcium in cardiomyocytes (Myo[Ca2+]) and activity of sarcoplosnic Ca2+-ATPase (SRCa2+-ATPase) in Adriamycin (ADR)-treated rats. Methods: Rats were intraperitoneally injected with ADR (2.5mg/kg every other day for 6 times) and then with different dosages of FDP (every other day for twenty-one times). Bi-antibodies sandwich Enzyme linked immune absorption assay (ELISA) was performed to detect serum level of cTnI. CK-MB was detected by monoclonal antibody, Myo[Ca2+] was detected by fluorescent spectrophotometry and the activity of SRCa2+-ATPase was detected by inorganic phosphate method. Results: FDP (300, 600, 1200 mg/kg) significantly reduced the serum levels of cTnI and CK-MB, while at the same time decreased calcium concentration and increased SRCa2+-ATPase activity in cardiomyocytes of ADR-treated rats (P<0.01). Conclusions: FDP might alleviate the cardiotoxic effects induced by ADR through decreasing calcium level as well as increasing SRCa2+-ATPase activity in cardiomyocytes.

  5. Halothane, isoflurane, xenon, and nitrous oxide inhibit calcium ATPase pump activity in rat brain synaptic plasma membranes.

    Science.gov (United States)

    Franks, J J; Horn, J L; Janicki, P K; Singh, G

    1995-01-01

    Perturbation of neuronal calcium homeostasis may alter neurotransmission in the brain, a phenomenon postulated to characterize the anesthetic state. Because of the central role of plasma membrane Ca(2+)-ATPase (PMCA) in maintaining Ca2+ homeostasis, the authors examined the effect of several inhalational anesthetics on PMCA function in synaptic plasma membranes (SPM) prepared from rat brain. Ca(2+)-ATPase pumping activity was assessed by measurement of ATP-dependent uptake of Ca2+ by SPM vesicles. ATPase hydrolytic activity was assessed by spectrophotometric measurement of inorganic phosphate (Pi) released from ATP. For studies of anesthetic effects on PMCA activity, Ca2+ uptake or Pi release was measured in SPM exposed to halothane, isoflurane, xenon, and nitrous oxide at partial pressures ranging from 0 to 1.6 MAC equivalents. Halothane and isoflurane exposures were carried out under a gassing hood. For xenon and nitrous oxide exposures, samples were incubated in a pressure chamber at total pressures sufficient to provide anesthetizing partial pressures for each agent. Dose-related inhibition of Ca(2+)-ATPase pumping activity was observed in SPM exposed to increasing concentrations of halothane and isoflurane, confirmed by ANOVA and multiple comparison testing (P Xenon and nitrous oxide also inhibited Ca2+ uptake by SPM vesicles. At partial pressures of these two gases equivalent to 1.3 MAC, PMCA was inhibited approximately 20%. Hydrolysis of ATP by SPM fractions was also inhibited in a dose-related fashion. An additive effect occurred when 1 vol% of halothane was added to xenon or nitrous oxide at partial pressures equivalent to 0-1.6 MAC for the latter two agents. Plasma membranes Ca(2+)-ATPase is significantly inhibited, in a dose-related manner, by clinically relevant partial pressures of halothane, isoflurane, xenon, and nitrous oxide. Furthermore, these anesthetics inhibit PMCA activity in accordance with their known potencies, and an additive effect was

  6. Pressure effects on the interactions of the sarcoplasmic reticulum calcium transport enzyme with calcium and dinitrophenyl phosphate.

    Science.gov (United States)

    Hasselbach, W

    1988-01-01

    The effect of hydrostatic pressure on the calcium-dependent hydrolysis of dinitrophenyl phosphate by the sarcoplasmic calcium transport enzyme has been studied. The magnesium dinitrophenyl phosphate complex is the true substrate of the enzyme (K = 7000 M-1) by which it is hydrolyzed at 20 degrees C with a turnover rate of 4 s-1. Activation by calcium ions occurs between 0.1 and 1 microM as observed for ATP hydrolysis. The activation volume of the enzyme saturated with both ligands exhibits pronounced pressure-dependence, rising from 25 ml/mol at atmospheric pressure to 80 ml/mol at 100 MPa. The apparent binding volumes for magnesium dinitrophenyl phosphate and calcium are likewise pressure-dependent. The volume changes connected with the binding of magnesium dinitrophenyl phosphate is quite small approaching zero at 100 MPa. The apparent binding volume for calcium greatly increases with pressure from 35 ml/mol at atmospheric pressure to 150 ml/mol at 70 MPa. A nearly constant binding volume of approximately 40 ml/mol results if the effect of pressure on the respective rate constants that contribute to the apparent binding constant, is taken into account. The pressure-dependence of enzyme activity at subsaturating calcium concentrations yields an activation volume of 250 ml/mol related to the rate of calcium binding indicating the occurrence of a transient large volume expansion of the enzyme complex. The volume changes observed for the calcium-dependent interaction of the enzyme with magnesium dinitrophenyl phosphate well agree with that found for magnesium p-nitrophenyl phosphate (W. Hasselbach and L. Stephan,Z. Naturforsch. 42 c, 641-652 (1987)) indicating that the found volume changes are intrinsic properties of the transport enzyme, independent of the respective energy donor.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    Science.gov (United States)

    Golovina, Vera A

    2005-05-01

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

  8. Reduction of calcium flux from the extracellular region and endoplasmic reticulum by amorphous nano-silica particles owing to carboxy group addition on their surface

    Directory of Open Access Journals (Sweden)

    Akira Onodera

    2017-03-01

    Full Text Available Several studies have reported that amorphous nano-silica particles (nano-SPs modulate calcium flux, although the mechanism remains incompletely understood. We thus analyzed the relationship between calcium flux and particle surface properties and determined the calcium flux route. Treatment of Balb/c 3T3 fibroblasts with nano-SPs with a diameter of 70 nm (nSP70 increased cytosolic calcium concentration, but that with SPs with a diameter of 300 or 1000 nm did not. Surface modification of nSP70 with a carboxy group also did not modulate calcium flux. Pretreatment with a general calcium entry blocker almost completely suppressed calcium flux by nSP70. Preconditioning by emptying the endoplasmic reticulum (ER calcium stores slightly suppressed calcium flux by nSP70. These results indicate that nSP70 mainly modulates calcium flux across plasma membrane calcium channels, with subsequent activation of the ER calcium pump, and that the potential of calcium flux by nano-SPs is determined by the particle surface charge.

  9. A continuous dietary supply of free calcium formate negatively affects the parietal cell population and gastric RNA expression for H+/K+-ATPase in weaning pigs.

    Science.gov (United States)

    Bosi, Paolo; Mazzoni, Maurizio; De Filippi, Sara; Trevisi, Paolo; Casini, Luisa; Petrosino, Gregorio; Lalatta-Costerbosa, Giovanna

    2006-05-01

    Baby formula acidification can be used to reduce diarrhea. Calcium formate is a dietary acidifier frequently used in animal weaning diets; it is also a source of available calcium. Gastric acidification reduces gastrin release and hydrochloric acid (HCl) secretion. To study the medium-term effects on fundic gastric mucosa, we fed weaning pigs control diets or diets supplemented with free or fat-protected calcium formate. We evaluated the following: 1) the number of HCl-secreting parietal cells, by immunohistochemistry using an antibody against H(+)/K(+)-ATPase; 2) the number of enteroendocrine cells immunohistochemically stained with chromogranin A (CGA), somatostatin, and histamine (HIS); and 3) the expression of the H(+)/K(+)-ATPase gene, by real-time RT-PCR in the oxyntic mucosa. Cells co-staining for CGA and HIS were defined as enterochromaffin-like (ECL) cells. Pigs fed calcium formate had fewer parietal cells and a lower expression of the H(+)/K(+)-ATPase gene than the controls (P < 0.05). This reduction did not occur in pigs fed fat-protected calcium formate. Somatostatin immune-reactive cells were also more numerous in pigs fed free calcium formate than in controls (P < 0.05). The number of ECL cells was not affected. Using covariance analysis, the number of parietal cells explained part of the differences in the expression of H(+)/K(+)-ATPase gene (positive correlation, r = 0.385, P < 0.01), and excluded the statistical significance of the diet. In the future, the effects on the oxyntic mucosa should be checked when the diet supplemented with calcium formate is discontinued. Furthermore, a reduction in the number of parietal cells could impair the absorption of vitamin B-12 due to a reduced secretion of the intrinsic factor by these cells.

  10. The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase.

    Science.gov (United States)

    Medda, P; Fassold, E; Hasselbach, W

    1987-06-01

    sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].

  11. [Calcium transport in sarcoplasmic reticulum in the presence of AR-L 115 BS].

    Science.gov (United States)

    Hasselbach, W

    1981-01-01

    2-[(2-Methoxy-4-methylsulfinyl)phenyl]-1H-imidazo[4,5-b]pyridine (AR-L 115 BS) is a substance with positive inotropic activity which does not influence the activity of the sarcoplasmic calcium pump. It can, therefore, be expected that AR-L 115 BS does not interfere with the distribution and movement of calcium in the resting and active muscle.

  12. A computational model of spatio-temporal cardiac intracellular calcium handling with realistic structure and spatial flux distribution from sarcoplasmic reticulum and t-tubule reconstructions.

    Directory of Open Access Journals (Sweden)

    Michael A Colman

    2017-08-01

    Full Text Available Intracellular calcium cycling is a vital component of cardiac excitation-contraction coupling. The key structures responsible for controlling calcium dynamics are the cell membrane (comprising the surface sarcolemma and transverse-tubules, the intracellular calcium store (the sarcoplasmic reticulum, and the co-localisation of these two structures to form dyads within which calcium-induced-calcium-release occurs. The organisation of these structures tightly controls intracellular calcium dynamics. In this study, we present a computational model of intracellular calcium cycling in three-dimensions (3-D, which incorporates high resolution reconstructions of these key regulatory structures, attained through imaging of tissue taken from the sheep left ventricle using serial block face scanning electron microscopy. An approach was developed to model the sarcoplasmic reticulum structure at the whole-cell scale, by reducing its full 3-D structure to a 3-D network of one-dimensional strands. The model reproduces intracellular calcium dynamics during control pacing and reveals the high-resolution 3-D spatial structure of calcium gradients and intracellular fluxes in both the cytoplasm and sarcoplasmic reticulum. We also demonstrated the capability of the model to reproduce potentially pro-arrhythmic dynamics under perturbed conditions, pertaining to calcium-transient alternans and spontaneous release events. Comparison with idealised cell models emphasised the importance of structure in determining calcium gradients and controlling the spatial dynamics associated with calcium-transient alternans, wherein the probabilistic nature of dyad activation and recruitment was constrained. The model was further used to highlight the criticality in calcium spark propagation in relation to inter-dyad distances. The model presented provides a powerful tool for future investigation of structure-function relationships underlying physiological and pathophysiological

  13. Inositol trisphosphate and thapsigargin discriminate endoplasmic reticulum stores of calcium in rat brain

    DEFF Research Database (Denmark)

    Verma, A; Hirsch, D J; Hanley, M R

    1990-01-01

    ATP dependent Ca2+ accumulation into oxalate-loaded rat brain microsomes is potently inhibited by thapsigargin with an IC50 of 2 nM and maximal inhibition at 10 nM. Approximately 15% of the total A23187-releasable microsomal calcium store is insensitive to thapsigargin concentrations up to 100 mi...

  14. Role of SERCA and the sarcoplasmic reticulum calcium content on calcium waves propagation in rat ventricular myocytes.

    Science.gov (United States)

    Salazar-Cantú, Ayleen; Pérez-Treviño, Perla; Montalvo-Parra, Dolores; Balderas-Villalobos, Jaime; Gómez-Víquez, Norma L; García, Noemí; Altamirano, Julio

    2016-08-15

    In Ca(2+)-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca(2+) level to trigger and sustain Ca(2+) waves, that propagate at constant rate (ʋwave). High luminal Ca(2+) sensitizes RyR2, thereby increasing Ca(2+) sparks frequency, and the larger RyR2-mediated SR Ca(2+) flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca(2+) reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca(2+) removal could instead impair RyR2 activation. We assessed whether rapid SR Ca(2+) uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca(2+) waves amplitude was largely unaffected. Furthermore, SR Ca(2+) also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca(2+) and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca(2+) determines ʋwave, while rapid SERCA Ca(2+) uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca(2+).

  15. Mapping the interactions between ATP and the sarcoplasmic reticulum Ca 2 + -ATPase with ATP and ATP analogs studied by Fourier transform infrared spectroscopy

    OpenAIRE

    Liu, Man

    2004-01-01

    Die Infrarotspektroskopie in Verbindung mit photoaktivierbaren Substraten wurde zur Untersuchung von Substrat-Protein-Wechselwirkungen eingesetzt. Dabei wurden Konformationsänderungen der Ca2+-ATPase des Sarkoplasmatischen Retikulums bei Bindung des Nukleotids, der Phosphorylierung der ATPase und der Hydrolyse des Phosphoenzyms beobachtet. Verwender wurden das native Substrat ATP und seine Analoga ADP, AMPPNP, 2'-deoxyATP, 3'-deoxyATP, ITP, AMP, Pyrophosphat, Ribosetriphosphat und TNP-AMP beo...

  16. 姜黄素对心力衰竭兔肌浆网钙泵表达的影响%Effects of curcumin on sarcoplasmic reticulum Ca~(2+) -ATPase in rabbits with heart failure

    Institute of Scientific and Technical Information of China (English)

    张艳; 林国生; 包明威; 武欣迎; 王澈; 杨波

    2010-01-01

    目的 探讨姜黄素对心力衰竭(心衰)兔肌浆网钙泵表达的影响.方法 采用主动脉瓣反流联合腹主动脉缩窄制作慢性心衰家兔模型.随机分为心衰姜黄素组、心衰安慰剂组、对照姜黄素组、对照安慰剂组.8周后计算心脏重量与体重比值,观察超微结构,检测肌浆网钙泵mRNA和蛋白的表达水平及活性.结果 心衰姜黄素组和心衰安慰剂组心脏重量与体重比值均大于对照组(P<0.05);且心衰姜黄素组比值小于心衰安慰剂组(P<0.05).电子显微镜显示心衰姜黄素组的心脏超微结构有所改善.心衰姜黄素组和心衰安慰剂组肌浆网钙泵mRNA、蛋白表达及活性均小于对照组(P<0.05),但心衰姜黄素组均显著高于心衰安慰剂组(P<0.05).结论 姜黄素能在mRNA水平和蛋白水平提高心衰家兔肌浆网钙泵的表达,提高肌浆网钙泵的活性,这可能是姜黄素改善心衰的机制之一.%Objective To investigate the effects of curcumin on sarcoplasmic reticulum Ca~(2+)-ATPase in heart failure rabbits.Methods Rabbit heart failure model was made with aortic regurgitation and abdominal aorta constriction and 40 rabbits were randomly divided into 4 groups including:(1) heart failure treated with curcumin;(2) heart failure treated with placebo;(3) healthy control treated with curcumin and (4) healthy control treated with placebo.All rabbits were administrated with curcumin capsules or placebo capsules 100 mg·kg~(-1)·d~(-1),respectively.All groups were sacrificed after eight weeks.Myocardial ultrastructural organization was detected by transmission electron microscope.RT-PCR and Western blot were used to measure the expression of sarcoplasmic reticulum Ca~(2+)-ATPase in mRNA and protein levels,respectively.Malachite green colorimetric assay was used to evaluate the activity of sarcoplasmic reticulum Ca~(2+) -ATPase.Results All detected parameters were similar between control curcumin group and control placebo

  17. Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

    Directory of Open Access Journals (Sweden)

    Lu Fang-hao

    2010-06-01

    Full Text Available Abstract Communication between the SR (sarcoplasmic reticulum, SR and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM. Although it has been demonstrated that CaR (calcium sensing receptor activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re, the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

  18. Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation.

    Science.gov (United States)

    Lu, Fang-hao; Tian, Zhiliang; Zhang, Wei-hua; Zhao, Ya-jun; Li, Hu-lun; Ren, Huan; Zheng, Hui-shuang; Liu, Chong; Hu, Guang-xia; Tian, Ye; Yang, Bao-feng; Wang, Rui; Xu, Chang-qing

    2010-06-17

    Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

  19. Immunocytochemical localization of Na+,K(+)-ATPase in the calcium-transporting sternal epithelium of the terrestrial isopod Porcellio scaber L. (Crustacea).

    Science.gov (United States)

    Ziegler, A

    1997-03-01

    Terrestrial isopods store large amounts of calcium carbonate between the epithelium and the old cuticle of the first four anterior sternites before molt. During the formation of these sternal CaCO3 deposits, large amounts of calcium are transported across the anterior sternal epithelium from the base to the apical side of the integument, and in the reverse direction during resorption of the deposit. A monoclonal antibody against the avian alpha-subunit of Na+,K(+)-ATPase was used to localize Na+,K(+)-ATPase in the anterior and the posterior sternal epithelium of Porcellio scaber. Semithin cryosections 0.5 micron thick were used for immunofluorescence microscopy and ultrathin cryosections for immunogold electron microscopy. The Na+,K(+)-ATPase was localized in the basolateral plasma membrane of the posterior and anterior sternal epithelium. The apical plasma membrane, including cytoplasmic extensions into the newly secreted cuticle, was virtually devoid of the enzyme. This pattern of immunolocalization was not affected by the direction of transepithelial calcium transport associated with the deposition and resorption phases of the molt cycle.

  20. Pressure effects on the binding of vanadate to the sarcoplasmic reticulum calcium-transport enzyme.

    Science.gov (United States)

    Ronzani, N; Stephan, L; Hasselbach, W

    1991-10-01

    The effect which hydrostatic pressure exerts on the binding of vanadate to the calcium-transport enzyme was determined. The recent unavailability of radioactive vanadate prevented direct measurements of vanadate binding. The vanadate-free enzyme fraction was instead monitored by phosphorylating it with ATP according to Medda and Hasselbach [Medda, P. & Hasselbach, W. (1983) Eur. J. Biochem. 137, 7-14]. Vanadate binding is reduced with rising pressure at first markedly and subsequently, above 30 MPa, relatively little. The biphasic pressure-binding relationship was analysed by applying a biexponential fitting procedure to the experimental data. The biphasicity of the pressure-binding relationship indicates that the description of vanadate binding requires at least a two-step reaction sequence. The volume increments which predominate at lower pressure values, range from 200-400 ml.mol-1 depending on the composition of the reaction medium containing 5 microM and 20 microM vanadate and no or 15% (by vol.) Me2SO. The binding volumes deduced for the higher pressure range amount to 20-40 ml.mol-1. Vanadate binding is reduced in the presence of 30 microM calcium, and simultaneously both binding volumes are diminished by 100 ml.mol-1 and 20 ml.mol-1 for the low and high pressure values, respectively, as one can expect for mutual interactions between the two ligands of the transport enzyme.

  1. Two-Dimensional Crystallization of the Ca(2+)-ATPase for Electron Crystallography.

    Science.gov (United States)

    Glaves, John Paul; Primeau, Joseph O; Young, Howard S

    2016-01-01

    Electron crystallography of two-dimensional crystalline arrays is a powerful alternative for the structure determination of membrane proteins. The advantages offered by this technique include a native membrane environment and the ability to closely correlate function and dynamics with crystalline preparations and structural data. Herein, we provide a detailed protocol for the reconstitution and two-dimensional crystallization of the sarcoplasmic reticulum calcium pump (also known as Ca(2+)-ATPase or SERCA) and its regulatory subunits phospholamban and sarcolipin.

  2. Structural studies of Ca2+-ATPase ligand and regulatory complexes

    DEFF Research Database (Denmark)

    Drachmann, Nikolaj Düring

    2015-01-01

    The Ca2+-ATPase (sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA is a part of the vital P-type ATPase family, which was first discovered in 1957 by Professor Jens Christian Skou. He was the first to describe the Na+,K+-ATPase and its role in generating the membrane potential across the axonal...

  3. Improvement of alcoholic fermentation by calcium ions under enological conditions involves the increment of plasma membrane H(+)-ATPase activity.

    Science.gov (United States)

    Li, Jingyuan; Huang, Weidong; Wang, Xiuqin; Tang, Tian; Hua, Zhaozhe; Yan, Guoliang

    2010-07-01

    The effect of Ca(2+) on alcoholic fermentation and plasma membrane H(+)-ATPase activity of wine yeast under enological conditions were investigated in this study. The results showed that fermentation rate, cell growth and ethanol production were improved by 0.5 and 1.5 mM Ca(2+) supplementation, which correlated well with the increment of ATPase activity and protein levels. Considering the important role of ATPase in the tolerance of yeast to ethanol, the improvement could be, at least partially, attributed to the increment of ATPase activity. No activation of ATPase by Ca(2+) was observed in the early phase of fermentation and the increment of activity was only observed when ethanol concentration exceeded 6.5%. Therefore, the enhancement of ATPase activity by Ca(2+) was ascribed to alleviating the inhibition of ATPase activity by ethanol through protection of membrane structure. Our results suggest that, besides maintenance of cell membrane structure, the increment of plasma membrane ATPase activity was also responsible for the improvement of alcoholic fermentation by Ca(2+) supplementation.

  4. Cytoplasmic Hsp70 promotes ubiquitination for endoplasmic reticulum-associated degradation of a misfolded mutant of the yeast plasma membrane ATPase, PMA1.

    Science.gov (United States)

    Han, Sumin; Liu, Yu; Chang, Amy

    2007-09-01

    Cells have a variety of strategies for dealing with misfolded proteins. Heat shock response involves transcriptional induction of chaperones to promote and/or correct folding, and also activation of the ubiquitin/proteasome system to degrade defective proteins. In the secretory pathway, it is primarily luminal misfolded or unassembled proteins that trigger the unfolded protein response which, like heat shock, induces chaperones and components of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. To understand cellular response to a misfolded polytopic membrane protein of the secretory pathway, we studied Pma1-D378S, a model ERAD substrate. Expression of misfolded Pma1 induces heat shock response in the absence of increased temperature. Overexpression of HSF1, the transcription factor that mediates heat shock response, increases degradation of Pma1-D378S without temperature upshift. Nevertheless, efficient Pma1-D378S degradation occurs in an hsf1 mutant that maintains basal transcription levels but cannot mediate transcriptional activation. Thus, heat shock protein induction enhances but is not necessary for ERAD. The Ssa group of cytoplasmic Hsp70 chaperones is required for ERAD of both Pma1-D378S and another transmembrane ERAD substrate, Ste6*. In the absence of Ssa chaperones, ubiquitination of both substrates is impaired, resulting in stabilization. We suggest a role for Hsp70 cytoplasmic chaperones in recognition by the endoplasmic reticulum-associated ubiquitination machinery.

  5. Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca(2+) release during the quasi-steady level of release in twitch fibers from frog skeletal muscle.

    Science.gov (United States)

    Fénelon, Karine; Lamboley, Cédric R H; Carrier, Nicole; Pape, Paul C

    2012-10-01

    Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits

  6. Coil-to-Helix Transition within Phospholamban Underlies Release of Ca-ATPase Inhibition in Response to β-Adrenergic Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Bigelow, Diana J.; Squier, Thomas C.

    2006-02-01

    Phospholamban (PLB) is a major target of beta-adrenergic signaling, whose phosphorylation results in enhanced rates of relaxation in the heart. Prior to phosphorylation, PLB functions to reduce the calcium sensitivity of the Ca-ATPase, resulting in slower rates of calcium resequestration into the sarcoplasmic reticulum after each contractile event. Recent structures indicate that the inhibitory interaction between PLB and the Ca-ATPase requires PLB to assume an extended structure, where the transmembrane and cytosolic portions of PLB undergo specific binding interactions with distant sites on the Ca-ATPase. In the extended conformation, PLB binding to the Ca-ATPase functions to inhibit the Ca-ATPase through a reduction in the rates of catalytically important motions involving the nucleotide binding domain. Phosphorylation of PLB at either Ser16 or Thr17 releases the inhibitory interaction between PLB and the Ca-ATPase. These sites of phosphorylation are within a hinge region in PLB that separates the highly structured transmembrane and cytosolic portions that associate with the Ca-ATPase. The helical content of the hinge region increases following the phosphorylation of PLB, which induces a shortening of the maximal dimensions of PLB and a release of the inhibitory interaction with the Ca-ATPase. Following phosphorylation, PLB remains associated with the Ca-ATPase in a more compact form that has no inhibitory capability. Thus, the conformational switch involving PLB regulation of the Ca-ATPase relies upon a physical mechanism, whereby the phosphorylation-dependent stabilization of the structure of PLB functions to destabilize the inhibitory interaction between PLB and the Ca-ATPase. Upon hydrolysis of the phosphoester linkages by endogenous phosphatases PLB is poised to reassume the inhibited state through re-association with inhibitory sites on the nucleotide binding domain of the Ca-ATPase.

  7. Structure-based drug design studies of the interactions of ent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparum sarco/endoplasmic reticulum Ca2+-ATPase PfATP6

    Directory of Open Access Journals (Sweden)

    Daniel Silqueira Martins Guimarães

    2015-04-01

    Full Text Available Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosa were tested in silico against the Plasmodium falciparum Ca2+-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.

  8. Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes.

    OpenAIRE

    Pierce, D H; Scarpa, A.; Trentham, D R; Topp, M. R.; Blasie, J K

    1983-01-01

    Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane...

  9. Altered calcium pump and secondary deficiency of gamma-sarcoglycan and microspan in sarcoplasmic reticulum membranes isolated from delta-sarcoglycan knockout mice.

    Science.gov (United States)

    Solares-Pérez, Alhondra; Alvarez, Rocío; Crosbie, Rachelle H; Vega-Moreno, Jesús; Medina-Monares, Joel; Estrada, Francisco J; Ortega, Alicia; Coral-Vazquez, Ramón

    2010-07-01

    Sarcoglycans (SGs) and sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and delta-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of delta-SG isoforms in TT and SR results in a secondary deficiency of gamma-SG and microSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the delta-SG isoforms may stabilize the expression of gamma-SG and microSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. Decreased agonist-stimulated mitochondrial ATP production caused by a pathological reduction in endoplasmic reticulum calcium content in human complex I deficiency.

    Science.gov (United States)

    Visch, Henk-Jan; Koopman, Werner J H; Leusink, Anouk; van Emst-de Vries, Sjenet E; van den Heuvel, Lambertus W P J; Willems, Peter H G M; Smeitink, Jan A M

    2006-01-01

    Although a large number of mutations causing malfunction of complex I (NADH:ubiquinone oxidoreductase) of the OXPHOS system is now known, their cell biological consequences remain obscure. We previously showed that the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP](M)) is significantly reduced in primary skin fibroblasts from a patient with an isolated complex I deficiency. The present work addresses the mechanism(s) underlying this impaired response. Luminometry of fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase revealed that the Bk-induced increase in [ATP](M) was significantly, but to a variable degree, decreased in 10 patients. The same variation was observed for the increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)), measured with mitochondria targeted aequorin, and cytosolic [Ca(2+)] ([Ca(2+)](C)), measured with fura-2, and for the Ca(2+) content of the endoplasmic reticulum (ER), calculated from the increase in [Ca(2+)](C) evoked by thapsigargin, an inhibitor of the ER Ca(2+) ATPase. Regression analysis revealed that the increase in [ATP](M) was directly proportional to the increases in [Ca(2+)](C) and [Ca(2+)](M) and to the ER Ca(2+) content. Our findings provide evidence that a pathological reduction in ER Ca(2+) content is the direct cause of the impaired Bk-induced increase in [ATP](M) in human complex I deficiency.

  11. Sarcoplasmic reticulum calcium mobilization in right ventricular pressure-overload hypertrophy in the ferret: relationships to diastolic dysfunction and a negative treppe.

    Science.gov (United States)

    Gwathmey, J K; Morgan, J P

    1993-03-01

    In a model of right-ventricular pressure-overload hypertrophy (POH) in the ferret, action potential duration (to 90% repolarization) was found to be significantly longer (228 +/- 11 vs 314 +/- 12 ms) with no change in amplitude (85 +/- 3 vs 85 +/- 2 mV) or resting membrane potential (-79 +/- 1.5 vs -79 +/- 1 mV) for control and POH, respectively. Peak sarcoplasmic reticulum Ca2+ release (expressed as the logarithm of the fractional luminescence, -4.2 +/- 0.1 vs -4.4 +/- 0.3) and resting calcium concentrations (-5.5 +/- 0.1 vs -5.7 +/- 0.1) were not different between the two groups (control vs POH respectively). Muscles from control and POH animals demonstrated a positive force/interval relationship in the presence of physiological extracellular [Ca2+]. However, unlike muscles from control animals, muscles from animals with POH subjected to increasing frequencies of contraction in the presence of increased extracellular [Ca2+] demonstrated further impairment of diastolic relaxation and a negative treppe. Exposure of muscles from POH animals to isoproterenol returned the slowed Ca2+ uptake by the sarcoplasmic reticulum as detected with aequorin to control values, although the relaxation phase of the isometric twitch remained prolonged compared to non-hypertrophied muscles. Exposure to milrinone also abbreviated the time course of the intracellular Ca2+ transient, but did not return it to that seen in normal myocardium. The exposure of non-hypertrophied isolated muscles to caffeine resulted in similar prolongation of the isometric twitch duration to that seen in hypertrophied myocardium. Results of these experiments suggest that impaired muscle relaxation in POH reflects changes at the level of the myofilaments. Thus, although slowed intracellular calcium mobilization contributes to diastolic relaxation abnormalities, it can not be the sole factor responsible for the slowed relaxation as has been suggested.

  12. Gossypol increases expression of the pro-apoptotic BH3-only protein NOXA through a novel mechanism involving phospholipase A2, cytoplasmic calcium, and endoplasmic reticulum stress.

    Science.gov (United States)

    Soderquist, Ryan S; Danilov, Alexey V; Eastman, Alan

    2014-06-06

    Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We demonstrated previously that gossypol failed to directly inhibit BCL2 in cells or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL, respectively, for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pretreatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress, and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity, indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154(+) stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells, suggesting that a combination of these drugs may be efficacious in the clinic.

  13. Association of cardiac injury with iron-increased oxidative and nitrative modifications of the SERCA2a isoform of sarcoplasmic reticulum Ca(2+)-ATPase in diabetic rats.

    Science.gov (United States)

    Li, Xueli; Li, Wenliang; Gao, Zhonghong; Li, Hailing

    2016-08-01

    The role of iron in the etiology of diabetes complications is not well established. Thus, this study was performed to test whether the iron-induced increase of oxidative/nitrative damage is involved in SERCA2a-related diabetic heart complication. Four randomly divided groups of rats were used: normal control group; iron overload group; diabetes group, and diabetic plus iron overload group. Iron supplementation stimulated cardiomyocyte hypertrophy and led to an increase in cardiac protein carbonyls, nitrotyrosine (3-NT) formation, and iNOS protein expression, thus resulting in abnormal myocardium calcium homeostasis of diabetic rats. The levels of SECA2a oxidation/nitration were significantly increased in the iron overload diabetic rats, along with a decrease in SECA2a expression and activity. In order to elucidate the possible role of iron in SERCA2a dysfunction, the effects of iron (Fe(3+) or hemin) on peroxynitrite (ONOO(-)) induced SERCA2a oxidation and nitration were further investigated in vitro. It was found that tyrosine nitration played more important role in SERCA2a inactivation than thiol oxidation. These results present a potential mechanism in which iron exacerbates the diabetes-induced oxidative/nitrative modification of SERCA2a, which may cause functional deficits in the myocyte associated with diabetic cardiac dysfunction. Our findings may help to further understand the role of iron in the pathogenesis of diabetic complications.

  14. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles. Volume I

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR, HSR) were isolated from rabbit leg muscle. They were then diluted and washed with sucrose or KCl and referred to as sucrose or KCl washed vesicles. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes where as the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material. The LSR consists of predominantly Ca2+ + Mg2+ ATPase (80 to 90%), a small amount of the high affinity Ca binding protein (5%), and a 5000 dalton proteolipid. The sucrose HSR vesicles contain the Ca2+ + Mg2+ ATPase (50%), Calsequestrin (25%), high affinity Ca binding protein (5%), one extrinsic 34,000 dalton protein (3%), one intrinsic 30,000 dalton protein (3%), a 9000 dalton proteolipid, and a 5000 dalton proteolipid. The sucrose--washed HSR vesicles contain greater than three times the calcium content of the sucrose washed LSR vesicles where as the KCl--washed vesicles contain less than 15 nmoles Ca2+ mg of protein each. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP. Exchange of methanesulfonate for chloride resulted in the release of calcium from both the light and heavy SR vesicles. Sucrose causes a slight inhibition of chloride--induced calcium release from the heavy SR vesicles but it greatly reduces the release of calcium from the light SR vesicles. Sodium dantrolene (20 uM) has no effect on the release of calcium from the light SR vesicles but it inhibits the release of calcium from the heavy SR vesicles. The results indicate that the chloride--induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization.

  15. 肌浆网/内质网钙ATP酶在大鼠肝再生过程中的表达和活性分析%Expression Patterns and Activity Analysis of the Sarco (endo) Plasmic Reticulum Ca2+-ATPase in the Liver Regeneration of Rats

    Institute of Scientific and Technical Information of China (English)

    吴灿; 侯国俊; 支樑健; 王颖; 叶波平

    2012-01-01

    肌浆网/内质网钙ATP酶(SERCA)主要位于内质网和肌浆网膜上,是生物体内重要的钙离子转运酶.为进一步探讨肝再生过程中SERCA的表达变化,该研究构建了大鼠2/3肝脏切除模型,用实时荧光定量RT-PCR和测定酶活性的方法研究该酶的两种亚型(SERCA2b和SERCA3)在肝切除后O,1,12,24,48,72,120,168,216 h共9个时间点的表达变化,结果发现肝再生早期两种亚型表达量均下调,但在一定时间内显著应激性上升,分别在12h和48h达到峰值,之后表达量下调至原始水平.同时,酶活性测定结果与基因表达变化相类似,在12~48 h酶活性达到峰值,以上结果表明SERCA参与了肝脏再生过程,可能的途径是调控内质网钙离子,进而影响细胞质的钙震荡和细胞周期.%Sarco ( endo) plasmic reticulum Ca2+-ATPase, SERCA, is thought to play an important role in Ca2+-transporting. It is mainly located in the membranes of sarco ( endo) plasmic reticulum. To characterize its role in liver regeneration, the expression patterns of its two isoforms( SERCA2b,SERCA3) at different time points of 0,1 ,12,24,48,72,120,168,216 h were studied after 2/3 partial hepatectomy(PH) by Real-time quantiatative PCR. And the total enzyme activity was also determined. The results showed that the mRNA level of the two isoforms was down-regulated at the early stage of liver regeneration. However they increased dramtically responding to the PH. SERCA2b peaked at 12 h and the SERCA3 peaked at 48 h. After reaching the peak,the level decreased to the normal. The change of total enzyme activity was consistent with the mRNA level and kept the highest level between the 12~48 h. All these implied that SERCA involved in the liver regeneration. It may play an important role in charging the calcium oscillatory in the cytoplasmic and cell cycle by regulateing the calcium in the ER.

  16. Biphasic contractions induced by milrinone at low temperature in ferret ventricular muscle: role of the sarcoplasmic reticulum and transmembrane calcium influx.

    Science.gov (United States)

    Malecot, C O; Bers, D M; Katzung, B G

    1986-08-01

    The effects of milrinone were studied in ferret papillary muscle stimulated at various rates and temperatures from 23 degrees to 36 degrees C. In voltage-clamp experiments, 50 micrograms/ml (0.237 mM) milrinone induced a 2.1-fold increase in calcium current at 28 degrees or 36 degrees C. At 50 micrograms/ml, milrinone transiently increased contractility in all muscles at 28 degrees C, but its steady-state effect was either increased (+50%) or decreased (-24.7%) steady-state twitch amplitude. A negative inotropic effect always occurred below 27 degrees C. Milrinone decreased the total twitch duration and split the twitch into two components (P1 and P2) in the absence of any evidence of aberrant conduction. Increasing milrinone concentration from 50 to 300 micrograms/ml decreased P1 and increased P2. Ryanodine (100 mM) or caffeine (10 mM) suppressed P1. Contractions elicited after 30 seconds of rest were also biphasic in the presence of milrinone, but not in its absence. P2 of post-rest contraction was increased by caffeine or calcium (10 mM) and decreased by cobalt (2 mM) when drugs were applied at the beginning of the rest. Ryanodine and caffeine also suppressed P1 of post-rest contraction. The evidence suggests that P1 may be caused by Ca release from the sarcoplasmic reticulum and P2 by increased Ca influx during the action potential via the calcium channel. It is also suggested that P2 may be present under control conditions, but to a lesser extent, and masked by a large P1.

  17. Rhein triggers apoptosis via induction of endoplasmic reticulum stress, caspase-4 and intracellular calcium in primary human hepatic HL-7702 cells

    Energy Technology Data Exchange (ETDEWEB)

    KoraMagazi, Arouna [Department of Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu (China); Wang, Dandan [Department of Pharmacology, China Pharmaceutical University, Nanjing, Jiangsu (China); Yousef, Bashir; Guerram, Mounia [Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, Jiangsu (China); Yu, Feng, E-mail: yufengcpu14@yahoo.com [Department of Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu (China); Department of Pharmacology, China Pharmaceutical University, Nanjing, Jiangsu (China); Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Nanjing, Jiangsu (China)

    2016-04-22

    Rhein is an active component of rhubarb; a traditional Chinese medicine reported to induce apoptosis and cause liver toxicity. However, rhein's apoptotic-inducing effects, as well as its molecular mechanisms of action on hepatic cells need to be further explored. In the present study, rhein was found to trigger apoptosis in primary human hepatic HL-7702 cells as showed by annexin V/PI double staining assay and nuclear morphological changes demonstrated by Hoechst 33258 staining. Moreover, it was observed that the mechanism implicated in rhein-induced apoptosis was caspase-dependent, presumably via ER-stress associated pathways, as illustrated by up-regulation of glucose-regulated protein 78 (GRP 78), PKR-like ER kinase (PERK), C-Jun N-terminal kinase (JNK) and CCAAT/enhancer-binding protein homologous protein (CHOP). Meanwhile, caspase-4 as a hallmark of ER-stress, was also showed to be activated following by caspase-3 activation. Furthermore, rhein also promoted intracellular elevation of calcium that contributed in apoptosis induction. Interestingly, pre-treatment with calpain inhibitor I reduced the effects of rhein on apoptosis induction and JNK activation. These data suggested that rhein-induced apoptosis through ER-stress and elevated intracellular calcium level in HL-7702 cells. - Highlights: • Rhein triggers apoptotic cell death on primary human hepatic HL-7702 cells. • Rhein leads to caspase-4 activation in HL-7702 cells. • Rhein induces endoplasmic reticulum stress pathways in HL-7702 cells. • Rhein causes elevation of intracellular calcium concentrations in HL-7702 cells.

  18. The reaction of N-(1-pyrene)maleimide with sarcoplasmic reticulum.

    Science.gov (United States)

    Papp, S; Kracke, G; Joshi, N; Martonosi, A

    1986-01-01

    The excimer fluorescence of the adduct of N-(1-pyrene)maleimide (PMI) with the Ca2+-ATPase was proposed as a probe of ATPase-ATPase interactions in sarcoplasmic reticulum (Lüdi and Hasselbach, Eur. J. Biochem., 1983, 130:5-8). We tested this proposition by analyzing the spectral properties and stoichiometry of the adducts of pyrenemaleimide with sarcoplasmic reticulum and with dithiothreitol and by comparing the effects of various detergents on the excimer fluorescence of the two adducts, with their influence on the sedimentation characteristics, ATPase activity, and light scattering of the pyrenemaleimide-labeled sarcoplasmic reticulum. These studies indicate that pyrenemaleimide reacts nearly randomly with several SH groups on the Ca2+-ATPase, and suggest that the observed excimer fluorescence of pyrenemaleimide-labeled sarcoplasmic reticulum may reflect intramolecular phenomena rather than ATPase-ATPase interactions. Further work is required to establish the relative contribution of intra- and intermolecular mechanisms to the excimer fluorescence. PMID:2937461

  19. Calcium in pollen-pistil interaction in `Petunia hybrida Hat`. Pt. 3. Localization of Ca{sup 2+} ions and Ca{sup 2+}-ATPase in pollinated pistil

    Energy Technology Data Exchange (ETDEWEB)

    Bednarska, E.; Butowt, R. [Uniwersytet Mikolaja Kopernika, Torun (Poland)

    1995-12-31

    Studies were carried out of Ca{sup 2+} and Ca{sup 2+}-ATPase localization in pollinated (6 and 48 h after pollination) pistils of `Petunia hybrida`. The results were confronted with Ca{sup 2+} localization in mature pollen grain and in unpollinated pistil. It has been found that after pollination the number of Ca{sup 2+} sequestered in the stigmal exudate and in the sporoderm of the pollen grain gets lower. That phenomenon was associated with the appearance of a large number of Sb/Ca precipitates in the submembrane cytoplasm of the germinating pollen. In the vacuolized pollen grain, i.e. grown into a pollen tube, there were only a few precipitates. In the pollen tube, Ca{sup 2+} were found in the organelles of the tip cytoplasm and in the external pectin cell wall. Studies with the use of {sup 45}Ca{sup 2+} have revealed that the source of calcium ions incorporated into the pollen tube tip and its pectin wall is the transmitting tract of the style. In the transmitting tract overgrown with pollen tubes, Ca{sup 2+} were localized in the intercellular matrix and in the transmitting cell. Sb/Ca precipitates occurred in the nuclei, around the secretary vesicles and on the plasmalemma in the transverse walls region. Elevated Ca{sup 2+} level was found in degenerating cells (inhibited pollen tubes, transmitting cells, nucellar cells). The progressing degeneration process of the cells of the transmitting tract of the pollinated pistil was associated with a decrease in the activity of plasmalemma Ca{sup 2+}-ATPase. (author). 30 refs, 19 figs.

  20. Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Binod [Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032 (India); Radiation and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Kumar, Amit [Radiation and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Ghosh, Subhalakshmi [Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032 (India); Pandey, Badri N., E-mail: bnp@barc.gov.in [Radiation and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Mishra, Kaushala P. [Radiation and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Hazra, Banasri, E-mail: banasrihazra@yahoo.co.in [Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032 (India)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Diospyrin diethylether (D7) caused oxidative stress-dependent activation of PC-PLC. Black-Right-Pointing-Pointer Activated PC-PLC induced a sustained-release of Ca{sup 2+} from endoplasmic reticulum. Black-Right-Pointing-Pointer The elevated cytosolic Ca{sup +2} led to the calpain-caspase12 dependent apoptosis. Black-Right-Pointing-Pointer D7-Induced Ca{sup +2} also found to accentuate the mitochondrial pathway of apoptosis. -- Abstract: Diospyrin diethylether (D7), a bisnaphthoquinonoid derivative, exhibited an oxidative stress-dependent apoptosis in several human cancer cells and tumor models. The present study was aimed at evaluation of the increase in cytosolic calcium [Ca{sup 2+}]{sub c} leading to the apoptotic cell death triggered by D7 in MCF7 human breast carcinoma cells. A phosphotidylcholine-specific phospholipase C (PC-PLC) inhibitor, viz. U73122, and an antioxidant, viz. N-acetylcysteine, could significantly prevent the D7-induced rise in [Ca{sup 2+}]{sub c} and PC-PLC activity. Using an endoplasmic reticulum (ER)-Ca{sup 2+} mobilizer (thapsigargin) and an ER-IP3R antagonist (heparin), results revealed ER as a major source of [Ca{sup 2+}]{sub c} which led to the activation of calpain and caspase12, and cleavage of fodrin. These effects including apoptosis were significantly inhibited by the pretreatment of Bapta-AM (a cell permeable Ca{sup 2+}-specific chelator), or calpeptin (a calpain inhibitor). Furthermore, D7-induced [Ca{sup 2+}]{sub c} was found to alter mitochondrial membrane potential and induce cytochrome c release, which was inhibited by either Bapta-AM or ruthenium red (an inhibitor of mitochondrial Ca{sup 2+} uniporter). Thus, these results provided a deeper insight into the D7-induced redox signaling which eventually integrated the calcium-dependent calpain/caspase12 activation and mitochondrial alterations to accentuate the induction of apoptotic cell death.

  1. Calcium-induced alteration of mitochondrial morphology and mitochondrial-endoplasmic reticulum contacts in rat brown adipocytes

    Directory of Open Access Journals (Sweden)

    I. Golic

    2014-09-01

    Full Text Available Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca2+]i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ® drinking or tap water (control drinking for three days. Alizarin Red S staining showed increased Ca2+ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca2+ diet affected mitochondrial fusion as mitofusin 1 (MFN1 and mitofusin 2 (MFN2 were increased, and mitochondrial fission as dynamin related protein 1 (DRP1 was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER. The level of uncoupling protein-1 (UCP1 was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes

  2. Calcium-induced alteration of mitochondrial morphology and mitochondrial-endoplasmic reticulum contacts in rat brown adipocytes.

