Sample records for ca2 mg2 atpase

  1. Effects of aqueous extract of Hibiscus sabdariffa on renal Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in Wistar rats. (United States)

    Olatunji, Lawrence A; Usman, Taofeek O; Adebayo, Joseph O; Olatunji, Victoria A


    To investigate the effects of oral administration of aqueous extract of Hibiscus sabdariffa on renal Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in rats. The 25 and 50 mg/(kg·d) of aqueous extracts of H. sabdariffa were respectively given to rats in the experimental groups for 28 d, and rats in the control group received an appropriate volume of distilled water as vehicle. Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in the kidney were assayed by spectrophotometric method. Administrations of 25 and 50 mg/(kg·d) of aqueous extract of H. sabdariffa significantly decreased the Ca(2+)-Mg(2+)-ATPase activity in the kidney of rats (Psabdariffa may preserve the renal function despite a decreased renal Ca(2+)-Mg(2+)-ATPase activity.

  2. Purification of the synaptosomal plasma membrane (Ca(2+) + Mg(2+))-ATPase from pig brain. (United States)

    Salvador, J M; Mata, A M


    The Ca(2+)-ATPase from the synaptosomal plasma membrane has been purified nearly to homogeneity from pig brain by a new procedure involving the calmodulin-affinity-chromatography technique. This is a convenient alternative to the standard methods for the purification of the plasma membrane Ca(2+)-ATPase from different sources that were unsuitable to purify the enzyme from pig brain. The main feature of this procedure is the use of 15% (v/v) glycerol as stabilizing agent, instead of acidic phospholipid. By using this protocol the enzyme was purified 36-fold with respect to the plasma membrane vesicle fraction, showing a specific activity of 2.3 i.u. in the presence of acidic phospholipid. In SDS/PAGE, it appears as a single protein band around Mr140 000 that can be phosphorylated by [gamma-(32)P]ATP in the presence of La(3+) and recognized by specific antibodies against the plasma membrane Ca(2+)-ATPase from pig antral smooth muscle. Calmodulin activates the enzyme 1.5-1.8-fold in the presence of phosphatidylcholine but not in the presence of phosphatidylserine.

  3. Activity of Ca(2+,Mg(2+-ATPase of sarcoplasmic reticulum and contraction strength of the frog skeletal muscles under the effect of organophosphorus insecticides

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    D. M. Nozdrenko


    Full Text Available The results of an experimental study of organo­phosphorus insecticides, including pirimiphosmethyl, diazinon and chlorpyrifos caused a decline of the contraction properties in m. tibialis anterior fiber bundles of Rana temporaria, as well as sarcoplasmic reticulum Ca2+,Mg2+-ATPase enzymatic activity reduction are outlined in this paper. Concentration-dependent strengths response diminishing in isolated skeletal muscle fiber bundles as a result of non-cholinergic influence of organophosphorus insecticides were found. A decrease of Ca2+,Mg2+-ATPase enzymatic activity in sarcoplasmic reticulum was observed after administration of each insecticide. The most significant inhibition of this enzyme was observed when using chlorpyrifos.

  4. Ca2+- and Mg2+-ATPase activities in winter wheat root plasma membranes as affected by NaCl stress during growth

    NARCIS (Netherlands)

    Mansour, MMF; van Hasselt, PR; Kuiper, PJC

    Winter wheat seedlings were grown in Hoagland nutrient solution with or without 100 mmol/L NaCl added. Plasma membranes from root cells were prepared by aqueous polymer two phase partitioning and the stimulation of plasma membrane ATPase activity by Mg2+ and Ca2+ was investigated. The enzyme was

  5. Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs of Lymnaea stagnalis and Bithynia tentaculata (Mollusca)

    NARCIS (Netherlands)

    D. Zivkovic (Dana); R. Créton (Robbert); G. Zwaan (Gideon); W.C. de Bruijn (Wim); M.R. Dohmen (M.René)


    textabstractDuring extrusion of the first polar body in eggs of Lymnaea stagnalis and Bithynia tentaculata a localized Ca2+ /Mg2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity

  6. Increased oxidative stress and decreased activities of Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase in the red blood cells of the hibernating black bear. (United States)

    Chauhan, Ved P S; Tsiouris, John A; Chauhan, Abha; Sheikh, Ashfaq M; Brown, W Ted; Vaughan, Michael


    During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P hibernation (P hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.

  7. Increased oxidative stress and decreased activities of Ca2+/Mg2+-ATPase and Na+/K+-ATPase in the red blood cells of the hibernating black bear (United States)

    Chauhan, V.P.S.; Tsiouris, J.A.; Chauhan, A.; Sheikh, A.M.; Brown, W. Ted; Vaughan, M.


    During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P hibernation (P hibernating state as compared to the active state. Na+/K+-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca2+/Mg2+-ATPase and Na+/K+-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression. ?? 2002 Elsevier Science Inc. All rights reserved.

  8. The inhibitory influence of calix[4]arene of C-90 on the activity of Ca(2+,Mg(2+-ATPases in plasma membrane and sarcoplasmic reticulum in myometrium сells

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    T. O. Veklich


    Full Text Available Our study on the plasma membrane vesicles and myometrium cells treated with 0.1% digitonin showed that inhibitory effect of calix[4]arene C-90 (5,11,17,23-tetra(trifluoromethyl(phenylsulphonylimino-methylamino-25,26,27,28-tetrapropoxy-calix[4]arene on the plasma membrane Ca2+,Mg2+-ATPase was more significant than on the Ca2+,Mg2+-ATPase in sarcoplasmic reticulum (the inhibition coefficient I0.5 values were 20.2 ± 0.5 µM and 57.0 ± 1.4 µM for the plasma membrane Ca2+,Mg2+-ATPase and Ca2+,Mg2+-ATPase in sarcoplasmic reticulum, respectively (n = 5. Inhibition kinetics of calix[4]arene C-90 effect on the Ca2+,Mg2+-ATPase activities in plasma membrane and sarcoplasmic reticulum were studied. This substance inhibited both pumps as complete noncompetitive inhibitor. Calix[4]arene C-90 caused the increase of intracellular Ca2+ concentration and decrease of hydrodynamic diameter in smooth muscle cells similar to the action of uterotonic drug oxytocin.

  9. Effects of anthropogenic sound on digging behavior, metabolism, Ca2+/Mg2+ ATPase activity, and metabolism-related gene expression of the bivalve Sinonovacula constricta (United States)

    Peng, Chao; Zhao, Xinguo; Liu, Saixi; Shi, Wei; Han, Yu; Guo, Cheng; Jiang, Jingang; Wan, Haibo; Shen, Tiedong; Liu, Guangxu


    Anthropogenic sound has increased significantly in the past decade. However, only a few studies to date have investigated its effects on marine bivalves, with little known about the underlying physiological and molecular mechanisms. In the present study, the effects of different types, frequencies, and intensities of anthropogenic sounds on the digging behavior of razor clams (Sinonovacula constricta) were investigated. The results showed that variations in sound intensity induced deeper digging. Furthermore, anthropogenic sound exposure led to an alteration in the O:N ratios and the expression of ten metabolism-related genes from the glycolysis, fatty acid biosynthesis, tryptophan metabolism, and Tricarboxylic Acid Cycle (TCA cycle) pathways. Expression of all genes under investigation was induced upon exposure to anthropogenic sound at ~80 dB re 1 μPa and repressed at ~100 dB re 1 μPa sound. In addition, the activity of Ca2+/Mg2+-ATPase in the feet tissues, which is directly related to muscular contraction and subsequently to digging behavior, was also found to be affected by anthropogenic sound intensity. The findings suggest that sound may be perceived by bivalves as changes in the water particle motion and lead to the subsequent reactions detected in razor clams. PMID:27063002

  10. Sensing mechanisms involved in Ca2+ and Mg2+ homeostasis

    NARCIS (Netherlands)

    Ferre, S.; Hoenderop, J.G.J.; Bindels, R.J.M.


    Calcium (Ca(2+)) and magnesium (Mg(2+)) ions are involved in many vital physiological functions. In the human body, Ca(2+) and Mg(2+) homeostatic systems rely on three components: (i) tissues (re)absorbing or storing Ca(2+) and Mg(2+), mainly kidney, intestine, and bone; (ii) hormones that modulate

  11. Epithelial Ca2+ and Mg2+ channels in kidney disease.

    NARCIS (Netherlands)

    Thebault, S.C.; Hoenderop, J.G.J.; Bindels, R.J.M.


    Many physiological functions rely on the precise maintenance of body calcium (Ca2+) and magnesium (Mg2+) balance, which is tightly regulated by the concerted actions of intestinal absorption, renal reabsorption, and exchange with bone. The kidney plays an important role in the homeostasis of

  12. Studies of Ca2+ ATPase (SERCA) inhibition. (United States)

    Inesi, Giuseppe; Hua, Suming; Xu, Cheng; Ma, Hailun; Seth, Malini; Prasad, Anand M; Sumbilla, Carlota


    The Ca(2+) transport ATPase of intracellular membranes (SERCA) can be inhibited by a series of chemical compounds such as Thapsigargin (TG), 2,5-di(tert-butyl)hydroquinone (DBHQ) and 1,3-dibromo-2,4,6-tris (methyl-isothio-uronium) benzene (Br(2)-TITU). These compounds have specific binding sites in the ATPase protein, and different mechanisms of inhibition. On the other hand, SERCA gene silencing offers a convenient and specific method for suppression of SERCA activity in cells. The physiological and pharmacological implications of SERCA inhibition are discussed.

  13. Hereditary tubular transport disorders: implications for renal handling of Ca2+ and Mg2+.

    NARCIS (Netherlands)

    Dimke, H.; Hoenderop, J.G.J.; Bindels, R.J.M.


    The kidney plays an important role in maintaining the systemic Ca2+ and Mg2+ balance. Thus the renal reabsorptive capacity of these cations can be amended to adapt to disturbances in plasma Ca2+ and Mg2+ concentrations. The reabsorption of Ca2+ and Mg2+ is driven by transport of other electrolytes,

  14. Segmental transport of Ca(2)(+) and Mg(2)(+) along the gastrointestinal tract.

    NARCIS (Netherlands)

    Lameris, A.L.L.; Nevalainen, P.I.; Reijnen, D.; Simons, E.; Eygensteyn, J.; Monnens, L.A.; Bindels, R.J.M.; Hoenderop, J.G.J.


    Calcium (Ca(2+)) and magnesium (Mg(2+)) ions are involved in many vital physiological functions. Since dietary intake is the only source of minerals for the body, intestinal absorption is essential for normal homeostatic levels. The aim of this study was to characterize the absorption of Ca(2+) as

  15. Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator (United States)

    Pham, Khoa; Dhulipala, Gangadhar; Gonzalez, Walter G; Gerstman, Bernard S; Regmi, Chola; Chapagain, Prem P; Miksovska, Jaroslava


    Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca2+ concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo-state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca2+/Mg2+ association to DREAM. Our data indicate a minor impact of Ca2+ association on the overall structure of the N- and C-terminal domains, although Ca2+ binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca2+ bound forms of the protein. Time-resolved fluorescence data indicate that Ca2+binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca2+ association to EF-hand 3 and EF-hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca2+ and Mg2+ suggesting that, under physiological conditions, Ca2+ free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions. PMID:25627705

  16. Effects of Mg2+ and Ca2+ on photoinduced Euglena flagellar responses (United States)


    The flagellar frequency and waveform of Euglena were analyzed under full illumination (420-700 nm) and in a restricted wavelength band (530- 700 nm) when the cells were in a medium containing Mg2+ or had been microinjected with Mg2+, Mn2+, or Ca2+ in solution. Magnesium abolished the change in flagellar frequency and the reversal in waveform that cells exhibit when illuminated by a 530-700 nm wavelength band. Under this restricted illumination, Ca2+ caused an increase in flagellar waveform reversal and a decrease in beating frequency. The flagellar motility of cells impaled on a microelectrode was examined in cells illuminated with various wavelengths. PMID:6769928

  17. Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte (United States)

    Michailova, A.; McCulloch, A.


    We extended the model of the ventricular myocyte by Winslow et al. (Circ. Res 1999, 84:571-586) by incorporating equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). The results indicate that, under normal conditions, Ca(2+) binding by low-affinity ATP and diffusion of CaATP may affect the amplitude and time course of intracellular Ca(2+) signals. The model also suggests that a fall in ATP/ADP ratio significantly reduces sarcoplasmic Ca(2+) content, increases diastolic Ca(2+), lowers systolic Ca(2+), increases Ca(2+) influx through L-type channels, and decreases the efficiency of the Na(+)/Ca(2+) exchanger in extruding Ca(2+) during periodic voltage-clamp stimulation. The analysis suggests that the most important reason for these changes during metabolic inhibition is the down-regulation of the sarcoplasmic Ca(2+)-ATPase pump by reduced diastolic MgATP levels. High Ca(2+) concentrations developed near the membrane might have a greater influence on Mg(2+), ATP, and ADP concentrations than that of the lower Ca(2+) concentrations in the bulk myoplasm. The model predictions are in general agreement with experimental observations measured under normal and pathological conditions.

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

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    Delia I. Chiarello


    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.

  19. Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition. (United States)

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


    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

  20. Epithelial Ca2+ and Mg2+ channels in health and disease.

    NARCIS (Netherlands)

    Hoenderop, J.G.J.; Bindels, R.J.M.


    A near constancy of the extracellular Ca(2+) and Mg(2+) concentration is required for numerous physiologic functions at the organ, tissue, and cellular levels. This suggests that minor changes in the extracellular concentration of these divalents must be detected to allow the appropriate correction

  1. Molecular basis of epithelial Ca2+ and Mg2+ transport: insights from the TRP channel family

    NARCIS (Netherlands)

    Dimke, H.; Hoenderop, J.G.J.; Bindels, R.J.M.


    Maintenance of plasma Ca(2+) and Mg(2+) levels is of vital importance for many physiological functions. This is achieved via a coordinated interplay between the intestine, bone and kidney by amending the rate of absorption, storage and excretion, respectively. Discovery of the transient receptor

  2. Ca 2 and Mg 2 binding induce conformational stability of Calfumirin ...

    Indian Academy of Sciences (India)

    Isothermal titration calorimetry (ITC) resultsshow two possible independent binding sites of comparable affinity for the metal ions. However, their binding to the EF-IV E helix-loop-F helix mutant apo-protein happens with different affinities. The present study demonstrates that Ca2+ or Mg2+ binding plays a possible role in the ...

  3. Ca2+/Mg2+ exchange in parvalbumin and other EF-hand proteins. A theoretical study. (United States)

    Allouche, D; Parello, J; Sanejouand, Y H


    A remarkable conformational rearrangement occurs upon Ca2+/Mg2+ exchange in the C-terminal EF-hand site (labelled site EF or EF-4) of parvalbumin, as initially established by X-ray crystallography. Such a conformational rearrangement is characterised as follows: (i) the co-ordination number decreases from seven oxygen atoms in the Ca-loaded form to six oxygen atoms in the Mg-loaded form, the heptaco-ordination of Ca2+ corresponding with a skewed pentagonal bipyramid configuration of the seven oxygen atoms, whereas the hexaco-ordination of Mg2+ corresponds with a regular octahedral configuration of the six oxygen atoms; and (ii) Glu101, at the relative position 12 in the EF-hand loop sequence (labelled "Glu12"), acts as a bidentate ligand in the Ca-loaded form and as a monodentate ligand in the Mg-loaded form. As part of the conformational rearrangement, the chi1 dihedral angle undergoes a gauche(+) to gauche(-) transition upon substitution of Ca2+ by Mg2+, whereas the chi2 angle remains practically unchanged and the chi3 angles in both forms adopt a nearly mirror image relationship. In order to understand the molecular mechanisms underlying such a conformational rearrangement, we undertook a theoretical study using the free energy perturbation (FEP) method, starting from high-resolution crystal structures of the same parvalbumin (pike 4. 10 isoform) differing by the substitution of their two cationic sites EF-3 (or CD) and EF-4 (or EF), i.e. the 1pal structure with EF-3(Ca2+) and EF-4(Ca2+), the 4pal structure with EF-3(Ca2+) and EF-4(Mg2+). When Mg2+ is "alchemically" transformed into Ca2+ within the EF-4 site of 4pal, the conformational rearrangement of Glu12 is correctly predicted by the FEP calculation. When Ca2+ is transformed into Mg2+ within the EF-3 site of 4pal, the FEP calculation predicts the topology of the fully Mg-loaded form for which no crystallographic data is presently available. As expected, Glu62 (at the relative position 12 in EF-3 loop) is

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

    DEFF Research Database (Denmark)

    Drachmann, Nikolaj Düring


    is regulated by the Sarcolipin homologue Phospholamban. They act upon binding by increasing the apparent Ca2+ affinity of the ATPases, thus regulating the activity in the physiologically relevant Ca2+ concentrations. In the first part of the thesis, a purification protocol of native SERCA2a from pig hearts...... is presented. The purified protein was used for X-ray crystallographic studies aiming at determining the three dimensional structure of the SERCA2a isoform in a Ca2+-free conformation. Crystals of the Ca2+ free state of SERCA2a stabilised by the inhibitor cyclopiazonic acid was obtained and a dataset...... was collected scaling to 3.26 Å resolution, allowing a preliminary structural analysis. The overall crystal structure is very similar to SERCA1a. Additionally, co-crystallisation studies have been initiated of SERCA2a and recombinantly expressed Phospholamban. Besides the above mentioned regulatory peptides...

  5. Peran Enzim Ca2+ - ATPase pada Membran Sel Darah Merah

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    Ferry P. Gultom


    Full Text Available Ca2+ - ATP ase enzyme is transmembrane protein which is found in membrane cell. This protein works as a pump in many cases such as thalassemia which causes leakage of the cell as there is oxidation of sulphidril chain from amino acid in membrane. The calcium-ion intake must be pumped out to get red blood cell homeostatic condition. The aim of this paper is to determine the activity of Ca2+ - ATPase enzyme as a pump of Ca ion in membrane cell.

  6. Toxicity of cypermethrin, effects on serum electrolytes (Ca+2, Mg+2 ...

    African Journals Online (AJOL)

    The LC50 values for cypermethrin 24, 48, 72 and 96 h were 5.43, 5.12, 4.82 and 4.56 μg/l, respectively. The Ca+2 levels decreased in the exposed fishes up to 96 h, whereas, Mg+2 and Pi recorded an increase. During recovery period the serum electrolytes recorded a pattern towards normalcy when compared with 96 h ...

  7. Inhibition of duodenal enterocyte Mg2+-ATPase by arachidonic acid is not mediated by an effect on protein kinase C. (United States)

    Haag, M; Leonard, F; Magada, O N; Kruger, M C


    Active absorption processes in the duodenal enterocyte are driven by various ATPases. It is known that the activity of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase can be modulated by polyunsaturated fatty acids of the n-6 series, for example by linoleic and gamma-linolenic acids. These effects may be achieved by protein phosphorylation via protein kinase C. The present study was undertaken to determine the effect of arachidonic acid on Mg2+-ATPase (measured colorimetrically) activity in basolateral membranes prepared from rat duodenum. It shows, for the first time, significant dose-dependent inhibition of Mg2+-ATPase (26-62%) by arachidonic acid (10-50 microg/ml) which already takes place after one minute of exposure, indicating involvement of a rapid signal transduction mechanism. Addition of the protein kinase C inhibitors bisimidolylmaleimide (2.5 microM) and calphostin (0.5 microM) did not influence the action of arachidonic acid on Mg2+-ATPase; protein kinase C involvement in this process is thus not indicated.

  8. Ultrastructural and Immunohistochemical Localization of the Plasma Membrane Ca2+-ATPase 4 (PMCA4) in Ca2+-Transporting Epithelia

    DEFF Research Database (Denmark)

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


    Plasma Membrane Ca(2+)-ATPase's (PMCA) participate in epithelial Ca(2+) transport and intracellular Ca(2+) signaling. The Pmca4 isoform is enriched in distal nephron isolates and decreased in mice lacking the epithelial Ca(2+) channel, Trpv5. We therefore hypothesized that Pmca4 plays a significa...... to a housekeeping function of the pump in Ca(2+) transporting epithelia. In conclusion, Pmca4 shows a divergent expression pattern in Ca(2+) transporting epithelia, inferring diverse roles for this isoform not limited to transepithelial Ca(2+) transport....

  9. Removal of K+, Na+, Ca2+, and Mg2+ from saline-alkaline water using the microalga Scenedesmus obliquus (United States)

    Yao, Zongli; Ying, Chengqi; Lu, Jianxue; Lai, Qifang; Zhou, Kai; Wang, Hui; Chen, Ling


    The capability of Scenedesmus obliquus to remove cations (K+, Na+, Ca2+, Mg2+) from saline-alkaline water was investigated at different salinities (0, 5, 10, 15, 20, 25) and carbonate alkalinities (0, 5, 10, 15, 20, 25, 30, 35 mmol/L). K+, Na+, Ca2+, and Mg2+ in saline-alkaline water were efficiently removed by S. obliquus. The maximum removal of the cations (29.37 mg for K+, 185.85 mg for Na+, 23.07 mg for Ca2+, 66.14 mg for Mg2+) occurred at salinity 25. The maximum removal of K+ (2.28 mg), Na+ (6.62 mg), Ca2+ (1.01 mg), and Mg2+ (0.62 mg) occurred at carbonate alkalinities of 25 mmol/L for K+, 35 mmol/L for Na+, 20 mmol/L for Ca2+, and 25 mmol/L for Mg2+, respectively. Under a salinity stress, the concentration of Na+ in S. obliquus increased significantly, while that of K+ decreased significantly. The concentrations of Ca2+ and Mg2+ decreased as well. The ratios of K+/Na+, Ca2+/Na+, and Mg2+/Na+ were significantly lower in all salinity treatments than those of the control. Under alkaline stress, the concentrations of Na+ and K+ in S. obliquus decreased significantly and the ratios of K+/Na+, Ca2+/Na+, and Mg2+/Na+ were significantly higher in all treatments than in the control. Moreover, the concentrations of Ca2+ and Mg2+ in S. obliquus at alkalinities of 5-10 mmol/L were significantly higher than those of the other treatments. The removal of Na+ by S. obliquus mainly occurs through biosorption, and Mg2+ and Ca2+ were removed through both biosorption and bioaccumulation.

  10. Kinetics of inhibitory effect of calix[4]arene С-90 on activity of transporting plasma membrane Cа(2+,Mg(2+-ATPase of smooth muscle cells

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    T. O. Veklich


    Full Text Available In experiments on the suspension of myometrium cell plasma membrane, processed by 0.1% digitonin, the inhibitory action of calix[4]arene C-90 (5,11,17,23-tetra(threeftormethyl(phenilsulphonilimino-methylamino-25,26, 27,28-tetrapropoxy-calix[4]arene on the activi­ty of Ca2+,Mg2+-ATPase was investigated. The authors also examined the influence of calix[4]arene in different concentration on affinity of enzyme (Ca2+,Mg2+-ATPase for the ATP and ions of Mg and Ca, and its influence on cooperative effect and maximum velocity of ATP hydrolysis. It is shown that calix[4]arene does not influence the affinity of Ca2+,Mg2+-ATPase for the ATP, which means that these two compounds have different binding centers­. Also calix[4]arene has no influence on affinity and cooperative effect of Ca ions, if it is used in concentration lower than 50 µM. Calix[4]arene slightly increases coefficient of Ca2+,Mg2+-ATPase activation by magnesium chloride. In all three cases, where ATP, Mg and Ca ions are used to test the impact of calix[4]arene, maximum velocity of ATP hydrolysis significantly decreases. All these results clarify that calix[4]arene implements its inhibitory action through mechanism of uncompetitive inhibition of Ca2+,Mg2+-ATPase activity.

  11. Interactions of Na+, K+, Mg2+, and Ca 2+ with benzene self-assembled monolayers

    DEFF Research Database (Denmark)

    Pedersen, Morten Rimmen; Matthiesen, Jesper; Bovet, Nicolas Emile


    measurements. The results of our studies clearly show that even a nonpolar, hydrophobic molecule, such as benzene, has a role to play in the behavior of aqueous solutions and that it interacts differently depending on which ions are present. Even ions from the same column in the periodic table behave......Interactions between cations and organic molecules are found throughout nature, from the functionality and structure of proteins in humans and animals to the exchange of ions in minerals in soil and oil reservoirs with the fluid phases. We have explored the behavior of the s-block elements...... that are most common in the natural world, namely, Na+, K+, Mg 2+, and Ca2+. Specifically, we investigated how these ions affect the interactions between surfaces covered by self-Assembled monolayers (SAMs) terminated with benzene molecules. We used a flat oxidized silicon substrate and an atomic force...

  12. Isotopic Fractionation of Mg2+(aq), Ca2+(aq), and Fe2+(aq) with Carbonate Minerals

    Energy Technology Data Exchange (ETDEWEB)

    Rustad, James R.; Casey, William H.; Yin, Qing-Zhu; Bylaska, Eric J.; Felmy, Andrew R.; Bogatko, Stuart A.; Jackson, Virgil E.; Dixon, David A.


    Density functional electronic structure calculations are used to compute the equilibrium constant (the isotope fractionation factor) for 26Mg/24Mg and 44Ca/40Ca isotope exchange between carbonate minerals and uncomplexed divalent aquo ions. The most reliable calculations at the B3LYP/6-311++G(2d,2p) level predict equilibrium constants K, reported as 103ln(K) at 25 °C, of -5.3, -1.1, and +1.1 for 26Mg/24Mg exchange between calcite (CaCO3), magnesite (MgCO3), and dolomite (Ca0.5Mg0.5CO3), respectively, and Mg2+(aq), with positive values indicating enrichment in the mineral phase. For 44Ca/40Ca exchange between calcite and Ca2+(aq), the calculations predict values of +1.5 for Ca2+(aq) in six-fold coordination and +4.1 for Ca2+(aq) in seven-fold coordination. We find that the reduced partition function ratios can be reliably computed from systems as small as M(CO3)610- and M2+(H2O)6 embedded in a set of fixed atoms representing the 2nd shell (and greater) coordination environment. We find that the aqueous cluster representing the aquo ion is much more sensitive to improvements in the basis set than the calculations on the mineral systems, and that fractionation factors should be computed using 2 the best possible basis set for the aquo complex, even if the reduced partition function ratio calculated with the same basis set is not available for the mineral system. The new calculations show that the previous discrepancies between theory and experiment for Fe3+-hematite and Fe2+-siderite fractionations arise from an insufficiently accurate reduced partition function ratio for the Fe3+(aq) and Fe2+(aq) species.

  13. Isotopic fractionation of Mg 2+(aq), Ca 2+(aq), and Fe 2+(aq) with carbonate minerals (United States)

    Rustad, James R.; Casey, William H.; Yin, Qing-Zhu; Bylaska, Eric J.; Felmy, Andrew R.; Bogatko, Stuart A.; Jackson, Virgil E.; Dixon, David A.


    Density-functional electronic structure calculations are used to compute the equilibrium constants for 26Mg/ 24Mg and 44Ca/ 40Ca isotope exchange between carbonate minerals and uncomplexed divalent aquo ions. The most reliable calculations at the B3LYP/6-311++G(2d,2p) level predict equilibrium constants K, reported as 10 3ln ( K) at 25 °C, of -5.3, -1.1, and +1.2 for 26Mg/ 24Mg exchange between calcite (CaCO 3), magnesite (MgCO 3), and dolomite (Ca 0.5Mg 0.5CO 3), respectively, and Mg 2+(aq), with positive values indicating enrichment of the heavy isotope in the mineral phase. For 44Ca/ 40Ca exchange between calcite and Ca 2+(aq) at 25 °C, the calculations predict values of +1.5 for Ca 2+(aq) in 6-fold coordination and +4.1 for Ca 2+(aq) in 7-fold coordination. We find that the reduced partition function ratios can be reliably computed from systems as small as M(CO)610- and M(HO)62+ embedded in a set of fixed atoms representing the second-shell (and greater) coordination environment. We find that the aqueous cluster representing the aquo ion is much more sensitive to improvements in the basis set than the calculations on the mineral systems, and that fractionation factors should be computed using the best possible basis set for the aquo complex, even if the reduced partition function ratio calculated with the same basis set is not available for the mineral system. The new calculations show that the previous discrepancies between theory and experiment for Fe 3+-hematite and Fe 2+-siderite fractionations arise from an insufficiently accurate reduced partition function ratio for the Fe 3+(aq) and Fe 2+(aq) species.

  14. Inhibitory effect of lidocaine on the sarcoplasmic reticulum Ca2+-dependent atpase from temporalis muscle. (United States)

    Sánchez, Gabriel A; Casadoumecq, Ana C; Alonso, Guillermo L; Takara, Delia


    Myotoxic effects of local anesthetics on skeletal musclefibers involve the inhibition ofsarcoplasmic reticulum Ca2+ -dependent ATPase activity and Ca2 transport. Lidocaine is a local anesthetic frequently used to relieve the symptoms of trigeminal neuralgia. The aim of this work was to test the inhibitory and/or stimulatory effect of lidocaine on sarcoplasmic reticulum Ca2+ -dependent ATPase isolated from rabbit temporalis muscle. Ca2+ -dependent ATPase activity was determined by a colorimetric method Calcium-binding to the Ca dependent ATPase, Ca2+ transport, and phosphorylation of the enzyme by ATP were determined with radioisotopic techniques. Lidocaine inhibited the Ca2+ -dependent ATPase activity in a concentration-dependent manner. The preincubation of the sarcoplasmic reticulum membranes with lidocaine enhanced the Ca2+ dependent ATPase activity in the absence of calcium ionophore. Lidocaine also inhibited both Ca2+ uptake and enzyme phosphorylation by ATP but had no effect on Ca2+ -binding to the enzyme. We conclude that the effect of lidocaine on the sarcoplasmic reticulum Ca2+ -dependent ATPase from temporalis muscle is due to the drug's direct interaction with the enzyme and the increased permeability of the sarcoplasmic reticulum membrane to Ca.

  15. Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA.

    Directory of Open Access Journals (Sweden)

    Sen Li

    Full Text Available Intracellular pH (pHi and Ca(2+ regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca(2+. The sources of the Ca(2+ increase are from the endoplasmic reticulum (ER Ca(2+ pools as well as from Ca(2+ influx. The store-mobilization component of the Ca(2+ increase induced by the pHi rise was not sensitive to antagonists for either IP(3-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca(2+-ATPase (SERCA, leading to depletion of the ER Ca(2+ store. We further showed that the physiological consequence of depletion of the ER Ca(2+ store by pHi rise is the activation of store-operated channels (SOCs of Orai1 and Stim1, leading to increased Ca(2+ influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca(2+ leak from ER pools followed by Ca(2+ influx via SOCs.


    DEFF Research Database (Denmark)

    Andersen, A.; Treiman, M.; Poulsen, J. C. J.


    The preparation of a nonionic desoxy-analogue of thapsigargin possessing a Ca2+-ATPase inhibitory potency similar to that of thapsioargin is described.......The preparation of a nonionic desoxy-analogue of thapsigargin possessing a Ca2+-ATPase inhibitory potency similar to that of thapsioargin is described....

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

    Directory of Open Access Journals (Sweden)

    Jiao Li

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

  18. Probing determinants of cyclopiazonic acid sensitivity of bacterial Ca2+-ATPases. (United States)

    Kotšubei, Aljona; Gorgel, Manuela; Morth, Jens P; Nissen, Poul; Andersen, Jacob L


    Cyclopiazonic acid (CPA) is a specific and potent inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase 1a (SERCA1a). Despite high sequence similarity to SERCA1a, Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1) is not inhibited by CPA. To test whether a CPA binding site could be created while maintaining the functionality of the ATPase we targeted four amino acid positions in LMCA1 for mutational studies based on a multiple sequence alignment of SERCA-like Ca(2+)-ATPases and structural analysis of the CPA site. The identification of CPA-sensitive gain-of-function mutants pinpointed key determinants of the CPA binding site. The importance of these determinants was further underscored by the characterization of the CPA sensitivity of two additional bacterial Ca(2+)-ATPases from Lactococcus lactis and Bacillus cereus. The CPA sensitivity was predicted from their sequence compared with the LMCA1 results, and this was experimentally confirmed. Interestingly, a cluster of Lactococcus bacteria applied in the production of fermented cheese display Ca(2+)-ATPases that are predictably CPA insensitive and may originate from their coexistence with CPA-producing Penicillum and Aspergillus fungi in the cheese. The differences between bacterial and mammalian binding pockets encompassing the CPA site suggest that CPA derivatives that are specific for bacteria or other pathogens can be developed. © 2013 FEBS.

  19. Penentuan Kesadahan Ca 2+ dan Mg 2+ Pada Air Baku dan Air Resevoir Di PDAM Tirtanadi Instalasi Delituamedan


    Harahap, Mahyusar


    Has conducted analysis on the raw water hardness and water reservoir in PDAM Tirtanadi Installation Delitua Medan. Mainly caused by water hardness ions - ions Ca2 + and Mg2+ , magnesium and calcium hardness is determined by titration method using Na2EDTA and indicators EBT to total hardness and mureksid indicator for the level of calcium. Magnesium hardness value is determined by the reduction in volume (ml) Na2EDTA titration the total hardness of CaCO3 with the volume (ml) Na2EDTA titration ...

  20. Influence of Ca2+ and Mg2+ on the thermotropic behaviour and permeability properties of liposomes prepared from dimyristoyl phosphatidylglycerol and mixtures of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine

    NARCIS (Netherlands)

    Dijck, P.W.M. van; Ververgaert, P.H.J.Th.; Verkleij, A.J.; Deenen, L.L.M. van; Gier, J. de


    1. 1. Calorimetric experiments showed a marked effect of Ca2+ and Mg2+ on the thermotropic behaviour of dimyristoyl phosphatidylglycerol. 2. 2. Concentrations of Ca2+ and Mg2+ lower than 1 ion to 2 molecules of phosphatidylglycerol produced a shift of the phase transition to higher temperatures

  1. Single-molecule, structural and functional studies of Listeria monocytogenes Ca2+-ATPase

    DEFF Research Database (Denmark)

    Dyla, Mateusz

    -ion transport (e.g. H+ for Ca2+-ATPases). P-type ATPases undergo major conformational changes during their functional cycle, as has been learned from a wealth of atomic-resolution X-ray crystallographic structures (4). In this work, single-molecule, structural and functional studies were employed to investigate...... of Cy3 and Cy5 in an optimized form of LMCA1 with reduced background labeling. LMCA1 was found to reside in the high-FRET E1 conformational state through most of its functional cycle, even in the absence of Ca2+. Binding of Ca2+ brought the cytoplasmic domains of LMCA1 closer together, whereas...... of single vesicles, providing complementary read-out of the single-molecule dynamics. Furthermore, the effects of metal fluorides on the ATPase activity of the pump were characterized to validate the possibility of trapping LMCA1 in specific functional states analogous to the well-studied sarco...

  2. Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase. (United States)

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


    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

  3. Ca2+ transport by reconstituted synaptosomal ATPase is associated with H+ countertransport and net charge displacement. (United States)

    Salvador, J M; Inesi, G; Rigaud, J L; Mata, A M


    The synaptosomal plasma membrane Ca2+-ATPase (PMCA) purified from pig brain was reconstituted with liposomes prepared by reverse phase evaporation at a lipid to protein ratio of 150/1 (w/w). ATP-dependent Ca2+ uptake and H+ ejection by the reconstituted proteoliposomes were demonstrated by following light absorption and fluorescence changes undergone by arsenazo III and 8-hydroxy-1,3, 6-pyrene trisulfonate, respectively. Ca2+ uptake was increased up to 2-3-fold by the H+ ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, consistent with relief of an inhibitory transmembrane pH gradient (i.e. lumenal alkalinization) generated by H+ countertransport. The stoichiometric ratio of Ca2+/H+ countertransport was 1.0/0.6, and the ATP/Ca2+ coupling stoichiometry was 1/1 at 25 degrees C. The electrogenic character of the Ca2+/H+ countertransport was demonstrated by measuring light absorption changes undergone by oxonol VI. It was shown that a 20 mV steady state potential (positive on the lumenal side) was formed as a consequence of net charge transfer associated with the 1/1 Ca2+/H+ countertransport. Calmodulin stimulated ATPase activity, Ca2+ uptake, and H+ ejection, demonstrating that these parameters are linked by the same mechanism of PMCA regulation.

  4. An improvement to the ligand optimisation method (LOM) for measuring the apparent dissociation constant and ligand purity in Ca2+ and Mg2+ buffer solutions. (United States)

    McGuigan, John A S; Kay, James W; Elder, Hugh Y


    In Ca(2+)/Mg(2+) buffers the calculated ionised concentrations ([X(2+)]) can vary by up to a factor of seven. Since there are no defined standards it is impossible to check calculated [X(2+)], making measurement essential. The ligand optimisation method (LOM) is an accurate method to measure [X(2+)] in Ca(2+)/Mg(2+) buffers; independent estimation of ligand purity extends the method to pK(/) buffers, to calculate electrode and buffer characteristics as a function of Σ. Ca(2+)-electrodes have a Σ buffers. These results demonstrated that it is pK(/) that is normally distributed. Until defined standards are available, [X(2+)] in Ca(2+)/Mg(2+) buffers have to be measured. The most appropriate method is to use Ca(2+)/Mg(2) electrodes combined with the Excel programs SALE or AEC. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Arabidopsis calmodulin-like protein CML36 is a calcium (Ca2+) sensor that interacts with the plasma membrane Ca2+-ATPase isoform ACA8 and stimulates its activity. (United States)

    Astegno, Alessandra; Bonza, Maria Cristina; Vallone, Rosario; La Verde, Valentina; D'Onofrio, Mariapina; Luoni, Laura; Molesini, Barbara; Dominici, Paola


    Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca2+)-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca2+ and Mg2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca2+/Mg2+ mixed binding sites and two low-affinity Ca2+-specific sites. Binding of Ca2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca2+ or Mg2+, stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca2+-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca2+ pumps. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Espinoza-Fonseca, L Michel


    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.

  7. Control of temperature and aqueous Mg2+/Ca2+ ratio on the (trans-)formation of ikaite (United States)

    Purgstaller, B.; Dietzel, M.; Baldermann, A.; Mavromatis, V.


    The calcium carbonate hexahydrate mineral ikaite (CaCO3 ṡ 6 H2O) has been documented in aquatic environments at near-freezing temperatures. An increase of the prevailing temperature in the depositional environment, results in the transformation of natural ikaite into less soluble calcium carbonate phases occasionally leaving calcite pseudomorphs in the sediments, which are considered as an indicator for primary cold water temperatures. Detailed understanding on the physicochemical parameters controlling ikaite (trans-)formation however, such as temperature and reactive solution chemical composition, are still under debate. In order to study the formation of ikaite, we conducted precipitation experiments under controlled physicochemical conditions (pH = 8.3 ± 0.1; T = 6, 12, and 18 ± 0.1 °C) at defined aqueous molar Mg/Ca ratios. The transformation of ikaite into anhydrous calcium carbonate polymorphs was investigated in solution and at air exposure. The obtained results reveal the formation of ikaite at temperatures up to 12 °C, whereas Mg-rich amorphous calcium carbonate precipitated at 18 °C. In contact with the reactive solution ikaite transformed into aragonite at aqueous molar Mg2+/Ca2+ ratios of ≥14. In contrast, ikaite separated from the Mg-rich solution and exposed to air transformed in all cases into calcite/vaterite. The herein obtained temperature limit of ≤12 for ikaite formation is significantly higher than formerly expected and most probably caused by (i) the high saturation degree of the solution with respect to ikaite and (ii) the slow dehydration of the aqueous Ca2+ ion at low temperatures. This result questions the suitability of calcite pseudomorphs (i.e. glendonites) as a proxy for near-freezing temperatures. Moreover, our findings show that the CaCO3 polymorph formed from ikaite is strongly controlled by the physicochemical conditions, such as aqueous molar Mg2+/Ca2+ ratio of the reactive fluid and H2O availability throughout the

  8. Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump? (United States)

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


    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.

  9. Inhibition of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase by thapsigargin analogs induces cell death via ER Ca2+ depletion and the unfolded protein response

    DEFF Research Database (Denmark)

    Sehgal, Pankaj; Szalai, Paula; Olesen, Claus


    Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA-inhibition induces cell death are incompletely...... and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response (UPR). We conclude that ER Ca2+ drainage and sustained UPR activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high...

  10. Sarco/Endoplasmic Reticulum Ca2+-ATPases (SERCA) Contribute to GPCR-Mediated Taste Perception (United States)

    Iguchi, Naoko; Ohkuri, Tadahiro; Slack, Jay P.; Zhong, Ping; Huang, Liquan


    The sense of taste is important for providing animals with valuable information about the qualities of food, such as nutritional or harmful nature. Mammals, including humans, can recognize at least five primary taste qualities: sweet, umami (savory), bitter, sour, and salty. Recent studies have identified molecules and mechanisms underlying the initial steps of tastant-triggered molecular events in taste bud cells, particularly the requirement of increased cytosolic free Ca2+ concentration ([Ca2+]c) for normal taste signal transduction and transmission. Little, however, is known about the mechanisms controlling the removal of elevated [Ca2+]c from the cytosol of taste receptor cells (TRCs) and how the disruption of these mechanisms affects taste perception. To investigate the molecular mechanism of Ca2+ clearance in TRCs, we sought the molecules involved in [Ca2+]c regulation using a single-taste-cell transcriptome approach. We found that Serca3, a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family that sequesters cytosolic Ca2+ into endoplasmic reticulum, is exclusively expressed in sweet/umami/bitter TRCs, which rely on intracellular Ca2+ release for signaling. Serca3-knockout (KO) mice displayed significantly increased aversive behavioral responses and greater gustatory nerve responses to bitter taste substances but not to sweet or umami taste substances. Further studies showed that Serca2 was mainly expressed in the T1R3-expressing sweet and umami TRCs, suggesting that the loss of function of Serca3 was possibly compensated by Serca2 in these TRCs in the mutant mice. Our data demonstrate that the SERCA family members play an important role in the Ca2+ clearance in TRCs and that mutation of these proteins may alter bitter and perhaps sweet and umami taste perception. PMID:21829714

  11. Sarco/Endoplasmic reticulum Ca2+-ATPases (SERCA contribute to GPCR-mediated taste perception.

    Directory of Open Access Journals (Sweden)

    Naoko Iguchi

    Full Text Available The sense of taste is important for providing animals with valuable information about the qualities of food, such as nutritional or harmful nature. Mammals, including humans, can recognize at least five primary taste qualities: sweet, umami (savory, bitter, sour, and salty. Recent studies have identified molecules and mechanisms underlying the initial steps of tastant-triggered molecular events in taste bud cells, particularly the requirement of increased cytosolic free Ca(2+ concentration ([Ca(2+](c for normal taste signal transduction and transmission. Little, however, is known about the mechanisms controlling the removal of elevated [Ca(2+](c from the cytosol of taste receptor cells (TRCs and how the disruption of these mechanisms affects taste perception. To investigate the molecular mechanism of Ca(2+ clearance in TRCs, we sought the molecules involved in [Ca(2+](c regulation using a single-taste-cell transcriptome approach. We found that Serca3, a member of the sarco/endoplasmic reticulum Ca(2+-ATPase (SERCA family that sequesters cytosolic Ca(2+ into endoplasmic reticulum, is exclusively expressed in sweet/umami/bitter TRCs, which rely on intracellular Ca(2+ release for signaling. Serca3-knockout (KO mice displayed significantly increased aversive behavioral responses and greater gustatory nerve responses to bitter taste substances but not to sweet or umami taste substances. Further studies showed that Serca2 was mainly expressed in the T1R3-expressing sweet and umami TRCs, suggesting that the loss of function of Serca3 was possibly compensated by Serca2 in these TRCs in the mutant mice. Our data demonstrate that the SERCA family members play an important role in the Ca(2+ clearance in TRCs and that mutation of these proteins may alter bitter and perhaps sweet and umami taste perception.

  12. Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes. (United States)

    Seth, M; Sumbilla, C; Mullen, S P; Lewis, D; Klein, M G; Hussain, A; Soboloff, J; Gill, D L; Inesi, G


    Transient elevations of cytosolic Ca2+ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) plays an important role in terminating Ca2+ transients by returning cytosolic Ca2+ to intracellular stores. Stored Ca2+ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca2+ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na+/Ca2+ exchanger, indicating that intracellular store deficiency was compensated for by Ca2+ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na+/Ca2+ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca2+ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca2+ signaling dynamics and in cardiac development and hypertrophy.

  13. Cellular mechanisms that control pulmonary vascular tone during hypoxia and normoxia. Possible role of Ca2+ATPases. (United States)

    Farrukh, I S; Michael, J R


    We investigated cellular mechanisms that may be involved in controlling cytosol calcium and pulmonary artery pressure during hypoxia and normoxia in isolated blood-perfused ferret lungs. Alveolar hypoxia in ferret lungs causes an active increase in pulmonary vascular resistance. Hypoxic pulmonary vasoconstriction directly correlates with extracellular calcium ([Ca2+]o), and the absence of [Ca2+]o in the perfusate markedly attenuates the hypoxemia-induced pulmonary vasoconstriction. Alveolar hypoxia does not potentiate the production of thromboxane B2 (TxB2) or 6-keto-PGF1 alpha. Vanadate, a widely used inhibitor of Ca2+ATPases, increases pulmonary arterial pressure (Ppa) in the presence or absence of [Ca2+]o and without affecting the production of TxB2 or 6-keto-PGF1 alpha. Vanadate and ouabain, an inhibitor of Na+/K+ATPase, produce synergistic increases in Ppa. Amiloride, an inhibitor of Na+/Ca2+ exchange, reverses the increase in Ppa caused by ouabain, but not the increase caused by vanadate. The additional effect produced by ouabain on Ppa after near maximal vanadate effect and the ability of amiloride to reverse the pulmonary vasoconstriction caused by ouabain, but not vanadate, suggests that vanadate does not inhibit Na+/K+ATPase in ferret lungs. In addition, cyclic GMP (cGMP), which has been reported to increase the activity of Ca2+ATPases in vascular smooth muscle, was able to reverse and prevent the effect of vanadate on Ppa, but not the effect of ouabain. Inhibition of Ca2+ATPases with vanadate in ferret lungs increases pulmonary vascular resistance during both normoxia and hypoxia. The Ca2+ entry mediated by alveolar hypoxia appears to overpower the ability of Ca2+ATPases and other membrane Ca2+ transport proteins to translocate [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Impact of elevated Ca(2+)/Mg(2+) concentrations of reverse osmosis membrane desalinated seawater on the stability of water pipe materials. (United States)

    Liang, Juan; Deng, Anqi; Xie, Rongjing; Gomez, Mylene; Hu, Jiangyong; Zhang, Jufang; Ong, Choon Nam; Adin, Avner


    Hardness and alkalinity are known factors influencing the chemical stability of desalinated water. This study was carried out to investigate the effect of Ca(2+) and Mg(2+) on corrosion and/or scale formation on the surface of different water distribution pipe materials under tropical conditions. The corrosion rates of ductile iron, cast iron and cement-lined ductile iron coupons were examined in reverse osmosis (RO) membrane desalinated seawater which was remineralised using different concentrations of Ca(2+) and Mg(2+). The changes in water characteristics and the coupon corrosion rates were studied before and after the post-treatment. The corrosion mechanisms and corrosion products were examined using scanning electron microscope and X-ray diffraction, respectively. We found that the combination of Ca(2+) and Mg(2+) (60/40 mg/L as CaCO3) resulted in lower corrosion rates than all other treatments for the three types of pipe materials, suggesting that Ca(2+)/Mg(2+) combination improves the chemical stability of desalinated seawater rather than Ca(2+) only.

  15. Epigallocatechin-3-Gallate Protects Erythrocyte Ca2+-ATPase and Na+/K+-ATPase Against Oxidative Induced Damage During Aging in Humans

    Directory of Open Access Journals (Sweden)

    Prabhanshu Kumar


    Full Text Available Purpose: The main purpose of this study was to investigate the protective role of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced oxidative damage in erythrocyte during aging in humans. Methods: Human erythrocyte membrane bound Ca2+-ATPase and Na+/K+-ATPase activities were determined as a function of human age. Protective role of epigallocatechin-3-gallate was evaluated by in vitro experiments by adding epigallocatechin-3-gallate in concentration dependent manner (final concentration range 10-7M to 10-4M to the enzyme assay medium. Oxidative stress was induced in vitro by incubating washed erythrocyte ghosts with tertiary butyl hydroperoxide (10-5 M final concentration. Results: We have reported concentration dependent effect of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced damage on activities of Ca2+-ATPase and Na+/K+-ATPase during aging in humans. We have detected a significant (p < 0.001 decreased activity of Ca2+-ATPase and Na+/K+ -ATPase as a function of human age. Epigallocatechin-3-gallate protected ATPases against tertiary butyl hydroperoxide induced damage in concentration dependent manner during aging in humans. Conclusion: Epigallocatechin-3-gallate is a powerful antioxidant that is capable of protecting erythrocyte Ca2+-ATPase and Na+/K+ -ATPase against oxidative stress during aging in humans. We may propose hypothesis that a high intake of catechin rich diet may provide some protection against development of aging and age related diseases.

  16. Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca(2+)-ATPase. (United States)

    Lenoir, Guillaume; Menguy, Thierry; Corre, Fabienne; Montigny, Cédric; Pedersen, Per A; Thinès, Denyse; le Maire, Marc; Falson, Pierre


    Large amounts of heterologous C-terminally his-tagged SERCA1a Ca(2+)-ATPase were expressed in yeast using a galactose-regulated promoter and purified by Ni(2+) affinity chromatography followed by Reactive red chromatography. Optimizing the number of galactose inductions and increasing the amount of Gal4p transcription factor improved expression. Lowering the temperature from 28 degrees C to 18 degrees C during expression enhanced the recovery of solubilized and active Ca(2+)-ATPase. In these conditions, a 4 l yeast culture produced 100 mg of Ca(2+)-ATPase, 60 and 22 mg being pelleted with the heavy and light membrane fractions respectively, representing 7 and 1.7% of total proteins. The Ca(2+)-ATPase expressed in light membranes was 100% solubilized with L-alpha-lysophosphatidylcholine (LPC), 50% with n-dodecyl beta-D-maltoside (DM) and 25% with octaethylene glycol mono-n-dodecyl ether (C(12)E(8)). Compared to LPC, DM preserved specific activity of the solubilized Ca(2+)-ATPase during the chromatographic steps. Starting from 1/6 (3.8 mg) of the total amount of Ca(2+)-ATPase expressed in light membranes, 800 microg could be routinely purified to 50% purity by metal affinity chromatography and then 200 microg to 70% with Reactive red chromatography. The purified Ca(2+)-ATPase displayed the same K(m) for calcium and ATP as the native enzyme but a reduced specific activity ranging from 4.5 to 7.3 micromol ATP hydrolyzed/min/mg Ca(2+)-ATPase. It was stable and active for several days at 4 degrees C or after removal of DM with Bio-beads and storage at -80 degrees C.

  17. Plasma membrane Ca2+-ATPase isoforms composition regulates cellular pH homeostasis in differentiating PC12 cells in a manner dependent on cytosolic Ca2+ elevations

    DEFF Research Database (Denmark)

    Boczek, Tomasz; Lisek, Malwina; Ferenc, Bozena


    cellular acidification during KCl-stimulated Ca2+ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu......+-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient......Plasma membrane Ca2+-ATPase (PMCA) by extruding Ca2+ outside the cell, actively participates in the regulation of intracellular Ca2+ concentration. Acting as Ca2+/H+ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four...

  18. Cloning and characterization of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish axial muscle. Sarco/Endoplasmic Reticulum Ca(2+)-ATPase. (United States)

    Zhang, Z; Chen, D; Wheatly, M G


    The discontinuous pattern of muscle growth during the moulting cycle of a freshwater crustacean (the crayfish Procambarus clarkii) was used as a model system to examine the regulation of the expression of Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). We describe the cloning, sequencing and characterization of a novel SERCA cDNA (3856 bp) obtained from crayfish axial abdominal muscle by reverse transcription/polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). This complete sequence contains a 145 base pair (bp) noncoding region at the 5' end, a 3006 bp open reading frame coding for 1002 amino acid residues with a molecular mass of 110 kDa and 705 bp of untranslated region at the 3' end. This enzyme contains all the conserved domains found in 'P'-type ATPases, and the hydropathy profile suggests a transmembrane organization typical of other SERCAs. It exhibits 80% amino acid identity with Drosophila melanogaster SERCA, 79% identity with Artemia franciscana SERCA, 72% identity with rabbit fast-twitch muscle neonatal isoform SERCA1b, 71% identity with slow-twitch muscle isoform SERCA2 and 67% identity with SERCA3. Sequence alignment revealed that regions anchoring the cytoplasmic domain in the membrane were highly conserved and that most differences were in the NH(2) terminus, the central loop region and the COOH terminus. Northern analysis of total RNA from crayfish tissues probed with the 460 bp fragment initially isolated showed four bands (7.6, 7.0, 5.8 and 4.5 kilobases) displaying tissue-specific expression. SERCA was most abundant in muscle (axial abdominal, cardiac and stomach), where it is involved in Ca(2+) resequestration during relaxation, and in eggs, where it may be implicated in early embryogenesis. The level of SERCA mRNA expression in axial abdominal muscle varied during the moulting cycle as determined by slot-blot analysis. SERCA expression was greatest during intermoult and decreased to approximately 50% of

  19. K+, Na+, Mg2+, Ca2+, and water contents in human skeletal muscle: correlations among these monovalent and divalent cations and their alterations in K+ -depleted subjects. (United States)

    Tavichakorntrakool, Ratree; Prasongwattana, Vitoon; Sriboonlue, Pote; Puapairoj, Anucha; Wongkham, Chaisiri; Wiangsimma, Thitichai; Khunkitti, Wattana; Triamjangarun, Sombat; Tanratanauijit, Maneewan; Chamsuwan, Amporn; Khunkitti, Wirut; Yenchitsomanus, Pa-Thai; Thongboonkerd, Visith


    None of previous studies had simultaneously analyzed the K(+), Na(+), Mg(2+), and Ca(2+) contents in human skeletal muscle. We examined extensively and simultaneously the levels of all these cations and examined water content in vastus lateralis and pectoralis major muscles in 30 northeastern Thai men who were apparently healthy but died from an accident. Specimen collection was performed within 6 h of death. We used atomic absorption or flame photometry to measure the level of muscle cation. Histopathology of muscle and kidney was also evaluated. K(+), Na(+), Mg(2+), and Ca(2+) contents in vastus lateralis were 84.74 +/- 1.50, 38.64 +/- 0.77, 7.58 +/- 0.17, and 0.94 +/- 0.06 micromol/g wet weight, respectively, whereas K(+), Na(+), and Mg(2+) contents in pectoralis major were 82.83 +/- 1.54, 37.57 +/- 0.72, and 7.30 +/- 0.17 micromol/g wet weight, respectively. The water component was comparable in vastus lateralis and pectoralis major (78.66 +/- 0.41 and 78.09 +/- 0.56 %, respectively). Based on muscle K(+) levels, we divided the subjects into 2 main groups: K(+)-depleted (KD) group (K(+) or = 80 micromol/g wet weight; n = 23). In the KD muscle, Na(+) and Ca(2+) levels were significantly higher, whereas the level of Mg(2+) was significantly lower. Linear regression analysis showed significant correlations of K(+) and Mg(2+) levels and between Na(+) and Ca(2+). However, K(+) and Mg(2+) had the negative correlation with Na(+) and Ca(2+). Histopathologic examination showed no change in the KD muscles, whereas 29% (2 of 7) of the KD kidneys had vacuolization in proximal renal tubular cells. Our study not only provided the descriptive data but also implied the balance or homeostasis of these monovalent and divalent cations in their muscle pools.

  20. Cloning of sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) from Caribbean spiny lobster Panulirus argus. (United States)

    Mandal, A; Arunachalam, S C; Meleshkevitch, E A; Mandal, P K; Boudko, D Y; Ahearn, G A


    We have previously reported on calcium transport mechanisms in American lobster, Homarus americanus, using (45)Ca(2+) coupled with vesicle preparations of hepatopancreatic endoplasmic reticulum. The active transport of calcium across membranes bordering calcium-sequestering stores such as sarcoplasmic or endoplasmic reticulum is catalyzed by membrane-spanning proteins, the sarco-endoplasmic Ca(2+)-ATPases (SERCAs). In the study described here we used advanced bioinformatics and molecular techniques to clone SERCA from the economically important Caribbean spiny lobster, Panulirus argus. We report the complete cloning of a full-length SERCA from P. argus antenna cDNA (GenBank accession number AY702617). This cDNA has a 1020-amino acid residue open reading frame which is 90% identical to published sequences of other crustacean SERCA proteins. Our data support the hypothesis that one crustacean and three vertebrate genes controlling calcium transport were derived from a common ancestral gene.

  1. Experiment and Optimization for Simultaneous Carbonation of Ca2+ and Mg2+ in A Two-phase System of Insoluble Diisobutylamine and Aqueous Solution (United States)

    Wang, Wenlong; Wang, Man; Liu, Xin; Wang, Peng; Xi, Zhenqian


    An optimized approach of CO2 fixation in Ca2+/Mg2+-rich aqueous solutions using insoluble amine as an enhancing medium was reported. Apparent basicity was verified to be an effective indicator for the selection and optimization of organic amine systems and finally the diisobutylamine + n-octanol system was selected to enhance the carbonation reactions of CO2 in an artificial Ca2+/Mg2+-rich solution. In our experiments, when the volume ratio of insoluble organic phase to aqueous one was 2:1 and the reaction temperature was 28 °C, 92% of Ca2+ and 80% of Mg2+ could be converted to calcium and magnesium carbonate precipitates within 5 min of reaction with the bubbling-in of CO2. The organic amine system could be regenerated by using carbide slag as the regeneration agent and could still show attractive enhancement performances after 7 rounds of carbonation-regeneration experiments. In this way, the CO2 capture and sequestration was realized within one single process, with value-added Ca/Mg carbonates being the byproducts. In view of the vast availability of Ca2+/Mg2+-rich aqueous solutions and the feasible technical coordination with desalination industry, this novel process may have a good application potential in the future.

  2. Coordinated regulation of cardiac Na(+)/Ca (2+) exchanger and Na (+)-K (+)-ATPase by phospholemman (FXYD1). (United States)

    Cheung, Joseph Y; Zhang, Xue-Qian; Song, Jianliang; Gao, Erhe; Chan, Tung O; Rabinowitz, Joseph E; Koch, Walter J; Feldman, Arthur M; Wang, JuFang


    Phospholemman (PLM) is the founding member of the FXYD family of regulators of ion transport. PLM is a 72-amino acid protein consisting of the signature PFXYD motif in the extracellular N terminus, a single transmembrane (TM) domain, and a C-terminal cytoplasmic tail containing three phosphorylation sites. In the heart, PLM co-localizes and co-immunoprecipitates with Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and L-type Ca(2+) channel. The TM domain of PLM interacts with TM9 of the α-subunit of Na(+)-K(+)-ATPase, while its cytoplasmic tail interacts with two small regions (spanning residues 248-252 and 300-304) of the proximal intracellular loop of Na(+)/Ca(2+) exchanger. Under stress, catecholamine stimulation phosphorylates PLM at serine(68), resulting in relief of inhibition of Na(+)-K(+)-ATPase by decreasing K(m) for Na(+) and increasing V(max), and simultaneous inhibition of Na(+)/Ca(2+) exchanger. Enhanced Na(+)-K(+)-ATPase activity lowers intracellular Na(+), thereby minimizing Ca(2+) overload and risks of arrhythmias. Inhibition of Na(+)/Ca(2+) exchanger reduces Ca(2+) efflux, thereby preserving contractility. Thus, the coordinated actions of PLM during stress serve to minimize arrhythmogenesis and maintain inotropy. In acute cardiac ischemia and chronic heart failure, either expression or phosphorylation of PLM or both are altered. PLM regulates important ion transporters in the heart and offers a tempting target for development of drugs to treat heart failure.

  3. Probabilidade de resposta da cana-de-açúcar à adubação potássica em razão da relação K+ (Ca2++Mg2+ -0,5 do solo Probability of sugarcane response to potassium fertilizer as a function of soil K+ (Ca2++Mg2+ -0,5 ratio

    Directory of Open Access Journals (Sweden)

    Roberto dos Anjos Reis Junior


    Full Text Available O objetivo deste trabalho foi avaliar a probabilidade de resposta da produção de cana-de-açúcar à adubação potássica, em razão da relação K+ (Ca2++Mg2+ -0,5 no solo. Foram compilados dados de 106 experimentos de adubação potássica na cana-de-açúcar. Em cada experimento foi registrado o ciclo de cultivo (cana-planta ou cana-soca, os teores de K, Ca e Mg do solo antes da adubação potássica, a relação K+ (Ca2++Mg2+ -0,5, e se houve, ou não, resposta estatisticamente significativa da produção à adubação potássica. Foi utilizado o método estatístico de regressão logística, efetuado pelo procedimento CATMOD do Statistical Analysis System. A característica ciclo de cultivo foi eliminada do modelo, pois esta se apresentou como não-significativa no ajuste estatístico. A relação K+ (Ca2++Mg2+ -0,5 do solo influenciou a probabilidade de resposta da produção de cana-de-açúcar à adubação potássica. À medida que a relação K+ (Ca2++Mg2+ -0,5 aumentou, a probabilidade de resposta da produção de cana-de-açúcar à adubação potássica diminuiu. A relação K+ (Ca2++Mg2+ -0,5 no solo foi classificada em baixa (0,3349. A relação K+ (Ca2++Mg2+ -0,5 no solo deve ser usada como mais um critério para orientar a adubação potássica na cultura da cana-de-açúcar.This study was carried out to evaluate the effect of soil K+ (Ca2++Mg2+ -0.5 ratio on sugarcane yield response probability to potassium fertilizer. Results of 106 experiments of potassium fertilizer on sugarcane and their soil exchangeable K+, Ca2+ and Mg2+ were studied, evaluating the K+ (Ca2++Mg2+ -0.5 ratio in each experiment. The statistical method of logistic regression was used, carried out through CATMOD procedure of Statistical Analysis System. There was no difference of behavior of this ratio between plant cane and ratoons; therefore this characteristic was not significant during the adjustment of the statistical model. Soil K+ (Ca2++Mg2+ -0

  4. Plasma membrane Ca2+-ATPase isoforms composition regulates cellular pH homeostasis in differentiating PC12 cells in a manner dependent on cytosolic Ca2+ elevations. (United States)

    Boczek, Tomasz; Lisek, Malwina; Ferenc, Bozena; Kowalski, Antoni; Stepinski, Dariusz; Wiktorska, Magdalena; Zylinska, Ludmila


    Plasma membrane Ca(2+)-ATPase (PMCA) by extruding Ca(2+) outside the cell, actively participates in the regulation of intracellular Ca(2+) concentration. Acting as Ca(2+)/H(+) counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca(2+) overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pHmito and pHcyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca(2+) clearance and partially attenuated cellular acidification during KCl-stimulated Ca(2+) influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca(2+) overload. Cyclosporin and bongkrekic acid prevented Ψm loss suggesting the involvement of Ca(2+)-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.

  5. Thermogenic activity of the Ca2+-ATPase from blue marlin heater organ: regulation by KCl and temperature. (United States)

    da Costa, Danielly Cristiny Ferraz; Landeira-Fernandez, Ana Maria


    This work shows that vesicles derived from the blue marlin heater organ retain a sarcoplasmic reticulum (SR) Ca(2+)-ATPase that can interconvert different forms of energy. During the hydrolysis of ATP part of the energy is always converted into heat, and the other part can be converted into work (Ca(2+) transport) or heat, depending on the temperature and the presence of KCl in the reaction medium. At 15 degrees C, where KCl stimulates the activity approximately threefold, measurements of the amount of heat released per mole of ATP hydrolyzed (DeltaH(cal)) show similar values (approximately -11 kcal/mol) in the presence or absence of a Ca(2+) gradient. At 25 degrees C, KCl activates the enzyme to the same extent as at 15 degrees C, but inhibits the production of extra heat by SR Ca(2+)-ATPase when a Ca(2+) gradient is built up across the membrane. The DeltaH(cal) values found in the presence of a Ca(2+)-gradient were -26.2 +/- 2.9 kcal/mol (n = 7) in control experiments and -16.1 +/- 1.5 (n = 14) in the presence of 100 mM KCl. At 35 degrees C, KCl has a smaller effect ( approximately 1.5-fold) on activating the enzyme. Similar to SR Ca(2+)-ATPase from mammals, at this temperature the enzyme produces almost twice the amount of heat per mole of ATP hydrolyzed in the presence of a Ca(2+) gradient and KCl has no effect at all on this increment. These data suggest that the marlin SR Ca(2+)-ATPase may play an important role in heater organ thermogenesis and that KCl has the potential for regulating the heat production catalyzed by the enzyme.

  6. Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes. (United States)

    Ma, Hailun; Sumbilla, Carlota M; Farrance, Iain K G; Klein, Michael G; Inesi, Giuseppe


    We evaluated various constructs to obtain cell-specific expression of the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) gene in cardiac myocytes after cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous enhanced green fluorescent protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. Whereas the cytomegalovirus (CMV) promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or beta-myosin heavy chain promoter segments. A double virus system for Cre-dependent expression under control of the CMV promoter and Cre expression under control of a cardiac-specific promoter yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immunostaining of exogenous SERCA1 and endogenous SERCA2 and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell-specific SERCA expression and a definite increase over endogenous Ca2+ -ATPase activity as well as faster removal of cytosolic calcium after membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell-specific expression of exogenous SERCA is wanted in cardiac myocytes after cDNA delivery to mixed cell populations.

  7. Characterization of the intracellular and the plasma membrane Ca2+-ATPases in fractionated pig brain membranes using calcium pump inhibitors. (United States)

    Salvador, J M; Mata, A M


    The Ca2+-ATPase activity of isolated membranes and purified plasma membrane ATPase from pig brain was measured in the presence of specific inhibitors. The inhibition of the enzymatic activity by vanadate presents a lower affinity in microsomes than in the synaptic plasma membrane vesicles, showing K0.5 of 0.4 and 0.2 microM, respectively. The purified enzyme showed a higher sensitivity to vanadate with a K0.5 of 0.10 microM. Thapsigargin (Tg) and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) were stronger inhibitors of the Ca2+-ATPase activity in microsomes than in the synaptic membrane vesicles. The activity of the purified enzyme was not affected by Tg and only partially by BHQ. Cyclopiazonic acid inhibited the enzymatic activity in all fractions, being more sensitive in microsomes. The microsome preparation incorporated 32P from [gamma-32P]ATP into two main proteins that appear at approx 110,000 and 140,000. According to the inhibition pattern, the lower phosphorylated band was identified as the sarco(endo)plasmic reticulum Ca2+-ATPase, being in a higher percentage than the upper band. Synaptic membrane vesicles also incorporated radioactive 32P into two protein bands. The 140,000 protein (upper band) shows the typical behavior of the purified plasma membrane Ca2+-ATPase, being more abundant in this preparation than the organellar Ca2+-pump (lower band). This study highlights the heterogeneous nature of the Ca2+-ATPase activity measured in brain membrane fractions. Copyright 1998 Academic Press.

  8. Secretory pathway Ca2+-ATPases promote in vitro microcalcifications in breast cancer cells. (United States)

    Dang, Donna; Prasad, Hari; Rao, Rajini


    Calcification of the breast is often an outward manifestation of underlying molecular changes that drive carcinogenesis. Up to 50% of all non-palpable breast tumors and 90% of ductal carcinoma in situ present with radiographically dense mineralization in mammographic scans. However, surprisingly little is known about the molecular pathways that lead to microcalcifications in the breast. Here, we report on a rapid and quantitative in vitro assay to monitor microcalcifications in breast cancer cell lines, including MCF7, MDA-MB-231, and Hs578T. We show that the Secretory Pathway Ca 2+ -ATPases SPCA1 and SPCA2 are strongly induced under osteogenic conditions that elicit microcalcifications. SPCA gene expression is significantly elevated in breast cancer subtypes that are associated with microcalcifications. Ectopic expression of SPCA genes drives microcalcifications and is dependent on pumping activity. Conversely, knockdown of SPCA expression significantly attenuates formation of microcalcifications. We propose that high levels of SPCA pumps may initiate mineralization in the secretory pathway by elevating luminal Ca 2+ . Our new findings offer mechanistic insight and functional implications on a widely observed, yet poorly understood radiographic signature of breast cancer. © 2017 Wiley Periodicals, Inc.

  9. A tomato ER-type Ca2+-ATPase, LCA1, has a low thapsigargin-sensitivity and can transport manganese

    DEFF Research Database (Denmark)

    Johnson, Neil A.; Liu, F; Weeks, P. D.


    investigate the differences between plant and animal Ca pumps utilizing comparisons between chicken and rabbit SERCA-type pumps with Arabidopsis (ECA1) and tomato plant (LCA1) Ca(2+)-ATPases. Enzyme function was confirmed by the ability of each Ca(2+)-ATPase to rescue K616 growth on EGTA-containing agar...... and directly via in vitro ATP hydrolysis. We found LCA1 to be approximately 300-fold less sensitive to thapsigargin than animal SERCAs, whereas ECA1 was thapsigargin-resistant. LCA1 showed typical pharmacological sensitivities to cyclopiazonic acid, vanadate, and eosin, consistent with it being a P...

  10. 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


    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...

  11. Neuropharmacological effects of lipoic acid and ubiquinone on δ-aminolevulinic dehydratase, Na(+) , K(+) -ATPase, and Mg(2+) -ATPase activities in rat hippocampus after pilocarpine-induced seizures. (United States)

    de Freitas, Rivelilson Mendes; Feng, Dejiang; Jordán, Joaquín


    In this study, we investigated the effects of lipoic acid (LA) in the hippocampus oxidative stress caused by pilocarpine-induced seizures in adult rats. Wistar rats were treated with 0.9% saline (i.p., control group), LA (10mg/kg, i.p., LA group), ubiquinone [20mg/kg, i.p., ubiquinone (UQ) group], pilocarpine (400mg/kg, i.p., P400 group), and the association of LA (10mg/kg, i.p.) plus pilocarpine (400mg/kg, i.p.) or UQ (20mg/kg, i.p.) plus pilocarpine (400mg/kg, i.p.), 30min before of administration of P400 (LA plus P400 group and UQ plus P400 group, respectively). After the treatments, all groups were observed for 1h. The enzyme activities (δ-aminolevulinic dehydratase (δ-ALA-D), Mg(2+) -ATPase, and Na(+) , K(+) -ATPase) were measured using spectrophotometric methods, and the results compared to values obtained from saline and pilocarpine-treated animals. Protective effects of LA and UQ were also evaluated on the same parameters. We reported here for the first time that Na(+) , K(+) -ATPase and δ-ALA-D activities inhibition and Mg(2+) -ATPase stimulation in the pilocarpine model are probably attributed to the oxidative stress caused by seizures in the rat hippocampus. The addition of the antioxidants LA and UQ may reverses the previously mentioned Na(+) , K(+) -ATPase and δ-ALA-D inhibitions and Mg(2+) -ATPase stimulation. The oxidative stress plays an important signaling role in pilocarpine-induced seizures, and antioxidant drugs might be considered as therapeutical tools in this pathology. © 2010 The Authors Fundamental and Clinical Pharmacology © 2010 Société Française de Pharmacologie et de Thérapeutique.

  12. Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle. (United States)

    Bowman, B J; Berenski, C J; Jung, C Y


    Using radiation inactivation, we have measured the size of the H+-ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H+-ATPase in situ was found to be approximately 2.3 X 10(5) daltons. We also used radiation inactivation to measure the size of the Ca2+-ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, we directly compared the sizes of these two ATPases and found them to be essentially the same. We conclude that both H+-ATPase and Ca2+-ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides.

  13. Localization of Ca2+ and Ca-ATPase on wet (Ruscus aculeatus and dry (Primula officinalis stigma surface

    Directory of Open Access Journals (Sweden)

    Elżbieta Bednarska


    Full Text Available Calcium is present in the surface cells of both types of stigma. The chlorotetracycline method was used to show that the papillae of the dry stigma of Primula officinalis are the source of fluorescence of the CTC-Ca2+ complex. The precipitation method (NHA localized calcium ions mainly in the pellicula surrounding these papillae. The NHA-Ca2+ precipitates were localized on the plasma membrane in the papillae of the wet stigma of Ruscus aculeatus. Ca-ATPase activity was found in both types of stigma in sites where the presence of Ca2+ had been determined.

  14. [Influence of omeprasole and lansoprasole on Na+, K+ -ATPase and Mg2+ -ATPase activity of the plasmatic membrane of myometrium smooth muscle cells]. (United States)

    Veklich, T O; Shkrabak, O A; Medvediev, V V; Kurs'kyĭ, M D; Kosterin, S O


    The paper deals with the influence of the proton pump inhibitors - omeprasole and lansoprasole on the enzymatic activity of the ouabain-sensitive Na+, K+ -ATPase and the ouabain-resistant Mg2+ - ATPase in the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution. It was found, that omeprasole and lansoprasole inhibited Na+, K+ -ATPase in the range from 10 to 100 microM. The maximal effect was observed at a concentration of 100 microM with the percentage of inhibition of 81 and 86% at an average as compared with the control for omeprasole and lansoprasole, respectively. The magnitudes of the inhibition coefficient I(0.5) for omeprasole and lansoprasole were 35.60 +/- 0.81 and 29.40 +/- 1.79 microM respectively. Meanwhile cooperative effects on the Na+, K+ - ATPase activity were not found, as the Hill coefficient n(H) for omeprasole was 1.00 +/- 0.08, while for lansoprasole it was 1.20 +/- 0.03. These substances had also insignificant influence on Mg2+ -ATPase: the enzymatic activity was decreased to 84 and 82% as compared with the control with omeprasole and lansoprasole, respectively, in concentration of 100 microM for each inhibitor. The inhibition of Na+, K+ -ATPase activity can evidence for the possible side effects of omeprasole and lansoprasole when they are used for treatment of acid-dependent diseases of the stomach. In addition, obtained experimental data can be useful for further research of the membrane mechanisms of omeprasole and lansoprasole action on cationic exchange in the smooth muscle cells.

  15. Adsorption of acetanilide herbicides on soil and its components. II. Adsorption and catalytic hydrolysis of diethatyl-ethyl on saturated Na(+)-, K(+)-, Ca(2+)-, and Mg(2+)-montmorillonite. (United States)

    Liu, W P; Fang, Z; Liu, H J; Yang, W C


    Adsorption and catalytic hydrolysis of the herbicide diethatyl-ethyl [N-chloroacetyl-N-(2,6-diethylphenyl)glycine ethyl ester] on homoionic Na(+)-, K(+)-, Ca(2+)-, and Mg(2+)-montmorillonite clays were investigated in water solution. The Freundlich adsorption coefficient, Ki, got from isotherms on clay followed the order of Na+ approximately K+ > Mg2+ approximately Ca2+. Analysis of FT-IR spectra of diethatyl-ethyl adsorbed on clay suggests probable bonding at the carboxyl and amide carbonyl groups of the herbicide. The rate of herbicide hydrolysis in homoionic clay suspensions followed the same order as that for adsorption, indicating that adsorption may have preceded and thus caused hydrolysis. Preliminary product identification showed that hydrolysis occurred via nucleophilic substitution at the carboxyl carbon, causing the cleavage of the ester bond and formation of diethatyl and its dechlorinated derivative, and at the amide carbon, yielding an ethyl ester derivative and its acid. These pathways also suggest that hydrolysis of diethatyl-ethyl was catalyzed by adsorption on the clay surface.

  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 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.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.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 tissue-specificity and inducible

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


    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

  18. Cysteine-674 oxidation and degradation of sarcoplasmic reticulum Ca(2+) ATPase in diabetic pig aorta. (United States)

    Ying, Jia; Sharov, Victor; Xu, Shanqin; Jiang, Bingbing; Gerrity, Ross; Schöneich, Christian; Cohen, Richard A


    The sarcoplasmic reticulum Ca2+ ATPase (SERCA) is redox-regulated by posttranslational thiol modifications of cysteine-674 to regulate smooth muscle relaxation and migration. To detect oxidation of cysteine-674 that irreversibly prevents redox regulation, a polyclonal, sequence-specific antibody was developed toward a peptide containing cysteine-674 sulfonic acid. The antibody stained intact 110-kDa SERCA in pig cardiac SR that was oxidized in vitro by peroxynitrite in a sequence-specific manner, and histochemically stained atherosclerotic pig and rabbit aorta. Surprisingly, immunoblots of the pig aorta failed to stain intact 110-kDa SERCA protein, but rather, higher molecular mass aggregates and lower molecular mass bands. Of the latter bands at 70 and 60 kDa, the largest were observed in diabetic, hyperlipidemic pigs, and coincided with the most positive histochemical staining. The 70- and 60-kDa molecular mass bands also coincided with the majority of the protein detected by a monoclonal total anti-SERCA antibody, which detected the intact 110-kDa protein in normal pigs. Mass spectrometry identified SERCA in all the major bands detected by the sulfonic acid antibody as well as the oxidation of cysteine-674 in the 70-kDa band. These studies demonstrate a sequence-specific antibody that detects partial degradation products of SERCA, which represent the majority of the protein in some diabetic hypercholesterolemic pig aortae. In addition, the results suggest an association between irreversible oxidation of SERCA and its degradation, and that an important portion of the oxidized protein in tissue samples may be partially degraded.

  19. Localization of Ca2+ and Ca-ATPase on wet (Ruscus aculeatus) and dry (Primula officinalis) stigma surface


    Elżbieta Bednarska


    Calcium is present in the surface cells of both types of stigma. The chlorotetracycline method was used to show that the papillae of the dry stigma of Primula officinalis are the source of fluorescence of the CTC-Ca2+ complex. The precipitation method (NHA) localized calcium ions mainly in the pellicula surrounding these papillae. The NHA-Ca2+ precipitates were localized on the plasma membrane in the papillae of the wet stigma of Ruscus aculeatus. Ca-ATPase activity was found in both types of...

  20. Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells. (United States)

    Seo, Jeong-Ah; Kim, Boyun; Dhanasekaran, Danny N; Tsang, Benjamin K; Song, Yong Sang


    Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca(2+) homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca(2+) concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca(2+) flux was evident after the curcumin treatment (15 µM). Treatment with Ca(2+) chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca(2+) concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca(2+) elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca(2+) homeostasis and thereby promotes apoptosis in ovarian cancer cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. Disponibilidad de Ca2+, Mg2+ y K+ en función de las propiedades del suelo, zona cafetera central de Colombia

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    Luz Adriana Lince Salazar


    Full Text Available La nutrición mineral se da a partir de los elementos que se encuentran en forma disponible en la solución del suelo, la cual se abastece principalmentede la fase de cambio iónico. Con el objetivo de determinar la disponibilidad de cationes en la solución del suelo y su relación con las propiedades edáficas, se realizó una investigación con suelos provenientes de cinco unidades cartográficas de la zona cafetera central de Colombia. Por cada unidad cartográfica se seleccionó un lote cafetero, en el que se tomaron muestras de suelo a diferentes profundidades y se analizaron las características físicas, químicas y mineralógicas, incluyendo la concentración de Ca2+, Mg2+ y K+ en la solución.En las cinco unidades el catión predominante fue Ca2+, seguido por Mg2+ y K+, y las concentraciones de éstos para la fase de cambio y la solución, enla mayoría fueron iguales en los primeros 30 cm de profundidad y diferentes entre unidades, de las cuales las unidades Catarina, Doscientos y Guamal,presentaron los valores más altos y Quindío y Chinchiná los más bajos, lo cual se relacionó con el material parental ya que las tres primeras provienende rocas máficas y ultramáficas y las dosúltimas de materiales de composición intermedia. Finalmente las concentraciones de Ca2+ en la solución se explicaron desde 36,97 % hasta 88,11 % por la fase de cambio, las de Mg2+ desde 32,23 % hasta 97,30 % y las de K+ desde 79,06 % hasta 94,68 %, mediante modelos lineales que incluyeron nutrientes del suelo y propiedades como CIC, pH y contenido de arcillas.

  2. Effect of spermine on the activity of synaptosomal plasma membrane Ca(2+)-ATPase reconstituted in neutral or acidic phospholipids. (United States)

    Palacios, Javier; Sepúlveda, M Rosario; Salvador, J M; Mata, Ana M


    The activity of purified plasma membrane Ca(2+)-ATPase (PMCA) from pig brain was inhibited by spermine (a naturally occurring and highly abundant polycation in brain). The level of inhibition was dependent on the phospholipid used for reconstitution as well as on the intact or truncated state of the enzyme. An IC(50) value of 12.5 mM spermine was obtained for both, the intact protein plus calmodulin and the trypsin-digested protein, reconstituted in phosphatidylcholine (PC). In the absence of calmodulin the intact Ca(2+)-ATPase gave an IC(50) of 27 mM. This form was more sensitive to spermine inhibition when it was reconstituted with phosphatidylserine (PS), showing an IC(50) value of 2.5 mM spermine. However, the truncated form was less responsive to spermine inhibition, having an IC(50) value of 12.5 mM. Spermine has no effect on the affinity of the PMCA for Ca(2+) or ATP, but its effect on the protein is pH-dependent. It is suggested that spermine could bind to negatively charged residues on the ATPase with different accessibility, depending on the structural rearrangement of the protein. Further, when the protein is reconstituted in PS, spermine also binds to the lipid.

  3. Study of the interactions of dissolved organic matter with zinc ion and the impact of competitive metal ions (Ca(2+) and Mg(2+)) by in situ absorbance. (United States)

    Zhang, Tianyu; Wang, Tao; Lu, Yujuan


    The bioavailability and toxicity of zinc to aquatic life depend on dissolved organic matter (DOM), such as Suwannee River Fulvic Acid (SRFA), which plays an important role in the speciation of zinc. This study examined reactions of SRFA with zinc at different concentrations from pH 3.0 to 9.0, and competitive binding of calcium/magnesium and zinc to SRFA at pH 6.0, using in situ absorbance. Interactions of Zn(2+) with SRFA chromophores were evidenced by the emergence of features in Zn-differential spectra. Among all Zn(2+)-SRFA systems, dominant peaks, located at 235, 275 and 385 nm, and the highest intensity at 235 nm indicated the replacement of protons by the bound Zn(2+). The Zn(2+) binding with SRFA could be quantified by calculating the changes of the slopes of Zn-differential log-transformed absorbance in the wavelength range of 350-400 nm (denoted as DS350-400) and by comparing the experimental data with predictions using the Non-Ideal Competitive Adsorption (NICA-Donnan) model. DS350-400 was correlated well with the bound Zn(2+) concentrations predicted by NICA-Donnan model with or without Ca(2+) or Mg(2+). Ca(2+) and Mg(2+) only affect intensity of the Zn-differential and Zn-differential log-transformed absorbance, not shape. In situ absorbance can be used to gain further information about Me(n+)-DOM interactions in the presence of various metals.

  4. Silencing of plasma membrane Ca2+-ATPase isoforms 2 and 3 impairs energy metabolism in differentiating PC12 cells. (United States)

    Boczek, Tomasz; Lisek, Malwina; Ferenc, Bozena; Kowalski, Antoni; Wiktorska, Magdalena; Zylinska, Ludmila


    A close link between Ca(2+), ATP level, and neurogenesis is apparent; however, the molecular mechanisms of this relationship have not been completely elucidated. Transient elevations of cytosolic Ca(2+) may boost ATP synthesis, but ATP is also consumed by ion pumps to maintain a low Ca(2+) in cytosol. In differentiation process plasma membrane Ca(2+) ATPase (PMCA) is considered as one of the major players for Ca(2+) homeostasis. From four PMCA isoforms, the fastest PMCA2 and PMCA3 are expressed predominantly in excitable cells. In the present study we assessed whether PMCA isoform composition may affect energy balance in differentiating PC12 cells. We found that PMCA2-downregulated cells showed higher basal O2 consumption, lower NAD(P)H level, and increased activity of ETC. These changes associated with higher [Ca(2+)]c resulted in elevated ATP level. Since PMCA2-reduced cells demonstrated greatest sensitivity to ETC inhibition, we suppose that the main source of energy for PMCA isoforms 1, 3, and 4 was oxidative phosphorylation. Contrary, cells with unchanged PMCA2 expression exhibited prevalence of glycolysis in ATP generation. Our results with PMCA2- or PMCA3-downregulated lines provide an evidence of a novel role of PMCA isoforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation.

  5. Characterization of ryanodine receptor and Ca2+-ATPase isoforms in the thermogenic heater organ of blue marlin (Makaira nigricans). (United States)

    Morrissette, Jeffery M; Franck, Jens P G; Block, Barbara A


    A thermogenic organ is found beneath the brain of billfishes (Istiophoridae), swordfish (Xiphiidae) and the butterfly mackerel (Scombridae). The heater organ has been shown to warm the brain and eyes up to 14 degrees C above ambient water temperature. Heater cells are derived from extraocular muscle fibers and express a modified muscle phenotype with an extensive transverse-tubule (T-tubule) network and sarcoplasmic reticulum (SR) enriched in Ca(2+)-ATPase (SERCA) pumps and ryanodine receptors (RyRs). Heater cells have a high mitochondria content but have lost most of the contractile myofilaments. Thermogenesis has been hypothesized to be associated with release and reuptake of Ca(2+). In this study, Ca(2+) fluxes in heater SR vesicles derived from blue marlin (Makaira nigricans) were measured using fura-2 fluorescence. Upon the addition of MgATP, heater SR vesicles rapidly sequestered Ca(2+). Uptake of Ca(2+) was thapsigargin sensitive, and maximum loading ranged between 0.8 micro mol Ca(2+) mg(-1) protein and 1.0 micro mol Ca(2+) mg(-1) protein. Upon the addition of 10 mmol l(-1) caffeine or 350 micro mol l(-1) ryanodine, heater SR vesicles released only a small fraction of the loaded Ca(2+). However, ryanodine could elicit a much larger Ca(2+) release event when the activity of the SERCA pumps was reduced. RNase protection assays revealed that heater tissue expresses an RyR isoform that is also expressed in fish slow-twitch skeletal muscle but is distinct from the RyR expressed in fish fast-twitch skeletal muscle. The heater and slow-twitch muscle RyR isoform has unique physiological properties. In the presence of adenine nucleotides, this RyR remains open even though cytoplasmic Ca(2+) is elevated, a condition that normally closes RyRs. The fast Ca(2+) sequestration by the heater SR, coupled with a physiologically unique RyR, is hypothesized to promote Ca(2+) cycling, ATP turnover and heat generation. A branch of the oculomotor nerve innervates heater organs

  6. Characterization of the Mg2+-activated ATPase activity in smooth-muscle membranes. NADH oxidase and adenylate kinase interfere with the NADH-coupled enzyme assay.


    Missiaen, L; Wuytack, F; Casteels, R


    The apparent Mg2+-activated ATPase activity measured by the continuous NADH-coupled enzyme assay was studied in a number of microsomal preparations obtained from smooth muscle of the myometrium from pregnant or 17 beta-oestradiol-pretreated rats, the bovine aorta, the guinea-pig taenia coli, the rabbit ear artery and pig antrum. It was shown that this ATPase assay is prone to the effects of a number of artefacts that are tissue-dependent. The apparent Mg2+-ATPase activity in microsomes (micro...

  7. Uso da eletrodiálise na eliminação de Ca2+ e Mg2+ e sua influência na reologia de dispersões de argilas bentoníticas da Paraíba Electrodialysis uses in Paraíba bentonite clay dispersions to eliminate Ca2+ and Mg2+ and its influence on rheology

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    L. V. Amorim


    Full Text Available Este trabalho avalia a influência dos cátions Ca2+ e Mg2+, através da eletrodiálise, na reologia de dispersões de argilas bentoníticas de Boa Vista, PB. As dispersões de argila foram preparadas com 4,86% em massa, e realizado o processo de eletrodiálise com uma membrana polimérica catiônica, em um reator de bancada, sendo utilizadas tensões de 7, 10 e 13 V e tempos de ensaios de 40, 80 e 120 min, segundo planejamento fatorial do tipo 2² + 3 experimentos no ponto central. Após a eletrodiálise, as dispersões foram tratadas com Na2CO3 e após cura, determinadas as viscosidades aparente e plástica, em viscosímetro Fann 35A e o volume de filtrado, em filtroprensa Fann. Para quantificar os cátions eliminados, foram determinados os cátions trocáveis antes e após o processo de eletrodiálise. Os resultados mostraram que a eletrodiálise poderá ser uma valiosa ferramenta no estudo dos fatores que determinam o comportamento reológico das dispersões de argilas bentoníticas, e ainda que os cátions Ca2+ e Mg2+, parcialmente eliminados, não interferem na reologia das dispersões estudadas.The aim of this work is to evaluate, by electrodialysis, the influence of Ca2+ and Mg2+ on the rheology of bentonite clay dispersions from Boa Vista, PB. Dispersions were prepared with 4.86 wt.% and submitted to electrodialysis process by using a cationic polymeric membrane in the batch reactor. The electrodialysis was set up according to 2² + 3 factorial design by using 7, 10 and 13 V and 40, 80 and 120 min. After electrodialysis, the dispersions were treated with Na2CO3 and after curing, the apparent and plastic viscosities were measured using a viscosimeter and water loss with a filter-press. Moreover, exchanged cations were determinated before and after the electrodialysis process. The results show that the electrodialysis can be a valuable tool to identify the factors that dictate the rheological behavior of bentonite clay dispersions, as

  8. The influence of smoking on semen quality, seminal microelements and Ca2+-ATPase activity among infertile and fertile men. (United States)

    Kumosani, T A; Elshal, M F; Al-Jonaid, A A; Abduljabar, H S


    Tobacco smoking is now increasing rapidly throughout the developing world and is one of the biggest threats to current and future world health. Several studies have addressed the role of cigarette smoking on semen quality, but the exact mechanisms remain inconclusive. In order to evaluate the detrimental effects of smoking on semen quality among Saudi subjects, the levels of different seminal parameters in smokers were compared to non-smokers. A total of 159 semen samples (61 smokers and 98 non-smokers) from men attending an infertility clinic for routine infertility workup were sub-grouped into fertile or infertile and were compared based on standard semen analysis (according to WHO guidelines), content of metals (magnesium, zinc and cadmium) and plasma membrane Ca(2+)-ATPase activity of sperms. Cadmium concentration was found significantly higher in smokers than in non-smokers either in fertile or infertile group (2.9+/-0.4 vs 1.4+/-0.7; 2.9+/-0.5 vs 1.3+/-0.7 microg L(-1); respectively). Together with this increase in seminal Cd a significant decrease in Ca(2+)-ATPase activity (21.5+/-2.8 vs 33.71+/-1.2; 20.7+/-1.5 vs 35.07+/-2.9 mmol min(-1) mg(-1) protein, p<0.05), decrease in seminal zinc (109.8+/-8.1 vs 189.7+/-9.9 mg L(-1), p<0.01) and decrease in sperm motility (41.9%+/-2.9 vs 46.01%+/-2.5; 9.8%+/-2.4 vs 15.3%+/-2.7, p<0.05) were found. Our data demonstrate that cigarette smoking affects both Ca(2+)-ATPase activity and motility of the spermatozoa. These effects may be attributed to increased seminal cadmium and reduced zinc concentrations.

  9. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments (United States)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul


    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  10. Effect of Ca(2+), Fe(2+) and Mg(2+) on rheological properties of new food matrix made of modified cell wall polysaccharides from apple. (United States)

    Mierczyńska, Joanna; Cybulska, Justyna; Sołowiej, Bartosz; Zdunek, Artur


    A new food matrix made of modified cell wall polysaccharides (MPS) from apple pomace was developed. In this experiment, an effect of metal divalent ions: calcium, magnesium and iron ions on rheological properties of MPS was studied. An increase of Ca(2+) or Fe(2+) concentration in MPS suspensions significantly increased viscosity as well as elastic (G') and viscous (G″) moduli. Contrary, Mg(2+) addition caused a significant decrease of viscosity, G' and G″. Herschel-Bulkley's model fitted to shear stress vs. shear strain (flow curves) showed that calcium and iron ions increased pseudoplasticity and viscosity proportionally to concentration. The addition of any studied metal ions to MPS increased thixotropic effect. A temperature at the gel point increased when concentration increased to 9mM, then the gel points appeared at lower temperatures again for higher concentrations. This study showed that the MPS is an effective texture modifier with a controlled function by metal ions. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Evidence of the presence of a calmodulin-sensitive plasma membrane Ca2+-ATPase in Trypanosoma equiperdum. (United States)

    Pérez-Gordones, María Carolina; Ramírez-Iglesias, José Rubén; Cervino, Vincenza; Uzcanga, Graciela L; Benaim, Gustavo; Mendoza, Marta


    Trypanosoma equiperdum belongs to the subgenus Trypanozoon, which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca 2+ regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca 2+ homeostasis. Therefore, the plasma membrane Ca 2+ -ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca 2+ translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1-18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1-18 motif. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. ADF/cofilin regulates secretory cargo sorting at the TGN via the Ca2+ ATPase SPCA1. (United States)

    von Blume, Julia; Alleaume, Anne-Marie; Cantero-Recasens, Gerard; Curwin, Amy; Carreras-Sureda, Amado; Zimmermann, Timo; van Galen, Josse; Wakana, Yuichi; Valverde, Miguel Angel; Malhotra, Vivek


    Actin-severing proteins ADF/cofilin are required for the sorting of secretory cargo at the trans-Golgi network (TGN) in mammalian cells. How do these cytoplasmic proteins interact with the cargoes in the lumen of the TGN? Put simply, how are these two sets of proteins connected across the TGN membrane? Mass spectrometry of cofilin1 immunoprecipitated from HeLa cells revealed the presence of actin and the Ca(2+) ATPase SPCA1. Moreover, cofilin1 was localized to the TGN and bound to SPCA1 via dynamic actin. SPCA1 knockdown, like ADF/cofilin1 knockdown, inhibited Ca(2+) uptake into the TGN and caused missorting of secretory cargo. These defects were rescued by the overexpression of the TGN-localized SPCA1. We propose that ADF/cofilin-dependent severing of actin filaments exposes and promotes the activation of SPCA1, which pumps Ca(2+) into the lumen of the TGN for the sorting of the class of secretory cargo that binds Ca(2+). Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Interactions of Phosphate Groups of ATP and Aspartyl Phosphate with the Sarcoplasmic Reticulum Ca2+-ATPase: An FTIR Study (United States)

    Liu, Man; Krasteva, Maria; Barth, Andreas


    Phosphate binding to the sarcoplasmic reticulum Ca2+-ATPase was studied by time-resolved Fourier transform infrared spectroscopy with ATP and isotopically labeled ATP ([β-18O2, βγ-18O]ATP and [γ-18O3]ATP). Isotopic substitution identified several bands that can be assigned to phosphate groups of bound ATP: bands at 1260, 1207, 1145, 1110, and 1085 cm−1 are affected by labeling of the β-phosphate, bands likely near 1154, and 1098–1089 cm−1 are affected by γ-phosphate labeling. The findings indicate that the strength of interactions of β- and γ- phosphate with the protein are similar to those in aqueous solution. Two bands, at 1175 and 1113 cm−1, were identified for the phosphate group of the ADP-sensitive phosphoenzyme Ca2E1P. They indicate terminal and bridging P-O bond strengths that are intermediate between those of ADP-insensitive phosphoenzyme E2P and the model compound acetyl phosphate in water. The bridging bond of Ca2E1P is weaker than for acetyl phosphate, which will facilitate phosphate transfer to ADP, but is stronger than for E2P, which will make the Ca2E1P phosphate less susceptible to attack by water. PMID:16169973

  14. A pK change of acidic residues contributes to cation countertransport in the Ca-ATPase of sarcoplasmic reticulum. Role of H+ in Ca(2+)-ATPase countertransport. (United States)

    Yu, X; Hao, L; Inesi, G


    Proteoliposomal vesicles reconstituted with sarcoplasmic reticulum ATPase and exogenous lipids sustain ATP-dependent Ca2+ uptake and H+ ejection, as well as net charge displacement by Ca2+. We have studied the effect of lumenal (inner) and medium (extravesicular) pH variations on the countertransport ratios of H+ and Ca2+. We find that the Ca2+/H+ molar ratio is approximately 1 when the lumenal and medium pH is near neutrality, but changes with a specific pattern when the medium pH is varied in the presence of a constant lumenal pH and when the lumenal pH is varied in the presence of a constant medium pH. Empirical analysis of the experimental data shows that the apparent pK of the residue(s) releasing H+ into the medium is approximately 6.1, whereas the apparent pK of the residue(s) binding lumenal H+ is approximately 7.7. Assuming that the same acidic residues are involved in H+ and Ca2+ countertransport, our findings suggest a lower affinity for H+ in their outward orientation (prevalent in the ground state of the enzyme) and a higher affinity for H+ in lumenal orientation (prevalent in the phosphorylated state of the enzyme). Cyclic pK changes, coupled to ATP utilization, promote cation exchange, Ca2+ uptake, and H+ ejection by the vesicles. The stoichiometry of countertransport and net charge displacement is matched by a corresponding electrogenic behavior. A calculation of voltage development related to initial rates of charge transfer (dV/dt = (dQ/dt)/Cm) is given as a corrective replacement of a previous steady state calculation.

  15. Effects of oestrogen on sarcoplasmic reticulum Ca2+-ATPase activity and gene expression in genioglossus in chronic intermittent hypoxia rat. (United States)

    Liu, Yue-Hua; Li, Wen; Song, Wei-Hua


    This study was designed to investigate the effects of oestrogen on sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity and gene expression in ovariectomised rats under the condition of chronic intermittent hypoxia (CIH). Thirty-two female Sprague-Dawley rats were randomly divided into four groups: the normal control group (NC), the CIH group (CIH), the CIH-ovariectomised group (CIH+OVX), and the group of CIH-ovariectomised rats receiving estradiol replacement (CIH+OVX+E(2)). Rats in the latter three groups were exposed to CIH for 5 weeks. The animals were killed before genioglossus (GG) was rapidly excised, and their body and uterus mass were determined. Estradiol level was detected by radioimmunoassay. SR Ca(2+)-ATPase (SERCA) activity was observed by detecting inorganic phosphorus ion, and the SERCA mRNA level was measured using real-time quantitative polymerase chain reaction (real-time PCR). It was found that, compared with the NC group, the SERCA activity and mRNA level were remarkably reduced (pSERCA activity and mRNA level were also significantly reduced (pSERCA activity and mRNA level significantly increased (pSERCA activity and mRNA expression, and oestrogen-deficiency could exacerbate this effect; whilst estradiol replacement can partially reverse the effect of CIH in ovariectomised rats.

  16. [Effects of desulfurization waste on calcium distribution, Ca(2+)-ATPase activity, and antioxidant characteristics of rice leaf under alkali stress]. (United States)

    Mao, Gui-Lian; Xu, Xing; Zeng, Jin; Yue, Zi-Hui; Yang, Shu-Juan


    To approach the action mechanisms of desulfurization waste on alleviating alkali stress-induced injury of rice, a pot experiment was conducted to study the variations of leaf total calcium content, calcium distribution, plasma membrane Ca(2+)-ATPase activity, and reactive oxygen content of rice seedlings under alkali stress after the application of desulfurization waste. In the control, a few calcium particulates scattered in the cell wall and chloroplasts, while applying desulfurization waste or CaSO4 increased the calcium particulates in the plasma membrane, intercellular space, cell wall, and vacuole significantly. With the increasing application rate of desulfurization waste or CaSO4, the leaf total calcium content increased, Ca(2+)-ATPase activity in plasma membrane and tonoplast presented an increasing trend, plasma membrane relative permeability, MDA content, and O2 production rate decreased, and SOD and POD activities increased. The desulfurization waste could relieve the alkali stress to rice in some extent, and the main reactive compound in the waste could be CaSO4.