    Science.gov (United States)

    Golic, I; Velickovic, K; Markelic, M; Stancic, A; Jankovic, A; Vucetic, M; Otasevic, V; Buzadzic, B; Korac, B; Korac, A

    2014-09-09

    Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca2+]i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ® drinking or tap water (control) drinking for three days. Alizarin Red S staining showed increased Ca2+ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca2+ diet affected mitochondrial fusion as mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were increased, and mitochondrial fission as dynamin related protein 1 (DRP1) was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER). The level of uncoupling protein-1 (UCP1) was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes.

  3. Role of the Calcium-Sensing Receptor in Cardiomyocyte Apoptosis via the Sarcoplasmic Reticulum and Mitochondrial Death Pathway in Cardiac Hypertrophy and Heart Failure

    Directory of Open Access Journals (Sweden)

    Fang-Hao Lu

    2013-05-01

    Full Text Available Aims: Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. Methods and Results: In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso. Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours

  4. Role of the calcium-sensing receptor in cardiomyocyte apoptosis via the sarcoplasmic reticulum and mitochondrial death pathway in cardiac hypertrophy and heart failure.

    Science.gov (United States)

    Lu, Fang-Hao; Fu, Song-Bin; Leng, Xiaoning; Zhang, Xinying; Dong, Shiyun; Zhao, Ya-Jun; Ren, Huan; Li, Hulun; Zhong, Xin; Xu, Chang-Qing; Zhang, Wei-Hua

    2013-01-01

    Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR) and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR) in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso). Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC) in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours, activation of CaR reduced [Ca(2+)]SR

  5. The Bcl-2 gene polymorphism rs956572AA increases inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticulum calcium release in subjects with bipolar disorder.

    Science.gov (United States)

    Machado-Vieira, Rodrigo; Pivovarova, Natalia B; Stanika, Ruslan I; Yuan, Peixiong; Wang, Yun; Zhou, Rulun; Zarate, Carlos A; Drevets, Wayne C; Brantner, Christine A; Baum, Amber; Laje, Gonzalo; McMahon, Francis J; Chen, Guang; Du, Jing; Manji, Husseini K; Andrews, S Brian

    2011-02-15

    Bipolar disorder (BPD) is characterized by altered intracellular calcium (Ca(2+)) homeostasis. Underlying mechanisms involve dysfunctions in endoplasmic reticulum (ER) and mitochondrial Ca(2+) handling, potentially mediated by B-cell lymphoma 2 (Bcl-2), a key protein that regulates Ca(2+) signaling by interacting directly with these organelles, and which has been implicated in the pathophysiology of BPD. Here, we examined the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular Ca(2+) dynamics in patients with BPD. Live cell fluorescence imaging and electron probe microanalysis were used to measure intracellular and intra-organelle free and total calcium in lymphoblasts from 18 subjects with BPD carrying the AA, AG, or GG variants of the rs956572 SNP. Analyses were carried out under basal conditions and in the presence of agents that affect Ca(2+) dynamics. Compared with GG homozygotes, variant AA-which expresses significantly reduced Bcl-2 messenger RNA and protein-exhibited elevated basal cytosolic Ca(2+) and larger increases in inositol 1,4,5-trisphosphate receptor-mediated cytosolic Ca(2+) elevations, the latter in parallel with enhanced depletion of the ER Ca(2+) pool. The aberrant behavior of AA cells was reversed by chronic lithium treatment and mimicked in variant GG by a Bcl-2 inhibitor. In contrast, no differences between SNP variants were found in ER or mitochondrial total Ca(2+) content or in basal store-operated Ca(2+) entry. These results demonstrate that, in patients with BPD, abnormal Bcl-2 gene expression in the AA variant contributes to dysfunctional Ca(2+) homeostasis through a specific ER inositol 1,4,5-trisphosphate receptor-dependent mechanism. Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  6. The Bcl-2 Gene Polymorphism rs956572AA Increases Inositol 1,4,5-Trisphosphate Receptor–Mediated Endoplasmic Reticulum Calcium Release in Subjects with Bipolar Disorder

    Science.gov (United States)

    Machado-Vieira, Rodrigo; Pivovarova, Natalia B.; Stanika, Ruslan I.; Yuan, Peixiong; Wang, Yun; Zhou, Rulun; Zarate, Carlos A.; Drevets, Wayne C.; Brantner, Christine A.; Baum, Amber; Laje, Gonzalo; McMahon, Francis J.; Chen, Guang; Du, Jing; Manji, Husseini K.; Andrews, S. Brian

    2011-01-01

    Background Bipolar disorder (BPD) is characterized by altered intracellular calcium (Ca2+) homeostasis. Underlying mechanisms involve dysfunctions in endoplasmic reticulum (ER) and mitochondrial Ca2+ handling, potentially mediated by B-cell lymphoma 2 (Bcl-2), a key protein that regulates Ca2+ signaling by interacting directly with these organelles, and which has been implicated in the pathophysiology of BPD. Here, we examined the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular Ca2+ dynamics in patients with BPD. Methods Live cell fluorescence imaging and electron probe microanalysis were used to measure intracellular and intra-organelle free and total calcium in lymphoblasts from 18 subjects with BPD carrying the AA, AG, or GG variants of the rs956572 SNP. Analyses were carried out under basal conditions and in the presence of agents that affect Ca2+ dynamics. Results Compared with GG homozygotes, variant AA—which expresses significantly reduced Bcl-2 messenger RNA and protein—exhibited elevated basal cytosolic Ca2+ and larger increases in inositol 1,4,5-trisphosphate receptor–mediated cytosolic Ca2+ elevations, the latter in parallel with enhanced depletion of the ER Ca2+ pool. The aberrant behavior of AA cells was reversed by chronic lithium treatment and mimicked in variant GG by a Bcl-2 inhibitor. In contrast, no differences between SNP variants were found in ER or mitochondrial total Ca2+ content or in basal store-operated Ca2+ entry. Conclusions These results demonstrate that, in patients with BPD, abnormal Bcl-2 gene expression in the AA variant contributes to dysfunctional Ca2+ homeostasis through a specific ER inositol 1,4,5-trisphosphate receptor–dependent mechanism. PMID:21167476

  7. Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged adenosine 5'-triphosphate.

    Science.gov (United States)

    Pierce, D H; Scarpa, A; Topp, M R; Blasie, J K

    1983-11-08

    The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.

  8. Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle.

    Science.gov (United States)

    Baylor, S M; Hollingworth, S

    2003-08-15

    Experiments were carried out to compare the amplitude and time course of Ca2+ release from the sarcoplasmic reticulum (SR) in intact slow-twitch and fast-twitch mouse fibres. Individual fibres within small bundles were injected with furaptra, a low-affinity, rapidly responding Ca2+ indicator. In response to a single action potential at 16 degrees C, the peak amplitude and half-duration of the change in myoplasmic free [Ca2+] (Delta[Ca2+]) differed significantly between fibre types (slow-twitch: peak amplitude, 9.4 +/- 1.0 microM (mean +/- S.E.M.); half-duration, 7.7 +/- 0.6 ms; fast-twitch: peak amplitude 18.5 +/- 0.5 microM; half-duration, 4.9 +/- 0.3 ms). SR Ca2+ release was estimated from Delta[Ca2+] with a computational model that calculated Ca2+ binding to the major myoplasmic Ca2+ buffers (troponin, ATP and parvalbumin); buffer concentrations and reaction rate constants were adjusted to reflect fibre-type differences. In response to an action potential, the total concentration of released Ca2+ (Delta[CaT]) and the peak rate of Ca2+ release ((d/dt)Delta[CaT]) differed about 3-fold between the fibre types (slow-twitch: Delta[CaT], 127 +/- 7 microM; (d/dt)Delta[CaT], 70 +/- 6 microM ms-1; fast-twitch: Delta[CaT], 346 +/- 6 microM; (d/dt)Delta[CaT], 212 +/- 4 microM ms-1). In contrast, the half-duration of (d/dt)Delta[CaT] was very similar in the two fibre types (slow-twitch, 1.8 +/- 0.1 ms; fast-twitch, 1.6 +/- 0.0 ms). When fibres were stimulated with a 5-shock train at 67 Hz, the peaks of (d/dt)Delta[CaT] in response to the second and subsequent shocks were much smaller than that due to the first shock; the later peaks, expressed as a fraction of the amplitude of the first peak, were similar in the two fibre types (slow-twitch, 0.2-0.3; fast-twitch, 0.1-0.3). The results support the conclusion that individual SR Ca2+ release units function similarly in slow-twitch and fast-twitch mammalian fibres.

  9. Crystal Structure of the Vanadate-Inhibited Ca2+-ATPase

    DEFF Research Database (Denmark)

    Clausen, Johannes D.; Bublitz, Maike; Arnou, Bertrand Jean-Paul;

    2016-01-01

    Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca2+-ATPase with bound vanadate in the absence of Ca2+. Vanadate is bound...

  10. Molecular Cloning and Distribution of a Plasma Membrane Calcium ATPase Homolog from the Pearl Oyster Pinctada fucata

    Institute of Scientific and Technical Information of China (English)

    WANG Xue; FAN Weimin; XIE Liping; ZHANG Rongqing

    2008-01-01

    Plasma membrane calcium ATPaso (PMCA) plays a critical role in transporting Ca2 out of the cy- tosol across the plasma membrane which is essential both in keeping intracellular Ca2+ homeostasis and in biomineralization.In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata.This PMCA shares similarity with other published PMCAs within the functional domains.Reverse transcdption-polymerase chain reaction analysis shows that it is expressed ubiquitously.Furthermore,in situ hybridization reveals that it is expressed in the inner epithelial calls of the outer fold and in the outer epithelial calls of the middle fold,as well as the edge near the shell,which suggests that PMCA may be involved in calcified layer formation.The identification and characterization of oyster PMCA can help to further under-stand the structural and functional properties of molluscan PMCA,as well as the mechanism of maintaining Ca2+ homeostasis and the mechanism of mineralization in pead oyster.

  11. Roles of transmembrane segment M1 of Na(+),K (+)-ATPase and Ca (2+)-ATPase, the gatekeeper and the pivot

    DEFF Research Database (Denmark)

    Einholm, Anja P.; Andersen, Jens Peter; Vilsen, Bente

    2007-01-01

    In this review we summarize mutagenesis work on the structure-function relationship of transmembrane segment M1 in the Na(+),K(+)-ATPase and the sarco(endo)plasmic reticulum Ca(2+)-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported...... cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca(2+) interaction/occlusion in Ca(2+)-ATPase and K(+) interaction/occlusion in Na(+),K(+)-ATPase. Leu(65) of the Ca(2+)-ATPase and Leu(99) of the Na(+),K(+)-ATPase, located...... of the extracytoplasmic gate in both the Ca(2+)-ATPase and the Na(+),K(+)-ATPase. Udgivelsesdato: 2007-Dec...

  12. Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) ATPases

    DEFF Research Database (Denmark)

    Vandecaetsbeek, Ilse; Christensen, Søren Brøgger; Liu, Huizhen

    2011-01-01

    The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus...

  13. Post-conditioning protects cardiomyocytes from apoptosis via PKC(epsilon)-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum-mitochondria crosstalk.

    Science.gov (United States)

    Dong, Shiyun; Teng, Zongyan; Lu, Fang-Hao; Zhao, Ya-Jun; Li, Hulun; Ren, Huan; Chen, He; Pan, Zhen-Wei; Lv, Yan-Jie; Yang, Bao-Feng; Tian, Ye; Xu, Chang-Qing; Zhang, Wei-Hua

    2010-08-01

    The intracellular Ca(2+) concentration ([Ca(2+)](i)) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca(2+)](i), results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca(2+)](i) via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP(3)) to increase the release of intracellular Ca(2+) from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca(2+)](i) through an interaction of PKC with CaR to alleviate [Ca(2+)](ER) depletion and [Ca(2+)](m) elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC(epsilon) translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca(2+)](i), the [Ca(2+)](ER) was not reduced and [Ca(2+)](m) was not increased in the PC and GdCl(3)-PC groups. Furthermore, there was no evident psi(m) collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl(3)-PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC(epsilon) interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the

  14. [The role of mitochondrial uniporter in calcium-homeostasis of the exorbital lacrimal gland secretory cells].

    Science.gov (United States)

    Kotliarova, A B; Merlavs'kyĭ, V M; Dorosh, O M; Man'ko, V V

    2014-01-01

    The role of mitochondrial calcium-uniporter in calcium-homeostasis maintenance and correlations of calcium-uniporter with other calcium-transport systems of the rat exorbital lacrimal gland secretory cells were studied. The experiments were performed on intact and digitonin-permeabilized cells. The interdependence of calcium-uniporter and other calcium-transporting systems functioning was estimated on the basis of additivity of their inhibitors/agonists effects, which was accompanied with a decrease in the Ca2+ content in the gland cells. It was found that in conditions of simultaneously inhibition of sarco endoplasmic reticulum Ca2+-ATPase (SERCA) and mitochondrial calcium-uniporter Ca2+ passively released from different calcium stores, because the effects of these calcium-transport systems inhibitors (thapsigargin and ruthenium red, respectively) were additive. Similarly, the processes of inositol-1,4,5-trisphosphate receptors (IP3Rs) activation and calcium-uniporter inhibition were additive. In contrast, the effects of ryanodine and ruthenium red on the Ca2+ content in cells were significantly non-additive. In addition, ryanodine at concentrations 1-3 μM reduced respiration rate of studied cells in dose-dependent manner, and this effect was persisted at cells preincubation with ruthenium red or tapsigargin. Thus, besides the activation of ryanodine receptors (RyRs) in endoplasmic reticulum, ryanodine inhibits Ca2+ influx to the mitochondrial matrix, that was insensitive to ruthenium red.

  15. The use of the indicator fluo-5N to measure sarcoplasmic reticulum calcium in single muscle fibres of the cane toad.

    Science.gov (United States)

    Kabbara, A A; Allen, D G

    2001-07-01

    1. Single fibres from the lumbrical muscles of the cane toad (Bufo marinus) were incubated in fluo-5N AM for 2 h at 35 degrees C in order to load the indicator into the sarcoplasmic reticulum. Fluo-5N is a low-affinity calcium indicator (K(Ca) 90 microM). Successful sarcoplasmic reticulum (SR) loading was indicated by a fluorescence signal that declined during contraction. 2. Confocal microscopy showed that the dye loaded principally in lines perpendicular to the long axis of the fibre that repeated each sarcomere. This is consistent with much of the dye residing in the SR. 3. To establish the site of loading, fibres were exposed to 30 mM caffeine in the presence of 20 microM 2,5-di(tert-butyl)1,4-hydroquinone (TBQ, an SR pump inhibitor) which should release most Ca(2+) from the SR; this procedure reduced the fluorescence to 46 +/- 4 % of the control value. To determine how much indicator was in the myoplasm, fibres were exposed to 100 microg ml(-1) saponin which permeabilizes the surface membrane; saponin treatment reduced the fluorescence to 51 +/- 2 % of the control value. 4. During maximally activated tetani (100 Hz stimulation rate, 22 degrees C) the component of signal from the SR declined by 33 +/- 4 %. During relaxation the SR signal recovered in two phases with time constants of 0.38 +/- 0.14 s and 10.1 +/- 1.7 s. Partially activated tetani (30 Hz stimulation rate) showed a smaller SR signal. Application of the SR Ca(2+) pump inhibitor TBQ slowed the rate of recovery of the SR signal. 5. Muscle fatigue was produced by repeated short tetani until tension was reduced to 50 %. The SR signal during the periods between tetani declined steadily and the SR Ca(2+) signal was eventually reduced to 71 +/- 8 % of the control signal. This signal recovered in two phases when the muscle was rested. An initial phase had a time constant of 1.7 +/- 0.2 s so that by 20 s of recovery the SR Ca(2+) signal was 86 +/- 7 % of control; the second phase was slower and by 5 min the

  16. Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

    National Research Council Canada - National Science Library

    Ren-Jie Tang Hua Liu Yang Yang Lei Yang Xiao-Shu Gao Veder J Garcia Sheng Luan Hong-Xia Zhang

    2012-01-01

    ...+) signals that are transmitted by diverse calcium sensors. The calcineurin B-like (CBL) protein family represents calcium sensors that decode calcium signals through specific interactions with a group of CBL-interacting protein kinases...

  17. Nitric oxide-dependent activation of CaMKII increases diastolic sarcoplasmic reticulum calcium release in cardiac myocytes in response to adrenergic stimulation.

    Directory of Open Access Journals (Sweden)

    Jerry Curran

    Full Text Available Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+ leak through ryanodine receptors. Beta-adrenergic (β-AR tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca(2+ waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+ leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+ leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca(2+ leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis

  18. Detection of plasma membrane Ca(2+)-ATPase activity in mouse T lymphocytes by flow cytometry using fluo-3-loaded vesicles.

    Science.gov (United States)

    Telford, W G; Miller, R A

    1996-07-01

    The plasma membrane Ca(2+)-ATPase (PMCA) is the primary means by which many cell types pump calcium out of the cytosol following release of calcium from internal stores, returning intracellular calcium concentrations to normal levels. Traditional methods for measuring PMCA activity utilizing isotopic calcium uptake into inside-out (IO) membrane vesicles have poor specificity for PMCA activity and require large numbers of cells. A flow cytometric method has been devised that allows the measurement of calcium uptake in IO vesicles using the fluorescent calcium chelator fluo-3. IO vesicles from mouse lymphocytes were loaded with fluo-3 pentapotassium salt and analyzed by flow cytometry following treatment with buffered calcium and/or ATP. IO vesicles appeared as a subpopulation of low forward-scatter/low side-scatter events, which were distinguishable from higher side-scatter debris. Treatment of vesicles with calcium and ATP resulted in a 5-fold to 30-fold increase in IO vesicle fluo-3 fluorescence. Measurement of uptake kinetics gave K0.5 values of approximately 0.2-0.8 microM and 2 mM for calcium- and ATP-stimulated PMCA activity, respectively, which were consistent with published values obtained by other methods. Broad specificity P-type ATPase inhibitors and more narrowly specific PMCA and calmodulin inhibitors all blocked calcium uptake, whereas thapsigargin (an endoplasmic/sarcoplasmic reticulum (ER/SR-AT-Pase) inhibitor) had no effect, indicating that the assay provides a specific measure of vesicular PMCA activity. Flow cytometric analysis, therefore, may represent a useful approach for quantifying PMCA activity in mammalian cells.

  19. Calcium

    Science.gov (United States)

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

  20. Selective upregulation of the expression of plasma membrane calcium ATPase isoforms upon differentiation and 1,25(OH)2D3-vitamin treatment of colon cancer cells.

    Science.gov (United States)

    Ribiczey, Polett; Papp, Béla; Homolya, László; Enyedi, Ágnes; Kovács, Tünde

    2015-08-14

    We have previously presented co-expression of the plasma membrane calcium ATPase isoforms 4b (PMCA4b) and 1b (PMCA1b) in colon carcinoma cells, and selective upregulation of PMCA4b during differentiation initiated by short chain fatty acids or post-confluent growth. Here we show that the induction of PMCA4b expression is a characteristic feature of the post-confluency-induced differentiation of both enterocyte-type and goblet cell-type colon cancer cells. Vitamin D3 (1,25(OH)2D3) is a well-known regulator of intestinal Ca(2+) absorption and of basic cell functions such as growth and differentiation in various cell types. As PMCA proteins are involved both in intestinal Ca(2+) absorption and adenocarcinoma cell differentiation, we investigated the effect of 1,25(OH)2D3 on PMCA expression in enterocyte-like colon carcinoma cells, and monitored its effect on the expression of various differentiation markers. 1,25(OH)2D3 stimulated PMCA1b, but not PMCA4b expression without modulating the expression of the majority of the differentiation markers examined. Caco-2 cells differentiated in post-confluent cultures present normal enterocyte-like intestinal epithelial phenotype. To better understand the role of PMCA proteins in vectorial Ca(2+) transport by enterocytes, we also studied their subcellular localization in mature polarized Caco-2 cells. Both PMCA isoforms were located to the basolateral membrane, and the PMCA-specific immunofluorescent signal was significantly higher in vitamin D3-treated cells, underlining the 1,25(OH)2D3-induced upregulation of PMCA (presumably 1b isoform) expression in differentiated Caco-2 cells. We suggest that while PMCA1b has a housekeeping function in colon cancer cells, PMCA4b participates in the reorganization of the Ca(2+) signalling machinery during cell differentiation. The subcellular localization of PMCA1b and its selective 1,25(OH)2D3-dependent upregulation indicate that this isoform may have a specific role in 1,25(OH)2D3

  1. Effect of Hypoxia on the Calcium and Magnesium Content, Lipid Peroxidation Level, and Ca2+-ATPase Activity of Syncytiotrophoblast Plasma Membranes from Placental Explants

    Directory of Open Access Journals (Sweden)

    Delia I. Chiarello

    2014-01-01

    Full Text Available In the current study the possible relationship between the Ca2+/Mg2+ ratio of human syncytiotrophoblast plasma membranes and their lipid peroxidation and Ca2+-ATPase activity was determined. Syncytiotrophoblast plasma membranes of placental explants cultured under hypoxia increased their lipid peroxidation and Ca2+ content, diminished their Ca2+-ATPase activity, and kept their Mg2+ content unchanged. Membranes preincubated with different concentrations of Ca2+ increased their Ca2+ content without changes in their Mg2+ content. There is a direct relationship between Ca2+ content and lipid peroxidation of the membranes, as well as an inverse relationship between their Ca2+ content and Ca2+-ATPase activity. On the contrary, preincubation of membranes with different concentrations of Mg2+ showed a higher Mg2+ content without changing their lipid peroxidation and Ca2+-ATPase activity. Explants cultured under hypoxia in the presence of 4 mM MgSO4 showed similar values of lipid peroxidation and Ca2+-ATPase activity of their membranes compared to those of explants cultured under normoxia. Increased Ca2+ content of the membranes by interacting with negatively charged phospholipids could result in destabilizing effects of the membrane structure, exposing hydrocarbon chains of fatty acids to the action of free radicals. Mg2+ might exert a stabilizing effect of the membranes, avoiding their exposure to free radicals.

  2. Rotary ATPases

    Science.gov (United States)

    Stewart, Alastair G.; Sobti, Meghna; Harvey, Richard P.; Stock, Daniela

    2013-01-01

    Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors. PMID:23369889

  3. Structural divergence between the two subgroups of P5 ATPases

    DEFF Research Database (Denmark)

    Sørensen, Danny Mollerup; Buch-Pedersen, Morten Jeppe; Palmgren, Michael Broberg

    2010-01-01

    been identified in the endoplasmic reticulum and seem to have basic functions in protein maturation and secretion. P5B ATPases localize to vacuolar/lysosomal or apical membranes and in animals play a role in hereditary neuronal diseases. Here we have used a bioinformatical approach to identify....... Together these findings indicate that P5A and P5B ATPases are structurally and functionally different....

  4. Excessive signal transduction of gain-of-function variants of the calcium-sensing receptor (CaSR are associated with increased ER to cytosol calcium gradient.

    Directory of Open Access Journals (Sweden)

    Marianna Ranieri

    Full Text Available In humans, gain-of-function mutations of the calcium-sensing receptor (CASR gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA and reducing expression of Plasma Membrane Calcium-ATPase (PMCA. Wild-type CaSR (hCaSR-wt and its gain-of-function (hCaSR-R990G; hCaSR-N124K variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate receptor inputs to cell function.

  5. Excessive signal transduction of gain-of-function variants of the calcium-sensing receptor (CaSR) are associated with increased ER to cytosol calcium gradient.

    Science.gov (United States)

    Ranieri, Marianna; Tamma, Grazia; Di Mise, Annarita; Vezzoli, Giuseppe; Soldati, Laura; Svelto, Maria; Valenti, Giovanna

    2013-01-01

    In humans, gain-of-function mutations of the calcium-sensing receptor (CASR) gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA) and reducing expression of Plasma Membrane Calcium-ATPase (PMCA). Wild-type CaSR (hCaSR-wt) and its gain-of-function (hCaSR-R990G; hCaSR-N124K) variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate) receptor inputs to cell function.

  6. On Allosteric Modulation of P-Type Cu+-ATPases

    DEFF Research Database (Denmark)

    Mattle, Daniel; Sitsel, Oleg; Autzen, Henriette Elisabeth

    2013-01-01

    -specific sequence motifs and structural elements that are linked to transport specificity and mechanistic modulation. Here we provide an overview of the Cu+-transporting ATPases (of subclass PIB) and compare them to the well-studied sarco(endo)plasmic reticulum Ca2 +-ATPase (of subclass PIIA). Cu+ ions in the cell...

  7. The E646D-ATP13A4 mutation associated with autism reveals a defect in calcium regulation.

    Science.gov (United States)

    Vallipuram, Janaki; Grenville, Jeffrey; Crawford, Dorota A

    2010-03-01

    ATP13A4 is a member of the subfamily of P5-type ATPases. P5-type ATPases are the least studied of the P-type ATPase subfamilies with no ion specificities assigned to them. In order to elucidate ATP13A4 function, we studied the protein's subcellular localization and tested whether it is involved in calcium regulation. The intracellular calcium concentration was measured in COS-7 cells over-expressing mouse ATP13A4 using ratiometric calcium imaging with fura-2 AM as a calcium indicator. The results of this study show that ATP13A4 is localized to the endoplasmic reticulum (ER). Furthermore, we demonstrate that over-expression of ATP13A4 in COS-7 cells caused a significant increase in the intracellular calcium level. Interestingly, over-expression of the sequence variant containing a substitution of aspartic acid for a glutamic acid (E646D), previously found in patients with autism spectrum disorder (ASD), did not increase the free cellular calcium likely due to the mutation. In this study, we also describe the expression of ATP13A4 during mouse embryonic development. Quantitative real-time PCR revealed that ATP13A4 was highly expressed at embryonic days 15-17, when neurogenesis takes place. The present study is the first to provide further insights into the biological role of a P5-type ATPase. Our results demonstrate that ATP13A4 may be involved in calcium regulation and that its expression is developmentally regulated. Overall, this study provides support for the hypothesis that ATP13A4 may play a vital role in the developing nervous system and its impairment can contribute to the symptoms seen in ASD.

  8. Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

    Directory of Open Access Journals (Sweden)

    D.V. Vassallo

    2008-09-01

    Full Text Available Lead (Pb2+ poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM to the bath. Changes in rate of stimulation (0.1-1.5 Hz, relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM, and the effect of isoproterenol (20 ng/mL were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

  9. Effect of roscovitine on intracellular calcium dynamics: differential enantioselective responses.

    Science.gov (United States)

    Tamma, Grazia; Ranieri, Marianna; Di Mise, Annarita; Spirlì, Alessia; Russo, Annamaria; Svelto, Maria; Valenti, Giovanna

    2013-12-02

    Cyclin-dependent kinases (CDKs) inhibitors have emerged as interesting therapeutic candidates. Of these, (S)-roscovitine has been proposed as potential neuroprotective molecule for stroke while (R)-roscovitine is currently entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis. In addition, (R)-roscovitine has been suggested as potential antihypertensive and anti-inflammatory drug. Dysfunction of intracellular calcium balance is a common denominator of these diseases, and the two roscovitine enantiomers (S and R) are known to modulate calcium voltage channel activity differentially. Here, we provide a detailed description of short- and long-term responses of roscovitine on intracellular calcium handling in renal epithelial cells. Short-term exposure to (S)-roscovitine induced a cytosolic calcium peak, which was abolished after stores depletion with cyclopiazonic acid (CPA). Instead, (R)-roscovitine caused a calcium peak followed by a small calcium plateau. Cytosolic calcium response was prevented after stores depletion. Bafilomycin, a selective vacuolar H(+)-ATPase inhibitor, abolished the small calcium plateau. Long-term exposure to (R)-roscovitine significantly reduced the basal calcium level compared to control and (S)-roscovitine treated cells. However, both enantiomers increased calcium accumulation in the endoplasmic reticulum (ER). Consistently, cells treated with (R)-roscovitine showed a significant increase in SERCA activity, whereas (S)-roscovitine incubation resulted in a reduced PMCA expression. We also found a tonic decreased ability to release calcium from the ER, likely via IP3 signaling, under treatment with (S)- or (R)-roscovitine. Together our data revealed that (S)-roscovitine and (R)-roscovitine exert distinct enantiospecific effects on intracellular calcium signaling in renal epithelial cells. This distinct pharmacological profile can be relevant for roscovitine clinical use.

  10. Response of Ca2+-ATPase to clinorotaion of pea seedlings. O. M. Nedukha and E. L. Kordyum

    Science.gov (United States)

    Nedukha, Olena

    2016-07-01

    The present study was aimed to reveal of response of Ca2+-ATPase activity of cortex cells in distal elongation zone of Pisum sativum root to slow clinorotation. Pea seedlings were grown on a horizontal clinostat (2 rpm) and in the stationary control for 6 days. The electron-cytochemical method was used to examine the effects of imitated microgravity on the distribution of Ca2+-ATPase in outer layers of root cortex. The quantitative analysis of the density of cytochemical reaction products was measured using the Image J program. Electron microscopy showed the presence of electron-dense lead phosphate precipitated grains, the enzymatic activity reaction products on the plasma membrane, membranes of vesicular structures, endoplasmic reticulum (ER) and on organelles envelope in both of samples of the stationary control and clinorotated seedlings. We revealed the sensitivity of Ca2+-ATPase to clinorotation. The quantitative analysis of the area and density of enzymatic activity reaction products revealed that clinorotation led to the decrease of 3.4 times the density of reaction products on the plasma membrane and the increase of reaction products density on endomembranes and organelles membranes, in particular: in 2.2 times on mitochondria membranes; in 1.3 times - on membranes of ER; in 2.5 times - on tonoplast; by an order of magnitude greater - on contacting membranes of organelles with plasma membrane in comparison with such in cells of control samples. The data analysis can indicate an intensification of calcium pump on endomembranes, on envelopes of cytoplasmic organelles and nucleus. The obtained data suggest that the redistribution of Ca2+-ATPase activity in cells can be mediated by the activation of certain isoforms of enzyme or/and by an activation of Ca2+/H+ antiporter in plasma membrane that helps to maintain optimal calcium balance in plant cells under imitated microgravity.

  11. Disruption of the vacuolar calcium-ATPases in arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway

    Science.gov (United States)

    Calcium (Ca2+) signals regulate many aspects of plant development, including the Hypersensitive Response (HR) that triggers a programmed cell death response to protect a plant from a pathogen. A transient increase in cytosolic Ca2+ ([Ca2+]cyt ) results from Ca2+ entry from the apoplast or release fr...

  12. Cardiac contraction and calcium transport function aftersevere burn injury in rats

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To examine the function change of myocardial calcium transports and determined what role the change plays in cardiac dysfunction after severe burn injury in rats. Methods: The contraction and relaxation properties of the left ventricle (LV) were studied in the isolated hearts preparations of Wistar rats at 3, 8, and 24 h after a 30%TBSA (total body surface area) full-thickness burn. The calcium transport function of the sarcoplasmic reticulum (SR) was measured by the millipore filtration technique. Results: The maximal rate of LV pressure (± dp/dtmax) of the burn group was significantly lower than that of the control group (P < 0.01). In addition, the calciumdependent ATPase activity and the coupling ratio of SR were also markedly depressed. Conclusions: It indicates that the decrease in the SR calcium transport function is one of the important mechanisms for the cardiac contractile dysfunction after severe burn injury.

  13. Regulation of Calcium Fluxes by GPX8, a Type-II Transmembrane Peroxidase Enriched at the Mitochondria-Associated Endoplasmic Reticulum Membrane.

    Science.gov (United States)

    Yoboue, Edgar Djaha; Rimessi, Alessandro; Anelli, Tiziana; Pinton, Paolo; Sitia, Roberto

    2017-09-20

    Glutathione peroxidases (GPXs) are enzymes that are present in almost all organisms with the primary function of limiting peroxide accumulation. In mammals, two of the eight members (GPX7 and GPX8) reside in the endoplasmic reticulum (ER). A peculiar feature of GPX8 is the concomitant presence of a conserved N-terminal transmembrane domain (TMD) and a C-terminal KDEL-like motif for ER localization. Investigating whether and how GPX8 impacts Ca(2+) homeostasis and signaling. We show that GPX8 is enriched in mitochondria-associated membranes and regulates Ca(2+) storage and fluxes. Its levels correlate with [Ca(2+)]ER, and cytosolic and mitochondrial Ca(2+) fluxes. GPX7, which lacks a TMD, does not share these properties. Deleting or replacing the GPX8 TMD with an unrelated N-terminal membrane integration sequence abolishes all effects on Ca(2+) fluxes, whereas appending the GPX8 TMD to GPX7 transfers the Ca(2+)-regulating properties. Innovation and Conclusion: The notion that the TMD of GPX8, in addition to its enzymatic activity, is essential for regulating Ca(2+) dynamics reveals a novel level of integration between redox-related proteins and Ca(2+) signaling/homeostasis. Antioxid. Redox Signal. 27, 583-595.

  14. Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol.

    Science.gov (United States)

    Breckenridge, David G; Stojanovic, Marina; Marcellus, Richard C; Shore, Gordon C

    2003-03-31

    Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

  15. The calcium binding protein ALG-2 binds and stabilizes Scotin, a p53-inducible gene product localized at the endoplasmic reticulum membrane

    DEFF Research Database (Denmark)

    Draeby, Ingrid; Woods, Yvonne L; la Cour, Jonas Marstrand

    2007-01-01

    ALG-2 (apoptosis linked gene 2 product) is a calcium binding protein for which no clear cellular function has been established. In this study we identified Scotin as a novel ALG-2 target protein containing 6 PXY and 4 PYP repeats, earlier identified in the ALG-2 binding regions of AIP1/ALIX and TSG......101, respectively. An in vitro synthesized C-terminal fragment of Scotin bound specifically to immobilized recombinant ALG-2 and tagged ALG-2 and Scotin were shown by immunoprecipitation to interact in MCF7 and U2OS cell lines. Furthermore ALG-2 bound to endogenous Scotin in extracts from mouse NIH3T3...

  16. Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex1

    Science.gov (United States)

    Glukhov, Alexey V.; Kalyanasundaram, Anuradha; Lou, Qing; Hage, Lori T.; Hansen, Brian J.; Belevych, Andriy E.; Mohler, Peter J.; Knollmann, Björn C.; Periasamy, Muthu; Györke, Sandor; Fedorov, Vadim V.

    2015-01-01

    Aims Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. Methods and results In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca2+ cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2−/−) mice. Casq2−/− mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca2+ cycling, including abnormal Ca2+ release, periods of significantly elevated diastolic Ca2+ levels leading to pauses and unstable pacemaker rate. Importantly, Ca2+ cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca2+ transient upstrokes throughout the atrial pacemaker complex. Conclusions Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca2+ release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients. PMID:24216388

  17. Fine structure and Ca-ATPase activity of the stratum intermedium cells during odontogenesis in gars, Lepisosteus, Actinopterygii.

    Science.gov (United States)

    Sasagawa, Ichiro; Ishiyama, Mikio

    2002-01-01

    This is the first report on the stratum intermedium in vertebrates other than mammals. The aim of this study is to elucidate the fine structure and cytochemical features of the stratum intermedium during the stages of enameloid formation in Lepisosteus. Inner dental epithelium, stratum intermedium, stellate reticulum, and outer dental epithelium are consistently present in the tooth germs of Lepisosteus. The stratum intermedium cells are oval in shape, contain elliptical nuclei, and extend many small processes. It is implied that the structure of the enamel organ is different among actinopterygians, and that constitution of the enamel organ in Lepisosteus resembles that in higher vertebrates. Marked Ca-ATPase activity is observed at the cell membrane of the stratum intermedium cells, suggesting that the cells are involved in calcium transport during the stages of enameloid formation.

  18. Calcium and Mitosis

    Science.gov (United States)

    Hepler, P.

    1983-01-01

    Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.

  19. Calcium and Mitosis

    Science.gov (United States)

    Hepler, P.

    1983-01-01

    Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.

  20. A conformation-specific interhelical salt bridge in the K+ binding site of gastric H,K-ATPase

    NARCIS (Netherlands)

    Koenderink, J.B.; Swarts, H.G.P.; Willems, P.; Krieger, E.; Pont, J.J.H.H.M. de

    2004-01-01

    Homology modeling of gastric H, K-ATPase based on the E-2 model of sarcoplasmic reticulum Ca2+-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835-839) revealed the presence of a single high-affinity binding site for K+ and an E-2 form-specific salt bridge between Glu(820) (M6) and Lys(791)

  1. Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling [v1; ref status: indexed, http://f1000r.es/32q

    Directory of Open Access Journals (Sweden)

    Nicola Fameli

    2014-04-01

    Full Text Available Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca2+ waves. In pulmonary artery smooth muscle cells (PASMCs it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP triggers increases in cytoplasmic Ca2+ via L-SR junctions, in a manner that requires initial Ca2+ release from lysosomes and subsequent Ca2+-induced Ca2+ release (CICR via ryanodine receptor (RyR subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca2+ signal information as input data. Simulations of NAADP-dependent junctional Ca2+ transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca2+ signals and simulated Ca2+ transients within L-SR junctions, we estimate that “trigger zones” comprising 60–100 junctions are required to confer a signal of similar magnitude. This is compatible with the 110 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional [Ca2+] such that there is a failure to breach the threshold for CICR via RyR3. L

  2. The effect of kinetin on cytochemical localization of Mg++ dependent ATP-ase in isolated lupine cotyledons

    Directory of Open Access Journals (Sweden)

    Roman Przymusiński

    2014-01-01

    Full Text Available ATP-ase activity stimulated with Mg++ ions was localized cytochermically in lupine cotyledons. Studies were also made of the effect of kinetin on this activity. Activity of Mg++ dependent ATP-ase was observed in plasmalemma, nucleus, nucleolus, endoplasmic reticulum, thylakoid membranes, prolamellar bodies, cell wall, and irilter-cellular spaces. Kinetin (6-furfurylaminpurine used in the experiment, stimulated ATP-ase activity, but dad not affect its localization.

  3. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.P.

    1978-01-01

    Light and heavy sarcoplasmic reticulum vesicles isolated from rabbit leg muscle have been used in a study of chloride-induced calcium release. The biochemical and morphological data indicate that light sarcoplasmic reticulum vesicles are derived from the longitudinal reticulum and heavy sarcoplasmic reticulum vesicles are derived from the terminal cisternae of the sarcoplasmic reticulum. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP to amounts greater than 100 nmoles Ca/sup + +/ per mg of protein in less than one minute. Light and heavy sarcoplasmic reticulum vesicles each had a biphasic time course of calcium uptake. The initial uptake was followed by a rapid release after approximately one minute, of 30 to 40% of the accumulated calcium, which was then followed by a slower phase of calcium accumulation. Results indicate that the chloride induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization. The release of calcium from the light SR vesicles is probably due to osmotic swelling and the release of calcium from the heavy SR vesicles is probably due to depolarization.

  4. Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

    Directory of Open Access Journals (Sweden)

    V.A. Vizotto

    2007-01-01

    Full Text Available We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2, phospholamban (PLB, and ryanodine channel (RYR2 mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01. The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively. Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

  5. Analysis of Ca(2+) uptake into the smooth endoplasmic reticulum of permeabilised sternal epithelial cells during the moulting cycle of the terrestrial isopod Porcellio scaber.

    Science.gov (United States)

    Hagedorn, Monica; Ziegler, Andreas

    2002-07-01

    In terrestrial isopods, large amounts of Ca(2+) are transported across anterior sternal epithelial cells during moult-related deposition and resorption of CaCO(3) deposits. Because of its toxicity and function as a second messenger, resting cytosolic Ca(2+) levels must be maintained below critical concentrations during epithelial Ca(2+) transport, raising the possibility that organelles play a role during Ca(2+) transit. We therefore studied the uptake of Ca(2+) into Ca(2+)-sequestering organelles by monitoring the formation of birefringent calcium oxalate crystals in permeabilised anterior and posterior sternal epithelium cells of Porcellio scaber during Ca(2+)-transporting and non-transporting stages of the moulting cycle using polarised-light microscopy. The results indicate ATP-dependent uptake of Ca(2+) into organelles. Half-maximal crystal growth at a Ca(2+) activity, a(Ca), of 0.4 micromol l(-1) and blockade by cyclopiazonic acid suggest Ca(2+) uptake into the smooth endoplasmic reticulum by the smooth endoplasmic reticulum Ca(2+)-ATPase. Analytical electron microscopical techniques support this interpretation by revealing the accumulation of Ca(2+)-containing crystals in smooth membranous intracellular compartments. A comparison of different moulting stages demonstrated a virtual lack of crystal formation in the early premoult stage and a significant fivefold increase between mid premoult and the Ca(2+)-transporting stages of late premoult and intramoult. These results suggest a contribution of the smooth endoplasmic reticulum as a transient Ca(2+) store during intracellular Ca(2+) transit.

  6. Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography

    Directory of Open Access Journals (Sweden)

    Geisler Markus

    1998-01-01

    Full Text Available In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993 et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 &mgr;M and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

  7. Origin and evolution of metal P-type ATPases in Plantae (Archaeplastida)

    OpenAIRE

    2014-01-01

    Metal ATPases are a subfamily of P-type ATPases involved in the transport of metal cations across biological membranes. They all share an architecture featuring eight transmembrane domains in pairs of two and are found in prokaryotes as well as in a variety of Eukaryotes. In Arabidopsis thaliana, eight metal P-type ATPases have been described, four being specific to copper transport and four displaying a broader metal specificity, including zinc, cadmium and possibly copper and calcium. So fa...

  8. Structure and function of CrACA1, the major PM-type Ca2+-ATPase, expressed at the peak of the gravity-directed trans-cell calcium current in spores of the fern Ceratopteris richardii.

    Science.gov (United States)

    Bushart, T J; Cannon, A; Clark, G; Roux, S J

    2014-01-01

    Spores of the fern Ceratopteris richardii have proven to be a valuable single-cell system for studying gravity responses. The earliest cellular change directed by gravity in these cells is a trans-cell calcium current, which peaks near 10 h after the spores are induced to germinate. This current is needed for gravity-directed axis alignment, and its peak is coincident with the time period when gravity polarises the direction of subsequent nuclear migration and rhizoid growth. Transcriptomic analysis of genes expressed at the 10-h time point revealed several that encode proteins likely to be key components that either drive the current or regulate it. Notable among these is a plasma membrane (PM)-type Ca(2+) ATPase, CrACA1, whose activity pumping Ca(2+) out of cells is regulated by gravity. This report provides an initial characterisation of the structure and expression of this protein, and demonstrates its heterologous function complementing the K616 mutant of yeast, which is deficient in PM-type Ca(2+) pump activity. Gravity-induced changes in the trans-cell Ca(2+) current occur within seconds, a result consistent with the hypothesis that the force of gravity can rapidly alter the post-translational state of the channels and pumps that drive this current across spore cells. This report identifies a transporter likely to be a key driver of the current, CrACA1, and characterises the role of this protein in early germination and gravity-driven polarity fixation through analysis of expression levels, functional complementation and pharmacological treatments. These data, along with newly available transcriptomic data obtained at the 10-h time point, indicate that CrACA1 is present, functional and likely a major contributing component of the trans-cell Ca(2+) efflux. CrACA1 is not necessary for polar axis alignment, but pharmacological perturbations of it disrupt rhizoid development. These data support and help refine the post-translational modification model for

  9. P4-ATPases

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Theorin, Lisa; Palmgren, Michael Broberg;

    2014-01-01

    Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Among these ATP-driven transporters, the P4 subfamily of P-type ATPases (P4-ATPases......) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4...... to include the regulation of membrane traffic, cytoskeletal dynamics, cell division, lipid metabolism, and lipid signaling. In this review, we will summarize the basic features of P4-ATPases and the physiological implications of their lipid transport activity in the cell. © 2013 The Author(s)....

  10. Calcium signaling of pancreatic acinar cells in the pathogenesis of pancreatitis.

    Science.gov (United States)

    Li, Jun; Zhou, Rui; Zhang, Jian; Li, Zong-Fang

    2014-11-21

    Pancreatitis is an increasingly common and sometimes severe disease that lacks a specific therapy. The pathogenesis of pancreatitis is still not well understood. Calcium (Ca(2+)) is a versatile carrier of signals regulating many aspects of cellular activity and plays a central role in controlling digestive enzyme secretion in pancreatic acinar cells. Ca(2+) overload is a key early event and is crucial in the pathogenesis of many diseases. In pancreatic acinar cells, pathological Ca(2+) signaling (stimulated by bile, alcohol metabolites and other causes) is a key contributor to the initiation of cell injury due to prolonged and global Ca(2+) elevation that results in trypsin activation, vacuolization and necrosis, all of which are crucial in the development of pancreatitis. Increased release of Ca(2+) from stores in the intracellular endoplasmic reticulum and/or increased Ca(2+) entry through the plasma membrane are causes of such cell damage. Failed mitochondrial adenosine triphosphate (ATP) production reduces re-uptake and extrusion of Ca(2+) by the sarco/endoplasmic reticulum Ca(2+)-activated ATPase and plasma membrane Ca(2+)-ATPase pumps, which contribute to Ca(2+) overload. Current findings have provided further insight into the roles and mechanisms of abnormal pancreatic acinar Ca(2+) signals in pancreatitis. The lack of available specific treatments is therefore an objective of ongoing research. Research is currently underway to establish the mechanisms and interactions of Ca(2+) signals in the pathogenesis of pancreatitis.

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

    Science.gov (United States)

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

    2006-09-01

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

  12. The structural basis of calcium transport by the calcium pump

    DEFF Research Database (Denmark)

    Olesen, Claus; Picard, Martin; Winther, Anne-Marie Lund

    2007-01-01

    The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding......, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset...... of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen...

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

    Science.gov (United States)

    Liang, Willmann

    2008-01-01

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

  14. Synthetic null-cysteine phospholamban analogue and the corresponding transmembrane domain inhibit the Ca-ATPase.

    Science.gov (United States)

    Karim, C B; Marquardt, C G; Stamm, J D; Barany, G; Thomas, D D

    2000-09-05

    Chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) have been used to analyze the structure and function of phospholamban (PLB), a 52-residue integral membrane protein that regulates the calcium pump (Ca-ATPase) in cardiac sarcoplasmic reticulum (SR). PLB exists in equilibrium between monomeric and pentameric forms, as observed by SDS-PAGE, EPR, and fluorescence. It has been proposed that inhibition of the pump is due primarily to the monomeric form, with both pentameric stability and inhibition dependent primarily on the transmembrane (TM) domain. To test these hypotheses, we have studied the physical and functional properties of a synthetic null-cysteine PLB analogue that is entirely monomeric on SDS-PAGE, and compared it with the synthetic null-cysteine TM domain (residues 26-52). The TM domain was found to be primarily oligomeric on SDS-PAGE, and boundary lipid spin label analysis in lipid bilayers verified that the isolated TM domain is more oligomeric than the full-length parent molecule. These results indicate that the stability of the PLB pentamer is due primarily to attractive interactions between hydrophobic TM domains, overcoming the repulsive electrostatic interactions between the cationic cytoplasmic domains (residues 1-25). When reconstituted into liposomes containing the Ca-ATPase, the null-cysteine TM domain had the same inhibitory function as that of the full-length parent molecule. We conclude that the TM domain of PLB is sufficient for inhibitory function, the oligomeric stability of PLB does not determine its inhibitory activity, and the three Cys residues in the TM domain are not required for inhibitory function.

  15. Endoplasmic Reticulum Stress and Associated ROS

    Directory of Open Access Journals (Sweden)

    Hafiz Maher Ali Zeeshan

    2016-03-01

    Full Text Available The endoplasmic reticulum (ER is a fascinating network of tubules through which secretory and transmembrane proteins enter unfolded and exit as either folded or misfolded proteins, after which they are directed either toward other organelles or to degradation, respectively. The ER redox environment dictates the fate of entering proteins, and the level of redox signaling mediators modulates the level of reactive oxygen species (ROS. Accumulating evidence suggests the interrelation of ER stress and ROS with redox signaling mediators such as protein disulfide isomerase (PDI-endoplasmic reticulum oxidoreductin (ERO-1, glutathione (GSH/glutathione disuphide (GSSG, NADPH oxidase 4 (Nox4, NADPH-P450 reductase (NPR, and calcium. Here, we reviewed persistent ER stress and protein misfolding-initiated ROS cascades and their significant roles in the pathogenesis of multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases.

  16. The minimal requirements to use calcium imaging to analyze ICRAC.

    Science.gov (United States)

    Alansary, Dalia; Kilch, Tatiana; Holzmann, Christian; Peinelt, Christine; Hoth, Markus; Lis, Annette

    2014-06-02

    Endogenous calcium release-activated channel (CRAC) currents are usually quite small and not always easy to measure using the patch-clamp technique. While we have, for instance, successfully recorded very small CRAC currents in primary human effector T cells, we have not yet managed to record CRAC in naïve primary human T cells. Many groups, including ours, therefore use Ca(2+) imaging technologies to analyze CRAC-dependent Ca(2+) influx. However, Ca(2+) signals are quite complex and depend on many different transporter activities; thus, it is not trivial to make quantitative statements about one single transporter, in this case CRAC channels. Therefore, a detailed patch-clamp analysis of ICRAC is always preferred. Since many laboratories use Ca(2+) imaging for ICRAC analysis, we detail here the minimal requirements for reliable measurements. Ca(2+) signals not only depend on the net Ca(2+) influx through CRAC channels but also depend on other Ca(2+) influx mechanisms, K(+) channels or Cl(-) channels (which determine the membrane potential), Ca(2+) export mechanisms like plasma membrane Ca(2+) ATPase (PMCA), sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) or Na(+)-Ca(2+) exchangers, and (local) Ca(2+) buffering often by mitochondria. In this protocol, we summarize a set of experiments that allow (quantitative) statements about CRAC channel activity using Ca(2+) imaging experiments, including the ability to rule out Ca(2+) signals from other sources.

  17. P4-ATPases

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Theorin, Lisa; Palmgren, Michael Broberg

    2014-01-01

    ) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4...

  18. beta-Adrenergic activation reveals impaired cardiac calcium handling at early stage of diabetes.

    Science.gov (United States)

    op den Buijs, Jorn; Miklós, Zsuzsanna; van Riel, Natal A W; Prestia, Christina M; Szenczi, Orsolya; Tóth, András; Van der Vusse, Ger J; Szabó, Csaba; Ligeti, László; Ivanics, Tamás

    2005-01-21

    Cardiac function is known to be impaired in diabetes. Alterations in intracellular calcium handling have been suggested to play a pivotal role. This study aimed to test the hypothesis that beta-adrenergic activation can reveal the functional derangements of intracellular calcium handling of the 4-week diabetic heart. Langendorff perfused hearts of 4-week streptozotocin-induced diabetic rats were subjected to the beta-adrenoceptor agonist isoproterenol. Cyclic changes in [Ca(2+)](i) levels were measured throughout the cardiac cycle using Indo-1 fluorescent dye. Based on the computational analysis of the [Ca(2+)](i) transient the kinetic parameters of the sarcoplasmic reticulum Ca(2+)-ATPase and the ryanodine receptor were determined by minimizing the squared error between the simulated and the experimentally obtained [Ca(2+)](i) transient. Under unchallenged conditions, hemodynamic parameters were comparable between control and diabetic hearts. Isoproterenol administration stimulated hemodynamic function to a greater extent in control than in diabetic hearts, which was exemplified by more pronounced increases in rate of pressure development and decline. Under unchallenged conditions, [Ca(2+)](i) amplitude and rate of rise and decline of [Ca(2+)](i) as measured throughout the cardiac cycle were comparable between diabetic and control hearts. Differences became apparent under beta-adrenoceptor stimulation. Upon beta-activation the rate-pressure product showed a blunted response, which was accompanied by a diminished rise in [Ca(2+)](i) amplitude in diabetic hearts. Computational analysis revealed a reduced function of the sarcoplasmic reticulum Ca(2+)-ATPase and Ca(2+)-release channel in response to beta-adrenoceptor challenge. Alterations in Ca(2+)(i) handling may play a causative role in depressed hemodynamic performance of the challenged heart at an early stage of diabetes.

  19. The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm

    DEFF Research Database (Denmark)

    Winther, Anne-Marie Lund; Bublitz, Maike; Karlsen, Jesper Lykkegaard

    2013-01-01

    The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca(2+), initiated by the release of Ca(2+) from the sarcoplasmic reticulum and terminated by re-sequestration of Ca(2+) into the sarcoplasmic reticulum as the main mechanism of Ca(2+) removal....... Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called...

  20. Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

    Science.gov (United States)

    Bravo, Roberto; Gutierrez, Tomás; Paredes, Felipe; Gatica, Damián; Rodriguez, Andrea E.; Pedrozo, Zully; Chiong, Mario; Parra, Valentina; Quest, Andrew F.G.; Rothermel, Beverly A.; Lavandero, Sergio

    2014-01-01

    Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER–mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders. PMID:22064245

  1. Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy.

    Science.gov (United States)

    Montague, Karli; Malik, Bilal; Gray, Anna L; La Spada, Albert R; Hanna, Michael G; Szabadkai, Gyorgy; Greensmith, Linda

    2014-07-01

    Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington's disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases.

  2. The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Zhang Wei-hua

    2011-02-01

    Full Text Available Abstract Background The extracellular calcium-sensing receptor (CaSR belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA is unknown. Methods The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca2+]i was detected by a laser-scanning confocal microscope. Results The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs and PAs. Increased levels of [Ca2+]o (extracellular calcium concentration or Gd3+ (an agonist of CaSR induced an increase of [Ca2+]i and PAs constriction in a concentration-dependent manner. In addition, the above-mentioned effects of Ca2+ and Gd3+ were inhibited by U73122 (specific inhibitor of PLC, 2-APB (specific antagonist of IP3 receptor, and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase. Conclusions CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca2+]i through G-PLC-IP3 pathway.

  3. Crystal Structure of the Vanadate-Inhibited Ca(2+)-ATPase.

    Science.gov (United States)

    Clausen, Johannes D; Bublitz, Maike; Arnou, Bertrand; Olesen, Claus; Andersen, Jens Peter; Møller, Jesper Vuust; Nissen, Poul

    2016-04-05

    Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca(2+)-ATPase with bound vanadate in the absence of Ca(2+). Vanadate is bound at the catalytic site as a planar VO3(-) in complex with water and Mg(2+) in a dephosphorylation transition-state-like conformation. Validating bound VO3(-) by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl(-) site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca(2+)-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3(-) structure with that in E2·BeF3(-) (E2P ground state analog) reveals multiple binding modes to the Ca(2+)-ATPase.

  4. Detergent resistant membrane fractions are involved in calcium signaling in Müller glial cells of retina.

    Science.gov (United States)

    Krishnan, Gopinath; Chatterjee, Nivedita

    2013-08-01

    Compartmentalization of the plasma membrane into lipid microdomains promotes efficient cellular processes by increasing local molecular concentrations. Calcium signaling, either as transients or propagating waves require integration of complex macromolecular machinery. Calcium waves represent a form of intercellular signaling in the central nervous system and the retina. We hypothesized that the mechanism for calcium waves would require effector proteins to aggregate at the plasma membrane in lipid microdomains. The current study shows that in Müller glia of the retina, proteins involved in calcium signaling aggregate in detergent resistant membranes identifying rafts and respond by redistributing on stimulation. We have investigated Purinoreceptor-1 (P2Y1), Ryanodine receptor (RyR), and Phospholipase C (PLC-β1). P2Y1, RyR and PLC-β1, redistribute from caveolin-1 and flotillin-1 positive fractions on stimulation with the agonists, ATP, 2MeS-ATP and Thapsigargin, an inhibitor of sarcoplasmic-endoplasmic reticulum Ca-ATPase (SERCA). Redistribution is absent on treatment with cyclopiazonic acid, another SERCA inhibitor. Disruption of rafts by removing cholesterol cause proteins involved in this machinery to redistribute and change agonist-induced calcium signaling. Cholesterol depletion from raft lead to increase in time to peak of calcium levels in agonist-evoked calcium signals in all instances, as seen by live imaging. This study emphasizes the necessity of a sub-population of proteins to cluster in specialized lipid domains. The requirement for such an organization at the raft-like microdomains may have implications on intercellular communication in the retina. Such concerted interaction at the rafts can regulate calcium dynamics and could add another layer of complexity to calcium signaling in cells.

  5. Ivermectin is a nonselective inhibitor of mammalian P-type ATPases.

    Science.gov (United States)

    Pimenta, Paulo Henrique Cotrim; Silva, Claudia Lucia Martins; Noël, François

    2010-02-01

    Ivermectin is a large spectrum antiparasitic drug that is very safe at the doses actually used. However, as it is being studied for new applications that would require higher doses, we should pay attention to its effects at high concentrations. As micromolar concentrations of ivermectin have been reported to inhibit the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), we decided to investigate its putative inhibitory effect on other two important P-type ATPases, namely the Na(+) , K(+)-ATPase and H(+)/K(+)-ATPase. We first extended the data on SERCA, using preparations from rat enriched in SERCA1a (extensor digitorum longus) and 1b (heart) isoforms. Secondly, we tested the effect of ivermectin in two preparations of rat Na(+), K(+)-ATPase in order to appreciate its putative selectivity towards the alpha(1) isoform (kidney) and the alpha(2)/alpha(3) isoforms (brain), and in an H(+)/K(+)-ATPase preparation from rat stomach. Ivermectin inhibited all these ATPases with similar IC(50) values (6-17 microM). With respect to the inhibition of the Na(+), K(+)-ATPase, ivermectin acts by a mechanism different from the classical cardiac glycosides, based on selectivity towards the isoforms, sensibility to the antagonistic effect of K(+) and to ionic conditions favoring different conformations of the enzyme. We conclude that ivermectin is a nonselective inhibitor of three important mammalian P-type ATPases, which is indicative of putative important adverse effects if this drug were used at high doses. As a consequence, we propose that novel analogs of ivermectin should be developed and tested both for their parasitic activity and in vitro effects on P-type ATPases.

  6. Towards defining the substrate of orphan P5A-ATPases

    DEFF Research Database (Denmark)

    Sørensen, Danny Mollerup; Holen, Henrik Waldal; Holemans, Tine

    2015-01-01

    Background P-type ATPases are ubiquitous ion and lipid pumps found in cellular membranes. P5A-ATPases constitute a poorly characterized subfamily of P-type ATPases present in all eukaryotic organisms but for which a transported substrate remains to be identified. Scope of review This review aims...... to discuss the available evidence which could lead to identification of possible substrates of P5A-ATPases. Major conclusions The complex phenotypes resulting from the loss of P5A-ATPases in model organisms can be explained by a role of the P5A-ATPase in the endoplasmic reticulum (ER), where loss of function...... of tail-anchored proteins in the ER membrane. A role for P5A-ATPases in vesicle formation would explain why sterol transport and distribution are affected in knock out cells, which in turn has a negative impact on the spontaneous insertion of tail-anchored proteins. It would also explain why secretory...

  7. Curcumin modulation of Na,K-ATPase: phosphoenzyme accumulation, decreased K+ occlusion, and inhibition of hydrolytic activity

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed

    2005-01-01

    Curcumin, the major constitute of tumeric, is an important nutraceutical that has been shown to be useful in the treatment of many diseases. As an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, curcumin was shown to correct cystic fibrosis (CF) defects in some model systems, whereas others...

  8. Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca{sup 2+}-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B., E-mail: korn@mail.ibch.ru [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Dmitriev, Ruslan I.; Kostina, Maria B. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Korneenko, Tatyana V. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shakhparonov, Mikhail I. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Full-length secretory pathway Ca-ATPase (SPCA2) cloned from rat duodenum. Black-Right-Pointing-Pointer ATP2C2 gene (encoding SPCA2) exists only in genomes of Tetrapoda. Black-Right-Pointing-Pointer Rat and pig SPCA2 are expressed in intestines, lung and some secretory glands. Black-Right-Pointing-Pointer Subcellular localization of SPCA2 may depend on tissue type. Black-Right-Pointing-Pointer In rat duodenum, SPCA2 is localized in plasma membrane-associated compartments. -- Abstract: Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2

  9. Cell Death and Survival Through the Endoplasmic Reticulum-Mitochondrial Axis

    Science.gov (United States)

    Bravo-Sagua, R.; Rodriguez, A.E.; Kuzmicic, J.; Gutierrez, T.; Lopez-Crisosto, C.; Quiroga, C.; Díaz-Elizondo, J.; Chiong, M.; Gillette, T.G.; Rothermel, B.A.; Lavandero, S.

    2014-01-01

    The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial–associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate. PMID:23228132

  10. Changes of Plasma Membrane H+-ATPase Activities of Glycine max Seeds by PEG Treatment

    Institute of Scientific and Technical Information of China (English)

    Yang Yong-qing; Wang Xiao-feng

    2005-01-01

    The soybean (Glycine max) Heihe No. 23 is sensitive to imbibitional chilling injury. Polyethylene glycol (PEG)treatment can improve chilling tolerance of soybean seeds to a certain extent. The changes of hydrolytic ATPase in plasma membranes and H+-pumping responses in soybean seeds were investigated during PEG treatments. Effects of exogenous calcium and exogenous ABA on the hydrolytic ATPase were also examined in order to understand the mechanism of chilling resistance. Highly purified plasma membranes were isolated by 6.0% aqueous two-phase partitioning from soybean seeds, as judged by the sensitivity of hydrolytic ATPase to sodium vanadate. PEG treatment resulted in a slight increase of the hydrolytic ATPase activity in 12 h. Then the activity decreased gradually, but still higher than the control. The H+-pumping activity increased steadily during PEG treatment.Exogenous calcium had both activating and inhibiting effects on the hydrolytic ATPase, but the activity was inhibited in soybean seeds treated with exogenous ABA. Results suggested that PEG treatment, not the exogenous calcium and ABA, up-regulated H+-ATPase activities in soybean seeds.

  11. Calmodulin modulates the delay period between release of calcium from internal stores and activation of calcium influx via endogenous TRP1 channels.

    Science.gov (United States)

    Vaca, Luis; Sampieri, Alicia

    2002-11-01

    In the present study we have explored the role of calmodulin (CaM) and inositol 1,4,5-trisphosphate receptor (IP(3)R) in the communication process activated after the release of calcium from the endoplasmic reticulum (ER) and the activation of calcium influx via endogenous TRP1 channels from Chinese hamster ovary cells. Experiments using combined rapid confocal calcium and electrophysiology measurements uncovered a consistent delay of around 900 ms between the first detectable calcium released from the ER and the activation of the calcium current. This delay was evident with two different methods used to release calcium from the ER: either the blockade of the microsomal calcium ATPase with thapsigargin or activation of bradykinin receptors linked to the IP(3) cascade. Direct application of IP(3) or a peptide from the NH(2)-terminal region of the IP(3)R activated store operated calcium, reducing the delay period. Introduction of CaM into the cell via the patch pipette increased the delay period from 900 +/- 100 ms to 10 +/- 2.1 s (n = 18). Furthermore, the use of selective CaM antagonists W7 and trifluoperazine maleate resulted in a substantial reduction of the delay period to 200 +/- 100 ms with 5 microm trifluoperazine maleate (n = 16) and 150 +/- 50 ms with 500 nm W7 (n = 22). CaM reduced also the current density activated by thapsigargin or brandykinin to about 60% from control. The CaM antagonists did not affect significantly the current density. The results presented here are consistent with an antagonistic effect of IP(3)R and CaM for the activation of store operated calcium after depletion of the ER. The functional competition between the activating effect of IP(3)R and the inhibiting effect of CaM may modulate the delay period between the release of calcium from the ER and the activation of calcium influx observed in different cells, as well as the amount of current activated after depletion of the ER.

  12. Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis

    OpenAIRE

    Jintao Zhang; Man Yi; Longying Zha; Siqiang Chen; Zhijia Li; Cheng Li; Mingxing Gong; Hong Deng; Xinwei Chu; Jiehua Chen; Zheqing Zhang; Limei Mao; Suxia Sun

    2016-01-01

    Purpose Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated au...

  13. Treatment with alpha-melanocyte stimulating hormone preserves calcium regulatory proteins in rat heart allografts.

    Science.gov (United States)

    Colombo, Gualtiero; Sordi, Andrea; Lonati, Caterina; Carlin, Andrea; Turcatti, Flavia; Leonardi, Patrizia; Gatti, Stefano; Catania, Anna

    2008-08-01

    Prevention of graft dysfunction is a major objective in transplantation medicine. Previous research on experimental heart transplantation indicated that treatment with the immunomodulatory peptide alpha-melanocyte stimulating hormone (alpha-MSH) improves histopathology, prolongs allograft survival, and reduces expression of the main tissue injury mediators. Because calcium-handling is critical in heart graft function, we determined the effects of transplantation injury and influences of alpha-MSH treatment on representative calcium regulatory proteins in rat heart allografts. Hearts from Brown Norway rats were transplanted heterotopically into MHC incompatible Lewis rats. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), protein kinase C epsilon (PKC epsilon), sarcoplasmic/endoplasmic reticulum calcium-ATPase 2 (SERCA2a), arrestin-beta1 (Arrb1), cholinergic receptor M2 (Chrm2), and inositol 1,4,5-triphosphate receptor 1 (InsP(3)R1) were examined in: (1) non-transplanted donor hearts; (2) allografts from saline-treated rats; and (3) allografts from rats treated with the synthetic alpha-MSH analog Nle4-DPhe7-alpha-MSH (NDP-alpha-MSH) (100 microg i.p. every 12h). Transplantation injury was associated with severe reduction in calcium regulatory protein transcription and expression level. NDP-alpha-MSH administration partly reversed inhibition of protein transcription and almost completely prevented protein loss. Finally, because certain effects of cyclic 3'-5'-adenosine monophosphate (cAMP) signaling on calcium handling in cardiac myocytes depend on activation of exchange protein directly activated by cAMP 1 (Epac1), we determined Epac1 mRNA and protein expression in heart allografts. Transplantation injury markedly reduced Epac1. NDP-alpha-MSH treatment significantly preserved both Epac1 protein and mRNA in the allografts. Administration of alpha-MSH or related melanocortins could reduce transplantation-induced dysfunction through protection of heart calcium

  14. The Na/K-ATPase-mediated signal transduction as a target for new drug development.

    Science.gov (United States)

    Xie, Zijian; Xie, Joe

    2005-09-01

    The Na/K-ATPase, or Na+ pump, is a member of the P-type ATPase superfamily. In addition to pumping ions, the Na/K-ATPase is a receptor that not only regulates the function of protein kinases, but also acts as a scaffold, capable of tethering different proteins into a signalplex. The signaling Na/K-ATPase resides in caveolae and forms a "binary receptor" with the tyrosine kinase Src. Endogenous cardiotonic steroids and digitalis drugs such as ouabain act as agonists and provoke this binary receptor, resulting in tyrosine phosphorylation of the proteins that are either associated with, or in close proximity to, the signaling Na/K-ATPase. Subsequently, this initiates protein kinase cascades including ERKs and PKC isozymes. It also increases mitochondrial production of reactive oxygen species (ROS) and regulates intracellular calcium concentration. Like other receptors, activation of the Na/K-ATPase/Src by ouabain induces the endocytosis of the plasma membrane Na/K-ATPase. Significantly, this newly appreciated signaling function of the Na/K-ATPase appears to play an important role in the pathogenesis of many cardiovascular diseases, therefore serving as an important target for development of novel therapeutic agents.

  15. Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin.

    Science.gov (United States)

    Tufi, R; Panaretakis, T; Bianchi, K; Criollo, A; Fazi, B; Di Sano, F; Tesniere, A; Kepp, O; Paterlini-Brechot, P; Zitvogel, L; Piacentini, M; Szabadkai, G; Kroemer, G

    2008-02-01

    Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.

  16. Dense-cored vesicles, smooth endoplasmic reticulum, and mitochondria are closely associated with non-specialized parts of plasma membrane of nerve terminals: implications for exocytosis and calcium buffering by intraterminal organelles.

    Science.gov (United States)

    Lysakowski, A; Figueras, H; Price, S D; Peng, Y Y

    1999-01-18

    To determine whether there are anatomical correlates for intraterminal Ca2+ stores to regulate exocytosis of dense-cored vesicles (DCVs) and whether these stores can modulate exocytosis of synaptic vesicles, we studied the spatial distributions of DCVs, smooth endoplasmic reticulum (SER), and mitochondria in 19 serially reconstructed nerve terminals in bullfrog sympathetic ganglia. On average, each bouton had three active zones, 214 DCVs, 26 SER fragments (SERFs), and eight mitochondria. DCVs, SERFs and mitochondria were located, on average, 690, 624, and 526 nm, respectively, away from active zones. Virtually no DCVs were within "docking" (i.e., similar to those for exocytosis of synaptic vesicles. Because there were virtually no SERFs or mitochondria within 50 nm of any active zone, Ca2+ modulation by these organelles is unlikely to affect ACh release evoked by a single action potential. In contrast, 30% of DCVs and 40% of SERFs were located within 50 nm of the nonspecialized regions of the plasma membrane. Because each bouton had at least one SERF within 50 nm of the plasma membrane and most of these SERFs had DCVs, but not mitochondria, near them, it is possible for Ca2+ release from the SER to provide the Ca2+ necessary for DCV exocytosis. The fact that 60% of the mitochondria had some part within 50 nm of the plasma membrane means that it is possible for mitochondrial Ca2+ buffering to affect DCV exocytosis.

  17. Endoplasmic reticulum stress and cardiovascular diseases

    Institute of Scientific and Technical Information of China (English)

    Xiaohui Duan; Yongfen Qi; Chaoshu Tang

    2009-01-01

    The endoplasmic reticulum (ER) serves several important functions, mainly post-translational modification, folding and assembly of newly synthesized secretary proteins, synthesizing lipids and cellular calcium storage. Various factors can disrupt ER homeostasis and disturb its functions, which leads to the accumulation of unfolded and misfolded proteins and to potential cellular dysfunction and pathological consequences, collectively termed ER stress. Recent progress suggests that ER stress plays a key role in the immune response, diabetes, tumor growth, and some neurodegenerative diseases. In particular, ER stress is involved in several processes of cardiovascular diseases, such as ischemia/reperfusion injury, cardiomyopathy, cardiac hypertrophy, heart failure, and atherosclerosis. Further research on the relation of ER stress to cardiovascular diseases will greatly enhance the understanding of these pathological processes and provide novel avenues to potential therapies.

  18. Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak

    Science.gov (United States)

    Sankaranarayanan, Rajiv; Li, Yatong; Greensmith, David J.; Eisner, David A.

    2016-01-01

    Key points Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay.In the presence of β‐adrenergic stimulation, RyR‐mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase.Two forms of Ca leak have been studied, Ca‐sensitising (induced by caffeine) and non‐sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient.Only Ca‐sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine.Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient. Abstract In heart failure, a reduction in Ca transient amplitude and contractile dysfunction can by caused by Ca leak through the sarcoplasmic reticulum (SR) Ca channel (ryanodine receptor, RyR) and/or decreased activity of the SR Ca ATPase (SERCA). We have characterised the effects of two forms of Ca leak (Ca‐sensitising and non‐sensitising) on calcium cycling and compared with those of SERCA inhibition. We measured [Ca2+]i with fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing SR leak with either caffeine (to sensitise the RyR to Ca activation) or ryanodine (non‐sensitising) had similar effects to SERCA inhibition: decreased systolic [Ca2+]i, increased diastolic [Ca2+]i and slowed decay. However, in the presence of isoproterenol, leak produced a biphasic decay of the Ca transient in the majority of cells while SERCA inhibition produced monophasic decay. Tetracaine reversed the effects of caffeine but not of ryanodine. When caffeine (1 mmol l−1) was added to a cell which displayed Ca waves, the wave frequency initially increased before waves disappeared and biphasic decay developed. Eventually (at higher caffeine concentrations), the

  19. Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion.

    Science.gov (United States)

    Hurne, Alanna M; O'Brien, Jennifer J; Wingrove, Douglas; Cherednichenko, Gennady; Allen, Paul D; Beam, Kurt G; Pessah, Isaac N

    2005-11-04

    Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

  20. Effects of C-terminal truncations on trafficking of the yeast plasma membrane H+-ATPase.

    Science.gov (United States)

    Mason, A Brett; Allen, Kenneth E; Slayman, Carolyn W

    2006-08-18

    Within the large family of P-type cation-transporting ATPases, members differ in the number of C-terminal transmembrane helices, ranging from two in Cu2+-ATPases to six in H+-, Na+,K+-, Mg2+-, and Ca2+-ATPases. In this study, yeast Pma1 H+-ATPase has served as a model to examine the role of the C-terminal membrane domain in ATPase stability and targeting to the plasma membrane. Successive truncations were constructed from the middle of the major cytoplasmic loop to the middle of the extended cytoplasmic tail, adding back the C-terminal membrane-spanning helices one at a time. When the resulting constructs were expressed transiently in yeast, there was a steady increase in half-life from 70 min in Pma1 delta452 to 348 min in Pma1 delta901, but even the longest construct was considerably less stable than wild-type ATPase (t(1/2) = 11 h). Confocal immunofluorescence microscopy showed that 11 of 12 constructs were arrested in the endoplasmic reticulum and degraded in the proteasome. The only truncated ATPase that escaped the ER, Pma1 delta901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 delta901 to be misfolded, however, resulting in premature delivery to the vacuole for degradation. As model substrates, this series of truncations affirms the importance of the entire C-terminal domain to yeast H+-ATPase biogenesis and defines a sequence element of 20 amino acids in the carboxyl tail that is critical to ER escape and trafficking to the plasma membrane.

  1. Distal Renal Tubular Acidosis and Calcium Nephrolithiasis

    Science.gov (United States)

    Moe, Orson W.; Fuster, Daniel G.; Xie, Xiao-Song

    2008-09-01

    Calcium stones are commonly encountered in patients with congenital distal renal tubular acidosis, a disease of renal acidification caused by mutations in either the vacuolar H+-ATPase (B1 or a4 subunit), anion exchanger-1, or carbonic anhydrase II. Based on the existing database, we present two hypotheses. First, heterozygotes with mutations in B1 subunit of H+-ATPase are not normal but may harbor biochemical abnormalities such as renal acidification defects, hypercalciuria, and hypocitraturia which can predispose them to kidney stone formation. Second, we propose at least two mechanisms by which mutant B1 subunit can impair H+-ATPase: defective pump assembly and defective pump activity.

  2. Calcium signalling and calcium channels: evolution and general principles.

    Science.gov (United States)

    Verkhratsky, Alexei; Parpura, Vladimir

    2014-09-15

    Calcium as a divalent cation was selected early in evolution as a signaling molecule to be used by both prokaryotes and eukaryotes. Its low cytosolic concentration likely reflects the initial concentration of this ion in the primordial soup/ocean as unicellular organisms were formed. As the concentration of calcium in the ocean subsequently increased, so did the diversity of homeostatic molecules handling calcium. This includes the plasma membrane channels that allowed the calcium entry, as well as extrusion mechanisms, i.e., exchangers and pumps. Further diversification occurred with the evolution of intracellular organelles, in particular the endoplasmic reticulum and mitochondria, which also contain channels, exchanger(s) and pumps to handle the homeostasis of calcium ions. Calcium signalling system, based around coordinated interactions of the above molecular entities, can be activated by the opening of voltage-gated channels, neurotransmitters, second messengers and/or mechanical stimulation, and as such is all-pervading pathway in physiology and pathophysiology of organisms.

  3. Dual mechanisms of sHA 14-1 in inducing cell death through endoplasmic reticulum and mitochondria.

    Science.gov (United States)

    Hermanson, David; Addo, Sadiya N; Bajer, Anna A; Marchant, Jonathan S; Das, Sonia Goutam Kumar; Srinivasan, Balasubramanian; Al-Mousa, Fawaz; Michelangeli, Francesco; Thomas, David D; Lebien, Tucker W; Xing, Chengguo

    2009-09-01

    HA 14-1 is a small-molecule Bcl-2 antagonist that promotes apoptosis in malignant cells, but its mechanism of action is not well defined. We recently reported that HA 14-1 has a half-life of only 15 min in vitro, which led us to develop a stable analog of HA 14-1 (sHA 14-1). The current study characterizes its mode of action. Because of the antiapoptotic function of Bcl-2 family proteins on the endoplasmic reticulum (ER) and mitochondria, the effect of sHA 14-1 on both organelles was evaluated. sHA 14-1 induced ER calcium release in human leukemic cells within 1 min, followed by induction of the ER stress-inducible transcription factor ATF4. Similar kinetics and stronger intensity of ER calcium release were induced by the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, accompanied by similar kinetics and intensity of ATF4 induction. sHA 14-1 directly inhibited SERCA enzymatic activity but had no effect on the inositol triphosphate receptor. Evaluation of the mitochondrial pathway showed that sHA 14-1 triggered a loss of mitochondrial transmembrane potential (Delta psi m) and weak caspase-9 activation, whereas thapsigargin had no effect. (R)-4-(3-Dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4'-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitrobenzenesulfonamide (ABT-737), a well established small-molecule Bcl-2 antagonist, rapidly induced loss of Delta psi m and caspase-9 activation but had no effect on the ER. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone had some protective effect on sHA 14-1-induced cell death. These collective results suggest a unique dual targeting mechanism of sHA 14-1 on the apoptotic resistance machinery of tumor cells that includes antiapoptotic Bcl-2 family proteins and SERCA proteins.

  4. The calcium signaling toolkit of the Apicomplexan parasites Toxoplasma gondii and Plasmodium spp.

    Science.gov (United States)

    Lourido, Sebastian; Moreno, Silvia N J

    2015-03-01

    Apicomplexan parasites have complex life cycles, frequently split between different hosts and reliant on rapid responses as the parasites react to changing environmental conditions. Calcium ion (Ca(2+)) signaling is consequently essential for the cellular and developmental changes that support Apicomplexan parasitism. Apicomplexan genomes reveal a rich repertoire of genes involved in calcium signaling, although many of the genes responsible for observed physiological changes remain unknown. There is evidence, for example, for the presence of a nifedipine-sensitive calcium entry mechanism in Toxoplasma, but the molecular components involved in Ca(2+) entry in both Toxoplasma and Plasmodium, have not been identified. The major calcium stores are the endoplasmic reticulum (ER), the acidocalcisomes, and the plant-like vacuole in Toxoplasma, or the food vacuole in Plasmodium spp. Pharmacological evidence suggests that Ca(2+) release from intracellular stores may be mediated by inositol 1,4,5-trisphosphate (IP3) or cyclic ADP ribose (cADPR) although there is no molecular evidence for the presence of receptors for these second messengers in the parasites. Several Ca(2+)-ATPases are present in Apicomplexans and a putative mitochondrial Ca(2+)/H(+) exchanger has been identified. Apicomplexan genomes contain numerous genes encoding Ca(2+)-binding proteins, with the notable expansion of calcium-dependent protein kinases (CDPKs), whose study has revealed roles in gliding motility, microneme secretion, host cell invasion and egress, and parasite differentiation. Microneme secretion has also been shown to depend on the C2 domain containing protein DOC2 in both Plasmodium spp. and Toxoplasma, providing further evidence for the complex transduction of Ca(2+) signals in these organisms. The characterization of these pathways could lead to the discovery of novel drug targets and to a better understanding of the role of Ca(2+) in these parasites. Copyright © 2014 Elsevier Ltd. All

  5. Effects of Weak Magnetic Field on Ca2+ Modulation of Skeletal Muscle Sarcoplamic Reticulum%弱磁场对骨骼肌肌质网钙调控作用的研究

    Institute of Scientific and Technical Information of China (English)

    刘仁臣; 吴永刚; 程和平; 谢国秋; 陆静; 夏子奂

    2011-01-01

    目的:探讨弱磁场对提取的骨骼肌肌质网系(SR)Ca2+转运、钙泵(Ca2 - Mg2+ -ATPase)及钙释放通道(RyR)活性的影响,从分子水平和细胞信号系统的角度来解释生物电磁效应.方法:利用动态光谱法检测0.4 mT弱磁场辐照过的SR Ca2+转运、Ca2 -ATPase活性,还原型辅酶(NADH)的氧化初速率和超氧(O2)产率,以及用同位素标记方法检测[3H] -Ryanodine与RyR的平衡结合度.结果:弱磁场辐照引起SR的Ca2+摄取功能和Ca2 -ATPase的活性明显下降,Ca2+释放和[3H] -Ryanodine平衡结合度上升,同时上调了NADH的氧化初速率和O2的产率.结论:提示0.4 mT弱磁场辐照30 min对SR Ca2 -ATPase活性有明显抑制,对RyR有一定的激活效果.%Objective: Discuss the effects of weak magnetic field on isolated sarcoplasmic reticulum (SR) Ca2+ modulation, calcium pump (Ca2+-Mg2+-ATPase) and Ca2 + release channel (Ryanodine Receptor, RyR) activity, so as to explain the mechanism of biological effects caused by electromagnetic fields in terms of molecular level and signal transduction system. Methods;The Ca + modulation and Ca + -ATPase activity, NADH oxidation initial rates and superoxide production of SR exposed to 0. 4 mT weak magnetic field ( MF) were investigated with dynamic Ca2 * dye spectrum method, [3 H ] -Ryanodine binding assay was investigated by isotope Labeling. Results: Weak MF exposure decreased SR Ca + uptake and Ca2 + -ATPase activity obviously, increased SR Ca2 + release and [3 H ] -Rryanodine binding, up-regulated the initial rates of NADH oxidation and the production of superoxide. Conclusion: It is indicated that 0. 4 mT weak magnetic field exposure for 30 min inhibited Ca2 + -ATPase activity and promoted the RyR channel function.

  6. αB-Crystallin R120G variant causes cardiac arrhythmias and alterations in the expression of Ca(2+) -handling proteins and endoplasmic reticulum stress in mice.

    Science.gov (United States)

    Jiao, Qibin; Sanbe, Atsushi; Zhang, Xingwei; Liu, Jun-Ping; Minamisawa, Susumu

    2014-08-01

    Mutations of αB-crystallin (CryαB), a small heat shock protein abundantly expressed in cardiac and skeletal muscles, are known to cause desmin-related myopathies. The CryαB R120G allele has been linked to a familial desminopathy and, in transgenic mice, causes a sudden death at about 28 weeks of age. To investigate the mechanisms of the sudden cardiac arrest of CryαB R120G transgenic mice, we prepared protein samples from left ventricular tissues of two different age groups (10 and 28 weeks) and examined Ca(2+) -handling proteins. Expression of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2, phospholamban, ryanodine receptor 2 and calsequestrin 2 was significantly decreased in 28- versus 10-week-old CryαB R120G transgenic mice. In addition, low heart rate variability, including heart rate, total power and low frequency, was observed and continuous electrocardiogram monitoring revealed cardiac arrhythmias, such as ventricular tachycardia, atrioventricular block and atrial flutter, in 28-week-old CryαB R120G transgenic mice. In contrast, expression of endoplasmic reticulum (ER) degradation enhancing α-mannosidase-like protein, inositol requirement 1 and X-box binding protein 1 were increased significantly in 28- versus 10-week-old CryαBR120G transgenic mice, suggesting that the CryαBR120G transgenic mice exhibit increased ER stress compared with wild-type mice. Together, the data suggest that the CryαB R120G dominant variant induces ER stress and impairs Ca(2+) regulation, leading to ageing-related cardiac dysfunction, arrhythmias and decreased autonomic tone with shortened lifespan.

  7. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR,HSR) were isolated from rabbit leg muscle using a combination of differential centrifugation and isopycnic zonal ultracentrifugation. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes whereas the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The sucrose HSR vesicles have an additional morphological feature which appears as membrane projections that resemble the SR feet. The freeze-fracture morphology of either type of SR reveals an asymmetric distribution of intramembraneous particles in the same orientation and distribution as the sarcoplasmic reticulum in vivo. Biochemical studies were made on the content of Ca, Mg, ATPase, and protein of the vesicles and phosphorylation of the vesicles. The biochemical and morphological data indicate that the LSR is derived from the longitudinal sarcoplasmic reticulum and the HSR is derived from the terminal cisternae of the sarcoplasmic reticulum, contains junctional SR membrane and has three unique proteins (calsequestrin, an intrinsic 30,000 dalton protein and a 9000 dalton proteolipid).

  8. ERp57 modulates mitochondrial calcium uptake through the MCU.

    Science.gov (United States)

    He, Jingquan; Shi, Weikang; Guo, Yu; Chai, Zhen

    2014-06-01

    ERp57 participates in the regulation of calcium homeostasis. Although ERp57 modulates calcium flux across the plasma membrane and the endoplasmic reticulum membrane, its functions on mitochondria are largely unknown. Here, we found that ERp57 can regulate the expression of the mitochondrial calcium uniporter (MCU) and modulate mitochondrial calcium uptake. In ERp57-silenced HeLa cells, MCU was downregulated, and the mitochondrial calcium uptake was inhibited, consistent with the effect of MCU knockdown. When MCU was re-expressed in the ERp57 knockdown cells, mitochondrial calcium uptake was restored. Thus, ERp57 is a potent regulator of mitochondrial calcium homeostasis.

  9. Identification of Domains within the V-ATPase Accessory Subunit Ac45 Involved in V-ATPase Transport and Ca2+-dependent Exocytosis

    Science.gov (United States)

    Jansen, Eric J. R.; van Bakel, Nick. H. M.; Loohuis, Nikkie F. M. Olde; Hafmans, Theo G. M.; Arentsen, Tim; Coenen, Anthon J. M.; Scheenen, Wim J. J. M.; Martens, Gerard J. M.

    2012-01-01

    The vacuolar (H+)-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V0-sector is involved in Ca2+-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V0-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca2+-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca2+-dependent-regulated exocytosis. PMID:22736765

  10. Identification of domains within the V-ATPase accessory subunit Ac45 involved in V-ATPase transport and Ca2+-dependent exocytosis.

    Science.gov (United States)

    Jansen, Eric J R; van Bakel, Nick H M; Olde Loohuis, Nikkie F M; Hafmans, Theo G M; Arentsen, Tim; Coenen, Anthon J M; Scheenen, Wim J J M; Martens, Gerard J M

    2012-08-10

    The vacuolar (H(+))-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V(0)-sector is involved in Ca(2+)-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V(0)-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca(2+)-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca(2+)-dependent-regulated exocytosis.

  11. Reduced sarcoplasmic reticulum content of releasable Ca2+ in rat soleus muscle fibres after eccentric contractions

    DEFF Research Database (Denmark)

    Nielsen, J S; Sahlin, K; Ørtenblad, N

    2007-01-01

    AIM: The purpose was to evaluate the effects of fatiguing eccentric contractions (EC) on calcium (Ca2+) handling properties in mammalian type I muscles. We hypothesized that EC reduces both endogenous sarcoplasmic reticulum (SR) content of releasable Ca2+ (eSRCa2+) and myofibrillar Ca2+ sensitivity...

  12. Effect of losartan on sarcoplasmic reticulum Ca2+ handing proteins in heart failure rabbit

    Institute of Scientific and Technical Information of China (English)

    姚艳

    2006-01-01

    Objective To investigate the effects of losartan on mRNA expression of myocardial sarcoplasmic reticulum calcium handling proteins (SERCA2, RyR2 and PLB) and the role of which in prevention of chronic heart failure in rabbit. Methods After chronic heart failure was

  13. The key target of neuroprotection after the onset of ischemic stroke:secretory pathway Ca2+-ATPase 1

    Institute of Scientific and Technical Information of China (English)

    Li-hua Li; Xiang-rong Tian; Zhi-ping Hu

    2015-01-01

    The regulatory mechanisms of cytoplasmic Ca2+ after myocardial infarction-induced Ca2+ over-load involve secretory pathway Ca2+-ATPase 1 and the Golgi apparatus and are well understood. However, the effect of Golgi apparatus on Ca2+ overload after cerebral ischemia and reperfusion remains unclear. Four-vessel occlusion rats were used as animal models of cerebral ischemia. The expression of secretory pathway Ca2+-ATPase 1 in the cortex and hippocampus was detected by immunoblotting, and Ca2+ concentrations in the cytoplasm and Golgi vesicles were determined. Results showed an overload of cytoplasmic Ca2+ during ischemia and reperfusion that reached a peak after reperfusion. Levels of Golgi Ca2+ showed an opposite effect. The expression of Gol-gi-specific secretory pathway Ca2+-ATPase 1 in the cortex and hippocampus decreased before ischemia and reperfusion, and increased after reperfusion for 6 hours. This variation was simi-lar to the alteration of calcium in separated Golgi vesicles. These results indicate that the Golgi apparatus participates in the formation and alleviation of calcium overload, and that secretory pathway Ca2+-ATPase 1 tightly responds to ischemia and reperfusion in nerve cells. Thus, we concluded that secretory pathway Ca2+-ATPase 1 plays an essential role in cytosolic calcium regu-lation and its expression can be used as a marker of Golgi stress, responding to cerebral ischemia and reperfusion. The secretory pathway Ca2+-ATPase 1 can be an important neuroprotective target of ischemic stroke.

  14. Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase.