  17. Distinct Roles of the C-terminal 11th Transmembrane Helix and Luminal Extension in the Partial Reactions Determining the High Ca2+ Affinity of Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2b (SERCA2b)

    DEFF Research Database (Denmark)

    Clausen, Johannes D; Vandecaetsbeek, Ilse; Wuytack, Frank


    of each of these parts and their interactions with the SERCA environment were examined by transient kinetic analysis of the partial reaction steps in the Ca2+ transport cycle in mutant and chimeric Ca2+-ATPase constructs. Manipulations to the LE of SERCA2b markedly increased the rate of Ca2+ dissociation...... from Ca2E1. Addition of the SERCA2b tail to SERCA1a slowed Ca2+ dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca2E1. The interaction of LE with L7/8 is also important for the low rate...... of the Ca2E1P-E2P conformational transition. These findings can be rationalized in terms of stabilization of the Ca2E1 and Ca2E1P forms by docking of the LE near L7/8. By contrast, low rates of E2P dephosphorylation and E2-E1 transition in SERCA2b depend critically on the TM11, but not at all on the LE...

  18. Molecular aspects of Mg2+ transport systems. (United States)

    Smith, D L; Maguire, M E


    The gram-negative bacterium Salmonella typhimurium possesses three distinct Mg2+ transport systems, encoded by the CorA, MgtA, and MgtB loci. The CorA transport system is the constitutive Mg2+ influx system but can also mediate efflux at high extracellular Mg2+ concentrations. The CorA protein lacks homology to any known protein, is an integral membrane protein containing 28% percent-charged amino acids, and has three carboxyl terminal membrane-spanning segments. Its properties indicate that it is a new class of membrane transport protein, likely found in all gram-negative bacteria and possibly other organisms. In contrast, the MgtA and MgtB Mg2+ transport systems are normally expressed only at low extracellular Mg2+ concentrations and can mediate only the influx of Mg2+. Both MgtA and MgtB system are P-type ATPases; they have relatively poor homology to other known prokaryotic P-type ATPases but are highly homologous to mammalian P-type ATPases, particularly reticular Ca(2+)-ATPases. Expression of both MgtA and MgtB is highly regulated by the concentration of extracellular Mg2+. Transcription of MgtB is increased about 1,000-fold by lowering Mg2+ from 1 mM to 1 microM, and, under growth conditions of limiting Mg2+, MgtB becomes the dominant Mg2+ influx system. However, it is unclear why the cells require the use of ATP to mediate the influx of Mg2+ down its electrochemical gradient. Study of these Mg2+ transport systems should lead to further understanding of cellular Mg2+ homeostasis and eventually to characterization of eukaryotic Mg2+ transport systems.

  19. Changes in plasmalemma K+Mg2+ -ATPase dephosphorylating activity and H+ transport in relation to seasonal growth and freezing tolerance of Festuca pratensis Huds. (United States)

    Darginaviciene, Jūrate; Pasakinskiene, Izolda; Maksimov, Gemir; Rognli, Odd Arne; Jurkoniene, Sigita; Sveikauskas, Vaidevutis; Bareikiene, Nijole


    Changes in plasmalemma K(+)Mg(2+)-ATPase dephosphorylating activity and H(+) transport were examined in freezing-tolerant and non-tolerant genotypes of the perennial grass species Festuca pratensis Huds. Enzyme activity and DeltamuH(+) were measured in plasmalemma fractions isolated from basal nodes and roots. Three types of experiments were undertaken: (i) a field experiment, utilizing the seasonal growth and cessation cycle of a perennial plant; (ii) a cold acclimation experiment in hydroponics; and (iii) an instant freezing test. A specific fluctuation in K(+)Mg(2+)-ATPase activity was found throughout the seasonal growth of the plants (i). The K(+)Mg(2+)-ATPase activity peaks for both the basal node and the root plasmalemma were determined early in the spring before the renewal of growth. The lowest activity values in roots occurred at the time approaching flowering, and in basal nodes at the transition into the growth cessation. The K(+)Mg(2+)-ATPase activity was approximately 50% lower in the basal node plasmalemma of freezing-tolerant plants than of non-tolerant ones, when assessed at the optimal growth stage in hydroponics. In hydroponics (ii) and in the freezing test (iii), temperature stress was followed by a more pronounced change in the level of K(+)Mg(2+)-ATPase activity than in that of H(+) transport, and this change was more clearly differentiated in the basal node plasmalemma of contrasting genotypes than in the roots. Stress response was manifested differently in freezing-tolerant and non-tolerant plants at cold acclimation (4-2 degrees C) and at freezing (-8 degrees C) temperatures. Proton transport regulation via coupled changes in the hydrolysed ATP/transported proton ratio, as an attribute of freezing-tolerant plants, is discussed.

  20. Paradoxical effects of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) activator gingerol on NG115-401L neuronal cells: failure to augment ER Ca(2+) uptake and protect against ER stress-induced cell death. (United States)

    Zhang, Changfeng; Bose, Diptiman D; Thomas, David W


    Perturbation of endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress are thought to underlie a spectrum of defects encompassing major societal diseases such as diabetes and neurodegeneration. In this report we used the NG115-401L neuronal cell line to test the hypothesis that neuroprotection against ER stress may be conferred by pharmacological stimulation of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pumps. We report that the SERCA activator gingerol stimulates SR microsomal Ca(2+)-ATPase activity and restores enzymatic function in the presence of potent SERCA blockers. Yet, enzyme protection in isolated membranes does not extend to protection from ER stress in intact NG115-401L cells. Surprisingly, gingerol not only failed to protect cells from SERCA blocker-induced ER stress and cell death, the compound itself potently induced cell death. Also, we report that gingerol failed to augment ER Ca(2+) uptake, a result contradictory to what has been observed in muscle. Unexpectedly, gingerol discharged ER Ca(2+) stores and coupled robustly to Ca(2+) influx pathways. These observations suggest that gingerol is not acting as a traditional SERCA blocker as thapsigargin mediated ER Ca(2+) store depletion fails to stimulate Ca(2+) influx in the NG115-401L cell phenotype. Moreover, cell death induced by gingerol, in contrast to the classic SERCA inhibitors, is not accompanied by increases in reactive oxygen species production or enzymatic caspase activity. These results argue for a finer regulatory control on SERCA function with gingerol's actions revealing potentially novel routes of coupling altered pump regulation to the assembly of functional Ca(2+) influx units and activation of cell death pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders. (United States)

    Kang, Soojeong; Dahl, Russell; Hsieh, Wilson; Shin, Andrew; Zsebo, Krisztina M; Buettner, Christoph; Hajjar, Roger J; Lebeche, Djamel


    Dysregulation of endoplasmic reticulum (ER) Ca(2+) homeostasis triggers ER stress leading to the development of insulin resistance in obesity and diabetes. Impaired function of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) has emerged as a major contributor to ER stress. We pharmacologically activated SERCA2b in a genetic model of insulin resistance and type 2 diabetes (ob/ob mice) with a novel allosteric activator, CDN1163, which markedly lowered fasting blood glucose, improved glucose tolerance, and ameliorated hepatosteatosis but did not alter glucose levels or body weight in lean controls. Importantly, CDN1163-treated ob/ob mice maintained euglycemia comparable with that of lean mice for >6 weeks after cessation of CDN1163 administration. CDN1163-treated ob/ob mice showed a significant reduction in adipose tissue weight with no change in lean mass, assessed by magnetic resonance imaging. They also showed an increase in energy expenditure using indirect calorimetry, which was accompanied by increased expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue. CDN1163 treatment significantly reduced the hepatic expression of genes involved in gluconeogenesis and lipogenesis, attenuated ER stress response and ER stress-induced apoptosis, and improved mitochondrial biogenesis, possibly through SERCA2-mediated activation of AMP-activated protein kinase pathway. The findings suggest that SERCA2b activation may hold promise as an effective therapy for type-2 diabetes and metabolic dysfunction. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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


    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. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Sarcoendoplasmic reticulum Ca(2+) ATPase. A critical target in chlorine inhalation-induced cardiotoxicity. (United States)

    Ahmad, Shama; Ahmad, Aftab; Hendry-Hofer, Tara B; Loader, Joan E; Claycomb, William C; Mozziconacci, Olivier; Schöneich, Christian; Reisdorph, Nichole; Powell, Roger L; Chandler, Joshua D; Day, Brian J; Veress, Livia A; White, Carl W


    Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.

  4. Sarcoendoplasmic Reticulum Ca2+ ATPase. A Critical Target in Chlorine Inhalation–Induced Cardiotoxicity (United States)

    Ahmad, Aftab; Hendry-Hofer, Tara B.; Loader, Joan E.; Claycomb, William C.; Mozziconacci, Olivier; Schöneich, Christian; Reisdorph, Nichole; Powell, Roger L.; Chandler, Joshua D.; Day, Brian J.; Veress, Livia A.; White, Carl W.


    Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation–induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration–approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia–reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure. PMID:25188881

  5. Ablation of plasma membrane Ca(2+)-ATPase isoform 4 prevents development of hypertrophy in a model of hypertrophic cardiomyopathy. (United States)

    Prasad, Vikram; Lorenz, John N; Lasko, Valerie M; Nieman, Michelle L; Jiang, Min; Gao, Xu; Rubinstein, Jack; Wieczorek, David F; Shull, Gary E


    The mechanisms linking the expression of sarcomeric mutant proteins to the development of pathological hypertrophy in hypertrophic cardiomyopathy (HCM) remain poorly understood. We investigated the role of the plasma membrane Ca(2+)-ATPase PMCA4 in the HCM phenotype using a transgenic model that expresses mutant (Glu180Gly) α-tropomyosin (Tm180) in heart. Immunoblot analysis revealed that cardiac PMCA4 expression was upregulated early in Tm180 disease pathogenesis. This was accompanied by an increase in levels of the L-type Ca(2+)-channel, which is implicated in pathological hypertrophy. When Tm180 mice were crossed with a PMCA4-null line, loss of PMCA4 caused the abrogation of hypertrophy in Tm180/PMCA4-null double mutant mice. RT-PCR analysis of Tm180/PMCA4-null hearts revealed blunting of the fetal program and reversion of pro-fibrotic Col1a1 and Col3a1 gene expression to wild-type levels. This was accompanied by evidence of reduced L-type Ca(2+)-channel expression, and diminished calcineurin activity. Expression of the metabolic substrate transporters glucose transporter 4 and carnitine palmitoyltransferase 1b was preserved and Tm180-related changes in mRNA levels of various contractile stress-related proteins including the cardiac ankyrin protein CARP and the N2B isoform of titin were reversed in Tm180/PMCA4-null hearts. cGMP levels were increased and phosphorylation of vasodilator-stimulated phosphoprotein was elevated in Tm180/PMCA4-null hearts. These changes were associated with a sharp reduction in left ventricular end-diastolic pressure in Tm180/PMCA4-null hearts, which occurred despite persistence of Tm180-related impairment of relaxation dynamics. These results reveal a novel and specific role for PMCA4 in the Tm180 hypertrophic phenotype, with the "protective" effects of PMCA4 deficiency encompassing multiple determinants of HCM-related hypertrophy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Effect of carticaine on the sarcoplasmic reticulum Ca2+-adenosine triphosphatase. II. Cations dependence. (United States)

    Takara, Delia; Sánchez, Gabriel A; Toma, Augusto F; Bonazzola, Patricia; Alonso, Guillermo L


    Ca2+-ATPase is a major intrinsic protein in the sarcoplasmic reticulum (SR) from skeletal muscles. It actively transports Ca2+ from the cytoplasm to the SR lumen, reducing cytoplasmic [Ca2+] to promote muscle relaxation. Carticaine is a local anesthetic widely used in operative dentistry. We previously showed that carticaine inhibits SR Ca2+-ATPase activity and the coupled Ca(2+) uptake by isolated SR vesicles, and increases the rate of Ca2+ efflux from preloaded vesicles. We also found that these effects were antagonized by divalent cations, and concluded that they were mainly due to the direct interaction of carticaine with the Ca2+-ATPase protein. Here we present additional results on the modulation of the above effects of carticaine by Ca2+ and Mg2+. The activating effect of Ca2+ on the ATPase activity is competitively inhibited by carticaine, indicating a decreased Ca2+ binding to the high affinity Ca2+ transport sites. The activating effect of Mg2+ on the phosphorylation of Ca2+-ATPase by orthophosphate is also inhibited by carticaine. The anesthetic does not affect the reaction mechanism of the cations acting as cofactors of ATP in the catalytic site. On the basis of the present and our previous results, we propose a model that describes the effect of carticaine on the Ca2+-ATPase cycle.

  7. Splice-site A choice targets plasma-membrane Ca2+-ATPase isoform 2 to hair bundles. (United States)

    Hill, Jennifer K; Williams, Diane E; LeMasurier, Meredith; Dumont, Rachel A; Strehler, Emanuel E; Gillespie, Peter G


    Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca2+-ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites (A and C) to hair-cell targeting of PMCA2. When PMCA2 isoforms were immunoprecipitated from purified hair bundles of rat utricle, 2w was the only site A variant detected; moreover, immunocytochemistry for 2w in rat vestibular and cochlear tissues indicated that this splice form was located solely in bundles. To demonstrate the necessity of the 2w sequence, we transfected hair cells with PMCA2 containing different variants at splice sites A and C. Although native hair bundles exclusively use the 2a form at splice-site C, epitope-tagged PMCA2w/a and PMCA2w/b were both concentrated in bundles, indicating that site C is not involved in bundle targeting. In contrast, PMCA2z/a was excluded from bundles and was instead targeted to the basolateral plasma membrane. Bundle-specific targeting of PMCA2w/a tagged with green fluorescent protein (GFP) was diminished, suggesting that GFP interfered with splice-site A. Together, these data demonstrate that PMCA2w/a is the hair-bundle isoform of PMCA in rat hair cells and that 2w targets PMCA2 to bundles. The 2w sequence is thus the first targeting signal identified for a hair-bundle membrane protein; moreover, the striking distribution of inner-ear PMCA isoforms dictated by selective targeting suggests a critical functional role for segregated pathways of Ca2+ transport.

  8. Distribution of the intracellular Ca(2+)-ATPase isoform 2b in pig brain subcellular fractions and cross-reaction with a monoclonal antibody raised against the enzyme isoform. (United States)

    Salvador, J M; Berengena, M; Sepúlveda, M R; Mata, A M


    The presence and distribution of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) isoform 2b in microsomes and other subcellular fractions isolated from pig brain has been demonstrated by the combined use of a specific antibody raised against the SERCA2b isoform and ATP phosphorylation experiments. All subcellular fractions show an approximately 110 kDa phosphorylated protein, the band intensity being stronger in microsomes. Preliminary treatment of the samples with trypsin generates two phosphorylated fragments of about 57 and 33 kDa in the presence of Ca(2+). The observed fragments are typical trypsinized products of the SERCA2b isoform. The monoclonal antibody Y/1F4 raised against the sarcoplasmic reticulum Ca(2+)-ATPase (isoform 1) binds to the 110 kDa band in membranes isolated from brain. The binding was stronger in microsomes than in other fractions. Furthermore, this antibody also recognizes a clear band at around 115 kDa. This band is always stronger in plasma membrane than in synaptosomes or microsomes and is unaffected by trypsin. Phosphorylation studies in the absence of Ca(2+) suggest that the 115 kDa protein is not a Ca(2+)-ATPase.

  9. Refined equation for description of the influence of intracellular Mg2+ on the activity of single Ca2+-activated K+ channels in Vero kidney cells. (United States)

    Kazachenko, V N; Kochetkov, K V


    A refined empirical equation relating the mean value of the logarithm of the apparent dissociation constant (K0.5) to intracellular Mg2+ concentration is given. The equation is derived based on a more precise adjustment of analytical expressions to experimental data.

  10. Pycnogenol® and Ginkgo biloba extract: effect on peroxynitrite-oxidized sarcoplasmic reticulum Ca2+-ATPase (United States)

    Žižková, Petronela; Viskupičová, Jana; Horáková, L'ubica


    The effect of two natural standardized plant extracts, Pycnogenol® and EGb 761, on sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity and posttranslational modifications induced by peroxynitrite was investigated to assess their possible protective role. EGb 761 was found to have a protective effect on SERCA activity in the concentration range of 5–40 µg/ml. On the other hand, Pycnogenol® caused a decrease of SERCA activity at concentrations of 25 µg/ml. EGb 761 did not prevent protein carbonyl formation upon oxidation with peroxynitrite. On the contrary, Pycnogenol® at the concentrations of 5 and 10 µg/ml significantly decreased the level of protein carbonyls by 44% and 54%, respectively. Neither Pycnogenol® nor EGb 761 exerted a protective effect against thiol group oxidation.The plant extracts studied modulated peroxynitrite-injured SERCA activity by different ways and failed to correlate with posttranslational modifications. Their effect seems to be associated with their ability to change SERCA conformation rather than by their antioxidant capacity. PMID:21331179

  11. Cloning and characterization of the heart muscle isoform of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) from crayfish. (United States)

    Chen, Dongdong; Zhang, Zhiping; Wheatly, Michele G; Gao, Yongping


    This paper describes the cloning and functional characterization of the heart muscle isoform of Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish Procambarus clarkii. The complete crayfish heart SERCA, identified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), consists of 4495 bp with a 3060 bp open reading frame, coding for 1020 amino acids. This isoform differs from the previously identified axial abdominal (tail) muscle SERCA solely in its C-terminal amino acids. The last nine amino acids of the tail muscle isoform are replaced by 27 hydrophobic amino acids in the heart isoform that have the potential to form an additional transmembrane domain. Consistent with other invertebrate studies, Southern blot analysis suggested that the heart and tail muscle isoforms are encoded from the same gene that is equally related to SERCA-1, -2 and -3 of vertebrates. The tissue distributions of these two isoforms have been assessed using isoform-specific probes and northern analysis. A cardiac-specific probe bound only to a 5.8 kb species in heart and had minimal cross-hybridization with 7.6 and 5.8 kb species in eggs and no hybridization with tail muscle. A tail-isoform-specific probe hybridized with a 4.5 kb species in tail muscle and cross-hybridized with a 4.5 kb species in eggs and 8.8 kb in heart muscle. Both isoforms are expressed in eggs suggesting that transcripts are formed early in development and are subsequently broadly expressed in all tissue types. Expression of the cardiac muscle SERCA isoform varied with the stage of moulting. Expression was high in intermoult and decreased in premoult. However, expression was restored rapidly in postmoult (within 2 days) unlike expression of tail muscle SERCA, which remained downregulated for weeks. Differences in contractility between the two muscle types in the postmoult period may explain these expression patterns.

  12. Inhibiting Na+/K+ ATPase can impair mitochondrial energetics and induce abnormal Ca2+ cycling and automaticity in guinea pig cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Qince Li

    Full Text Available Cardiac glycosides have been used for the treatment of heart failure because of their capabilities of inhibiting Na+/K+ ATPase (NKA, which raises [Na+]i and attenuates Ca2+ extrusion via the Na+/Ca2+ exchanger (NCX, causing [Ca2+]i elevation. The resulting [Ca2+]i accumulation further enhances Ca2+-induced Ca2+ release, generating the positive inotropic effect. However, cardiac glycosides have some toxic and side effects such as arrhythmogenesis, confining their extensive clinical applications. The mechanisms underlying the proarrhythmic effect of glycosides are not fully understood. Here we investigated the mechanisms by which glycosides could cause cardiac arrhythmias via impairing mitochondrial energetics using an integrative computational cardiomyocyte model. In the simulations, the effect of glycosides was mimicked by blocking NKA activity. Results showed that inhibiting NKA not only impaired mitochondrial Ca2+ retention (thus suppressed reactive oxygen species (ROS scavenging but also enhanced oxidative phosphorylation (thus increased ROS production during the transition of increasing workload, causing oxidative stress. Moreover, concurrent blocking of mitochondrial Na+/Ca2+ exchanger, but not enhancing of Ca2+ uniporter, alleviated the adverse effects of NKA inhibition. Intriguingly, NKA inhibition elicited Ca2+ transient and action potential alternans under more stressed conditions such as severe ATP depletion, augmenting its proarrhythmic effect. This computational study provides new insights into the mechanisms underlying cardiac glycoside-induced arrhythmogenesis. The findings suggest that targeting both ion handling and mitochondria could be a very promising strategy to develop new glycoside-based therapies in the treatment of heart failure.

  13. 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.


    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.

  14. Transcription of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase type 3 gene, ATP2A3, is regulated by the calcineurin/NFAT pathway in endothelial cells. (United States)

    Hadri, Lahouaria; Pavoine, Catherine; Lipskaia, Larissa; Yacoubi, Sabrina; Lompré, Anne-Marie


    Histamine, known to induce Ca2+ oscillations in endothelial cells, was used to alter Ca2+ cycling. Treatment of HUVEC (human umbilical-vein endothelial cell)-derived EA.hy926 cells with histamine for 1-3 days increased the levels of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 3, but not of SERCA 2b, transcripts and proteins. Promoter-reporter gene assays demonstrated that this increase in expression was due to activation of SERCA 3 gene transcription. The effect of histamine was abolished by mepyramine, but not by cimetidine, indicating that the H1 receptor, but not the H2 receptor, was involved. The histamine-induced up-regulation of SERCA 3 was abolished by cyclosporin A and by VIVIT, a peptide that prevents calcineurin and NFAT (nuclear factor of activated T-cells) from interacting, indicating involvement of the calcineurin/NFAT pathway. Histamine also induced the nuclear translocation of NFAT. NFAT did not directly bind to the SERCA 3 promoter, but activated Ets-1 (E twenty-six-1), which drives the expression of the SERCA 3 gene. Finally, cells treated with histamine and loaded with fura 2 exhibited an improved capacity in eliminating high cytosolic Ca2+ concentrations, in accordance with an increase in activity of a low-affinity Ca2+-ATPase, like SERCA 3. Thus chronic treatment of endothelial cells with histamine up-regulates SERCA 3 transcription. The effect of histamine is mediated by the H1R (histamine 1 receptor) and involves activation of the calcineurin/NFAT pathway. By increasing the rate of Ca2+ sequestration, up-regulation of SERCA 3 counteracts the cytosolic increase in Ca2+ concentration.

  15. Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase. (United States)

    Moreno, S N; Mason, R P; Docampo, R


    At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.

  16. Evaluation of a two-site, three-barrier model for permeation in Ca(V)3.1 (alpha1G) T-type calcium channels: Ca (2+), Ba (2+), Mg (2+), and Na (+). (United States)

    Lopin, Kyle V; Obejero-Paz, Carlos A; Jones, Stephen W


    We explored the ability of a two-site, three-barrier (2S3B) Eyring model to describe recently reported data on current flow through open Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide range (100 nM: -110 mM: ) while recording whole-cell currents over a wide voltage range (-150 mV to +100 mV) from channels stably expressed in HEK 293 cells. Effects on permeation were isolated using instantaneous current-voltage relationships (IIV) after strong, brief depolarizations to activate channels with minimal inactivation. Most experimental results were reproduced by a 2S3B model. The model described the IIV relationships, apparent affinities for permeation and block for Ca(2+) and Ba(2+), and shifts in reversal potential between Ca(2+) and Ba(2+). The fit to block by 1 mM Mg(2+)(i) was reasonable, but block by Mg(2+)(0) was described less well. Surprisingly, fits were comparable with strong ion-ion repulsion, with no repulsion, or with intermediate values. With weak repulsion, there was a single high-affinity site, with a low-affinity site near the cytoplasmic side of the pore. With strong repulsion, the net charge of ions in the pore was near +2 over a relatively wide range of concentration and voltage, suggesting a knockoff mechanism. With strong repulsion, Ba(2+) preferred the inner site, while Ca(2+) preferred the outer site, potentially explaining faster entry of Ni(2+) and other pore blockers when Ba(2+) is the charge carrier.

  17. Raman spectroscopy of DNA-metal complexes. II. The thermal denaturation of DNA in the presence of Sr2+, Ba2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+.


    Duguid, J G; Bloomfield, V A; Benevides, J M; Thomas, G J


    Differential scanning calorimetry, laser Raman spectroscopy, optical densitometry, and pH potentiometry have been used to investigate DNA melting profiles in the presence of the chloride salts of Ba2+, Sr2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+. Metal-DNA interactions have been observed for the molar ratio [M2+]/[PO2-] = 0.6 in aqueous solutions containing 5% by weight of 160 bp mononucleosomal calf thymus DNA. All of the alkaline earth metals, plus Mn2+, elevate the melting temperature of ...

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

    Directory of Open Access Journals (Sweden)

    Angel Zarain-Herzberg


    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.

  19. Presence of cardiac alpha-myosin correlates with histochemical myosin Ca2+ ATPase activity in rabbit masseter muscle

    NARCIS (Netherlands)

    Bredman, J. J.; Weijs, W. A.; Moorman, A. F.


    A combined enzyme-histochemical (ATPase reactivity) and immunohistochemical study has been performed on sections of rabbit masseter muscle. The majority of the fibres previously designated as type IIC and/or type I according to their ATPase activity were found to contain 'cardiac' alpha-myosin heavy

  20. Nandrolone decanoate treatment affects sarcoplasmic reticulum Ca(2+) ATPase function in skinned rat slow- and fast-twitch fibres. (United States)

    Bouhlel, Aicha; Joumaa, Wissam H; Léoty, Claude


    The effects of anabolic-androgenic steroid administration on the function of the sarcoplasmic reticulum (SR) pump were investigated in chemically skinned fibres from the extensor digitorum longus (EDL) and soleus muscles of sedentary rats. Twenty male rats were divided into two groups, one group received an intramuscular injection of nandrolone decanoate (15 mg x kg(-1)) weekly for 8 weeks, the second received similar weekly doses of vehicle (sterile peanut oil). Compared with control muscles, nandrolone decanoate treatment reduced SR Ca(2+) loading in EDL and soleus fibres by 49% and 29%, respectively. In control and treated muscles, the rate of Ca(2+) leakage depended on the quantity of Ca(2+) loaded. Furthermore, for similar SR Ca(2+) contents, the Ca(2+) leakage rate was not significantly modified by nandrolone decanoate treatment. Nandrolone decanoate treatment thus affects Ca (2+) uptake by the SR in a fibre-type dependent manner.

  1. [Comparative study of calixarene effect on Mg2+ -dependent ATP-hydrolase enzymatic systems from smooth muscle cells of the uterus]. (United States)

    Labyntseva, R D; Slinchenko, N M; Veklich, T O; Rodik, R V; Cherenok, S O; Boĭko, V I; Kal'chenko, V I; Kosterin, S O


    Investigation the influence of calyx[4]arenes C-90, C-91, C-97 and C-99 (codes are indicated) on the enzymatic activity of four functionally different Mg2+ -dependent ATPases from smooth muscle of the uterus: actomyosin ATPase, transporting Ca2+, Mg2+ -ATPase, ouabain-sensible Na+, K+ -ATPase and basal Mg2+ -ATPase. It was shown that calixarenes C-90 and C-91 in concentration 100 microM act multidirectionally on the functionally different Mg2+ -dependent ATP-hydrolase enzymatic systems. These compounds activate effectively the actomyosin ATPase (Ka = 52 +/- 11 microM [Ukrainian character: see text] 8 +/- 2 microM, accordingly), at the same time calixarene C-90 inhibited effectively activity of transporting Ca2+, Mg2+ -ATPase of plasmatic membranes (I(0,5) = 34.6 +/- 6.4 microM), but influence on membrane-bound Na+, K+ -ATPase and basal Mg2+ -ATPase. Calixarene C-91 reduce effectively basal Mg2+ -ATPase activity, insignificantly activating Na+, K+ -ATPase but has no influence on transporting Ca2+, Mg2+ -ATPase activity of plasmatic membranes. Calixarenes C-97 and C-99 (100 microM), which have similar structure, have monodirectional influence on activity of three functionally different Mg2+-dependent ATPases of the myometrium: actomyosin ATPase and two ATPases, that related to the ATP-hydrolases of P-type--Ca2+, Mg2+ -ATPase and Na+, K+ -ATPase of plasmatic membranes. Basal Mg2+ -ATPase is resistant to the action of these two connections. Results of comparative experiments that were obtained by catalytic titration of calixarenes C-97 and C-99 by actomyosin ATPase (I(0,5) = 88 +/- 9 and 86 +/- 8 microM accordingly) and Na+, K+ -ATPase from plasmatic membranes (I(0,5) = 33 +/- 4 and 98 +/- 8 nM accordingly) indicate to the considerably more sensitiveness of Na+, K+ -ATP-ase to these calixarenes than ATPase of contractile proteins. Thus, it is shown that calixarenes have influence on activity of a number of important enzymes, involved in functioning of the smooth muscle

  2. Interactions between small ankyrin 1 and sarcolipin coordinately regulate activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1). (United States)

    Desmond, Patrick F; Labuza, Amanda; Muriel, Joaquin; Markwardt, Michele L; Mancini, Allison E; Rizzo, Mark A; Bloch, Robert J


    SERCA1, the sarco(endo)plasmic reticulum Ca(2+)-ATPase of skeletal muscle, is essential for muscle relaxation and maintenance of low resting Ca(2+) levels in the myoplasm. We recently reported that small ankyrin 1 (sAnk1) interacts with the sarco(endo)plasmic reticulum Ca(2+)-ATPase in skeletal muscle (SERCA1) to inhibit its activity. We also showed that this interaction is mediated at least in part through sAnk1's transmembrane domain in a manner similar to that of sarcolipin (SLN). Earlier studies have shown that SLN and phospholamban, the other well studied small SERCA-regulatory proteins, oligomerize either alone or together. As sAnk1 is coexpressed with SLN in muscle, we sought to determine whether these two proteins interact with one another when coexpressed exogenously in COS7 cells. Coimmunoprecipitation (coIP) and anisotropy-based FRET (AFRET) assays confirmed this interaction. Our results indicated that sAnk1 and SLN can associate in the sarcoplasmic reticulum membrane and after exogenous expression in COS7 cells in vitro but that their association did not require endogenous SERCA2. Significantly, SLN promoted the interaction between sAnk1 and SERCA1 when the three proteins were coexpressed, and both coIP and AFRET experiments suggested the formation of a complex consisting of all three proteins. Ca(2+)-ATPase assays showed that sAnk1 ablated SLN's inhibition of SERCA1 activity. These results suggest that sAnk1 interacts with SLN both directly and in complex with SERCA1 and reduces SLN's inhibitory effect on SERCA1 activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. K+ Transport by the OsHKT2;4 Transporter from Rice with Atypical Na+ Transport Properties and Competition in Permeation of K+ over Mg2+ and Ca2+ Ions1[C][W][OA (United States)

    Horie, Tomoaki; Brodsky, Dennis E.; Costa, Alex; Kaneko, Toshiyuki; Lo Schiavo, Fiorella; Katsuhara, Maki; Schroeder, Julian I.


    Members of class II of the HKT transporters, which have thus far only been isolated from grasses, were found to mediate Na+-K+ cotransport and at high Na+ concentrations preferred Na+-selective transport, depending on the ionic conditions. But the physiological functions of this K+-transporting class II of HKT transporters remain unknown in plants, with the exception of the unique class II Na+ transporter OsHKT2;1. The genetically tractable rice (Oryza sativa; background Nipponbare) possesses two predicted K+-transporting class II HKT transporter genes, OsHKT2;3 and OsHKT2;4. In this study, we have characterized the ion selectivity of the class II rice HKT transporter OsHKT2;4 in yeast and Xenopus laevis oocytes. OsHKT2;4 rescued the growth defect of a K+ uptake-deficient yeast mutant. Green fluorescent protein-OsHKT2;4 is targeted to the plasma membrane in transgenic plant cells. OsHKT2;4-expressing oocytes exhibited strong K+ permeability. Interestingly, however, K+ influx in OsHKT2;4-expressing oocytes did not require stimulation by extracellular Na+, in contrast to other class II HKT transporters. Furthermore, OsHKT2;4-mediated currents exhibited permeabilities to both Mg2+ and Ca2+ in the absence of competing K+ ions. Comparative analyses of Ca2+ and Mg2+ permeabilities in several HKT transporters, including Arabidopsis thaliana HKT1;1 (AtHKT1;1), Triticum aestivum HKT2;1 (TaHKT2;1), OsHKT2;1, OsHKT2;2, and OsHKT2;4, revealed that only OsHKT2;4 and to a lesser degree TaHKT2;1 mediate Mg2+ transport. Interestingly, cation competition analyses demonstrate that the selectivity of both of these class II HKT transporters for K+ is dominant over divalent cations, suggesting that Mg2+ and Ca2+ transport via OsHKT2;4 may be small and would depend on competing K+ concentrations in plants. PMID:21610181

  4. Effect of major cations (Ca2+, Mg2+, Na+, K+) and anions (SO4(2-), Cl- , NO3-) on Ni accumulation and toxicity in aquatic plant (Lemna minor L.): implications For Ni risk assessment. (United States)

    Gopalapillai, Yamini; Hale, Beverley; Vigneault, Bernard


    The effect of major cation activity (Ca(2+) , Mg(2+) , Na(+) , K(+) ) on Ni toxicity, with dose expressed as exposure (total dissolved Ni concentration NiTot ) or free Ni ion activity (in solution Ni(2+) ), or as tissue residue (Ni concentration in plant tissue NiTiss ) to the aquatic plant Lemna minor L. was examined. In addition, Ni accumulation kinetics was explored to provide mechanistic insight into current approaches of toxicity modeling, such as the tissue residue approach and the biotic ligand model (BLM), and the implications for plant Ni risk assessment. Major cations did not inhibit Ni accumulation via competitive inhibition as expected by the BLM framework. For example, Ca(2+) and Mg(2+) (sulfate as counter-anion) had an anticompetitive effect on Ni accumulation, suggesting that Ca or Mg forms a ternary complex with Ni-biotic ligand. The counter-anion of the added Ca (sulfate, chloride, or nitrate) affected plant response (percentage of root growth inhibition) to Ni. Generally, sulfate and chloride influenced plant response while nitrate did not, even when compared within the same range of Ca(2+) , which suggests that the anion dominated the observed plant response. Overall, although an effect of major cations on Ni toxicity to L. minor L. was observed at a physiological level, Ni(2+) or NiTot alone modeled plant response, generally within a span of twofold, over a wide range of water chemistry. Thus, consideration of major cation competition for improving Ni toxicity predictions in risk assessment for aquatic plants may not be necessary. Copyright © 2013 SETAC.

  5. Endomembrane Ca2+-AtPases play a significant role in virus-induced adaptation to oxidative stress

    DEFF Research Database (Denmark)

    Shabala, Sergey; Bækgaard, Lone; Shabala, Lana


    in adaptive responses to oxidative stress by removing excessive Ca2+ from the cytosol, and that their functional expression is significantly altered in PVX-inoculated plants. These findings highlight the crucial role of Ca2+ efflux systems in acquired tolerance to oxidative stress and open up prospects...... for practical applications in agriculture, after in-depth comprehension of the fundamental mechanisms involved in common responses to environmental factors at the genomic, cellular and organismal levels....

  6. Some commonly used brominated flame retardants cause Ca2+-ATPase inhibition, beta-amyloid peptide release and apoptosis in SH-SY5Y neuronal cells.

    Directory of Open Access Journals (Sweden)

    Fawaz Al-Mousa

    Full Text Available Brominated flame retardants (BFRs are chemicals commonly used to reduce the flammability of consumer products and are considered pollutants since they have become widely dispersed throughout the environment and have also been shown to bio-accumulate within animals and man. This study investigated the cytotoxicity of some of the most commonly used groups of BFRs on SH-SY5Y human neuroblastoma cells. The results showed that of the BFRs tested, hexabromocyclododecane (HBCD, tetrabromobisphenol-A (TBBPA and decabromodiphenyl ether (DBPE, all are cytotoxic at low micromolar concentrations (LC(50 being 2.7 ± 0.7 µM, 15 ± 4 µM and 28 ± 7 µM, respectively. They induced cell death, at least in part, by apoptosis through activation of caspases. They also increased intracellular [Ca(2+] levels and reactive-oxygen-species within these neuronal cells. Furthermore, these BFRs also caused rapid depolarization of the mitochondria and cytochrome c release in these neuronal cells. Elevated intracellular [Ca(2+] levels appear to occur through a mechanism involving microsomal Ca(2+-ATPase inhibition and this maybe responsible for Ca(2+-induced mitochondrial dysfunction. In addition, µM levels of these BFRs caused β-amyloid peptide (Aβ-42 processing and release from these cells with a few hours of exposure. These results therefore shows that these pollutants are both neurotoxic and amyloidogenic in-vitro.

  7. 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


    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 microar......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......, 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...

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

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


    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.

  9. bcpmr1 encodes a P-type Ca(2+)/Mn(2+)-ATPase mediating cell-wall integrity and virulence in the phytopathogen Botrytis cinerea. (United States)

    Plaza, Verónica; Lagües, Yanssuy; Carvajal, Mauro; Pérez-García, Luis A; Mora-Montes, Hector M; Canessa, Paulo; Larrondo, Luis F; Castillo, Luis


    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Function and evolution of a MicroRNA that regulates a Ca2+-ATPase and triggers the formation of phased small interfering RNAs in tomato reproductive growth. (United States)

    Wang, Ying; Itaya, Asuka; Zhong, Xuehua; Wu, Yang; Zhang, Jianfeng; van der Knaap, Esther; Olmstead, Richard; Qi, Yijun; Ding, Biao


    MicroRNAs (miRNAs) regulate a wide variety of biological processes in most eukaryotes. We investigated the function and evolution of miR4376 in the family Solanaceae. We report that the 22-nucleotide miR4376 regulates the expression of an autoinhibited Ca(2+)-ATPase, tomato (Solanum lycopersicum) ACA10, which plays a critical role in tomato reproductive growth. Deep phylogenetic mapping suggested (1) an evolution course of MIR4376 loci and posttranscriptional processing of pre-miR4376 as a likely limiting step for the evolution of miR4376, (2) an independent phylogenetic origin of the miR4376 target site in ACA10 homologs, and (3) alternative splicing as a possible mechanism of eliminating such a target in some ACA10 homologs. Furthermore, miR4376 triggers the formation of phased small interfering RNAs (siRNAs) from Sl ACA10 and its Solanum tuberosum homolog. Together, our data provide experimental evidence of miRNA-regulated expression of universally important Ca(2+)-ATPases. The miR4376-regulated expression of ACA10 itself, and possibly also the associated formation of phased siRNAs, may function as a novel layer of molecular mechanisms underlying tomato reproductive growth. Finally, our data suggest that the stochastic emergence of a miRNA-target gene combination involves multiple molecular events at the genomic, transcriptional, and posttranscriptional levels that may vary drastically in even closely related species.

  11. The ataxia related G1107D mutation of the plasma membrane Ca2+ ATPase isoform 3 affects its interplay with calmodulin and the autoinhibition process. (United States)

    Calì, Tito; Frizzarin, Martina; Luoni, Laura; Zonta, Francesco; Pantano, Sergio; Cruz, Carlos; Bonza, Maria Cristina; Bertipaglia, Ilenia; Ruzzene, Maria; De Michelis, Maria Ida; Damiano, Nunzio; Marin, Oriano; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Lopreiato, Raffaele; Carafoli, Ernesto


    The plasma membrane Ca2+ ATPases (PMCA pumps) have a long, cytosolic C-terminal regulatory region where a calmodulin-binding domain (CaM-BD) is located. Under basal conditions (low Ca2+), the C-terminal tail of the pump interacts with autoinhibitory sites proximal to the active center of the enzyme. In activating conditions (i.e., high Ca2+), Ca2+-bound CaM displaces the C-terminal tail from the autoinhibitory sites, restoring activity. We have recently identified a G1107D replacement within the CaM-BD of isoform 3 of the PMCA pump in a family affected by X-linked congenital cerebellar ataxia. Here, we investigate the effects of the G1107D replacement on the interplay of the mutated CaM-BD with both CaM and the pump core, by combining computational, biochemical and functional approaches. We provide evidence that the affinity of the isolated mutated CaM-BD for CaM is significantly reduced with respect to the wild type (wt) counterpart, and that the ability of CaM to activate the pump in vitro is thus decreased. Multiscale simulations support the conclusions on the detrimental effect of the mutation, indicating reduced stability of the CaM binding. We further show that the G1107D replacement impairs the autoinhibition mechanism of the PMCA3 pump as well, as the introduction of a negative charge perturbs the contacts between the CaM-BD and the pump core. Thus, the mutation affects both the ability of the pump to optimally transport Ca2+ in the activated state, and the autoinhibition mechanism in its resting state. Copyright © 2016. Published by Elsevier B.V.

  12. Acid-gastric antisecretory effect of the ethanolic extract from Arctium lappa L. root: role of H+, K+-ATPase, Ca2+influx and the cholinergic pathway. (United States)

    da Silva, Luisa Mota; Burci, Ligia de Moura; Crestani, Sandra; de Souza, Priscila; da Silva, Rita de Cássia Melo Vilhena de Andrade Fonseca; Dartora, Nessana; de Souza, Lauro Mera; Cipriani, Thales Ricardo; da Silva-Santos, José Eduardo; André, Eunice; Werner, Maria Fernanda de Paula


    Arctium lappa L., popularly known as burdock, is a medicinal plant used worldwide. The antiulcer and gastric-acid antisecretory effects of ethanolic extract from roots of Arctium lappa (EET) were already demonstrated. However, the mechanism by which the extract reduces the gastric acid secretion remains unclear. Therefore, this study was designed to evaluate the antisecretory mode of action of EET. The effects of EET on H + , K + -ATPase activity were verified in vitro, whereas the effects of the extract on cholinergic-, histaminergic- or gastrinergic-acid gastric stimulation were assessed in vivo on stimulated pylorus ligated rats. Moreover, ex vivo contractility studies on gastric muscle strips from rats were also employed. The incubation with EET (1000 µg/ml) partially inhibited H + , K + -ATPase activity, and the intraduodenal administration of EET (10 mg/kg) decreased the volume and acidity of gastric secretion stimulated by bethanechol, histamine, and pentagastrin. EET (100-1000 µg/ml) did not alter the gastric relaxation induced by histamine but decreased acetylcholine-induced contraction in gastric fundus strips. Interestingly, EET also reduced the increase in the gastric muscle tone induced by 40 mM KCl depolarizing solution, as well as the maximum contractile responses evoked by CaCl 2 in Ca 2+ -free depolarizing solution, without impairing the effect of acetylcholine on fundus strips maintained in Ca 2+ -free nutritive solution. Our results reinforce the gastric antisecretory properties of preparations obtained from Arctium lappa, and indicate that the mechanisms involved in EET antisecretory effects include a moderate reduction of the H + , K + -ATPase activity associated with inhibitory effects on calcium influx and of cholinergic pathways in the stomach muscle.

  13. The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA Which Displays Light-Dependent Gene and Protein Expressions

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    Yuen K. Ip


    Full Text Available Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification in light. Light-enhanced calcification requires an increase in the supply of Ca2+ to, and simultaneously an augmented removal of H+ from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca2+-ATPase (PMCA from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa. The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca2+ in exchange for H+. More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA/PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam.

  14. An exposed carboxyl group on sialic acid is essential for gangliosides to inhibit calcium uptake via the sarco/endoplasmic reticulum Ca2+-ATPase: relevance to gangliosidoses. (United States)

    Ginzburg, Luba; Li, Su-Chen; Li, Yu-Teh; Futerman, Anthony H


    We previously observed that gangliosides GM2, GM1, and GM3 inhibit Ca2+-uptake via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in neurons and in brain microsomes. We now systematically examine the effect of various gangliosides and their analogs on Ca2+-uptake via SERCA and demonstrate that an exposed carboxyl group on the ganglioside sialic acid residue is required for inhibition. Thus, asialo-GM2 and asialo-GM1 have no inhibitory effect, and modifications of the carboxyl group of GM1 and GM2 into a hydroxymethyl residue (CH2OH), a methyl ester (COOCH3) or a taurine-conjugated amide (CONHCH2CH2SO3H) drastically diminish their inhibitory activities. We also demonstrate that the saccharides must be attached to a ceramide backbone in order to inhibit SERCA as the ceramide-free ganglioside saccharides only inhibit SERCA to a minimal extent. Finally, we attempted to use the ceramide-free ganglioside saccharides to antagonize the effects of the gangliosides on SERCA; although some reversal was observed, the inhibitory effects of the gangliosides were not completely abolished.

  15. The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA) Which Displays Light-Dependent Gene and Protein Expressions. (United States)

    Ip, Yuen K; Hiong, Kum C; Goh, Enan J K; Boo, Mel V; Choo, Celine Y L; Ching, Biyun; Wong, Wai P; Chew, Shit F


    Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification) in light. Light-enhanced calcification requires an increase in the supply of Ca2+ to, and simultaneously an augmented removal of H+ from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca2+-ATPase (PMCA) from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa. The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca2+ in exchange for H+. More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA/PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam.

  16. Tuning the structural coupling between the transmembrane and cytoplasmic domains of phospholamban to control sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) function. (United States)

    Ha, Kim N; Gustavsson, Martin; Veglia, Gianluigi


    Phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), the integral membrane enzyme responsible for 70 % of the removal of Ca(2+) from the cytosol, inducing cardiac muscle relaxation in humans. Dysfunctions in SERCA:PLN interactions have been implicated as having a critical role in cardiac disease, and targeting Ca(2+) transport has been demonstrated to be a promising avenue in treating conditions of heart failure. Here, we designed a series of new mutants able to tune SERCA function, targeting the loop sequence that connects the transmembrane and cytoplasmic helices of PLN. We found that a variable degree of loss of inhibition mutants is attainable by engineering glycine mutations along PLN's loop domain. Remarkably, a double glycine mutation results in a complete loss-of-function mutant, fully mimicking the phosphorylated state of PLN. Using nuclear magnetic resonance spectroscopy, we rationalized the effects of these mutations in terms of entropic control on PLN function, whose inhibitory function can be modulated by increasing its conformational dynamics. However, if PLN mutations go past a threshold set by the phosphorylated state, they break the structural coupling between the transmembrane and cytoplasmic domains, resulting in a species that behaves as the inhibitory transmembrane domain alone. These studies provide new potential candidates for gene therapy to reverse the effects of heart failure.

  17. Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca(2+)-ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation. (United States)

    Adebayo, Olusegun L; Khera, Alka; Sandhir, Rajat; Adenuga, Gbenga A


    The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca(2+)-ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein-undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l(-1), respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) activity, Ca(2+)/CaM activated EGM Ca(2+) ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca(2+)/CaM to activate EGM Ca(2+)-ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Slowed relaxation of diaphragm in septic rats is associated with reduced expression of sarco-endoplasmic reticulum CA2+ -ATPase genes SERCA1 and SERCA2. (United States)

    Wu, Jin; Zhang, Jian You; Gong, Yuan; Li, Shi Tong


    The aim of this study was to study the effects of sepsis on diaphragm relaxation properties and the associated expression of sarco-endoplasmic reticulum Ca2+ -ATPase genes SERCA1 and SERCA2. Rats were randomized to undergo either sham surgery or cecal ligation and puncture (CLP). Diaphragm isometric relaxation parameters were measured after 24 h. The mRNA expression and protein content of SERCA1 and SERCA2 in diaphragm muscles were determined. Both diaphragm maximal twitch and tetanus relaxation rates were reduced. Twitch half-relaxation time was prolonged after normalization to half of peak twitch tension. The mRNA expression and protein content of SERCA1 and SERCA2 were decreased. Slowed relaxation of the diaphragm in septic rats was associated with reduced expression of SERCA1 and SERCA2. Muscle Nerve 54: 1108-1113, 2016. © 2016 Wiley Periodicals, Inc.

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

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    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.

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

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    Wei XIN


    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.

  1. Analysis of Functional Motions in Brownian Molecular Machines with an Efficient Block Normal Mode Approach: Myosin-II and Ca2+-ATPase (United States)

    Li, Guohui; Cui, Qiang


    The structural flexibilities of two molecular machines, myosin and Ca2+-ATPase, have been analyzed with normal mode analysis and discussed in the context of their energy conversion functions. The normal mode analysis with physical intermolecular interactions was made possible by an improved implementation of the block normal mode (BNM) approach. The BNM results clearly illustrated that the large-scale conformational transitions implicated in the functional cycles of the two motor systems can be largely captured with a small number of low-frequency normal modes. Therefore, the results support the idea that structural flexibility is an essential part of the construction principle of molecular motors through evolution. Such a feature is expected to be more prevalent in motor proteins than in simpler systems (e.g., signal transduction proteins) because in the former, large-scale conformational transitions often have to occur before the chemical events (e.g., ATP hydrolysis in myosin and ATP binding/phosphorylation in Ca2+-ATPase). This highlights the importance of Brownian motions associated with the protein domains that are involved in the functional transitions; in this sense, Brownian molecular machines is an appropriate description of molecular motors, although the normal mode results do not address the origin of the ratchet effect. The results also suggest that it might be more appropriate to describe functional transitions in some molecular motors as intrinsic elastic motions modulating local structural changes in the active site, which in turn gets stabilized by the subsequent chemical events, in contrast with the conventional idea of local changes somehow getting amplified into larger-scale motions. In the case of myosin, for example, we favor the idea that Brownian motions associated with the flexible converter propagates to the Switch I/II region, where the salt-bridge formation gets stabilized by ATP hydrolysis, in contrast with the textbook notion that ATP

  2. OsACA6, a P-type 2B Ca(2+) ATPase functions in cadmium stress tolerance in tobacco by reducing the oxidative stress load. (United States)

    Shukla, Devesh; Huda, Kazi Md Kamrul; Banu, Mst Sufara Akhter; Gill, Sarvajeet Singh; Gill, Sarvjeet Singh; Tuteja, Renu; Tuteja, Narendra


    The present study demonstrates the first direct evidence of the novel role of OsACA6 in providing Cd (2+) stress tolerance in transgenic tobacco by maintaining cellular ion homeostasis and modulating ROS-scavenging pathway. Cadmium, a non-essential toxic heavy metal, interferes with the plant growth and development. It reaches the leaves through xylem and may become part of the food chain, thus causing detrimental effects to human health. Therefore, there is an urgent need to develop strategies for engineering plants for Cd(2+) tolerance and less accumulation. The members of P-type ATPases family transport metal ions including Cd(2+), and thus play important role an ion homeostasis. The present study elucidates the role of P-type 2B Ca(2+) ATPase (OsACA6) in Cd(2+) stress tolerance. The transcript levels of OsACA6 were up-regulated upon Cd(2+), Zn(2+) and Mn(2+) exposure. Transgenic tobacco expressing OsACA6 showed tolerance towards Cd(2+) stress as demonstrated by several physiological indices including root length, biomass, chlorophyll, malondialdehyde and hydrogen peroxide content. The roots of the transgenic lines accumulated more Cd(2+) as compared to shoot. Further, confocal laser scanning microscopy showed that Cd(2+) exposure altered Ca(2+) uptake in OsACA6 transgenic plants. OsACA6 expression in tobacco also protected the transgenic plants from oxidative stress by enhancing the activity of enzymatic (SOD, CAT, APX, GR) and non-enzymatic (GSH and AsA) antioxidant machinery. Transgenic lines also tolerated Zn(2+) and Mn(2+) stress; however, tolerance for these ions was not as significant as observed for Cd(2+) exposure. Thus, overexpression of OsACA6 confers Cd(2+) stress tolerance in transgenic lines by maintaining cellular ion homeostasis and modulating reactive oxygen species (ROS)-scavenging pathway. The results of the present study will help to develop strategies for engineering Cd(2+) stress tolerance in economically important crop plants.