    Science.gov (United States)

    Song, Jianliang; Zhang, Xue-Qian; Wang, JuFang; Cheskis, Ellina; Chan, Tung O; Feldman, Arthur M; Tucker, Amy L; Cheung, Joseph Y

    2008-10-01

    Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct

  15. Evolution of plant P-type ATPases

    Directory of Open Access Journals (Sweden)

    Christian N.S. Pedersen

    2012-02-01

    Full Text Available Five organisms having completely sequenced genomes and belonging to all major branches of green plants (Viridiplantae were analyzed with respect to their content of P-type ATPases encoding genes. These were the chlorophytes Ostreococcus tauria and Chlamydomonas reinhardtii, and the streptophytes Physcomitrella patens (a moss, Selaginella moellendorffii (a primitive vascular plant, and Arabidopsis thaliana (a model flowering plant. Each organism contained sequences for all five subfamilies of P-type ATPases. Our analysis demonstrates when specific subgroups of P-type ATPases disappeared in the evolution of Angiosperms. Na/K-pump related P2C ATPases were lost with the evolution of streptophytes whereas Na+ or K+ pumping P2D ATPases and secretory pathway Ca2+-ATPases remained until mosses. An N-terminally located calmodulin binding domain in P2B ATPases can only be detected in pumps from Streptophytae, whereas, like in animals, a C-terminally localized calmodulin binding domain might be present in chlorophyte P2B Ca2+-ATPases. Chlorophyte genomes encode P3A ATPases resembling protist plasma membrane H+-ATPases and a C-terminal regulatory domain is missing. The complete inventory of P-type ATPases in the major branches of Viridiplantae is an important starting point for elucidating the evolution in plants of these important pumps.

  16. Origin and evolution of metal p-Type ATPases in Plantae (Archaeplastida

    Directory of Open Access Journals (Sweden)

    Marc eHanikenne

    2014-01-01

    Full Text Available Metal ATPases are a subfamily of P-type ATPases involved in the transport of metal cations across biological membranes. They all share an architecture featuring eight transmembrane domains in pairs of two and are found in prokaryotes as well as in a variety of Eukaryotes. In Arabidopsis thaliana, eight metal P-type ATPases have been described, four being specific to copper transport and four displaying a broader metal specificity, including zinc, cadmium and possibly copper and calcium. So far, few efforts have been devoted to elucidating the origin and evolution of these proteins in Eukaryotes. In this work, we use large-scale phylogenetics to show that metal P-type ATPases form a homogenous group among P-type ATPases and that their specialisation into either monovalent (Cu or divalent (Zn, Cd… metal transport stems from a gene duplication that took place early in the evolution of Life. Then, we demonstrate that the four subgroups of plant metal ATPases all have a different evolutionary origin and a specific taxonomic distribution, only one tracing back to the cyanobacterial progenitor of the chloroplast. Finally, we examine the subsequent evolution of these proteins in green plants and conclude that the genes thoroughly characterised in model organisms are often the result of lineage-specific gene duplications, which calls for caution when attempting to infer function from sequence similarity alone in non-model organisms.

  17. A putative plant aminophospholipid flippase, the Arabidopsis P4 ATPase ALA1, localizes to the plasma membrane following association with a β-subunit.

    Directory of Open Access Journals (Sweden)

    Rosa L López-Marqués

    Full Text Available Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner leaflet and sphingolipids in the outer leaflet. This unequal distribution of lipids between leaflets is, amongst several proposed functions, hypothesized to be a prerequisite for endocytosis. P4 ATPases, belonging to the P-type ATPase superfamily of pumps, are involved in establishing lipid asymmetry across plasma membranes, but P4 ATPases have not been identified in plant plasma membranes. Here we report that the plant P4 ATPase ALA1, which previously has been connected with cold tolerance of Arabidopsis thaliana, is targeted to the plasma membrane and does so following association in the endoplasmic reticulum with an ALIS protein β-subunit.

  18. Alterations in mitochondria and sarcoplasmic reticulum from heart and skeletal muscle of horizontally casted primates

    Science.gov (United States)

    Sordahl, L. A.; Stone, H. L.

    1982-01-01

    Horizontally body-casted rhesus monkeys are used as an animal model in order to study the physiological changes known as cardiovascular deconditioning which occur during weightless conditions. No difference was found between the experimental and control animals in heart mitochondrial oxidative phosphorylation which indicates that no apparent changes occurred in the primary energy-producing system of the heart. A marked increase in cytochrome oxidase activity was observed in the casted primate heart mitochondria compared to controls, while a 25% decrease in respiratory substrate-supported calcium uptake was found in casted primate heart mitochondria compared to controls. Sacroplasmic reticulum isolated from the primate hearts revealed marked changes in calcium transport activities. It is concluded that the marked depression in cardiac sarcoplasmic reticulum functions indicates altered calcium homeostasis in the casted-primate heart which could be a factor in cardiovascular deconditioning.

  19. Effect of Qingyitang on activity of intracellular Ca2+-Mg2+-ATPase in rats with acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Ying Qiu; Yong-Yu Li; Shu-Guang Li; Bo-Gen Song; Gui-Fen Zhao

    2004-01-01

    AIM: To study the change of intracellular calcium-magnesium ATPase (Ca2+-Mg2+-ATPase) activity in pancreas, liver and kidney tissues of rats with acute pancreatitis (AP), and to investigate the effects of Qingyitang (QYT) (Decoction for clearing the pancreas) and tetrandrine (Tet) and vitamin E (VitE) on the activity of Ca2+-Mg2+-ATPase.METHODS: One hundred and five Sprague-Dawley rats were randomly divided into: normal control group, AP group,treatment group with QYT (1 mi/100 g) or Tet (0.4 ml/L00 g)or VitE (100 mg/kg). AP model was prepared by a retrograde injection of sodium taurocholate into the pancreatic duct.Tissues of pancreas, liver and kidney of the animals were taken at 1 h, 5 h, 10 h respectively after AP induction, and the activity of Ca2+-Mg2+-ATPase was studied using enzymehistochemistry staining. Meanwhile, the expression of Ca2+-Mg2+-ATPase of the tissues was studied by RT-PCR.RESULTS: The results showed that the positive rate of Ca2+-Mg2+-ATPase in AP group (8.3%, 25%, 29.2%) was lower than that in normal control group (100%) in all tissues (P<0.01), the positive rate of Ca2+-Mg2+-ATPase in treatment group with QYT (58.3%, 83.3%, 83.3%), Tet (50.0%,70.8%, 75.0%) and VitE (54.2%, 75.0%, 79.2%) was higher than that in AP group (8.3%, 25.0%, 29.2%) in all tissues (P<0.01). RT-PCR results demonstrated that in treatment groups Ca2+-Mg2+-ATPase gene expression in pancreas tissue was higher than that in AP group at the observing time points, and the expression at 5 h was higher than that at L h. The expression of Ca2+-Mg2+-ATPase in liver tissue was positive, but without significant difference between different groups.CONCLUSION: The activity and expression of intracellular Ca2+-Mg2+-ATPase decreased in rats with AP, suggesting that Ca2+-Mg2+-ATPase may contribute to the occurrence and development of cellular calcium overload in AP. QYT, Tet and VitE can increase the activity and expression of Ca2+-Mg2+-ATPase and may relieve intracellular calcium

  20. Endoplasmic Reticulum Stress and Diabetic Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Jiancheng Xu

    2012-01-01

    Full Text Available The endoplasmic reticulum (ER is an organelle entrusted with lipid synthesis, calcium homeostasis, protein folding, and maturation. Perturbation of ER-associated functions results in an evolutionarily conserved cell stress response, the unfolded protein response (UPR that is also called ER stress. ER stress is aimed initially at compensating for damage but can eventually trigger cell death if ER stress is excessive or prolonged. Now the ER stress has been associated with numerous diseases. For instance, our recent studies have demonstrated the important role of ER stress in diabetes-induced cardiac cell death. It is known that apoptosis has been considered to play a critical role in diabetic cardiomyopathy. Therefore, this paper will summarize the information from the literature and our own studies to focus on the pathological role of ER stress in the development of diabetic cardiomyopathy. Improved understanding of the molecular mechanisms underlying UPR activation and ER-initiated apoptosis in diabetic cardiomyopathy will provide us with new targets for drug discovery and therapeutic intervention.

  1. On the thermodynamic efficiency of Ca²⁺-ATPase molecular machines.

    Science.gov (United States)

    Lervik, Anders; Bresme, Fernando; Kjelstrup, Signe; Rubí, J Miguel

    2012-09-19

    Experimental studies have shown that the activity of the reconstituted molecular pump Ca(2+)-ATPase strongly depends on the thickness of the supporting bilayer. It is thus expected that the bilayer structure will have an impact on the thermodynamic efficiency of this nanomachine. Here, we introduce a nonequilibrium-thermodynamics theoretical approach to estimate the thermodynamic efficiency of the Ca(2+)-ATPase from analysis of available experimental data about ATP hydrolysis and Ca(2+) transport. We find that the entropy production, i.e., the heat released to the surroundings under working conditions, is approximately constant for bilayers containing phospholipids with hydrocarbon chains of 18-22 carbon atoms. Our estimates for the heat released during the pump operation agree with results obtained from separate calorimetric experiments on the Ca(2+)-ATPase derived from sarcoplasmic reticulum. We show that the thermodynamic efficiency of the reconstituted Ca(2+)-ATPase reaches a maximum for bilayer thicknesses corresponding to maximum activity. Surprisingly, the estimated thermodynamic efficiency is very low, ∼12%. We discuss the significance of this result as representative of the efficiency of other nanomachines, and we address the influence of the experimental set-up on such a low efficiency. Overall, our approach provides a general route to estimate thermodynamic efficiencies and heat dissipation in experimental studies of nanomachines.

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

    Science.gov (United States)

    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.

  3. Phylogenetic analysis of P5 P-type ATPases, a eukaryotic lineage of secretory pathway pumps

    DEFF Research Database (Denmark)

    Møller, Annette; Asp, Torben; Holm, Preben Bach

    2008-01-01

    Eukaryotes encompass a remarkable variety of organisms and unresolved lineages. Different phylogenetic analyses have lead to conflicting conclusions as to the origin and associations between lineages and species. In this work, we investigated evolutionary relationship of a family of cation pumps...... exclusive for the secretory pathway of eukaryotes by combining the identification of lineage-specific genes with phylogenetic evolution of common genes. Sequences of P5 ATPases, which are regarded to be cation pumps in the endoplasmic reticulum (ER), were identified in all eukaryotic lineages but not in any...

  4. Disease causing mutations of calcium channels.

    Science.gov (United States)

    Lorenzon, Nancy M; Beam, Kurt G

    2008-01-01

    Calcium ions play an important role in the electrical excitability of nerve and muscle, as well as serving as a critical second messenger for diverse cellular functions. As a result, mutations of genes encoding calcium channels may have subtle affects on channel function yet strongly perturb cellular behavior. This review discusses the effects of calcium channel mutations on channel function, the pathological consequences for cellular physiology, and possible links between altered channel function and disease. Many cellular functions are directly or indirectly regulated by the free cytosolic calcium concentration. Thus, calcium levels must be very tightly regulated in time and space. Intracellular calcium ions are essential second messengers and play a role in many functions including, action potential generation, neurotransmitter and hormone release, muscle contraction, neurite outgrowth, synaptogenesis, calcium-dependent gene expression, synaptic plasticity and cell death. Calcium ions that control cell activity can be supplied to the cell cytosol from two major sources: the extracellular space or intracellular stores. Voltage-gated and ligand-gated channels are the primary way in which Ca(2+) ions enter from the extracellular space. The sarcoplasm reticulum (SR) in muscle and the endoplasmic reticulum in non-muscle cells are the main intracellular Ca(2+) stores: the ryanodine receptor (RyR) and inositol-triphosphate receptor channels are the major contributors of calcium release from internal stores.

  5. Effect of propranolol on rat brain synaptosomal Na(+)-K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase.

    Science.gov (United States)

    Gopalaswamy, U V; Satav, J G; Katyare, S S; Bhattacharya, R K

    1997-01-24

    The beta blocker drug propranolol (PPL) significantly inhibited Na(+)-K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase activities in a concentration dependent manner in rat brain synaptosomes. The concentrations required for 50% inhibition (IC50) in the activity of these enzymes were 1.5-1.8 mM. The double-reciprocal plot of ATP-stimulated Na(+)-K(+)-ATPase activity in the presence of PPL showed apparent decrease in K(m) and Vmax and the inhibition was of an uncompetitive type with respect to ATP. The nature of inhibition by PPL of Na(+)-activated Na(+)-K(+)-ATPase activity was of a mixed type showing an increase in Km and decrease in Vmax. Potassium activation kinetics of Na(+)-K(+)-ATPase displayed uncompetitive type of inhibition with PPL since Km and Vmax were decreased. Magnesium activation of Mg(2+)-ATPase showed decrease in Vmax with no apparent change in Km in the presence of PPL. The drug inhibited synaptosomal Ca(2+)-ATPase in an uncompetitive manner. The observed inhibition of synaptosomal ATPases indicates possible alterations in the synaptic transmission by the beta blocker drug PPL.

  6. Functional Analysis of P4-ATPases

    DEFF Research Database (Denmark)

    Theorin, Lisa

    studies have identified P4-ATPases as phospholipid transporters that interact and cooperate with cytosolic coat proteins to drive vesicle formation at the trans-Golgi network, endosome compartment and plasma membrane. Our hypothesis is that by flipping a lipid the P4-ATPases either provide essential......Across membranes of the late secretory pathway in eukaryotic cells an asymmetric lipid distribution is maintained, with the lipids phosphatidylserine and phosphatidylethanolamine restricted to the cytoplasmic leaflet of the membrane. In recent years a subgroup of P-type ATPases, P4-ATPases, has...... and mammalian P4-ATPases have been studied extensively and the physiological function is mostly known, while the exact biochemistry and specific activity is mostly unknown. Even though the plant Arabidopsis thaliana has 12 P4-ATPases, not much is known about their function. In this study, the biochemical...

  7. 大鼠 H9c2心肌细胞缺氧/复氧损伤模型肌浆网钙调控相关蛋白表达及当归补血汤的干预作用%Danggui Buxue Decoction on expression of sarcoplasmic reticulum calcium regulatory protein in H9c2 myocardial cell model with hypoxia/reoxygenation damage

    Institute of Scientific and Technical Information of China (English)

    周春刚; 汤加; 李卿; 徐辰; 张志斌

    2016-01-01

    Objective To establish the model of hypoxia / reoxygenation damage in H9c2 rat myocardial cell and observe the expression of myocardial sarcoplasmic reticulum calcium regulatory protein and the effect of Danggui Bux-ue Decoction on calcium overload in H9c2 cells with hypoxia / reoxygenation injury. Methods JC-1 staining was used to detect the mitochondrial membrane potential by flow cytometry,Fluo-3 AM calcium fluorescence probe for the detection of intracellular calcium concentration,The expression level of sarcoplasmic reticulum Ca transport ATP en-zyme(SERCA2a),L type calcium channel(CAV1. 3),ryanodine receptor(RyR1,RyR2),phospholamban(PLB), calsequestrin(CASQ)mRNA were detected by RT-PCR. Results Compared with the normal control group,the early apoptosis rate and intracellular calcium concentration in model group increased significantly(P 0. 05),compared with the model group. Con-clusion There are obvious calcium overload and sarcoplasmic reticulum calcium regulating proteins adjustment disor-der in the model of H9c2 rat myocardial cell with hypoxia / reoxygenation damage,Danggui Buxue Decoction can in-crease the expression of the sarcoplasmic reticulumcalcium regulating protein SERCA2a and CASQ mRNA after reoxy-genation,alleviate calcium overload,and significantly reduce the early apoptosis rate of reoxygenation damage cells.%目的:建立缺氧/复氧大鼠 H9c2心肌细胞损伤模型,观察心肌细胞肌浆网钙调控相关蛋白表达及当归补血汤对 H9c2细胞缺氧/复氧损伤钙超载的影响。方法 JC-1染色流式细胞仪检测细胞线粒体膜电位, Fluo-3 AM 钙离子荧光探针检测细胞内钙离子浓度,RT-PCR 荧光相对定量检测大鼠心肌肌浆网 Ca 转运 ATP酶(SERCA2a)、L 型钙通道(CAV1.3)、兰尼碱受体(RyR1,RyR2)、受磷蛋白(PLB)、肌集钙蛋白( CASQ)的mRNA 表达水平。结果与正常对照组比较,模型组细胞早期凋亡显著增加(P <0.05),细胞内

  8. 钙通道阻滞剂改善内质网应激抑制压力过负荷高血压大鼠的心肌重构%Protective effect of calcium channel blocker on hypertensive rat myocardial remodeling of pressure overload induced by endoplasmic reticulum stress

    Institute of Scientific and Technical Information of China (English)

    槐勇; 赵连友; 李炜; 郑强荪; 刘静; 郭丽; 丁璐

    2012-01-01

    目的:研究在压力过负荷应激状态下钙通道阻滞剂拉西地平对内质网应激(endoplasmic reticulum stress,ERS)分子钙网蛋白(calreticulin,CRT)和细胞凋亡效应分子-12(caspase-12)在大鼠心肌组织中的表达及对心肌重构的影响.方法:将30只雄性Sprague-Dawly(SD)大鼠随机分为3组,即假手术组(Sham组)、腹主动脉缩窄组(TAC组)及TAC+拉西地平干预组(简称拉西地平组),每组10只.通过颈动脉插管测定各组大鼠的血流动力学,心脏超声心动图检查左心室的形态结构.采用免疫组化染色法检测大鼠心肌中CRT及caspase-12蛋白的表达水平,TUNEL荧光染色法检测心肌细胞的凋亡率.结果:与Sham组相比较,TAC组大鼠的平均动脉压(MAP)、室间隔厚度(IVST)、左室后壁厚度(LVPWT)、左室质量指数(LVWI)、CRT和caspase-12蛋白表达水平及心肌细胞的凋亡率比较,均有显著升高(P<0.01).拉西地平组大鼠MAP、IVST、LVPWT、LVWI、CRT和caspase-12蛋白的表达水平及心肌细胞的凋亡率较TAC组明显下降(P<0.05).结论:内质网应激可能参与了压力过负荷高血压大鼠心肌重构的过程.钙通道阻滞剂拉西地平可能通过降低CRT及caspase-12的表达及减少心肌细胞的凋亡干预ERS介导的压力负荷所致高血压大鼠心肌肥厚的信号通路,从而通过改善内质网应激发挥对心脏的保护作用.%AIM: To investigate the influence of calcium channel blocker lacidipine on the expression of calreticulin (CRT) and caspase-12 in rat cardiac myocytes and the effects of lacidipine against myocardium remodeling under pressure overload conditions. METHODS: Thirty male Sprague Dawley rats were randomly divided into TAC group (transverse aortic constriction) , control group ( sham group) and TAC + lacidipine group with ten rats in each group. Invasive hemodynamics were measured through carotid cannulation and left ventricular morphology was monitored by echocardiography

  9. T-tubule disruption promotes calcium alternans in failing ventricular myocytes: mechanistic insights from computational modeling.

    Science.gov (United States)

    Nivala, Michael; Song, Zhen; Weiss, James N; Qu, Zhilin

    2015-02-01

    In heart failure (HF), T-tubule (TT) disruption contributes to dyssynchronous calcium (Ca) release and impaired contraction, but its role in arrhythmogenesis remains unclear. In this study, we investigate the effects of TT disruption and other HF remodeling factors on Ca alternans in ventricular myocytes using computer modeling. A ventricular myocyte model with detailed spatiotemporal Ca cycling modeled by a coupled Ca release unit (CRU) network was used, in which the L-type Ca channels and the ryanodine receptor (RyR) channels were simulated by random Markov transitions. TT disruption, which removes the L-type Ca channels from the associated CRUs, results in "orphaned" RyR clusters and thus provides increased opportunity for spark-induced Ca sparks to occur. This effect combined with other HF remodeling factors promoted alternans by two distinct mechanisms: 1) for normal sarco-endoplasmic reticulum Ca ATPase (SERCA) activity, alternans was caused by both CRU refractoriness and coupling. The increased opportunity for spark-induced sparks by TT disruption combined with the enhanced CRU coupling by Ca elevation in the presence or absence of increased RyR leakiness facilitated spark synchronization on alternate beats to promote Ca alternans; 2) for down-regulated SERCA, alternans was caused by the sarcoplasmic reticulum (SR) Ca load-dependent mechanism, independent of CRU refractoriness. TT disruption and increased RyR leakiness shifted and steepened the SR Ca release-load relationship, which combines with down-regulated SERCA to promote Ca alternans. In conclusion, the mechanisms of Ca alternans for normal and down-regulated SERCA are different, and TT disruption promotes Ca alternans by both mechanisms, which may contribute to alternans at different stages of HF.

  10. Glial calcium signaling in physiology and pathophysioilogy

    Institute of Scientific and Technical Information of China (English)

    Alexei VERKHRASKY

    2006-01-01

    Neuronal-glial circuits underlie integrative processes in the nervous system.Function of glial syncytium is,to a very large extent,regulated by the intracellular calcium signaling system.Glial calcium signals are triggered by activation of multiple receptors,expressed in glial membrane,which regulate both Ca2+ entry and Ca2+ release from the endoplasmic reticulum.The endoplasmic reticulum also endows glial cells with intracellular excitable media,which is able to produce and maintain long-ranging signaling in a form of propagating Ca2+ waves.In pathological conditions,calcium signals regulate glial response to injury,which might have both protective and detrimental effects on the nervous tissue.

  11. ATPase Activity Measurements Using Radiolabeled ATP

    NARCIS (Netherlands)

    Swarts, H.G.; Koenderink, J.B.

    2016-01-01

    ATP provides the energy that is essential for all P-type ATPases to actively transport their substrates against an existing gradient. This ATP hydrolysis can be measured using different methods. Here, we describe a method that uses radiolabeled [gamma-(32)P]ATP, which is hydrolyzed by P-type ATPases

  12. SwissProt search result: AK109037 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109037 002-154-C10 (Q8R429) Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (...EC 3.6.3.8) (Calcium pump 1) (SERCA1) (SR Ca(2+)-ATPase 1) (Calcium-transporting ATPase sarcoplasmic reticul

  13. [Experiments on the mechanism of action of vascular spasmolytics. 4. Effect of nitroprusside sodium, nitroglycerin, prenylamine and verapamil on the calcium uptake of microsomes of the smooth bascular muscles].

    Science.gov (United States)

    Klinner, U; Ehlers, D; Fermum, R; Meisel, P

    1977-01-01

    Nitroprusside-sodium, nitroglycerol, and verapamil had no effect on the calcium uptake by microsomes from the carotid artery of cattle. Prenylamine reduced the passive binding and the active uptake and released already bound calcium. The basal Mg-dependendent ATPase and Ca-stimulatable Mg-ATPase were inhibited by prenylamine.

  14. Calcium Uptake in Crude Tissue Preparation

    Science.gov (United States)

    Bidwell, Philip A.; Kranias, Evangelia G.

    2016-01-01

    SUMMARY The various isoforms of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) are responsible for the Ca2+ uptake from the cytosol into the endoplasmic or sarcoplasmic reticulum (ER/SR). In some tissues, the activity of SERCA can be modulated by binding partners, such as phospholamban and sarcolipin. The activity of SERCA can be characterized by its apparent affinity for Ca2+ as well as maximal enzymatic velocity. Both parameters can be effectively determined by the protocol described here. Specifically, we describe the measurement of the rate of oxalate-facilitated 45Ca uptake into the SR of crude mouse ventricular homogenates. This protocol can easily be adapted for different tissues and animal models as well as cultured cells. PMID:26695031

  15. Calcium in plant cells

    Directory of Open Access Journals (Sweden)

    V. V. Schwartau

    2014-04-01

    Full Text Available The paper gives the review on the role of calcium in many physiological processes of plant organisms, including growth and development, protection from pathogenic influences, response to changing environmental factors, and many other aspects of plant physiology. Initial intake of calcium ions is carried out by Ca2+-channels of plasma membrane and they are further transported by the xylem owing to auxins’ attractive ability. The level of intake and selectivity of calcium transport to ove-ground parts of the plant is controlled by a symplast. Ca2+enters to the cytoplasm of endoderm cells through calcium channels on the cortical side of Kaspary bands, and is redistributed inside the stele by the symplast, with the use of Ca2+-АТPases and Ca2+/Н+-antiports. Owing to regulated expression and activity of these calcium transporters, calclum can be selectively delivered to the xylem. Important role in supporting calcium homeostasis is given to the vacuole which is the largest depo of calcium. Regulated quantity of calcium movement through the tonoplast is provided by a number of potential-, ligand-gated active transporters and channels, like Ca2+-ATPase and Ca2+/H+ exchanger. They are actively involved in the inactivation of the calcium signal by pumping Ca2+ to the depo of cells. Calcium ATPases are high affinity pumps that efficiently transfer calcium ions against the concentration gradient in their presence in the solution in nanomolar concentrations. Calcium exchangers are low affinity, high capacity Ca2+ transporters that are effectively transporting calcium after raising its concentration in the cell cytosol through the use of protons gradients. Maintaining constant concentration and participation in the response to stimuli of different types also involves EPR, plastids, mitochondria, and cell wall. Calcium binding proteins contain several conserved sequences that provide sensitivity to changes in the concentration of Ca2+ and when you

  16. Effect of exercise on gene expression of calcium modulin in myocardial sarcoplasmic reticulum of diabetic rat%运动对1型糖尿病大鼠心肌肌浆网钙调控蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    江卫华; 罗达亚; 余乐涵; 段荣; 万福生

    2008-01-01

    目的 观察运动对1型糖尿病大鼠心肌肌浆网钙调控蛋白表达的影响,并探讨其机制.方法 选用健康Spragne-Dawley大鼠,随机分为正常对照组、运动对照组、糖尿病组和糖尿病+运动组,每组10只.用腹腔注射链脲佐菌素(55 mg/ks)法复制糖尿病模型,糖尿病+运动组大鼠在糖尿病模型建成后第4天开始跑台运动,于运动第4周末心脏采血,制备血清.采用放射免疫法测定血清胰岛素水平,采用RT-PCR法检测肌浆网Ca结果-ATP酶(SERCA)、磷酸受纳蛋白(PLB)和Ryanodine受体-2(RyR2)mRNA的表达,采用Western-blotting法检测SERCA2、PLB蛋白的表达.结果 与正常对照组相比,糖尿病组血糖、糖化血清蛋白及低密度脂蛋白水平升高,胰岛素和高密度脂蛋白水平降低,心肌SERCA2、PLB、RyR2 mRNA和SERCA2、PLB蛋白表达差异无统计学意义;糖尿病+运动组血糖水平升高,胰岛素水平降低,心肌SERCA2、PLB和RyR2的mRNA表达水平提高,SERCA2、PLB蛋白表达量增加,差异有统计学意义(P -ATPase (SERCA2) and phaspholamban (PLB). Results The level of biochemical indicator of exercisegroup is not affected when comparing with that of the control group, but significantly changed in diabetic group ( P <0. 01 ) ; The level of blood glucose, insulin, blood fat and glycosylated serum protein were ameliorated in diabetic rats inthe exercise training group. No significant changes in mRNA level of SERCA2, PLB and ryanodine receptor type 2(RYR2) were observed between control and diabetic group, the same to protein expression of SERCA2 and PLB. Butexpression of calcium modulin mRNA was significantly increased in exercise group and diabetic rats in the exercisetraining group comparing with that of the control and diabetic groups ( P < 0.01 ), the same to protein expression ofSERCA2 and PLB. Conclusion Exercise exerted good protective effects on the myocardial injury with 1 type diabetesrat, which might attribute to the

  17. The Contribution of Candida albicans Vacuolar ATPase Subunit V1B, Encoded by VMA2, to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

    Science.gov (United States)

    Rane, Hallie S.; Bernardo, Stella M.; Hayek, Summer R.; Binder, Jessica L.; Parra, Karlett J.

    2014-01-01

    Candida albicans vacuoles are central to many critical biological processes, including filamentation and in vivo virulence. The V-ATPase proton pump is a multisubunit complex responsible for organellar acidification and is essential for vacuolar biogenesis and function. To study the function of the V1B subunit of C. albicans V-ATPase, we constructed a tetracycline-regulatable VMA2 mutant, tetR-VMA2. Inhibition of VMA2 expression resulted in the inability to grow at alkaline pH and altered resistance to calcium, cold temperature, antifungal drugs, and growth on nonfermentable carbon sources. Furthermore, V-ATPase was unable to fully assemble at the vacuolar membrane and was impaired in proton transport and ATPase-specific activity. VMA2 repression led to vacuolar alkalinization in addition to abnormal vacuolar morphology and biogenesis. Key virulence-related traits, including filamentation and secretion of degradative enzymes, were markedly inhibited. These results are consistent with previous studies of C. albicans V-ATPase; however, differential contributions of the V-ATPase Vo and V1 subunits to filamentation and secretion are observed. We also make the novel observation that inhibition of C. albicans V-ATPase results in increased susceptibility to osmotic stress. Notably, V-ATPase inhibition under conditions of nitrogen starvation results in defects in autophagy. Lastly, we show the first evidence that V-ATPase contributes to virulence in an acidic in vivo system by demonstrating that the tetR-VMA2 mutant is avirulent in a Caenorhabditis elegans infection model. This study illustrates the fundamental requirement of V-ATPase for numerous key virulence-related traits in C. albicans and demonstrates that the contribution of V-ATPase to virulence is independent of host pH. PMID:25038082

  18. Restoring the impaired cardiac calcium homeostasis and cardiac function in iron overload rats by the combined deferiprone and N-acetyl cysteine

    Science.gov (United States)

    Wongjaikam, Suwakon; Kumfu, Sirinart; Khamseekaew, Juthamas; Chattipakorn, Siriporn C.; Chattipakorn, Nipon

    2017-01-01

    Intracellular calcium [Ca2+]i dysregulation plays an important role in the pathophysiology of iron overload cardiomyopathy. Although either iron chelators or antioxidants provide cardioprotection, a comparison of the efficacy of deferoxamine (DFO), deferiprone (DFP), deferasirox (DFX), N-acetyl cysteine (NAC) or a combination of DFP plus NAC on cardiac [Ca2+]i homeostasis in chronic iron overload has never been investigated. Male Wistar rats were fed with either a normal diet or a high iron (HFe) diet for 4 months. At 2 months, HFe rats were divided into 6 groups and treated with either a vehicle, DFO (25 mg/kg/day), DFP (75 mg/kg/day), DFX (20 mg/kg/day), NAC (100 mg/kg/day), or combined DFP plus NAC. At 4 months, the number of cardiac T-type calcium channels was increased, whereas cardiac sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA) was decreased, leading to cardiac iron overload and impaired cardiac [Ca2+]i homeostasis. All pharmacological interventions restored SERCA levels. Although DFO, DFP, DFX or NAC alone shared similar efficacy in improving cardiac [Ca2+]i homeostasis, only DFP + NAC restored cardiac [Ca2+]i homeostasis, leading to restoring left ventricular function in the HFe-fed rats. Thus, the combined DFP + NAC was more effective than any monotherapy in restoring cardiac [Ca2+]i homeostasis, leading to restored myocardial contractility in iron-overloaded rats. PMID:28287621

  19. Molecular transformations in sarcoplasmic reticulum of fast-twitch muscle by electro-stimulation.

    Science.gov (United States)

    Heilmann, C; Pette, D

    1979-02-01

    Chronic electro-stimulation of fast-twitch rabbit muscle with the frequency pattern received by a slow-twitch muscle induces a progressive transformation of the sarcoplasmic reticulum. After 2 days stimulation activities of Ca2+-dependent ATPase and of Ca2+ transport begin to decrease, and are paralleled by a progressive decrease in Ca2+-dependent and Ca2+, Mg2+-dependent phosphoprotein formation, reduced rate of dephosphorylation and a rearrangement of the electrophoretic polypeptide and phosphoprotein patterns. These findings suggest a transformation of the sarcoplasmic reticulum to resemble that of a slow-twitch muscle. This transformation is paralleled by increase in time-to-peak of twitch contraction and half relaxation time and occurs before conversion of the myosin light chain pattern is observed. The parallel time course of changes in contractile properties of stimulated muscle and the molecular and functional properties of the sarcoplasmic reticulum emphasizes the definitive role of the latter in determining the twitch characteristics of fast and slow twitch muscles.

  20. Endoplasmic reticulum stress in lung disease

    Directory of Open Access Journals (Sweden)

    Stefan J. Marciniak

    2017-06-01

    Full Text Available Exposure to inhaled pollutants, including fine particulates and cigarette smoke is a major cause of lung disease in Europe. While it is established that inhaled pollutants have devastating effects on the genome, it is now recognised that additional effects on protein folding also drive the development of lung disease. Protein misfolding in the endoplasmic reticulum affects the pathogenesis of many diseases, ranging from pulmonary fibrosis to cancer. It is therefore important to understand how cells respond to endoplasmic reticulum stress and how this affects pulmonary tissues in disease. These insights may offer opportunities to manipulate such endoplasmic reticulum stress pathways and thereby cure lung disease.

  1. Mitochondria and endoplasmic reticulum crosstalk in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Manfredi, Giovanni; Kawamata, Hibiki

    2016-06-01

    Physical and functional interactions between mitochondria and the endoplasmic reticulum (ER) are crucial for cell life. These two organelles are intimately connected and collaborate to essential processes, such as calcium homeostasis and phospholipid biosynthesis. The connections between mitochondria and endoplasmic reticulum occur through structures named mitochondria associated membranes (MAMs), which contain lipid rafts and a large number of proteins, many of which serve multiple functions at different cellular sites. Growing evidence strongly suggests that alterations of ER-mitochondria interactions are involved in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a devastating and rapidly fatal motor neuron disease. Mutations in proteins that participate in ER-mitochondria interactions and MAM functions are increasingly being associated with genetic forms of ALS and other neurodegenerative diseases. This evidence strongly suggests that, rather than considering the two organelles separately, a better understanding of the disease process can derive from studying the alterations in their crosstalk. In this review we discuss normal and pathological ER-mitochondria interactions and the evidence that link them to ALS. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Kinetic properties of the mitochondrial F1FO-ATPase activity elicited by Ca(2+) in replacement of Mg(2).

    Science.gov (United States)

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pirini, Maurizio; Pagliarani, Alessandra

    2017-09-01

    The mitochondrial F-ATPase can be activated either by the classical cofactor Mg(2+) or, with lower efficiency, by Ca(2+). The latter may play a role when calcium concentration rises in mitochondria, a condition associated with cascade events leading to cell death. Common and distinctive features of these differently activated mitochondrial ATPases were pointed out in swine heart mitochondria. When Ca(2+) replaces the natural cofactor Mg(2+), the enzyme responsiveness to the transmembrane electrochemical gradient and to the classical F-ATPase inhibitors DCCD and oligomycin as well as the oligomycin sensitivity loss by thiol oxidation, are maintained. Consistently, the two mitochondrial ATPases apparently share the F1FO complex basic structure and mechanism. Peculiar cation-dependent properties, which may affect the F1 catalytic mechanism and/or the FO proton binding site features, may be linked to a different physiological role of the mitochondrial Ca-activated F-ATPase with respect to the Mg-activated F-ATPase. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  3. Vitamin D-enhanced duodenal calcium transport.

    Science.gov (United States)

    Wongdee, Kannikar; Charoenphandhu, Narattaphol

    2015-01-01

    For humans and rodents, duodenum is a very important site of calcium absorption since it is exposed to ionized calcium released from dietary complexes by gastric acid. Calcium traverses the duodenal epithelium via both transcellular and paracellular pathways in a vitamin D-dependent manner. After binding to the nuclear vitamin D receptor, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] upregulates the expression of several calcium transporter genes, e.g., TRPV5/6, calbindin-D9k, plasma membrane Ca(2+)-ATPase1b, and NCX1, thereby enhancing the transcellular calcium transport. This action has been reported to be under the regulation of parathyroid-kidney-intestinal and bone-kidney-intestinal axes, in which the plasma calcium and fibroblast growth factor-23 act as negative feedback regulators, respectively. 1,25(OH)2D3 also modulates the expression of tight junction-related genes and convective water flow, presumably to increase the paracellular calcium permeability and solvent drag-induced calcium transport. However, vitamin D-independent calcium absorption does exist and plays an important role in calcium homeostasis under certain conditions, particularly in neonatal period, pregnancy, and lactation as well as in naturally vitamin D-impoverished subterranean mammals.

  4. Sodium, potassium-atpases in algae and oomycetes.

    Science.gov (United States)

    Barrero-Gil, Javier; Garciadeblás, Blanca; Benito, Begoña

    2005-08-01

    We have investigated the presence of K(+)-transporting ATPases that belong to the phylogenetic group of animal Na(+),K(+)-ATPases in the Pythium aphanidermatum Stramenopile oomycete, the Porphyra yezoensis red alga, and the Udotea petiolata green alga, by molecular cloning and expression in heterologous systems. PCR amplification and search in EST databases allowed one gene to be identified in each species that could encode ATPases of this type. Phylogenetic analysis of the sequences of these ATPases revealed that they cluster with ATPases of animal origin, and that the algal ATPases are closer to animal ATPases than the oomycete ATPase is. The P. yezoensis and P. aphanidermatum ATPases were functionally expressed in Saccharomyces cerevisiae and Escherichia coli alkali cation transport mutants. The aforementioned cloning and complementary searches in silicio for H(+)- and Na(+),K(+)-ATPases revealed a great diversity of strategies for plasma membrane energization in eukaryotic cells different from typical animal, plant, and fungal cells.

  5. Cellular mechanisms by which oxytocin mediates uterine prostaglandin F2 alpha synthesis in bovine endometrium: role of calcium.

    Science.gov (United States)

    Burns, P D; Hayes, S H; Silvia, W J

    1998-11-01

    The objective of these experiments was to determine the role of Ca2+ during oxytocin-stimulated prostaglandin (PG) F2 alpha release from bovine endometrial tissue in vitro. Uteri were collected from dairy cows on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to determine phospholipase C activity or PGF2 alpha release. A23,187 (a calcium ionophore) and maitotoxin (an activator of voltage-gated L-type calcium channels) stimulated release of PGF 2 alpha in a concentration-dependent manner (P < 0.05). Thapsigargin (induces accumulation of Ca2+ in the cytoplasm by inhibiting endoplasmic reticulum Ca2+/ATPase pumps) stimulated release of PGF2 alpha in a concentration-dependent manner as well (P < 0.13). Oxytocin (10(-6) M), AIF4- (a nonspecific activator of G-proteins; 10(-5) M), A23,187 (10(-5) M), and melittin (a stimulator of phospholipase A2; 10(-4) M) stimulated PGF2 alpha release when explants were incubated in Ca(2+)-free medium (P < 0.10); however, oxytocin, A23,187, or melittin were unable to stimulate PGF2 alpha release when explants were incubated in Ca(2+)-free medium containing the calcium chelator EGTA (P < 0.10). This treatment did not prevent oxytocin or AIF4- from stimulating phospholipase C activity (P < 0.08). CoCl2 (a nonspecific Ca2+ channel blocker) and methoxyverapamil (a specific voltage-gated L-type Ca2+ channel blocker) prevented oxytocin from stimulating PGF2 alpha release (P < 0.05). Our results suggest that both extracellular and intracellular Ca2+ may be required for oxytocin to stimulate PGF2 alpha secretion in bovine endometrial tissue.

  6. Cloning and expression of vacuolar proton-pumping ATPase subunits in the follicular epithelium of the bullfrog endolymphatic sac

    OpenAIRE

    Yajima, Shinya; Kubota, Makoto; Nakakura, Takashi; Hasegawa, Takahiro; Katagiri, Nobuto; Tomura, Hideaki; Sasayama, Yuichi; Suzuki, Masakazu; Tanaka, Shigeyasu

    2007-01-01

    In an investigation aimed at clarifying the mechanism of crystal dissolution of the calcium carbonate lattice in otoconia (the mineral particles embedded in the otolithic membrane) of the endolymphatic sac (ELS) of the bullfrog, cDNAs encoding the A- and E-subunits of bullfrog vacuolar proton-pumping ATPase (V-ATPase) were cloned and sequenced. The cDNA of the A-subunit consisted of an 11-bp 5′-untranslated region (UTR), a 1,854-bp open reading frame (ORF) encoding a protein comprising 617 am...

  7. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    赵文; 姜志胜; 倪菊华; 陈光慧; 刘乃奎; 汤健; 贾弘褆; 唐朝枢

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sar-coplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  8. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  9. [ATPase and phosphatase activity of drone brood].

    Science.gov (United States)

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae.

  10. Functional Analysis of P4-ATPases

    DEFF Research Database (Denmark)

    Theorin, Lisa

    and mammalian P4-ATPases have been studied extensively and the physiological function is mostly known, while the exact biochemistry and specific activity is mostly unknown. Even though the plant Arabidopsis thaliana has 12 P4-ATPases, not much is known about their function. In this study, the biochemical...... properties, with a focus on the lipid requirements, of the Aminophospholipid ATPase 2 (ALA2), a P4-ATPase from A. thaliana, were characterized. Heterologous expression of ALA2 together with its subunit, the Cdc50 homolog ALA Interacting Subunit 5 (ALIS5), in the yeast Saccharomyces cerevisiae allowed...... is specific for phosphatidylserine and that binding of the lipid to the substrate binding site requires a unique spatial configuration of the lipid head group. Detailed information on the substrate requirements lead the way towards the full function and transport pathway of lipid flippases in plants. Recent...

  11. Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C

    DEFF Research Database (Denmark)

    Kristensen, B; Birkelund, Svend; Jørgensen, PL

    2003-01-01

    . Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation......Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent...... along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein...

  12. Growth at high pH and sodium and potassium tolerance in media above the cytoplasmic pH depend on ENA ATPases in Ustilago maydis.

    Science.gov (United States)

    Benito, Begoña; Garciadeblás, Blanca; Pérez-Martín, José; Rodríguez-Navarro, Alonso

    2009-06-01

    Potassium and Na(+) effluxes across the plasma membrane are crucial processes for the ionic homeostasis of cells. In fungal cells, these effluxes are mediated by cation/H(+) antiporters and ENA ATPases. We have cloned and studied the functions of the two ENA ATPases of Ustilago maydis, U. maydis Ena1 (UmEna1) and UmEna2. UmEna1 is a typical K(+) or Na(+) efflux ATPase whose function is indispensable for growth at pH 9.0 and for even modest Na(+) or K(+) tolerances above pH 8.0. UmEna1 locates to the plasma membrane and has the characteristics of the low-Na(+)/K(+)-discrimination ENA ATPases. However, it still protects U. maydis cells in high-Na(+) media because Na(+) showed a low cytoplasmic toxicity. The UmEna2 ATPase is phylogenetically distant from UmEna1 and is located mainly at the endoplasmic reticulum. The function of UmEna2 is not clear, but we found that it shares several similarities with Neurospora crassa ENA2, which suggests that endomembrane ENA ATPases may exist in many fungi. The expression of ena1 and ena2 transcripts in U. maydis was enhanced at high pH and at high K(+) and Na(+) concentrations. We discuss that there are two modes of Na(+) tolerance in fungi: the high-Na(+)-content mode, involving ENA ATPases with low Na(+)/K(+) discrimination, as described here for U. maydis, and the low-Na(+)-content mode, involving Na(+)-specific ENA ATPases, as in Neurospora crassa.

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

    Science.gov (United States)

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

    2011-01-01

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

  14. Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions.

    Science.gov (United States)

    Liu, Yi; Gianinazzi-Pearson, Vivienne; Arnould, Christine; Wipf, Daniel; Zhao, Bin; van Tuinen, Diederik

    2013-01-01

    Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca(2+) in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca(2+)-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca(2+)-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca(2+) ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca(2+)-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots. Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  15. Transport Properties of the Tomato Fruit Tonoplast : III. Temperature Dependence of Calcium Transport.

    Science.gov (United States)

    Joyce, D C; Cramer, G R; Reid, M S; Bennett, A B

    1988-12-01

    Calcium transport into tomato (Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca(2+) transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca(2+). A low affinity Ca(2+) uptake system (K(m) > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H(+)/Ca(2+) antiport. A high affinity Ca(2+) uptake system (K(m) = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca(2+) transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12 degrees C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca(2+)/H(+) antiport activity could only by partially ascribed to an effect of low temperature on H(+)-ATPase activity, ATP-dependent H(+) transport, passive H(+) fluxes, or passive Ca(2+) fluxes. These results suggest that low temperature directly affects Ca(2+)/H(+) exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca(2+)/H(+) exchange protein or by an indirect effect of temperature on lipid interactions with the Ca(2+)/H(+) exchange protein.

  16. Vanadate oligomer inhibition of passive and active Ca2+ translocation by the Ca2+ pump of sarcoplasmic reticulum.

    Science.gov (United States)

    Aureliano, M

    2000-05-30

    'Monovanadate' containing mainly monomeric, dimeric and tetrameric vanadate species or 'decavanadate', containing mainly decameric vanadate species inhibits the passive and the active efflux of Ca2+ through the sarcoplasmic reticulum calcium pump. When the efflux of Ca2+ by sarcoplasmic reticulum vesicles is not associated with ATP synthesis both vanadate solutions inhibit the passive efflux of Ca2+. However, only 'decavanadate' exerts noticeable effects when the efflux of Ca2+ is associated with ATP synthesis being the active efflux of Ca2+ almost completely inhibited by decameric species concentration as low as 40 microM.

  17. P160L mutation in the Ca(2+) ATPase 2A domain in a patient with severe Darier disease.

    Science.gov (United States)

    Godic, Aleksandar; Glavac, Damjan; Korosec, Branka; Miljković, Jovan; Potocnik, Marko; Kansky, Aleksej

    2004-01-01

    Darier disease (DD) is caused by mutations of the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2). The mutations affect protein expression, degradation and activity. We report a patient with severe sporadic DD, who did not respond adequately to repeated courses of orally administered acitretin and isotretinoin. He was found to harbor the missense P160L mutation of the ATP2A2 gene in a heterozygous state in the A domain of SERCA2 and polymorphism in intron 18 (2741 + 54 G --> A). The A domain plays a key role in translocation of Ca(2+) from cytoplasm to endoplasmic reticulum lumen, thus establishing a low intracellular Ca(2+) concentration.

  18. The coupling ATPase complex: an evolutionary view.

    Science.gov (United States)

    Harris, D A

    1981-01-01

    Phospholipid micelles and vesicles, present in the primordial soup, formed both primitive (surface) catalyst and primitive replicative life forms. With the adoption of a common energy source, ATP, integrated biochemical systems within these vesicles became possible - cells. Fermentation within these primitive cells was favoured by the evolution, first of ion channels allowing protons to leak out, and then of an active ATP-driven pump. In the prokaryotic/mitochondria/chloroplast line, the proton channel was such as to be blocked by dicyclohexylcarbodiimide and the adenosine 5' triphosphate phosphohydrolase (ATPase) by 4-chloro 7-nitrobenzofurazan (Nbf-C1). The ATPase was initially simple (4 subunits) but later, possibly concomitant with its evolution to an ATP synthetase, became more complex (8 subunits). One of the steps in evolution probably involved gene duplication and divergence of 2 subunits (alpha and beta) from the largest of the ATPase subunits. From this stage, the general form of the ATPase was fixed, although sensitivity to, for example, oligomycin involved later, after divergence of the mitochondrial and chloroplast lines. A regulatory protein, the ATPase inhibitor, is found associated with a wide spectrum of coupling ATPases.

  19. Alendronate affects calcium dynamics in cardiomyocytes in vitro.

    Science.gov (United States)

    Kemeny-Suss, Naomi; Kasneci, Amanda; Rivas, Daniel; Afilalo, Jonathan; Komarova, Svetlana V; Chalifour, Lorraine E; Duque, Gustavo

    2009-01-01

    Therapy with bisphosphonates, including alendronate (ALN), is considered a safe and effective treatment for osteoporosis. However, recent studies have reported an unexpected increase in serious atrial fibrillation (AF) in patients treated with bisphosphonates. The mechanism that explains this side effect remains unknown. Since AF is associated with an altered sarcoendoplasmic reticulum calcium load, we studied how ALN affects cardiomyocyte calcium homeostasis and protein isoprenylation in vitro. Acute and long-term (48h) treatment of atrial and ventricular cardiomyocytes with ALN (10(-8)-10(-6)M) was performed. Changes in calcium dynamics were determined by both fluorescence measurement of cytosolic free Ca(2+) concentration and western blot analysis of calcium-regulating proteins. Finally, effect of ALN on protein farnesylation was also identified. In both atrial and ventricular cardiomyocytes, ALN treatment delayed and diminished calcium responses to caffeine. Only in atrial cells, long-term exposure to ALN-induced transitory calcium oscillations and led to the development of oscillatory component in calcium responses to caffeine. Changes in calcium dynamics were accompanied by changes in expression of proteins controlling sarcoendoplasmic reticulum calcium. In contrast, ALN minimally affected protein isoprenylation in these cells. In summary, treatment of atrial cardiomyocytes with ALN-induced abnormalities in calcium dynamics consistent with induction of a self-stimulatory, pacemaker-like behavior, which may contribute to the development of cardiac side effects associated with these drugs.

  20. Contribution of plasma membrane Ca2+ ATPase to cerebellar synapse function

    Institute of Scientific and Technical Information of China (English)

    Helena; Huang; Raghavendra; Y; Nagaraja; Molly; L; Garside; Walther; Akemann; Thomas; Knpfel; Ruth; M; Empson

    2010-01-01

    The cerebellum expresses one of the highest levels of the plasma membrane Ca2+ATPase,isoform 2 in the mammalian brain.This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex;i.e. the Purkinje neurons(PNs) .Here we review recent evidence,including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2(PMCA2) knockout mouse,to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour.These studies have also revealed that deletionof PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development,they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs.

  1. Diazeniumdiolate mediated nitrosative stress alters nitric oxide homeostasis through intracellular calcium and S-glutathionylation of nitric oxide synthetase.

    Directory of Open Access Journals (Sweden)

    Yefim Manevich

    Full Text Available BACKGROUND: PABA/NO is a diazeniumdiolate that acts as a direct nitrogen monoxide (NO donor and is in development as an anticancer drug. Its mechanism of action and effect on cells is not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: We used HPLC and mass spectrometry to identify a primary nitroaromatic glutathione metabolite of PABA/NO and used fluorescent assays to characterize drug effects on calcium and NO homeostasis, relating these to endothelial nitric oxide synthase (eNOS activity. Unexpectedly, the glutathione conjugate was found to be a competitive inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA presumably at the same site as thapsigargin, increasing intracellular Ca2+ release and causing auto-regulation of eNOS through S-glutathionylation. CONCLUSIONS/SIGNIFICANCE: The initial direct release of NO after PABA/NO was followed by an eNOS-mediated generation of NO as a consequence of drug-induced increase in Ca2+ flux and calmodulin (CaM activation. PABA/NO has a unique dual mechanism of action with direct intracellular NO generation combined with metabolite driven regulation of eNOS activation.

  2. Differential expression of genes involved in the calcium homeostasis in masticatory muscles of MDX mice.

    Science.gov (United States)

    Kunert-Keil, C H; Gredes, T; Lucke, S; Botzenhart, U; Dominiak, M; Gedrange, T

    2014-04-01

    Duchenne Muscular Dystrophy (DMD) and its murine model, mdx, are characterized by Ca(2+) induced muscle damage and muscle weakness followed by distorted dentofacial morphology. In both, DMD patients and in mdx mice, could be proven so far that only the extraocular muscles (EOM) are not affected by muscular dystrophy. The EOMs are protected against calcium overload by enhanced expression of genes involved in the Ca(2+) homeostasis. We could recently demonstrate that masticatory muscles of mdx mice are differentially affected by muscle dystrophy. The dystrophic masseter and temporalis shows muscle histology comparable to all other skeletal muscles in this animal model, whereas dystrophic tongue muscles seem to develop a milder phenotype. Due to this fact it is to hypothesize that an altered Ca(2+) homeostasis seems to underlie the mdx masticatory muscle pathology. Aim of this study was to examine the mRNA and protein levels of the sarcoplasmic reticulum Ca(2+) ATPases SERCA1 and SERCA2, the plasma membrane Ca(2+) ATPases Atp2b1 and Atp2b4, the sodium/calcium exchanger NCX1, the ryanodine receptor 1, parvalbumin, sarcolipin, phospholamban and the L-type Ca(2+) channel alpha-1 subunit (Cacna1s) in Musculus masseter, temporalis, and tongue of 100 day old control and mdx mice. In mdx masseter muscle significant increased mRNA levels of NCX1 and Cacna1s were found compared to control mice. In contrast, the mRNA amount of RYR1 was significant reduced in mdx temporalis muscle, whereas ATP2b4 was significant increased. In mdx tongue a down-regulation of the ATP2b1, sarcolipin and parvalbumin mRNA expression was found, whereas the phospholamban mRNA level was significantly increased compared to controls. These data were verified by western blot analyses. Our findings revealed that mdx masticatory muscles showed an unequally altered expression of genes involved in the Ca(2+) homeostasis that can support the differences in masticatory muscles response to dystrophin deficiency.

  3. Studies on the Endoplasmic Reticulum

    Science.gov (United States)

    Porter, Keith R.; Machado, Raul D.

    1960-01-01

    Cells of onion and garlic root tips were examined under the electron and phase contrast microscopes after fixation in KMnO4. Special attention was focused on the distribution and behavior of the endoplasmic reticulum (ER) during the several phases of mitosis. Slender profiles, recognized as sections through thin lamellar units of the ER (most prominent in KMnO4-fixed material), are distributed more or less uniformly in the cytoplasm of interphase cells and show occasional continuity with the nuclear envelope. In late prophase the nuclear envelope breaks down and its remnants plus cytoplasmic elements of the ER, which are morphologically identical, surround the spindle in a zone from which mitochondria, etc., are excluded. During metaphase these ER elements persist and concentrate as two separate systems in the polar caps or zones of the spindle. At about this same time they begin to proliferate and to invade the ends of the spindle. The invading lamellar units form drape-like partitions between the anaphase chromosomes. In late anaphase, their advancing margins reach the middle zone of the spindle and begin to fray out. Finally, in telophase, while elements of the ER in the poles of the spindle coalesce around the chromosomes to form the new envelope, the advancing edges of those in the middle zone reticulate at the level of the equator to form a close lattice of tubular elements. Within this, which is identified as the phragmoplast, the earliest signs of the cell plate appear in the form of small vesicles. These subsequently grow and fuse to complete the separation of the two protoplasts. Other morphological units apparently participating in mitosis are described. Speculation is provided on the equal division or not of the nuclear envelope and the contribution the envelope fragments make to the ER of the new cell. PMID:14434278

  4. Essential Role for Pro(21) in Phospholamban for Optimal Inhibition of the Ca-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jinhui; Boschek, Curt B.; Xiong, Yijia; Sacksteder, Colette A.; Squier, Thomas C.; Bigelow, Diana J.

    2005-12-13

    We have investigated the functional role of the flexible hinge region centered near the sequence TIEMP21, which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attaining optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro21 was mutated to an alanine (P21A-PLBC); in this construct all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes. In agreement with prior measurements in cardiac SR microsomes, the calcium concentration associated with half-maximal activation (Ca1/2) of the Ca-ATPase, 290 ? 10 nM, is shifted to 580 ? 20 nM when co-reconstituted with PLBC (Pro21) as a result of a 75% reduction in the rate of formation of the second high-affinity calcium binding site associated with calcium activation. In comparison, there is a 43% reduction in ?Ca1/2 upon reconstitution of the Ca-ATPase with P21A-PLBC, which can be simulated by decreasing the rate constant associated with calcium activation by 50%. The diminished inhibitory action of P21A-PLBC is associated with alterations in the structure of the hinge element, as evidenced by the diminished solvent accessibility of FMal relative to the native structure. Likewise, increases in the ?-helical content and decreases in the mobility of the carboxyl-terminal domain of P21A-PLBC are observed using circular dichroism and fluorescence spectroscopy. Collectively, these results indicate that the overall dimensions of the carboxyl-terminal domain of PLB are increased through a stabilization of secondary structural elements upon mutation in P21A-PLBC that result in a reduction in the ability of the

  5. Regulation of V-ATPase assembly and function of V-ATPases in tumor cell invasiveness.

    Science.gov (United States)

    McGuire, Christina; Cotter, Kristina; Stransky, Laura; Forgac, Michael

    2016-08-01

    V-ATPases are ATP-driven proton pumps that function within both intracellular compartments and the plasma membrane in a wide array of normal physiological and pathophysiological processes. V-ATPases are composed of a peripheral V(1) domain that hydrolyzes ATP and an integral V(0) domain that transports protons. Regulated assembly of the V-ATPase represents an important mechanism of regulating V-ATPase activity in response to a number of environmental cues. Our laboratory has demonstrated that glucose-dependent assembly of the V-ATPase complex in yeast is controlled by the Ras/cAMP/PKA pathway. By contrast, increased assembly of the V-ATPase during dendritic cell maturation involves the PI-3 kinase and mTORC1 pathways. Recently, we have shown that amino acids regulate V-ATPase assembly in mammalian cells, possibly as a means to maintain adequate levels of amino acids upon nutrient starvation. V-ATPases have also been implicated in cancer cell survival and invasion. V-ATPases are targeted to different cellular membranes by isoforms of subunit a, with a3 targeting V-ATPases to the plasma membrane of osteoclasts. We have shown that highly invasive human breast cancer cell lines express higher levels of the a3 isoform than poorly invasive lines and that knockdown of a3 reduces both expression of V-ATPases at the plasma membrane and in vitro invasion of breast tumor cells. Moreover, overexpression of a3 in a non-invasive breast epithelial line increases both plasma membrane V-ATPases and in vitro invasion. Finally, specific ablation of plasma membrane V-ATPases in highly invasive human breast cancer cells using either an antibody or small molecule approach inhibits both in vitro invasion and migration. These results suggest that plasma membrane and a3-containing V-ATPases represent a novel and important target in the development of therapeutics to limit breast cancer metastasis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics

  6. A Molecular Web: Endoplasmic Reticulum Stress, Inflammation and Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Namrata eChaudhari

    2014-07-01

    Full Text Available Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded protein response (UPR through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS. Toxic accumulation of ROS within ER and mitochondria disturb fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways has been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease and others. In this review we have discussed the UPR signaling pathways, and networking between ER stress induced inflammatory pathways, oxidative stress and mitochondrial signaling events which further induce or exacerbate ER stress.

  7. Recording of calcium transient and analysis of calcium removal mechanisms in cardiac myocytes from rats and ground squirrels

    Institute of Scientific and Technical Information of China (English)

    王世强; 周曾铨; 钱洪

    2000-01-01

    With confocal microscopy, we recorded calcium transients and analyzed calcium removal rate at different temperatures in cardiac myocytes from the rat, a non-hibernator, and the ground squirrel, a hibernator. The results showed a remarkable increase of the diastolic level of calcium transients in the rat but no detectable change in the ground squirrel. Calcium transient of the ground squirrel, compared with that of the rat at the same temperature, had a shorter duration and showed a faster calcium removal. As indicated by the pharmacological effect of cyclopiazonic acid, calcium uptake by sarcoplasmic reticulum (SR) was the major mechanism of calcium removal, and was faster in the ground squirrel than in the rat. Our results confirmed the essential role of SR in hypothermia-tolerant adaptation, and negated the importance of Na-Ca exchange. We postulated the possibility to improve hypothermia-tolerance of the cardiac tissue of non-hibernating mammals.

  8. Newinsightsintostore-independentCa21entry:secretory pathwaycalciumATPase2innormalphysiologyandcancer

    Institute of Scientific and Technical Information of China (English)

    Ming-Ye Feng; Rajini Rao

    2013-01-01

    Recent studies in secretory pathway calcium ATPases (SPCA) revealed novel functions of SPCA2 in interacting with store-operated Ca21 channel Orai1 and inducing Ca21 influx at the cell surface. Importantly, SPCA2-mediated Ca21 signaling is uncoupled from its conventional role of Ca21-ATPase and independent of store-operated Ca21 signaling pathway. SPCA2-induced store-independent Ca21 entry (SICE) plays essential roles in many important physiological processes, while unbalanced SICE leads to enhanced cell proliferation and tumorigenesis. Finally, we have summarized the clinical implication of SICE in oral cancer prognosis and treatment. Inhibition of SICE may be a new target for the development of cancer therapeutics.

  9. Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole cell oscillations in smooth muscle cells

    DEFF Research Database (Denmark)

    Jacobsen, Jens Christian Brings; Aalkjær, Christian; Nilsson, Holger;

    2007-01-01

    waves sweeping through the cytoplasm when the sarcoplasmic reticulum (SR) is stimulated to release calcium. A rise in cGMP leads to the experimentally observed transition from waves to whole cell calcium oscillations. At the same time, membrane potential starts to oscillate and the frequency...... approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes a uniform opening of L-type calcium...

  10. An Involvement of Oxidative Stress in Endoplasmic Reticulum Stress and Its Associated Diseases

    Directory of Open Access Journals (Sweden)

    Bidur Bhandary

    2012-12-01

    Full Text Available The endoplasmic reticulum (ER is the major site of calcium storage and protein folding. It has a unique oxidizing-folding environment due to the predominant disulfide bond formation during the process of protein folding. Alterations in the oxidative environment of the ER and also intra-ER Ca2+ cause the production of ER stress-induced reactive oxygen species (ROS. Protein disulfide isomerases, endoplasmic reticulum oxidoreductin-1, reduced glutathione and mitochondrial electron transport chain proteins also play crucial roles in ER stress-induced production of ROS. In this article, we discuss ER stress-associated ROS and related diseases, and the current understanding of the signaling transduction involved in ER stress.

  11. ECA3, a Golgi-localized P2A-type-ATPase, plays a crucial role in manganese nutrition in Arabidopsis

    DEFF Research Database (Denmark)

    Mills, Rebecca F.; Doherty, Melissa Louise; Lopez Marques, Rosa Laura

    2008-01-01

    ECA3, a Golgi-localized P2A-type ATPase, plays a crucial role in manganese nutrition in Arabidopsis. Mills RF , Doherty ML , López-Marqués RL , Weimar T , Dupree P , Palmgren MG , Pittman JK , Williams LE . Calcium (Ca) and manganese (Mn) are essential nutrients required for normal plant growth...

  12. Secretory pathway Ca2+/Mn2+-ATPase isoform 2 and lactation: specific localization of plasmalemmal and secretory pathway Ca2+ pump isoforms in the mammary gland

    Energy Technology Data Exchange (ETDEWEB)

    Faddy, Helen M.; Smart, Chanel E.; Xu, Ren; Lee, Genee Y.; Kenny, Paraic A.; Feng, Mingye; Rao, Rajini; Brown, Melissa A.; Bissell, Mina J.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.

    2008-04-09

    The supply of calcium to the developing neonate via milk is an important physiological process. Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely mediated via the secretory pathway. However, recent studies suggest that a specific isoform of the plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during different stages of development. SPCA2 levels increased over 35 fold during lactation, while SPCA1 increased only a modest two fold. The potential importance of SPCA2 in lactation was also highlighted by its localization to luminal secretory cells of the mammary gland during lactation, while SPCA1 was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1 during lactation. Using the SCp2 mouse mammary epithelial cell 3D culture model, differences in the sub-cellular distribution of PMCA2 and PMCA1 were clear. These studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation, and link the recently characterized SPCA2 calcium pump to the supply of calcium into milk and the regulation of Golgi resident enzymes important in lactation. They also indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.

  13. Effect of bacoside A on membrane-bound ATPases in the brain of rats exposed to cigarette smoke.

    Science.gov (United States)

    Anbarasi, K; Vani, G; Balakrishna, K; Devi, C S Shyamala

    2005-01-01

    Membrane-bound enzymes play a vital role in neuronal function through maintenance of membrane potential and impulse propagation. We have evaluated the harmful effects of chronic cigarette smoking on membrane-bound ATPases and the protective effect of Bacoside A in rat brain. Adult male albino rats were exposed to cigarette smoke for a period of 12 weeks and simultaneously administered with Bacoside A (the active principle isolated from Bacopa monniera) at a dosage of 10 mg/kg b.w/day, p.o. The levels of lipid peroxides as marker for evaluating the extent of membrane damage, the activities of Na+/K+-ATPase, Ca2+-ATPase and Mg2+-ATPase, and associated cations sodium (Na+), potassium (K+), calcium (Ca2+), and magnesium (Mg2+) were investigated in the brain. Neuronal membrane damage was evident from the elevated levels of lipid peroxides and decreased activities of membrane-bound enzymes. Disturbances in the electrolyte balance with accumulation of Na+ and Ca2+ and depletion of K+ and Mg2+ were also observed. Administration of Bacoside A inhibited lipid peroxidation, improved the activities of ATPases, and maintained the ionic equilibrium. The results of our study indicate that Bacoside A protects the brain from cigarette smoking induced membrane damage.

  14. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

      The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded H+-ATPases extrude protons from cells...... of plants and fungi to generate electrochemical proton gradients. A recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Together with biochemical and structural data presented in this thesis we are now able...... to describe the basic molecular components that allow the plasma membrane proton H+-ATPase to carry out proton transport against large membrane potentials. Moreover, a completely new paradigm for post-translational activation of these proteins is presented. The talk will focus on the following themes...

  15. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

      The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded H+-ATPases extrude protons from cells...... of plants and fungi to generate electrochemical proton gradients. A recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Together with biochemical and structural data presented in this thesis we are now able...... to describe the basic molecular components that allow the plasma membrane proton H+-ATPase to carry out proton transport against large membrane potentials. Moreover, a completely new paradigm for post-translational activation of these proteins is presented. The talk will focus on the following themes...

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

    Science.gov (United States)

    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

  17. Effect of Indole Butyric Acid on the Transportation of Stored Calcium in Malus hupehensis Rhed. Seedling

    Institute of Scientific and Technical Information of China (English)

    LI Jia; YANG Hong-qiang; YAN Tian-li; SHU Huai-rui

    2006-01-01

    Calcium (Ca) plays an important role in the metabolism of higher plants. Recently, research on Ca2+ in plants has been focused especially at the cellular and molecular levels. Uptake, transport, and distribution are also very important for Ca to accomplish its function at the whole-plant level. In this experiment, one-year-old apple seedlings (M. hupehensis Rehd.) were investigated to determine the distribution of stored Ca, the different forms of Ca, and Ca2+-ATPase activity after treatment with indole butyric acid (IBA). The results showed that the total Ca measured in mature leaves and Ca2+-ATPase activity in tender leaves were higher compared with those in the control (CK). Calcium nitrate and calcium chloride (ALe-Ca) and calcium phosphate and calcium carbonate (HAC-Ca) decreased in both mature leaves and shoots,whereas water-soluble calcium (H2O-Ca), calcium pectate (NaCl-Ca), and calcium oxalate (HCl-Ca) increased. The percentage of active calcium, calcium pectate, and water-soluble calcium increased, whereas the percentage of calcium phosphate and calcium carbonate decreased. When treated with IBA, calcium fractions and percentage of the different forms of Ca was enhanced in 40 part per million (ppm) IBA compared with 20 ppm IBA and water. The results indicated that IBA increased the percentage of both active calcium (NaCl-Ca and H2O-Ca) in tender shoots and boosted the transportation of stored Ca in plants. IBA promoted Ca2+-ATPase activity and Ca2+ uptake in tender shoots of M. hupehensis. It can improve the total Ca contents and the relative percentage of Ca.

  18. Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells.

    Science.gov (United States)

    Perry, Jacob L; Ramachandran, Nina K; Utama, Budi; Hyser, Joseph M

    2015-11-15

    Calcium signaling is a ubiquitous and versatile process involved in nearly every cellular process, and exploitation of host calcium signals is a common strategy used by viruses to facilitate replication and cause disease. Small molecule fluorescent calcium dyes have been used by many to examine changes in host cell calcium signaling and calcium channel activation during virus infections, but disadvantages of these dyes, including poor loading and poor long-term retention, complicate analysis of calcium imaging in virus-infected cells due to changes in cell physiology and membrane integrity. The recent expansion of genetically-encoded calcium indicators (GECIs), including blue and red-shifted color variants and variants with calcium affinities appropriate for calcium storage organelles like the endoplasmic reticulum (ER), make the use of GECIs an attractive alternative for calcium imaging in the context of virus infections. Here we describe the development and testing of cell lines stably expressing both green cytoplasmic (GCaMP5G and GCaMP6s) and red ER-targeted (RCEPIAer) GECIs. Using three viruses (rotavirus, poliovirus and respiratory syncytial virus) previously shown to disrupt host calcium homeostasis, we show the GECI cell lines can be used to detect simultaneous cytoplasmic and ER calcium signals. Further, we demonstrate the GECI expression has sufficient stability to enable long-term confocal imaging of both cytoplasmic and ER calcium during the course of virus infections.

  19. Low-Level Vagus Nerve Stimulation Reverses Cardiac Dysfunction and Subcellular Calcium Handling in Rats With Post-Myocardial Infarction Heart Failure.

    Science.gov (United States)

    Zhang, Yunhe; Chen, Ao; Song, Lei; Li, Min; Luo, Zhangyuan; Zhang, Wenzan; Chen, Yingmin; He, Ben

    2016-05-25

    Vagus nerve stimulation (VNS), targeting the imbalanced autonomic nervous system, is a promising therapeutic approach for chronic heart failure (HF). Moreover, calcium cycling is an important part of cardiac excitation-contraction coupling (ECC), which also participates in the antiarrhythmic effects of VNS. We hypothesized that low-level VNS (LL-VNS) could improve cardiac function by regulation of intracellular calcium handling properties. The experimental HF model was established by ligation of the left anterior descending coronary artery (LAD). Thirty-two male Sprague-Dawley rats were divided into 3 groups as follows; control group (sham operated without coronary ligation, n = 10), HF-VNS group (HF rats with VNS, n = 12), and HF-SS group (HF rats with sham nerve stimulation, n = 10). After 8 weeks of treatment, LL-VNS significantly improved left ventricular ejection fraction (LVEF) and attenuated myocardial interstitial fibrosis in the HF-VNS group compared with the HF-SS group. Elevated plasma norepinephrine and dopamine, but not epinephrine, were partially reduced by LL-VNS. Additionally, LL-VNS restored the protein and mRNA levels of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a), Na(+)-Ca(2+) exchanger 1 (NCX1), and phospholamban (PLB) whereas the expression of ryanodine receptor 2 (RyR2) as well as mRNA level was unaffected. Thus, our study results suggest that the improvement of cardiac performance by LL-VNS is accompanied by the reversal of dysfunctional calcium handling properties including SERCA2a, NCX1, and PLB which may be a potential molecular mechanism of VNS for HF.

  20. Disturbances of the sarcoplasmic reticulum and transverse tubular system in 24-h electrostimulated fast-twitch skeletal muscle

    DEFF Research Database (Denmark)

    Frías, J A; Cadefau, J A; Prats, C;

    2005-01-01

    -migration of terminal cisternae and t-tubules from R3 to R4, indicating the presence of triads. This density change may be associated with calcium overload of the sarcoplasmic reticulum, since total calcium rose three- to fourfold in stimulated muscle homogenates. These changes correlate well with ultrastructural...... damage to longitudinal sarcoplasmic reticulum and swelling of t-tubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may......Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigue-resistant fibres of rabbit...

  1. Impaired relaxation despite upregulated calcium-handling protein atrial myocardium from type 2 diabetic patients with preserved ejection fraction.

    Science.gov (United States)

    Lamberts, Regis R; Lingam, Shivanjali J; Wang, Heng-Yu; Bollen, Ilse A E; Hughes, Gillian; Galvin, Ivor F; Bunton, Richard W; Bahn, Andrew; Katare, Rajesh; Baldi, J Chris; Williams, Michael J A; Saxena, Pankaj; Coffey, Sean; Jones, Peter P

    2014-04-05

    Diastolic dysfunction is a key factor in the development and pathology of cardiac dysfunction in diabetes, however the exact underlying mechanism remains unknown, especially in humans. We aimed to measure contraction, relaxation, expression of calcium-handling proteins and fibrosis in myocardium of diabetic patients with preserved systolic function. Right atrial appendages from patients with type 2 diabetes mellitus (DM, n = 20) and non-diabetic patients (non-DM, n = 36), all with preserved ejection fraction and undergoing coronary artery bypass grafting (CABG), were collected. From appendages, small cardiac muscles, trabeculae, were isolated to measure basal and β-adrenergic stimulated myocardial function. Expression levels of calcium-handling proteins, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB), and of β1-adrenoreceptors were determined in tissue samples by Western blot. Collagen deposition was determined by picro-sirius red staining. In trabeculae from diabetic samples, contractile function was preserved, but relaxation was prolonged (Tau: 74 ± 13 ms vs. 93 ± 16 ms, non-DM vs. DM, p = 0.03). The expression of SERCA2a was increased in diabetic myocardial tissue (0.75 ± 0.09 vs. 1.23 ± 0.15, non-DM vs. DM, p = 0.007), whereas its endogenous inhibitor PLB was reduced (2.21 ± 0.45 vs. 0.42 ± 0.11, non-DM vs. DM, p = 0.01). Collagen deposition was increased in diabetic samples. Moreover, trabeculae from diabetic patients were unresponsive to β-adrenergic stimulation, despite no change in β1-adrenoreceptor expression levels. Human type 2 diabetic atrial myocardium showed increased fibrosis without systolic dysfunction but with impaired relaxation, especially during β-adrenergic challenge. Interestingly, changes in calcium-handling protein expression suggests accelerated active calcium re-uptake, thus improved relaxation, indicating a compensatory calcium-handling mechanism in diabetes

  2. Glycoprotein folding in the endoplasmic reticulum

    NARCIS (Netherlands)

    Braakman, L.J.; Benham, A.M.

    2000-01-01

    Our understanding of eukaryotic protein folding in the endoplasmic reticulum has increased enormously over the last 5 years. In this review, we summarize some of the major research themes that have captivated researchers in this field during the last years of the 20th century. We follow the path of

  3. Protein transport into the human endoplasmic reticulum

    NARCIS (Netherlands)

    Dudek, Johanna; Pfeffer, Stefan; Lee, Po-Hsien; Jung, Martin; Cavalié, Adolfo; Helms, Volkhard; Förster, Friedrich; Zimmermann, Richard

    2015-01-01

    Protein transport into the endoplasmic reticulum (ER) is essential for all eukaryotic cells and evolutionary related to protein transport into and across the cytoplasmic membrane of eubacteria and archaea. It is based on amino-terminal signal peptides in the precursor polypeptides plus various trans

  4. BAP, a mammalian BiP-associated protein, is a nucleotide exchange factor that regulates the ATPase activity of BiP.

    Science.gov (United States)

    Chung, Kyung Tae; Shen, Ying; Hendershot, Linda M

    2002-12-01

    We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an approximately 54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.

  5. Cylindrical Spirals in Skeletal Muscles Originate From the Longitudinal Sarcoplasmic Reticulum.

    Science.gov (United States)

    Xu, Jing-Wen; Liu, Fu-Chen; Li, Wei; Zhao, Yu-Ying; Zhao, Dan-Dan; Luo, Yue-Bei; Lu, Jian-Qiang; Yan, Chuan-Zhu

    2016-02-01

    Cylindrical spirals (CSs) are rare but distinct subsarcolemmal accumulations in skeletal muscle fibers. To date, CSs have been reported in only 16 patients with a variety of neuromuscular conditions. The origin and composition of CSs are unknown, although there are some morphologic similarities between CSs and tubular aggregates (TAs). To clarify the nature of CSs, we characterized the sarcoplasmic reticulum (SR) and other intracellular membrane system proteins in CSs of muscle biopsies from 2 adult Chinese siblings. Immunohistochemical studies revealed subsarcolemmal immunoreactivity for sarco/endoplasmic reticulum Ca2þ-ATPase 1 (SERCA 1) in the longitudinal SR, but no immunoreactivity for calsequestrin in the terminal cisternae or type 1 ryanodine receptor (RYR1) in the junctional SR. Muscles biopsied from 2 patients with TAs showed immunoreactivity not only for SERCA1 but also for other SR proteins, including calsequestrin and RYR1. CSs exhibited no immunoreactivity for the Golgi apparatus marker GM130, the nuclear membrane emerin, desmin, the autophagosome marker LC3, the lysosomal membrane marker LAMP2, dystrophin, or myosin. Our results suggest CSs may originate only from the longitudinal SR, whereas TAs are composed of both the junctional and longitudinal SR. Immunochemical staining with antibodies against calsequestrin and RYR1 help to distinguish these 2 pathological alterations.

  6. Effect of bafilomycin and NAADP on membrane-associated ATPases and respiration of isolated mitochondria of the murine Nemeth-Kellner lymphoma.

    Science.gov (United States)

    Hreniukh, V; Bychkova, S; Kulachkovsky, O; Babsky, A

    2016-12-01

    The goal of the study was to estimate the effect of a selective V-type H(+) -ATPase inhibitor bafilomycin A1 and nicotinic acid adenine dinucleotide phosphate (NAADP) on energetic processes in NK/Ly cell by directly measuring the respiration of isolated mitochondria and ATPase activities. NAADP (7 μM) increased the activity of Na(+) /K(+) -ATPase in the postmitochondrial fraction of NK/Ly cells, but lower concentration of NAADP decreased it (0.1 and 1 μM). The increase the activity of plasma membrane Ca(2)(+) ATPase (PMCA) under NAADP application (1 and 7 μM) was observed. However, NAADP (1 μM) decreased activities of sarcoendoplasmic reticulum Ca(2)(+) -ATPase (SERCA) and basal Mg(2)(+) -ATPase. Bafilomycin A1 (1 μM) increased the activity of Na(+) /K(+) -ATPase and potentiated the effect of NAADP (1 μM) on this pump. At the same time, bafilomycin A1 (1 μM) completely prevented all effects of NAADP (1 μM) on activities of PMCA, SERCA, and basal Mg(2)(+) -ATPase, confirming that these effects are dependent on acidic stores. Bafilomycin A1 or NAADP decreased respiratory and oxidative phosphorylation rates in NK/Ly mitochondria when α-ketoglutarate was used as substrate in contrast to succinate. Thus, α-ketoglutarate oxidation is more sensitive to bafilomycin A1 and NAADP influences compared with succinate oxidation. However, bafilomycin A1 + NAADP and any of these compounds separately lead to full uncoupling of mitochondria after ADP addition irrespectively to substrate used. Bafilomycin A1 affects isolated tumor mitochondria more effectively in combination with NAADP. Bafilomycin and NAADP alter some membrane-associated ATPases and inhibit respiration in mitochondria of the Nemeth-Kellner lymphoma. Bafilomycin A1 potentiates the effect of NAADP by inhibiting the mitochondrial energetic process in lymphoma cells and activity of Na(+) /K(+) -ATPase. The obtained data show promising possibility to use bafilomycin A1 and NAADP as chemotherapeutic

  7. Ouabain, a steroid hormone that signals with slow calcium oscillations

    OpenAIRE

    2001-01-01

    The plant-derived steroid, digoxin, a specific inhibitor of Na,K-ATPase, has been used for centuries in the treatment of heart disease. Recent studies demonstrate the presence of a digoxin analog, ouabain, in mammalian tissue, but its biological role has not been elucidated. Here, we show in renal epithelial cells that ouabain, in doses causing only partial Na,K-ATPase inhibition, acts as a biological inducer of regular, low-frequency intracellular calcium ([Ca2+]i) oscillations that elicit a...

  8. Characterization and partial purification of a proton translocating ATPase from corn coleoptile tonoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Mandala, S.M.

    1985-01-01

    ATP-dependent proton translocating activity in microsomal membranes from corn coleoptiles was characterized. Proton pumping activity, detected by either /sup 14/C-methylamine uptake or quinacrine fluorescence quenching, had a broad optimum at pH 7.5, and was substrate specific for MgATP. N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and protonophores were found to inhibit proton transport, while vanadate and oligomycin had little effect. Proton pumping activity was stimulated 10 fold with Cl/sup -/ but was almost completely inhibited by 50 mM, KNO/sub 3/. Uptake studies with /sup 36/Cl/sup -/ indicated the Cl/sup -/ was transported into the microsomal vesicles in response to the pH gradient. ATP-stimulated proton pumping activity was localized on linear density gradients. On sucrose gradients, the activity cosedimented with the marker for endoplasmic reticulum at 1.11 g/cc. Sucrose gradients prepared in the presence of MgCl/sub 2/ were used to shift the ER marker to a heavier density, away from proton pumping activity. Linear dextran gradients also resulted in a clear separation of ATP-stimulated methylamine, thiocyanate, and /sup 36/Cl/sup -/ uptake, from markers for ER, Golgi, mitochondria, and plasma membranes. The tonoplast ATPase was solubilized with octylglucoside and partially purified on linear sucrose gradients. The specific activity of the KNO/sub 3/-sensitive ATPase increased 30-fold during purification.

  9. The p97 ATPase associates with EEA1 to regulate the size of early endosomes

    Institute of Scientific and Technical Information of China (English)

    Harish N Ramanathan; Yihong Ye

    2012-01-01

    The AAA ((A)TPase-(a)ssociated with various cellular (a)ctivities) ATPase p97 acts on diverse substrate proteins to partake in various cellular processes such as membrane fusion and endoplasmic reticulum-associated degradation (ERAD).In membrane fusion,p97 is thought to function in analogy to the related ATPase NSF (N-ethylmaleimidesensitive fusion protein),which promotes membrane fusion by disassembling a SNARE complex.In ERAD,p97 dislocates misfolded proteins from the ER membrane to facilitate their turnover by the proteasome.Here,we identify a novel function of p97 in endocytic trafficking by establishing the early endosomal autoantigen 1 (EEA1) as a new p97 substrate.We demonstrate that a fraction of p97 is localized to the early endosome membrane,where it binds EEA1 via the N-terminal C2H2 zinc finger domain.Inhibition of p97 either by siRNA or a pharmacological inhibitor results in clustering and enlargement of early endosomes,which is associated with an altered trafficking pattern for an endocytic cargo.Mechanistically,we show that p97 inhibition causes increased EEA1 self-association at the endosome membrane.We propose that p97 may regulate the size of early endosomes by governing the oligomeric state of EEA1.

  10. The impact of ovariectomy on calcium homeostasis and myofilament calcium sensitivity in the aging mouse heart.

    Directory of Open Access Journals (Sweden)

    Elias Fares

    Full Text Available This study determined whether deficiency of ovarian estrogen starting very early in life promoted age-associated Ca(2+ dysregulation and contractile dysfunction in isolated ventricular myocytes. Myocytes were isolated from anesthetized C57BL/6 female mice. Animals received an ovariectomy or sham-operation at one month and were aged to ~24 months. Excitation-contraction coupling parameters were compared in fura-2 loaded myocytes (37°C. While Ca(2+ transients were larger and faster in field-stimulated myocytes from ovariectomized mice, ovariectomy had no effect on peak fractional shortening. Similarly, ovariectomy had no effect on fractional shortening measured in vivo by echocardiography (values were 60.5 ± 2.9 vs. 60.3 ± 2.5% in sham and ovariectomized, respectively; n=5 mice/group. Ovariectomy did decrease myofilament Ca(2+ sensitivity, as evidenced by a 26% increase in the Ca(2+ required to activate actomyosin MgATPase in ovariectomized hearts. Larger Ca(2+ transients were attributable to a 48% increase in peak Ca(2+ current, along with an increase in the amplitude, width and frequency of Ca(2+ sparks measured in fluo-4 loaded myocytes. These changes in Ca(2+ handling were not due to increased expression of Ca(2+ channels (Cav1.2, sarcoplasmic reticulum Ca(2+ ATPase (SERCA2 or Na(+-Ca(2+ exchanger in ovariectomized hearts. However, ovariectomy increased sarcoplasmic reticulum Ca(2+ stores by ~90% and promoted spontaneous Ca(2+ release from the sarcoplasmic reticulum when compared to sham controls. These observations demonstrate that long-term ovariectomy promotes intracellular Ca(2+ dysregulation, reduces myofilament Ca(2+ sensitivity and increases spontaneous Ca(2+ release in the aging female heart.

  11. Involvement of inositol 1,4,5-trisphosphate in nicotinic calcium responses in dystrophic myotubes assessed by near-plasma membrane calcium measurement.

    Science.gov (United States)

    Basset, Olivier; Boittin, François-Xavier; Dorchies, Olivier M; Chatton, Jean-Yves; van Breemen, Cornelis; Ruegg, Urs T

    2004-11-05

    In skeletal muscle cells, plasma membrane depolarization causes a rapid calcium release from the sarcoplasmic reticulum through ryanodine receptors triggering contraction. In Duchenne muscular dystrophy (DMD), a lethal disease that is caused by the lack of the cytoskeletal protein dystrophin, the cytosolic calcium concentration is known to be increased, and this increase may lead to cell necrosis. Here, we used myotubes derived from control and mdx mice, the murine model of DMD, to study the calcium responses induced by nicotinic acetylcholine receptor stimulation. The photoprotein aequorin was expressed in the cytosol or targeted to the plasma membrane as a fusion protein with the synaptosome-associated protein SNAP-25, thus allowing calcium measurements in a restricted area localized just below the plasma membrane. The carbachol-induced calcium responses were 4.5 times bigger in dystrophic myotubes than in control myotubes. Moreover, in dystrophic myotubes the carbachol-mediated calcium responses measured in the subsarcolemmal area were at least 10 times bigger than in the bulk cytosol. The initial calcium responses were due to calcium influx into the cells followed by a fast refilling/release phase from the sarcoplasmic reticulum. In addition and unexpectedly, the inositol 1,4,5-trisphosphate receptor pathway was involved in these calcium signals only in the dystrophic myotubes. This surprising involvement of this calcium release channel in the excitation-contraction coupling could open new ways for understanding exercise-induced calcium increases and downstream muscle degeneration in mdx mice and, therefore, in DMD.

  12. Obstacle Effects on One-Dimensional Translocation of ATPase

    Institute of Scientific and Technical Information of China (English)

    WANG Xian-Ju; AI Bao-Quan; LIU Liang-Gang

    2002-01-01

    We apply a general random walk model to the study of the ATPase's one-dimensional translocation along obstacle biological environment, and show the effects of random obstacles on the ATPase translocation along single stranded DNA. We find that the obstacle environment can reduce the lifetime of ATPase lattice-bound state which results in the inhibition of ATPase activity. We also carry out the ranges of rate constant of ATPase unidirectonal translocation and bidirectional translocation. Our results are consistent with the experiments and relevant theoretical consideration, and can be used to explain some physiological phenomena.

  13. Disturbed calcium signaling in spinocerebellar ataxias and Alzheimer's disease.

    Science.gov (United States)

    Egorova, Polina; Popugaeva, Elena; Bezprozvanny, Ilya

    2015-04-01

    Neurodegenerative disorders, such as spinocerebellar ataxias (SCAs) and Alzheimer's disease (AD) represent a huge scientific and medical question, but the molecular mechanisms of these diseases are still not clear. There is increasing evidence that neuronal calcium signaling is abnormal in many neurodegenerative disorders. Abnormal neuronal calcium release from the endoplasmic reticulum may result in disturbances of cell homeostasis, synaptic dysfunction, and eventual cell death. Neuronal loss is observed in most cases of neurodegenerative diseases. Recent experimental evidence supporting the role of neuronal calcium signaling in the pathogenesis of SCAs and AD is discussed in this review.

  14. Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity.

    Science.gov (United States)

    Raychaudhuri, Sumana; Espenshade, Peter J

    2015-06-01

    Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1-Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

  15. Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes.

    Science.gov (United States)

    Inesi, G; Lewis, D; Sumbilla, C; Nandi, A; Strock, C; Huff, K W; Rogers, T B; Johns, D C; Kessler, P D; Ordahl, C P

    1998-03-01

    Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.

  16. Diversity of proton pumps in osteoclasts: V-ATPase with a3 and d2 isoforms is a major form in osteoclasts.

    Science.gov (United States)

    Matsumoto, Naomi; Daido, Shun; Sun-Wada, Ge-Hong; Wada, Yoh; Futai, Masamitsu; Nakanishi-Matsui, Mayumi

    2014-06-01

    Osteoclasts acidify bone resorption lacunae through proton translocation by plasma membrane V-ATPase (vacuolar-type ATPase) which has an a3 isoform, one of the four isoforms of the trans-membrane a subunit (Toyomura et al., J. Biol. Chem., 278, 22023-22030, 2003). d2, a kidney- and epididymis-specific isoform of the d subunit, was also induced in osteoclast-like cells derived from the RAW264.7 line, and formed V-ATPase with a3. The amount of d2 in osteoclasts was 4-fold higher than that of d1, a ubiquitous isoform. These results indicate that V-ATPase with d2/a3 is a major osteoclast proton pump. Essentially the same results were obtained with osteoclasts derived from mouse spleen macrophages. Macrophages from a3-knock-out mice could differentiate into multi-nuclear cells with osteoclast-specific enzymes. In these cells, the d2 isoform was also induced and assembled in V-ATPase with the a1 or a2 isoform. However, they did not absorb calcium phosphate, indicating that V-ATPase with d2/a1 or d2/a2 could not perform the function of that with d2/a3.

  17. An optimized micro-assay of myosin Ⅱ ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors

    Institute of Scientific and Technical Information of China (English)

    CHEN Hong-Lin; ZHAO Jing; ZHANG Guan-Jun; KOU Jun-Ping; YU Bo-Yang

    2016-01-01

    Myosin Ⅱ plays multiple roles in physiological and pathological functions through its ATPase activity.The present study was designed to optimize a micro-assay of myosin Ⅱ ATPase activity based on molybdenum blue method,using a known myosin Ⅱ ATPase inhibitor,blebbistatin.Several parameters were observed in the enzymatic reaction procedure,including the concentrations of the substrate (ATP) and calcium chloride,pH,and the reaction and incubation times.The proportion of coloration agent was also investigated.The sensitivity of this assay was compared with the malachite green method and bioluminescence method.Additionally,20 natural compounds were studied for myosin Ⅱ ATPase inhibitory activity using the optimized method.Our results showed that ATP at the concentration of 5 mmol·L-1 and ammonium molybdate:stannous chloride at the ratio of 15 ∶ 1 could greatly improve the sensitivity of this method.The IC50 of blebbistatin obtained by this method was consistent with literature.Compound 8 was screened with inhibitory activity on myosin Ⅱ ATPase.The optimized method showed similar accuracy,lower detecting limit,and wider linear range,which could be a promising approach to screening myosin Ⅱ ATPase inhibitors in vitro.