  3. Nifetepimine, a dihydropyrimidone, ensures CD4+ T cell survival in a tumor microenvironment by maneuvering sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). (United States)

    Ghosh, Swatilekha; Adhikary, Arghya; Chakraborty, Samik; Nandi, Pinki; Mohanty, Suchismita; Chakraborty, Supriya; Bhattacharjee, Pushpak; Mukherjee, Sanhita; Putatunda, Salil; Chakraborty, Srabasti; Chakraborty, Arijit; Sa, Gaurisankar; Das, Tanya; Sen, Parimal C


    Multiple mechanisms have been proposed by which tumors induce T cell apoptosis to circumvent tumor immune-surveillance. Although sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) have long been known to regulate intracellular Ca(2+) homeostasis, few studies have examined the role of SERCA in processes of T lymphocyte survival and activation. In this context it remains largely unexplored as to how tumors jeopardize SERCA function to disable T cell-mediated anti-tumor immunity. Here, we show that human CD4(+) T cells in the presence of tumor conditions manifested an up-regulation of SERCA3 expression that resulted in development of endoplasmic reticulum stress leading to CD4(+) T cell apoptosis. Prostaglandin E(2) produced by the tumor cell plays a critical role in up-regulating SERCA3 by enhancing the binding of its transcription factor Sp1. Gene manipulation and pharmacological approaches further established that an increase in SERCA expression also resulted in subsequent inhibition of PKCα and -θ and retention of NFκB in the cytosol; however, down-modulation of SERCA3 expression by a dihydropyrimidone derivative, ethyl-4-(3-nitro)-phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate (nifetepimine), protected the CD4(+) T cells from tumor-induced apoptosis. In fact, nifetepimine-mediated restoration of PKC activity resulted in nuclear translocation of p65NFκB, thereby ensuring its survival. Studies further undertaken in a tumor-bearing mice model revalidated the immunoprotective role of nifetepimine. Our present study thus strongly suggests that imbalance in cellular calcium homeostasis is an important factor leading to CD4(+) T cell death during cancer and holds promise that nifetepimine may have the potential to be used as an immunorestoring agent in cancer bearers.

  4. Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC. (United States)

    Mandal, Amritlal; Das, Sudip; Chakraborti, Tapati; Kar, Pulak; Ghosh, Biswarup; Chakraborti, Sajal


    The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured

  5. Raman spectroscopy of DNA-metal complexes. II. The thermal denaturation of DNA in the presence of Sr2+, Ba2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+. (United States)

    Duguid, J G; Bloomfield, V A; Benevides, J M; Thomas, G J


    Differential scanning calorimetry, laser Raman spectroscopy, optical densitometry, and pH potentiometry have been used to investigate DNA melting profiles in the presence of the chloride salts of Ba2+, Sr2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+. Metal-DNA interactions have been observed for the molar ratio [M2+]/[PO2-] = 0.6 in aqueous solutions containing 5% by weight of 160 bp mononucleosomal calf thymus DNA. All of the alkaline earth metals, plus Mn2+, elevate the melting temperature of DNA (Tm > 75.5 degrees C), whereas the transition metals Co2+, Ni2+, and Cd2+ lower Tm. Calorimetric (delta Hcal) and van't Hoff (delta HVH) enthalpies of melting range from 6.2-8.7 kcal/mol bp and 75.6-188.6 kcal/mol cooperative unit, respectively, and entropies from 17.5 to 24.7 cal/K mol bp. The average number of base pairs in a cooperative melting unit () varied from 11.3 to 28.1. No dichotomy was observed between alkaline earth and transition DNA-metal complexes for any of the thermodynamic parameters other than their effects on Tm. These results complement Raman difference spectra, which reveal decreases in backbone order, base unstacking, distortion of glycosyl torsion angles, and rupture of hydrogen bonds, which occur after thermal denaturation. Raman difference spectroscopy shows that transition metals interact with the N7 atom of guanine in duplex DNA. A broader range of interaction sites with single-stranded DNA includes ionic phosphates, the N1 and N7 atoms of purines, and the N3 atom of pyrimidines. For alkaline earth metals, very little interaction was observed with duplex DNA, whereas spectra of single-stranded complexes are very similar to those of melted DNA without metal. However, difference spectra reveal some metal-specific perturbations at 1092 cm-1 (nPO2-), 1258 cm-1 (dC, dA), and 1668 cm-1 (nC==O, dNH2 dT, dG, dC). Increased spectral intensity could also be observed near 1335 cm-1 (dA, dG) for CaDNA. Optical densitometry, employed to detect DNA

  6. Mercuric chloride-induced inhibition of different ATPases in the intestine of mudskipper, Boleophthalmus dentatus. (United States)

    Lakshmi, R; Kundu, R; Thomas, E; Mansuri, A P


    This paper deals with the toxicity of mercuric chloride to different ATPases in the intestine of mudskipper (Boleophthalmus dentatus). Mudskippers were exposed to four sublethal concentrations of mercuric chloride for three durations. The specific activities of Na+, K(+)-ATPase, Ca(2+)-ATPase, Mg(2+)-ATPase, Ca2+, HCO3(-)-ATPase, and Mg2+, HCO3(-)-ATPase were estimated. There was linear inhibition of all the enzymes with increasing mercuric chloride concentration as well as exposure duration. The Na+,K(+)-ATPase was found to be the enzyme most affected, followed by other ion-dependent ATPases. Inhibition of all the enzymes indicates severe damage to the intestinal cells, resulting in a blockage of the transport of substances across the membrane.

  7. Constitutive cardiac overexpression of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase delays myocardial failure after myocardial infarction in rats at a cost of increased acute arrhythmias. (United States)

    Chen, Ying; Escoubet, Brigitte; Prunier, Fabrice; Amour, Julien; Simonides, Warner S; Vivien, Benoît; Lenoir, Christophe; Heimburger, Michèle; Choqueux, Christine; Gellen, Barnabas; Riou, Bruno; Michel, Jean-Baptiste; Franz, Wolfgang M; Mercadier, Jean-Jacques


    Heart failure often complicates myocardial infarction (MI), and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is underexpressed in the failing myocardium. We examined the effect of preexisting cardiac SERCA2a protein overexpression on rat survival and left ventricular (LV) remodeling after MI. Baseline myocardial SERCA2a expression was 37% higher in transgenic (TG) rats than in their wild-type (WT) controls, consistent with enhanced myocardial function. The mortality rate of TG rats during the 24 hours after surgical MI was higher than that of WT rats (71% versus 35%, P<0.001), associated with a higher frequency of ventricular arrhythmias, and was normalized by lidocaine treatment. The increased acute-phase mortality in TG rats was not accompanied by increased 6-month mortality. Function of the noninfarcted myocardium, as assessed by tissue Doppler imaging, was higher in TG rats than in WT rats for up to 1 month after MI, a beneficial effect no longer observed at 3 months. LV remodeling and global function were similar in TG and WT rats. No difference in papillary muscle function was found at 6 months. Constitutive cardiac SERCA2a overexpression has a transient beneficial effect on remote myocardium function in rat MI, with no improvement in LV global function or prevention of LV remodeling and failure. This benefit is associated with a higher risk of acute mortality, which is prevented by lidocaine treatment.

  8. Different sensitivity of Na(+,K(+-ATPase and Mg(2+-АТРase to ethanol and arachidonic acid in rat colon smooth muscle under pretreatment of cellular membranes with Ds-Na

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    A. A. Kaplia


    Full Text Available The methodological procedure provides the detection of the relatively high Na+,K+-ATPase functional activity in the crude cellular membranes of rat colon smooth muscle (CSM following standard detergent pretreatment (with Ds-Na vs digitonin. It includes the essential discrete steps: detergent membrane permeabilization under optimal detergent/protein ratio and active site protection by ATP (for Ds-Na prior enzymatic reaction with substantial detergent dilution far below critical micelle concentration in the ATPase medium. The high level of the Na+,K+-ATPase activity, originally detected in CSM, did not differ for two detergents and was comparable with ouabain-resistant Mg2+,АТР-hydrolase activity, The features of ATPase protein-lipid complexes were evaluated by the enzyme sensitivity to the effect of ethanol and arachidonic acid with different membrane disordering effectiveness. The long-chain fatty acid is a more effective inhibitor as compared with aliphatic alcohol for both ATPases. Mg2+,АТР-hydrolase appeared to be much more resistant to inactivation than Na+,K+-ATPase. The data reflect the possible differences in lipid dependence of two enzymatic systems due to the peculiarities of the structural arrangement in membrane and importance of the hydrophobic microenvironment for mechanism of catalysis. Thus, the data represent the approach to the simple and reliable Na+,K+-АТРase activity determination in nonpurified CSM membranes, acceptable for different tissues and appropriate for quantitative comparison in pathophysiological studies and for testing the impact of diverse effectors on Na+,K+-АТРase.

  9. Are there different water requirements in different steps of a catalytic cycle? Hydration effects at the E1 and E2 conformers of sarcoplasmic reticulum Ca(2+)-ATPase studied in organic solvents with low amounts of water. (United States)

    Barrabin, H; Scofano, H M; de Gómez-Puyou, M T; Gómez-Puyou, A


    The Ca(2+)-ATPase from sarcoplasmic reticulum was transferred in an active form to a low-water system composed of toluene, phospholipids, and Triton X-100 (TPT). The Ca(2+)-ATPase activity in the TPT system with 4.0% water (by vol. was about 50% of the activity observed in all-aqueous mixtures. Phosphate formation was linear with time up to 20% of ATP hydrolysis and, as expected from an enzyme-catalysed reaction, activity was linear with protein concentration. No ATPase activity was detected in the presence of 3 mM EGTA, indicating that the enzyme retained its Ca2+ dependence in the TPT system. A hyperbolic response to ATP concentration was observed with a Km of 0.15 mM. There was no detectable ATPase activity at water concentrations below 1.5% (by vol.). With 2.0% water, activity became detectable and increased as the water content was progressively raised to 7.0% (by vol.). Higher amounts of water produced unstable emulsions. Enzyme phosphorylation by ATP and dephosphorylation took place in the TPT system. The velocities of both enzyme phosphorylation and dephosphorylation increased with increments in the water content. The enzyme could also be phosphorylated in the TPT system by inorganic phosphate. However, in comparison to ATP, phosphorylation by phosphate took place with significantly lower amounts of water. It is suggested that at low amounts of water, the enzyme is in a relatively rigid conformation and, as the water content is increased, the ATPase acquires more flexibility and, hence, the capacity to carry out catalysis at higher rates. Nevertheless, the release of conformational constraints of the catalytic site of the E2 conformer takes place at water concentrations much lower than those needed for the expression of catalytic activity by the E1 conformer.

  10. β-Arrestin2 Improves Post-Myocardial Infarction Heart Failure via Sarco(endo)plasmic Reticulum Ca2+-ATPase-Dependent Positive Inotropy in Cardiomyocytes. (United States)

    McCrink, Katie A; Maning, Jennifer; Vu, Angela; Jafferjee, Malika; Marrero, Christine; Brill, Ava; Bathgate-Siryk, Ashley; Dabul, Samalia; Koch, Walter J; Lymperopoulos, Anastasios


    Heart failure is the leading cause of death in the Western world, and new and innovative treatments are needed. The GPCR (G protein-coupled receptor) adapter proteins βarr (β-arrestin)-1 and βarr-2 are functionally distinct in the heart. βarr1 is cardiotoxic, decreasing contractility by opposing β 1 AR (adrenergic receptor) signaling and promoting apoptosis/inflammation post-myocardial infarction (MI). Conversely, βarr2 inhibits apoptosis/inflammation post-MI but its effects on cardiac function are not well understood. Herein, we sought to investigate whether βarr2 actually increases cardiac contractility. Via proteomic investigations in transgenic mouse hearts and in H9c2 rat cardiomyocytes, we have uncovered that βarr2 directly interacts with SERCA2a (sarco[endo]plasmic reticulum Ca 2+ -ATPase) in vivo and in vitro in a β 1 AR-dependent manner. This interaction causes acute SERCA2a SUMO (small ubiquitin-like modifier)-ylation, increasing SERCA2a activity and thus, cardiac contractility. βarr1 lacks this effect. Moreover, βarr2 does not desensitize β 1 AR cAMP-dependent procontractile signaling in cardiomyocytes, again contrary to βarr1. In vivo, post-MI heart failure mice overexpressing cardiac βarr2 have markedly improved cardiac function, apoptosis, inflammation, and adverse remodeling markers, as well as increased SERCA2a SUMOylation, levels, and activity, compared with control animals. Notably, βarr2 is capable of ameliorating cardiac function and remodeling post-MI despite not increasing cardiac βAR number or cAMP levels in vivo. In conclusion, enhancement of cardiac βarr2 levels/signaling via cardiac-specific gene transfer augments cardiac function safely, that is, while attenuating post-MI remodeling. Thus, cardiac βarr2 gene transfer might be a novel, safe positive inotropic therapy for both acute and chronic post-MI heart failure. © 2017 American Heart Association, Inc.

  11. Islet secretory defect in insulin receptor substrate 1 null mice is linked with reduced calcium signaling and expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2b and -3. (United States)

    Kulkarni, Rohit N; Roper, Michael G; Dahlgren, Gabriella; Shih, David Q; Kauri, Lisa M; Peters, Jennifer L; Stoffel, Markus; Kennedy, Robert T


    Mice with deletion of insulin receptor substrate (IRS)-1 (IRS-1 knockout [KO] mice) show mild insulin resistance and defective glucose-stimulated insulin secretion and reduced insulin synthesis. To further define the role of IRS-1 in islet function, we examined the insulin secretory defect in the knockouts using freshly isolated islets and primary beta-cells. IRS-1 KO beta-cells exhibited a significantly shorter increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) than controls when briefly stimulated with glucose or glyceraldehyde and when l-arginine was used to potentiate the stimulatory effect of glucose. These changes were paralleled by a lower number of exocytotic events in the KO beta-cells in response to the same secretagogues, indicating reduced insulin secretion. Furthermore, the normal oscillations in intracellular Ca(2+) and O(2) consumption after glucose stimulation were dampened in freshly isolated KO islets. Semiquantitative RT-PCR showed a dramatically reduced islet expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b and -3 in the mutants. These data provide evidence that IRS-1 modulation of insulin secretion is associated with Ca(2+) signaling and expression of SERCA-2b and -3 genes in pancreatic islets and provides a direct link between insulin resistance and defective insulin secretion.

  12. [ATPase and phosphatase activity of drone brood]. (United States)

    Bodnarchuk, L I; Stakhman, O S


    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.

  13. The functional importance of the extreme C-terminal tail in the gene 2 organellar Ca(2+)-transport ATPase (SERCA2a/b).


    Verboomen, H; Wuytack, F; van den Bosch, L; Mertens, L.; Casteels, R


    Ca(2+)-uptake experiments in microsomal fractions from transfected COS-1 cells have revealed a functional difference between the non-muscle SERCA2b Ca2+ pump and its muscle-specific SERCA2a splice variant. Structurally, the two pumps differ only in their C-terminal tail. The last four amino acids of SERCA2a are replaced in SERCA2b by a 49-residue-long peptide chain containing a very hydrophobic stretch which could be an additional transmembrane segment. The functionally important subdomains i...

  14. β2-Adrenergic stimulation enhances Ca2+ release and contractile properties of skeletal muscles, and counteracts exercise-induced reductions in Na+–K+-ATPase Vmax in trained men (United States)

    Hostrup, M; Kalsen, A; Ørtenblad, N; Juel, C; Mørch, K; Rzeppa, S; Karlsson, S; Backer, V; Bangsbo, J


    The aim of the present study was to examine the effect of β2-adrenergic stimulation on skeletal muscle contractile properties, sarcoplasmic reticulum (SR) rates of Ca2+ release and uptake, and Na+–K+-ATPase activity before and after fatiguing exercise in trained men. The study consisted of two experiments (EXP1, n = 10 males, EXP2, n = 20 males), where β2-adrenoceptor agonist (terbutaline) or placebo was randomly administered in double-blinded crossover designs. In EXP1, maximal voluntary isometric contraction (MVC) of m. quadriceps was measured, followed by exercise to fatigue at 120% of maximal oxygen uptake (). A muscle biopsy was taken after MVC (non-fatigue) and at time of fatigue. In EXP2, contractile properties of m. quadriceps were measured with electrical stimulations before (non-fatigue) and after two fatiguing 45 s sprints. Non-fatigued MVCs were 6 ± 3 and 6 ± 2% higher (P fatigue. After sprints, MVC declined (P fatigue, but declined (P fatigue. Na+–K+-ATPase activity was unaffected by terbutaline compared with placebo at non-fatigue, but terbutaline counteracted exercise-induced reductions in maximum rate of activity (Vmax) at time of fatigue. In conclusion, increased contractile force induced by β2-adrenergic stimulation is associated with enhanced rate of Ca2+ release in humans. While β2-adrenergic stimulation elicits positive inotropic and lusitropic effects on non-fatigued m. quadriceps, these effects are blunted when muscles fatigue. PMID:25344552

  15. 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


    , for presynaptic and postsynaptic Ca2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium...

  16. 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


    , for presynaptic and postsynaptic Ca(2+) regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium...

  17. Estudio teorico de las interacciones de dos modelos de ácidos húmicos con los cationes Al3+, Ca2+, Mg2+, Zn2+, K+ y NH4+ a un nivel de calculo DFT y un modelo de solvatación PCM

    Directory of Open Access Journals (Sweden)

    Eduardo Espinosa-Fuentes

    Full Text Available In this paper, two models of humic acids, the Temple-Northeastern-Birmingham (TNB and Kolla models, were studied. Also, the complexation reaction of the structures formed by the interaction of the TNB and Kolla models with Al3+, Ca2+, Mg2+, Zn2+, K+ and NH4+ cations, common in agricultural soils, was studied. These calculations were made for the complexes, at PM6 and DFT / LANL2DZ level of theory, both in vacuum and in aqueous medium. We found a strong affinity between Kolla and TNB models, and Al3+, Ca2+, Mg2+, Zn2+, K+ and NH4+ cations, influenced by the solvent that affected the interaction sites; the solvent increased the rate of reactivity and affinity for the cations in nucleophilic regions and decreased it in electrophilic regions of the structures. Calculations of molecular electronic potential, MEP and atomic charges, the local smoothness, Fukui functions and the HSAB principle adequately described the HA/cations interactions which were affected by the number of hydrogen bonds. The most reactive sites were the hydroxyl, phenolic, carbonyls oxygens and nitrogens at both vacuum and aqueous medium, especially carbonyl oxygens. These results are consistent with the properties of HA that make them attractive as components of agricultural soils.

  18. Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a during murine estrus: association with oviductal exosomes and uptake in sperm.

    Directory of Open Access Journals (Sweden)

    Amal A Al-Dossary

    Full Text Available PMCA4, a membrane protein, is the major Ca(2+ efflux pump in murine sperm where its deletion leads to a severe loss of hyperactivated motility and to male infertility. We have previously shown that the PMCA4b splice variant interacts with CASK (Ca(2+/CaM-dependent serine kinase in regulating sperm Ca(2+. More recently we detected that PMCA4a isoform, in addition to its presence in testis, is secreted in the epididymal luminal fluid and transferred to sperm. Here we show that Pmca4 mRNA is expressed in both the 4a and 4b variants in the vagina, uterus, and oviduct. Immunofluorescence reveals that PMCA4a is similarly expressed and is elevated during estrus, appearing in the glandular and luminal epithelia. Western analysis detected PMCA4a in all tissues and in the luminal fluids (LF of the vagina (VLF, uterus (ULF, and the oviduct (OLF collected during estrus. It was ~9- and 4-fold higher in OLF than in VLF and ULF, and only marginally present in LF collected at metestrus/diestrus. Fractionation of the LF collected at estrus, via ultracentrifugation, revealed that 100% of the PMCA4a resides in the vesicular fraction of the ULF and OLF. Transmission electron microscopy (TEM revealed that OLF vesicles have an exosomal orientation (with the cytoplasmic-side inward, a size range of 25-100 nm, with the characteristic CD9 biomarker. Thus, we dubbed these vesicles "oviductosomes", to which PMCA4a was immunolocalized. Incubation of caudal sperm in the combined LF or exosomes resulted in up to a ~3-fold increase of sperm PMCA4a, as detected by flow cytometry, indicating in vitro uptake. Our results are consistent with the increased requirement of Ca(2+ efflux in the oviduct. They show for the first time the presence of oviductal exosomes and highlight their role, along with uterosomes and vaginal exosomes, in post-testicular sperm acquisition of PMCA4a which is essential for hyperactivated motility and fertility.

  19. Materiales de Al2O3 - MgAl2O4 - CaAl12O19 - Ca2Mg2Al28O46 obtenidos mediante un proceso de sinterización reactiva entre Al2O3 y CaMg(CO32

    Directory of Open Access Journals (Sweden)

    Pena, P.


    Full Text Available Taking into account the system Al2O3-MgO-CaO, refractory materials containing Al2O3-MgAl2O4-CaAl12O19-Ca2Mg2Al28O46 has been obtained by reaction-sintering of appropriate mixtures of Al2O3 and CaMg(CO32. The study of the mechanism of reaction was carried out by differential thermal analysis (DTA and thermogravimetry (TG, followed by dilatometric and xray diffraction (XRD studies. A static study of the reaction was performed at different temperatures. After each thermal treatment a XRD analysis of the phases present was made, as well as a microestructural study by scanning electron microscopy (SEM coupled with energy-dispersive spectroscopy (EDS. From the results obtained, and taking into account the free energy of formation of the different compounds, the mechanism of reaction was established.Utilizado la información suministrada por el diagrama de equilibrio de fases Al2O3-MgO-CaO se ha diseñado y obtenido un material de Al2O3-MgAl2O4-CaAl12O19-Ca2Mg2Al28O46 mediante sinterización reactiva de una mezcla de Al2O3 y CaMg(CO32. Las reacciones que tienen lugar en la mezcla durante el proceso se han estudiado usando técnicas de análisis térmico diferencial, termogravimetrico y dilatometría. Muestras reaccionadas a temperaturas seleccionadas se han estudiado por difracción de rayos X y microscopía electrónica de barrido con microanálisis mediante dispersión de energías. Los resultados obtenidos revelan que la reacción entre las materias primas tiene lugar en varias etapas con formación de fases transitorias, dando lugar a una microestructura final con granos aciculares de CaAl12O19 y partículas de la fase ternaria Ca2Mg2Al28O46 formadas en aquellos puntos en los que entran en contacto las fases de MgAl2O4 y CaAl12O19.

  20. 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


    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.

  1. A New Approach of Short Wave Protection against Middle Cerebral Artery Occlusion/Reperfusion Injury via Attenuation of Golgi Apparatus Stress by Inhibition of Downregulation of Secretory Pathway Ca(2+)-ATPase Isoform 1 in Rats. (United States)

    Fan, Yongmei; Zhang, Changjie; Li, Ting; Peng, Wenna; Yin, Jing; Li, Xiaofao; Kong, Ying; Lan, Chunna; Wang, Rumi; Hu, Zhiping


    Short wave (SW), a pattern of electromagnetic therapy, achieves an oscillating electromagnetic field. It has been reported that it may have a potential effect on cerebral injury. The present study was designed to investigate the potential role and possible mechanism of SW in focal cerebral ischemia/reperfusion (I/R) injury in rats. Secretory pathway Ca(2+)/Mn(2+) ATPase isoform 1 is a major component of Golgi apparatus stress. It has been reported as representative of Golgi apparatus stress. Up to 120 minutes of middle cerebral artery occlusion (MCAO) and reperfusion injury was induced in male Sprague-Dawley rats. Different sessions of SW daily were administered over head after reperfusion from day 1 to day 7. Functional recovery scores, survival rates, infarct volume analysis, electron microscope test, and western blotting studies were used to analyze the therapy. SW protected against neuronal death and apoptosis in cornu ammon 1 region of hippocampus by reducing neuronal deficit, infarct volume, and ultrastructure. SW partly inhibited upregulation of caspase3. In addition, the expression of secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) was upregulated by SW. Our data indicate that SW can be protected against focal cerebral I/R injury, and the influence on Golgi apparatus stress might provide us a new perspective in further study. To the authors' knowledge, this is the first report using SW to increase expression of SPCA1 indicating modulate Golgi apparatus stress in MCAO and reperfusion model. Copyright © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  2. Targeted ablation of plasma membrane Ca2+-ATPase (PMCA) 1 and 4 indicates a major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4. (United States)

    Okunade, Gbolahan W; Miller, Marian L; Pyne, Gail J; Sutliff, Roy L; O'Connor, Kyle T; Neumann, Jonathan C; Andringa, Anastasia; Miller, Daniel A; Prasad, Vikram; Doetschman, Thomas; Paul, Richard J; Shull, Gary E


    The relative importance of plasma membrane Ca2+-ATPase (PMCA) 1 and PMCA4 was assessed in mice carrying null mutations in their genes (Atp2b1 and Atp2b4). Loss of both copies of the gene encoding PMCA1 caused embryolethality, whereas heterozygous mutants had no overt disease phenotype. Despite widespread and abundant expression of PMCA4, PMCA4 null (Pmca4-/-) mutants exhibited no embryolethality and appeared outwardly normal. Loss of PMCA4 impaired phasic contractions and caused apoptosis in portal vein smooth muscle in vitro; however, this phenotype was dependent on the mouse strain being employed. Pmca4-/- mice on a Black Swiss background did not exhibit the phenotype unless they also carried a null mutation in one copy of the Pmca1 gene. Pmca4-/- male mice were infertile but had normal spermatogenesis and mating behavior. Pmca4-/- sperm that had not undergone capacitation exhibited normal motility but could not achieve hyperactivated motility needed to traverse the female genital tract. Ultrastructure of the motility apparatus in Pmca4-/- sperm tails was normal, but an increased incidence of mitochondrial condensation indicated Ca2+ overload. Immunoblotting and immunohistochemistry showed that PMCA4 is the most abundant isoform in testis and sperm and that it is localized to the principle piece of the sperm tail, which is also the location of the major Ca2+ channel (CatSper) required for sperm motility. These results are consistent with an essential housekeeping or developmental function for PMCA1, but not PMCA4, and show that PMCA4 expression in the principle piece of the sperm tail is essential for hyperactivated motility and male fertility.

  3. Loss of the Atp2c1 secretory pathway Ca(2+)-ATPase (SPCA1) in mice causes Golgi stress, apoptosis, and midgestational death in homozygous embryos and squamous cell tumors in adult heterozygotes. (United States)

    Okunade, Gbolahan W; Miller, Marian L; Azhar, Mohamad; Andringa, Anastasia; Sanford, L Philip; Doetschman, Thomas; Prasad, Vikram; Shull, Gary E


    Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.

  4. Sarco/endoplasmic reticulum Ca2+ (SERCA)-pumps: link to heart beats and calcium waves. (United States)

    Misquitta, C M; Mack, D P; Grover, A K


    Mobilization of endoplasmic reticulum Ca2+ is pivotal to the ability of a cell to send or respond to stimuli. Ca(2+)-Mg(2+)-ATPases, termed SERCA pumps, sequester Ca2+ into the sarco/endoplasmic reticulum. There are several SERCA protein isoforms encoded by three genes. This paper summarizes the structure, function, tissue and subcellular distribution, and regulation of various SERCA isoforms. Then it attempts to link divergence in the signal transduction processes of cells to the types and levels of SERCA proteins they express and to how the cells regulate their SERCA pump activity. The paper examines possible linkages between SERCA pumps and receptor-activated Ca2+ entry, SERCA isoform localization and Ca(2+)-waves, and the role of SERCA pumps in nuclear Ca2+ in cell proliferation and apoptosis. Then it uses available information on cardiac function and chronic stimulation of the fast-twitch muscle to answer a series of basic questions on the regulation of SERCA activity and expression and their linkage to signal transduction. Finally, it discusses the possibility that neurons exhibit complex Ca(2+)-waves whose interactions have the potential to explain the operational basis of neural networks. A series of unanswered questions emerge based on this synthesis, including the unsettling issue of whether all the isoforms are needed to achieve the divergence in signal transduction or if there is a degree of redundancy in the system.

  5. Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Lunde, Per; Levin, Kasper


    -977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P RyR. No significant differences in Ca(2+) uptake rate and Ca(2+)-ATPase capacity were...

  6. Identification of the Mg2+-binding site in the P-type ATPase and phosphatase members of the HAD (haloacid dehalogenase) superfamily by structural similarity to the response regulator protein CheY

    NARCIS (Netherlands)

    Ridder, Ivo S.; Dijkstra, Bauke W.


    The large HAD (haloacid dehalogenase) superfamily of hydrolases comprises P-type ATPases, phosphatases, epoxide hydrolases and L-2-haloacid dehalogenases. A comparison of the three-dimensional structure of L-2-haloacid dehalogenase with that of the response regulator protein CheY allowed the

  7. An Inhibitory Effect of Extracellular Ca2+ on Ca2+-Dependent Exocytosis (United States)

    Wang, Yeshi; Chen, Xiaowei; Sun, Lei; Guo, Ning; Zheng, Hui; Zheng, Lianghong; Ruat, Martial; Han, Weiping; Zhang, Claire Xi; Zhou, Zhuan


    Aim Neurotransmitter release is elicited by an elevation of intracellular Ca2+ concentration ([Ca2+]i). The action potential triggers Ca2+ influx through Ca2+ channels which causes local changes of [Ca2+]i for vesicle release. However, any direct role of extracellular Ca2+ (besides Ca2+ influx) on Ca2+-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis. Results Using photolysis of caged Ca2+ and caffeine-induced release of stored Ca2+, we found that extracellular Ca2+ inhibited exocytosis following moderate [Ca2+]i rises (2–3 µM). The IC50 for extracellular Ca2+ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca2+ concentration ([Ca2+]o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca2+]o. The calcimimetics Mg2+, Cd2+, G418, and neomycin all inhibited exocytosis. The extracellular Ca2+-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE. Conclusion/Significance As an extension of the classic Ca2+ hypothesis of synaptic release, physiological levels of extracellular Ca2+ play dual roles in evoked exocytosis by providing a source of Ca2+ influx, and by directly regulating quantal size and release probability in neuronal cells. PMID:22028769

  8. [Kinetic regularities of the proceeding and possible reaction mechanism of Mg2+-dependent enzymatic hydrolysis of ATP in the fraction of plasmatic membranes of the smooth muscle]. (United States)

    Danylovych, H V; Kosterin, S O


    Kinetic regularities of the reaction of Ca2+-independent Mg2+-dependent enzymatic hydrolysis of ATP catalyzed by the so-called "basal" Mg2+-ATPase localized in the plasmatic membrane of the uterus smooth-muscle cells have been studied using the methods of kinetic analysis performed under the equilibrium conditions. The analysis was based on the study of the concentration dependence of initial velocity of nucleoside triphosphate hydrolysis in EGTA-containing medium under the change of general concentrations of ATP [ATP]o and Mg2+[Mg2+]o in conditions of their equimolar ratio ([ATP]o/ [Mg2+]o)= 1; here the ratio between the concentrations of free reagents ([ATP4-]o/[Mg2+]o) was equal to 1.25. The obtained concentration dependence was interpreted in terms of two practically possible alternative mechanisms of Mg2+-dependent ATP-hydrolase enzymatic reaction. Mechanism I. Two separate independent centres of Mg ions and ATP binding by the enzymatic protein are supposed to exist, while Mg2+-dependent ATP-hydrolase enzymatic reaction proceeds independent of the equilibrium reaction of Mg ions chelatization of muscleside triphosphate. Mechanism II. The existence of the only centre of the chelate complex Mg2+ATP2- binding is postulated on the enzymatic protein; this process is also realized independent of the binding of Mg2+ and ATP-hydralase reaction catalized by it.

  9. Role of Ca2+-activated K+ channels and Na+-K+-ATPase in prostaglandin E1- and E2-induced inhibition of the adrenergic response in human vas deferens


    Medina Bessó, Pascual; Segarra Irles, Gloria Vicenta; Mauricio Aviñó, María Dolores; Vila Salinas, José María; Chuan Nuez, Pascual; Lluch López, Salvador


    Abstract We studied the role of K+ channels and Na+,K+-ATPase in the presynaptic inhibitory effects of prostaglandin E1 (PGE1) and PGE2 on the adrenergic responses of human vas deferens. Furthermore, we determined the effects of increasing extracellular K+ concentrations ([K+]o) and inhibition of Na+,K+-ATPase on neurogenic and norepinephrine -induced contractile responses. Ring segments of the epididymal part of the vas deferens were taken from 45 elective vasectomies and mounted ...

  10. Effects of flour bleaching agent on mice liver antioxidant status and ATPases. (United States)

    Jia, Xiaojing; Wu, Yangxinwei; Liu, Ping


    Benzoyl peroxide (BPO) is a strong oxidizing agent and widely used as flour bleaching agent. However their potential risk of liver damage is unknown. The aim of this study was to investigate the effects of BPO on mice liver antioxidant status and ATPases according to the actual amount of BPO in flour from Jinan, China. The results showed that the maximum concentration of BPO reached up to 284.6 mg/kg and content of BPO mainly ranged from 0 to 240 mg/kg. Therefore, four groups of mice were gavaged daily with BPO at doses of 0, 50, 100, 200mg/kg b.w./d for 42 days, respectively. In liver tissue, superoxide dismutase (SOD) activity was significantly decreased, while the content of malondialdehyde (MDA) significantly increased following BPO exposure at 200mg/kg b.w. BPO significantly decreased the Mg(2+)-ATPase and Ca(2+)-ATPase activities of the liver at 200mg/kg b.w. BPO, at all of the doses assayed, produced non-significant effects on glutathione peroxidase (GSH-Px) and Na(+)K(+)-ATPase activities. Experimental results suggested that BPO had certain adverse effects on antioxidant status and the activities of Mg(2+)-ATPase and Ca(2+)-ATPase of liver tissue. Copyright © 2011 Elsevier B.V. All rights reserved.


    Directory of Open Access Journals (Sweden)

    Shahdevi Nandar Kurniawan


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

  12. Sarcoplasmic reticulum Ca2+ uptake and leak properties, and SERCA isoform expression, in type I and type II fibres of human skeletal muscle. (United States)

    Lamboley, C R; Murphy, R M; McKenna, M J; Lamb, G D


    The Ca(2+) uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca(2+)] heavily buffered in the pCa range (-log10[Ca(2+)]) 7.3-6.0 for set times and the amount of net SR Ca(2+) accumulation determined from the force response elicited upon emptying the SR of all Ca(2+). Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.6. Maximal Ca(2+) uptake rate was ∼0.18 and ∼0.21 mmol Ca(2+) (l fibre)(-1) s(-1) in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg(2+)] from 1 to 3 mM had no significant effect on the net Ca(2+) uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca(2+) release channels during uptake in either fibre type. Ca(2+) leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca(2+) leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca(2+) uptake in the type I human fibres can account for the higher relative level of SR Ca(2+) loading observed in type I compared to type II fibres, and the SR Ca(2+) leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate.

  13. ATP-ases of synaptic plasma membranes in striatum: enzymatic systems for synapses functionality by in vivo administration of L-acetylcarnitine in relation to Parkinson's Disease. (United States)

    Villa, R F; Ferrari, F; Gorini, A


    The maximum rate (Vmax) of some enzymatic activities related to energy consumption was evaluated in synaptic plasma membranes from rat brain striatum, the synaptic energy state being a crucial factor in neurodegenerative diseases etiopathogenesis. Two types of synaptic plasma membranes were isolated from rats subjected to in vivo treatment with L-acetylcarnitine at two different doses (30 and 60 mg × kg(-1) i.p., 28 days, 5 days/week). The following enzyme activities were evaluated: acetylcholinesterase (AChE); Na(+), K(+), Mg(2+)-ATP-ase; ouabain insensitive Mg(2+)-ATP-ase; Na(+), K(+)-ATP-ase; direct Mg(2+)-ATP-ase; Ca(2+), Mg(2+)-ATP-ase; and low- and high-affinity Ca(2+)-ATP-ase. In control (vehicle-treated) animals, enzymatic activities are differently expressed in synaptic plasma membranes type I (SPM1) with respect to synaptic plasma membranes type II (SPM2), the evaluated enzymatic activities being higher in SPM2. Subchronic treatment with L-acetylcarnitine decreased AChE on SPM1 and SPM2 at the dose of 30 mg × kg(-1). Pharmacological treatment decreased ouabain insensitive Mg(2+)-ATP-ase activity and high affinity Ca(2+)-ATP-ase activity at the doses of 30 and 60 mg × kg(-1) respectively on SPM1, while it decreased Na(+), K(+)-ATP-ase, direct Mg(2+)-ATP-ase and Ca(2+), Mg(2+)-ATP-ase activities at the dose of 30 mg × kg(-1) on SPM2. These results suggest that the sensitivity to drug treatment is different between these two populations of synaptic plasma membranes from the striatum, confirming the micro-heterogeneity of these subfractions, possessing different metabolic machinery with respect to energy consumption and utilization and the regional selective effect of L-acetylcarnitine on cerebral tissue, depending on the considered area. The drug potential effect at the synaptic level in Parkinson's Disease neuroprotection is also discussed with respect to acetylcholine and energy metabolism. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights

  14. Methyl tert-butyl ether (MTBE) induced Ca(2+)-dependent cytotoxicity in isolated rabbit tracheal epithelial cells. (United States)

    Wang, Yajing; Chen, Chang; Wu, Tao; Xu, Jing; Han, Xiaodong


    As a volatile synthetic organic chemical, methyl tert-butyl ether (MTBE) was the most common gasoline additive. The increasing use of MTBE raised concern over its health safety. Inhalation was the principle route of exposure for the general population. This study used a model of rabbit tracheal epithelial cells (RTEs) in primary culture to investigate the cytotoxic effects induced by MTBE and the potential mechanism. RTEs were incubated with medium alone (control), 0.5, 50, 5000ppm MTBE respectively. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo liumbromide) assay, staining with fluorescein diacetate, propidium iodide and lactate dehydrogenase leakage ratio were used to assess MTBE cytotoxicity on cells. We also observed a significant elevation in cytosolic Ca2+ by fluorescence probe Fluo-3AM at 3, 6 and 12h following exposure to MTBE. Loss of mitochondrial membrane potential (MMP) was detected following 12 and 24h treatment of NP and assessment by rhodamine 123 (Rh123) staining. Activity changes of the Ca(2+)-ATPase, Ca(2+)-Mg(2+)-ATPase following MTBE treatment displayed a similar trend, suggesting an initial elevation before 6h and subsequent dramatic decrease at 12h. Our results demonstrated that induction of cell injury, associated with mitochondrial dysfunction, and alterations in cytosolic Ca2+ in RTEs represent key mechanisms by which MTBE exerts its cytotoxic effects.

  15. Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles. (United States)

    Datiles, Manuel J; Johnson, Eric A; McCarty, Richard E


    Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory epsilon subunit (CF1-epsilon) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-epsilon or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-epsilon from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the alpha/beta heterohexamer.

  16. Role of matrix metalloprotease-2 in oxidant activation of Ca 2 ...

    Indian Academy of Sciences (India)

    Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H2O2 (1 mM) stimulated Ca2+ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca2+ATPase activity by H2O2 in the smooth muscle plasma membrane. The smooth ...

  17. Ca2+ ionophores trigger membrane remodeling without a need for store-operated Ca2+ entry. (United States)

    Galitzine, Marie; Capiod, Thierry; Le Deist, Françoise; Meyer, Dominique; Freyssinet, Jean-Marie; Kerbiriou-Nabias, Danièle


    Calcium (Ca2+) ionophores are the most effective agents able to elicit rapid membrane remodeling in vitro. This process exposes aminophospholipids at the surface of platelets and blood cells, thus providing a catalytic surface for coagulation. To explore the underlying mechanism, we examined if cytosolic Ca2+ ([Ca2+]i) increase through store-operated Ca2+ entry (SOCE) was necessary for the potent effect of ionophores. Recent studies have demonstrated that the Ca2+-ATPase inhibitor thapsigargin, although able to elevate [Ca2+]i through SOCE, does not trigger the rapid membrane remodeling. However, it was not known if the additional effect of ionophores to promote the process required SOCE or could it occur independently. We took advantage of two mutant B lymphoblast cell lines, characterized either by defective SOCE or altered membrane remodeling, to simultaneously assess [Ca2+]i increase and membrane remodeling in the presence of ionophores or thapsigargin. Results imply that ionophores trigger membrane remodeling without the requirement for a functional SOCE.

  18. Ca(2+-dependent regulation of the Ca(2+ concentration in the myometrium mitochondria. II. Ca(2+ effects on mitochondria membranes polarization and [Ca(2+](m

    Directory of Open Access Journals (Sweden)

    L. G. Babich


    Full Text Available It is known that Ca2+ accumulation in the mitochondria undergoes complex regulation by Ca2+ itself. But the mechanisms of such regulation are still discussed. In this paper we have shown that Ca ions directly or indirectly regulate the level of myometrium mitochondria membranes polarization. The additions of 100 µM Ca2+ were accompanied by depolarization of the mitochondria membranes. The following experiments were designed to study the impact of Ca2+ on the myometrium mitochondria [Ca2+]m. Isolated myometrium mitochondria were preincubated without or with 10 μM Са2+ followed by 100 μM Са2+ addition. Experiments were conducted in three mediums: without ATP and Mg2+ (0-medium, in the presence of 3 mM Mg2+ (Mg-medium and 3 mM Mg2+ + 3 mM ATP (Mg,ATP-medium. It was shown that the effects of 10 μM Са2+ addition were different in different mediums, namely in 0- and Mg-medium the [Ca2+]m values increased, whereas in Mg,ATP-medium statistically reliable changes were not registered. Preincubation of mitochondria with 10 μM Са2+ did not affect the [Ca2+]m value after the addition of 100 μM Са2+. The [Ca2+]m values after 100 μM Са2+ addition were the same in 0- and Mg,ATP-mediums and somewhat lower in Mg-medium. Preliminary incubation of mitochondria with 10 μM Са2+ in 0- and Mg-mediums reduced changes of Fluo 4 normalized fluorescence values that were induced by 100 μM Са2+ additions, but in Mg,ATP-medium such differences were not recorded. It is concluded that Са2+ exchange in myometrium mitochondria is regulated by the concentration of Ca ions as in the external medium, so in the matrix of mitochondria. The medium composition had a significant impact on the [Са2+]m values in the absence of exogenous cation. It is suggested that light increase of [Са2+]m before the addition of 100 μM Са2+ may have a positive effect on the functional activity of the mitochondria.

  19. Correlation between cyanide-induced decreases in ocular Ca 2+ ...

    African Journals Online (AJOL)

    The effect of cyanide toxicity on Ca2+-ATPase activities were significantly decreased in the lens and vitreous humour of the cyanide-fed rabbits (p< 0.05), while the corneal enzyme was unaffected. Ophthalmoscopic examination of the cyanide-exposed rabbits revealed adverse morphological changes, including pale fundus ...

  20. Effects of high pressure treatment on Ca2+ release and Ca2+ uptake of sarcoplasmic reticulum. (United States)

    Okamoto, A; Suzuki, A; Ikeuchi, Y; Saito, M


    To clarify the mechanism of pressure-induced meat tenderization or acceleration of meat conditioning, the pressure-induced morphological and biochemical changes in sarcoplasmic reticulum (SR), and Ca2+ release from SR in the rabbit skeletal muscle treated with high pressure (100-300 MPa, 5 min) were investigated in comparison with those of the SR from conditioned muscle. The destruction of the membrane structure of the SR expanded with increasing pressure applied to the muscle. Significant changes in the SDS-PAGE profile were not observed in the SR from the pressurized muscle up to 200 MPa, but a marked decrease of the ATPase protein and high-affinity Ca(2+)-binding protein were observed in the SR from the pressurized muscle at 300 MPa. The ATPase activities increased in the SR isolated from the muscle exposed to high pressure up to 200 MPa. When the muscle was pressurized at 300 MPa, the ATPase activity dropped to the same level with that of the SR from the untreated muscle. Ca2+ uptake ability of the SR vesicles measured using a fluorescent chelating reagent decreased with increasing pressure applied to the muscle. Ultrastructural studies showed that Ca2+, which was mainly localized in the SR region of the untreated fiber bundles, was translocated into myofibrillar space in the pressurized muscle. It is clear that a brief exposure of the muscle to high pressure causes considerable changes in membrane structure and biochemical function of SR as compared with those of SR in the muscle induced by conditioning.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Insulin resistance vs. hyperinsulinemia in hypertension: insulin regulation of Ca2+ transport and Ca(2+)-regulation of insulin sensitivity. (United States)

    Zemel, M B


    Hypertension in obesity and insulin resistance has been attributed to insulin stimulation of sympathetic neural output and renal sodium retention. However, recent data demonstrates a significant vasodilatory effect of insulin and suggests that vascular smooth muscle resistance to this action may instead be the cause of hypertension in insulin resistance. This concept is supported by the observation that pharmacological amplification of peripheral insulin sensitivity results in reduced arterial pressure. Insulin attenuates vasoconstrictor responses to pressor agonists and accelerates vascular smooth muscle relaxation, while these effects are blunted in obesity and insulin resistance. Insulin regulation of vasoconstriction and vascular relaxation appears to be secondary to regulation of intracellular Ca2+ ([Ca2+]i), as insulin attenuates both voltage- and receptor-mediated Ca2+ influx and stimulates both the transcription and activity of Ca(2+)-ATPase in vascular smooth muscle cells. Further, these effects are also blunted in insulin resistance. Although [Ca2+]i plays a poorly understood role in insulin signalling, increases beyond an optimal range results in impaired insulin sensitivity, possibly by Ca(2+)-inhibition of insulin-induced dephosphorylation of insulin-sensitive substrates. Consistent with this concept, ectopic overexpression of the agouti gene in the viable yellow (Avy) mouse results in increased skeletal myocyte [Ca2+]i. Accordingly, increased [Ca2+]i in primary insulin target tissues appears to result in peripheral insulin resistance which then results in aberrant regulation of vascular smooth muscle [Ca2+]i and increases in arterial pressure.

  2. Divergence of Ca(2+) selectivity and equilibrium Ca(2+) blockade in a Ca(2+) release-activated Ca(2+) channel. (United States)

    Yamashita, Megumi; Prakriya, Murali


    Prevailing models postulate that high Ca(2+) selectivity of Ca(2+) release-activated Ca(2+) (CRAC) channels arises from tight Ca(2+) binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca(2+) binding for Ca(2+) selectivity in recombinant Orai3 channels, which function as highly Ca(2+)-selective channels when gated by the endoplasmic reticulum Ca(2+) sensor STIM1 or as poorly Ca(2+)-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca(2+) blocked Na(+) currents in both gating modes with a similar inhibition constant (Ki; ~25 µM). Thus, equilibrium binding as set by the Ki of Ca(2+) blockade cannot explain the differing Ca(2+) selectivity of the two gating modes. Unlike STIM1-gated channels, Ca(2+) blockade in 2-APB-gated channels depended on the extracellular Na(+) concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na(+) pore occupancy. Moreover, the second-order rate constants of Ca(2+) blockade were eightfold faster in 2-APB-gated channels than in STIM1-gated channels. A four-barrier, three-binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca(2+) and Na(+) to simulate the faster rate constants of 2-APB-gated channels qualitatively reproduces their low Ca(2+) selectivity, suggesting that ion entry and exit rates strongly affect Ca(2+) selectivity. Noise analysis indicated that the unitary Na(+) conductance of 2-APB-gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (Po; ~0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high Po state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca

  3. Targeting Cardiomyocyte Ca2+ Homeostasis in Heart Failure (United States)

    Røe, Åsmund T.; Frisk, Michael; Louch, William E.