  18. Novel Sulfur Metabolites of Garlic Attenuate Cardiac Hypertrophy and Remodeling through Induction of Na+/K+-ATPase Expression

    Science.gov (United States)

    Khatua, Tarak N.; Borkar, Roshan M.; Mohammed, Soheb A.; Dinda, Amit K.; Srinivas, R.; Banerjee, Sanjay K.

    2017-01-01

    Epidemiologic studies show an inverse correlation between garlic consumption and progression of cardiovascular disease. However, the molecular basis for the beneficial effect of garlic on the heart is not known. Therefore, the objective of the present study was to (1) investigate the effect of raw garlic on isoproterenol (Iso) induced cardiac hypertrophy (2) find the active metabolites of garlic responsible for the beneficial effect. Cardiac hypertrophy was induced in rats by subcutaneous single injection of Iso 5 mg kg-1 day-1 for 15 days and the effect of garlic (250 mg/kg/day orally) was evaluated. Garlic metabolites in in vivo were identified by LC/MS study. The effect of garlic and its metabolites were evaluated against hypertrophy in H9C2 cells. Garlic normalized cardiac oxidative stress after Iso administration. Cardiac pathology and mitochondrial enzyme activities were improved in hypertrophy heart after garlic administration. Decreased Na+/K+-ATPase protein level that observed in hypertrophy heart was increased after garlic administration. We identified three garlic metabolites in rat serum. To confirm the role of garlic metabolites on cardiac hypertrophy, Na+/K+-ATPase expression and intracellular calcium levels were measured after treating H9C2 cells with raw garlic and two of its active metabolites, allyl methyl sulfide and allyl methyl sulfoxide. Raw garlic and both metabolites increased Na+/K+-ATPase protein level and decreased intracellular calcium levels and cell size in Iso treated H9C2 cells. This antihypertrophic effect of garlic and its sulfur metabolites were lost in H9C2 cells in presence of Na+/K+-ATPase inhibitor. In conclusion, garlic and its active metabolites increased Na+/K+-ATPase in rat heart, and attenuated cardiac hypertrophy and associated remodeling. Our data suggest that identified new garlic metabolites may be useful for therapeutic intervention against cardiac hypertrophy. PMID:28194108

  19. Calcium in diet

    Science.gov (United States)

    ... D is needed to help your body use calcium. Milk is fortified with vitamin D for this reason. ... of calcium dietary supplements include calcium citrate and calcium carbonate. Calcium citrate is the more expensive form of ...

  20. Endoplasmic reticulum stress is chronically activated in chronic pancreatitis.

    Science.gov (United States)

    Sah, Raghuwansh P; Garg, Sushil K; Dixit, Ajay K; Dudeja, Vikas; Dawra, Rajinder K; Saluja, Ashok K

    2014-10-03

    The pathogenesis of chronic pancreatitis (CP) is poorly understood. Endoplasmic reticulum (ER) stress has now been recognized as a pathogenic event in many chronic diseases. However, ER stress has not been studied in CP, although pancreatic acinar cells seem to be especially vulnerable to ER dysfunction because of their dependence on high ER volume and functionality. Here, we aim to investigate ER stress in CP, study its pathogenesis in relation to trypsinogen activation (widely regarded as the key event of pancreatitis), and explore its mechanism, time course, and downstream consequences during pancreatic injury. CP was induced in mice by repeated episodes of acute pancreatitis (AP) based on caerulein hyperstimulation. ER stress leads to activation of unfolded protein response components that were measured in CP and AP. We show sustained up-regulation of unfolded protein response components ATF4, CHOP, GRP78, and XBP1 in CP. Overexpression of GRP78 and ATF4 in human CP confirmed the experimental findings. We used novel trypsinogen-7 knock-out mice (T(-/-)), which lack intra-acinar trypsinogen activation, to clarify the relationship of ER stress to intra-acinar trypsinogen activation in pancreatic injury. Comparable activation of ER stress was seen in wild type and T(-/-) mice. Induction of ER stress occurred through pathologic calcium signaling very early in the course of pancreatic injury. Our results establish that ER stress is chronically activated in CP and is induced early in pancreatic injury through pathologic calcium signaling independent of trypsinogen activation. ER stress may be an important pathogenic mechanism in pancreatitis that needs to be explored in future studies.

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

    Science.gov (United States)

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

    2014-09-01

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

  2. Calcium regulation of HCN channels supports persistent activity in a multiscale model of neocortex.

    Science.gov (United States)

    Neymotin, S A; McDougal, R A; Bulanova, A S; Zeki, M; Lakatos, P; Terman, D; Hines, M L; Lytton, W W

    2016-03-01

    Neuronal persistent activity has been primarily assessed in terms of electrical mechanisms, without attention to the complex array of molecular events that also control cell excitability. We developed a multiscale neocortical model proceeding from the molecular to the network level to assess the contributions of calcium (Ca(2+)) regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in providing additional and complementary support of continuing activation in the network. The network contained 776 compartmental neurons arranged in the cortical layers, connected using synapses containing AMPA/NMDA/GABAA/GABAB receptors. Metabotropic glutamate receptors (mGluR) produced inositol triphosphate (IP3) which caused the release of Ca(2+) from endoplasmic reticulum (ER) stores, with reuptake by sarco/ER Ca(2+)-ATP-ase pumps (SERCA), and influence on HCN channels. Stimulus-induced depolarization led to Ca(2+) influx via NMDA and voltage-gated Ca(2+) channels (VGCCs). After a delay, mGluR activation led to ER Ca(2+) release via IP3 receptors. These factors increased HCN channel conductance and produced firing lasting for ∼1min. The model displayed inter-scale synergies among synaptic weights, excitation/inhibition balance, firing rates, membrane depolarization, Ca(2+) levels, regulation of HCN channels, and induction of persistent activity. The interaction between inhibition and Ca(2+) at the HCN channel nexus determined a limited range of inhibition strengths for which intracellular Ca(2+) could prepare population-specific persistent activity. Interactions between metabotropic and ionotropic inputs to the neuron demonstrated how multiple pathways could contribute in a complementary manner to persistent activity. Such redundancy and complementarity via multiple pathways is a critical feature of biological systems. Mediation of activation at different time scales, and through different pathways, would be expected to protect against disruption, in

  3. Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells

    Science.gov (United States)

    Yoon, Mi Na; Kim, Dong Kwan; Kim, Se Hoon

    2017-01-01

    Intracellular calcium (Ca2+) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H2O2) on intracellular Ca2+ accumulation in mouse pancreatic acinar cells. Perfusion of H2O2 at 300 µM resulted in additional elevation of intracellular Ca2+ levels and termination of oscillatory Ca2+ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca2+. Antioxidants, catalase or DTT, completely prevented H2O2-induced additional Ca2+ increase and termination of Ca2+ oscillation. In Ca2+-free medium, H2O2 still enhanced CCh-induced intracellular Ca2+ levels and thapsigargin (TG) mimicked H2O2-induced cytosolic Ca2+ increase. Furthermore, H2O2-induced elevation of intracellular Ca2+ levels was abolished under sarco/endoplasmic reticulum Ca2+ ATPase-inactivated condition by TG pretreatment with CCh. H2O2 at 300 µM failed to affect store-operated Ca2+ entry or Ca2+ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca2+ uniporter blocker, failed to attenuate H2O2-induced intracellular Ca2+ elevation. These results provide evidence that excessive generation of H2O2 in pathological conditions could accumulate intracellular Ca2+ by attenuating refilling of internal Ca2+ stores rather than by inhibiting Ca2+ extrusion to extracellular fluid or enhancing Ca2+ mobilization from extracellular medium in mouse pancreatic acinar cells.

  4. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Bækgaard, Lone

    2010-01-01

    -bound calmodulin (Ca(2+)-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space......Plasma-membrane Ca(2+)-ATPases (PMCAs) are calcium pumps that expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. They are of major physiological importance, with different isoforms being essential, for example...

  5. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Palmgren, Michael Broberg

    2010-01-01

    -bound calmodulin (Ca2+-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space......Plasma-membrane Ca2+-ATPases (PMCAs) are calcium pumps that expel Ca2+ from eukaryotic cells to maintain overall Ca2+ homoeostasis and to provide local control of intracellular Ca2+ signalling. They are of major physiological importance, with different isoforms being essential, for example...

  6. Calcium supplements

    Science.gov (United States)

    ... Related Bone Diseases National Resource Center. Calcium and vitamin D: Important at every age. NIAMS.NIH.gov website. www.niams.nih.gov/Health_Info/Bone/Bone_Health/Nutrition . Updated May 2015. Accessed March ...

  7. Calcium Electroporation

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; Gibot, Laure; Madi, Moinecha;

    2015-01-01

    BACKGROUND: Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill...... efficacy-and normal cell sensitivity. METHODS: Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29), a bladder transitional cell carcinoma (SW780......), and a breast adenocarcinoma (MDA-MB231), as well as on primary normal human dermal fibroblasts (HDF-n). RESULTS: The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p

  8. Stress responses from the endoplasmic reticulum in cancer

    Directory of Open Access Journals (Sweden)

    Hironori eKato

    2015-04-01

    Full Text Available The endoplasmic reticulum (ER is a dynamic organelle that is essential for multiple cellular functions. During cellular stress conditions, including nutrient deprivation and dysregulation of protein synthesis, unfolded/misfolded proteins accumulate in the ER lumen, resulting in activation of the unfolded protein response (UPR. The UPR also contributes to the regulation of various intracellular signalling pathways such as calcium signalling and lipid signalling. More recently, the mitochondria-associated ER membrane (MAM, which is a site of close contact between the ER and mitochondria, has been shown to function as a platform for various intracellular stress responses including apoptotic signalling, inflammatory signalling, the autophagic response, and the UPR. Interestingly, in cancer, these signalling pathways from the ER are often dysregulated, contributing to cancer cell metabolism. Thus, the signalling pathway from the ER may be a novel therapeutic target for various cancers. In this review, we discuss recent research on the roles of stress responses from the ER, including the MAM.

  9. The Endoplasmic Reticulum: A Social Network in Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Jun Chen; Caitlin Doyle; Xingyun Qi; Huanquan Zheng

    2012-01-01

    The endoplasmic reticulum (ER) is an interconnected network comprised of ribosome-studded sheets and smooth tubules.The ER plays crucial roles in the biosynthesis and transport of proteins and lipids,and in calcium (Ca2+) regulation in compartmentalized eukaryotic cells including plant cells.To support its well-segregated functions,the shape of the ER undergoes notable changes in response to both developmental cues and outside influences.In this review,we will discuss recent findings on molecular mechanisms underlying the unique morphology and dynamics of the ER,and the importance of the interconnected ER network in cell polarity.In animal and yeast cells,two family proteins,the reticulons and DP1/Yop1,are required for shaping high-curvature ER tubules,while members of the atlastin family of dynamin-like GTPases are involved in the fusion of ER tubules to make an interconnected ER network.In plant cells,recent data also indicate that the reticulons are involved in shaping ER tubules,while RHD3,a plant member of the atlastin GTPases,is required for the generation of an interconnected ER network.We will also summarize the current knowledge on how the ER interacts with other membrane-bound organelles,with a focus on how the ER and Golgi interplay in plant cells.

  10. Endoplasmic reticulum-mitochondria junction is required for iron homeostasis.

    Science.gov (United States)

    Xue, Yong; Schmollinger, Stefan; Attar, Narsis; Campos, Oscar A; Vogelauer, Maria; Carey, Michael F; Merchant, Sabeeha S; Kurdistani, Siavash K

    2017-08-11

    The endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) is a protein complex that physically tethers the two organelles to each other and creates the physical basis for communication between them. ERMES functions in lipid exchange between the ER and mitochondria, protein import into mitochondria, and maintenance of mitochondrial morphology and genome. Here, we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of known ERMES roles in calcium regulation, phospholipid biosynthesis, or effects on mitochondrial morphology. A mutation in the vacuolar protein sorting 13 (VPS13) gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our findings reveal that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Overproduction of PIB-Type ATPases

    DEFF Research Database (Denmark)

    Liu, Xiangyu; Sitsel, Oleg; Wang, Kaituo

    2016-01-01

    Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction and purifi...... and purification strategy of a bacterial PIB-type ATPase for isolation of milligrams of target protein per liter Escherichia coli cell culture, with a final quality of the sample which is sufficient for generating high-resolution crystals....

  12. Overproduction of PIB-Type ATPases.

    Science.gov (United States)

    Liu, Xiangyu; Sitsel, Oleg; Wang, Kaituo; Gourdon, Pontus

    2016-01-01

    Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction and purification strategy of a bacterial PIB-type ATPase for isolation of milligrams of target protein per liter Escherichia coli cell culture, with a final quality of the sample which is sufficient for generating high-resolution crystals.

  13. Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.

    Directory of Open Access Journals (Sweden)

    Jintao Zhang

    Full Text Available Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells.Human colorectal cancer cell lines (HCT-116 and HT-29 were treated with sodium butyrate at concentrations ranging from 0.5-5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining, and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot.Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II, beclin-1, and autophagocytosis-associated protein (Atg3. The autophagy inhibitors 3-methyladenine (3-MA and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin and genetic

  14. Store-Operated Calcium Channels.

    Science.gov (United States)

    Prakriya, Murali; Lewis, Richard S

    2015-10-01

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

  15. Role of platelet plasma membrane Ca2+-ATPase in health and disease

    Institute of Scientific and Technical Information of China (English)

    William; L; Dean

    2010-01-01

    Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.On the other hand,hyperactive platelets lead to heart attack and stroke.Calcium is a major second messenger in platelet activation,and elevated intracellular calcium leads to hyperactive platelets.Elevated platelet calcium has been documented in hypertension and diabetes;both conditions increase the likelihood of heart attack and stroke. Thus,proper regulation of calcium metabolism in the platelet is extremely important.Plasma membrane Ca2+-ATPase(PMCA)is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux.In keeping with the important role of calcium in platelet function,PMCA is a highly regulated transporter.In human platelets,PMCA is activated by Ca2+/calmodulin,by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide.It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis.In addition,the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation.Rapid regulation by phosphorylation results in changes in the rate of platelet activation,whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction.In hypertension and diabetes,PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation.Clearly,a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.

  16. Vacuolar-ATPase (V-ATPase) Mediates Progesterone-Induced Uterine Fluid Acidification in Rats.

    Science.gov (United States)

    Karim, Kamarulzaman; Giribabu, Nelli; Muniandy, Sekaran; Salleh, Naguib

    2016-04-01

    We hypothesized that progesterone-induced decrease in uterine fluid pH involves V-ATPase. In this study, expression and functional activity of V-ATPase in uterus were investigated under progesterone influence. Ovariectomized adult female rats received subcutaneous injection of estradiol-17β (1 µg/kg/day) or progesterone (20 mg/kg/day) for 3 days or 3 days estradiol-17β followed by 3 days vehicle, progesterone, or estradiol-17β plus progesterone. Mifepristone, a progesterone receptor blocker, was concomitantly given to the rats which received progesterone. A day after last injection, rate of uterine fluid secretion, its HCO3 (-) concentration, and pH were determined via in vivo uterine perfusion in rats under anesthesia. V-ATPase inhibitor, bafilomycin, was introduced into the perfusion buffer, and changes in these parameters were observed. Expression of V-ATPase A1 and B1/2 proteins and mRNAs in uterus were quantified by Western blotting and real-time PCR, respectively. Distribution of these proteins was observed by immunohistochemistry. Our findings showed that under progesterone influence, uterine fluid secretion rate, HCO3 (-) concentration, and pH were significantly reduced. Administration of bafilomycin did not cause significant changes in fluid secretion rate; however, HCO3 (-) concentration and pH were significantly elevated. In parallel with these changes, expression of V-ATPase A1 and B1/2 proteins and mRNAs were significantly increased with these proteins highly distributed in uterine luminal and glandular epithelia. In conclusion, increased expression and functional activity of V-ATPase were most likely responsible for the decreased in uterine fluid pH observed under progesterone influence.

  17. Dependency of calcium alternans on ryanodine receptor refractoriness.

    Directory of Open Access Journals (Sweden)

    Enric Alvarez-Lacalle

    Full Text Available BACKGROUND: Rapid pacing rates induce alternations in the cytosolic calcium concentration caused by fluctuations in calcium released from the sarcoplasmic reticulum (SR. However, the relationship between calcium alternans and refractoriness of the SR calcium release channel (RyR2 remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: To investigate how ryanodine receptor (RyR2 refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We used a mathematical rabbit cardiomyocyte model to study the beat-to-beat calcium response as a function of RyR2 activation and inactivation. Bi-dimensional maps were constructed depicting the beat-to-beat response. When alternans was observed, a novel numerical clamping protocol was used to determine whether alternans was caused by oscillations in SR calcium loading or by RyR2 refractoriness. Using this protocol, we identified regions of RyR2 gating parameters where SR calcium loading or RyR2 refractoriness underlie the induction of calcium alternans, and we found that at the onset of alternans both mechanisms contribute. At low inactivation rates of the RyR2, calcium alternans was caused by alternation in SR calcium loading, while at low activation rates it was caused by alternation in the level of available RyR2s. CONCLUSIONS/SIGNIFICANCE: We have mapped cardiomyocyte beat-to-beat responses as a function of RyR2 activation and inactivation, identifying domains where SR calcium load or RyR2 refractoriness underlie the induction of calcium alternans. A corollary of this work is that RyR2 refractoriness due to slow recovery from inactivation can be the cause of calcium alternans even when alternation in SR calcium load is present.

  18. Analysis of F1F0-ATPase from Helicobacter pylori.

    OpenAIRE

    1997-01-01

    The adaptive mechanisms that permit Helicobacter species to survive within the gastric mucosa are not well understood. The proton-translocating F1F0-ATPase is an important enzyme for regulating intracellular pH or synthesizing ATP in many other enteric bacteria; therefore, we used degenerate primers derived from conserved bacterial F1F0-ATPase sequences to PCR amplify and clone the gene (atpD) encoding the H. pylori F1F0-ATPase beta subunit. The deduced amino acid sequences of the F1F0-ATPase...

  19. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

  20. Variation in expression of calcium-handling proteins is associated with inter-individual differences in mechanical performance of rat (Rattus norvegicus) skeletal muscle.

    Science.gov (United States)

    James, Rob S; Walter, Isabel; Seebacher, Frank

    2011-11-01

    An important constraint on locomotor performance is the trade-off between sprint and endurance performance. One intuitive explanation for this trade-off is that an individual muscle cannot excel at generating both maximal force/power and high fatigue resistance. The underlying reasons for this muscle trade-off are poorly defined. The aim of this study was to test the hypothesis that inter-individual variation in muscle mechanics is associated with inter-individual differences in metabolic capacities and expression of calcium-handling proteins. Lateral gastrocnemius muscles were isolated from 20 rats (Rattus norvegicus) and analysed to determine metabolic capacity, sarco/endoplasmic reticulum calcium ATPase (SERCA)1 protein concentration, total SERCA activity, and mRNA concentrations of SERCA1, SERCA2, troponin I and ryanodine receptors. Isometric studies of lateral gastrocnemius muscles at 30°C showed that muscles with higher sprint performance had lower fatigue resistance. More rapid muscle contraction was correlated with higher lactate dehydrogenase activity and increased expression of ryanodine receptor 1. More rapid muscle relaxation was correlated with increased expression of troponin I type 2 (fast) isoform and decreased expression of SERCA2 (slow) isoform. Treating muscles with dantrolene confirmed that ryanodine receptor activity is important in determining tetanus force and muscle contraction rates, but has no effect on fatigue resistance. Thapsigargin treatment revealed that SERCA activity determines fatigue resistance but does not affect maximal muscle force or contraction rates. We conclude that the opposing roles of SERCA activity and expression of ryanodine receptors in determining fatigue resistance and force production, respectively, at least partly explain differences in sprint and endurance performance in isolated rat gastrocnemius muscle.

  1. 肝细胞膜钠泵活性变化在胆红素钙结石成石过程中的作用%Effects of Activities of Na+-K+-ATPase in Plasma Membranes of Hepatocytes in the Formation of Calcium Bilirubinate Gallstone

    Institute of Scientific and Technical Information of China (English)

    庄文; 肖路加; 左凤琼; 东云华; 吴忠舜; 吴兆锋

    2002-01-01

    目的 探讨肝细胞膜钠泵(Na+-K+-ATPase)活性变化在胆红素钙结石成石过程中的作用.方法采用胆红素钙结石兔模型进行研究,对照组28只,胆道不全梗阻组(BO组)36只,胆道不全梗阻加感染组(BOI组)39只,各组再按术后处死时间3 d、7 d、14 d及20 d时相分组.动态测定各组各时相肝细胞膜钠泵活性、肝细胞钙及胆汁中胆汁酸含量. 结果 BO组及BOI组肝细胞膜钠泵活性及胆汁中胆汁酸含量进行性下降,肝细胞钙含量呈进行性增多,与对照组相比差异有显著性意义(P<0.01),但BOI组上述变化较BO组明显(P<0.05).结论胆红素钙结石成石过程中存在肝细胞膜钠泵活性的进行性下降,钠泵活性下降引起肝细胞钙超负荷及胆汁中胆汁酸含量下降,从而促进成石.细菌感染加重上述变化,使结石形成增多.

  2. Association of immunoproteasomes with the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Brooks, P.; Murray, R.Z.; Mason, G.G.F.;

    2000-01-01

    pathway. Two of the gamma-interferon inducible subunits, LMP2 and LMP7, are encoded within the MHC class II region adjacent to the two TAP (transporter associated with antigen presentation) genes. We have investigated the localization of immunoproteasomes using monoclonal antibodies to LMP2 and LMP7...... that immunoproteasomes are strongly enriched at the endoplasmic reticulum, where they may be located close to the TAP transporter to provide efficient transport of peptides into the lumen of the endoplasmic recticulum for association with MHC class I molecules....

  3. Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

    Energy Technology Data Exchange (ETDEWEB)

    Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.

    1986-01-01

    The H translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ approx. = 70,000 and approx. = 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-( UC)ethylmaleimide and 7-chloro-4-nitro( UC)benzo-2-oxa-1,3-diazole, labeled the M/sub r/ approx. = 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-( UC)dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ approx. = 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10V, 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ approx. = 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F0F1 ATPases.

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

    Institute of Scientific and Technical Information of China (English)

    陈立华; 于嘉; 刘丽旭; 曹美鸿

    2002-01-01

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

  5. Calcium and bones

    Science.gov (United States)

    Bone strength and calcium ... calcium (as well as phosphorus) to make healthy bones. Bones are the main storage site of calcium in ... your body does not absorb enough calcium, your bones can get weak or will not grow properly. ...

  6. Phosphorylation of the Na+,K+-ATPase and the H+,K+-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Morth, Jens Preben; Jensen, Jan Egebjerg;

    2010-01-01

    Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na(+),K(+)- and H(+),K(+)-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two...... as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations....

  7. Calcium Carbonate

    Science.gov (United States)

    ... doctor if you have or have ever had kidney disease or stomach conditions.tell your doctor if you are pregnant, plan to become pregnant, or are breast-feeding. If you become pregnant while taking calcium carbonate, call your doctor.

  8. Calcium Test

    Science.gov (United States)

    ... if a person has symptoms of a parathyroid disorder , malabsorption , or an overactive thyroid. A total calcium level is often measured as part of a routine health screening. It is included in the comprehensive metabolic panel (CMP) and the basic metabolic panel (BMP) , ...

  9. Ouabain, a steroid hormone that signals with slow calcium oscillations.

    Science.gov (United States)

    Aizman, O; Uhlén, P; Lal, M; Brismar, H; Aperia, A

    2001-11-06

    The plant-derived steroid, digoxin, a specific inhibitor of Na,K-ATPase, has been used for centuries in the treatment of heart disease. Recent studies demonstrate the presence of a digoxin analog, ouabain, in mammalian tissue, but its biological role has not been elucidated. Here, we show in renal epithelial cells that ouabain, in doses causing only partial Na,K-ATPase inhibition, acts as a biological inducer of regular, low-frequency intracellular calcium ([Ca(2+)](i)) oscillations that elicit activation of the transcription factor, NF-kappa B. Partial inhibition of Na,K-ATPase using low extracellular K(+) and depolarization of cells did not have these effects. Incubation of cells in Ca(2+)-free media, inhibition of voltage-gated calcium channels, inositol triphosphate receptor antagonism, and redistribution of actin to a thick layer adjacent to the plasma membrane abolished [Ca(2+)](i) oscillations, indicating that they were caused by a concerted action of inositol triphosphate receptors and capacitative calcium entry via plasma membrane channels. Blockade of ouabain-induced [Ca(2+)](i) oscillations prevented activation of NF-kappa B. The results demonstrate a new mechanism for steroid signaling via plasma membrane receptors and underline a novel role for the steroid hormone, ouabain, as a physiological inducer of [Ca(2+)](i) oscillations involved in transcriptional regulation in mammalian cells.

  10. MAPK-mediated enhanced expression of vacuolar H(+)-ATPase confers the improved adaption to NaCl stress in a halotolerate peppermint (Mentha piperita L.).

    Science.gov (United States)

    Li, Zhe; Zhen, Zhen; Guo, Kai; Harvey, Paul; Li, Jishun; Yang, Hetong

    2016-03-01

    Vacuolar H(+)-ATPase (V-H(+)-ATPase) has been proved to be of importance in maintenance of ion homeostasis inside plant cells under NaCl stress. In this study, the expression levels and salt-tolerate function of V-H(+)-ATPase genes were investigated in the roots and leaves of a halotolerate peppermint (Mentha × piperita L.) Keyuan-1 treated with different concentrations of NaCl. Results showed that the expressions of V-H(+)-ATPase in the transcriptional, protein and activity levels were significantly enhanced in the halotolerate peppermint Keyuan-1 compared to the wild-type (WT) peppermint under 50, 100, and 150 mM NaCl treatment. Moreover, inhibition experiments exhibited that V-H(+)-ATPase activity played vital roles in the salt tolerance of peppermint Keyuan-1 to 150 mM NaCl stress through increasing the vacuolar H(+) pumping activity and Na(+) compartmentalization capacity. Furthermore, results of Western blots showed that the activity of a mitogen-activated protein kinase (MAPK) was significantly increased under different concentrations of NaCl in the halotolerate peppermint Keyuan-1, which was much higher than that of WT peppermint. Further experiments with inhibitors suggested that this MAPK protein was responsible for the enhanced expression of V-H(+)-ATPase in the halotolerate peppermint Keyuan-1. In response to NaCl stress, increase of cytoplasmic calcium concentration ([Ca(2+)]cyt) occurred upstream of MAPK activation in the halotolerate peppermint Keyuan-1. In all, these findings demonstrated that increased V-H(+)-ATPase activity was positively correlated with the enhanced salt tolerance in the halotolerate peppermint Keyuan-1, providing the theoretic basis for the further development and utilization of peppermint in saline areas.

  11. Endoplasmic reticulum stress and diabetic retinopathy

    Directory of Open Access Journals (Sweden)

    Toshiyuki Oshitari

    2008-02-01

    Full Text Available Toshiyuki Oshitari1,2, Natsuyo Hata1, Shuichi Yamamoto11Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba City, Chiba, Japan; 2Department of Ophthalmology, Kimitsu Central Hospital, Kisarazu City, Chiba, JapanAbstract: Endoplasmic reticulum (ER stress is involved in the pathogenesis of several diseases including Alzheimer disease and Parkinson disease. Many recent studies have shown that ER stress is related to the pathogenesis of diabetes mellitus, and with the death of pancreatic β-cells, insulin resistance, and the death of the vascular cells in the retina. Diabetic retinopathy is a major complication of diabetes and results in death of both neural and vascular cells. Because the death of the neurons directly affects visual function, the precise mechanism causing the death of neurons in early diabetic retinopathy must be determined. The ideal therapy for preventing the onset and the progression of diabetic retinopathy would be to treat the factors involved with both the vascular and neuronal abnormalities in diabetic retinopathy. In this review, we present evidence that ER stress is involved in the death of both retinal neurons and vascular cells in diabetic eyes, and thus reducing or blocking ER stress may be a potential therapy for preventing the onset and the progression of diabetic retinopathy.Keywords: endoplasmic reticulum stress, diabetic retinopathy, vascular cell death, neuronal cell death

  12. Isolation of rat cardiac sarcoplasmic reticulum with improved Ca2+ uptake and ryanodine binding.

    Science.gov (United States)

    Feher, J J; Davis, M D

    1991-03-01

    The instability of the oxalate-supported Ca2+ uptake activity of rat cardiac sarcoplasmic reticulum (CSR) in ventricular homogenates most likely accounts for the low specific activity of the rate of oxalate-supported Ca2+ uptake in previously reported fractions of isolated rat CSR. We have found that CSR vesicles with improved Ca2+ transport capabilities can be isolated if 1 M KCl is used to stabilize the CSR activity and to allow the extraction of the CSR from the cellular debris. The average rate of Ca2+ uptake by the isolated rat CSR in the presence of 10 mM oxalate at 37 degrees C was 0.45 mumols/min-mg in the absence of CSR Ca2+ channel blockers and 0.87 mumols/min-mg in the presence of 10 microM ruthenium red. The Ca(2+)-dependent ATPase activity under the conditions of oxlate-supported uptake was 1.25 mumols/min-mg and 0.84 mumols/min-mg in the absence and presence of 10 microM ruthenium red, respectively. The rat CSR vesicles bound 3H-ryanodine with a Kd of 1.45 nM and a Bmax of 3.7 pmol mg. The level of phosphorylated intermediate was 0.30 nmol/mg. The values Bmax, EP and Ca(2+)-ATPase activity are from one-third to one-half of those previously reported for isolated canine CSR vesicles. These results suggest that the isolated rat CSR may be quite similar to dog CSR.

  13. Regulation of the Plasma Membrane H+-ATPase

    DEFF Research Database (Denmark)

    Falhof, Janus

    The plasma membrane (PM) H+-ATPase is responsible for generating the electrochemical gradientthat drives the secondary transport of nutrients across the cellular membrane. It belongs to a familyof cation and lipid transporters that are vital to many organisms. PM H+-ATPases are Type P3AATPases...

  14. Evolution of copper transporting ATPases in eukaryotic organisms.

    Science.gov (United States)

    Gupta, Arnab; Lutsenko, Svetlana

    2012-04-01

    Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases in prokaryotic kingdom. In eukaryotes, in addition to removing excess copper from the cell, Copper-ATPases have another equally important function - to supply copper to copper dependent enzymes within the secretory pathway. This review focuses on the origin and diversification of Copper ATPases in eukaryotic organisms. From a single Copper ATPase in protozoans, a divergence into two functionally distinct ATPases is observed with the evolutionary appearance of chordates. Among the key functional domains of Copper-ATPases, the metal-binding N-terminal domain could be responsible for functional diversification of the copper ATPases during the course of evolution.

  15. Purification and Properties of an ATPase from Sulfolobus solfataricus

    Science.gov (United States)

    Hochstein, Lawrence I.; Stan-Lotter, Helga

    1992-01-01

    A sulfite-activated ATPase isolated from Sulfolobus solfataricus had a relative molecular mass of 370,000. It was composed of three subunits whose relative molecular masses were 63,000, 48,000, and 24,000. The enzyme was inhibited by the vacuolar ATPase inhibitors nitrate and N-ethylmaleimide; 4-chloro-7-nitrobenzo-furazan (NBD-Cl) was also inhibitory. N-Ethylmaleimide was predominately bound to the largest subunit while NBD-CL was bound to both subunits. ATPase activity was inhibited by low concentrations of p-chloromercuri-phenyl sulfonate and the inhibition was reversed by cysteine which suggested that thiol groups were essential for activity. While the ATPase from S. solfataricus shared several properties with the ATPase from S. acidocaldarius there were significant differences. The latter enzyme was activated by sulfate and chloride and was unaffected by N-ethylmaleimide, whereas the S. solfataricus ATPase was inhibited by these anions as well as N-ethyimaleimide. These differences as well as differences that occur in other vacuolar-like ATPases isolated from the methanogenic and the extremely halophilic bacteria suggest the existence of several types of archaeal ATPases, none of which have been demonstrated to synthesize ATP.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  17. Calcium paradox and calcium entry blockers

    NARCIS (Netherlands)

    Ruigrok, T.J.C.; Slade, A.M.; Nayler, W.G.; Meijler, F.L.

    1984-01-01

    Reperfusion of isolated hearts with calcium-containing solution after a short period of calcium-free perfusion results in irreversible cell damage (calcium paradox). This phenomenon is characterized by an excessive influx of calcium into the cells, the rapid onset of myocardial contracture, exhausti

  18. Effects of strictosamide on mouse brain and kidney Na+, K+-ATPase and Mg2+-ATPase activities.

    Science.gov (United States)

    Candeias, M F; Abreu, P; Pereira, A; Cruz-Morais, J

    2009-01-12

    Present study reports on the general bioactivity of strictosamide and on its effects on Na(+),K(+)-ATPase and Mg(2+)-ATPase activities of Charles River male mouse. Strictosamide is the main glycoalkaloid of Sarcocephalus latifolius (Rubiaceae) leaves and roots, used as medicinal plant in folk medicine. In this work, we studied the in vitro effects of various concentrations of strictosamide (0.25, 0.5, 1 or 2 mg/mL) and the in vivo effects of single doses (50, 100 or 200 mg/kg, i.p.) of this compound on kidney and brain Na(+),K(+)-ATPase and Mg(2+)-ATPase activities. Results of general study showed that strictosamide is slightly toxic to Charles River mouse (LD(50)=723.17 mg/kg), producing CNS depression and kidney toxicity, but the exact mechanism of these effects could not be defined. Strictosamide inhibited the in vitro and in vivo Mg(2+)-ATPase activity on kidney but had nonsignificant effect on brain. Furthermore, strictosamide had nonsignificant in vitro and in vivo effect on kidney Na(+),K(+)-ATPase activity but produced an in vivo increase of Na(+),K(+)-ATPase activity of brain, these findings suggesting that strictosamine may be related to the induction of alpha(2) isoform of Na(+),K(+)-ATPase and may account for the folk use of Sarcocephalus latifolius root infusion on hypertension.

  19. Cellular mechanisms of reduced sarcoplasmic reticulum Ca2+ content in L-thyroxin-induced rat ventricular hypertrophy

    Institute of Scientific and Technical Information of China (English)

    Lai-jing SONG; Guan-lei WANG; Jie LIU; Qin-ying QIU; Jing-hua OU; Yong-yuan GUAN

    2008-01-01

    Aim:To examine how the sarcoplasmic reticulum (SR) Ca2+ content changes and the underlying mechanism in L-thyroxin-induced cardiac hypertrophy. Methods:Echocardiography was used to confirm the establishment of the cardiac hypertro-phy model. The confocal microscopy and fluorescent indicator Fluo-3 was ap-plied to examine the intracellular Ca2+ concentration ([Ca2+]I), the Ca2+ sparks, and the caffeine-induced Ca2+ transient in freshly isolated cardiac ventricular myocytes. The activity of sarcolemmal and SR Ca2+-ATPase 2a (SERCA2a) in the ventricular tissue was also measured, respectively. Results:L-thyroxin (1 mg/kg injection for 10 d) induces left ventricular cardiac hypertrophy with normal myocardial function. The decreased caffeine-induced Ca2+ transient in the Ca2+-free solution was detected. The spontaneous Ca2+ sparks in hypertrophied myocytes occurred more frequently than in normal cells, with similar duration and spatial spread, but smaller amplitude. Then the basal [Ca2+]I increase was observed in quiescent left ventricular myocytes from hyperthyroidism rats. The activity of sarcolemmal and SR Ca2+-ATPase was decreased in the hypertrophied ventricle tissue. Conclusion:The results suggested that the reduced SR Ca2+ content may be associated with an increased Ca2+ leak and reduced SERCA2a activity, contributing to abnormal intracellular Ca2+ handling during hypertrophy in hyperthyroidism rats.

  20. Effects of prior exercise and a low-carbohydrate diet on muscle sarcoplasmic reticulum function during cycling in women.

    Science.gov (United States)

    Duhamel, T A; Green, H J; Perco, J G; Ouyang, J

    2006-09-01

    The effects of exercise and diet on sarcoplasmic reticulum Ca(2+)-cycling properties in female vastus lateralis muscle were investigated in two groups of women following four different conditions. The conditions were 4 days of a low-carbohydrate (Lo CHO) and glycogen-depleting exercise plus a Lo CHO diet (Ex + Lo CHO) (experiment 2) and 4 days of normal CHO (Norm CHO) and glycogen-depleting exercise plus Norm CHO (Ex + Norm CHO) (experiment 1). Peak aerobic power (Vo2peak)) was 38.1 +/- 1.4 (SE); n = 9 and 35.6 +/- 1.4 ml.kg(-1).min(-1); n = 9, respectively. Sarcoplasmic reticulum properties measured in vitro in homogenates (micromol.g protein(-1).min(-1)) indicated exercise-induced reductions (P 30, 60 min > fatigue), Ca(2+) uptake (0 > 30 > 60 min, fatigue), and Ca(2+) release, both phase 1 (0, 30 > 60 min, fatigue) and phase 2 (0 > 30, 60 min, fatigue; 30 min > fatigue) in Norm CHO. Exercise was without effect in altering the Hill slope (n(H)), defined as the slope of relationship between Ca(2+)-ATPase activity and Ca(2+) concentration. No differences were observed between Norm CHO and Ex+Norm CHO. Compared with Norm CHO, Lo CHO resulted in a lower (P cycling and that, with the exception of Ca(2+) release, a glycogen-depleting session of exercise before Lo CHO can reverse the effects.

  1. Activation of AMP-activated protein kinase inhibits oxidized LDL-triggered endoplasmic reticulum stress in vivo.

    Science.gov (United States)

    Dong, Yunzhou; Zhang, Miao; Wang, Shuangxi; Liang, Bin; Zhao, Zhengxing; Liu, Chao; Wu, Mingyuan; Choi, Hyoung Chul; Lyons, Timothy J; Zou, Ming-Hui

    2010-06-01

    The oxidation of LDLs is considered a key step in the development of atherosclerosis. How LDL oxidation contributes to atherosclerosis remains poorly defined. Here we report that oxidized and glycated LDL (HOG-LDL) causes aberrant endoplasmic reticulum (ER) stress and that the AMP-activated protein kinase (AMPK) suppressed HOG-LDL-triggered ER stress in vivo. ER stress markers, sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity and oxidation, and AMPK activity were monitored in cultured bovine aortic endothelial cells (BAECs) exposed to HOG-LDL or in isolated aortae from mice fed an atherogenic diet. Exposure of BAECs to clinically relevant concentrations of HOG-LDL induced prolonged ER stress and reduced SERCA activity but increased SERCA oxidation. Chronic administration of Tempol (a potent antioxidant) attenuated both SERCA oxidation and aberrant ER stress in mice fed a high-fat diet in vivo. Likewise, AMPK activation by pharmacological (5'-aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside, metformin, and statin) or genetic means (adenoviral overexpression of constitutively active AMPK mutants) significantly mitigated ER stress and SERCA oxidation and improved the endothelium-dependent relaxation in isolated mouse aortae. Finally, Tempol administration markedly attenuated impaired endothelium-dependent vasorelaxation, SERCA oxidation, ER stress, and atherosclerosis in ApoE(-/-) and ApoE(-/-)/AMPKalpha2(-/-) fed a high-fat diet. We conclude that HOG-LDL, via enhanced SERCA oxidation, causes aberrant ER stress, endothelial dysfunction, and atherosclerosis in vivo, all of which are inhibited by AMPK activation.

  2. ATF6 as a Transcription Activator of the Endoplasmic Reticulum Stress Element: Thapsigargin Stress-Induced Changes and Synergistic Interactions with NF-Y and YY1

    OpenAIRE

    Li, Mingqing; Baumeister, Peter; Roy, Binayak; Phan, Trevor; Foti, Dolly; Luo, Shengzhan; Lee, Amy S.

    2000-01-01

    ATF6, a member of the leucine zipper protein family, can constitutively induce the promoter of glucose-regulated protein (grp) genes through activation of the endoplasmic reticulum (ER) stress element (ERSE). To understand the mechanism of grp78 induction by ATF6 in cells subjected to ER calcium depletion stress mediated by thapsigargin (Tg) treatment, we discovered that ATF6 itself undergoes Tg stress-induced changes. In nonstressed cells, ATF6, which contains a putative short transmembrane ...

  3. Endoplasmic Reticulum (ER) Stress and Endocrine Disorders

    Science.gov (United States)

    Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro

    2017-01-01

    The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article. PMID:28208663

  4. Endoplasmic Reticulum (ER) Stress and Endocrine Disorders.

    Science.gov (United States)

    Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro

    2017-02-11

    The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the "unfolded protein response" (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article.

  5. Targeting endoplasmic reticulum stress in insulin resistance.

    Science.gov (United States)

    Salvadó, Laia; Palomer, Xavier; Barroso, Emma; Vázquez-Carrera, Manuel

    2015-08-01

    The endoplasmic reticulum (ER) is involved in the development of insulin resistance and progression to type 2 diabetes mellitus (T2DM). Disruption of ER homeostasis leads to ER stress, which activates the unfolded protein response (UPR). This response is linked to different processes involved in the development of insulin resistance (IR) and T2DM, including inflammation, lipid accumulation, insulin biosynthesis, and β-cell apoptosis. Understanding the mechanisms by which disruption of ER homeostasis leads to IR and its progression to T2DM may offer new pharmacological targets for the treatment and prevention of these diseases. Here, we examine ER stress, the UPR, and downstream pathways in insulin sensitive tissues, and in IR, and offer insights towards therapeutic strategies.

  6. Endoplasmic Reticulum (ER Stress and Endocrine Disorders

    Directory of Open Access Journals (Sweden)

    Daisuke Ariyasu

    2017-02-01

    Full Text Available The endoplasmic reticulum (ER is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR, which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI, Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2 are discussed in this article.

  7. Endoplasmic Reticulum Stress and Ethanol Neurotoxicity

    Directory of Open Access Journals (Sweden)

    Fanmuyi Yang

    2015-10-01

    Full Text Available Ethanol abuse affects virtually all organ systems and the central nervous system (CNS is particularly vulnerable to excessive ethanol exposure. Ethanol exposure causes profound damages to both the adult and developing brain. Prenatal ethanol exposure induces fetal alcohol spectrum disorders (FASD which is associated with mental retardation and other behavioral deficits. A number of potential mechanisms have been proposed for ethanol-induced brain damage; these include the promotion of neuroinflammation, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, and thiamine deficiency. The endoplasmic reticulum (ER regulates posttranslational protein processing and transport. The accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress and induces unfolded protein response (UPR which are mediated by three transmembrane ER signaling proteins: pancreatic endoplasmic reticulum kinase (PERK, inositol-requiring enzyme 1 (IRE1, and activating transcription factor 6 (ATF6. UPR is initiated to protect cells from overwhelming ER protein loading. However, sustained ER stress may result in cell death. ER stress has been implied in various CNS injuries, including brain ischemia, traumatic brain injury, and aging-associated neurodegeneration, such as Alzheimer’s disease (AD, Huntington’s disease (HD, Amyotrophic lateral sclerosis (ALS, and Parkinson’s disease (PD. However, effects of ethanol on ER stress in the CNS receive less attention. In this review, we discuss recent progress in the study of ER stress in ethanol-induced neurotoxicity. We also examine the potential mechanisms underlying ethanol-mediated ER stress and the interaction among ER stress, oxidative stress and autophagy in the context of ethanol neurotoxicity.

  8. [Nature of membrane ATPase inactivation in an Escherichia coli mutant with genetically impaired ATPase].

    Science.gov (United States)

    Chetkauskaĭte, A V; Planutis, D L; Zimkus, A Z; Akimenko, V K; Grinius, L L

    1980-07-01

    Homogeneous preparations of F1 possessing identical subunit composition have been isolated from the mutant strain of E. coli AN 120 with genetically impaired membrane ATPase and from the wild strain of AN 180. Using ion-exchange chromatography, the subunits alpha and beta of F1 were isolated. It was shown that the alpha- and beta-subunits of both active and genetically impaired F1 have similar molecular weights and total electrical charges.

  9. Red blood cell calcium homeostasis in patients with end-stage renal disease

    Energy Technology Data Exchange (ETDEWEB)

    Gafter, U.; Malachi, T.; Barak, H.; Djaldetti, M.; Levi, J. (Hasharon Hospital, Petah-Tiqva (Israel))

    1989-09-01

    Low cell calcium level is essential for preservation of red blood cell (RBC) membrane deformability and survival. RBCs from patients with end-stage renal disease (ESRD) demonstrate reduction in membrane deformability, possibly as a result of increased RBC cellular calcium level. To evaluate calcium homeostasis in RBCs from patients with ESRD, we measured cell calcium level, basal and calmodulin-stimulated calcium-stimulated Mg-dependent ATPase (CaATPase) activity, and calcium 45 efflux were measured before and after hemodialysis. The in vitro effect of uremic plasma and of urea on CaATPase activity of normal RBCs was tested, and 45Ca influx into RBCs of patients undergoing hemodialysis also was determined. A morphologic evaluation of red cells from patients with ESRD was performed with a scanning electron microscope. RBC calcium level in patients (mean +/- SEM 21.2 +/- 2.8 mumol/L of cells; n = 28) was higher than in controls (4.9 +/- 0.3 mumol/L of cells; n = 24; p less than 0.001). Hemodialysis had no effect on cell calcium level. Both basal and calmodulin-stimulated RBC CaATPase activities in patients with ESRD (n = 9) were reduced by approximately 50% (p less than 0.01), but after hemodialysis, enzyme activity returned to normal. 45Ca efflux from calcium-loaded cells, which was 2574.0 +/- 217.0 mumol/L of cells per 0.5 hours before hemodialysis, increased to 3140.7 +/- 206.8 mumol/L of cells per 0.5 hours after hemodialysis (p less than 0.005). In vitro incubation of normal RBCs with uremic plasma depressed CaATPase activity, but incubation with urea had no effect. RBCs of patients with ESRD revealed increased 45Ca influx, 7.63 +/- 1.15 mumol/L of cells per hour versus 4.61 +/- 0.39 mumol/L of cells per hour (p less than 0.025). RBCs of patients revealed a high incidence of spherocytosis and echynocytosis, which correlated with a high cell calcium level (r = 0.894, p less than 0.01).

  10. Regulation of vacuolar H(+)-ATPase in microglia by RANKL.

    Science.gov (United States)

    Serrano, Eric M; Ricofort, Ryan D; Zuo, Jian; Ochotny, Noelle; Manolson, Morris F; Holliday, L Shannon

    2009-11-01

    Vacuolar H(+)-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor kappaB-ligand (RANKL). We found that Receptor Activator of Nuclear Factor kappaB (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  11. Regulation of Vacuolar H+-ATPase in Microglia by RANKL

    Science.gov (United States)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian; Ochotny, Noelle; Manolson, Morris F.; Holliday, L. Shannon

    2009-01-01

    Vacuolar H+-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPase play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor κ B -ligand (RANKL). We found that Receptor Activator of Nuclear Factor κ B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia. PMID:19715671

  12. Kinetic characterization of the ATPase and actin-activated ATPase activities of Acanthamoeba castellanii myosin-2.

    Science.gov (United States)

    Heissler, Sarah M; Liu, Xiong; Korn, Edward D; Sellers, James R

    2013-09-13

    Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

  13. 黄芪总黄酮与丹参酮ⅡA磺酸钠对病毒性心肌炎小鼠心肌细胞内质网伴侣蛋白及L-型钙通道表达的作用%Effect of total flavonoids of astragalus and sodium Tanshinone Ⅱ-A sulfonate on gene expression of L-type calcium channels and expression level of endoplasmic reticulum haperone in mouse myocardium with myocarditis caused by coxsackievirus B3

    Institute of Scientific and Technical Information of China (English)

    周晓敏; 陶谢鑫; 杨洋; 王伊林; 柴花; 黄侠; 赵雅君; 赵明

    2015-01-01

    目的:观察黄芪总黄酮(TFA)与丹参酮ⅡA磺酸钠对病毒性心肌炎(VMC)小鼠心肌细胞内质网伴侣蛋白GRP78及L-型钙通道(LCCs) mRNA表达的影响.方法:48只雄性Balb/c小鼠分为对照组、VMC组、TFA组和丹参酮ⅡA磺酸钠治疗组(丹参组),每组12只.注射柯萨奇(CV)B3病毒10 d处死小鼠取心脏行心肌病理观察,采用半定量逆转录聚合酶链式反应(RT-PCR)检测对照组、VMC组、TFA组、丹参组心肌细胞LCCs a1亚单位mRNA表达量,采用Western blotting技术检测4组心肌细胞内质网伴侣蛋白GRP78表达量.结果:①与对照组比较,VMC组心室肌LCCsa1亚单位的mRNA表达量明显增多(P<0.01);与VMC组比较,TFA可使VMC小鼠心室肌细胞LCCsa1亚单位的mRNA表达量减少(P<0.01),而丹参酮ⅡA磺酸钠可使VMC小鼠心室肌细胞LCCsa1亚单位的mRNA表达量减少(P<0.01).②与对照组比较,VMC组心室肌细胞内质网伴侣蛋白GRP78表达量明显增多(P<0.01);与VMC组比较,TFA可使VMC小鼠心室肌细胞内质网伴侣蛋白GRP78表达量减少(P<0.01),而丹参酮ⅡA磺酸钠可使VMC小鼠心室肌细胞内质网伴侣蛋白GRP78表达量减少(P<0.05).结论:TFA与丹参酮ⅡA磺酸钠可以降低VMC小鼠心室肌细胞LCCsa1亚单位的mR-NA表达量,缓解VMC小鼠心肌细胞内质网应激,抑制心肌细胞钙超载介导的内质网应激,从而改善心肌损伤.%Objective:To observe the effects of total flavonoids of astragalus (TFA) and sodium Tanshinone Ⅱ-A sulfonate(Tanshinone Ⅱ-A) on gene expression of L-type calcium channels(LCC) and expression of endoplasmic reticulum chaperones glucose-regulated protein(GRP)78 in viral myocarditis.Method:Forty eight male Balb/c mice were randomly divided into 4 groups:control group(n=12),viral infection control group(n =12),TFA group(n=12),Tanshinone Ⅱ-A group(n=12).When all the mice were sacrificed on day 10,the gene expression of LCCα1 subunit was monitored by RT-PCR and the

  14. Metal Fluoride Complexes of Na,K-ATPase

    Science.gov (United States)

    Cornelius, Flemming; Mahmmoud, Yasser A.; Toyoshima, Chikashi

    2011-01-01

    The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K+·Pi with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na+, after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na+ demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway. PMID:21708939

  15. Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells.

    Science.gov (United States)

    Maruyama, K; MacLennan, D H

    1988-01-01

    Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis. Images PMID:2966962

  16. KUS121, a VCP modulator, attenuates ischemic retinal cell death via suppressing endoplasmic reticulum stress

    Science.gov (United States)

    Hata, Masayuki; Ikeda, Hanako O.; Kikkawa, Chinami; Iwai, Sachiko; Muraoka, Yuki; Hasegawa, Tomoko; Kakizuka, Akira; Yoshimura, Nagahisa

    2017-01-01

    Ischemic neural damages cause several devastating diseases, including brain stroke and ischemic retinopathies, and endoplasmic reticulum (ER) stress has been proposed to be the underlying mechanism of the neuronal cell death of these conditions. We previously synthesized Kyoto University substances (KUSs) as modulators of valosin-containing protein (VCP); KUSs inhibit VCP ATPase activity and protect cells from different cell death-inducing insults. Here, we examined the efficacy of KUS121 in a rat model of retinal ischemic injury. Systemic administration of KUS121 to rats with ischemic retinal injury significantly suppressed inner retinal thinning and death of retinal ganglion and amacrine cells, with a significant functional maintenance of visual functions, as judged by electroretinography. Furthermore, intravitreal injection of KUS121, which is the clinically preferred route of drug administration for retinal diseases, appeared to show an equal or better neuroprotective efficacy in the ischemic retina compared with systemic administration. Indeed, induction of the ER stress marker C/EBP homologous protein (CHOP) after the ischemic insult was significantly suppressed by KUS121 administration. Our study suggests VCP modulation by KUS as a promising novel therapeutic strategy for ischemic neuronal diseases. PMID:28317920

  17. Vanadate oligoanions interact with the proton ejection by the Ca2+ pump of sarcoplasmic reticulum.

    Science.gov (United States)

    Aureliano, M; Madeira, V M

    1994-11-30

    Decameric vanadate differs from other oligomeric vanadate species in inhibiting Ca2+ uptake and H+ ejection promoted by sarcoplasmic reticulum ATPase. A decavanadate solution, 2 mM in total vanadium, containing about 200 microM decameric species, inhibits by about 50% the uptake of Ca2+ and by 75% the H+ ejection, whereas 2 mM nominal monovanadate slightly increases the uptake of Ca2+ and inhibits the ejection of H+ by 25%. Moreover, decavanadate linearly increases the Ca2+/H+ ratio, whereas monovanadate mimicks decavanadate behavior only at concentrations up to 1.2 mM. For higher concentrations of monovanadate, this effect is reversed probably due to the formation of metavanadates, namely tetravandate. It is concluded that Ca2+ uptake is tightly coupled to proton ejection through molecular events that are sensitive to the interaction of vanadate species. Apparently, the stoichiometry is variable and modulated by molecular events involved in vanadate interaction suggesting alterations in the energetic coupling associated with Ca2+ translocation.

  18. Development of Endoplasmic Reticulum Stress during Experimental Oxalate Nephrolithiasis.

    Science.gov (United States)

    Motin, Yu G; Lepilov, A V; Bgatova, N P; Zharikov, A Yu; Motina, N V; Lapii, G A; Lushnikova, E L; Nepomnyashchikh, L M

    2016-01-01

    Morphological and ultrastructural study of the kidney was performed in rats with oxalate nephrolithiasis. Specific features of endoplasmic reticulum stress were evaluated during nephrolithiasis and treatment with α-tocopherol. We observed the signs of endoplasmic reticulum stress with activation of proapoptotic pathways and injury to the cell lining in nephron tubules and collecting ducts. Ultrastructural changes were found in the organelles, nuclei, and cell membranes of epitheliocytes. A relationship was revealed between endoplasmic reticulum stress and oxidative damage, which developed at the early state of lithogenesis.

  19. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Directory of Open Access Journals (Sweden)

    Qingxi Yue

    Full Text Available Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the

  20. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Science.gov (United States)

    Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

    2016-01-01

    Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase

  1. Relevance of Endoplasmic Reticulum Stress Cell Signaling in Liver Cold Ischemia Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Emma Folch-Puy

    2016-05-01

    Full Text Available The endoplasmic reticulum (ER is involved in calcium homeostasis, protein folding and lipid biosynthesis. Perturbations in its normal functions lead to a condition called endoplasmic reticulum stress (ERS. This can be triggered by many physiopathological conditions such as alcoholic steatohepatitis, insulin resistance or ischemia-reperfusion injury. The cell reacts to ERS by initiating a defensive process known as the unfolded protein response (UPR, which comprises cellular mechanisms for adaptation and the safeguarding of cell survival or, in cases of excessively severe stress, for the initiation of the cell death program. Recent experimental data suggest the involvement of ERS in ischemia/reperfusion injury (IRI of the liver graft, which has been considered as one of major problems influencing outcome after liver transplantation. The purpose of this review is to summarize updated data on the molecular mechanisms of ERS/UPR and the consequences of this pathology, focusing specifically on solid organ preservation and liver transplantation models. We will also discuss the potential role of ERS, beyond the simple adaptive response and the regulation of cell death, in the modification of cell functional properties and phenotypic changes.

  2. Impact of high cholesterol and endoplasmic reticulum stress on metabolic diseases: An updated mini-review

    Directory of Open Access Journals (Sweden)

    Erdi Sozen

    2017-08-01

    Full Text Available Endoplasmic reticulum (ER is the major site of protein folding and calcium storage. Beside the role of ER in protein homeostasis, it controls the cholesterol production and lipid-membrane biosynthesis as well as surviving and cell death signaling mechanisms in the cell. It is well-documented that elevated plasma cholesterol induces adverse effects in cardiovascular diseases (CVDs, liver disorders, such as non-alcoholic fatty liver disease (NAFLD, non-alcoholic steatosis hepatitis (NASH, and metabolic diseases which are associated with oxidative and ER stress. Recent animal model and human studies have showed high cholesterol and ER stress as an emerging factors involved in the development of many metabolic diseases. In this review, we will summarize the crucial effects of hypercholesterolemia and ER stress response in the pathogenesis of CVDs, NAFLD/NASH, diabetes and obesity which are major health problems in western countries.

  3. Mechanisms of Alcohol-Induced Endoplasmic Reticulum Stress and Organ Injuries

    Directory of Open Access Journals (Sweden)

    Cheng Ji

    2012-01-01

    Full Text Available Alcohol is readily distributed throughout the body in the blood stream and crosses biological membranes, which affect virtually all biological processes inside the cell. Excessive alcohol consumption induces numerous pathological stress responses, part of which is endoplasmic reticulum (ER stress response. ER stress, a condition under which unfolded/misfolded protein accumulates in the ER, contributes to alcoholic disorders of major organs such as liver, pancreas, heart, and brain. Potential mechanisms that trigger the alcoholic ER stress response are directly or indirectly related to alcohol metabolism, which includes toxic acetaldehyde and homocysteine, oxidative stress, perturbations of calcium or iron homeostasis, alterations of S-adenosylmethionine to S-adenosylhomocysteine ratio, and abnormal epigenetic modifications. Interruption of the ER stress triggers is anticipated to have therapeutic benefits for alcoholic disorders.

  4. Conserved V-ATPase c subunit plays a role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

    Science.gov (United States)

    Zhou, Aimin; Bu, Yuanyuan; Takano, Tetsuo; Zhang, Xinxin; Liu, Shenkui

    2016-01-01

    In plant cells, the vacuolar-type H(+)-ATPases (V-ATPase) are localized in the tonoplast, Golgi, trans-Golgi network and endosome. However, little is known about how V-ATPase influences plant growth, particularly with regard to the V-ATPase c subunit (VHA-c). Here, we characterized the function of a VHA-c gene from Puccinellia tenuiflora (PutVHA-c) in plant growth. Compared to the wild-type, transgenic plants overexpressing PutVHA-c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V-ATPase activity. Consistently, the Arabidopsis atvha-c5 mutant shows reduced V-ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA-c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V-ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA-c-GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling-1 (AtRGS1). These findings suggest that the decrease in V-ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA-c plays an important role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

  5. Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase) for anti-fungal therapy

    Science.gov (United States)

    Hayek, Summer R.; Lee, Samuel A.; Parra, Karlett J.

    2014-01-01

    Vacuolar proton-translocating ATPase (V-ATPase) is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens. PMID:24478704

  6. Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase for anti-fungal therapy

    Directory of Open Access Journals (Sweden)

    Summer R. Hayek

    2014-01-01

    Full Text Available Vacuolar proton-translocating ATPase (V-ATPase is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae (S. cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans (C. albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens.

  7. Role of the Na+/K+-ATPase in regulating the membrane potential in rat peritoneal mast cells

    DEFF Research Database (Denmark)

    Friis, U G; Praetorius, Birger Hans; Knudsen, T;

    1997-01-01

    1. The aim of this study was to investigate the effect of the Na+/K+-ATPase on the membrane potential of peritoneal mast cells isolated from male Sprague-Dawley SPF-rats. 2. Experiments were performed at 22-26 degrees C in the tight-seal whole-cell configuration of the patch-clamp technique by use...... current (low-pass filtered at 500 Hz). 3. Na+/K+-ATPase activity was measured as the ouabain-sensitive change in the zero-current potential. The zero-current potential in rat peritoneal mast cells measured 2 min after obtaining whole-cell configuration amounted to 1.7 +/- 2.5 mV (n = 21). Ouabain (5 m......M), a Na+/K+-ATPase-inhibitor, had only a very minor effect upon the membrane potential under resting conditions (n = 3). 4. When mast cells were superfused with nominal calcium-free external solution, the cells hyperpolarized (delta mV: 20.2 +/- 3.8 mV (n = 5)). In addition, when the mast cells were...

  8. Detailed search for protein kinase(s) involved in plasma membrane H+-ATPase activity regulation of yeast cells.

    Science.gov (United States)

    Pereira, Renata R; Castanheira, Diogo; Teixeira, Janaina A; Bouillet, Leoneide E M; Ribeiro, Erica M C; Trópia, Maria M J; Alvarez, Florencia; Correa, Lygia F M; Mota, Bruno E F; Conceição, Luis Eduardo F R; Castro, Ieso M; Brandão, Rogelio L

    2015-03-01

    This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.

  9. Accessibility of myofilament cysteines and effects on ATPase depend on the activation state during exposure to oxidants.

    Directory of Open Access Journals (Sweden)

    Sean M Gross

    Full Text Available Signaling by reactive oxygen species has emerged as a major physiological process. Due to its high metabolic rate, striated muscle is especially subject to oxidative stress, and there are multiple examples in cardiac and skeletal muscle where oxidative stress modulates contractile function. Here we assessed the potential of cysteine oxidation as a mechanism for modulating contractile function in skeletal and cardiac muscle. Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species. To refine this list of cysteines to those that may modulate function, we estimated the accessibility of oxidants to cysteine residues using protein crystal structures, and then sharpened these estimates using fluorescent labeling of cysteines in cardiac and skeletal myofibrils. We demonstrate that cysteine accessibility to oxidants and ATPase rates depend on the contractile state in which preparations are exposed. Oxidant exposure of skeletal and cardiac myofibrils in relaxing solution exposes myosin cysteines not accessible in rigor solution, and these modifications correspond to a decrease in maximum ATPase. Oxidant exposure under rigor conditions produces modifications that increase basal ATPase and calcium sensitivity in ventricular myofibrils, but these effects were muted in fast twitch muscle. These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types.

  10. The plasma membrane Ca2+ ATPase of animal cells: structure, function and regulation.

    Science.gov (United States)

    Di Leva, Francesca; Domi, Teuta; Fedrizzi, Laura; Lim, Dmitry; Carafoli, Ernesto

    2008-08-01

    Most important processes in cell life are regulated by calcium (Ca2+). A number of mechanisms have thus been developed to maintain the concentration of free Ca2+ inside cells at the level (100-200nM) necessary for the optimal operation of the targets of its regulatory function. The systems that move Ca2+ back and forth across membranes are important actors in its control. The plasma membrane calcium ATPase (PMCA pump) which ejects Ca2+ from all eukaryotic cell types will be the topic of this contribution. The pump uses a molecule of ATP to transport one molecule of Ca2+ from the cytosol to the external environment. It is a P-type ATPase encoded by four genes (ATP2B1-4), the transcripts of which undergo different types of alternative splicing. Many pump variants thus exist. Their multiplicity is best explained by the specific Ca2+ demands in different cell types. In keeping with these demands, the isoforms are differently expressed in tissues and cell types and have differential Ca2+ extruding properties. At very low Ca2+ concentrations the PMCAs are nearly inactive. They must be activated by calmodulin, by acid phospholipids, by protein kinases, and by other means, e.g., a dimerization process. Other proteins interact with the PMCAs (i.e., MAGUK and NHERF at the PDZ domain and calcineurin A in the main intracellular domain) to sort them to specific regions of the cell membrane or to regulate their function. In some cases the interaction is isoform, or even splice variant specific. PMCAs knock out (KO) mice have been generated and have contributed information on the importance of PMCAs to cells and organisms. So far, only one human genetic disease, hearing loss, has been traced back to a PMCA defect.

  11. TRPV4 in porcine lens epithelium regulates hemichannel-mediated ATP release and Na-K-ATPase activity.

    Science.gov (United States)

    Shahidullah, Mohammad; Mandal, Amritlal; Delamere, Nicholas A

    2012-06-15

    In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic

  12. 精-甘-天冬-丝氨酸对脓毒性休克大鼠心肌肌浆网钙转运的影响%Effects of Arg-Gly-Asp-Ser on Ca2 + transport of myocardial sarcoplasmic reticulum in rat septic shock

    Institute of Scientific and Technical Information of China (English)

    吉勇; 赵明; 齐鹰; 董林旺; 吴立玲; 彭师奇; 苏静怡

    1996-01-01

    To study the effects of Arg-Gly-Asp-Ser (RGDS), a synthetic short peptide of fibrinogen degradation, on the Ca2 + transport function of cardiac sarcoplasmic reticulum in rat septic shock.4 h and 14 h after cecal ligation and puncture (CLP) operation on rats. Highly purified membrane of sarcoplasmic reticulum (SR) was prepared from rat hearts. Assays were made of ATP-dependent Ca2 + uptake by cardiac SR and [3H] ryanodine binding to SR. RESULTS: The initial rate and the capacity of SR Ca2 + uptake were increased by 104 % (P<0.01) and 12 % (P<0.05), respectively, paralleled by an increase in Ca2 +-ATPase activity and a decrease in calcium accumulation of myo- cardium of septic rats, whereas the Bmax and Kd values of Ca2+ activated [3H]ryanodine binding to SR were unaffected after RGDS administration. CONCLUTIONS: The results indicated that RGDS have cardioprotective effects of maintaining Ca2+ homeostasis of cardiac myocytes by enhancing SR Ca2 + uptake in rat septic shock.%探讨一种人工合成的纤维蛋白原降解肽片段RGDS对脓毒性休克大鼠心肌肌浆网钙转运功能的影响.方法:大鼠盲肠结扎穿孔术后4 h和14 h分两次尾静脉注射RGDS 5 μmol·kg-1.制备大鼠心肌肌浆网(SR)膜;测定SR Ca2+摄取和[3H]ryanodine受体结合功能.结果:RGDS组大鼠心肌SR摄Ca2+率及摄Ca2+量分别较休克组提高104%(P<0.01)和12%(P<0.05),而心肌SR钙释放通道-[3H]ryanodine受体结合Bmax和Ka值没有明显变化.同时RGDS还可以减轻休克大鼠心肌组织钙聚积.结论:RGDS提高休克大鼠心肌SR Ca2+摄取功能,维持心肌细胞钙稳态,具有心肌保护作用.

  13. Construction, expression and characterization of a plasmid-encoded Na(+)-specific ATPase hybrid consisting of Propionigenium modestum F0-ATPase and Escherichia coli F1-ATPase.

    Science.gov (United States)

    Kaim, G; Dimroth, P

    1994-06-01

    The Escherichia coli strain DK8, a deletion mutant lacking the complete unc operon, was transformed with a plasmid containing the genes encoding the a, b, c, delta and part of the alpha subunit of the Na(+)-dependent ATPase of Propionigenium modestum and the genes encoding the alpha, gamma, beta and epsilon subunits of the H(+)-dependent E. coli ATPase. The transformants showed Na(+)-dependent growth on succinate as non-fermentable carbon source. The functionally expressed hybrid ATPase was activated 13-fold at pH 7.5 by the addition of Na+ and inhibited by 1,3-dicyclohexylcarbodiimide, azide and tributyltin chloride. At pH 7.5 and pH 9.0, the hybrid enzyme was protected from inhibition by 1,3-dicyclohexylcarbodiimide in the presence of 50 mM NaCl and 5 mM NaCl, respectively. The hybrid ATPase was reconstituted into proteoliposomes and catalyzed the transport of Na+ upon ATP addition. ATP-dependent fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine proved that the ATPase hybrid was able to pump protons in the absence of Na+. Furthermore, ATP synthesis could be measured under conditions where a valinomycin-mediated K+ diffusion potential (delta psi) and a Na+ concentration gradient (delta p Na+) were imposed.

  14. Regulation of endoplasmic reticulum turnover by selective autophagy

    NARCIS (Netherlands)

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K.; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Huebner, Christian A.; Dikic, Ivan

    2015-01-01

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication(1). Constant ER turnover and modulation is needed to meet different cellular requirements

  15. An endoplasmic reticulum (ER)-directed fusion protein comprising a ...

    African Journals Online (AJOL)

    An endoplasmic reticulum (ER)-directed fusion protein comprising a bacterial subtilisin ... which are used for the commercial production of therapeutic proteins. ... expression platforms) to purify recombinant proteins in crude plant extracts.

  16. Regulation of endoplasmic reticulum turnover by selective autophagy

    NARCIS (Netherlands)

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

    2015-01-01

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and

  17. Regulation of endoplasmic reticulum turnover by selective autophagy

    NARCIS (Netherlands)

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K.; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Huebner, Christian A.; Dikic, Ivan

    2015-01-01

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication(1). Constant ER turnover and modulation is needed to meet different cellular requirements

  18. Calcium D-saccharate

    DEFF Research Database (Denmark)

    Garcia, André Castilho; Hedegaard, Martina Vavrusova; Skibsted, Leif Horsfelt

    2016-01-01

    K-1. Equilibria in supersaturated solutions of calcium d-saccharate seem only to adjust slowly, as seen from calcium activity measurements in calcium d-saccharate solutions made supersaturated by cooling. Solutions formed by isothermal dissolution of calcium d-gluconate in aqueous potassium d......-saccharate becomes spontaneously supersaturated with both d-gluconate and d-saccharate calcium salts, from which only calcium d-saccharate slowly precipitates. Calcium d-saccharate is suggested to act as a stabilizer of supersaturated solutions of other calcium hydroxycarboxylates with endothermic complex formation...

  19. Evolution of Copper Transporting ATPases in Eukaryotic Organisms

    OpenAIRE

    Gupta, Arnab; Lutsenko, Svetlana

    2012-01-01

    Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases i...

  20. The ER mitochondria calcium cycle and ER stress response as therapeutic targets in amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Vedrana eTadic

    2014-05-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. Although the etiology remains unclear, disturbances in calcium homoeostasis and protein folding are essential features of neurodegeneration in this disorder. Here, we review recent research findings on the interaction between endoplasmic reticulum (ER and mitochondria, and its effect on calcium signaling and oxidative stress. We further provide insights into studies, providing evidence that structures of the ER mitochondria calcium cycle (ERMCC serve as a promising targets for therapeutic approaches for treatment of ALS.

  1. Activity-dependent accumulation of calcium in Purkinje cell dendritic spines.

    OpenAIRE

    Andrews, S.B.; Leapman, R D; Landis, D M; Reese, T S

    1988-01-01

    The calcium content of synapses of parallel fibers on Purkinje cell dendritic spines was determined by electron probe x-ray microanalysis of freeze-dried cryosections from directly frozen slices of mouse cerebellar cortex. In fresh slices frozen within 20-30 sec of excision, calcium concentrations ranging from 0.8 to 18.6 mmol/kg of dry weight were measured in cisterns of smooth endoplasmic reticulum within Purkinje cell dendritic spines. The average calcium content of spine cisterns in rapid...

  2. Calcium efflux systems in stress signalling and adaptation in plants

    Directory of Open Access Journals (Sweden)

    Jayakumar eBose

    2011-12-01

    Full Text Available Transient cytosolic calcium ([Ca2+]cyt elevation is an ubiquitous denominator of the signalling network when plants are exposed to literally every known abiotic and biotic stress. These stress-induced [Ca2+]cyt elevations vary in magnitude, frequency and shape, depending on the severity of the stress as well the type of stress experienced. This creates a unique stress-specific calcium signature that is then decoded by signal transduction networks. While most published papers have been focused predominantly on the role of Ca2+ influx mechanisms in shaping [Ca2+]cyt signatures, restoration of the basal [Ca2+]cyt levels is impossible without both cytosolic Ca2+ buffering and efficient Ca2+ efflux mechanisms removing excess Ca2+ from cytosol, to reload Ca2+ stores and to terminate Ca2+ signalling. This is the topic of the current review. The molecular identity of two major types of Ca2+ efflux systems, Ca2+-ATPase pumps and Ca2+/H+ exchangers, is described, and their regulatory modes are analysed in detail. The spatial and temporal organisation of calcium signalling networks is described, and the importance of existence of intracellular calcium microdomains is discussed. Experimental evidence for the role of Ca2+ efflux systems in plant responses to a range of abiotic and biotic factors is summarised. Contribution of Ca2+-ATPase pumps and Ca2+/H+ exchangers in shaping [Ca2+]cyt signatures is then modelled by using a four-component model (plasma- and endo- membrane-based Ca2+-permeable channels and efflux systems taking into account the cytosolic Ca2+ buffering. It is concluded that physiologically relevant variations in the activity of Ca2+-ATPase pumps and Ca2+/H+ exchangers are sufficient to fully describe all the reported experimental evidence and determine the shape of [Ca2+]cyt signatures in response to environmental stimuli, emphasising the crucial role these active efflux systems play in plant adaptive responses to environment.

  3. Endoplasmic reticulum stress in periimplantation embryos.

    Science.gov (United States)

    Michalak, Marek; Gye, Myung Chan

    2015-03-01

    Stress coping mechanisms are critical to minimize or overcome damage caused by ever changing environmental conditions. They are designed to promote cell survival. The unfolded protein response (UPR) pathway is mobilized in response to the accumulation of unfolded proteins, ultimately in order to regain endoplasmic reticulum (ER) homeostasis. Various elements of coping responses to ER stress including Perk, Ask1, Bip, Chop, Gadd34, Ire1, Atf4, Atf6, and Xbp1 have been identified and were found to be inducible in oocytes and preimplantation embryos, suggesting that, as a normal part of the cellular adaptive mechanism, these coping responses, including the UPR, play a pivotal role in the development of preimplantation embryos. As such, the UPR-associated molecules and pathways may become useful markers for the potential diagnosis of stress conditions for preimplantation embryos. After implantation, ER stress-induced coping responses become physiologically important for a normal decidual response, placentation, and early organogenesis. Attenuation of ER stress coping responses by tauroursodeoxycholate and salubrinal was effective for prevention of cell death of cultured embryos. Further elucidation of new and relevant ER stress coping responses in periimplantation embryos might contribute to a comprehensive understanding of the regulation of normal development of embryonic development and potentiation of embryonic development in vitro.

  4. Effects of a high intake of unsaturated and saturated oils on intestinal transference of calcium and calcium mobilization from bone in an ovariectomized rat model of osteoporosis.

    Science.gov (United States)

    Chanda, S; Islam, N; Ghosh, T K; Mitra, C

    1999-06-01

    Intestinal transference of calcium and rate of bone turnover were evaluated in ovariectomized rats fed for 15 days with a high amount (30%) of lipid enriched with monounsaturated (groundnut oil), polyunsaturated (sunflower oil) and saturated (coconut oil) fatty acids. The results were compared with those for sham-operated control and ovariectomized groups fed a normal diet (7% groundnut oil). Irrespective of the saturation and unsaturation characteristics, all lipids (edible oils) used in our study considerably decreased the rate of in situ intestinal transference of calcium. Likewise, the activities of intestinal mucosal enzymes, alkaline phosphatase (AP) and calcium ATPase (Ca2+-ATPase) were decreased significantly in all the segments of the small intestine in a descending gradient. Significant changes in bone turnover and bone calcium (Ca) mobilization were confirmed in these animals by marked alterations in plasma AP activity, urinary calcium and phosphate excretion and calcium to creatinine (Ca:creatinine) ratio. Lipid supplementation (30%) in such ovariectomized rats using groundnut oil (monounsaturated), sunflower oil (polyunsaturated) or coconut oil (saturated) for 15 days further enhanced all of the above observed parameters. These results suggest that the intake of high amounts of lipids with different unsaturation and saturation characteristics may be an important factor in determining bone loss in ovariectomized rats.

  5. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe

    2011-01-01

    Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary tran......(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps....

  6. F1FO ATPase vesicle preparation and technique for performing patch clamp recordings of submitochondrial vesicle membranes.

    Science.gov (United States)

    Sacchetti, Silvio; Alavian, Kambiz N; Lazrove, Emma; Jonas, Elizabeth A

    2013-05-04

    Mitochondria are involved in many important cellular functions including metabolism, survival(1), development and, calcium signaling(2). Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death(3). The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase(4-5). In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol(6). In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL(7) interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity(8) and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion

  7. V-type ATPase proton pump expression during enamel formation.

    Science.gov (United States)

    Sarkar, Juni; Wen, Xin; Simanian, Emil J; Paine, Michael L

    2016-01-01

    Several diseases such as proximal and distal renal tubular acidosis and osteoporosis are related to intracellular pH dysregulation resulting from mutations in genes coding for ion channels, including proteins comprising the proton-pumping V-type ATPase. V-type ATPase is a multi-subunit protein complex expressed in enamel forming cells. V-type ATPase plays a key role in enamel development, specifically lysosomal acidification, yet our understanding of the relationship between the endocytotic activities and dental health and disease is limited. The objective of this study is to better understand the ameloblast-associated pH regulatory networks essential for amelogenesis. Quantitative RT-PCR was performed on tissues from secretory-stage and maturation-stage enamel organs to determine which of the V-type ATPase subunits are most highly upregulated during maturation-stage amelogenesis: a time when ameloblast endocytotic activity is highest. Western blot analyses, using specific antibodies to four of the V-type ATPase subunits (Atp6v0d2, Atp6v1b2, Atp6v1c1 and Atp6v1e1), were then applied to validate much of the qPCR data. Immunohistochemistry using these same four antibodies was also performed to identify the spatiotemporal expression profiles of individual V-type ATPase subunits. Our data show that cytoplasmic V-type ATPase is significantly upregulated in enamel organ cells during maturation-stage when compared to secretory-stage. These data likely relate to the higher endocytotic activities, and the greater need for lysosomal acidification, during maturation-stage amelogenesis. It is also apparent from our immunolocalization data, using antibodies against two of the V-type ATPase subunits (Atp6v1c1 and Atp6v1e1), that significant expression is seen at the apical membrane of maturation-stage ameloblasts. Others have also identified this V-type ATPase expression profile at the apical membrane of maturation ameloblasts. Collectively, these data better define the

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

    Science.gov (United States)

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

    2007-10-01

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

  9. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

    Science.gov (United States)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly ( P P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

  10. Experimental determination of control by the H+-ATPase in Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole; Westerhoff, H. V.

    1995-01-01

    and oxygen consumption of these mutants was increased compared to the wild-type rates. In order to analyze the importance of the H+-ATPase at its physiological level, the cellular concentration of H+-ATPase was modulated around the wild-type level, using genetically manipulated strains. The control...... coefficient by the H+-ATPase with respect to growth rate and catabolic fluxes was measured. Control on growth rate was absent at the wildtype concentration of H+-ATPase, independent of whether the substrate for growth was glucose or succinate. Control by the H+-ATPase on the catabolic fluxes, including...... respiration, was negative at the wild-type H+-ATPase level. Moreover, the turnover number of the individual H+-ATPase enzymes increased as the H+-ATPase concentration was lowered. The negative control by the H+-ATPase on catabolism may thus be involved in a homeostatic control of ATP synthesis and, to some...

  11. Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex

    Directory of Open Access Journals (Sweden)

    Kluge Christoph

    2004-08-01

    Full Text Available Abstract Background Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET. Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i a less conserved cytoplasmically orientated N-terminus and (ii a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP in transfected cells. Conclusions All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA

  12. Protective effect of fish oil on changes in the activities of membrane-bound ATPases and mineral status in experimentally induced myocardial infarction in Wistar rats.

    Science.gov (United States)

    Padma, Viswanadha Vijaya; Devi, Chennam Srinivasulu Shyamala; Kalaiselvi, Palaniswamy

    2010-12-01

    The present study evaluated the protective effect of fish oil in isoproterenol-induced myocardial infarction in rats. The results of the present study indicate that the IPH administration decreases the activities of membrane-bound ATPases compared to control animals. Fish oil pretreatment brought about significant increase in the activity of these membrane-bound ATPases in IPH (isoproterenol hydrochloride)-treated animals. Significant increase in serum potassium level with concomitant decrease in the values of sodium, magnesium, and calcium were observed in IPH-treated rats compared to control rats, fish oil pretreatment reversed these changes to near normal. Significant elevation of sodium and calcium levels with concomitant decrease in the levels of potassium and magnesium were observed in the myocardial tissue of IPH-administered rats compared to control rats, fish oil pretreatment followed by IPH administration brought these levels to near normal. The levels of lipid peroxidation (LPO) in both serum and tissue were increased in IPH-treated rats compared with control rats, whereas pretreatment with fish oil in IPH-treated rats maintained near-normal LPO levels. The results of the present study reveals that the pretreatment of fish maintains the activities of membrane-bound ATPases and the mineral levels at near normal by the inhibition of lipid peroxidation.

  13. Calcium sensing in exocytosis

    DEFF Research Database (Denmark)

    Gustavsson, Natalia; Wu, Bingbing; Han, Weiping

    2012-01-01

    an increase in intracellular calcium levels. Besides the triggering role, calcium signaling modulates the precise amount and kinetics of vesicle release. Thus, it is a central question to understand the molecular machineries responsible for calcium sensing in exocytosis. Here we provide an overview of our...... current understanding of calcium sensing in neurotransmitter release and hormone secretion....

  14. A simplified model for V-ATPase H+ extrusion.

    Science.gov (United States)

    Luo, Chuan; Clark, John W; Heming, Thomas A; Bidani, Akhil

    2004-12-01

    An analytical model of V-type H+-translocating ATPase (V-ATPase) was developed based on an approximation to the mechanochemical model of Grabe et al. (Biophys. J., pp. 2798-2813, vol. 78, 2000). Grabe's work utilizes structural information and physiological assumptions to construct a detailed mechanochemical model of the V-ATPase. Due to the complexity of their model, it does not give a readily usable mathematical expression for the V-ATPase current. Based on their analysis of the structure of the proton pump, we develop a two-compartment model of the V-ATPase, which contains a membrane "half-channel" for proton translocation separated by a hydrophilic strip and a hydrophobic wall from the cytoplasm. Using the Langevin equation to describe proton transport across the membrane, we simplify the model based on their assumptions on the molecular structure of the pump and arrive at a general form of solution to the proton pump flux driven by ATP hydrolysis based on assumptions on the physiological properties of the strip and the wall, as well as the two fluid compartments. In this process of simplification, we explicitly relate V-ATPase structure, stoichiometry, pump efficiency, and ATP hydrolysis energy to the active pump current. The simplified model is used to provide model-generated approximations to measured data from a variety of laboratories. In addition, it provides a very compact characterization of V-ATPase, which can be used as a proton extruder in a variety of different cell membranes, as well as in the membranes of intracellular organelles. Index Terms-Electrophysiology, mechanochemstry, molecular motors, proton extrusion

  15. Archazolid and apicularen: Novel specific V-ATPase inhibitors

    Directory of Open Access Journals (Sweden)

    Zeeck Axel

    2005-08-01

    Full Text Available Abstract Background V-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic membranes, they energize many different transport processes. Since their malfunction is correlated with various diseases in humans, the elucidation of the properties of this enzyme for the development of selective inhibitors and drugs is one of the challenges in V-ATPase research. Results Archazolid A and B, two recently discovered cytotoxic macrolactones produced by the myxobacterium Archangium gephyra, and apicularen A and B, two novel benzolactone enamides produced by different species of the myxobacterium Chondromyces, exerted a similar inhibitory efficacy on a wide range of mammalian cell lines as the well established plecomacrolidic type V-ATPase inhibitors concanamycin and bafilomycin. Like the plecomacrolides both new macrolides also prevented the lysosomal acidification in cells and inhibited the V-ATPase purified from the midgut of the tobacco hornworm, Manduca sexta, with IC50 values of 20–60 nM. However, they did not influence the activity of mitochondrial F-ATPase or that of the Na+/K+-ATPase. To define the binding sites of these new inhibitors we used a semi-synthetic radioactively labelled derivative of concanamycin which exclusively binds to the membrane Vo subunit c. Whereas archazolid A prevented, like the plecomacrolides concanamycin A, bafilomycin A1 and B1, labelling of subunit c by the radioactive I-concanolide A, the benzolactone enamide apicularen A did not compete with the plecomacrolide derivative. Conclusion The myxobacterial antibiotics archazolid and apicularen are highly efficient and specific novel inhibitors of V-ATPases. While archazolid at least partly shares a common binding site with the plecomacrolides bafilomycin and concanamycin, apicularen adheres to an independent binding site.

  16. Imaging calcium in neurons.

    Science.gov (United States)

    Grienberger, Christine; Konnerth, Arthur

    2012-03-08

    Calcium ions generate versatile intracellular signals that control key functions in all types of neurons. Imaging calcium in neurons is particularly important because calcium signals exert their highly specific functions in well-defined cellular subcompartments. In this Primer, we briefly review the general mechanisms of neuronal calcium signaling. We then introduce the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices that are used in freely moving animals. We provide an overview of the classical chemical fluorescent calcium indicators and of the protein-based genetically encoded calcium indicators. Using application examples, we introduce new developments in the field, such as calcium imaging in awake, behaving animals and the use of calcium imaging for mapping single spine sensory inputs in cortical neurons in vivo. We conclude by providing an outlook on the prospects of calcium imaging for the analysis of neuronal signaling and plasticity in various animal models.

  17. The reticulons: Guardians of the structure and function of the endoplasmic reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Di Sano, Federica; Bernardoni, Paolo [Department of Biology, University of Rome ' Tor Vergata' , Via della Ricerca Scientifica 1, 00133 Rome (Italy); Piacentini, Mauro, E-mail: mauro.piacentini@uniroma2.it [Department of Biology, University of Rome ' Tor Vergata' , Via della Ricerca Scientifica 1, 00133 Rome (Italy); National Institute for Infectious Diseases, IRCCS ' L. Spallanzani' , Via Portuense, 00149 Rome (Italy)

    2012-07-01

    The endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of tubules and membrane sheets. The tubules are shaped by a specific class of curvature stabilizing proteins, the reticulons and DP1; however it is still unclear how the sheets are assembled. The ER is the cellular compartment responsible for secretory and membrane protein synthesis. The reducing conditions of ER lead to the intra/inter-chain formation of new disulphide bonds into polypeptides during protein folding assessed by enzymatic or spontaneous reactions. Moreover, ER represents the main intracellular calcium storage site and it plays an important role in calcium signaling that impacts many cellular processes. Accordingly, the maintenance of ER function represents an essential condition for the cell, and ER morphology constitutes an important prerogative of it. Furthermore, it is well known that ER undergoes prominent shape transitions during events such as cell division and differentiation. Thus, maintaining the correct ER structure is an essential feature for cellular physiology. Now, it is known that proper ER-associated proteins play a fundamental role in ER tubules formation. Among these ER-shaping proteins are the reticulons (RTN), which are acquiring a relevant position. In fact, beyond the structural role of reticulons, in very recent years new and deeper functional implications of these proteins are emerging in relation to their involvement in several cellular processes.

  18. Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration.

    Science.gov (United States)

    Feng, Juan; Lü, Silin; Ding, Yanhong; Zheng, Ming; Wang, Xian

    2016-06-01

    Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretion in T cells. However, whether homocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivo and in vitro studies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition of ER stress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.

  19. New insights in the role of Bcl-2 Bcl-2 and the endoplasmic reticulum.

    Science.gov (United States)

    Rudner, J; Jendrossek, V; Belka, C

    2002-10-01

    The oncogenic protein Bcl-2 which is expressed in membranes of different subcellular organelles protects cells from apoptosis induced by endogenic stimuli. Most of the results published so far emphasise the importance of Bcl-2 at the mitochondria. Several recent observations suggest a role of Bcl-2 at the endoplasmic reticulum (ER). Bcl-2 located at the ER was shown to interfere with apoptosis induction by Bax, ceramides, ionising radiation, serum withdrawal and c-myc expression. Although the detailed functions of Bcl-2 at the ER remain elusive, several speculative mechanisms may be supposed. For instance, Bcl-2 at the ER may regulate calcium fluxes between the ER and the mitochondria. In addition, Bcl-2 is able to interact with the endoplasmic protein Bap31 thus avoiding caspase activation at the ER. Bcl-2 may also abrogate the function of ER located pro-apoptotic Bcl-2 like proteins by heterodimerization. Current data on the function of Bcl-2 at the ER, its role for the modulation of calcium fluxes and its influence on caspase activation at the ER are reviewed.

  20. Endoplasmic reticulum Ca2+-homeostasis is altered in small and non-small cell lung cancer cell lines

    Directory of Open Access Journals (Sweden)

    Tufman Amanda

    2009-02-01

    Full Text Available Abstract Background Knowledge of differences in the cellular physiology of malignant and non-malignant cells is a prerequisite for the development of cancer treatments that effectively kill cancer without damaging normal cells. Calcium is a ubiquitous signal molecule that is involved in the control of proliferation and apoptosis. We aimed to investigate if the endoplasmic reticulum (ER Ca2+-homeostasis is different in lung cancer and normal human bronchial epithelial (NHBE cells. Methods The intracellular Ca2+-signaling was investigated using fluorescence microscopy and the expression of Ca2+-regulating proteins was assessed using Western Blot analysis. Results In a Small Cell Lung Cancer (H1339 and an Adeno Carcinoma Lung Cancer (HCC cell line but not in a Squamous Cell Lung Cancer (EPLC and a Large Cell Lung Cancer (LCLC cell line the ER Ca2+-content was reduced compared to NHBE. The reduced Ca2+-content correlated with a reduced expression of SERCA 2 pumping calcium into the ER, an increased expression of IP3R releasing calcium from the ER, and a reduced expression of calreticulin buffering calcium within the ER. Lowering the ER Ca2+-content with CPA led to increased proliferation NHBE and lung cancer cells. Conclusion The significant differences in Ca2+-homeostasis between lung cancer and NHBE cells could represent a new target for cancer treatments.