    Improved treatments for heart failure patients will require the development of novel therapeutic strategies that target basal disease mechanisms. Disrupted cardiomyocyte Ca2+ homeostasis is recognized as a major contributor to the heart failure phenotype, as it plays a key role in systolic and diastolic dysfunction, arrhythmogenesis, and hypertrophy and apoptosis signaling. In this review, we outline existing knowledge of the involvement of Ca2+ homeostasis in these deficits, and identify four promising targets for therapeutic intervention: the sarcoplasmic reticulum Ca2+ ATPase, the Na+-Ca2+ exchanger, the ryanodine receptor, and t-tubule structure. We discuss experimental data indicating the applicability of these targets that has led to recent and ongoing clinical trials, and suggest future therapeutic approaches. PMID:25483944

  4. Control mechanisms of mitochondrial Ca(2+) uptake - feed-forward modulation of aldosterone secretion. (United States)

    Szanda, Gergö; Rajki, Anikó; Spät, András


    Mitochondrial Ca(2+) signal activates metabolism by boosting pyridine nucleotide reduction and ATP synthesis or, if Ca(2+) sequestration is supraphysiological, may even lead to apoptosis. Although the molecular background of mitochondrial Ca(2+) uptake has recently been elucidated, the regulation of Ca(2+) handling is still not properly clarified. In human adrenocortical H295R cells we found a regulatory mechanism involving p38 MAPK and novel-type PKC isoforms. Upon stimulation with angiotensin II (AII) these kinases are activated typically prior to the release of Ca(2+) and - most probably by reducing the Ca(2+) permeation through the outer mitochondrial membrane - attenuate mitochondrial Ca(2+) uptake in a feed-forward manner. The biologic significance of the kinase-mediated reduction of mitochondrial Ca(2+) signal is also reflected by the attenuation of AII-mediated aldosterone secretion. As another feed-forward mechanism, we found in HEK-293T and H295R cells that Ca(2+) signal evoked either by IP(3) or by voltage-gated influx is accompanied by a concomitant cytosolic Mg(2+) signal. In permeabilized HEK-293T cells Mg(2+) was found to be a potent inhibitor of mitochondrial Ca(2+) uptake in the physiologic [Mg(2+)] and [Ca(2+)] range. Thus, these inhibitory mechanisms may serve not only as protection against mitochondrial Ca(2+) overload and subsequent apoptosis but also have the potential to substantially alter physiological responses. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+ Signaling (United States)

    El-Najjar, Nahed; Kulkarni, Rashmi P.; Nader, Nancy; Hodeify, Rawad


    Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+ signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+ signaling, including most prominently an inhibition of the passive ER Ca2+ leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+ leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+ signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+ signaling machinery are different. PMID:28713824

  6. Evolution of plant P-type ATPases

    Directory of Open Access Journals (Sweden)

    Christian N.S. Pedersen


    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.

  7. Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation

    Directory of Open Access Journals (Sweden)

    Bassani R.A.


    Full Text Available Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A, Na+-Ca2+ exchanger (NCX and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake. To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW. Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 µM, whereas SR-dependent flux was lower with TG-TW (77 vs 81 µM. The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.

  8. Mitochondrial Ca2+ homeostasis during Ca2+ influx and Ca2+ release in gastric myocytes from Bufo marinus (United States)

    Drummond, Robert M; Mix, T Christian H; Tuft, Richard A; Walsh, John V; Fay, Fredric S


    The Ca2+-sensitive fluorescent indicator rhod-2 was used to monitor mitochondrial Ca2+ concentration ([Ca2+]m) in gastric smooth muscle cells from Bufo marinus. In some studies, fura-2 was used in combination with rhod-2, allowing simultaneous measurement of cytoplasmic Ca2+ concentration ([Ca2+]i) and [Ca2+]m, respectively. During a short train of depolarizations, which causes Ca2+ influx from the extracellular medium, there was an increase in both [Ca2+]i and [Ca2+]m. The half-time (t½) to peak for the increase in [Ca2+]m was considerably longer than the t½ to peak for the increase in [Ca2+]i. [Ca2+]m remained elevated for tens of seconds after [Ca2+]i had returned to its resting value. Stimulation with caffeine, which causes release of Ca2+ from the sarcoplasmic reticulum (SR), also produced increases in both [Ca2+]i and [Ca2+]m. The values of t½ to peak for the increase in [Ca2+] in both cytoplasm and mitochondria were similar; however, [Ca2+]i returned to baseline values much faster than [Ca2+]m. Using a wide-field digital imaging microscope, changes in [Ca2+]m were monitored within individual mitochondria in situ, during stimulation of Ca2+ influx or Ca2+ release from the SR. Mitochondrial Ca2+ uptake during depolarizing stimulation caused depolarization of the mitochondrial membrane potential. The mitochondrial membrane potential recovered considerably faster than the recovery of [Ca2+]m. This study shows that Ca2+ influx from the extracellular medium and Ca2+ release from the SR are capable of increasing [Ca2+]m in smooth muscle cells. The efflux of Ca2+ from the mitochondria is a slow process and appears to be dependent upon the amount of Ca2+ in the SR. PMID:10713963

  9. The Rice High-Affinity K+ Transporter OsHKT2;4 Mediates Mg2+ Homeostasis under High-Mg2+ Conditions in Transgenic Arabidopsis

    Directory of Open Access Journals (Sweden)

    Chi Zhang


    Full Text Available Rice (Oryza sativa; background Nipponbare contains nine HKT (high-affinity K+ transport-like genes encoding membrane proteins belonging to the superfamily of Ktr/TRK/HKT. OsHKTs have been proposed to include four selectivity filter-pore-forming domains homologous to the bacterial K+ channel KcsA, and are separated into OsHKT1s with Na+-selective activity and OsHKT2s with Na+-K+ symport activity. As a member of the OsHKT2 subfamily, OsHKT2;4 renders Mg2+ and Ca2+ permeability for yeast cells and Xenopus laevis oocytes, besides K+ and Na+. However, physiological functions related to Mg2+in planta have not yet been identified. Here we report that OsHKT2;4 from rice (O. sativa; background Nipponbare functions as a low-affinity Mg2+ transporter to mediate Mg2+ homeostasis in plants under high-Mg2+ environments. Using the functional complementation assay in Mg2+-uptake deficient Salmonella typhimurium strains MM281 and electrophysiological analysis in X. laevis oocytes, we found that OsHKT2;4 could rescue the growth of MM281 in Mg2+-deficient conditions and induced the Mg2+ currents in oocytes at millimolar range of Mg2+. Additionally, overexpression of OsHKT2;4 to Arabidopsis mutant lines with a knockout of AtMGT6, a gene encoding the transporter protein necessary for Mg2+ adaptation in Arabidopsis, caused the Mg2+ toxicity to the leaves under the high-Mg2+ stress, but not under low-Mg2+ environments. Moreover, this Mg2+ toxicity symptom resulted from the excessive Mg2+ translocation from roots to shoots, and was relieved by the increase in supplemental Ca2+. Together, our results demonstrated that OsHKT2;4 is a low-affinity Mg2+ transporter responsible for Mg2+ transport to aerials in plants under high-Mg2+ conditions.

  10. [ATPase activity and processes of calcium transport in membranes of sarcoplasmic reticulum of skeletal muscles with E-avitaminotic dystrophy]. (United States)

    Kurskiĭ, M D; Grigor'eva, V A; Medovar, E N; Meshkova, L I


    Peculiarities of functioning of the sarcoplasmic reticulum muscles membranes with E-avitaminotic distrophy were studied. It was determined that the level of ATP-dependent consumption of Ca2+, value of the Mg2+, Ca2+-ATPase activity and an amount of the intermediate phosphorylated product forming in the reaction of ATP hydrolysis decrease. The rate of this product formation in the sarcoplasmic reticulum of the distrophic muscles is inhibited as compared to normalcy. Elimination of Ca2+ into calcium-free medium from the vesicular membranes of the reticulum preliminarily loaded with Ca2+ occurs more rapidly under dystrophy than in normalcy. The data obtained evidence for a disturbance of mechanism of Ca2+ active transport and for an increase in the membrane permeability for Ca2+ in the membranes of the dystrophic muscles sarcoplasmic reticulum. A problem is considered on a dependence of the skeletal muscles observed in the reticulum under dystrophy of the functional changes on the membrane structure, in particular on their lipid composition.

  11. Characterization of the sarcoplasmic reticulum Ca-ATPase from rabbit temporalis muscle. (United States)

    Sánchez, Gabriel Antonio; Di Croce, Daniel Eduardo; Casadoumecq, Ana Clara; Richard, Susana Beatriz; Takara, Delia


    The aim of this work was to isolate the sarcoplasmic reticulum (SR) Ca-ATPase from rabbit temporalis muscle and to determine the optimal conditions for calcium transport and enzymatic activity. SR vesicles were isolated from rabbit temporalis muscle by differential centrifugation, the protein composition analyzed by electrophoresis and compared to fast-twitch muscle membrane suspensions. ELISA was used to determine the sarcoendoplasmic reticulum Ca-ATPase (SERCA) isoform. Ca-ATPase activity was determined by a colorimetric method. Calcium-binding to the Ca-ATPase, calcium uptake, calcium efflux and phosphorylation by P(i) were determined with radioisotopic techniques. Sixty five percent of the total protein concentration of SR membranes suspensions from rabbit temporalis corresponded to SERCA. Of the total SERCA protein, 64% was SERCA 2, 35% was SERCA 1 and less than 1% was SERCA 3. The optimal conditions of the SERCA isolated from rabbit temporalis muscle were: pH 7.2, 5 μM Ca(2+), 100 μM EGTA, 90 μM Mg(2+), 3mM ATP and 100mM KCl and did not differ from fast-twitch skeletal muscle. The temporalis maximal calcium uptake and Ca-ATPase activity were lower but the sensitivity to the specific Ca-ATPase inhibitor thapsigargin was higher. Calcium-binding to the enzyme and calcium efflux were similar while the phosphorylation of the enzyme by P(i) was lower. The lower enzymatic activity and calcium transport capability of the Ca-ATPase isolated from rabbit temporalis, and the higher sensitivity to inhibitory drugs are consistent with the presence of a substantial proportion of SERCA 2, which can be expected in other rabbit masticatory muscles. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Two isoforms of sarco/endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans. (United States)

    Cho, J H; Bandyopadhyay, J; Lee, J; Park, C S; Ahnn, J


    SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), a membrane bound Ca(2+)- /Mg(2+)- dependent ATPase that sequesters Ca(2+) into the SR/ER lumen, is one of the essential components for the maintenance of intracellular Ca(2+) homeostasis. Here we describe the identification and functional characterization of a C. elegans SERCA gene (ser-1). ser-1 is a single gene alternatively spliced at its carboxyl terminus to form two isoforms (SER-1A and SER-1B) and displays a high homology (70% identity, 80% similarity) with mammalian SERCAs. Green fluorescent protein (GFP) and whole-mount immunostaining analyses reveal that SER-1 expresses in neuronal cells, body-wall muscles, pharyngeal and vulval muscles, excretory cells, and vulva epithelial cells. Furthermore, SER-1::GFP expresses during embryonic stages and the expression is maintained through the adult stages. Double-stranded RNA injection (also known as RNAi) targeted to each SER-1 isoform results in severe phenotypic defects: ser-1A(RNAi) animals show embryonic lethality, whereas ser-1B(RNAi) results in L1 larval arrest phenotype. These findings suggest that both isoforms of C. elegans SERCA, like in mammals, are essential for embryonic development and post-embryonic growth and survival.

  13. Upregulation of Ca2+ removal in human skeletal muscle: a possible role for Ca2+-dependent priming of mitochondrial ATP synthesis.

    NARCIS (Netherlands)

    Koopman, W.J.H.; Renders, M.; Oosterhof, A.; Kuppevelt, A.H.M.S.M. van; Engelen, B.G.M. van; Willems, P.H.G.M.


    In muscle, ATP is required for the powerstroke of the myosin head, the detachment of actin and myosin filaments, and the reuptake of Ca2+ into the sarcoplasmic reticulum. During contraction-relaxation, large amounts of ATP are consumed at the sites of action of the myosin-ATPase and sarcoplasmic

  14. Environmental and cortisol-mediated control of Ca(2+) uptake in tilapia (Oreochromis mossambicus). (United States)

    Lin, Chia-Hao; Kuan, Wei-Chun; Liao, Bo-Kai; Deng, Ang-Ni; Tseng, Deng-Yu; Hwang, Pung-Pung


    Ca(2+) is a vital element for many physiological processes in vertebrates, including teleosts, which live in aquatic environments and acquire Ca(2+) from their surroundings. Ionocytes within the adult gills or larval skin are critical sites for transcellular Ca(2+) uptake in teleosts. The ionocytes of zebrafish were found to contain transcellular Ca(2+) transporters, epithelial Ca(2+) channel (ECaC), plasma membrane Ca(2+)-ATPase 2 (PMCA2), and Na(+)/Ca(2+) exchanger 1b (NCX1b), providing information about the molecular mechanism of transcellular Ca(2+) transports mediated by ionocytes in fish. However, more evidence is required to establish whether or not a similar mechanism of transcellular Ca(2+) transport also exists in others teleosts. In the present study, ecac, pmca2, and ncx1 were found to be expressed in the branchial ionocytes of tilapia, thereby providing further support for the mechanism of transcellular Ca(2+) transport through ionocytes previously proposed for zebrafish. In addition, we also reveal that low Ca(2+) water treatment of tilapia stimulates Ca(2+) uptake and expression of ecac and cyp11b (the latter encodes a cortisol-synthesis enzyme). Treatment of tilapia with exogenous cortisol (20 mg/l) enhanced both Ca(2+) influx and ecac expression. Therefore, increased cyp11b expression is suggested to enhance Ca(2+) uptake capacity in tilapia exposed to low Ca(2+) water. Furthermore, the application of cortisol receptor antagonists revealed that cortisol may regulate Ca(2+) uptake through glucocorticoid and/or mineralocorticoid receptor (GR and/or MR) in tilapia. Taken together, the data suggest that cortisol may activate GR and/or MR to execute its hypercalcemic action by stimulating ecac expression in tilapia.


    Chen, Chung-Ho; Greenawalt, John W.; Lehninger, Albert L.


    Mitochondria isolated from the hepatopancreas of the blue crab Callinectes sapidus show up to 12-fold stimulation of respiration on addition of Ca2+, which is accompanied by Ca2+ accumulation (Ca2+:site = 1.9) and H+ ejection (H+:Ca2+ = 0.85). Sr2+ and Mn2+ are also accumulated; Mg2+ is not. A strongly hypertonic medium (383 mosM), Mg2+, and phosphate are required for maximal Ca2+ uptake. Ca2+ uptake takes precedence over oxidative phosphorylation of ADP for respiratory energy. Once Ca2+ is accumulated by the crab mitochondria, it is stable and only very slowly released, even by uncoupling agents. ATP hydrolysis also supports Ca2+ uptake. Respiration-inhibited crab hepatopancreas mitochondria show both high-affinity and low-affinity Ca2+-binding sites, which are inactive in the presence of uncoupling agents. Crab hepatopancreas mitochondria have an enormous capacity for accumulation of Ca2+, up to 5,500 ng-atoms Ca2+ per mg protein, with an equivalent amount of phosphate. Freshly isolated mitochondria contain very large amounts of Ca2+, Mg2+, phosphate, K+, and Na+; their high Ca2+ content is a reflection of the vary large amount of extra-mitochondrial Ca2+ in the whole tissue. Electron microscopy of crab mitochondria loaded with Ca2+ and phosphate showed large electron-dense deposits, presumably of precipitated calcium phosphate. They consisted of bundles of needle-like crystals, whereas Ca2+-loaded rat liver mitochondria show only amorphous deposits of calcium phosphate under similar conditions. The very pronounced capacity of crab hepatopancreas mitochondria for transport of Ca2+ appears to be adapted to a role in the storage and release of Ca2+ during the molting cycle of this crustacean. PMID:4827906

  16. Characterization of Ca2+ transport in rat renal brush-border membranes and its modulation by phosphatidic acid.


    Somermeyer, M G; Knauss, T C; Weinberg, J M; Humes, H D


    The Ca2+ transport process by isolated renal brush-border membranes was characterized and the influence of the acidic phospholipid phosphatidic acid (PtdA) on this transport process was assessed. Ca2+ uptake by brush-border membranes exhibited saturation kinetics. It was inhibitable by a variety of multivalent cations, as well as by Ca2+-entry inhibitors, including verapamil, Ruthenium Red and gentamicin. It was selective for Ca2+ compared with Mg2+. This process was also electrophoretic sinc...

  17. Effect of bafilomycin and NAADP on membrane-associated ATPases and respiration of isolated mitochondria of the murine Nemeth-Kellner lymphoma. (United States)

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


    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

  18. Ca2+signaling and emesis: Recent progress and new perspectives. (United States)

    Zhong, Weixia; Picca, Andrew J; Lee, Albert S; Darmani, Nissar A


    Cisplatin-like chemotherapeutics cause vomiting via calcium (Ca 2+ )-dependent release of multiple neurotransmitters (dopamine, serotonin, substance P, etc.) from the gastrointestinal enterochromaffin cells and/or the brainstem. Intracellular Ca 2+ signaling is triggered by activation of diverse emetic receptors (including tachykininergic NK 1 , serotonergic 5-HT 3 , dopaminergic D 2 , cholinergic M 1 , or histaminergic H 1 ) , whose activation in vomit-competent species can evoke emesis. Other emetogens such as cisplatin, rotavirus NSP4 protein and bacterial toxins can also induce intracellular Ca 2+ elevation. Netupitant is a highly selective neurokinin NK 1 receptor (NK 1 R) antagonist and palonosetron is a selective second-generation serotonin 5-HT 3 receptor (5-HT 3 R) antagonist with a distinct pharmacological profile. An oral fixed combination of netupitant/palonosetron (NEPA; Akynzeo(®)) with >85% antiemetic efficacy is available for use in the prevention of acute and delayed chemotherapy-induced nausea and vomiting (CINV). Cannabinoid CB 1 receptor agonists possess broad-spectrum antiemetic activity since they prevent vomiting caused by a variety of emetic stimuli including the chemotherapeutic agent cisplatin, 5-HT 3 R agonists, and D 2 R agonists. Our findings demonstrate that application of the L-type Ca 2+ channel (LTCC) agonist FPL 64176 and the intracellular Ca 2+ mobilizing agent thapsigargin (a sarco/endoplasmic reticulum Ca 2+ -ATPase inhibitor) cause vomiting in the least shrew. On the other hand, blockade of LTCCs by corresponding antagonists (nifedipine or amlodipine) not only provide broad-spectrum antiemetic efficacy against diverse agents that specifically activate emetogenic receptors such as 5-HT 3 , NK 1 , D 2 , and M 1 receptors, but can also potentiate the antiemetic efficacy of palonosetron against the non-specific emetogen, cisplatin. In this review, we will provide an overview of Ca 2+ involvement in the emetic process; discuss the

  19. TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption.

    NARCIS (Netherlands)

    Voets, T.; Nilius, B.; Hoefs, S.J.G.; Kemp, J.W.C.M. van der; Droogmans, G.; Bindels, R.J.M.; Hoenderop, J.G.J.


    Mg2+ is an essential ion involved in a multitude of physiological and biochemical processes and a major constituent of bone tissue. Mg2+ homeostasis in mammals depends on the equilibrium between intestinal Mg2+ absorption and renal Mg2+ excretion, but little is known about the molecular nature of

  20. [Intervention of Qi-activating and Spleen-strengthening Herbs on Ca2+/CaMK II Signaling Pathways Key Factors in Skeletal Muscle Tissue of Rats with Spleen-qi Deficiency]. (United States)

    Duan, Yong-qiang; Cheng, Ying-xia; Liang, Yu-jie; Cheng, Wei-dong; Du, Juan; Yang, Xiao-yi; Wang, Yan


    To observe changes of [Ca2+]i concentration and CaM, CaMK II and p-CaMK II of Ca2+/CaMK II signaling pathways in skeletal muscle tissue of rats with spleen-qi deficiency and intervention of Sijunzi decoction and extract of Hedysarum polybotrys. Rats were randomized into four groups: normal control group, spleen-qi deficient model group, extract from Hedysarum polybotrys group and Sijunzi decoction group, ten rats in each group. After the spleen-qi deficient models were built by comprehensive application of rhubarb, exhaustive and hungry methods, and treatment groups were treated with extract from Hedysarum polybotrys at 6 g/(kg . d) or Sijunzi decoction at 20 g/(kg . d) for 21 d. Then, general existence,gastrointestinal hormones GAS and MOT levels, and activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase of skeletal muscle were evaluated. Also, confocal laser technology was used to test cellular[Ca2+]i concentrations in skeletal muscle and Western blotting technique was used to test CaM, CaMK II and p-CaMK 11 expression in intestinal tissue of spleen-qi deficient model rats. Compared with normal group, general condition was poor, levels of GAS and MOT decreased (P CaMK II and p-CaMK II in skeletal muscle decreased significantly (P CaMK II in skeletal muscle tissue increased (P CaMK II in skeletal muscle tissue increased in the rats of Sijunzi decoction group (P < 0. 05). Sijunzi decoction and extract of Hedysarum polybotrys can be applied to treat spleen-qi deficiency syndrome through the mechanism of regulating GAS and MOT secretion and raising expression of Ca2+ /CaM signaling pathways key factors in skeletal muscle tissue. Sijunzi decoction has the better effect

  1. Live microbial cells adsorb Mg2+ more effectively than lifeless organic matter (United States)

    Qiu, Xuan; Yao, Yanchen; Wang, Hongmei; Duan, Yong


    The Mg2+ content is essential in determining different Mg-CaCO3 minerals. It has been demonstrated that both microbes and the organic matter secreted by microbes are capable of allocating Mg2+ and Ca2+ during the formation of Mg-CaCO3, yet detailed scenarios remain unclear. To investigate the mechanism that microbes and microbial organic matter potentially use to mediate the allocation of Mg2+ and Ca2+ in inoculating systems, microbial mats and four marine bacterial strains (Synechococcus elongatus, Staphylococcus sp., Bacillus sp., and Desulfovibrio vulgaris) were incubated in artificial seawater media with Mg/Ca ratios ranging from 0.5 to 10.0. At the end of the incubation, the morphology of the microbial mats and the elements adsorbed on them were analyzed using scanning electronic microscopy (SEM) and energy diffraction spectra (EDS), respectively. The content of Mg2+ and Ca2+ adsorbed by the extracellular polysaccharide substances (EPS) and cells of the bacterial strains were analyzed with atomic adsorption spectroscopy (AAS). The functional groups on the surface of the cells and EPS of S. elongatus were estimated using automatic potentiometric titration combined with a chemical equilibrium model. The results show that live microbial mats generally adsorb larger amounts of Mg2+ than Ca2+, while this rarely is the case for autoclaved microbial mats. A similar phenomenon was also observed for the bacterial strains. The living cells adsorb more Mg2+ than Ca2+, yet a reversed trend was observed for EPS. The functional group analysis indicates that the cell surface of S. elongatus contains more basic functional groups (87.24%), while the EPS has more acidic and neutral functional groups (83.08%). These features may be responsible for the different adsorption behavior of Mg2+ and Ca2+ by microbial cells and EPS. Our work confirms the differential Mg2+ and Ca2+ mediation by microbial cells and EPS, which may provide insight into the processes that microbes use to

  2. Characterization of Bovine Brain ATPase (United States)


    with a complex structure (presence of galactose, vannose and sialic acid ). This structure raises the possibility of immobilizing via immobilized...SYNAPIIC The (Ca2+ + M9*2)- dependent ATPase-Calcitu Channel Complex is found at the synaptic termini of neurons ( 1). With the exception of Mojave toxin...for 60% inhibition. We examined the effects of various phospholipids on catalytic activity (Table IX). With the exception of phosphatidic acid , all

  3. Role of matrix metalloprotease-2 in oxidant activation of Ca ATPase ...

    Indian Academy of Sciences (India)


    Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H2O2. (1 mM) stimulated Ca2+ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca2+ATPase activity by H2O2 in the smooth muscle plasma membrane. The smooth ...

  4. MagFRET: the first genetically encoded fluorescent Mg2+ sensor.

    Directory of Open Access Journals (Sweden)

    Laurens H Lindenburg

    Full Text Available Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg(2+, MagFRET-1. This sensor is based on the high-affinity Mg(2+ binding domain of human centrin 3 (HsCen3, which undergoes a transition from a molten-globular apo form to a compactly-folded Mg(2+-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg(2+ affinity (K d = 148 µM with a 50% increase in emission ratio upon Mg(2+ binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg(2+ affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg(2+ concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg(2+ concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca(2+, this affinity is sufficiently attenuated (K d of 10 µM to make the sensor insensitive to known Ca(2+ stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg(2+ imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg(2+ sensors could prove very useful in understanding intracellular Mg(2+ homeostasis and signaling.

  5. Kinetic and mesoscopic non-equilibrium description of the Ca2+ pump : A comparison

    NARCIS (Netherlands)

    Lervik, A.; Bedeaux, D.; Kjelstrup, S.H.


    We analyse the operation of the Ca2?-ATPase ion pump using a kinetic cycle diagram. Using the methodology of Hill, we obtain the cycle fluxes, entropy production and efficiency of the pump. We compare these results with a mesoscopic non-equilibrium description of the pump and show that the kinetic

  6. Superstoichiometric Ca2+ uptake supported by hydrolysis of endogenous ATP in rat liver mitochondria. (United States)

    Brand, M D; Lehninger, A L


    The nature of the energy store causing rapid superstoichiometric leads to H+/2e minus ejection and leads to Ca2+/2e minus uptake ratios in rat liver mitochondria pulsed with Ca2+ has been investigated. The extent and the rate of the initial fast superstoichiometric phase of H plus ejection were greatly reduced by oligomycin and other ATPase inhibitors; the subsequent shoichiometric phase was unaffected. No such inhibition was seen with atractyloside. Similarly, the initial fast phase of Ca2+ uptake was reduced in extent by oligomycin, whereas the slower stoichiometric phase was unaffected. Moreover, the ATP content of mitochondria previously incubated with succinate decreased by about 80% within 5 s after pulsing with Ca2+. The energy store for superstoichiometric Ca2+ uptake and H plus injection is thus identified as endogenous ATP.

  7. Imaging Ca2+ with a Fluorescent Rhodol. (United States)

    Contractor, Alisha A; Miller, Evan W


    Ca2+ mediates a host of biochemical and biophysical signaling processes in cells. The development of synthetic, Ca2+-sensitive fluorophores has played an instrumental role in our understanding of the temporal and spatial dynamics of Ca2+. Coupling Ca2+-selective ligands to fluorescent reporters has provided a wealth of excellent indicators that span the visible excitation and emission spectrum and possess Ca2+ affinities suited to a variety of cellular contexts. One underdeveloped area is the use of hybrid rhodamine/fluorescein fluorophores, or rhodols, in the context of Ca2+ sensing. Rhodols are bright and photostable and have good two-photon absorption cross sections (σTPA), making them excellent candidates for incorporation into Ca2+-sensing scaffolds. Here, we present the design, synthesis, and application of rhodol Ca2+ sensor 1 (RCS-1), a chlorinated pyrrolidine-based rhodol. RCS-1 possesses a Ca2+ binding constant of 240 nM and a 10-fold turn response to Ca2+. RCS-1 effectively absorbs infrared light and has a σTPA of 76 GM at 840 nm, 3-fold greater than that of its fluorescein-based counterpart. The acetoxy-methyl ester of RCS-1 stains the cytosol of live cells, enabling observation of Ca2+ fluctuations and cultured neurons using both one- and two-photon illumination. Together, these results demonstrate the utility of rhodol-based scaffolds for Ca2+ sensing using two-photon illumination in neurons.

  8. Ca2+ Dependence of Synaptic Vesicle Endocytosis. (United States)

    Leitz, Jeremy; Kavalali, Ege T


    Ca(2+)-dependent synaptic vesicle recycling is essential for structural homeostasis of synapses and maintenance of neurotransmission. Although, the executive role of intrasynaptic Ca(2+) transients in synaptic vesicle exocytosis is well established, identifying the exact role of Ca(2+) in endocytosis has been difficult. In some studies, Ca(2+) has been suggested as an essential trigger required to initiate synaptic vesicle retrieval, whereas others manipulating synaptic Ca(2+) concentrations reported a modulatory role for Ca(2+) leading to inhibition or acceleration of endocytosis. Molecular studies of synaptic vesicle endocytosis, on the other hand, have consistently focused on the roles of Ca(2+)-calmodulin dependent phosphatase calcineurin and synaptic vesicle protein synaptotagmin as potential Ca(2+) sensors for endocytosis. Most studies probing the role of Ca(2+) in endocytosis have relied on measurements of synaptic vesicle retrieval after strong stimulation. Strong stimulation paradigms elicit fusion and retrieval of multiple synaptic vesicles and therefore can be affected by several factors besides the kinetics and duration of Ca(2+) signals that include the number of exocytosed vesicles and accumulation of released neurotransmitters thus altering fusion and retrieval processes indirectly via retrograde signaling. Studies monitoring single synaptic vesicle endocytosis may help resolve this conundrum as in these settings the impact of Ca(2+) on synaptic fusion probability can be uncoupled from its putative role on synaptic vesicle retrieval. Future experiments using these single vesicle approaches will help dissect the specific role(s) of Ca(2+) and its sensors in synaptic vesicle endocytosis. © The Author(s) 2015.

  9. Ca2+ homeostasis in microvascular endothelial cells from an insulin-dependent diabetic model: role of endosomes/lysosomes (United States)

    Sanka, Shankar C.; Bennett, David C.; Rojas, Jose D.; Tasby, Geraldine B.; Meininger, Cynthia J.; Wu, Guoyao; Wesson, Donald E.; Pfarr, Curtis M.; Martinez-Zaguilan, Raul


    Cytosolic Ca2+ ([Ca2+]cyt) regulates several cellular functions, e.g. cell growth, contraction, secretion, etc. In many cell types, ion homeostasis appears to be coupled with glucose metabolism. In certain cell types, a strict coupling between glycolysis and the activity of Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPases (SERCA) has been suggested. Glucose metabolism is altered in diabetes. We hypothesize that: (1) Ca2+ homeostasis is altered in microvascular endothelial cells from diabetic animals due to the dysfunction of glycolysis coupling the activity of SERCA; (2) endosomal/lysosomal compartments expressing SERCA are involved in the dysfunction associated with diabetes.

  10. Alternative splicing alterations of Ca2+ handling genes are associated with Ca2+ signal dysregulation in myotonic dystrophy type 1 (DM1) and type 2 (DM2) myotubes. (United States)

    Santoro, Massimo; Piacentini, Roberto; Masciullo, Marcella; Bianchi, Maria Laura Ester; Modoni, Anna; Podda, Maria Vittoria; Ricci, Enzo; Silvestri, Gabriella; Grassi, Claudio


    The pathogenesis of myotonic dystrophy type 1 (DM1) and type 2 (DM2) has been related to the aberrant splicing of several genes, including those encoding for ryanodine receptor 1 (RYR1), sarcoplasmatic/endoplasmatic Ca(2+)-ATPase (SERCA) and α1S subunit of voltage-gated Ca(2+) channels (Cav 1.1). The aim of this study is to determine whether alterations of these genes are associated with changes in the regulation of intracellular Ca(2+) homeostasis and signalling. We analysed the expression of RYR1, SERCA and Cav 1.1 and the intracellular Ca(2+) handling in cultured myotubes isolated from DM1, DM2 and control muscle biopsies by semiquantitative RT-PCR and confocal Ca(2+) imaging respectively. (i) The alternative splicing of RYR1, SERCA and Cav 1.1 was more severely affected in DM1 than in DM2 myotubes; (ii) DM1 myotubes exhibited higher resting intracellular Ca(2+) levels than DM2; (iii) the amplitude of intracellular Ca(2+) transients induced by sustained membrane depolarization was higher in DM1 myotubes than in controls, whereas DM2 showed opposite behaviour; and (iv) in both DM myotubes, Ca(2+) release from sarcoplasmic reticulum through RYR1 was lower than in controls. The aberrant splicing of RYR1, SERCA1 and Cav 1.1 may alter intracellular Ca(2+) signalling in DM1 and DM2 myotubes. The differing dysregulation of intracellular Ca(2+) handling in DM1 and DM2 may explain their distinct sarcolemmal hyperexcitabilities. © 2013 British Neuropathological Society.

  11. Multiple Ca2+ sensors in secretion

    DEFF Research Database (Denmark)

    Walter, Alexander M; Groffen, Alexander J; Sørensen, Jakob Balslev


    Regulated neurotransmitter secretion depends on Ca(2+) sensors, C2 domain proteins that associate with phospholipids and soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) complexes to trigger release upon Ca(2+) binding. Ca(2+) sensors are thought to prevent spontaneous...... fusion at rest (clamping) and to promote fusion upon Ca(2+) activation. At least eight, often coexpressed, Ca(2+) sensors have been identified in mammals. Accumulating evidence suggests that multiple Ca(2+) sensors interact, rather than work autonomously, to produce the complex secretory response...... observed in neurons and secretory cells. In this review, we present several working models to describe how different sensors might be arranged to mediate synchronous, asynchronous and spontaneous neurotransmitter release. We discuss the scenario that different Ca(2+) sensors typically act on one shared...

  12. Atrial myocyte function and Ca2+ handling is associated with inborn aerobic capacity.

    Directory of Open Access Journals (Sweden)

    Anne Berit Johnsen

    Full Text Available Although high aerobic capacity is associated with effective cardiac function, the effect of aerobic capacity on atrial function, especially in terms of cellular mechanisms, is not known. We aimed to investigate whether rats with low inborn maximal oxygen uptake (VO2 max had impaired atrial myocyte contractile function when compared to rats with high inborn VO2 max.Atrial myocyte function was depressed in Low Capacity Runners (LCR relative to High Capacity Runners (HCR which was associated with impaired Ca(2+ handling. Fractional shortening was 52% lower at 2 Hz and 60% lower at 5 Hz stimulation while time to 50% relengthening was 43% prolonged and 55% prolonged, respectively. Differences in Ca(2+ amplitude and diastolic Ca(2+ level were observed at 5 Hz stimulation where Ca(2+ amplitude was 70% lower and diastolic Ca(2+ level was 11% higher in LCR rats. Prolonged time to 50% Ca(2+ decay was associated with reduced sarcoplasmic reticulum (SR Ca(2+ ATPase function in LCR (39%. Na(+/Ca(2+ exchanger activity was comparable between the groups. Diastolic SR Ca(2+ leak was increased by 109%. This could be partly explained by increased ryanodine receptors phosphorylation at the Ca(2+-calmodulin-dependent protein kinase-II specific Ser-2814 site in LCR rats. T-tubules were present in 68% of HCR cells whereas only 33% LCR cells had these structures. In HCR, the significantly higher numbers of cells with T-tubules were combined with greater numbers of myocytes where Ca(2+ release in the cell occurred simultaneously in central and peripheral regions, giving rise to faster and more spatial homogenous Ca(2+-signal onset.This data demonstrates that contrasting for low or high aerobic capacity leads to diverse functional and structural remodelling of atrial myocytes, with impaired contractile function in LCR compared to HCR rats.

  13. Mg2+-dependent gating of bacterial MgtE channel underlies Mg2+ homeostasis (United States)

    Hattori, Motoyuki; Iwase, Norihiko; Furuya, Noritaka; Tanaka, Yoshiki; Tsukazaki, Tomoya; Ishitani, Ryuichiro; Maguire, Michael E; Ito, Koichi; Maturana, Andres; Nureki, Osamu


    The MgtE family of Mg2+ transporters is ubiquitously distributed in all phylogenetic domains. Recent crystal structures of the full-length MgtE and of its cytosolic domain in the presence and absence of Mg2+ suggested a Mg2+-homeostasis mechanism, in which the MgtE cytosolic domain acts as a ‘Mg2+ sensor' to regulate the gating of the ion-conducting pore in response to the intracellular Mg2+ concentration. However, complementary functional analyses to confirm the proposed model have been lacking. Moreover, the limited resolution of the full-length structure precluded an unambiguous characterization of these regulatory divalent-cation-binding sites. Here, we showed that MgtE is a highly Mg2+-selective channel gated by Mg2+ and elucidated the Mg2+-dependent gating mechanism of MgtE, using X-ray crystallographic, genetic, biochemical, and electrophysiological analyses. These structural and functional results have clarified the control of Mg2+ homeostasis through cooperative Mg2+ binding to the MgtE cytosolic domain. PMID:19798051

  14. Ca2+ homeostasis regulates Xenopus oocyte maturation. (United States)

    Sun, Lu; Hodeify, Rawad; Haun, Shirley; Charlesworth, Amanda; MacNicol, Angus M; Ponnappan, Subramaniam; Ponnappan, Usha; Prigent, Claude; Machaca, Khaled


    In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte's competence to undergo maturation in preparation for fertilization and embryonic development.

  15. Temporal monitoring of intracellular Ca2+ signaling and origins of Ca2+ oscillations


    Webb, Dominic-Luc


    This thesis examined parameters influencing stimulated cytoplasmic free Ca2+ concentration ([Ca2+]i) oscillations in hepatocytes and pancreatic beta-cells. Hepatic glucose output is regulated in part by hormones such as vasopressin that act through [Ca 2+]i oscillations. Pulsatile [Ca2+]i in beta-cells parallels insulin secretion and this results in potently controlled blood glucose homeostasis. Employing temporal [Ca2+]i measurements and related biochemical assays, efforts ...

  16. Cell Biology of Ca2+-Triggered Exocytosis


    Pang, Zhiping P.; Südhof, Thomas C.


    Ca2+ triggers many forms of exocytosis in different types of eukaryotic cells, for example synaptic vesicle exocytosis in neurons, granule exocytosis in mast cells, and hormone exocytosis in endocrine cells. Work over the last two decades has shown that synaptotagmins function as the primary Ca2+-sensors for most of these forms of exocytosis, and that synaptotagmins act via Ca2+-dependent interactions with both the fusing phospholipid membranes and the membrane fusion machinery. However, some...

  17. Characterization of cholera toxin B subunit-induced Ca(2+) influx in neuroblastoma cells: evidence for a voltage-independent GM1 ganglioside-associated Ca(2+) channel. (United States)

    Fang, Yu; Xie, Xin; Ledeen, Robert W; Wu, Gusheng


    The role of endogenous GM1 ganglioside in neurite outgrowth has been studied in N18 and NG108-15 neuroblastoma cells with the GM1-specific ligand cholera toxin B subunit (Ctx B), which stimulates Ca(2+) influx together with neuritogenesis. Our primary goal has been to identify the nature of the calcium channel that is modulated by GM1. An L-type voltage-operated Ca(2+) channel (VOCC) was previously proposed as the mediator of this phenomenon. This investigation, employing fura-2 fluorescent measurements and specific channel blockers and other agents, revealed that GM1 modulates a hitherto unidentified Ca(2+) channel not of the L type. It was opened by Ctx B; was permeable to Ca(2+) and Ba(2+) but not Mn(2+); and was blocked by Ni(2+), Cd(2+), and La(3+). Although most dihydropyridines inhibited Ctx B-induced Ca(2+) influx as well as neurite outgrowth at higher concentrations, they and other VOCC blockers at normally employed concentrations failed to do so, suggesting uninvolvement of VOCC. In addition, Ca(2+) influx induced by Ctx B was not mediated by cGMP-dependent or G-protein-coupled nonselective cation channels, as demonstrated by the cGMP antagonist Rp-cGMPS or the G-protein/receptor uncoupling agent suramin, respectively. Finally, Ca(2+) influx was unlikely to be due to inhibition or reversal of Na(+)-Ca(2+) exchanger via Ctx B induction of Na(+) uptake, insofar as no effect was seen on blocking Na(+) channels, inhibiting Na(+)-K(+)-ATPase, or eliminating extracellular Na(+). The results suggest that this novel channel is gated by interaction with GM1, which, when associated with the channel and bound by appropriate ligand, promotes Ca(2+) influx. This in turn induces signaling for the onset of neuritogenesis. Copyright 2002 Wiley-Liss, Inc.

  18. Sperm parameters and epididymis function in transgenic rats overexpressing the Ca2+-binding protein regucalcin: a hidden role for Ca2+ in sperm maturation? (United States)

    Correia, S; Oliveira, P F; Guerreiro, P M; Lopes, G; Alves, M G; Canário, A V M; Cavaco, J E; Socorro, Sílvia


    Sperm undergo maturation acquiring progressive motility and the ability to fertilize oocytes through exposure to the components of the epididymal fluid (EF). Although the establishment of a calcium (Ca(2+)) gradient along the epididymis has been described, its direct effects on epididymal function remain poorly explored. Regucalcin (RGN) is a Ca(2+)-binding protein, regulating the activity of Ca(2+)-channels and Ca(2+)-ATPase, for which a role in male reproductive function has been suggested. This study aimed at comparing the morphology, assessed by histological analysis, and function of epididymis, by analysis of sperm parameters, antioxidant potential and Ca(2+) fluxes, between transgenic rats overexpressing RGN (Tg-RGN) and their wild-type littermates. Tg-RGN animals displayed an altered morphology of epididymis and lower sperm counts and motility. Tissue incubation with (45)Ca(2+) showed also that epididymis of Tg-RGN displayed a diminished rate of Ca(2+)-influx, indicating unbalanced Ca(2+) concentrations in the epididymal lumen. Sperm viability and the frequency of normal sperm, determined by the one-step eosin-nigrosin staining technique and the Diff-Quik staining method, respectively, were higher in Tg-RGN. Moreover, sperm of Tg-RGN rats showed a diminished incidence of tail defects. Western blot analysis demonstrated the presence of RGN in EF as well as its higher expression in the corpus region. The results presented herein demonstrated the importance of maintaining Ca(2+)-levels in the epididymal lumen and suggest a role for RGN in sperm maturation. Overall, a new insight into the molecular mechanisms driving epididymal sperm maturation was obtained, which could be relevant to development of better approaches in male infertility treatment and contraception.

  19. SERCA pump level is a critical determinant of Ca(2+)homeostasis and cardiac contractility. (United States)

    Periasamy, M; Huke, S


    The control of intracellular calcium is central to regulation of cardiac contractility. A defect in SR Ca(2+)transport and SR Ca(2+)ATPase pump activity and expression level has been implicated as a major player in cardiac dysfunction. However, a precise cause-effect relationship between alterations in SERCA pump level and cardiac contractility could not be established from these studies. Progress in transgenic mouse technology and adenoviral gene transfer has provided new tools to investigate the role of SERCA pump level in the heart. This review focuses on how alterations in SERCA level affect Ca(2+)homeostasis and cardiac contractility. It discusses the consequences of altered SERCA pump levels for the expression and activity of other Ca(2+)handling proteins. Furthermore, the use of SERCA pump as a therapeutic target for gene therapy of heart failure is evaluated. Copyright 2001 Academic Press.

  20. Proton-gated Ca(2+)-permeable TRP channels damage myelin in conditions mimicking ischaemia. (United States)

    Hamilton, Nicola B; Kolodziejczyk, Karolina; Kougioumtzidou, Eleni; Attwell, David


    The myelin sheaths wrapped around axons by oligodendrocytes are crucial for brain function. In ischaemia myelin is damaged in a Ca(2+)-dependent manner, abolishing action potential propagation. This has been attributed to glutamate release activating Ca(2+)-permeable N-methyl-D-aspartate (NMDA) receptors. Surprisingly, we now show that NMDA does not raise the intracellular Ca(2+) concentration ([Ca(2+)]i) in mature oligodendrocytes and that, although ischaemia evokes a glutamate-triggered membrane current, this is generated by a rise of extracellular [K(+)] and decrease of membrane K(+) conductance. Nevertheless, ischaemia raises oligodendrocyte [Ca(2+)]i, [Mg(2+)]i and [H(+)]i, and buffering intracellular pH reduces the [Ca(2+)]i and [Mg(2+)]i increases, showing that these are evoked by the rise of [H(+)]i. The H(+)-gated [Ca(2+)]i elevation is mediated by channels with characteristics of TRPA1, being inhibited by ruthenium red, isopentenyl pyrophosphate, HC-030031, A967079 or TRPA1 knockout. TRPA1 block reduces myelin damage in ischaemia. These data suggest that TRPA1-containing ion channels could be a therapeutic target in white matter ischaemia.

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

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    Glushakova Svetlana


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

  2. Rotary ATPases (United States)

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


    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. A Transient Rise in Free Mg2+Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation. (United States)

    Maeshima, Kazuhiro; Matsuda, Tomoki; Shindo, Yutaka; Imamura, Hiromi; Tamura, Sachiko; Imai, Ryosuke; Kawakami, Syoji; Nagashima, Ryosuke; Soga, Tomoyoshi; Noji, Hiroyuki; Oka, Kotaro; Nagai, Takeharu


    For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1-5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6-17], free divalent cations such as Mg 2+ and Ca 2+ , which condense chromatin or chromosomes in vitro [18-28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like "beads on a string" by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg 2+ , have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg 2+ indicator that monitors free Mg 2+ dynamics throughout the cell cycle. By combining this indicator with Ca 2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg 2+ , but not Ca 2+ , increase during mitosis. The Mg 2+ increase is coupled with a decrease in ATP, which is normally bound to Mg 2+ in the cell [33]. ATP inhibited Mg 2+ -dependent chromatin condensation in vitro. Chelating Mg 2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg 2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg 2+ -ATP balance. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  4. Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2+ level in SH-SY5Y cells (United States)

    Chen, Yi-ching; Jinn, Tzyy-rong; Chung, Tse-yu; Li, Feng-yin; Fan, Ruey-jane; Tzen, Jason TC


    Aim: To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na+/K+-ATPase, leads to the elevation of intracellular Ca2+ level as observed in cells treated with cardiac glycosides. Methods: Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca2+ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na+/Ca2+ exchanger inhibitor) and 2-APB (IP3 receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na+/K+-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope. Results: severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca2+ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na+/K+-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells. Conclusion: Comparable to ouabain, MLB leads to the elevation of intracellular Ca2+ level presumably via the same mechanism by inhibiting Na+/K+-ATPase. The elevated Ca2+ levels seem to be supplied by Ca2+ influx through the reversed mode of the Na+/Ca2+ exchanger and intracellular release from endoplasmic reticulum. PMID:20686517

  5. Magnesium lithospermate B extracted from Salvia miltiorrhiza elevates intracellular Ca(2+) level in SH-SY5Y cells. (United States)

    Chen, Yi-Ching; Jinn, Tzyy-Rong; Chung, Tse-Yu; Li, Feng-Yin; Fan, Ruey-Jane; Tzen, Jason Tc


    To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na(+)/K(+)-ATPase, leads to the elevation of intracellular Ca(2+) level as observed in cells treated with cardiac glycosides. Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca(2+) levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na(+)/Ca(2+) exchanger inhibitor) and 2-APB (IP(3) receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na(+)/K(+)-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope. severe toxicity was observed in cells treated with ouabain of concentration higher than 1 micromol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca(2+) levels were substantially elevated by MLB (1 micromol/L) and ouabain (1 micromol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 micromol/L) or 2-APB (100 micromol/L). Equivalent interaction with the binding cavity of Na(+)/K(+)-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 micromol/L), but not MLB (1 mumol/L), induced dendritic shrink of SH-SY5Y cells. Comparable to ouabain, MLB leads to the elevation of intracellular Ca(2+) level presumably via the same mechanism by inhibiting Na(+)/K(+)-ATPase. The elevated Ca(2+) levels seem to be supplied by Ca(2+) influx through the reversed mode of the Na(+)/Ca(2+) exchanger and intracellular release from endoplasmic reticulum.

  6. When Isolated at Full Receptivity, in Vitro Fertilized Wheat (Triticum aestivum, L. Egg Cells Reveal [Ca2+]cyt Oscillation of Intracellular Origin

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    Zsolt Pónya


    Full Text Available During in vitro fertilization of wheat (Triticum aestivum, L. in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca2+]cyt were observed. The dynamics of [Ca2+]cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca2+]cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca2+]cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER Ca2+-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca2+]cyt in the absence of extracellular Ca2+, suggesting that an influx pathway for Ca2+ is activated by thapsigargin. The [Ca2+]cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl3 to the fusion medium, which prevented [Ca2+]cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca2+]cyt depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and this enhanced Ca2+ influx results in [Ca2+]cyt oscillation.

  7. SERCA and PMCA pumps contribute to the deregulation of Ca2+ homeostasis in human CF epithelial cells. (United States)

    Philippe, Réginald; Antigny, Fabrice; Buscaglia, Paul; Norez, Caroline; Becq, Frédéric; Frieden, Maud; Mignen, Olivier


    Cystic Fibrosis (CF) disease is caused by mutations in the CFTR gene (CF transmembrane conductance regulator). F508 deletion is the most represented mutation, and F508del-CFTR is absent of plasma membrane and accumulates into the endoplasmic reticulum (ER) compartment. Using specific Ca2+ genetics cameleon probes, we showed in the human bronchial CF epithelial cell line CFBE that ER Ca2+ concentration was strongly increased compared to non-CF (16HBE) cells, and normalized by the F508del-CFTR corrector agent, VX-809. We also showed that ER F508del-CFTR retention increases SERCA (Sarcoplasmic/Reticulum Ca2+ ATPase) pump activity whereas PMCA (Plasma Membrane Ca2+ ATPase) activities were reduced in these CF cells compared to corrected CF cells (VX-809) and non-CF cells. We are showing for the first time CFTR/SERCA and CFTR/PMCA interactions that are modulated in CF cells and could explain part of Ca2+ homeostasis deregulation due to mislocalization of F508del-CFTR. Using ER or mitochondria genetics Ca2+ probes, we are showing that ER Ca2+ content, mitochondrial Ca2+ uptake, SERCA and PMCA pump, activities are strongly affected by the localization of F508del-CFTR protein. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Ca(2+) signalling in the Golgi apparatus. (United States)

    Pizzo, Paola; Lissandron, Valentina; Capitanio, Paola; Pozzan, Tullio


    The Golgi apparatus plays a central role in lipid and protein post-translational modification and sorting. Morphologically the organelle is heterogeneous and it is possible to distinguish stacks of flat cysternae (cis- and medial Golgi), tubular-reticular networks and vesicles (trans-Golgi). These morphological differences parallel a distinct functionality with a selective distribution and complementary roles of the enzymes found in the different compartments. The Golgi apparatus has been also shown to be involved in Ca(2+) signalling: it is indeed endowed with Ca(2+) pumps, Ca(2+) release channels and Ca(2+) binding proteins and is thought to participate in determining the spatio-temporal complexity of the Ca(2+) signal within the cell, though this role is still poorly understood. Recently, it has been demonstrated that the organelle is heterogeneous in terms of Ca(2+) handling and selective reduction of Ca(2+) concentration, both in vitro and in a genetic human disease, within one of its sub-compartment results in alterations of protein trafficking within the secretory pathway and of the entire Golgi morphology. In this paper we review the available information on the Ca(2+) toolkit within the Golgi, its heterogeneous distribution in the organelle sub-compartments and discuss the implications of these characteristics for the physiopathology of the Golgi apparatus. 2011 Elsevier Ltd. All rights reserved.

  9. Changes in taste receptor cell [Ca2+]i modulate chorda tympani responses to bitter, sweet, and umami taste stimuli. (United States)

    Desimone, John A; Phan, Tam-Hao T; Ren, Zuojun; Mummalaneni, Shobha; Lyall, Vijay


    The relationship between taste receptor cell (TRC) intracellular Ca(2+) ([Ca(2+)](i)) and rat chorda tympani (CT) nerve responses to bitter (quinine and denatonium), sweet (sucrose, glycine, and erythritol), and umami [monosodium glutamate (MSG) and MSG + inosine 5'-monophosphate (IMP)] taste stimuli was investigated before and after lingual application of ionomycin (Ca(2+) ionophore) + Ca(2+), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; Ca(2+) chelator), U73122 (phospholipase C blocker), thapsigargin (Ca(2+)-ATPase blocker), and diC8-PIP(2) (synthetic phosphatidylinositol 4,5-bisphosphate). The phasic CT response to quinine was indifferent to changes in [Ca(2+)](i). However, a decrease in [Ca(2+)](i) inhibited the tonic part of the CT response to quinine. The CT responses to sweet and umami stimuli were indifferent to changes in TRC [Ca(2+)](i). However, a decrease in [Ca(2+)](i) attenuated the synergistic effects of ethanol on the CT response to sweet stimuli and of IMP on the glutamate CT response. U73122 and thapsigargin inhibited the phasic and tonic CT responses to bitter, sweet, and umami stimuli. Although diC8-PIP(2) increased the CT response to bitter and sweet stimuli, it did not alter the CT response to glutamate but did inhibit the synergistic effect of IMP on the glutamate response. The results suggest that bitter, sweet, and umami taste qualities are transduced by [Ca(2+)](i)-dependent and [Ca(2+)](i)-independent mechanisms. Changes in TRC [Ca(2+)](i) in the BAPTA-sensitive cytosolic compartment regulate quality-specific taste receptors and ion channels that are involved in the neural adaptation and mixture interactions. Changes in TRC [Ca(2+)](i) in a separate subcompartment, sensitive to inositol trisphosphate and thapsigargin but inaccessible to BAPTA and ionomycin + Ca(2+), are associated with neurotransmitter release.

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

    DEFF Research Database (Denmark)

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


    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 at the cat...... nucleotide analogs in the E2·VO3− structure with that in E2·BeF3− (E2P ground state analog) reveals multiple binding modes to the Ca2+-ATPase....

  11. Post mortem changes in Ca2+ transporting proteins of sarcoplasmic reticulum in dependence on malignant hyperthermia status in pigs. (United States)

    Küchenmeister, U; Kuhn, G; Wegner, J; Nürnberg, G; Ender, K


    Meat quality of pigs is dependent on biochemical and biophysical processes in the time course post mortem (p.m.) and is associated with the intracellular Ca2+ homeostasis. However, there is little known about changes in the Ca2+ transporting proteins controlling the Ca2+ uptake of sarcoplasmic reticulum (SR) in the time course p.m. In this study changes in the Ca2+ transporting proteins were investigated in homogenates of longissimus muscles of 4 malignant hyperthermia susceptible (MHS) and 6 malignant hyperthermia resistant (MHR) Pietrain pigs. Muscle samples were obtained at different time intervals: biopsy 2 h prior slaughtering and from the carcass immediately after exsanguination (0 h), 45 min, 4 h, and 22 h p.m. The SR Ca2+ uptake rate was measured immediately after homogenization with closed calcium release channel (CRC), with opened CRC and without manipulation of CRC. Additionally the SR Ca2+ ATPase activity was determined. The results show: (i) The ability of SR to sequester Ca2+ declined to about 60% in the first 45 min p.m. in MHS samples irrespective of CRC state, whereas in MHR samples this decline was about 5%; (ii) Ca2+ uptake and Ca2+ ATPase activity were not different between the biopsy and 0 h samples, i.e. the stress of slaughter was of no immediate influence; (iii) The Ca2+ ATPase activity of the SR declined at about the same rate as the Ca2+ uptake in both MHS and MHR pig samples in the course of time p.m.; (iv) In samples, taken immediately after exsanguination, the Ca2+ ATPase activity of MHS pigs was higher than that of MHR pigs. However, in samples taken 4 h p.m. Ca2+ ATPase activity of MHS pigs has declined to about 30% of the value at 0 h; (v) The CRC can be closed and opened in all samples up to 22 h p.m. and seems to be fully functional at all sampling times; (vi) The CRC of MHS pigs is almost fully open, whereas the CRC of MHR pigs is only partially open at all sampling times; (vii) The permeability of the SR membrane to Ca2

  12. Calcium-ATPases: Gene disorders and dysregulation in cancer. (United States)

    Dang, Donna; Rao, Rajini


    Ca(2+)-ATPases belonging to the superfamily of P-type pumps play an important role in maintaining low, nanomolar cytoplasmic Ca(2+) levels at rest and priming organellar stores, including the endoplasmic reticulum, Golgi, and secretory vesicles with high levels of Ca(2+) for a wide range of signaling functions. In this review, we introduce the distinct subtypes of Ca(2+)-ATPases and their isoforms and splice variants and provide an overview of their specific cellular roles as they relate to genetic disorders and cancer, with a particular emphasis on recent findings on the secretory pathway Ca(2+)-ATPases (SPCA). Mutations in human ATP2A2, ATP2C1 genes, encoding housekeeping isoforms of the endoplasmic reticulum (SERCA2) and secretory pathway (SPCA1) pumps, respectively, confer autosomal dominant disorders of the skin, whereas mutations in other isoforms underlie various muscular, neurological, or developmental disorders. Emerging evidence points to an important function of dysregulated Ca(2+)-ATPase expression in cancers of the colon, lung, and breast where they may serve as markers of differentiation or novel targets for therapeutic intervention. We review the mechanisms underlying the link between calcium homeostasis and cancer and discuss the potential clinical relevance of these observations. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. A link of Ca2+ to cAMP oscillations in Dictyostelium: the calmodulin antagonist W-7 potentiates cAMP relay and transiently inhibits the acidic Ca2+-store

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    Malchow Dieter


    Full Text Available Abstract Background During early differentiation of Dictyostelium the attractant cAMP is released periodically to induce aggregation of the cells. Here we pursue the question whether pulsatile cAMP signaling is coupled to a basic Ca2+-oscillation. Results We found that the calmodulin antagonist W-7 transiently enhanced cAMP spikes. We show that W-7 acts on an acidic Ca2+-store: it abolished ATP-dependent vesicular acidification, inhibited V-type H+ATPase activity more potently than the weaker antagonist W-5 and caused vesicular Ca2+-leakage. Concanamycin A, an inhibitor of the V-type H+-pump, blocked the Ca2+-leakage elicited by W-7 as well as cAMP-oscillations in the presence of W-7. Concanamycin A caused an increase of the cytosolic Ca2+-concentration whereas W-7 did not. In case of the latter, Ca2+ was secreted by the cells. In accord with our hypothesis that the link from Ca2+ to cAMP synthesis is mediated by a Ca2+-dependent phospholipase C we found that W-7 was not active in the phospholipase C knockout mutant. Conclusion We conclude that the potentiation of cAMP relay by W-7 is due to a transient inhibition of the acidic Ca2+-store. The inhibition of the proton pump by W-7 causes a leakage of Ca2+ that indirectly stimulates adenylyl cyclase activity via phospholipase C.

  14. Functional characterization and Me2+ ion specificity of a Ca2+-citrate transporter from Enterococcus faecalis

    NARCIS (Netherlands)

    Blancato, Victor S.; Magni, Christian; Lolkema, Juke S.


    Secondary transporters of the bacterial CitMHS family transport citrate in complex with a metal ion. Different members of the family are specific for the metal ion in the complex and have been shown to transport Mg2+-citrate, Ca2+-citrate or Fe3+-citrate. The Fe3+-citrate transporter of

  15. Effect of preparations Methyure and Ivine on Са(2+)-ATPases activity in plasma and vacuolar membrane of corn seedling roots under salt stress conditions. (United States)

    Rudnytska, M V; Palladina, T A


    Ca2+-ATPases regulate the functioning of Ca2+-dependent signaling pathway SOS which provides removal of Na+ from the cytoplasm of cells via Na+/H+-antiporters in saline conditions. The influence of synthetic preparations Methyure and Ivine on the Ca2+-ATPase activity was investigated. It was shown that exposition of corn seedlings in the presence of 0.1 M NaCl rather enhanced hydrolytic than transport activity of Ca2+-ATPases in plasma and vacuolar membrane of root cells. It was found that seed treatment with such preparations, especially Methyure, caused intensification of the both activities of Ca2+-ATPases, mainly in vacuolar membrane. The results indicate than salt protective activity of preparations, especially Methyure, is associated with increased Ca2+-ATPase activity, which regulates the functioning of Na+/H+-antiporters.

  16. Juvenile Hippocampal CA2 Region Expresses Aggrecan

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    Asako Noguchi


    Full Text Available Perineuronal nets (PNNs are distributed primarily around inhibitory interneurons in the hippocampus, such as parvalbumin-positive interneurons. PNNs are also present around excitatory neurons in some brain regions and prevent plasticity in these neurons. A recent study demonstrated that PNNs also exist around mouse hippocampal pyramidal cells, which are the principle type of excitatory neurons, in the CA2 subregion and modulate the excitability and plasticity of these neurons. However, the development of PNNs in the CA2 region during postnatal maturation was not fully investigated. This study found that a main component of PNNs, aggrecan, existed in the pyramidal cell layer of the putative CA2 subarea prior to the appearance of the CA2 region, which was defined by the CA2 marker protein regulator of G protein signaling 14 (RGS14. We also found that aggrecan immunoreactivity was more evident in the anterior sections of the CA2 area than the posterior sections, which suggests that the function of CA2 PNNs varies along the anterior-posterior axis.

  17. Preparation of pure and intact Plasmodium falciparum plasma membrane vesicles and partial characterisation of the plasma membrane ATPase

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    Smith Pete J


    Full Text Available Abstract Background In host erythrocytes, the malaria parasite must contend with ion and drug transport across three membranes; its own plasma membrane, the parasitophorous membrane and the host plasma membrane. Isolation of pure and intact Plasmodium falciparum plasma membrane would provide a suitable model to elucidate the possible role played by the parasite plasma membrane in ion balance and drug transport. Results This study describes a procedure for isolating parasite plasma membrane from P. falciparum-infected erythrocytes. With this method, the trophozoites released by saponin treatment were cleansed of erythrocyte membranes using anti-erythrocyte antibodies fixed to polystyrene beads. These trophozoites were then biotinylated and the parasite plasma membrane was disrupted by nitrogen cavitation. This process allows the membranes to reform into vesicles. The magnetic streptavidin beads bind specifically to the biotinylated parasite plasma membrane vesicles facilitating their recovery with a magnet. These vesicles can then be easily released from the magnetic beads by treatment with dithiotreithol. The parasite plasma membrane showed optimal ATPase activity at 2 mM ATP and 2 mM Mg2+. It was also found that Ca2+ could not substitute for Mg2+ ATPase activity in parasite plasma membranes whereas activity was completely preserved when Mn2+ was used instead of Mg2+. Other nucleoside triphosphates tested were hydrolysed as efficiently as ATP, while the nucleoside monophosphate AMP was not. Conclusions We have described the successful isolation of intact P. falciparum plasma membrane vesicles free of contaminating organelles and determined the experimental conditions for optimum ATPase activity.

  18. [Effect of polyamines on Mg(2+)-dependent ATP-hydrolase activity in plasmatic membrane of myometrium cells]. (United States)

    Veklich, T O; Shkrabak, O A; Kosterin, S O


    Influence of aliphatic polyamines of spermine and spermidine on the enzymatic activity of the ouabain-sensitive Na+,K(+)-ATPase and the ouabain-resistant basal Mg(2+)-ATPase (specific activity--10.6 +/- 0.9 and 18.1 +/- 1.2 microM P(i)/hour on 1 mg of protein accordingly, n = 7) has been studied in the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution. It was found, that the polyamine spermine in concentration of 1 and 10 mM activated the Na+,K(+)-ATPase by 54 and 64% on the average relative to control value. Spermidine also stimulated the Na+,K(+)-ATPase activity, however it did it less efficiently than spermine: by 8 and 20% on the average at concentration of 1 and 10 mM, accordingly. Similarly, polyamines had affect on the basal Mg(2+)-ATPase: spermine in concentration of 1 and 10 mM activated it by 26 and 39% relative to control value; spermidine in concentration of 1 and 10 mM activated it by 10 and 32% relative to control. The magnitudes of the apparent activation constant K(a) of spermine were 0.35 +/- 0.07 and 0.10 +/- 0.02 mM for Na+,K(+)-ATPase and basal Mg(2+)-ATPase, accordingly (M +/- m, n = 5). It is supposed, that the obtained experimental data can be useful in the further research of the membrane mechanisms underlying of the cationic exchange in the smooth muscles, in particular, when investigating the role of the plasmatic membrane in providing electromechanical coupling in them, and also in regulation of ionic homeostasis in the smooth muscle cells.

  19. Modulation of sarcoplasmic reticulum Ca2+ release by glycolysis in cat atrial myocytes. (United States)

    Kockskämper, Jens; Zima, Aleksey V; Blatter, Lothar A


    In cardiac myocytes, glycolysis and excitation-contraction (E-C) coupling are functionally coupled. We studied the effects of inhibitors (2-deoxy-D-glucose (2-DG), iodoacetate (IAA)), intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), phosphoenolpyruvate (PEP)) and products (pyruvate, L-lactate) of glycolysis on sarcoplasmic reticulum (SR) Ca(2+) release and uptake in intact and permeabilized cat atrial myocytes. In field-stimulated (0.5-0.7 Hz) intact myocytes, 2-DG (10 mm) and IAA (1 mm) caused elevation of diastolic [Ca(2+)](i) and [Ca(2+)](i) transient alternans (Ca(2+) alternans) followed by a decrease of the amplitude of the [Ca(2+)](i) transient. Focal application of 2-DG resulted in local Ca(2+) alternans that was confined to the region of exposure. 2-DG and IAA slowed the decay kinetics of the [Ca(2+)](i) transient and delayed its recovery (positive staircase) after complete SR depletion, suggesting impaired activity of the SR Ca(2+)-ATPase (SERCA). 2-DG and IAA reduced the rate of reuptake of Ca(2+) into the SR which was accompanied by a 15-20% decrease of SR Ca(2+) load. Major changes of mitochondrial redox state (measured as FAD autofluorescence) were not observed after inhibition of glycolysis. Pyruvate (10 mm) and L-lactate (10 mm) elicited similar changes of the [Ca(2+)](i) transient. Pyruvate, L-lactate and IAA - but not 2-DG - induced intracellular acidosis. Recording of single channel activity of ryanodine receptors (RyRs) incorporated into lipid bilayers revealed complex modulation by glycolytic intermediates and products (1 mm each): some were without effect (G6P, PEP, L-lactate) while others either increased (F6P, +40%; FBP, +265%) or decreased (pyruvate, -58%) the open probability of the RyR. Consistent with these findings, spontaneous SR Ca(2+) release (Ca(2+) sparks) in permeabilized myocytes was facilitated by FBP and inhibited by pyruvate. The results indicate that in atrial myocytes

  20. HCl Treatment for Preventing Diapause Causes Ca2+ Efflux in Bombyx mori Eggs. (United States)

    Kitta, Ryo; Okawa, Sin-ichiro; Saito, Miho; Mase, Keisuke; Sawada, Hiroshi


    To elucidate the mechanism for preventing entry into embryonic diapause or breakdown of diapause in Bombyx mori by HCl and dimethyl sulfoxide (DMSO) treatment or a combination of cold and HCl treatment, we performed quantitative analysis of Ca2+ and Mg2+ in the chorion and egg content using inductively coupled plasma atomic emission spectrometry (ICP-AES). When diapause eggs that had been incubated at 25°C for 2 days from oviposition and at 4°C for an additional six days were treated with HCl solution, the amount of Ca2+ in the chorion and egg content after HCl treatment was reduced to one-seventh, as compared with the amount before treatment. In contrast, there was no change in the amount of Mg2+ with HCl treatment. The amount of Ca2+ in the HCl solution after the diapause eggs were treated increased 7.5-fold, as compared with that of eggs treated with water. Even when 17-day-old diapausing eggs were treated with HCl, which did not break diapause, the amount of Ca2+ in the chorion and egg content was reduced to one-fifth, as compared with the control. Meanwhile, changes in Ca2+ and Mg2+ contents were not observed in 12-hr-old diapause-destined eggs before or after treatment with DMSO, which effectively prevents diapause. These data may suggest that Ca2+ efflux from diapause eggs by HCl is not directly associated with preventing entry into diapause or breaking of diapause. In addition, we discovered that the amount of Ca2+ in diapause-destined eggs was more than 2.4-fold larger than in non-diapause-destined eggs.

  1. An increase in [Ca2+]i activates basolateral chloride channels and inhibits apical sodium channels in frog skin epithelium

    DEFF Research Database (Denmark)

    Brodin, Birger; Rytved, K A; Nielsen, R


    absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 microM) increased [Ca2+]i from 66 +/- 9 to 137 +/- 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride...

  2. Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger* (United States)

    Andrikopoulos, Petros; Kieswich, Julius; Harwood, Steven M.; Baba, Akemichi; Matsuda, Toshio; Barbeau, Olivier; Jones, Keith; Eccles, Suzanne A.; Yaqoob, Muhammad M.


    Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na+/Ca2+ exchanger (NCX) operating in the Ca2+-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca2+ influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na+-K+-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving

  3. NO-sGC Pathway Modulates Ca2+ Release and Muscle Contraction in Zebrafish Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Zhou Xiyuan


    Full Text Available Vertebrate skeletal muscle contraction and relaxation is a complex process that depends on Ca2+ ions to promote the interaction of actin and myosin. This process can be modulated by nitric oxide (NO, a gas molecule synthesized endogenously by (nitric oxide synthase NOS isoforms. At nanomolar concentrations NO activates soluble guanylate cyclase (sGC, which in turn activates protein kinase G via conversion of GTP into cyclic GMP. Alternatively, NO post-translationally modifies proteins via S-nitrosylation of the thiol group of cysteine. However, the mechanisms of action of NO on Ca2+ homeostasis during muscle contraction are not fully understood and we hypothesize that NO exerts its effects on Ca2+ homeostasis in skeletal muscles mainly through negative modulation of Ca2+ release and Ca2+ uptake via the NO-sGC-PKG pathway. To address this, we used 5–7 days-post fecundation-larvae of zebrafish, a well-established animal model for physiological and pathophysiological muscle activity. We evaluated the response of muscle contraction and Ca2+ transients in presence of SNAP, a NO-donor, or L-NAME, an unspecific NOS blocker in combination with specific blockers of key proteins of Ca2+ homeostasis. We also evaluate the expression of NOS in combination with dihydropteridine receptor, ryanodine receptor and sarco/endoplasmic reticulum Ca2+ ATPase. We concluded that endogenous NO reduced force production through negative modulation of Ca2+ transients via the NO-sGC pathway. This effect could be reversed using an unspecific NOS blocker or sGC blocker.

  4. Identification of calcium-transporting ATPases of Entamoeba histolytica and cellular localization of the putative SERCA. (United States)

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


    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. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling. (United States)

    Baumgart, Joel P; Zhou, Zhen-Yu; Hara, Masato; Cook, Daniel C; Hoppa, Michael B; Ryan, Timothy A; Hemmings, Hugh C


    Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca(2+) influx without significantly altering the Ca(2+) sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca(2+)]i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca(2+) ([Ca(2+)]e). Lowering external Ca(2+) to match the isoflurane-induced reduction in Ca(2+) entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca(2+) entry without significant direct effects on Ca(2+)-exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca(2+) influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system.

  6. A Model-Independent Algorithm to Derive Ca2+ Fluxes Underlying Local Cytosolic Ca2+ Transients (United States)

    Ventura, Alejandra C.; Bruno, Luciana; Demuro, Angelo; Parker, Ian; Ponce Dawson, Silvina


    Local intracellular Ca2+ signals result from Ca2+ flux into the cytosol through individual channels or clusters of channels. To gain a mechanistic understanding of these events we need to know the magnitude and spatial distribution of the underlying Ca2+ flux. However, this is difficult to infer from fluorescence Ca2+ images because the distribution of Ca2+-bound dye is affected by poorly characterized processes including diffusion of Ca2+ ions, their binding to mobile and immobile buffers, and sequestration by Ca2+ pumps. Several methods have previously been proposed to derive Ca2+ flux from fluorescence images, but all require explicit knowledge or assumptions regarding these processes. We now present a novel algorithm that requires few assumptions and is largely model-independent. By testing the algorithm with both numerically generated image data and experimental images of sparklets resulting from Ca2+ flux through individual voltage-gated channels, we show that it satisfactorily reconstructs the magnitude and time course of the underlying Ca2+ currents. PMID:15681645

  7. Ca2+ cycling in heart cells from ground squirrels: adaptive strategies for intracellular Ca2+ homeostasis.

    Directory of Open Access Journals (Sweden)

    Xiao-Chen Li

    Full Text Available Heart tissues from hibernating mammals, such as ground squirrels, are able to endure hypothermia, hypoxia and other extreme insulting factors that are fatal for human and nonhibernating mammals. This study was designed to understand adaptive mechanisms involved in intracellular Ca(2+ homeostasis in cardiomyocytes from the mammalian hibernator, ground squirrel, compared to rat. Electrophysiological and confocal imaging experiments showed that the voltage-dependence of L-type Ca(2+ current (I(Ca was shifted to higher potentials in ventricular myocytes from ground squirrels vs. rats. The elevated threshold of I(Ca did not compromise the Ca(2+-induced Ca(2+ release, because a higher depolarization rate and a longer duration of action potential compensated the voltage shift of I(Ca. Both the caffeine-sensitive and caffeine-resistant components of cytosolic Ca(2+ removal were more rapid in ground squirrels. Ca(2+ sparks in ground squirrels exhibited larger amplitude/size and much lower frequency than in rats. Due to the high I(Ca threshold, low SR Ca(2+ leak and rapid cytosolic Ca(2+ clearance, heart cells from ground squirrels exhibited better capability in maintaining intracellular Ca(2+ homeostasis than those from rats and other nonhibernating mammals. These findings not only reveal adaptive mechanisms of hibernation, but also provide novel strategies against Ca(2+ overload-related heart diseases.

  8. Plasma membrane nadh dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species. (United States)

    Miner, C; López-Burillo, S; García-Sancho, J; Herreros, B


    Ca2+-dependent K+ transport and plasma membrane NADH dehydrogenase activities have been studied in several 'high-K+' (human, rabbit and guinea pig) and 'low-K+' (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca2+-dependent K+ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome c reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca2+-dependent K+ channel activities in the species studied. Nor were any of the above activities correlated with (Na+ + K+)-ATPase activity.

  9. Changes in taste receptor cell [Ca2+]i modulate chorda tympani responses to salty and sour taste stimuli (United States)

    DeSimone, John A.; Ren, ZuoJun; Phan, Tam-Hao T.; Heck, Gerard L.; Mummalaneni, Shobha


    The relationship between taste receptor cell (TRC) Ca2+ concentration ([Ca2+]i) and rat chorda tympani (CT) nerve responses to salty [NaCl and NaCl+benzamil (Bz)] and sour (HCl, CO2, and acetic acid) taste stimuli was investigated before and after lingual application of ionomycin+Ca2+, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), U73122 (phospholipase C blocker), and thapsigargin (Ca2+-ATPase inhibitor) under open-circuit or lingual voltage-clamp conditions. An increase in TRC [Ca2+]i attenuated the tonic Bz-sensitive NaCl CT response and the apical membrane Na+ conductance. A decrease in TRC [Ca2+]i enhanced the tonic Bz-sensitive and Bz-insensitive NaCl CT responses and apical membrane Na+ conductance but did not affect CT responses to KCl or NH4Cl. An increase in TRC [Ca2+]i did not alter the phasic response but attenuated the tonic CT response to acidic stimuli. A decrease in [Ca2+]i did not alter the phasic response but attenuated the tonic CT response to acidic stimuli. In a subset of TRCs, a positive relationship between [H+]i and [Ca2+]i was obtained using in vitro imaging techniques. U73122 inhibited the tonic CT responses to NaCl, and thapsigargin inhibited the tonic CT responses to salty and sour stimuli. The results suggest that salty and sour taste qualities are transduced by [Ca2+]i-dependent and [Ca2+]i-independent mechanisms. Changes in TRC [Ca2+]i in a BAPTA-sensitive cytosolic compartment regulate ion channels and cotransporters involved in the salty and sour taste transduction mechanisms and in neural adaptation. Changes in TRC [Ca2+]i in a separate subcompartment, sensitive to inositol trisphosphate and thapsigargin but inaccessible to BAPTA, are associated with neurotransmitter release. PMID:22956787

  10. Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2. (United States)

    Luyten, Tomas; Welkenhuyzen, Kirsten; Roest, Gemma; Kania, Elzbieta; Wang, Liwei; Bittremieux, Mart; Yule, David I; Parys, Jan B; Bultynck, Geert


    Previous work revealed that intracellular Ca2+ signals and the inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP3Rs and Ca2+ signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca2+-chelating agent. To elucidate the IP3R's role in this process, we employed the recently established HEK 3KO cells lacking all three IP3R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP3R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca2+ or by knocking out IP3Rs. Finally, we investigated whether resveratrol by itself induced Ca2+ release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca2+ ATPase (SERCA) activity nor the IP3-induced Ca2+ release nor the basal Ca2+ leak from the ER. Also, prolonged (4 h) treatment with 100μM resveratrol did not affect subsequent IP3-induced Ca2+ release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca2+ signals by itself, it acutely decreased the ER Ca2+-store content irrespective of the presence or absence of IP3Rs, leading to a dampened agonist-induced Ca2+ signaling. In conclusion, these results reveal that IP3Rs and cytosolic Ca2+ signaling are fundamentally important for driving autophagic flux, not only in response to m

  11. Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients. (United States)

    Corrales, Alexandra; Montoya G, José V; Sutachan, Jhon-Jairo; Cornillez-Ty, Genoveve; Garavito-Aguilar, Zayra; Xu, Fang; Blanck, Thomas J J; Recio-Pinto, Esperanza


    Transient increases in extracellular K+ are observed under various conditions, including repetitive neuronal firing, anoxia, ischemia and hypoglycemic coma. We studied changes in cytoplasmic Ca2+ ([Ca2+]cyt) evoked by pulses of KCl in human neuroblastoma SH-SY5Y cells and rat dorsal root ganglia (DRG) neurons at 37 degrees C. A "pulse" of KCl evoked two transient increases in [Ca2+]cyt, one upon addition of KCl (K+on) and the other upon removal of KCl (K+off). The K+on transient has been described in many cell types and is initiated by the activation of voltage-dependent Ca2+ channels followed by Ca2+-evoked Ca2+ release from intracellular Ca2+ stores. The level of KCl necessary to evoke the K+off transient depends on the type of neuron, in SH-SY5Y cells it required 100 mM KCl, in most (but not all) of dorsal root ganglia neurons it could be detected with 100-200 mM KCl and in a very few dorsal root ganglia neurons it was detectable at 20-50 mM KCl. In SH-SY5Y cells, reduction of extracellular Ca2+ inhibited the K+on more strongly than the K+off and slowed the decay of K+off. Isoflurane (1 mM) reduced the K+on)- but not the K+off-peak. However, isoflurane slowed the decay of K+off. The nonspecific cationic channel blocker La3+ (100 microM) had an effect similar to that of isoflurane. Treatment with thapsigargin (TG) at a concentration known to only deplete IP3-sensitive Ca2+ stores did not affect K+on or K+off, suggesting that Ca2+ release from the IP3-sensitive Ca2+ stores does not contribute to K+on and K+off transients and that the thapsigargin-sensitive Ca2+ ATPases do not contribute significantly to the rise or decay rates of these transients. These findings indicate that a pulse of extracellular K+ produces two distinct transient increases in [Ca2+]cyt.

  12. The relative contribution of NMDARs to excitatory postsynaptic currents is controlled by Ca2+-induced inactivation.

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    Fliza eValiullina


    Full Text Available NMDA receptors (NMDARs are important mediators of excitatory synaptic transmission and plasticity. A hallmark of these channels is their high permeability to Ca2+. At the same time, they are themselves inhibited by the elevation of intracellular Ca2+ concentration. It is unclear however, whether the Ca2+ entry associated with single NMDAR mediated synaptic events is sufficient to self-inhibit their activation. Such auto-regulation would have important effects on the dynamics of synaptic excitation in several central networks. Therefore, we studied NMDAR-mediated synaptic currents in mouse hippocampal CA1 pyramidal neurons. Postsynaptic responses to subthreshold Schaffer collateral stimulation depended strongly on the absence or presence of intracellular Ca2+ buffers. Loading of pyramidal cells with exogenous Ca2+ buffers increased the amplitude and decay time of NMDAR mediated EPSCs (EPSP and prolonged the time window for action potential generation.Our data indicate that the Ca2+ influx mediated by unitary synaptic events is sufficient to produce detectable self-inhibition of NMDARs even at a physiological Mg2+ concentration. Therefore, the contribution of NMDARs to synaptic excitation is strongly controlled by both previous synaptic activity as well as by the Ca2+ buffer capacity of postsynaptic neurons.

  13. Crystal structure of λ exonuclease in complex with DNA and Ca(2+). (United States)

    Zhang, Jinjin; Pan, Xinlei; Bell, Charles E


    Bacteriophage λ exonuclease (λexo) is a ring-shaped homotrimer that resects double-stranded DNA ends in the 5'-3' direction to generate a long 3'-overhang that is a substrate for recombination. λexo is a member of the type II restriction endonuclease-like superfamily of proteins that use a Mg(2+)-dependent mechanism for nucleotide cleavage. A previous structure of λexo in complex with DNA and Mg(2+) was determined using a nuclease defective K131A variant to trap a stable complex. This structure revealed the detailed coordination of the two active site Mg(2+) ions but did not show the interactions involving the side chain of the conserved active site Lys-131 residue. Here, we have determined the crystal structure of wild-type (WT) λexo in complex with the same DNA substrate, but in the presence of Ca(2+) instead of Mg(2+). Surprisingly, there is only one Ca(2+) bound in the active site, near the position of Mg(A) in the structure with Mg(2+). The scissile phosphate is displaced by 2.2 Å relative to its position in the structure with Mg(2+), and the network of interactions involving the attacking water molecule is broken. Thus, the structure does not represent a catalytic configuration. However, the crystal structure does show clear electron density for the side chain of Lys-131, which is held in place by interactions with Gln-157 and Glu-129. By combining the K131A-Mg(2+) and WT-Ca(2+) structures, we constructed a composite model to show the likely interactions of Lys-131 during catalysis. The implications with regard to the catalytic mechanism are discussed.

  14. Ca(2+) leakage out of the sarcoplasmic reticulum is increased in type I skeletal muscle fibres in aged humans. (United States)

    Lamboley, C R; Wyckelsma, V L; McKenna, M J; Murphy, R M; Lamb, G D


    The amount of Ca(2+) stored in the sarcoplasmic reticulum (SR) of muscle fibres is decreased in aged individuals, and an important question is whether this results from increased Ca(2+) leakage out through the Ca(2+) release channels (ryanodine receptors; RyRs). The present study examined the effects of blocking the RyRs with Mg(2+), or applying a strong reducing treatment, on net Ca(2+) accumulation by the SR in skinned muscle fibres from Old (∼70 years) and Young (∼24 years) adults. Raising cytoplasmic [Mg(2+)] and reducing treatment increased net SR Ca(2+) accumulation in type I fibres of Old subjects relative to that in Young. The densities of RyRs and dihydropyridine receptors were not significantly changed in the muscle of Old subjects. These findings indicate that oxidative modification of the RyRs causes increased Ca(2+) leakage from the SR in muscle fibres in Old subjects, which probably deleteriously affects normal muscle function both directly and indirectly. The present study examined whether the lower Ca(2+) storage levels in the sarcoplasmic reticulum (SR) in vastus lateralis muscle fibres in Old (70 ± 4 years) relative to Young (24 ± 4 years) human subjects is the result of increased leakage of Ca(2+) out of the SR through the Ca(2+) release channels/ryanodine receptors (RyRs) and due to oxidative modification of the RyRs. SR Ca(2+) accumulation in mechanically skinned muscle fibres was examined in the presence of 1, 3 or 10 mm cytoplasmic Mg(2+) because raising [Mg(2+)] strongly inhibits Ca(2+) efflux through the RyRs. In type I fibres of Old subjects, SR Ca(2+) accumulation in the presence of 1 mm Mg(2+) approached saturation at shorter loading times than in Young subjects, consistent with Ca(2+) leakage limiting net uptake, and raising [Mg(2+)] to 10 mm in such fibres increased maximal SR Ca(2+) accumulation. No significant differences were seen in type II fibres. Treatment with dithiothreitol (10 mm for 5 min), a strong reducing agent, also

  15. Non–Ca2+-conducting Ca2+ channels in fish skeletal muscle excitation-contraction coupling (United States)

    Schredelseker, Johann; Shrivastav, Manisha; Dayal, Anamika; Grabner, Manfred


    During skeletal muscle excitation-contraction (EC) coupling, membrane depolarizations activate the sarcolemmal voltage-gated L-type Ca2+ channel (CaV1.1). CaV1.1 in turn triggers opening of the sarcoplasmic Ca2+ release channel (RyR1) via interchannel protein–protein interaction to release Ca2+ for myofibril contraction. Simultaneously to this EC coupling process, a small and slowly activating Ca2+ inward current through CaV1.1 is found in mammalian skeletal myotubes. The role of this Ca2+ influx, which is not immediately required for EC coupling, is still enigmatic. Interestingly, whole-cell patch clamp experiments on freshly dissociated skeletal muscle myotubes from zebrafish larvae revealed the lack of such Ca2+ currents. We identified two distinct isoforms of the pore-forming CaV1.1α1S subunit in zebrafish that are differentially expressed in superficial slow and deep fast musculature. Both do not conduct Ca2+ but merely act as voltage sensors to trigger opening of two likewise tissue-specific isoforms of RyR1. We further show that non-Ca2+ conductivity of both CaV1.1α1S isoforms is a common trait of all higher teleosts. This non-Ca2+ conductivity of CaV1.1 positions teleosts at the most-derived position of an evolutionary trajectory. Though EC coupling in early chordate muscles is activated by the influx of extracellular Ca2+, it evolved toward CaV1.1-RyR1 protein–protein interaction with a relatively small and slow influx of external Ca2+ in tetrapods. Finally, the CaV1.1 Ca2+ influx was completely eliminated in higher teleost fishes. PMID:20212109

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

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    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.

  17. Intercellular Ca2+ Waves: Mechanisms and Function (United States)

    Sanderson, Michael J.


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

  18. A quantitative description of tubular system Ca(2+) handling in fast- and slow-twitch muscle fibres. (United States)

    Cully, Tanya R; Edwards, Joshua N; Murphy, Robyn M; Launikonis, Bradley S


    Current methods do not allow a quantitative description of Ca(2+) movements across the tubular (t-) system membrane without isolating the membranes from their native skeletal muscle fibre. Here we present a fluorescence-based method that allows determination of the t-system [Ca(2+) ] transients and derivation of t-system Ca(2+) fluxes in mechanically skinned skeletal muscle fibres. Differences in t-system Ca(2+) -handling properties between fast- and slow-twitch fibres from rat muscle are resolved for the first time using this new technique. The method can be used to study Ca(2+) handling of the t-system and allows direct comparisons of t-system Ca(2+) transients and Ca(2+) fluxes between groups of fibres and fibres from different strains of animals. The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca(2+) gradient and exchanges Ca(2+) between the extracellular and intracellular environments. Little is known of the Ca(2+) -handling properties of the t-system as the small Ca(2+) fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system-trapped rhod-5N inside skinned fibres from rat and [Ca(2+) ]t-sys , allowing confocal measurements of Ca(2+) -dependent changes in rhod-5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca(2+) ] transients in the t-system ([Ca(2+) ]t-sys (t)). Furthermore, t-system Ca(2+) -buffering power was determined so that t-system Ca(2+) fluxes could be derived from [Ca(2+) ]t-sys (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca(2+) induced a robust store-operated Ca(2+) entry (SOCE) in fast- and slow-twitch fibres, reducing [Ca(2+) ]t-sys to fibre types. Abruptly introducing internal solutions with 1 mm Mg(2+) and [Ca(2+) ]cyto (28 nm-1.3 μm) to Ca(2+) -depleted fibres generated t-system Ca(2+) uptake rates dependent on [Ca(2

  19. A quantitative description of tubular system Ca2+ handling in fast‐ and slow‐twitch muscle fibres (United States)

    Cully, Tanya R.; Edwards, Joshua N.; Murphy, Robyn M.


    Key points Current methods do not allow a quantitative description of Ca2+ movements across the tubular (t‐) system membrane without isolating the membranes from their native skeletal muscle fibre.Here we present a fluorescence‐based method that allows determination of the t‐system [Ca2+] transients and derivation of t‐system Ca2+ fluxes in mechanically skinned skeletal muscle fibres. Differences in t‐system Ca2+‐handling properties between fast‐ and slow‐twitch fibres from rat muscle are resolved for the first time using this new technique.The method can be used to study Ca2+ handling of the t‐system and allows direct comparisons of t‐system Ca2+ transients and Ca2+ fluxes between groups of fibres and fibres from different strains of animals. Abstract The tubular (t‐) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca2+ gradient and exchanges Ca2+ between the extracellular and intracellular environments. Little is known of the Ca2+‐handling properties of the t‐system as the small Ca2+ fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t‐system‐trapped rhod‐5N inside skinned fibres from rat and [Ca2+]t‐sys, allowing confocal measurements of Ca2+‐dependent changes in rhod‐5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca2+] transients in the t‐system ([Ca2+]t‐sys (t)). Furthermore, t‐system Ca2+‐buffering power was determined so that t‐system Ca2+ fluxes could be derived from [Ca2+]t‐sys (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca2+ induced a robust store‐operated Ca2+ entry (SOCE) in fast‐ and slow‐twitch fibres, reducing [Ca2+]t‐sys to fibre types. Abruptly introducing internal solutions with 1 mm Mg2+ and [Ca2+]cyto (28 nm–1.3 μm) to Ca2+‐depleted fibres generated t‐system Ca2+ uptake rates

  20. Crystal structure of a copper-transporting PIB-type ATPase

    DEFF Research Database (Denmark)

    Gourdon, Pontus; Liu, Xiang-Yu; Skjørringe, Tina


    copper entry point at the intracellular interface. Comparisons to Ca(2+)-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B...... proteins associated with Menkes' and Wilson's diseases....

  1. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+ homeostasis

    Directory of Open Access Journals (Sweden)

    Yuan Liu


    Full Text Available Heat shock protein 70 (HSP70 maintains Ca2+ homeostasis in PC12 cells, which may protect against apoptosis; however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca2+ levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overexpression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca2+ concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA, but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R, thereby leading to decreased intracellular Ca2+ concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca2+ homeostasis, by upregulating SERCA expression and by downregulating IP3R expression.

  2. Swelling-activated Ca2+ channels trigger Ca2+ signals in Merkel cells.

    Directory of Open Access Journals (Sweden)

    Henry Haeberle


    Full Text Available Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute

  3. STIM1 and STIM2 are located in the acidic Ca2+ stores and associates with Orai1 upon depletion of the acidic stores in human platelets. (United States)

    Zbidi, Hanene; Jardin, Isaac; Woodard, Geoffrey E; Lopez, Jose J; Berna-Erro, Alejandro; Salido, Ginés M; Rosado, Juan A


    Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.

  4. [Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria]. (United States)

    Veklich, T O; Kosterin, S O; Shynlova, O P


    In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process.

  5. Mitochondrial Ca2+ uptake in skeletal muscle health and disease

    CERN Document Server

    Zhou, Jingsong; Yi, Jianxun


    Muscle uses Ca2+ as a messenger to control contraction and relies on ATP to maintain the intracellular Ca2+ homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca2+ from their surroundings, a process called mitochondrial Ca2+ uptake. Under physiological conditions, Ca2+ uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca2+ overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca2+ uptake could shape spatio-temporal patterns of intracellular Ca2+ signaling. Malfunction of mitochondrial Ca2+ uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca2+ levels. Besides the sudden elevation of Ca2+ level induced by action potentials, Ca2+ transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as min...

  6. Ca2+ extrusion via Na+-Ca2+ exchangers in rat odontoblasts. (United States)

    Tsumura, Maki; Okumura, Reijiro; Tatsuyama, Shoko; Ichikawa, Hideki; Muramatsu, Takashi; Matsuda, Toshio; Baba, Akemichi; Suzuki, Keiko; Kajiya, Hiroshi; Sahara, Yoshinori; Tokuda, Masayuki; Momose, Yasunori; Tazaki, Masakazu; Shimono, Masaki; Shibukawa, Yoshiyuki


    Intracellular Ca(2+) is essential to many signal transduction pathways, and its level is tightly regulated by the Ca(2+) extrusion system in the plasma membrane, which includes the Na(+)-Ca(2+) exchanger (NCX). Although expression of NCX1 isoforms has been demonstrated in odontoblasts, the detailed properties of NCX remain to be clarified. In this study, we investigated localization and ion-transporting/pharmacologic properties of NCX isoforms in rat odontoblasts. We characterized both the reverse and forward modes of NCX activity in odontoblasts in a dental pulp slice preparation. Ca(2+) influx by reverse NCX activity was measured by fura-2 fluorescence. Ca(2+) efflux by forward NCX activity elicited inward Na(+) current as measured by perforated-patch clamp recording. For immunohistochemical analysis, cryostat sections of incisors were incubated with antibodies against NCX. Immunohistochemical observation revealed localization of NCX1 and NCX3 in the distal membrane of odontoblasts. Inward currents by forward NCX activity showed dependence on external Na(+). Fura-2 fluorescence measurement revealed that Ca(2+) influx by reverse NCX activity depended on extracellular Ca(2+) concentration, and that this influx was blocked by NCX inhibitor KB-R7943 in a concentration-dependent manner. However, Ca(2+) influx by NCX showed a slight sensitivity to SEA0400 (a potent NCX1 inhibitor), indicating that expression potencies in odontoblasts were NCX3 > NCX1. These results suggest that odontoblasts express NCX1 and NCX3 at the distal membrane, and that these isoforms play an important role in the Ca(2+) extrusion system as well as in the directional Ca(2+) transport pathway from the circulation to the dentin-mineralizing front. Copyright (c) 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  7. Activation of Na+-K+-ATPase with DRm217 attenuates oxidative stress-induced myocardial cell injury via closing Na+-K+-ATPase/Src/Ros amplifier. (United States)

    Yan, Xiaofei; Xun, Meng; Dou, Xiaojuan; Wu, Litao; Zhang, Fujun; Zheng, Jin


    Reduced Na+-K+-ATPase activity has close relationship with cardiomyocyte death. Reactive oxygen species (ROS) also plays an important role in cardiac cell damage. It has been proved that Na+-K+-ATPase and ROS form a feed-forward amplifier. The aim of this study was to explore whether DRm217, a proved Na+/K+-ATPase's DR-region specific monoclonal antibody and direct activator, could disrupt Na+-K+-ATPase/ROS amplifier and protect cardiac cells from ROS-induced injury. We found that DRm217 protected myocardial cells against hydrogen peroxide (H2O2)-induced cardiac cell injury and mitochondrial dysfunction. DRm217 also alleviated the effect of H2O2 on inhibition of Na+-K+-ATPase activity, Na+-K+-ATPase cell surface expression, and Src phosphorylation. H2O2-treatment increased intracellular ROS, mitochondrial ROS and induced intracellular Ca2+, mitochondrial Ca2+ overload. DRm217 closed Na+-K+-ATPase/ROS amplifier, alleviated Ca2+ accumulation and finally inhibited ROS and mitochondrial ROS generation. These novel results may help us to understand the important role of the Na+-K+-ATPase in oxidative stress and oxidative stress-related disease.

  8. Modeling the contributions of Ca2+ flows to spontaneous Ca2+ oscillations and cortical spreading depression-triggered Ca2+ waves in astrocyte networks.

    Directory of Open Access Journals (Sweden)

    Bing Li

    Full Text Available Astrocytes participate in brain functions through Ca(2+ signals, including Ca(2+ waves and Ca(2+ oscillations. Currently the mechanisms of Ca(2+ signals in astrocytes are not fully clear. Here, we present a computational model to specify the relative contributions of different Ca(2+ flows between the extracellular space, the cytoplasm and the endoplasmic reticulum of astrocytes to the generation of spontaneous Ca(2+ oscillations (CASs and cortical spreading depression (CSD-triggered Ca(2+ waves (CSDCWs in a one-dimensional astrocyte network. This model shows that CASs depend primarily on Ca(2+ released from internal stores of astrocytes, and CSDCWs depend mainly on voltage-gated Ca(2+ influx. It predicts that voltage-gated Ca(2+ influx is able to generate Ca(2+ waves during the process of CSD even after depleting internal Ca(2+ stores. Furthermore, the model investigates the interactions between CASs and CSDCWs and shows that the pass of CSDCWs suppresses CASs, whereas CASs do not prevent the generation of CSDCWs. This work quantitatively analyzes the generation of astrocytic Ca(2+ signals and indicates different mechanisms underlying CSDCWs and non-CSDCWs. Research on the different types of Ca(2+ signals might help to understand the ways by which astrocytes participate in information processing in brain functions.

  9. Can Endocrine disrupters interfere with Ca2+ homeostasis in invertebrate cells?

    Directory of Open Access Journals (Sweden)

    L. Canesi


    Full Text Available A wide range of environmental chemicals have been shown to alter the endocrine system of both wildlife and humans. There is increasing evidence that many of these endocrine disruptors (EDs, in particular estrogenic chemicals, can rapidly affect cellular homeostasis and signaling in mammalian Ca2+ systems. In this work, in vitro and in vivo data are summarised on the effects of different compounds known or suspected as EDs on homeostasis in Ca2+ marine invertebrate, the blue mussel Mytilus spp. Both synthetic estrogens and different EDs (DES, BPA, NP, PCB congeners, etc. rapidly increased sytosolic [Ca2+] in mussel hemosytes, as evaluated by FURA2 single cell fluorescence microscopy. The observed [Ca2+] increase was unaffected by the antiestrogen Tamoxifen and was due to either increased influx or release from Ca2+ intracellular stores, depending on the compound. Moreover, different ED,s including the brominated flame retardant TBBPA (tetrabromo bisphenol A induced a dose-dependent inhibition of the plasma membrane Ca2+ -ATPase (PMCA activity from mussel gills in vitro, this supporting a direct effect on membrane pumps. The in vitro effects of EDs were observed at concentrations generally higher than those of E2. However, in vivo, mussel exposure to environmetal concentrations of Bisphenol A (BPA and of the polybrominated diphenyl ether TBDE-47 resulted in large inhibition of PMCA activity in the digestive gland. The results indicate that, in invertebrate like in mammalian systems, interference with Ca2+ homeostasis may represent a significant mode of action of a variety of EDs.

  10. Neuronal Ca(2+) dyshomeostasis in Huntington disease. (United States)

    Giacomello, Marta; Oliveros, Juan C; Naranjo, Jose R; Carafoli, Ernesto


    The expansion of the N-terminal poly-glutamine tract of the huntingtin (Htt) protein is responsible for Huntington disease (HD). A large number of studies have explored the neuronal phenotype of HD, but the molecular aethiology of the disease is still very poorly understood. This has hampered the development of an appropriate therapeutical strategy to at least alleviate its symptoms. In this short review, we have focused our attention on the alteration of a specific cellular mechanism common to all HD models, either genetic or induced by treatment with 3-NPA, i.e. the cellular dyshomeostasis of Ca(2+). We have highlighted the direct and indirect (i.e. transcriptionally mediated) effects of mutated Htt on the maintenance of the intracellular Ca(2+) balance, the correct modulation of which is fundamental to cell survival and the disturbance of which plays a key role in the death of the cell.

  11. Role of arginine residues in the stimulation of the smooth-muscle plasma-membrane Ca2+ pump by negatively charged phospholipids.


    Missiaen, L; Raeymaekers, L; Droogmans, G; Wuytack, F; Casteels, R


    Negatively charged phospholipids strongly stimulate the purified plasma membrane Ca2+ pump of erythrocytes [Enyedi, Flura, Sarkadi, Gardos & Carafoli (1987) J. Biol. Chem. 262, 6425-6430] and of smooth muscle [Missiaen, Raeymaekers, Wuytack, Vrolix, De Smedt & Casteels, (1989) Biochem. J. 263, 687-694]. We have investigated the role of arginine residues in the interaction of these acidic phospholipids with the smooth-muscle Ca2+ transport ATPase. The arginine-modifying reagent phenylglyoxal i...