  1. Influence of hexavanadates on Na+/K+- ATPase activity

    Directory of Open Access Journals (Sweden)

    Zdravković Aleksandra

    2016-01-01

    Full Text Available Introduction: There is a great interest in use of polioximetalates in clinical medicine, primary as antibacterial, antiviral and antitumoral agents. Considering the key role of Na+/ K+- ATPase in normal functioning of most animal cells, as well as pivotal roles in cancer cell migration, the aim of this paper was to examine the influence of new synthesized hexavandates [V6-CH3][Na]2, [V6-NO2][TBA]2, [V6-C3][H]2, [V6-C5d][TBA]2 on Na+/K+- ATPase activity. Material and methods: The enzymatic activity of porcine cerebral cortex Na+/K+- ATPase was followed in both the absence and presence of increasing concentration of [V6-CH3] [Na]2, [V6-NO2][TBA]2, [V6-C3][H]2, [V6-C5d][TBA]2 (within the range 10-8 - 10-3 mol/L. The released Pi, liberated from the enzymatic hydrolysis of ATP, was determined by spectrophotometric method, using Perkin Elmer Lambda 35 UV-VIS spectrophotometer. Results: Investigated compounds inhibit the activity of Na+/K+ ATPase in dose-dependent manner within the investigated range. Obtained results indicate that all investigated compounds inhibit the Na+/K+ ATPase activity, but with different inhibiting power. [V6-NO2] [TBA]2 (IC50 = 1,87 × 10-5 mol/L was the most potent inhibitor of Na+/K+ ATPase, while [V6-C5d][TBA]2 showed the least potent inhibiting power (IC50 = 1,31 × 10-4 mol/L . The results are consistent with previously published concentration-dependent inhibitory effect of polyoxometalates (including polioxovandates on ATPase activity from different model syistems. Conclusion: Based on the results, we can conclude that the examined compounds inhibit Na+/K+- ATPase activity in a dose-dependent manner. Inhibiting power of tested hexavanadates are different, and weaker than inhibiting power of decavanadates (tested earlier on Na+/K+- ATPase activity, which is probably due to differences in charge, size and shape of these polioxometalates. Considering the role of this enzymes in the functioning of healthy cells and the

  2. Role of Ryanodine Receptor Subtypes in Initiation and Formation of Calcium Sparks in Arterial Smooth Muscle: Comparison with Striated Muscle

    Directory of Open Access Journals (Sweden)

    Maik Gollasch

    2009-01-01

    Full Text Available Calcium sparks represent local, rapid, and transient calcium release events from a cluster of ryanodine receptors (RyRs in the sarcoplasmic reticulum. In arterial smooth muscle cells (SMCs, calcium sparks activate calcium-dependent potassium channels causing decrease in the global intracellular [Ca2+] and oppose vasoconstriction. This is in contrast to cardiac and skeletal muscle, where spatial and temporal summation of calcium sparks leads to global increases in intracellular [Ca2+] and myocyte contraction. We summarize the present data on local RyR calcium signaling in arterial SMCs in comparison to striated muscle and muscle-specific differences in coupling between L-type calcium channels and RyRs. Accordingly, arterial SMC Cav1.2 L-type channels regulate intracellular calcium stores content, which in turn modulates calcium efflux though RyRs. Downregulation of RyR2 up to a certain degree is compensated by increased SR calcium content to normalize calcium sparks. This indirect coupling between Cav1.2 and RyR in arterial SMCs is opposite to striated muscle, where triggering of calcium sparks is controlled by rapid and direct cross-talk between Cav1.1/Cav1.2 L-type channels and RyRs. We discuss the role of RyR isoforms in initiation and formation of calcium sparks in SMCs and their possible molecular binding partners and regulators, which differ compared to striated muscle.

  3. Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Peter Rohloff

    Full Text Available Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+/H(+ countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.

  4. A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases.

    Directory of Open Access Journals (Sweden)

    Gianluca Bartolommei

    Full Text Available The detection of small amounts (nanomoles of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases, that produce inorganic phosphate by cleavage of the γ-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III oxide tartrate (originally employed for phosphate detection in environmental analysis to allow its use with phosphatase enzymes. In particular, the method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening.

  5. Exposure to extremely low frequency electromagnetic fields alters the calcium dynamics of cultured entorhinal cortex neurons.

    Science.gov (United States)

    Luo, Fen-Lan; Yang, Nian; He, Chao; Li, Hong-Li; Li, Chao; Chen, Fang; Xiong, Jia-Xiang; Hu, Zhi-An; Zhang, Jun

    2014-11-01

    Previous studies have revealed that extremely low frequency electromagnetic field (ELF-EMF) exposure affects neuronal dendritic spine density and NMDAR and AMPAR subunit expressions in the entorhinal cortex (EC). Although calcium signaling has a critical role in control of EC neuronal functions, however, it is still unclear whether the ELF-EMF exposure affects the EC neuronal calcium homeostasis. In the present study, using whole-cell recording and calcium imaging, we record the whole-cell inward currents that contain the voltage-gated calcium currents and show that ELF-EMF (50Hz, 1mT or 3mT, lasting 24h) exposure does not influence these currents. Next, we specifically isolate the high-voltage activated (HVA) and low-voltage activated (LVA) calcium channels-induced currents. Similarly, the activation and inactivation characteristics of these membrane calcium channels are also not influenced by ELF-EMF. Importantly, ELF-EMF exposure reduces the maximum amplitude of the high-K(+)-evoked calcium elevation in EC neurons, which is abolished by thapsigargin, a Ca(2+) ATPase inhibitor, to empty the intracellular calcium stores of EC neurons. Together, these findings indicate that ELF-EMF exposure specifically influences the intracellular calcium dynamics of cultural EC neurons via a calcium channel-independent mechanism.

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

    Science.gov (United States)

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

    2016-10-01

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

  7. Ist2 in the yeast cortical endoplasmic reticulum promotes trafficking of the amino acid transporter Bap2 to the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Wendelin Wolf

    Full Text Available The equipment of the plasma membrane in Saccharomyces cerevisiae with specific nutrient transporters is highly regulated by transcription, translation and protein trafficking allowing growth in changing environments. The activity of these transporters depends on a H(+ gradient across the plasma membrane generated by the H(+-ATPase Pma1. We found that the polytopic membrane protein Ist2 in the cortical endoplasmic reticulum (ER is required for efficient leucine uptake during the transition from fermentation to respiration. Experiments employing tandem fluorescence timer protein tag showed that Ist2 was necessary for efficient trafficking of newly synthesized leucine transporter Bap2 from the ER to the plasma membrane. This finding explains the growth defect of ist2Δ mutants during nutritional challenges and illustrates the important role of physical coupling between cortical ER and plasma membrane.

  8. The mechanism of hetero-synaptic interaction based on spatiotemporal intracellular calcium dynamics.

    Directory of Open Access Journals (Sweden)

    Daiki Futagi

    2014-03-01

    Full Text Available In recent physiological experiments focusing on synaptic plasticity, it is shown that synaptic modifications induced at one synapse are accompanied by hetero-synaptic changes at neighbor sites (Bi, 2002. These evidences imply that the hetero-synaptic interaction plays an important role in reconfiguration of synaptic connections to form and maintain functional neural circuits (Takahashi et al., 2012. Although the mechanism of the interaction is still unclear, some physiological studies suggest that the hetero-synaptic interaction could be caused by propagation of intracellular calcium signals (Nishiyama et al., 2000. Concretely, a spike-triggered calcium increase initiates calcium ion propagation along a dendrite through activation of molecular processes at neighboring sites. Here we hypothesized that the mechanism of the hetero-synaptic interaction was based on the intracellular calcium signaling, which is regulated by interactions between NMDA receptors (NMDARs, voltage-dependent calcium channels (VDCCs and Ryanodine receptors (RyRs on endoplasmic reticulum (ER. To assess realizability of the hypothesized interaction mechanism, we simulated intracellular calcium dynamics at a cellular level, using the computational model that integrated the model of intracellular calcium dynamics (Keizer and Levine, 1996 and the multi-compartment neuron model (Poirazi et al., 2003. Using the proposed computational model, we induced calcium influxes at a local site in postsynaptic dendrite by controlling the spike timings of pre- and postsynaptic neurons. As a result, synchronized calcium influxes through NMDARs and VDCCs caused calcium release from ER. According to the phase plane analysis, RyR-mediated calcium release occurred when the calcium concentration in cytoplasm sufficiently increased under the condition of a high calcium concentration in ER. An NMDAR-mediated calcium influx was slow and persistent, consequently responsible for maintaining a high

  9. Lipid Transport between the Endoplasmic Reticulum and Mitochondria

    Science.gov (United States)

    Flis, Vid V.

    2013-01-01

    Mitochondria are partially autonomous organelles that depend on the import of certain proteins and lipids to maintain cell survival and membrane formation. Although phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine are synthesized by mitochondrial enzymes, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and sterols need to be imported from other organelles. The origin of most lipids imported into mitochondria is the endoplasmic reticulum, which requires interaction of these two subcellular compartments. Recently, protein complexes that are involved in membrane contact between endoplasmic reticulum and mitochondria were identified, but their role in lipid transport is still unclear. In the present review, we describe components involved in lipid translocation between the endoplasmic reticulum and mitochondria and discuss functional as well as regulatory aspects that are important for lipid homeostasis. PMID:23732475

  10. Role of the Na+/K+-ATPase in regulating the membrane potential in rat peritoneal mast cells.

    Science.gov (United States)

    Friis, U G; Praetorius, H A; Knudsen, T; Johansen, T

    1997-10-01

    1. The aim of this study was to investigate the effect of the Na+/K+-ATPase on the membrane potential of peritoneal mast cells isolated from male Sprague-Dawley SPF-rats. 2. Experiments were performed at 22-26 degrees C in the tight-seal whole-cell configuration of the patch-clamp technique by use of Sylgard-coated patch pipettes (3-6 M[omega]). High-resolution membrane currents were recorded with an EPC-9 patch-clamp amplifier controlled by the 'E9SCREEN' software. In addition, a charting programme on another computer synchronously recorded at low resolution (2 Hz) membrane potential and holding current (low-pass filtered at 500 Hz). 3. Na+/K+-ATPase activity was measured as the ouabain-sensitive change in the zero-current potential. The zero-current potential in rat peritoneal mast cells measured 2 min after obtaining whole-cell configuration amounted to 1.7 +/- 2.5 mV (n = 21). Ouabain (5 mM), a Na+/K+-ATPase-inhibitor, had only a very minor effect upon the membrane potential under resting conditions (n = 3). 4. When mast cells were superfused with nominal calcium-free external solution, the cells hyperpolarized (delta mV: 20.2 +/- 3.8 mV (n = 5)). In addition, when the mast cells were preincubated in nominal calcium-free external solution for 12 +/- 1.6 min before whole-cell configuration, the membrane potential amounted to -53.7 +/- 9.8 mV (n = 8). A subsequent superfusion with ouabain (5 mM) depolarized the membrane potential (ouabain-sensitive hyperpolarization (delta mV): 23.0 +/- 8.4 mV (n = 8)). 5. A high intracellular concentration of Na+ ([Na+]i) (26.6 mM) also resulted in hyperpolarization (delta mV: 20.2 +/- 9.1 mV (n = 7)), but only when ATP was present. A subsequent superfusion with ouabain (5 mM) repolarized these cells to -1.2 +/- 14 mV (ouabain-sensitive hyperpolarization (delta mV): 19.7 +/- 7.7 mV (n = 7)). 6. The size of the [Na+]i-dependent hyperpolarization was dose-dependent. Low [Na+]i (1 mM) had no effect on membrane potential and these

  11. Calcium-dependent Dimerization of Human Soluble Calcium Activated Nucleotidase: Characterization of the Dimer Interface

    Energy Technology Data Exchange (ETDEWEB)

    Yang,M.; Horii, K.; Herr, A.; Kirley, T.

    2006-01-01

    Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile{sup 170}, Ser{sup 172}, and Ser{sup 226} with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys{sup 30}, located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi.

  12. Regulatory Mechanisms in the P4-ATPase Complex

    DEFF Research Database (Denmark)

    Costa, Sara

    Eukaryotic cell membranes are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Several relevant physiological processes depend on trans-bilayer phospholipid asymmetry, including vesiculation...... of their activity and regulation remain to be elucidated. Therefore, these studies focus on the role of the catalytic and CDC50 β-subunit in the phospholipid translocation and the regulation processes behind it. Recent studies suggested that P4-ATPase complex functionality is highly dependent on the conformation...... autoinhibitory domains in both N- and C-terminus of the P4-ATPase catalytic subunit from yeast. They regulate flippase activity in a coordinated manner which suggests the presence of a cross-talk between both protein termini. Furthermore, characterizing P4-ATPase activity is not a trivial task...

  13. Characterization of ATPase Activity of Recombinant Human Pif1

    Institute of Scientific and Technical Information of China (English)

    Yu HUANG; Deng-Hong ZHANG; Jin-Qiu ZHOU

    2006-01-01

    Saccharomyces cerevisiae Pif1p helicase is the founding member of the Pif1 subfamily that is conserved from yeast to human. The potential human homolog of the yeast PIF1 gene has been cloned from the cDNA library of the Hek293 cell line. Here, we described a purification procedure of glutathione Stransferase (GST)-fused N terminal truncated human Pif1 protein (hPif1△N) from yeast and characterized the enzymatic kinetics of its ATP hydrolysis activity. The ATPase activity of human Pif1 is dependent on divalent cation, such as Mg2+, Ca2+ and single-stranded DNA. Km for ATP for the ATPase activity is approximately 200 μM. As the ATPase activity is essential for hPif1's helicase activity, these results will facilitate the further investigation on hPif1.

  14. Vacuolar ATPase in phago(lyso)some biology.

    Science.gov (United States)

    Kissing, Sandra; Saftig, Paul; Haas, Albert

    2017-08-31

    Many eukaryotic cells ingest extracellular particles in a process termed phagocytosis which entails the generation of a new intracellular compartment, the phagosome. Phagosomes change their composition over time and this maturation process culminates in their fusion with acidic, hydrolase-rich lysosomes. During the maturation process, degradation and, when applicable, killing of the cargo may ensue. Many of the events that are pathologically relevant depend on strong acidification of phagosomes by the 'vacuolar' ATPase (V-ATPase). This protein complex acidifies the lumen of some intracellular compartments at the expense of ATP hydrolysis. We discuss here the roles and importance of V-ATPase in intracellular trafficking, its distribution, inhibition and activities, its role in the defense against microorganisms and the counteractivities of pathogens. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Glucose-6-phosphate Reduces Calcium Accumulation in Rat Brain Endoplasmic Reticulum

    Science.gov (United States)

    2012-04-01

    clinically relevant neu- ropathologies. For example, diabetes and hyperglycemia are well- documented to cause significantly worse outcome for patients who...enhanced in microsomes from liver, brain, and heart. Diabetes 47, 874. Dong, Z., Saikumar, P., Weinberg, J. M., and Venkatachalam, M. A. (2006...Pompella, A., and Benedetti, A. (1990). Glucose 6-phosphate stimulation of MgATP- dependent Ca2+ uptake by rat kid - ney microsomes. Biochim. Biophys. Acta

  16. Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

    Directory of Open Access Journals (Sweden)

    Kazi Md Kamrul Huda

    Full Text Available BACKGROUND: Plasma membrane Ca(2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+ from the cell, hence regulating Ca(2+ level within cells. Though plant Ca(2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. RESULTS: The 1478 bp promoter sequence of rice plasma membrane Ca(2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. CONCLUSIONS: The rice plasma membrane Ca(2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the

  17. Study on the changes in the levels of membrane-bound ATPases ...

    African Journals Online (AJOL)

    USER

    Significant (p<0.05) decline in the activities of membrane-bound ATPases and mineral status was observed .... important to cell function and integrity. λ cyhalothrin- induced .... of plasma membrane magnesium ion ATPase activities in normal.

  18. Ultracytochemical Localization and Functional Analysis of ATPase During the Endosperm Development in Oryza sativa L.

    Institute of Scientific and Technical Information of China (English)

    WEI Cun-xu; LAN Sheng-yin; XU Zhen-xiu

    2003-01-01

    Ultracytochemical localization of ATPase during development of rice endosperm was performed using a lead phosphate precipitation technique. The results indicated that, at the coenocyte and ceilularization stages, active ATPase was mainly distributed in an embryo sac wall, nucleus, and plasma membrane. At the early stage of development and differentiation, active ATPase was observed in the plasma membrane. At the grain filling stage, ATPase was highly active in the plasma membrane, intercellular space, and plasmodesmata in aleurone, moderately active on the plasma membrane in subaleurone. In starchy endosperm, ATPase was localized in the plasma membrane and degenerated nucleus. ATPase activity also appeared around vacuole and protein body in endosperm cell. The relationships between the ultracytochemical localization of ATPase and its function during the development of rice endosperm were discussed. Overall, ATPase was involved in the process of nutrition absorption and protein synthesis.

  19. Access of torsinA to the inner nuclear membrane is activity dependent and regulated in the endoplasmic reticulum

    Science.gov (United States)

    Goodchild, Rose E.; Buchwalter, Abigail L.; Naismith, Teresa V.; Holbrook, Kristen; Billion, Karolien; Dauer, William T.; Liang, Chun-Chi; Dear, Mary Lynn; Hanson, Phyllis I.

    2015-01-01

    ABSTRACT TorsinA (also known as torsin-1A) is a membrane-embedded AAA+ ATPase that has an important role in the nuclear envelope lumen. However, most torsinA is localized in the peripheral endoplasmic reticulum (ER) lumen where it has a slow mobility that is incompatible with free equilibration between ER subdomains. We now find that nuclear-envelope-localized torsinA is present on the inner nuclear membrane (INM) and ask how torsinA reaches this subdomain. The ER system contains two transmembrane proteins, LAP1 and LULL1 (also known as TOR1AIP1 and TOR1AIP2, respectively), that reversibly co-assemble with and activate torsinA. Whereas LAP1 localizes on the INM, we show that LULL1 is in the peripheral ER and does not enter the INM. Paradoxically, interaction between torsinA and LULL1 in the ER targets torsinA to the INM. Native gel electrophoresis reveals torsinA oligomeric complexes that are destabilized by LULL1. Mutations in torsinA or LULL1 that inhibit ATPase activity reduce the access of torsinA to the INM. Furthermore, although LULL1 binds torsinA in the ER lumen, its effect on torsinA localization requires cytosolic-domain-mediated oligomerization. These data suggest that LULL1 oligomerizes to engage and transiently disassemble torsinA oligomers, and is thereby positioned to transduce cytoplasmic signals to the INM through torsinA. PMID:26092934

  20. Lack of conventional ATPase properties in CFTR chloride channel gating.

    Science.gov (United States)

    Schultz, B D; Bridges, R J; Frizzell, R A

    1996-05-01

    CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for

  1. Structural plasticity of the nuclear envelope and the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Sheval E. V.

    2014-09-01

    Full Text Available The nuclear envelope is a double membrane structure, continuous with endoplasmic reticulum, and the morphological organization of both these structures is quite conservative. However, nuclear envelope and endoplasmic reticulum demonstrate distinct structural plasticity, i. e., based on common organization, cells may form various non-canonical membrane structures that are observed only in specialized types of cells or appear in different pathologies. In this review, we will discuss the mechanisms of the biogenesis of such non-canonical structures, and the possible role of this plasticity in the development of pathological processes.

  2. Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation.

    Science.gov (United States)

    Stolz, Alexandra; Besser, Stefanie; Hottmann, Heike; Wolf, Dieter H

    2013-09-17

    Quality control and degradation of misfolded proteins are essential processes of all cells. The endoplasmic reticulum (ER) is the entry site of proteins into the secretory pathway in which protein folding occurs and terminally misfolded proteins are recognized and retrotranslocated across the ER membrane into the cytosol. Here, proteins undergo polyubiquitination by one of the membrane-embedded ubiquitin ligases, in yeast Hrd1/Der3 (HMG-CoA reductase degradation/degradation of the ER) and Doa10 (degradation of alpha), and are degraded by the proteasome. In this study, we identify cytosolic Ubr1 (E3 ubiquitin ligase, N-recognin) as an additional ubiquitin ligase that can participate in ER-associated protein degradation (ERAD) in yeast. We show that two polytopic ERAD substrates, mutated transporter of the mating type a pheromone, Ste6* (sterile), and cystic fibrosis transmembrane conductance regulator, undergo Ubr1-dependent degradation in the presence and absence of the canonical ER ubiquitin ligases. Whereas in the case of Ste6* Ubr1 is specifically required under stress conditions such as heat or ethanol or in the absence of the canonical ER ligases, efficient degradation of human cystic fibrosis transmembrane conductance regulator requires function of Ubr1 already in wild-type cells under standard growth conditions. Together with the Hsp70 (heat shock protein) chaperone Ssa1 (stress-seventy subfamily A) and the AAA-type ATPase Cdc48 (cell division cycle), Ubr1 directs the substrate to proteasomal degradation. These data unravel another layer of complexity in ERAD.

  3. Upregulation of the SERCA-type Ca2+ pump activity in response to endoplasmic reticulum stress in PC12 cells

    Directory of Open Access Journals (Sweden)

    Frandsen Aase

    2001-04-01

    Full Text Available Abstract Background Ca2+-ATPases of endoplasmic reticulum (SERCAs are responsible for maintenance of the micro- to millimolar Ca2+ ion concentrations within the endoplasmic reticulum (ER of eukaryotic cells. This intralumenal Ca2+ storage is important for the generation of Ca2+ signals as well as for the correct folding and posttranslational processing of proteins entering ER after synthesis. ER perturbations such as depletion of Ca2+ or abolishing the oxidative potential, inhibition of glycosylation, or block of secretory pathway, activate the Unfolded Protein Response, consisting of an upregulation of a number of ER-resident chaperones/stress proteins in an effort to boost the impaired folding capacity. Results We show here that in PC12 cells, depletion of ER Ca2+ by EGTA, as well as inhibition of disulphide bridge formation within the ER by dithiotreitol or inhibition of N-glycosylation by tunicamycin, led to a 2- to 3-fold increase of the SERCA-mediated 45Ca2+ transport to microsomes isolated from cells exposed to these stress agents. The time course of this response corresponded to that for transcriptional upregulation of ER stress proteins, as well as to the increase in the SERCA2b mRNA, as we recently observed in an independent study. Conclusions These findings provide the first functional evidence for the increase of SERCA pumping capacity in cells subjected to the ER stress. Since at least three different and unrelated mechanisms of eliciting the ER stress response were found to cause this functional upregulation of Ca2+ transport into the ER, these results support the existence of a coupling between the induction of the UPR pathway in general, and the regulation of expression of at least one of the SERCA pump isoforms.

  4. Autoinhibitory Regulation of Plasma Membrane H+-ATPases

    DEFF Research Database (Denmark)

    Pedersen, Jesper Torbøl

    Electrochemical gradients across cell membranes are essential for nutrient uptake. In plant and fungal cells the electrochemical gradient across the plasma membrane (PM) can build much higher than in mammalian cells. The protein responsible for this gradient is the essential PM H+-ATPase that uses...... in mammalian cells and it has been speculated if they have a similar function in plants. In this thesis we show, that plant PM H+-ATPases are receptors for lysophospholipids and the autoinhibitory terminal inhibition is released upon lysophospholipid binding. Finally, we have used a group of stabilizing...

  5. Regulation of the Plasma Membrane H+-ATPase

    DEFF Research Database (Denmark)

    Falhof, Janus

    and that their green algae ancestor does not have this mechanism of regulation. Thisindicates that with the development of more complex organisms the need for a tighter regulation ofthe H+- ATPase activity has also evolved.Since the late 1980`s it have been known that lysophospholipids upregulate the activity...... and are generally perceived to be present in Archaea, algae, fungi and plants. The everexpanding sequence databases give us an opportunity to reevaluate our current knowledge of theevolutionary origin of PM H+-ATPases. In a phylogenetic study we found that a number of bacterialsequences grouped together with known...

  6. H+ countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes.

    Science.gov (United States)

    Yu, X; Carroll, S; Rigaud, J L; Inesi, G

    1993-04-01

    The Ca2+ transport adenosine triphosphatase of sarcoplasmic reticulum was reconstituted in unilamellar liposomes prepared by reverse-phase evaporation. The size of the resulting proteoliposomes was similar to that of native sarcoplasmic reticulum vesicles, but their protein content was much lower, with a protein/lipid ratio (wt/wt) of 1:40-160, as compared with 1:1 in the native membrane. The proteoliposomes sustained adenosine triphosphate-dependent Ca2+ uptake at rates proportional to the protein content (1-2 mumol Ca2+/mg protein/min), reaching asymptotic levels corresponding to a lumenal calcium concentration of 10-20 mM. The low permeability of the proteoliposomes permitted direct demonstration of Ca2+/H+ countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of one H+ per one Ca2+ was demonstrated, and inhibition of the Ca2+ pump by lumenal alkalinization was relieved by the H+ ionophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Consistent with the countertransport stoichiometry, net positive charge displacement was produced by Ca2+ transport, as revealed by a rapid oxonol VI absorption rise. The initial rise and the following steady-state level of oxonol absorption were highest when SO4(2-) was the prevalent anion and lowest in the presence of the lipophilic anion SCN-. The influence of anions was attributed to potential driven counterion compensation. The absorption rise was rapidly collapsed by addition of valinomycin in the presence of K+. Experimentation with Ca2+ and H+ ionophores was consistent with a primary role of Ca2+ and H+ in net charge displacement. The estimated value of the steady-state electrical potential observed under optimal conditions was approximately 50 mV and was accounted for by the estimated charge transfer associated with Ca2+ and H+ countertransport under the same conditions.

  7. Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

    Science.gov (United States)

    Restrepo, Simon; Basler, Konrad

    2016-08-01

    Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. We probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Here we discover that wing discs display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. Our results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies.

  8. Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves.

    Science.gov (United States)

    Restrepo, Simon; Basler, Konrad

    2016-08-09

    Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. We probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Here we discover that wing discs display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. Our results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies.

  9. Structure determination using poorly diffracting membrane-protein crystals: the H+-ATPase and Na+,K+-ATPase case history.

    Science.gov (United States)

    Pedersen, Bjørn P; Morth, J Preben; Nissen, Poul

    2010-03-01

    An approach is presented for the structure determination of membrane proteins on the basis of poorly diffracting crystals which exploits molecular replacement for heavy-atom site identification at 6-9 A maximum resolution and improvement of the heavy-atom-derived phases by multi-crystal averaging using quasi-isomorphous data sets. The multi-crystal averaging procedure allows real-space density averaging followed by phase combination between non-isomorphous native data sets to exploit crystal-to-crystal nonisomorphism despite the crystals belonging to the same space group. This approach has been used in the structure determination of H(+)-ATPase and Na(+),K(+)-ATPase using Ca(2+)-ATPase models and its successful application to the Mhp1 symporter using LeuT as a search model is demonstrated.

  10. Surface vacuolar ATPase in ameloblastoma contributes to tumor invasion of the jaw bone.

    Science.gov (United States)

    Yoshimoto, Shohei; Morita, Hiromitsu; Matsubara, Ryota; Mitsuyasu, Takeshi; Imai, Yuko; Kajioka, Shunichi; Yoneda, Masahiro; Ito, Yushi; Hirofuji, Takao; Nakamura, Seiji; Hirata, Masato

    2016-03-01

    Ameloblastoma is the most common benign odontogenic tumor in Japan. It is believed that it expands in the jaw bone through peritumoral activation of osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) released from the ameloblastoma, as in bone metastases of cancer cells. However, the clinical features of ameloblastoma, including its growth rate and patterns of invasion, are quite different from those of bone metastasis of cancer cells, suggesting that different underlying mechanisms are involved. Therefore, in the present study, we examined the possible mechanisms underlying the invasive expansion of ameloblastoma in the jaw bone. Expression levels of RANKL assessed by western blotting were markedly lower in ameloblastoma (AM-1) cells than in highly metastatic oral squamous cell carcinoma (HSC-3) cells. Experiments coculturing mouse macrophages (RAW264.7) with AM-1 demonstrated low osteoclastogenic activity, as assessed by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell formation, probably because of low release of RANKL, whereas cocultures of RAW264.7 with HSC-3 cells exhibited very high osteoclastogenic activity. Thus, RANKL release from AM-1 appeared to be too low to generate osteoclasts. However, AM-1 cultured directly on calcium phosphate-coated plates formed resorption pits, and this was inhibited by application of bafilomycin A1. Furthermore, vacuolar-type H+-ATPase (V-ATPase) and H+/Cl- exchange transporter 7 (CLC-7) were detected on the surface of AM-1 cells by plasma membrane biotinylation and immunofluorescence analysis. Immunohistochemical analysis of clinical samples of ameloblastoma also showed plasma membrane-localized V-ATPase and CLC-7 in the epithelium of plexiform, follicular and basal cell types. The demineralization activity of AM-1 was only 1.7% of osteoclasts demineralization activity, and the growth rate was 20% of human normal skin keratinocytes and HSC-3 cells. These results suggest that the

  11. Elucidation of the topography of the thapsigargin binding site in the sarco-endoplasmic calcium ATPase

    DEFF Research Database (Denmark)

    Skytte, Dorthe Mondrup; Møller, Jesper Vuust; Liu, Huizhen;

    2010-01-01

    of voluminous stiff residues caused dramatic reduction of the affinity. The results can be rationalized on the basis of the interactions of thapsigargin with the SERCA1a pump as revealed from 3D X-ray structural models of thapsigargin bound to the SERCA1a. In conclusion the results confirm and elaborate...

  12. Na+,K+-ATPase Na+ affinity in rat skeletal muscle fiber types

    DEFF Research Database (Denmark)

    Kristensen, Michael; Juel, Carsten

    2010-01-01

    Previous studies in expression systems have found different ion activation of the Na(+)/K(+)-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na(+),K(+)-ATPase activity, and the Na(+) affinity of Na(+),K(+)-ATPase...

  13. Subunit composition, structure, and distribution of bacterial V-type ATPases

    NARCIS (Netherlands)

    Lolkema, Juke S.; Chaban, Yuriy; Boekema, Egbert J.

    The overall structure of V-ATPase complexes resembles that of F-type ATPases, but the stalk region is different and more complex. Database searches followed by sequence analysis of the five water-soluble stalk region subunits C-G revealed that (i) to date V-ATPases are found in 16 bacterial species,

  14. Excess capacity of H+ ATPase and inverse respiratory control in Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Westerhoff, Hans V.; Michelsen, Ole

    1993-01-01

    the growth rate much less than proportionally; the H+-ATPase controlled growth rate by lt 10%. This lack of control reflected excess capacity: the rate of ATP synthesis per H+-ATPase (the turnover number) increased by 60% when the number of enzymes was decreased by 40%. At 15% H+-ATPase, the enzyme became...

  15. Structural and functional studies of a Cu+-ATPase from Legionella pneumophila

    DEFF Research Database (Denmark)

    Mattle, Daniel

    During his studies, Daniel Mattle explored the copper(I) export mechanism of a P-type Cu+ ATPase from Legionella pneumophila – a homologue to the human Cu+ ATPases. Cu+ ATPases are responsible for the homeostatic control of the physiological relevant – but toxic – copper(I) cations. To assess...

  16. Integumentary loss of calcium.

    Science.gov (United States)

    Chu, J Y; Margen, S; Calloway, D H; Costa, F M

    1979-08-01

    Integumentary calcium loss was studied in 16 healthy young men. The daily loss by the 16 ambulatory but relatively sedentary young men in 52 determinations of 6-day periods each was 8.7 +/- 1.9 mg/m2 per day (average 15.8 mg/man per day). The amount lost was not influenced by calcium intake (0.1 to 2.3 g/day). In contrast to urinary calcium excretion, which is directly related to protein intake, there was no significant change in integumentary calcium loss with varying protein intakes (1 to 96 g nitrogen per day). No compensatory relationship between urinary and integumentary calcium excretion was noted. During strenuous exercise calcium loss increased to an average of 25 mg in 40 min. There was no compensatory decrease in urinary excretion on the day of strenuous exercise. It was also noted that integumentary calcium loss was not affected by general calcium balance.

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

    Science.gov (United States)

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

    2015-04-01

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

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

    Science.gov (United States)

    Lewis, Richard S

    2007-03-15

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

  19. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe

    2011-01-01

    Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary...... transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na...

  20. Calcium - Function and effects

    NARCIS (Netherlands)

    Liang, Jianfen; He, Yifan; Gao, Qian; Wang, Xuan; Nout, M.J.R.

    2016-01-01

    Rice is the primary food source for more than half of the world population. Levels of calcium contents and inhibitor - phytic acid are summarized in this chapter. Phytic acid has a very strong chelating ability and it is the main inhibit factor for calcium in rice products. Calcium contents in br

  1. Calcium en cardioplegie

    NARCIS (Netherlands)

    Ruigrok, T.J.C.; Meijler, F.L.

    1985-01-01

    Coronary perfusion with a calcium-free solution, followed by reperfusion with a calcium containing solution, may result in acute myocardial cell death and in irreversible loss of the e1ectrical and mechanical activity of the heart. This phenomenon is known as the calcium paradox. A number of cardiop

  2. Folding of viral envelope glycoproteins in the endoplasmic reticulum

    NARCIS (Netherlands)

    Braakman, L.J.; Anken, E. van

    2000-01-01

    Viral glycoproteins fold and oligomerize in the endoplasmic reticulum of the host cell. They employ the cellular machinery and receive assistance from cellular folding factors. During the folding process, they are retained in the compartment and their structural quality is checked by the quality con

  3. A luminal flavoprotein in endoplasmic reticulum-associated degradation

    DEFF Research Database (Denmark)

    Riemer, Jan; Appenzeller-Herzog, Christian; Johansson, Linda

    2009-01-01

    The quality control system of the endoplasmic reticulum (ER) discriminates between native and nonnative proteins. The latter are degraded by the ER-associated degradation (ERAD) pathway. Whereas many cytosolic and membrane components of this system are known, only few luminal players have been id...

  4. Structural changes of CF 1-ATPase in solution

    Science.gov (United States)

    Calmettes, P.; Pezennec, S.; Berger, G.; Girault, G.

    1992-06-01

    Small changes in neutron scattering spectra were observed when spinach CF 1-ATPase is activated by dithiothreitol or allowed to bind to a nucleotide. It is shown that activation induces a significant conformation change whereas substrate binding does not. In solution, MgATP or MgAMP mainly modifies the interactions between the solute molecules.

  5. On the Thermodynamic Efficiency of Ca2+-ATPase Molecular Machines

    NARCIS (Netherlands)

    Lervik, A.; Bresme, F.; Kjelstrup, S.H.; Rubi, J.M.

    2012-01-01

    Experimental studies have shown that the activity of the reconstituted molecular pump Ca2+-ATPase strongly depends on the thickness of the supporting bilayer. It is thus expected that the bilayer structure will have an impact on the thermodynamic efficiency of this nanomachine. Here, we introduce a

  6. Autoinhibitory Regulation of Plasma Membrane H+-ATPases

    DEFF Research Database (Denmark)

    Pedersen, Jesper Torbøl

    Electrochemical gradients across cell membranes are essential for nutrient uptake. In plant and fungal cells the electrochemical gradient across the plasma membrane (PM) can build much higher than in mammalian cells. The protein responsible for this gradient is the essential PM H+-ATPase that uses...

  7. V-ATPase, ScNhxlp and Yeast Vacuole Fusion

    Institute of Scientific and Technical Information of China (English)

    Quan-Sheng Qiu

    2012-01-01

    Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos.It is a central cellular reaction that plays important roles in signal transduction,protein sorting and subcellular compartmentation.Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summanzed in this article.It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhxlp are key components of the vacuole fusion machinery in yeast.Yeast ScNhxlp regulates vacuole fusion by controlling the luminal pH.V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast.Fission defects are epistatic to fusion defects.Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast,the fusion reaction does not need the transport activity but requires the physical presence of the proton pump.Vo,the membrane-integral sector of the V-ATPase,forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the Vo trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.

  8. Roles and mechanisms of copper transporting ATPases in cancer pathogenesis.

    Science.gov (United States)

    Zhang, Yuqing; Li, Min; Yao, Qizhi; Chen, Changyi

    2009-01-01

    Copper (Cu) is an essential trace element for cell metabolism as a cofactor to many key metabolic enzymes. Numerous physiological processes rely on the adequate and timely transport of copper ions mediated by copper-transporting ATPases (Cu-ATPases), which are essential for human cell growth and development. Inherited gene mutations of ATP7A and ATP7B result in clinical diseases related to damage in the multiple organ systems. Increased expression of these genes has been recently observed in some human cancer specimens, and may be associated with tumorigenesis and chemotherapy resistance. However, underlying mechanisms of Cu-ATPases in human cancer progression and treatment are largely unknown. In this review, we summarize current progress on the copper transport system, the structural and functional properties of the Cu-ATPases, ATP7A and ATP7B, in copper homeostasis, and their roles in anti-tumor drug resistance and cancer metastasis. This review provides valuable information for clinicians and researchers who want to recognize the newest advances in this new field and identify possible lines of investigation in copper transport as important mediators in human physiology and cancer.

  9. Characterization and ATPase activity of human platelet actomyosin

    NARCIS (Netherlands)

    Lindemans, J.; Bouma, B.N.; Sixma, J.J.

    1974-01-01

    Platelet actomyosin, partially purified by successive precipitation had a specific viscosity of 0,15 and a sensitivity to ATP of 60 %. The enzyme preparation was separated into the actin and myosin components and some myosin fragments by SDS-polyacrylamide gel electrophoresis. The ATPase activity of

  10. Pharmacological evidence that potentiation of plasmalemmal Ca(2+)-extrusion is functionally coupled to inhibition of SR Ca(2+)-ATPases in vascular smooth muscle cells.

    Science.gov (United States)

    Zhang, Wen-Bo; Kwan, Chiu-Yin

    2016-04-01

    Cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic reticulum (SR) Ca(2+)-ATPases, causes slowly developing and subsequently diminishing characteristic contractions in vascular smooth muscle, and the second application of CPA has incompletely repeatable effects, depending on the vessel type. The objective of the present study was to examine the mechanisms underlying the significant decrease of CPA-induced contractions upon the second application. A pharmacological intervention of Ca(2+) extrusion process as a strategy was performed to modulate vasoconstrictor effects of CPA in rat aortic ring preparations. CPA-induced contractions, expressed as percentages of the contractions induced by KCl (80 mM), were significantly decreased from 44.1 ± 5.7 to 7.6 ± 1.8 % (P Ca(2+) exchangers, but not of KBR7943, an inhibitor of the reverse mode of Na(+)/Ca(2+) exchangers. Our findings indicate that CPA by inducing a transient rise in cytosolic Ca(2+) level causes a long-lasting upregulation of plasma membrane (PM) Ca(2+) extruders and thus leads to a diminished contraction upon its second application in blood vessels. This suggests that there is a functional coupling between PM Ca(2+) extruders and SR Ca(2+)-ATPases in rat aortic smooth muscle cells.

  11. bcpmr1 encodes a P-type Ca(2+)/Mn(2+)-ATPase mediating cell-wall integrity and virulence in the phytopathogen Botrytis cinerea.

    Science.gov (United States)

    Plaza, Verónica; Lagües, Yanssuy; Carvajal, Mauro; Pérez-García, Luis A; Mora-Montes, Hector M; Canessa, Paulo; Larrondo, Luis F; Castillo, Luis

    2015-03-01

    The cell wall of fungi is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. These usually contain both N- and O-linked oligosaccharides, coupled to the proteins by stepwise addition of mannose residues by mannosyltransferases in the endoplasmic reticulum and the Golgi apparatus. In yeast, an essential luminal cofactor for these mannosyltransferases is Mn(2+) provided by the Ca(2+)/Mn(2+)-ATPase known as Pmr1. In this study, we have identified and characterized the Botrytis cinerea pmr1 gene, the closest homolog of yeast PMR1. We hypothesized that bcpmr1 also encodes a Ca(2+)/Mn(2+)-ATPase that plays an important role in the protein glycosylation pathway. Phenotypic analysis showed that bcpmr1 null mutants displayed a significant reduction in conidial production, radial growth and diameter of sclerotia. Significant alterations in hyphal cell wall composition were observed including a 83% decrease of mannan levels and an increase in the amount of chitin and glucan. These changes were accompanied by a hypersensitivity to cell wall-perturbing agents such as Calcofluor white, Congo red and zymolyase. Importantly, the Δbcpmr1 mutant showed reduced virulence in tomato (leafs and fruits) and apple (fruits) and reduced biofilm formation. Together, our results highlight the importance of bcpmr1 for protein glycosylation, cell wall structure and virulence of B. cinerea.

  12. Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum

    Science.gov (United States)

    Ferguson, Scott A.; Cook, Gregory M.; Montgomery, Martin G.; Leslie, Andrew G. W.

    2016-01-01

    The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a “down” state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an “up” state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme’s hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual “open” conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis. PMID:27621435

  13. Functional coupling of V-ATPase and CLC-5

    Science.gov (United States)

    Satoh, Nobuhiko; Suzuki, Masashi; Nakamura, Motonobu; Suzuki, Atsushi; Horita, Shoko; Seki, George; Moriya, Kyoji

    2017-01-01

    Dent’s disease is an X-linked renal tubulopathy characterized by low molecular weight proteinuria, hypercalciuria and progressive renal failure. Disease aetiology is associated with mutations in the CLCN5 gene coding for the electrogenic 2Cl-/H+ antiporter chloride channel 5 (CLC-5), which is expressed in the apical endosomes of renal proximal tubules with the vacuolar type H+-ATPase (V-ATPase). Initially identified as a member of the CLC family of Cl- channels, CLC-5 was presumed to provide Cl- shunt into the endosomal lumen to dissipate H+ accumulation by V-ATPase, thereby facilitating efficient endosomal acidification. However, recent findings showing that CLC-5 is in fact not a Cl- channel but a 2Cl-/H+ antiporter challenged this classical shunt model, leading to a renewed and intense debate on its physiological roles. Cl- accumulation via CLC-5 is predicted to play a critical role in endocytosis, as illustrated in mice carrying an artificial Cl- channel mutation E211A that developed defective endocytosis but normal endosomal acidification. Conversely, a recent functional analysis of a newly identified disease-causing Cl- channel mutation E211Q in a patient with typical Dent’s disease confirmed the functional coupling between V-ATPase and CLC-5 in endosomal acidification, lending support to the classical shunt model. In this editorial, we will address the current recognition of the physiological role of CLC-5 with a specific focus on the functional coupling of V-ATPase and CLC-5. PMID:28101447

  14. Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival

    NARCIS (Netherlands)

    Sun, S.; Shi, Guojun; Han, X.; Francisco, A.; Ji, Y.; Kersten, A.H.

    2014-01-01

    Suppressor/Enhancer of Lin-12-like (Sel1L) is an adaptor protein for the E3 ligase hydroxymethylglutaryl reductase degradation protein 1 (Hrd1) involved in endoplasmic reticulum-associated degradation (ERAD). Sel1L’s physiological importance in mammalian ERAD, however, remains to be established. Her

  15. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance.

    Science.gov (United States)

    Schep, Daniel G; Zhao, Jianhua; Rubinstein, John L

    2016-03-22

    Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases.

  16. Endoplasmic reticulum stress and membranous nephropathy%内质网应激与膜性肾病

    Institute of Scientific and Technical Information of China (English)

    李静

    2011-01-01

    Endoplasmic reticulum ( ER) is an intracellular compartment that plays a critical role in the processing, folding and assembling of newly synthesized proteins. A scress-induced impairment of ER membrane integrity can be injurious by causing leakage of calcium ions and other ER luminal components and interfering with protein transport to Golgi apparatus. This in tum initiates the unfolded protein response ( UPR) . an integrated intracellular signaling pathway that responds to ER stress. As a defense mechanism,UPR can protect the cells from being impaired by ER stress. Also, UPR can activate pathways of cell death in response to prolonged or severe ER stress. This review highlights the current knowledge of ER stress in membranous nephropathy.%内质网(endoplasmic reticulum,ER)是调节蛋白质合成、折叠及组装的重要场所.各种原因如ER中Ca2+缺乏均可引起ER功能紊乱,使蛋白质从ER向高尔基体的转运受阻[1],最终引发内质网应激(endoplasmic reticulum stress,ERS).细胞通过激活未折叠蛋白反应(the unfolded protein response,UPR)保护ERS引起损伤的细胞,强烈或持久的ERS又可启动UPR的促凋亡信号.肾病尤其是膜性肾病(membranous nephropathy,MN)的发生发展与ERS密切相关,文中主要阐述ERS在MN发病机制中的作用.

  17. Calcium signaling and epilepsy.

    Science.gov (United States)

    Steinlein, Ortrud K

    2014-08-01

    Calcium signaling is involved in a multitude of physiological and pathophysiological mechanisms. Over the last decade, it has been increasingly recognized as an important factor in epileptogenesis, and it is becoming obvious that the excess synchronization of neurons that is characteristic for seizures can be linked to various calcium signaling pathways. These include immediate effects on membrane excitability by calcium influx through ion channels as well as delayed mechanisms that act through G-protein coupled pathways. Calcium signaling is able to cause hyperexcitability either by direct modulation of neuronal activity or indirectly through calcium-dependent gliotransmission. Furthermore, feedback mechanisms between mitochondrial calcium signaling and reactive oxygen species are able to cause neuronal cell death and seizures. Unravelling the complexity of calcium signaling in epileptogenesis is a daunting task, but it includes the promise to uncover formerly unknown targets for the development of new antiepileptic drugs.