  12. Puerarin Enhances Ca2+ Reuptake and Ca2+ Content of Sarcoplasmic Reticulum in Murine Embryonic Stem Cell-Derived Cardiomyocytes via Upregulation of SERCA2a

    Directory of Open Access Journals (Sweden)

    Lu Wang


    Full Text Available Background/Aims: The embryonic stem cell-derived cardiomyocytes (ES-CMs serve as potential sources for cardiac regenerative therapy. However, the immature sarcoplasmic reticulum (SR function of ES-CMs prevents its application. In this report, we examined the effect of puerarin, an isoflavone compound, on SR function of murine ES-CMs. Methods: Murine ES-CMs were harvested by embryoid body-based differentiation method. Confocal calcium imaging and whole-cell patch clamps were performed to assess the function of SR. The mRNA expression levels of SR-related genes were examined by quantitative PCR. The protein expression of sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a was evaluated by immunofluorescent and western blot. Results: Long-term application of puerarin promotes basic properties of spontaneous calcium transient with increased amplitude, decay velocity, and decreased duration. Puerarin fails to alter ICa,L but increases the Ca2+ content of SR. Puerarin-treated ES-CMs have intact SR Ca2+ cycling with more SR Ca2+ reuptake. Long-term application of puerarin asynchronously upregulates the mRNA and protein expression of SERCA2a, as well as the transcripts of calsequestrin and triadin in developing ES-CMs. Application of puerarin during the stage of post-cardiac differentiation upregulates dose-dependently the transcripts of SERCA2a, phospholamban and tridin which can be reversed by the inhibitors of the PI3K/Akt and MAPK/ERK signaling pathways, but shows no effect on the protein expression of SERCA2a. Conclusion: This study demonstrates that long-term puerarin treatment enhances Ca2+ reuptake and Ca2+ content via upregulation of SERCA2a.

  13. A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations* (United States)

    Calì, Tito; Lopreiato, Raffaele; Shimony, Joshua; Vineyard, Marisa; Frizzarin, Martina; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Shinawi, Marwan; Carafoli, Ernesto


    The particular importance of Ca2+ signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca2+ ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca2+. A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca2+ ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca2+ transients generated by cell stimulation and impairs its Ca2+ extrusion function under conditions of low resting cytosolic Ca2+ as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca2+-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca2+ homeostasis and the previous finding that PMCAs act as digenic modulators in Ca2+-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype. PMID:25953895

  14. A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations. (United States)

    Calì, Tito; Lopreiato, Raffaele; Shimony, Joshua; Vineyard, Marisa; Frizzarin, Martina; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Shinawi, Marwan; Carafoli, Ernesto


    The particular importance of Ca(2+) signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca(2+) ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca(2+). A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca(2+) ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca(2+) transients generated by cell stimulation and impairs its Ca(2+) extrusion function under conditions of low resting cytosolic Ca(2+) as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca(2+)-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca(2+) homeostasis and the previous finding that PMCAs act as digenic modulators in Ca(2+)-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. SPCA2 regulates Orai1 trafficking and store independent Ca2+ entry in a model of lactation.

    Directory of Open Access Journals (Sweden)

    Brandie M Cross

    Full Text Available An unconventional interaction between SPCA2, an isoform of the Golgi secretory pathway Ca(2+-ATPase, and the Ca(2+ influx channel Orai1, has previously been shown to contribute to elevated Ca(2+ influx in breast cancer derived cells. In order to investigate the physiological role of this interaction, we examined expression and localization of SPCA2 and Orai1 in mouse lactating mammary glands. We observed co-induction and co-immunoprecipitation of both proteins, and isoform-specific differences in the localization of SPCA1 and SPCA2. Three-dimensional cultures of normal mouse mammary epithelial cells were established using lactogenic hormones and basement membrane. The mammospheres displayed elevated Ca(2+ influx by store independent mechanisms, consistent with upregulation of both SPCA2 and Orai1. Knockdown of either SPCA2 or Orai1 severely depleted Ca(2+ influx and interfered with mammosphere differentiation. We show that SPCA2 is required for plasma membrane trafficking of Orai1 in mouse mammary epithelial cells and that this function can be replaced, at least in part, by a membrane-anchored C-terminal domain of SPCA2. These findings clearly show that SPCA2 and Orai1 function together to regulate Store-independent Ca(2+ entry (SICE, which mediates the massive basolateral Ca(2+ influx into mammary epithelia to support the large calcium transport requirements for milk secretion.

  16. Difference in photosynthetic performance among three peach ...

    African Journals Online (AJOL)

    RuBPCase), Ca2+- adenosinetriphosphatase (Ca2+-ATPase) and Mg2+- adenosine triphosphatase (Mg2+-ATPase) activities, together with nicotinamide adenine dinucleotide phosphate (NADPH) content and photosynthetic O2 evolution rate ...

  17. Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca2+ by mobilization of cellular Ca2+ and activation of Ca2+ influx

    DEFF Research Database (Denmark)

    Bouchelouche, P; Belhage, B; Frandsen, A


    The Ca2+ sensitive fluorescent probe, fura-2 has been used to monitor cytosolic free calcium levels in mature primary cultures of cerebellar granule cells during exposure to L-glutamate and other excitatory amino acids: quisqualate (QA) kainate (KA) and N-methyl-D-aspartate (NMDA). Glutamate...... at micromolar concentrations produced a prompt and dose-related increase in the intracellular concentration of free Ca2+, ([Ca2+]i), whereas QA, KA and NMDA had no effect. This increase was also seen in the absence of extracellular Ca2+, suggesting that L-glutamate promotes mobilization of Ca2+ from...

  18. Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus.

    Directory of Open Access Journals (Sweden)

    Yuanwei Zhang


    Full Text Available Finely tuned changes in cytosolic free calcium ([Ca2+]c mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited. Here, we demonstrate that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in Aspergillus nidulans, regulates [Ca2+]c homeostasis. Deletion of akrA showed marked defects in hyphal growth and conidiation under low calcium conditions which were similar to the effects of deleting components of the high-affinity calcium uptake system (HACS. The [Ca2+]c dynamics in living cells expressing the calcium reporter aequorin in different akrA mutant backgrounds were defective in their [Ca2+]c responses to high extracellular Ca2+ stress or drugs that cause ER or plasma membrane stress. All of these effects on the [Ca2+]c responses mediated by AkrA were closely associated with the cysteine residue of the AkrA DHHC motif, which is required for palmitoylation by AkrA. Using the acyl-biotin exchange chemistry assay combined with proteomic mass spectrometry, we identified protein substrates palmitoylated by AkrA including two new putative P-type ATPases (Pmc1 and Spf1 homologs, a putative proton V-type proton ATPase (Vma5 homolog and three putative proteins in A. nidulans, the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner. Thus, our findings provide strong evidence that the AkrA protein regulates [Ca2+]c homeostasis by palmitoylating these protein candidates and give new insights the role of palmitoylation in the regulation of calcium-mediated responses to extracellular, ER or plasma membrane stress.

  19. Ca2+-Citrate Uptake and Metabolism in Lactobacillus casei ATCC 334 (United States)

    Mortera, Pablo; Pudlik, Agata; Magni, Christian; Alarcón, Sergio


    The putative citrate metabolic pathway in Lactobacillus casei ATCC 334 consists of the transporter CitH, a proton symporter of the citrate-divalent metal ion family of transporters CitMHS, citrate lyase, and the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Resting cells of Lactobacillus casei ATCC 334 metabolized citrate in complex with Ca2+ and not as free citrate or the Mg2+-citrate complex, thereby identifying Ca2+-citrate as the substrate of the transporter CitH. The pathway was induced in the presence of Ca2+ and citrate during growth and repressed by the presence of glucose and of galactose, most likely by a carbon catabolite repression mechanism. The end products of Ca2+-citrate metabolism by resting cells of Lb. casei were pyruvate, acetate, and acetoin, demonstrating the activity of the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Following pyruvate, the pathway splits into two branches. One branch is the classical citrate fermentation pathway producing acetoin by α-acetolactate synthase and α-acetolactate decarboxylase. The other branch yields acetate, for which the route is still obscure. Ca2+-citrate metabolism in a modified MRS medium lacking a carbohydrate did not significantly affect the growth characteristics, and generation of metabolic energy in the form of proton motive force (PMF) was not observed in resting cells. In contrast, carbohydrate/Ca2+-citrate cometabolism resulted in a higher biomass yield in batch culture. However, also with these cells, no generation of PMF was associated with Ca2+-citrate metabolism. It is concluded that citrate metabolism in Lb. casei is beneficial when it counteracts acidification by carbohydrate metabolism in later growth stages. PMID:23709502

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

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed


    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...... have reported no or little effects on CF after curcumin treatment, suggesting that curcumin effect is not due to simple inhibition of the Ca2+-ATPase. We tested the hypothesis that curcumin may modulate other members of the P2-type ATPase superfamily by studying the effects of curcumin on the activity...... and kinetic properties of the Na,K-ATPase. Curcumin treatment inhibited Na,K-ATPase activity in a dose-dependent manner (K0.514.6 M). Curcumin decreased the apparent affinity of Na,K-ATPase for K+ and increased it for Na+ and ATP. Kinetic analyses indicated that curcumin induces a three-fold reduction...

  1. Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C.

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    Myriam A Badr

    Full Text Available Cardiac troponin C (cTnC is a key effector in cardiac muscle excitation-contraction coupling as the Ca2+ sensing subunit responsible for controlling contraction. In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP and an acceptor fluorescent protein (YFP. The sensors report Ca2+ and Mg2+ binding, and relay global structural information about the structural relationship between cTnC's N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca2+ binding in the presence or absence of Mg2+. The sensor that exhibited the largest responses in end point titrations, CTV-TnC, (Cerulean, TnC, and Venus was characterized more extensively. Most of the divalent cation-dependent FRET signal originates from the high affinity C-terminal EF hands. CTV-TnC reconstitutes into skinned fiber preparations indicating proper assembly of troponin complex, with only ~0.2 pCa unit rightward shift of Ca2+-sensitive force development compared to WT-cTnC. Affinity of CTV-TnC for divalent cations is in agreement with known values for WT-cTnC. Analytical ultracentrifugation indicates that CTV-TnC undergoes compaction as divalent cations bind. C-terminal sites induce ion-specific (Ca2+ versus Mg2+ conformational changes in cTnC. Our data also provide support for the presence of additional, non-EF-hand sites on cTnC for Mg2+ binding. In conclusion, we successfully generated a novel FRET-Ca2+ sensor based on full length cTnC with a variety of cellular applications. Our sensor reveals global structural information about cTnC upon divalent cation binding.

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

    DEFF Research Database (Denmark)

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


    protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated...


    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  4. Identification and characterization of a novel mammalian Mg2+ transporter with channel-like properties

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    Quamme Gary A


    Full Text Available Abstract Background Intracellular magnesium is abundant, highly regulated and plays an important role in biochemical functions. Despite the extensive evidence for unique mammalian Mg2+ transporters, few proteins have been biochemically identified to date that fulfill this role. We have shown that epithelial magnesium conservation is controlled, in part, by differential gene expression leading to regulation of Mg2+ transport. We used this knowledge to identify a novel gene that is regulated by magnesium. Results Oligonucleotide microarray analysis was used to identify a novel human gene that encodes a protein involved with Mg2+-evoked transport. We have designated this magnesium transporter (MagT1 protein. MagT1 is a novel protein with no amino acid sequence identity to other known transporters. The corresponding cDNA comprises an open reading frame of 1005 base pairs encoding a protein of 335 amino acids. It possesses five putative transmembrane (TM regions with a cleavage site, a N-glycosylation site, and a number of phosphorylation sites. Based on Northern analysis of mouse tissues, a 2.4 kilobase transcript is present in many tissues. When expressed in Xenopus laevis oocytes, MagT1 mediates saturable Mg2+ uptake with a Michaelis constant of 0.23 mM. Transport of Mg2+ by MagT1 is rheogenic, voltage-dependent, does not display any time-dependent inactivation. Transport is very specific to Mg2+ as other divalent cations did not evoke currents. Large external concentrations of some cations inhibited Mg2+ transport (Ni2+, Zn2+, Mn2+ in MagT1-expressing oocytes. Ca2+and Fe2+ were without effect. Real-time reverse transcription polymerase chain reaction and Western blot analysis using a specific antibody demonstrated that MagT1 mRNA and protein is increased by about 2.1-fold and 32%, respectively, in kidney epithelial cells cultured in low magnesium media relative to normal media and in kidney cortex of mice maintained on low magnesium diets

  5. Selective detection of Mg2+ ions via enhanced fluorescence emission using Au–DNA nanocomposites

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    Tanushree Basu


    Full Text Available The biophysical properties of DNA-modified Au nanoparticles (AuNPs have attracted a great deal of research interest for various applications in biosensing. AuNPs have strong binding capability to the phosphate and sugar groups in DNA, rendering unique physicochemical properties for detection of metal ions. The formation of Au–DNA nanocomposites is evident from the observed changes in the optical absorption, plasmon band, zeta potential, DLS particle size distribution, as well as TEM and AFM surface morphology analysis. Circular dichroism studies also revealed that DNA-functionalized AuNP binding caused a conformational change in the DNA structure. Due to the size and shape dependent plasmonic interactions of AuNPs (33–78 nm with DNA, the resultant Au–DNA nanocomposites (NCs exhibit superior fluorescence emission due to chemical binding with Ca2+, Fe2+ and Mg2+ ions. A significant increase in fluorescence emission (λex = 260 nm of Au–DNA NCs was observed after selectively binding with Mg2+ ions (20–800 ppm in an aqueous solution where a minimum of 100 ppm Mg2+ ions was detected based on the linearity of concentration versus fluorescence intensity curve (λem = 400 nm. The effectiveness of Au–DNA nanocomposites was further verified by comparing the known concentration (50–120 ppm of Mg2+ ions in synthetic tap water and a real life sample of Gelusil (300–360 ppm Mg2+, a widely used antacid medicine. Therefore, this method could be a sensitive tool for the estimation of water hardness after careful preparation of a suitably designed Au–DNA nanostructure.

  6. Selective detection of Mg2+ions via enhanced fluorescence emission using Au-DNA nanocomposites. (United States)

    Basu, Tanushree; Rana, Khyati; Das, Niranjan; Pal, Bonamali


    The biophysical properties of DNA-modified Au nanoparticles (AuNPs) have attracted a great deal of research interest for various applications in biosensing. AuNPs have strong binding capability to the phosphate and sugar groups in DNA, rendering unique physicochemical properties for detection of metal ions. The formation of Au-DNA nanocomposites is evident from the observed changes in the optical absorption, plasmon band, zeta potential, DLS particle size distribution, as well as TEM and AFM surface morphology analysis. Circular dichroism studies also revealed that DNA-functionalized AuNP binding caused a conformational change in the DNA structure. Due to the size and shape dependent plasmonic interactions of AuNPs (33-78 nm) with DNA, the resultant Au-DNA nanocomposites (NCs) exhibit superior fluorescence emission due to chemical binding with Ca 2+ , Fe 2+ and Mg 2+ ions. A significant increase in fluorescence emission (λ ex = 260 nm) of Au-DNA NCs was observed after selectively binding with Mg 2+ ions (20-800 ppm) in an aqueous solution where a minimum of 100 ppm Mg 2+ ions was detected based on the linearity of concentration versus fluorescence intensity curve (λ em = 400 nm). The effectiveness of Au-DNA nanocomposites was further verified by comparing the known concentration (50-120 ppm) of Mg 2+ ions in synthetic tap water and a real life sample of Gelusil (300-360 ppm Mg 2+ ), a widely used antacid medicine. Therefore, this method could be a sensitive tool for the estimation of water hardness after careful preparation of a suitably designed Au-DNA nanostructure.

  7. Removal of Mg2+, K+, SO4-2 Ions from Seawater by Precipitation Method

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    Pujiastuti Caecilia


    Full Text Available Removal of Mg2+, K+, and SO4-2 ions in seawater has been successfully done by precipitation in a mixing tank method. This study aims to remove the content of magnesium ions (Mg+, potassium (K+ and sulfate (SO4-2 in sea water with the addition of chemicals disodium phosphate (1.2 % volume, calcium chloride (2 % volume and sodium hydroxide (2% volume. Stirring is performed at 100 rpm and the pH solution is adjusted to 9. Disodium phosphate serves to bind magnesium ions and potassium, CaCl2 serves to bind the sulfate ion, while sodium hydroxide is used to adjust the pH of the solution mixture and also reacted with magnesium ions. In total, the removal efficiencies of Mg2+, K+ and SO4-2 ions in seawater were 97%, 96%, and 92%, respectively. The precipitated solids contains component of PO4- (14.5%, Mg2+(13.8%, SO4-2 (28.2%, Ca2+ (24.1% and K+ (1.9% ions.

  8. Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+ -induced glucagon exocytosis in pancreas

    DEFF Research Database (Denmark)

    Gustavsson, Natalia; Wei, Shun-Hui; Hoang, Dong Nhut


    secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly...

  9. Mechanical isolation, and measurement of force and myoplasmic free [Ca(2+)] in fully intact single skeletal muscle fibers. (United States)

    Cheng, Arthur J; Westerblad, Håkan


    Mechanical dissection of single intact mammalian skeletal muscle fibers permits real-time measurement of intracellular properties and contractile function of living fibers. A major advantage of mechanical over enzymatic fiber dissociation is that single fibers can be isolated with their tendons remaining attached, which allows contractile forces (in the normal expected range of 300-450 kN/m(2)) to be measured during electrical stimulation. Furthermore, the sarcolemma of single fibers remains fully intact after mechanical dissection, and hence the living fibers can be studied with intact intracellular milieu and normal function and metabolic properties, as well as ionic control. Given that Ca(2+) is the principal regulator of the contractile force, measurements of myoplasmic free [Ca(2+)] ([Ca(2+)]i) can be used to further delineate the intrinsic mechanisms underlying changes in skeletal muscle function. [Ca(2+)]i measurements are most commonly performed in intact single fibers using ratiometric fluorescent indicators such as indo-1 or fura-2. These Ca(2+) indicators are introduced into the fiber by pressure injection or by using the membrane-permeable indo-1 AM, and [Ca(2+)]i is measured by calculating a ratio of the fluorescence at specific wavelengths emitted for the Ca(2+)-free and Ca(2+)-bound forms of the dye. We describe here the procedures for mechanical dissection, and for force and [Ca(2+)]i measurement in intact single fibers from mouse flexor digitorum brevis (FDB) muscle, which is the most commonly used muscle in studies using intact single fibers. This technique can also be used to isolate intact single fibers from various muscles and from various species. As an alternative to Ca(2+) indicators, single fibers can also be loaded with fluorescent indicators to measure, for instance, reactive oxygen species, pH, and [Mg(2+)], or they can be injected with proteins to change functional properties. The entire protocol, from dissection to the start of an

  10. Abnormal Ca2+ homeostasis, atrial arrhythmogenesis and sinus node dysfunction in murine hearts modelling RyR2 modification

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    Yanmin eZhang


    Full Text Available RyR2 mutations are implicated in catecholaminergic polymorphic ventricular tachycardia thought to result from altered myocyte Ca2+ homeostasis reflecting inappropriate ‘leakiness’ of RyR2-Ca2+ release channels arising from increases in their basal activity, alterations in their phosphorylation, or defective interactions with other molecules or ions. The latter include calstabin, calsequestrin-2, Mg2+, and extraluminal or intraluminal Ca2+. Recent clinical studies additionally associate RyR2 abnormalities with atrial arrhythmias including atrial tachycardia, fibrillation and standstill, and sinus node dysfunction. Some RyR2 mutations associated with CPVT in mouse models also show such arrhythmias that similarly correlate with altered Ca2+ homeostasis. Some examples show evidence for increased Ca2+/calmodulin-dependent protein kinase II phosphorylation of RyR2. A homozygotic RyR2-P2328S variant demonstrates potential arrhythmic substrate resulting from reduced conduction velocity in addition to delayed afterdepolarizations and ectopic action potential firing. Finally, one model with an increased RyR2 activity in the sino-atrial node shows decreased automaticity in the presence of Ca2+-dependent decreases in ICa,L and diastolic sarcoplasmic reticular Ca2+ depletion.

  11. Structural basis for ion selectivity revealed by high-resolution crystal structure of Mg2+ channel MgtE. (United States)

    Takeda, Hironori; Hattori, Motoyuki; Nishizawa, Tomohiro; Yamashita, Keitaro; Shah, Syed T A; Caffrey, Martin; Maturana, Andrés D; Ishitani, Ryuichiro; Nureki, Osamu


    Magnesium is the most abundant divalent cation in living cells and is crucial to several biological processes. MgtE is a Mg(2+) channel distributed in all domains of life that contributes to the maintenance of cellular Mg(2+) homeostasis. Here we report the high-resolution crystal structures of the transmembrane domain of MgtE, bound to Mg(2+), Mn(2+) and Ca(2+). The high-resolution Mg(2+)-bound crystal structure clearly visualized the hydrated Mg(2+) ion within its selectivity filter. Based on those structures and biochemical analyses, we propose a cation selectivity mechanism for MgtE in which the geometry of the hydration shell of the fully hydrated Mg(2+) ion is recognized by the side-chain carboxylate groups in the selectivity filter. This is in contrast to the K(+)-selective filter of KcsA, which recognizes a dehydrated K(+) ion. Our results further revealed a cation-binding site on the periplasmic side, which regulate channel opening and prevents conduction of near-cognate cations.

  12. Store-operated Ca2+ entry supports contractile function in hearts of hibernators.

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    Olga V Nakipova

    Full Text Available Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA. We found that the specific SERCA inhibitor cyclopiazonic acid (CPA, in contrast to its effect in papillary muscles (PM from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS. In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC. Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C-7°C-30°C of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states.

  13. Store-operated Ca2+ entry supports contractile function in hearts of hibernators (United States)

    Nakipova, Olga V.; Averin, Alexey S.; Evdokimovskii, Edward V.; Pimenov, Oleg Yu.; Kosarski, Leonid; Ignat’ev, Dmitriy; Anufriev, Andrey; Kokoz, Yuri M.; Reyes, Santiago; Terzic, Andre; Alekseev, Alexey E.


    Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA). We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C–7°C–30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states. PMID:28531217

  14. Chloroquine Inhibits Ca2+ Signaling in Murine CD4+ Thymocytes

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    Jin-Chao Xu


    Full Text Available Background/Aims: Bitter-tasting chloroquine can suppress T cell activation by inhibiting Ca2+ signaling. However, the mechanism of inhibition remains largely unclear. Methods: In this study, CD4+ T cells were isolated from the thymus, and the calcium content of CD4+ thymocytes was measured using fura-2 AM and a TILL imaging system. Pyrazole-3 (Pyr3, thapsigargin (TG, and caffeine were used to assess the effects of chloroquine on the intracellular Ca2+ content of CD4+ T cells. Results: In murine CD4+ thymocytes, chloroquine decreased the TG-triggered intracellular Ca2+ increase in a dose-dependent manner. In the absence of chloroquine under Ca2+-free conditions (0 mM Ca2+ and 0.5 mM EGTA, TG induced a transient Ca2+ increase. After restoration of the extracellular Ca2+ concentration to 2 mM, a dramatic Ca2+ increase occurred. This elevation was completely blocked by chloroquine and was markedly inhibited by Pyr3, a selective antagonist of transient receptor potential C3 (TRPC3 channel and stromal interaction molecule (STIM/Orai channel. Furthermore, the TG-induced transient Ca2+ increase under Ca2+-free conditions was eliminated in the presence of chloroquine. Chloroquine also blocked the dialyzed inositol-1,4,5-trisphosphate (IP3-induced intracellular Ca2+ increase. However, chloroquine was not able to decrease the caffeine-induced Ca2+ increase. Conclusion: These data indicate that chloroquine inhibits the elevation of intracellular Ca2+ in thymic CD4+ T cells by inhibiting IP3 receptor-mediated Ca2+ release from intracellular stores and TRPC3 channel-mediated and/or STIM/Orai channel-mediated Ca2+ influx.

  15. Synthesis and Characterization of a Mg2+-Selective Fluorescent Probe

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    Chunwei Yu


    Full Text Available A new Mg2+-selective fluorescent probe P was synthesized and characterized. With optimal conditions, the proposed probe P showed good selectivity to Mg2+ compared to other common metal ions, and worked in a wide linear range of 5.0 × 10−7–6.0 × 10−6 M with a detection limit of 1.7 × 10−7 M Mg2+ in ethanol-water solution (9:1, v/v, 20 mM HEPES, pH = 10.0.

  16. Ca(2+) homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca(2+) storage. (United States)

    D'hooge, Petra; Coun, Catherina; Van Eyck, Vincent; Faes, Liesbeth; Ghillebert, Ruben; Mariën, Lore; Winderickx, Joris; Callewaert, Geert


    Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca(2+) signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca(2+) signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca(2+) transporters, combined with manipulations to estimate intracellular Ca(2+) storage, we evaluated the contribution of individual transport systems to intracellular Ca(2+) homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca(2+) homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca(2+) uptake and Ca(2+) influx across the plasma membrane. In the pmr1Δ strain, these effects were insensitive to calcineurin activity, independent of Cch1/Mid1 Ca(2+) channels and Pmc1 but required Vcx1. By contrast, in the cod1Δ strain increased vacuolar Ca(2+) uptake was not affected by Vcx1 deletion but was largely dependent on Pmc1 activity. Our analysis further corroborates the distinct roles of Vcx1 and Pmc1 in vacuolar Ca(2+) uptake and point to the existence of not-yet identified Ca(2+) influx pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport

    DEFF Research Database (Denmark)

    Sahoo, Sanjaya K; Shaikh, Sana A; Sopariwala, Danesh H


    The sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) is responsible for intracellular Ca(2+) homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca(2+)-bound SERCA, SLN continues...

  18. Leptin triggers Ca(2+) imbalance in monocytes of overweight subjects. (United States)

    Padra, János Tamás; Seres, Ildikó; Fóris, Gabriella; Paragh, György; Kónya, Gabriella; Paragh, György


    Obesity is a major risk factor in numerous diseases, in which elevated intracellular Ca(2+) plays a major role in increased adiposity. We examined the difference between Ca(2+) signals in monocytes of lean and overweight subjects and the relationship between leptin induced NADPH oxidase activation and intracellular calcium concentration [Ca(2+)](i) homeostasis. Our results are as follows: (1) The basal level of [Ca(2+)](i) in resting monocytes of overweight subjects (OW monocytes) was higher than that in control cells, whereas the leptin-induced peak of the Ca(2+) signal was lower and the return to basal level was delayed. (2) Ca(2+) signals were more pronounced in OW monocytes than in control cells. (3) Using different inhibitors of cellular signaling, we found that in control cells the Ca(2+) signals originated from intracellular pools, whereas in OW cells they were generated predominantly by Ca(2+)-influx from medium. Finally, we found correlation between leptin induced superoxide anion generation and Ca(2+) signals. The disturbed [Ca(2+)](i) homeostasis in OW monocytes was fully restored in the presence of fluvastatin. Statins have pleiotropic effects involving the inhibition of free radical generation that may account for its beneficial effect on elevated [Ca(2+)](i) and consequently on the pathomechanism of obesity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Strain engineering on thermoelectric performance of Mg2Si (United States)

    Kaur, Kulwinder; Dhiman, Shobhna; Kumar, Ranjan


    In this work, we propose to boost the thermoelectric performance of bulk magnesium silicide using isotropic strain. The effect of strain on the electronic and thermoelectric properties of Mg2Si is analyzed using first principles calculations combined with semi-classical Boltzmann theory. The Seebeck coefficient, power factor and electrical conductivity are strongly modified with strain. The lattice thermal conductivity is also tuned with strain. However, the strain effect on the lattice thermal conductivity of Mg2Si has not yet been systematically studied. The effect of strain on lattice thermal conductivity, specific heat, phonon dispersion curves and phonon density of states of Mg2Si are studied for the first time in this paper. The lattice thermal conductivity of bulk Mg2Si is shown to decrease continuously when applied strain changes from compressive to tensile. The results obtained in this paper have great importance in the thermoelectric field.

  20. Mitochondria Maintain Distinct Ca2+Pools in Cone Photoreceptors. (United States)

    Giarmarco, Michelle M; Cleghorn, Whitney M; Sloat, Stephanie R; Hurley, James B; Brockerhoff, Susan E


    Ca 2+ ions have distinct roles in the outer segment, cell body, and synaptic terminal of photoreceptors. We tested the hypothesis that distinct Ca 2+ domains are maintained by Ca 2+ uptake into mitochondria. Serial block face scanning electron microscopy of zebrafish cones revealed that nearly 100 mitochondria cluster at the apical side of the inner segment, directly below the outer segment. The endoplasmic reticulum surrounds the basal and lateral surfaces of this cluster, but does not reach the apical surface or penetrate into the cluster. Using genetically encoded Ca 2+ sensors, we found that mitochondria take up Ca 2+ when it accumulates either in the cone cell body or outer segment. Blocking mitochondrial Ca 2+ uniporter activity compromises the ability of mitochondria to maintain distinct Ca 2+ domains. Together, our findings indicate that mitochondria can modulate subcellular functional specialization in photoreceptors. SIGNIFICANCE STATEMENT Ca 2+ homeostasis is essential for the survival and function of retinal photoreceptors. Separate pools of Ca 2+ regulate phototransduction in the outer segment, metabolism in the cell body, and neurotransmitter release at the synaptic terminal. We investigated the role of mitochondria in compartmentalization of Ca 2+ We found that mitochondria form a dense cluster that acts as a diffusion barrier between the outer segment and cell body. The cluster is surprisingly only partially surrounded by the endoplasmic reticulum, a key mediator of mitochondrial Ca 2+ uptake. Blocking the uptake of Ca 2+ by mitochondria causes redistribution of Ca 2+ throughout the cell. Our results show that mitochondrial Ca 2+ uptake in photoreceptors is complex and plays an essential role in normal function. Copyright © 2017 the authors 0270-6474/17/372061-12$15.00/0.

  1. Simulation of Ca2+-activated Cl- current of cardiomyocytes in rabbit pulmonary vein: implications of subsarcolemmal Ca2+ dynamics. (United States)

    Leem, Chae Hun; Kim, Won Tae; Ha, Jeong Mi; Lee, Yoon Jin; Seong, Hyeon Chan; Choe, Han; Jang, Yeon Jin; Youm, Jae Boum; Earm, Yung E


    In recent studies, we recorded transiently activated outward currents by the application of three-step voltage pulses to induce a reverse mode of Na+-Ca2+ exchange (NCX). We found that these currents were mediated by a Ca2+-activated Cl- current. Based on the recent reports describing the atrial Ca2+ transients, the Ca2+ transient at the subsarcolemmal space was initiated and then diffused into the cytosolic space. Because the myocardium in the pulmonary vein is an extension of the atrium, the Ca2+-activated Cl- current may reflect the subsarcolemmal Ca2+ dynamics. We tried to predict the subsarcolemmal Ca2+ dynamics by simulating these current traces. According to recent reports on the geometry of atrial myocytes, we assumed that there were three compartments of sarcoplasmic reticulum (SR): a network SR, a junctional SR and a central SR. Based on these structures, we also divided the cytosolic space into three compartments: the junctional, subsarcolemmal and cytosolic spaces. Geometry information and cellular capacitance suggested that there were essentially no T-tubules in these cells. The basic physical data, such as the compartmental volumes, the diffusion coefficients and the stability coefficients of the Ca2+ buffers, were obtained from the literature. In the simulation, we incorporated the NCX, the L-type Ca2+ channel, the rapid activating outward rectifier K+ channel, the Na+-K+ pump, the SR Ca2+-pump, the ryanodine receptor, the Ca2+-activated Cl- channel and the dynamics of Na+, K+, Ca2+ and Cl-. In these conditions, we could successfully reconstruct the Ca2+-activated Cl- currents. The simulation allowed estimation of the Ca2+ dynamics of each compartment and the distribution of the Ca2+-activated Cl- channel and the NCX in the sarcolemma on the junctional or subsarcolemmal space.

  2. Sensory Neurons Derived from Diabetic Rats Have Diminished Internal Ca2+ Stores Linked to Impaired Re-uptake by the Endoplasmic Reticulum

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    Elena Zherebitskaya


    Full Text Available Distal symmetrical sensory neuropathy in diabetes involves the dying back of axons, and the pathology equates with axonal dystrophy generated under conditions of aberrant Ca2+ signalling. Previous work has described abnormalities in Ca2+ homoeostasis in sensory and dorsal horn neurons acutely isolated from diabetic rodents. We extended this work by testing the hypothesis that sensory neurons exposed to long-term Type 1 diabetes in vivo would exhibit abnormal axonal Ca2+ homoeostasis and focused on the role of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase. DRG (dorsal root ganglia sensory neurons from age-matched normal and 3–5-month-old STZ (streptozotocin-diabetic rats (an experimental model of Type 1 diabetes were cultured. At 1–2 days in vitro an array of parameters were measured to investigate Ca2+ homoeostasis including (i axonal levels of intracellular Ca2+, (ii Ca2+ uptake by the ER (endoplasmic reticulum, (iii assessment of Ca2+ signalling following a long-term thapsigargin-induced blockade of SERCA and (iv determination of expression of ER mass and stress markers using immunocytochemistry and Western blotting. KCl- and caffeine-induced Ca2+ transients in axons were 2-fold lower in cultures of diabetic neurons compared with normal neurons indicative of reduced ER calcium loading. The rate of uptake of Ca2+ into the ER was reduced by 2-fold (P<0.05 in diabetic neurons, while markers for ER mass and ER stress were unchanged. Abnormalities in Ca2+ homoeostasis in diabetic neurons could be mimicked via long-term inhibition of SERCA in normal neurons. In summary, axons of neurons from diabetic rats exhibited aberrant Ca2+ homoeostasis possibly triggered by suboptimal SERCA activity that could contribute to the distal axonopathy observed in diabetes.

  3. Mg2Sn heterostructures on Si(111) substrate (United States)

    Dózsa, L.; Galkin, N. G.; Pécz, B.; Osváth, Z.; Zolnai, Zs.; Németh, A.; Galkin, K. N.; Chernev, I. M.; Dotsenko, S. A.


    Thin un-doped and Al doped polycrystalline Mg-stannide films consisting mainly of Mg2Sn semiconductor phase have been grown by deposition of Sn-Mg multilayers on Si(111) p-type wafers at room temperature and annealing at 150 °C. Rutherford backscattering measurement spectroscopy (RBS) were used to determine the amount of Mg and Sn in the structures. Raman spectroscopy has shown the layers contain Mg2Sn phase. Cross sectional transmission electron microscopy (XTEM) measurements have identified Mg2Sn nanocrystallites in hexagonal and cubic phases without epitaxial orientation with respect to the Si(111) substrate. Significant oxygen concentration was found in the layer both by RBS and TEM. The electrical measurements have shown laterally homogeneous conductivity in the grown layer. The undoped Mg2Sn layers show increasing resistivity with increasing temperature indicating the scattering process dominates the resistance of the layers, i.e. large concentration of point defects was generated in the layer during the growth process. The Al doped layer shows increase of the resistance at low temperature caused by freeze out of free carriers in the Al doped Mg2Sn layer. The measurements indicate the necessity of protective layer grown over the Mg2Sn layers, and a short time delay between sample preparation and cross sectionalTEM analysis, since the unprotected layer is degraded by the interaction with the ambient.

  4. Revisiting Mg–Mg2Ni System from Electronic Perspective

    Directory of Open Access Journals (Sweden)

    Zhao Qian


    Full Text Available Both Mg and Mg2Ni are promising electrode materials in conversion-type secondary batteries. Earlier studies have shown their single-phase prospects in electro-devices, while in this work, we have quantitatively reported the electronic properties of their dual-phase materials, that is, Mg–Mg2Ni alloys, and analyzed the underlying reasons behind the property changes of materials. The hypoeutectic Mg–Mg2Ni alloys are found to be evidently more conductive than the hypereutectic Mg–Mg2Ni system. The density functional theory (DFT calculations give the intrinsic origin of electronic structures of both Mg2Ni and Mg. The morphology of quasi-nanoscale eutectics is another factor that can affect the electronic properties of the investigated alloy system; that is, the electrical property change of the investigated alloys system is due to a combination of the intrinsic property difference between the two constituting phases and the change of eutectic microstructures that affect electron scattering. In addition, regarding the Mg–Mg2Ni alloy design for device applications, the electronic property and mechanical aspect should be well balanced.

  5. Functional involvement of Ca(2+) and Ca(2+)-activated K(+) channels in anethol-induced changes in Ca(2+) dependent excitability of F1 neurons in Helix aspersa. (United States)

    Ghasemi, Zahra; Hassanpour-Ezatti, Majid; Kamalinejad, Mohammad; Janahmadi, Mahyar


    The effects of anethol, the major component of anise oil, on the Ca(2+)-dependent excitability and afterhyperpolarization (AHP) in snail neurons were examined using intracellular recording. Anethol (0.5%) significantly broadened the spike, reduced the firing frequency and enhanced the AHP amplitude. In contrast, anethol (2%) significantly increased the firing frequency and decreased the AHP. Blockade of Ca(2+) channels after anethol application depolarized the membrane potential and significantly reduced the firing rate. Furthermore, in the presence of anethol (0.5%) a significant decrease in the AHP was observed by Ca(2+) channels blockage. Here, anethol-induced functional modification of Ca(2+) and Ca(2+)-activated K(+) channels is suggested. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Two inhibitors of store operated Ca2+ entry suppress excitation contraction coupling in frog skeletal muscle. (United States)

    Olivera, J Fernando; Fernando Olivera, J; Pizarro, Gonzalo


    Two drugs, 2-APB and SKF-96365, commonly used to block Store Operated Ca(2+) Entry (SOCE) were found to have inhibitory effects at different levels of the Excitation Contraction Coupling (ECC) process in frog skeletal muscle fibers. Treatment with either drug suppressed Ca(2+) release from the Sarcoplasmic Reticulum, but this effect was not due to inhibition of SOCE as it occurred in Ca(2+)-free conditions. 2-APB applied extracellularly at 100 microM, the usual concentration to suppress SOCE, reversibly reduced the charge movement elicited by pulses in the range between -45 and -35 mV from 7.99 +/- 0.73 nC/microF (N = 17) before drug application to 6.27 +/- 0.68 nC/microF in the presence of 2-APB. This effect was mostly on the delayed Q(gamma) component. In fibers treated with the SERCA ATPase inhibitor CPA the Q(gamma) component disappeared, under this condition the application of 2-APB did not suppress the remaining charge movement. Thus the effect of 2-APB on charge movement currents seemed to be secondary to the suppression of Ca(2+) release, likely occurring directly on the release channels. No significant suppression of ECC was observed for concentration below 20 muM. 2-APB also inhibited the L-type Ca(2+) current (20 +/- 4%, N = 8). On the other hand SKF-96365 had a direct effect on the voltage sensor promoting its voltage dependent inactivation. Applied at 20 muM, a typical concentration used for inhibiting SOCE, to fibers held at -80 mV inhibited the charge moved in response to pulses ranging -45 to -30 mV from 7.95 +/- 2.59 nC/microF to 3.48 +/- 0.9 nC/microF (N = 12). A parallel reduction of Ca(2+) release was observed. Wash out was drastically increased by hyperpolarization of the holding potential to -100 mV. SKF-96365 also inhibited the L-type Ca(2+) current (41 +/- 8%, N = 4) and increased its rate of inactivation.

  7. The mitochondrial Na+/Ca2+ exchanger upregulates glucose dependent Ca2+ signalling linked to insulin secretion.

    Directory of Open Access Journals (Sweden)

    Iulia I Nita

    Full Text Available Mitochondria mediate dual metabolic and Ca(2+ shuttling activities. While the former is required for Ca(2+ signalling linked to insulin secretion, the role of the latter in β cell function has not been well understood, primarily because the molecular identity of the mitochondrial Ca(2+ transporters were elusive and the selectivity of their inhibitors was questionable. This study focuses on NCLX, the recently discovered mitochondrial Na(+/Ca(2+ exchanger that is linked to Ca(2+ signalling in MIN6 and primary β cells. Suppression either of NCLX expression, using a siRNA construct (siNCLX or of its activity, by a dominant negative construct (dnNCLX, enhanced mitochondrial Ca(2+ influx and blocked efflux induced by glucose or by cell depolarization. In addition, NCLX regulated basal, but not glucose-dependent changes, in metabolic rate, mitochondrial membrane potential and mitochondrial resting Ca(2+. Importantly, NCLX controlled the rate and amplitude of cytosolic Ca(2+ changes induced by depolarization or high glucose, indicating that NCLX is a critical and rate limiting component in the cross talk between mitochondrial and plasma membrane Ca(2+ signalling. Finally, knockdown of NCLX expression was followed by a delay in glucose-dependent insulin secretion. These findings suggest that the mitochondrial Na(+/Ca(2+ exchanger, NCLX, shapes glucose-dependent mitochondrial and cytosolic Ca(2+ signals thereby regulating the temporal pattern of insulin secretion in β cells.

  8. P4-ATPases

    DEFF Research Database (Denmark)

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


    ) 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......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...

  9. Ca2+ Homeostasis Regulates Xenopus Oocyte Maturation1 (United States)

    Sun, Lu; Hodeify, Rawad; Haun, Shirley; Charlesworth, Amanda; MacNicol, Angus M.; Ponnappan, Subramaniam; Ponnappan, Usha; Prigent, Claude; Machaca, Khaled


    In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte’s competence to undergo maturation in preparation for fertilization and embryonic development. PMID:18094360

  10. Ca2+ oscillations, Ca2+ sensitization, and contraction activated by protein kinase C in small airway smooth muscle. (United States)

    Mukherjee, Seema; Trice, Jacquelyn; Shinde, Paurvi; Willis, Ray E; Pressley, Thomas A; Perez-Zoghbi, Jose F


    Protein kinase C (PKC) has been implicated in the regulation of smooth muscle cell (SMC) contraction and may contribute to airway hyperresponsiveness. Here, we combined optical and biochemical analyses of mouse lung slices to determine the effects of PKC activation on Ca(2+) signaling, Ca(2+) sensitivity, protein phosphorylation, and contraction in SMCs of small intrapulmonary airways. We found that 10 µM phorbol-12-myristate-13-acetate or 1 µM phorbol 12,13-dibutyrate induced repetitive, unsynchronized, and transient contractions of the SMCs lining the airway lumen. These contractions were associated with low frequency Ca(2+) oscillations in airway SMCs that resulted from Ca(2+) influx through L-type voltage-gated Ca(2+) channels and the subsequent release of Ca(2+) from intracellular stores through ryanodine receptors. Phorbol ester stimulation of lung slices in which SMC intracellular Ca(2+) concentration ([Ca(2+)](i)) was "clamped" at a high concentration induced strong airway contraction, indicating that PKC mediated sensitization of the contractile response to [Ca(2+)](i). This Ca(2+) sensitization was accompanied by phosphorylation of both the PKC-potentiated PP1 inhibitory protein of 17 kD (CPI-17) and the regulatory myosin light chain. Thrombin, like the phorbol esters, induced a strong Ca(2+) sensitization that was inhibited by the PKC inhibitor GF-109203X and also potentiated airway contraction to membrane depolarization with KCl. In conclusion, we suggest that PKC activation in small airways leads to both the generation of Ca(2+) oscillations and strong Ca(2+) sensitization; agents associated with airway inflammation, such as thrombin, may activate this pathway to sensitize airway smooth muscle to agonists that cause membrane depolarization and Ca(2+) entry and induce airway hyperresponsiveness.

  11. Apical acidity decreases inhibitory effect of omeprazole on Mg2+ absorption and claudin-7 and -12 expression in Caco-2 monolayers (United States)

    Krishnamra, Nateetip


    Clinical studies reported hypomagnesaemia in long-term omeprazole usage that was probably due to intestinal Mg2+ wasting. Our previous report demonstrated the inhibitory effect of omeprazole on passive Mg2+ transport across Caco-2 monolayers. The present study aimed to identify the underlying mechanism of omeprazole suppression of passive Mg2+ absorption. By using Caco-2 monolayers, we demonstrated a potent inhibitory effect of omeprazole on passive Mg2+, but not Ca2+, transport across Caco-2 monolayers. Omeprazole shifted the %maximum passive Mg2+ transport-Mg2+ concentration curves to the right, and increased the half maximal effective concentration of those dose-response curves, indicating a lower Mg2+ affinity of the paracellular channel. By continually monitoring the apical pH, we showed that omeprazole suppressed apical acid accumulation. Neomycin and spermine had no effect on passive Mg2+ transport of either control or omeprazole treated monolayers, indicating that omeprazole suppressed passive Mg2+ transport in a calcium sensing receptor (CaSR)-independent manner. The results of western blot analysis showed that omeprazole significantly suppressed claudin (Cldn)-7 and -12, but not Cldn-2, expression in Caco-2 cells. By using apical solution of pH 5.5, 6.0, 6.5, and 7.0, we found that apical acidity markedly increased passive Mg2+ transport, Mg2+ affinity of the paracellular channel, and Cldn-7 and -12 expression in Caco-2 monolayers. Apical acidity abolished the inhibitory effect of omeprazole on passive Mg2+ transport and Cldn-7 and -12 expression. Our results provided the evidence for the regulation of intestinal passive Mg2+ absorption by luminal acidity-induced increase in Cldn-7 and -12 expression. PMID:22940736

  12. Inhibition of DNA ejection from bacteriophage by Mg+2 counterions (United States)

    Lee, Sell; Tran, C. V.; Nguyen, T. T.


    The problem of inhibiting viral DNA ejection from bacteriophages by multivalent counterions, specifically Mg+2 counterions, is studied. Experimentally, it is known that MgSO4 salt has a strong and nonmonotonic effect on the amount of DNA ejected. There exists an optimal concentration at which the minimum amount of DNA is ejected from the virus. At lower or higher concentrations, more DNA is ejected from the capsid. We propose that this phenomenon is the result of DNA overcharging by Mg+2 multivalent counterions. As Mg+2 concentration increases from zero, the net charge of DNA changes from negative to positive. The optimal inhibition corresponds to the Mg+2 concentration where DNA is neutral. At lower/higher concentrations, DNA genome is charged. It prefers to be in solution to lower its electrostatic self-energy, which consequently leads to an increase in DNA ejection. By fitting our theory to available experimental data, the strength of DNA-DNA short range attraction energies, mediated by Mg+2, is found to be -0.004 kBT per nucleotide base. This and other fitted parameters agree well with known values from other experiments and computer simulations. The parameters are also in agreement qualitatively with values for tri- and tetravalent counterions.

  13. Lentil seed aquaporins form a hetero-oligomer which is phosphorylated by a Mg(2+)-dependent and Ca(2+)-regulated kinase.


    Harvengt, P; Vlerick, A; Fuks, B.; Wattiez, R.; Ruysschaert, J M; Homble, F.


    In plants, aquaporins regulate the water flow through membranes during growth, development and stress responses. We have isolated two isoforms of the aquaporin family from the protein-storage vacuoles of lentil (Lens culinaris Med.) seeds. Chemical cross-linking experiments showed that both isoforms belong to the same oligomer in the membrane and are phosphorylated by a membrane-bound protein kinase. We assigned the kinase activity to a 52 kDa protein that is magnesium-dependent and calcium-r...

  14. Ca2+-regulatory proteins in cardiomyocytes from the right ventricle in children with congenital heart disease

    Directory of Open Access Journals (Sweden)

    Wu Yihe


    Full Text Available Abstract Background Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease (CHD which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Methods Right ventricle tissues were collected from children with tetralogy of Fallot [n = 25, hypoxia and hypertrophy group (HH group], pulmonary stenosis [n = 25, hypertrophy group (H group], or small isolated ventricular septal defect [n = 25, control group (C group] during open-heart surgery. Paraffin sections of tissues were stained with 3,3′-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a, ryanodine receptor (RyR2, sodiumcalcium exchanger (NCX, sarcolipin (SLN and phospholamban (PLN] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase (PP1 were evaluated using western blot. Results Mild cardiomyocyte hypertrophy of the right ventricle in H and HH groups was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P16-phosphorylated PLN was down-regulated (PP Conclusions The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser16 dephosphorylation. Increased PP1 could result in the decreased PLN-Ser16 and inhibition of PP1 is a potential therapeutic target for heart dysfunction in pediatrics.

  15. EF-hand protein Ca2+ buffers regulate Ca2+ influx and exocytosis in sensory hair cells. (United States)

    Pangršič, Tina; Gabrielaitis, Mantas; Michanski, Susann; Schwaller, Beat; Wolf, Fred; Strenzke, Nicola; Moser, Tobias


    EF-hand Ca(2+)-binding proteins are thought to shape the spatiotemporal properties of cellular Ca(2+) signaling and are prominently expressed in sensory hair cells in the ear. Here, we combined genetic disruption of parvalbumin-α, calbindin-D28k, and calretinin in mice with patch-clamp recording, in vivo physiology, and mathematical modeling to study their role in Ca(2+) signaling, exocytosis, and sound encoding at the synapses of inner hair cells (IHCs). IHCs lacking all three proteins showed excessive exocytosis during prolonged depolarizations, despite enhanced Ca(2+)-dependent inactivation of their Ca(2+) current. Exocytosis of readily releasable vesicles remained unchanged, in accordance with the estimated tight spatial coupling of Ca(2+) channels and release sites (effective "coupling distance" of 17 nm). Substitution experiments with synthetic Ca(2+) chelators indicated the presence of endogenous Ca(2+) buffers equivalent to 1 mM synthetic Ca(2+)-binding sites, approximately half of them with kinetics as fast as 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Synaptic sound encoding was largely unaltered, suggesting that excess exocytosis occurs extrasynaptically. We conclude that EF-hand Ca(2+) buffers regulate presynaptic IHC function for metabolically efficient sound coding.

  16. Effect of vanadate and of removal of extracellular Ca2+ and Na+ on tension development and 45Ca efflux in rat and frog myocardium

    DEFF Research Database (Denmark)

    Gesser, H; Bonefeld-Jørgensen, Eva Cecilie


    Vanadate in the range 0-5 mM has positive inotropic effects on myocardial strips of frog and to a lesser extent on those of rat. Inhibiting the sarcolemmal Na+, Ca2+ exchange by a solution free of Ca2+ and Na+ caused a drop in 45Ca efflux and a transient increase in resting tension. These effects...... were more expressed for the frog than for the rat myocardium, which suggests that the Na+ for Ca2+ exchange across the cell membrane is more important in the frog than in the rat myocardium. A subsequent addition of vanadate at 2 or 5 mM had no effect on 45Ca efflux, while it increased the resting...... tension. This increase was higher for the frog than for the rat myocardium. These results suggest that the inotropic effects of vanadate may be due to an effect on membrane-bound Ca2+-ATPase....

  17. A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity (United States)

    Breaker, Ronald R.; Joyce, Gerald F.


    Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

  18. Spectral components of cytosolic [Ca2+] spiking in neurons

    DEFF Research Database (Denmark)

    Kardos, J; Szilágyi, N; Juhász, G


    into evolutionary spectra of a characteristic set of frequencies. Non-delayed small spikes on top of sustained [Ca2+]c were synthesized by a main component frequency, 0.132+/-0.012 Hz, showing its maximal amplitude in phase with the start of depolarization (25 mM KCI) combined with caffeine (10 mM) application......We show here, by means of evolutionary spectral analysis and synthesis of cytosolic Ca2+ ([Ca2+]c) spiking observed at the single cell level using digital imaging fluorescence microscopy of fura-2-loaded mouse cerebellar granule cells in culture, that [Ca2+]c spiking can be resolved...

  19. Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity

    DEFF Research Database (Denmark)

    Bublitz, Maike; Kjellerup, Lasse; Cohrt, Karen O.Hanlon


    We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles...... inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H+-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca2+-ATPase (SERCA) and Na......+,K+-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 Å resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model...

  20. Role of STIM1- and Orai1-mediated Ca2+ entry in Ca2+-induced epidermal keratinocyte differentiation. (United States)

    Numaga-Tomita, Takuro; Putney, James W


    The uppermost thin layer on the surface of the skin, called the epidermis, is responsible for the barrier function of the skin. The epidermis has a multilayered structure in which each layer consists of keratinocytes (KCs) of different differentiation status. The integrity of KC differentiation is crucial for the function of skin and its loss causes or is accompanied by skin diseases. Intracellular and extracellular Ca(2+) is known to play important roles in KC differentiation. However, the molecular mechanisms underlying Ca(2+) regulation of KC differentiation are still largely unknown. Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most non-excitable cells. SOCE is evoked in response to a fall in Ca(2+) concentration in the endoplasmic reticulum. Two proteins have been identified as essential components of SOCE: STIM1, a Ca(2+) sensor in the ER, and Orai1, a subunit of Ca(2+) channels in the plasma membrane. In this study, we analyzed the contribution of SOCE to KC growth and differentiation using RNAi knockdown of STIM1 and Orai1 in the human keratinocyte cell line, HaCaT. KC differentiation was induced by a switch in extracellular Ca(2+) concentration from low (0.03 mM; undifferentiated KCs) to high (1.8 mM; differentiated KCs). This Ca(2+) switch triggers phospholipase-C-mediated intracellular Ca(2+) signals (Ca(2+)-switch-induced Ca(2+) response), which would probably involve the activation of SOCE. Knockdown of either STIM1 or Orai1 strongly suppressed SOCE and almost completely abolished the Ca(2+)-switch-induced Ca(2+) responses, resulting in impaired expression of keratin1, an early KC differentiation marker. Furthermore, loss of either STIM1 or Orai1 suppressed normal growth of HaCaT cells in low Ca(2+) and inhibited the growth arrest in response to a Ca(2+) switch. These results demonstrate that SOCE plays multiple crucial roles in KC differentiation and function.

  1. Ca2+-stores in sperm: their identities and functions (United States)

    Costello, Sarah; Michelangeli, Francesco; Nash, Kate; Lefievre, Linda; Morris, Jennifer; Machado-Oliveira, Gisela; Barratt, Christopher; Kirkman-Brown, Jackson; Publicover, Stephen


    Intracellular Ca2+ stores play a central role in the regulation of cellular [Ca2+]i and the generation of complex [Ca2+] signals such as oscillations and waves. Ca2+ signalling is of particular significance in sperm cells, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis and acrosome reaction) yet mature sperm lack endoplasmic reticulum and several other organelles which serve as Ca2+ stores in somatic cells. Here we review (i) the evidence for the expression in sperm of the molecular components (pumps and channels) which are functionally significant in the activity of Ca2+ stores of somatic cells and (ii) the evidence for the existence of functional Ca2+ stores in sperm. This evidence supports the existence of at least two storage organelles in mammalian sperm, one in the acrosomal region and another in the region of the sperm neck and midpiece. We then go on to discuss the likely identity of these organelles and their discrete functions: regulation by the acrosome of its own secretion and regulation by membranous organelles at the sperm neck (and possibly by the mitochondria) of flagellar activity and hyperactivation. Finally we consider the ability of the sperm discretely to control mobilisation of these stores and the functional interaction of stored Ca2+ at the sperm neck/midpiece with CatSper channels in the principal piece in regulation of the activities of mammalian sperm. PMID:19542252

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

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    Gregor Moenke

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

  3. Purification and biochemical characterization of a Ca2+- ...

    African Journals Online (AJOL)

    On the other hand, Ca+2 and EDTA (1 to 5 mM) did not significantly affect the enzyme activity. The main substrate for the enzyme was found to be starch but it could also hydrolyze raw starch, dextrin, γ-cyclodextrin and pullulan. Key words: Ca2+-independent, Bacillus sp, thermostable α-amylases, low pH profile, enzyme, ...

  4. Spectral components of cytosolic [Ca2+] spiking in neurons

    DEFF Research Database (Denmark)

    Kardos, J; Szilágyi, N; Juhász, G


    . Delayed complex responses of large [Ca2+]c spiking observed in cells from a different set of cultures were synthesized by a set of frequencies within the range 0.018-0.117 Hz. Differential frequency patterns are suggested as characteristics of the [Ca2+]c spiking responses of neurons under different...

  5. Characterization of the Ca2+ Channels Involved in the Progesterone ...

    African Journals Online (AJOL)

    There is evidence that intracellular Ca2+ concentration plays significant roles in sperm function such as motility and acrosome reaction. Many calcium channels have been identified in the plasma membrane of sperm. Progesterone (P4) stimulates Ca2+ influx and acrosome reaction in human spermatozoa. The effects of ...

  6. Low extracellular Ca2+ conditions induce an increase in brain endothelial permeability that involves intercellular Ca2+ waves. (United States)

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


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

  7. A yeast expression system for functional and pharmacological studies of the malaria parasite Ca2+/H+ antiporter

    Directory of Open Access Journals (Sweden)

    Salcedo-Sora J


    Full Text Available Abstract Background Calcium (Ca2+ signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca2+ is tightly regulated through the co-ordinated action of primary and secondary Ca2+ transporters. Identifying selective inhibitors of Ca2+ transporters is key towards understanding their physiological role as well as having therapeutic potential, therefore screening systems to facilitate the search for potential inhibitors are a priority. Here, the methodology for the expression of a Calcium membrane transporter that can be scaled to high throughputs in yeast is presented. Methods The Plasmodium falciparum Ca2+/H+ antiporter (PfCHA was expressed in the yeast Saccharomyces cerevisiae and its activity monitored by the bioluminescence from apoaequorin triggered by divalent cations, such as calcium, magnesium and manganese. Results Bioluminescence assays demonstrated that PfCHA effectively suppressed induced cytoplasmic peaks of Ca2+, Mg2+ and Mn2+ in yeast mutants lacking the homologue yeast antiporter Vcx1p. In the scalable format of 96-well culture plates pharmacological assays with a cation antiporter inhibitor allowed the measurement of inhibition of the Ca2+ transport activity of PfCHA conveniently translated to the familiar concept of fractional inhibitory concentrations. Furthermore, the cytolocalization of this antiporter in the yeast cells showed that whilst PfCHA seems to locate to the mitochondrion of P. falciparum, in yeast PfCHA is sorted to the vacuole. This facilitates the real-time Ca2+-loading assays for further functional and pharmacological studies. Discussion The functional expression of PfCHA in S. cerevisiae and luminescence-based detection of cytoplasmic cations as presented here offer a tractable system that facilitates functional and pharmacological studies in a high-throughput format. PfCHA is shown to behave as a divalent

  8. A Ca2(+ )release-activated Ca2(+) (CRAC) modulatory domain (CMD) within STIM1 mediates fast Ca2(+)-dependent inactivation of ORAI1 channels. (United States)

    Derler, Isabella; Fahrner, Marc; Muik, Martin; Lackner, Barbara; Schindl, Rainer; Groschner, Klaus; Romanin, Christoph


    STIM1 and ORAI1, the two limiting components in the Ca(2+) release-activated Ca(2+) (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2-3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca(2+), a decrease in intracellular Ca(2+) chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba(2+) substitution for extracellular Ca(2+) completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca(2+) entry by triggering fast Ca(2+)-dependent inactivation of ORAI/CRAC channels.

  9. Mice carrying a conditional Serca2flox allele for the generation of Ca2+ handling-deficient mouse models


    Andersson, Kristin B.; Alexandra V Finsen; Sjåland, Cecilie; Winer, Lisbeth H; Sjaastad, Ivar; Ødegaard, Annlaug; Louch, William E.; Wang, Yibin; Chen, Ju; Chien, Kenneth R; Sejersted, Ole M; Christensen, Geir


    Sarco(endo)plasmic reticulum calcium ATPases (SERCA) are cellular pumps that transport Ca2+ into the sarcoplasmic reticulum (SR). Serca2 is the most widely expressed gene family member. The very early embryonic lethality of Serca2null mouse embryos has precluded further evaluation of loss of Serca2 function in the context of organ physiology. We have generated mice carrying a conditional Serca2flox allele which allows disruption of the Serca2 gene in an organ-specific and/or inducible manner....

  10. Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

    KAUST Repository

    Fornander, Louise H


    Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

  11. 45Ca2+movements induced by Ca2+chloride in isolated rat aorta under K+-free conditions

    NARCIS (Netherlands)

    Wermelskirchen, D.; Nebel, U.; Wirth, A.; Wilffert, B.


    Increasing the extracellular Ca2+concentration induced a dihydropyridine-insensitive contraction in the isolated rat aorta bathed in K+-free solution. To obtained further insight into the mechanisms of this contraction45Ca2+uptake measurements were carried out with isolated rat aorta. Increasing the

  12. The plasma membrane Ca2+ pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells. (United States)

    Hegedũs, Luca; Garay, Tamás; Molnár, Eszter; Varga, Karolina; Bilecz, Ágnes; Török, Szilvia; Padányi, Rita; Pászty, Katalin; Wolf, Matthias; Grusch, Michael; Kállay, Enikõ; Döme, Balázs; Berger, Walter; Hegedũs, Balázs; Enyedi, Agnes


    Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca2+ signaling is a well-known regulator of tumor progression, the crosstalk between Ca2+ signaling and the Ras-BRAF-MEK-ERK pathway is much less explored. Here we show that in BRAF mutant melanoma cells the abundance of the plasma membrane Ca2+ ATPase isoform 4b (PMCA4b, ATP2B4) is low at baseline but markedly elevated by treatment with the mutant BRAF specific inhibitor vemurafenib. In line with these findings gene expression microarray data also shows decreased PMCA4b expression in cutaneous melanoma when compared to benign nevi. The MEK inhibitor selumetinib-similarly to that of the BRAF-specific inhibitor-also increases PMCA4b levels in both BRAF and NRAS mutant melanoma cells suggesting that the MAPK pathway is involved in the regulation of PMCA4b expression. The increased abundance of PMCA4b in the plasma membrane enhances [Ca2+ ]i clearance from cells after Ca2+ entry. Moreover we show that both vemurafenib treatment and PMCA4b overexpression induce marked inhibition of migration of BRAF mutant melanoma cells. Importantly, reduced migration of PMCA4b expressing BRAF mutant cells is associated with a marked decrease in their metastatic potential in vivo. Taken together, our data reveal an important crosstalk between Ca2+ signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor. © 2016 UICC.

  13. Decoding Dynamic Ca2+ Signaling in the Vascular Endothelium

    Directory of Open Access Journals (Sweden)

    Mark Stephen Taylor


    Full Text Available Although acute and chronic vasoregulation is inherently driven by endothelial Ca2+, control and targeting of Ca2+-dependent signals are poorly understood. Recent studies have revealed localized and dynamic endothelial Ca2+ events comprising an intricate signaling network along the vascular intima. Discrete Ca2+ transients emerging from both internal stores and plasmalemmal cation channels couple to specific membrane K+ channels, promoting endothelial hyperpolarization and vasodilation. The spatiotemporal tuning of these signals, rather than global Ca2+ elevation, appear to direct endothelial functions under physiologic conditions. In fact, altered patterns of dynamic Ca2+ signaling may underlie essential endothelial dysfunction in a variety of cardiovascular diseases. Advances in imaging approaches and analyses in recent years have allowed for detailed detection, quantification, and evaluation of Ca2+ dynamics in intact endothelium. Here, we discuss recent insights into these signals, including their sources of origination and their functional encoding. We also address key aspects of data acquisition and interpretation, including broad applications of automated high-content analysis.

  14. Intracellular Mg2+ interacts with structural determinants of the narrow constriction contributed by the NR1-subunit in the NMDA receptor channel. (United States)

    Wollmuth, L P; Kuner, T; Sakmann, B


    1. N-methyl-D-aspartate (NMDA) receptor channels are blocked by intracellular Mg2+ in a voltage-dependent manner. Amino acid residues positioned at or near the narrow constriction that interact with intracellular Mg2+ were identified in recombinant NR1-NR2A channels expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. 2. In the absence of extracellular Ca2+, the block of wild-type channel by intracellular Mg2+ measured using macroscopic currents showed a voltage dependence (delta) of around 0.38 and a voltage-independent affinity for the channel of 4 mM. These parameters were independent of the Mg2+ concentration (0.05-10mM), and were indistinguishable from those found for the reduction of single channel amplitudes under the same ionic conditions. Under bionic conditions with high intracellular Mg2+ and K+ extracellularly, Mg2+ was weakly permeant. Mg2+ efflux, however, attenuated the block only at positive potentials (> +80 mV). 3. Substitutions of the N-site asparagine in the NR1-subunit altered intracellular Mg2+ block over physiological membrane potentials (+10 to +50 mV). Substitution of glycine, glutamine or serine attenuated the extent of block whereas the negatively charged aspartate enhanced it, consistent with the side chain of the native asparagine at this position contributing to a blocking site for intracellular Mg2+. 4. Substitutions of the N-site or N + 1 site asparagine in the NR2A-subunit, which form a blocking site for extracellular Mg2+, also altered the block by intracellular Mg2+. However, for the NR2A-subunit N-site asparagine, the block was reduced but only at non-physiological high potentials (> +70 mV). 5. The NR2A-subunit N + 1 site asparagine, which together with NR1-subunit N-site asparagine forms the narrow constriction of the channel, also contributed to a blocking site for intracellular Mg2+. However, it did so to lesser extent than the NR1-subunit N-site and in a manner different from its contribution to a blocking

  15. Inhibition of Brain Swelling after Ischemia-Reperfusion by β-Adrenergic Antagonists: Correlation with Increased K+ and Decreased Ca2+ Concentrations in Extracellular Fluid

    Directory of Open Access Journals (Sweden)

    Dan Song


    Full Text Available Infarct size and brain edema following ischemia/reperfusion are reduced by inhibitors of the Na+, K+, 2Cl−, and water cotransporter NKCC1 and by β1-adrenoceptor antagonists. NKCC1 is a secondary active transporter, mainly localized in astrocytes, driven by transmembrane Na+/K+ gradients generated by the Na+,K+-ATPase. The astrocytic Na+,K+-ATPase is stimulated by small increases in extracellular K+ concentration and by the β-adrenergic agonist isoproterenol. Larger K+ increases, as occurring during ischemia, also stimulate NKCC1, creating cell swelling. This study showed no edema after 3 hr medial cerebral artery occlusion but pronounced edema after 8 hr reperfusion. The edema was abolished by inhibitors of specifically β1-adrenergic pathways, indicating failure of K+-mediated, but not β1-adrenoceptor-mediated, stimulation of Na+,K+-ATPase/NKCC1 transport during reoxygenation. Ninety percent reduction of extracellular Ca2+ concentration occurs in ischemia. Ca2+ omission abolished K+ uptake in normoxic cultures of astrocytes after addition of 5 mM KCl. A large decrease in ouabain potency on K+ uptake in cultured astrocytes was also demonstrated in Ca2+-depleted media, and endogenous ouabains are needed for astrocytic K+ uptake. Thus, among the ionic changes induced by ischemia, the decrease in extracellular Ca2+ causes failure of the high-K+-stimulated Na+,K+-ATPase/NKCC1 ion/water uptake, making β1-adrenergic activation the only stimulus and its inhibition effective against edema.

  16. Structural and magnetic properties of the layered compound Ca2 ...

    Indian Academy of Sciences (India)

    The brownmillerite-type layered compound Ca2.375La0.125Sr0.5GaMn2O8 has ... La3+ is doped at the Ca2+ site in the parent compound Ca2.5Sr0.5GaMn2O8. In .... 0.248(14). 1.03(4). 0.99(9) spins. Along the b-axis, the Mn spin is found to be ferromagnetically coupled to the nearest-neighbour Mn spin in the other MnO6 ...

  17. Requirement of extracellular Ca2+binding to specific amino acids for heat-evoked activation of TRPA1. (United States)

    Kurganov, Erkin; Saito, Shigeru; Tanaka Saito, Claire; Tominaga, Makoto


    We found that extracellular Ca 2+ , but not other divalent cations (Mg 2+ and Ba 2+ ) or intracellular Ca 2+ , is involved in heat-evoked activation of green anole (ga) TRPA1. Heat-evoked activation of chicken (ch) and rat snake (rs) TRPA1 does not depend solely on extracellular Ca 2+ . Neutralization of acidic amino acids on the outer surface of TRPA1 by extracellular Ca 2+ is important for heat-evoked large activation of gaTRPA1, chTRPA1 and rsTRPA1. Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non-selective cation-permeable channel that has six transmembrane domains and cytoplasmic N- and C-termini. The N-terminus is characterized by an unusually large number of ankyrin repeats. Although the 3-dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature-dependent TRPA1 activation is unclear. We previously reported that extracellular Ca 2+ , but not intracellular Ca 2+ , plays an important role in heat-evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca 2+ -dependent heat sensitivity of gaTRPA1 by comparing gaTRPA1 with heat-activated TRPA1 channels from rat snake (rsTRPA1) and chicken (chTRPA1). In the absence of extracellular Ca 2+ , rsTRPA1 and chTRPA1 are activated by heat and generate small inward currents. A comparison of extracellular amino acids in TRPA1 identified three negatively charged amino acid residues (glutamate and aspartate) near the outer pore vestibule that are involved in heat-evoked TRPA1 activation in the presence of extracellular Ca 2+ . These results suggest that neutralization of acidic amino acids by extracellular Ca 2+ is important for heat-evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat-evoked channel activation. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

  18. Envejecimiento Neuronal y Transporte de Ca2+ Intracelular


    Hernando Pérez, Mª Elena


    El envejecimiento promueve pérdida cognitiva y susceptibilidad a enfermedades neurodegenerativas, lo que se ha relacionado con la dishomeostasis del Ca2+ intracelular. Para investigar esta hipótesis se utiliza el cultivo a largo plazo de neuronas de hipocampo de rata, modelo de envejecimiento neuronal in vitro. Mediante imagen de fluorescencia se han estudiado cambios en transporte de Ca2+ en neuronas a lo largo del envejecimiento in vitro. Las neuronas se han identificado mediante inmunofluo...

  19. Shock states of solid Mg2SiO4 (United States)

    Townsend, Joshua; Shulenburger, Luke


    To date there have been thousands of planets discovered outside our solar system. Forsterite, the magnesium end-member of olivine, ((Mg , Fe) 2SiO4) is abundant in the Earth's mantle, and is likely a common planetary building block throughout the galaxy. Despite extensive investigation under terrestrial pressure and temperature regimes, the behavior of the Mg2SiO4 system at higher pressures and temperatures (P>100 GPa, T>4000 K) remains poorly understood. To better understand the behavior of planetary impact processes and the structure of massive planets we investigated the high pressure and high temperature properties of Mg2SiO4 using combined shock compression experiments on the Z-machine at Sandia National Laboratories, and ab-initio molecular dynamics simulations. We compare our results to other recent experiments on shocked forsterite. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under Contract No. DE-AC04-94AL85000. SAND2017-1987 C.

  20. Unraveling Mg2+-RNA binding with atomistic molecular dynamics. (United States)

    Cunha, Richard A; Bussi, Giovanni


    Interaction with divalent cations is of paramount importance for RNA structural stability and function. We report here a detailed molecular dynamics study of all the possible binding sites for Mg2+ on an RNA duplex, including both direct (inner sphere) and indirect (outer sphere) binding. In order to tackle sampling issues, we develop a modified version of bias-exchange metadynamics, which allows us to simultaneously compute affinities with previously unreported statistical accuracy. Results correctly reproduce trends observed in crystallographic databases. Based on this, we simulate a carefully chosen set of models that allows us to quantify the effects of competition with monovalent cations, RNA flexibility, and RNA hybridization. Our simulations reproduce the decrease and increase of Mg2+ affinity due to ion competition and hybridization, respectively, and predict that RNA flexibility has a site-dependent effect. This suggests a nontrivial interplay between RNA conformational entropy and divalent cation binding. © 2017 Cunha and Bussi; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  1. Retinoschisin is linked to retinal Na/K-ATPase signaling and localization. (United States)

    Plössl, Karolina; Royer, Melanie; Bernklau, Sarah; Tavraz, Neslihan N; Friedrich, Thomas; Wild, Jens; Weber, Bernhard H F; Friedrich, Ulrike


    Mutations in the RS1 gene cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy. We recently showed that retinoschisin, the protein encoded by RS1 , regulates ERK signaling and apoptosis in retinal cells. In this study, we explored an influence of retinoschisin on the functionality of the Na/K-ATPase, its interaction partner at retinal plasma membranes. We show that retinoschisin binding requires the β2-subunit of the Na/K-ATPase, whereas the α-subunit is exchangeable. Our investigations revealed no effect of retinoschisin on Na/K-ATPase-mediated ATP hydrolysis and ion transport. However, we identified an influence of retinoschisin on Na/K-ATPase-regulated signaling cascades and Na/K-ATPase localization. In addition to the known ERK deactivation, retinoschisin treatment of retinoschisin-deficient ( Rs1h -/Y ) murine retinal explants decreased activation of Src, an initial transmitter in Na/K-ATPase signal transduction, and of Ca 2+ signaling marker Camk2. Immunohistochemistry on murine retinae revealed an overlap of the retinoschisin-Na/K-ATPase complex with proteins involved in Na/K-ATPase signaling, such as caveolin, phospholipase C, Src, and the IP3 receptor. Finally, retinoschisin treatment altered Na/K-ATPase localization in photoreceptors of Rs1h -/Y retinae. Taken together, our results suggest a regulatory effect of retinoschisin on Na/K-ATPase signaling and localization, whereas Na/K-ATPase-dysregulation caused by retinoschisin deficiency could represent an initial step in XLRS pathogenesis. © 2017 Plössl et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (

  2. Cyclic AMP directs inositol (1,4,5)-trisphosphate-evoked Ca2+ signalling to different intracellular Ca2+ stores


    Tovey, Stephen C.; Taylor, Colin W.


    Cholesterol depletion reversibly abolishes carbachol-evoked Ca2+ release from inositol (1,4,5)-trisphosphate (IP3)-sensitive stores, without affecting the distribution of IP3 receptors (IP3R) or endoplasmic reticulum, IP3 formation or responses to photolysis of caged IP3. Receptors that stimulate cAMP formation do not alone evoke Ca2+ signals, but they potentiate those evoked by carbachol. We show that these potentiated signals are entirely unaffected by cholesterol depletion and that, within...

  3. Ca2+-independent and Ca2+/GTP-binding protein-controlled exocytosis in a plant cell. (United States)

    Homann, U; Tester, M


    Exocytosis allows the release of secretory products and the delivery of new membrane material to the plasma membrane. So far, little is known about the underlying molecular mechanism and its control in plant cells. We have used the whole-cell patch-clamp technique to monitor changes in membrane capacitance to study exocytosis in barley aleurone protoplasts. To investigate the involvement of Ca2+ and GTP-binding proteins in exocytosis, protoplasts were dialyzed with very low (Ca2+ and nonhydrolyzable guanine nucleotides guanosine 5'-gamma-thio]triphosphate (GTP[gammaS]) or guanosine 5'-[beta-thio]diphosphate (GDP[betaS]). With less than 2 nM cytoplasmic free Ca2+, the membrane capacitance increased significantly over 20 min. This increase was not altered by GTP[gammaS] or GDP[betaS]. In contrast, dialyzing protoplasts with 1 microM free Ca2+ resulted in a large increase in membrane capacitance that was slightly reduced by GTP[gammaS] and strongly inhibited by GDP[betaS]. We conclude that two exocytotic pathways exist in barley aleurone protoplasts: one that is Ca2+-independent and whose regulation is currently not known and another that is stimulated by Ca2+ and modulated by GTP-binding proteins. We suggest that Ca2+-independent exocytosis may be involved in cell expansion in developing protoplasts. Ca2+-stimulated exocytosis may play a role in gibberellic acid-stimulated alpha-amylase secretion in barley aleurone and, more generally, may be involved in membrane resealing in response to cell damage.

  4. The mechanism of epipodophyllotoxin-induced thymocyte apoptosis: possible role of a novel Ca(2+)-independent protein kinase. (United States)

    Ye, X; Georgoff, I; Fleisher, S; Coffman, F D; Cohen, S; Fresa, K L


    The epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), inhibit topoisomerase II activity by stabilization of the cleavable complex between the enzyme and DNA and formation of protein-bound double-stranded DNA breaks. While it is thought that these agents are cytotoxic by preventing cells from completing the S phase or undergoing mitosis, recent evidence suggests that these agents are also potent inducers of programmed cell death or apoptosis in both normal and malignant cells. We have examined the intracellular pathway leading to epipodophyllotoxin-induced apoptosis in normal mouse thymocytes. Epipodophyllotoxin-induced apoptosis may proceed via a mechanism that is independent of inhibition of topoisomerase activity per se because novobiocin and coumermycin, which inhibit the ATPase subunit of topoisomerase II, were relatively inefficient inducers of apoptosis in these cells, under conditions where strong apoptosis by the epipodophyllotoxins and dexamethasone could be observed. In addition, camptothecin, which inhibits topoisomerase I by stabilization of the cleavable complex between that enzyme and DNA, was also a poor inducer of apoptosis in these cells. Our data suggest that epipodophyllotoxin-induced mouse thymocyte apoptosis, like that induced by dexamethasone, proceeds via a mechanism that involves protein kinase C (PKC) or a similar enzyme. Apoptosis induced by VM-26 or by dexamethasone was inhibited by 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine dihydrochloride (H7), an inhibitor of both PKC and cAMP-dependent protein kinases, but was relatively unaffected by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor of cAMP-dependent protein kinases. A more specific inhibitor of PKC, sangivamycin, also inhibited both VM-26-induced and dexamethasone-induced apoptosis. Both VM-26- and dexamethasone-induced apoptosis were unaffected by EGTA, a calcium (Ca2+) chelator, under conditions that inhibited apoptosis induced by

  5. Non-stimulated, agonist-stimulated and store-operated Ca2+ influx in MDA-MB-468 breast cancer cells and the effect of EGF-induced EMT on calcium entry.

    Directory of Open Access Journals (Sweden)

    Felicity M Davis

    Full Text Available In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER Ca(2+ reserves, molecular components of the store-operated Ca(2+ entry pathway regulate breast cancer metastasis. A process implicated in cancer metastasis that describes the conversion to a more invasive phenotype is epithelial-mesenchymal transition (EMT. In this study we show that EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a reduction in agonist-stimulated and store-operated Ca(2+ influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca(2+ influx. The potential roles for specific Ca(2+ channels in these pathways were assessed by siRNA-mediated silencing of ORAI1 and transient receptor potential canonical type 1 (TRPC1 channels in MDA-MB-468 breast cancer cells. Non-stimulated, agonist-stimulated and store-operated Ca(2+ influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca(2+ influx in a manner dependent on Ca(2+ influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca(2+ release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca(2+-ATPase (time to peak [Ca(2+](CYT = 188.7 ± 34.6 s (TRPC1 siRNA versus 124.0 ± 9.5 s (non-targeting siRNA; P<0.05. These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca(2+ influx, which may be due to altered ORAI1 and/or TRPC1 channel function. Our findings also suggest that TRPC1 channels in MDA-MB-468 cells contribute to ORAI1-mediated Ca(2+ influx in non-stimulated cells.

  6. Cyclothiazide prolongs low [Mg2+]-induced seizure-like events. (United States)

    Lasztóczi, Bálint; Kardos, Julianna


    Here we address the effects of cyclothiazide (CTZ), an allosteric inhibitor of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor desensitization, on low [Mg(2+)]-induced seizure-like events (SLEs) recorded from the CA3 pyramidal layer of juvenile rat hippocampal slices. CTZ (100 microM) made the period of tonic-like discharges (161 +/- 18% of control) and the whole SLE (151 +/- 15% of control) longer (in 7 of 9 slices) or induced endless SLE by stabilizing clonic-like bursting (in 2 of 9 slices). CTZ (30 microM) had no significant effects on SLE dynamics (n = 4), whereas 300 microM CTZ induced endless SLEs in four of eight slices. Co-application of CTZ (100 microM) with 100 microM GYKI-52466, the allosteric inhibitor of AMPA receptor function, restrained the effects of CTZ and shortened SLEs and their tonic phases to 37 +/- 4.2 and 47 +/- 4.2% of the control, respectively. Effects of GYKI-52466 and GYKI-52466 with CTZ on SLE dynamics were indistinguishable. 4-aminopyridine (4-AP; 50 microM) alone (n = 5) or in combination with CTZ (n = 6) transformed recurrent SLE pattern into incessant epileptiform activity with patterns distinguishable from those under 100 microM CTZ application. The effect of 4-AP may suggest a role for facilitated presynaptic glutamate release in disrupting recurrent dynamics. In contrast, the self-similar slow-down of low [Mg(2+)]-induced SLE dynamics by CTZ indicate AMPA receptor desensitization as a parameter shaping SLEs.

  7. Cinética de absorção e eficiência nutricional de K+, Ca2+ e Mg2+ em plantas jovens de quatro clones de eucalipto Uptake kinetics and nutritional efficiency for K+, Ca2+ and Mg2+ in four eucalypt clones seedlings

    Directory of Open Access Journals (Sweden)

    Augusto Miguel Nascimento Lima


    Full Text Available Modelos mecanísticos baseados em princípios de transporte de solutos pode ser de grande utilidade para prever os impactos do cultivo de florestas plantadas, por exemplo, com eucalipto sobre o capital e fluxo de nutrientes no solo. Dentre as variáveis de entrada demandadas por tais modelos, tem-se os valores das constantes Vmax, Km e Cmin da cinética de absorção iônica. Assim, os objetivos do presente trabalho foram determinar os valores de Vmax, Km e Cmin para K, Ca e Mg, bem como avaliar as respectivas eficiências nutricionais de clones de eucalipto. O estudo consistiu de três ensaios em solução nutritiva (um para cada cátion, sendo utilizadas mudas propagadas vegetativamente de um híbrido de E. grandis x E. urophylla (clone 1213 e de três híbridos de E. grandis (clones 7074, 57 e 129. Com base nos teores de cada um desses nutrientes nas soluções de depleção, em cada tempo de amostragem, no volume inicial e final de solução nos vasos e no peso de matéria fresca de raízes, foram obtidos os valores das constantes cinéticas. Para K, o clone 7074 apresentou o menor valor de Vmax em relação aos demais clones, os quais não diferiram entre si, e em relação ao Km e Cmin, os clones não diferiram estatisticamente. Para Ca, os clones estudados diferiram quanto ao valor de Vmax e Km, não diferindo, entretanto, para o Cmin. Os menores valores de Km para Mg foram verificados para os clones 57 e 7074, ou seja, as proteínas transportadoras de Mg na membrana plasmática das células radiculares apresentaram maior afinidade para esse nutriente. Contudo, os valores de Vmax e Cmin não diferiram entre os clones estudados. Diferenças na eficiência nutricional dos clones estudados quanto a K e a Ca foram devidas às diferenças na eficiência de absorção, e para Mg às diferenças na eficiência de absorção e de utilização.The use of mechanistic models based on principles of solute transport may be of great utility to estimate, for example, the impact of eucalypt forest cultivation on the pools and fluxes of nutrient in soils. Among the parameters required by such models are the values of the ion absorption kinetics constants Vmax, Km and Cmin. Thus, the objective of this study was to determine the values of Vmax, Km and Cmin for K, Ca and Mg, as well as to evaluate the nutritional efficiency of eucalypt clones to these nutrients. The present study consisted of three experiments in solution culture (one for each cation. It was employed four genetic materials (clones 7074, 57, 1213 and 129 originated from clonal propagation. Clone 1213 is an Eucalyptus grandis x Eucalyptus urophylla hybrid, whereas the others are natural hybrids of E. grandis. The concentration of each nutrient being studied in the depletion solution at specific sampling times, initial and final pot solution volume and fresh root weight were used in order to obtain the value of the kinetics constants. For K, clone 7074 showed the lowest Vmax value in relation to the other clones, which did not differ among themselves. All clones did not differ statistically in relation to Km and Cmin for K. For Ca, the studied clones differed in relation to the values of Vmax and Km but, they did not differ for Cmin. The lower values of Km for Mg were observed for the clones 57 and 7074, that is, the carrier proteins at the root cells plasma membrane have a high affinity for this nutrient. Nevertheless, the values of Vmax and Cmin did not differ among the studied clones. Differences in nutritional efficiency of the studied clones in relation to K and Ca were due to differences in the absorption efficiency and for Mg were related with differences in the nutrient use efficiency and absorption efficiency.

  8. Ca2+ signaling in pancreatic acinar cells: physiology and pathophysiology

    Directory of Open Access Journals (Sweden)

    O.H. Petersen


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

  9. Synaptically activated Ca2+ waves and NMDA spikes locally suppress voltage-dependent Ca2+ signalling in rat pyramidal cell dendrites. (United States)

    Manita, Satoshi; Miyazaki, Kenichi; Ross, William N


    Postsynaptic [Ca(2+)](i) changes contribute to several kinds of plasticity in pyramidal neurons. We examined the effects of synaptically activated Ca(2+) waves and NMDA spikes on subsequent Ca(2+) signalling in CA1 pyramidal cell dendrites in hippocampal slices. Tetanic synaptic stimulation evoked a localized Ca(2+) wave in the primary apical dendrites. The [Ca(2+)](i) increase from a backpropagating action potential (bAP) or subthreshold depolarization was reduced if it was generated immediately after the wave. The suppression had a recovery time of 30-60 s. The suppression only occurred where the wave was generated and was not due to a change in bAP amplitude or shape. The suppression also could be generated by Ca(2+) waves evoked by uncaging IP(3), showing that other signalling pathways activated by the synaptic tetanus were not required. The suppression was proportional to the amplitude of the [Ca(2+)](i) change of the Ca(2+) wave and was not blocked by a spectrum of kinase or phosphatase inhibitors, consistent with suppression due to Ca(2+)-dependent inactivation of Ca(2+) channels. The waves also reduced the frequency and amplitude of spontaneous, localized Ca(2+) release events in the dendrites by a different mechanism, probably by depleting the stores at the site of wave generation. The same synaptic tetanus often evoked NMDA spike-mediated [Ca(2+)](i) increases in the oblique dendrites where Ca(2+) waves do not propagate. These NMDA spikes suppressed the [Ca(2+)](i) increase caused by bAPs in those regions. [Ca(2+)](i) increases by Ca(2+) entry through voltage-gated Ca(2+) channels also suppressed the [Ca(2+)](i) increases from subsequent bAPs in regions where the voltage-gated [Ca(2+)](i) increases were largest, showing that all ways of raising [Ca(2+)](i) could cause suppression.

  10. Activation of gill Ca2+-sensing receptor as a protective pathway to reduce Ca2+-induced cytotoxicity. (United States)

    Gu, J; Law, A Y S; Yeung, B H Y; Wong, C K C


    The expression of the Ca(2) (+)-sensing receptor (Casr) in the endocrine gland known as the corpuscle of Stannius (CS) regulates the secretion of the hypocalcemic hormone stanniocalcin-1 (STC1) to inhibit gill Ca(2) (+) uptake. Although numerous studies have reported the branchial expression of Casr and Stc1, the functions of these proteins in gills have not been elucidated yet. On the basis of recent findings regarding the autocrine/paracrine functions of STC1 in mammalian models, we proposed the hypothesis that branchial CaSR has an in situ 'sensing' function to regulate STC1 that maintains local Ca(2) (+) homeostasis. In this study, we investigated Casr-mediated signaling and its regulation of Stc1 and cyclooxygenase-2 (Cox2) expression/function using a primary gill-cell culture model. The biochemical responses of gill cells isolated from Japanese eels to an increasing concentration of extracellular Ca(2) (+) (0.1-1 mM) were tested. This stimulation led to a transient increase in phosphatidylcholine-phospholipase C (PC-PLC) activity, followed by activation of ERK and inositol 1,4,5-trisphosphate-Ca(2) (+)/calmodulin-dependent protein kinase 2 (CaMK2) signaling pathways. Cotreatment with the calcimimetic R467 caused synergistic effects on Ca(2) (+)-stimulated PC-PLC activity, ERK signaling, and CaMK2 signaling. The activation of the CaSR-PLC-ERK pathway was associated with increased expression levels of Stc1 and Cox2 as confirmed by the inhibition of Erk using a chemical inhibitor, PD98059. Functionally, Ca(2) (+)/R-467 pretreatment was found to protect cells from thapsigargin-induced cell death. Inhibition of COX2 activity using NS398 abolished this protection, while transduction of STC1 lentiviral particles in the gill cells increased the protective effects. Collectively, our data revealed the expression of functional CaSR in gill tissues. The identification of the CaSR-STC1/COX2-mediated protective pathway in gill cells sheds light on a possible cellular

  11. Delta(9)-tetrahydrocannabinol activates [Ca2+], increases partly sensitive to capacitative store refilling

    NARCIS (Netherlands)

    Filipeanu, CM; deZeeuw, D; Nelemans, SA


    Delta(9)-Tetrahydrocannabinol induces [Ca2+](i) increases in DDT1MF-2 smooth muscle cells. Both Ca2+ entry and release from intracellular Ca2+ stores were concentration dependently activated. The Ca2+ entry component contributed most to the increases in [Ca2+](i). Stimulation with

  12. Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET (United States)

    Ernst, Stefan; Batisse, Claire; Zarrabi, Nawid; Böttcher, Bettina; Börsch, Michael


    We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

  13. Cell surface topology creates high Ca2+ signalling microdomains

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B


    of a smooth cell surface predicts only moderate localized effects, the more realistic "wrinkled" surface topology predicts that Ca2+ concentrations up to 80 microM can persist within the folds of membranes for significant times. This intra-wrinkle location may account for 5% of the total cell volume. Using...... different geometries of wrinkles, our simulations show that high Ca2+ microdomains will be generated most effectively by long narrow membrane wrinkles of similar dimensions to those found experimentally. This is a new concept which has not previously been considered, but which has ramifications as the intra-wrinkle...

  14. Minocycline chelates Ca2+, binds to membranes, and depolarizes mitochondria by formation of Ca2+-dependent ion channels (United States)

    Antonenko, Yuri N.; Rokitskaya, Tatyana I.; Cooper, Arthur J. L.


    Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca2+-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates Ca2+ ions. Secondly, minocycline, in a Ca2+-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show that minocycline, in the presence of Ca2+, induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca2+-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition. PMID:20180001

  15. Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte (United States)

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


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

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

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


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

  17. Ventricular performance and Na+-K+ ATPase activity are reduced early and late after myocardial infarction in rats

    Directory of Open Access Journals (Sweden)

    I. Stefanon


    Full Text Available Myocardial infarction leads to compensatory ventricular remodeling. Disturbances in myocardial contractility depend on the active transport of Ca2+ and Na+, which are regulated by Na+-K+ ATPase. Inappropriate regulation of Na+-K+ ATPase activity leads to excessive loss of K+ and gain of Na+ by the cell. We determined the participation of Na+-K+ ATPase in ventricular performance early and late after myocardial infarction. Wistar rats (8-10 per group underwent left coronary artery ligation (infarcted, Inf or sham-operation (Sham. Ventricular performance was measured at 3 and 30 days after surgery using the Langendorff technique. Left ventricular systolic pressure was obtained under different ventricular diastolic pressures and increased extracellular Ca2+ concentrations (Ca2+e and after low and high ouabain concentrations. The baseline coronary perfusion pressure increased 3 days after myocardial infarction and normalized by 30 days (Sham 3 = 88 ± 6; Inf 3 = 130 ± 9; Inf 30 = 92 ± 7 mmHg; P < 0.05. The inotropic response to Ca2+e and ouabain was reduced at 3 and 30 days after myocardial infarction (Ca2+ = 1.25 mM; Sham 3 = 70 ± 3; Inf 3 = 45 ± 2; Inf 30 = 29 ± 3 mmHg; P < 0.05, while the Frank-Starling mechanism was preserved. At 3 and 30 days after myocardial infarction, ventricular Na+-K+ ATPase activity and contractility were reduced. This Na+-K+ ATPase hypoactivity may modify the Na+, K+ and Ca2+ transport across the sarcolemma resulting in ventricular dysfunction.

  18. Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients. (United States)

    Pelzl, Lisann; Elsir, Bhaeldin; Sahu, Itishri; Bissinger, Rosi; Singh, Yogesh; Sukkar, Basma; Honisch, Sabina; Schoels, Ludger; Jemaà, Mohamed; Lang, Elisabeth; Storch, Alexander; Hermann, Andreas; Stournaras, Christos; Lang, Florian


    The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment. © 2017 The

  19. Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients

    Directory of Open Access Journals (Sweden)

    Lisann Pelzl


    Full Text Available Background: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A causes chorea-acanthocytosis (ChAc, a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE. Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. Methods: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA inhibitor thapsigargin (1 µM, and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. Results: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. Conclusions: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts

  20. Cuscuta reflexa invasion induces Ca2+ release in its host

    NARCIS (Netherlands)

    Albert, M.; Krol, van der A.R.; Kaldenhoff, R.


    Cuscuta reflexa induces a variety of reaction in its hosts. Some of these are visual reactions, and it is clear that these morphological changes are preceded by events at the molecular level, where signal transduction is one of the early processes. Calcium (Ca(2+)) release is the major second

  1. Enhanced antioxidant defense after exogenous application of Ca 2+ ...

    African Journals Online (AJOL)

    Both of these nutrients play an important role in ameliorating drought stress in crop plants. This experiment was designed to study whether exogenous application of Ca2+ and K+ before the drought could enhance the potential of plants to survive under limiting water conditions. Brassica napus L. cv Bulbul-98 seedlings ...

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

    Directory of Open Access Journals (Sweden)

    F.R. Giachini


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

  3. A model of cardiac ryanodine receptor gating predicts experimental Ca2+-dynamics and Ca2+-triggered arrhythmia in the long QT syndrome (United States)

    Wilson, Dan; Ermentrout, Bard; Němec, Jan; Salama, Guy


    Abnormal Ca2+ handling is well-established as the trigger of cardiac arrhythmia in catecholaminergic polymorphic ventricular tachycardia and digoxin toxicity, but its role remains controversial in Torsade de Pointes (TdP), the arrhythmia associated with the long QT syndrome (LQTS). Recent experimental results show that early afterdepolarizations (EADs) that initiate TdP are caused by spontaneous (non-voltage-triggered) Ca2+ release from Ca2+-overloaded sarcoplasmic reticulum (SR) rather than the activation of the L-type Ca2+-channel window current. In bradycardia and long QT type 2 (LQT2), a second, non-voltage triggered cytosolic Ca2+ elevation increases gradually in amplitude, occurs before overt voltage instability, and then precedes the rise of EADs. Here, we used a modified Shannon-Puglisi-Bers model of rabbit ventricular myocytes to reproduce experimental Ca2+ dynamics in bradycardia and LQT2. Abnormal systolic Ca2+-oscillations and EADs caused by SR Ca2+-release are reproduced in a modified 0-dimensional model, where 3 gates in series control the ryanodine receptor (RyR2) conductance. Two gates control RyR2 activation and inactivation and sense cytosolic Ca2+ while a third gate senses luminal junctional SR Ca2+. The model predicts EADs in bradycardia and low extracellular [K+] and cessation of SR Ca2+-release terminate salvos of EADs. Ca2+-waves, systolic cell-synchronous Ca2+-release, and multifocal diastolic Ca2+ release seen in subcellular Ca2+-mapping experiments are observed in the 2-dimensional version of the model. These results support the role of SR Ca2+-overload, abnormal SR Ca2+-release, and the subsequent activation of the electrogenic Na+/Ca2+-exchanger as the mechanism of TdP. The model offers new insights into the genesis of cardiac arrhythmia and new therapeutic strategies.

  4. Distinct contributions of Orai1 and TRPC1 to agonist-induced [Ca(2+](i signals determine specificity of Ca(2+-dependent gene expression.

    Directory of Open Access Journals (Sweden)

    Hwei Ling Ong

    Full Text Available Regulation of critical cellular functions, including Ca(2+-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca(2+ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca(2+](i due to intracellular Ca(2+ release and Ca(2+ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca(2+](i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca(2+](i increases in human submandibular gland (HSG cells. However, only Orai1-mediated Ca(2+ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca(2+ store depletion in these cells, it is not clear how the cells decode individual Ca(2+ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh-induced [Ca(2+](i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca(2+ entry generates [Ca(2+](i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca(2+ entry generates sustained [Ca(2+](i elevation at high [CCh] and contributes to frequency of [Ca(2+](i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca(2+ dependent gene expression pathways, consistent with the different patterns of [Ca(2+](i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display "all-or-none" activation that is exclusively driven by local [Ca(2+](i generated by Orai1, independent of global [Ca(2+](i changes or TRPC1-mediated Ca(2+ entry. In contrast, Ca(2+ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca(2+ signals

  5. Tracking Ca2+-dependent and Ca2+-independent conformational transitions in syntaxin 1A during exocytosis in neuroendocrine cells. (United States)

    Greitzer-Antes, Dafna; Barak-Broner, Noa; Berlin, Shai; Oron, Yoram; Chikvashvili, Dodo; Lotan, Ilana


    A key issue for understanding exocytosis is elucidating the various protein interactions and the associated conformational transitions underlying soluble N-ethylmeleimide-sensitive factor attachment protein receptor (SNARE) protein assembly. To monitor dynamic changes in syntaxin 1A (Syx) conformation along exocytosis, we constructed a novel fluorescent Syx-based probe that can be efficiently incorporated within endogenous SNARE complexes, support exocytosis, and report shifts in Syx between 'closed' and 'open' conformations by fluorescence resonance energy transfer analysis. Using this probe we resolve two distinct Syx conformational transitions during membrane depolarization-induced exocytosis in PC12 cells: a partial 'opening' in the absence of Ca(2+) entry and an additional 'opening' upon Ca(2+) entry. The Ca(2+)-dependent transition is abolished upon neutralization of the basic charges in the juxtamembrane regions of Syx, which also impairs exocytosis. These novel findings provide evidence of two conformational transitions in Syx during exocytosis, which have not been reported before: one transition directly induced by depolarization and an additional transition that involves the juxtamembrane region of Syx. The superior sensitivity of our probe also enabled detection of subtle Syx conformational changes upon interaction with VAMP2, which were absolutely dependent on the basic charges of the juxtamembrane region. Hence, our results further suggest that the Ca(2+)-dependent transition in Syx involves zippering between the membrane-proximal juxtamembrane regions of Syx and VAMP2 and support the recently implied existence of this zippering in the final phase of SNARE assembly to catalyze exocytosis.

  6. Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function. (United States)

    Concepcion, Axel R; Vaeth, Martin; Wagner, Larry E; Eckstein, Miriam; Hecht, Lee; Yang, Jun; Crottes, David; Seidl, Maximilian; Shin, Hyosup P; Weidinger, Carl; Cameron, Scott; Turvey, Stuart E; Issekutz, Thomas; Meyts, Isabelle; Lacruz, Rodrigo S; Cuk, Mario; Yule, David I; Feske, Stefan


    Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release-activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel-deficient patients and mice with ectodermal tissue-specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

  7. The effect of sulfate on CaCO_{3} mineralogy and the incorporation of Mg^{2+} (United States)

    Goetschl, Katja Elisabeth; Mavromatis, Vasileios; Dietzel, Martin


    Sulfate is the second most concentrated anion in seawater, and it forms strong complexes with Ca2+ and Mg2+ in aqueous solutions. However, up to now not much is known about the effect of dissolved SO42- on the formation of calcium carbonate minerals. For example, it has earlier been documented that the primary CaCO3 polymorph in marine sediments was oscillating between calcite and aragonite throughout the Phanerozoic, reflecting changes in seawater chemistry, among them SO42- concentration. Most of the previous experimental work, however, has focused mainly on the Mg/Ca ratio in seawater as the major driver for a preferential formation of calcite or aragonite. It has thus been shown that Mg substitution affects the thermodynamic stability of calcite, causing precipitation of aragonite instead of calcite at an aqueous Mg/Ca ratio higher than ˜1.3 [1]. To shed light on the mechanisms controlling the calcium carbonate mineral formation in the presence of sulfate, we performed steady-state precipitation experiments with calcite seed material at 25 ˚ C. Furthermore in this study we examine the Mg partitioning and isotope fractionation during its incorporation in calcite. For this, throughout the experimental runs, the aqueous Mg concentration was kept constant at different growth rates and different concentrations of dissolved SO42- have been used. Our data show that the presence of sulfate induces the formation of aragonite on calcite seeds at elevated growth rates, whereas we see no formation of aragonite in the experiments performed in the absence of sulfate, but at the same Mg/Ca ratio. On a further step the effect of sulfate on Mg partitioning and stable isotope fractionation of Mg in calcite will be examined. These results will improve the general understanding of the controls of solution chemistry on the formation of CaCO3 minerals and in specific the effect of sulfate on the incorporation of Mg into the crystal lattice. [1] Morse et al., 1997, Geology

  8. High extracellular Ca2+ stimulates Ca2+-activated Cl- currents in frog parathyroid cells through the mediation of arachidonic acid cascade.

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    Yukio Okada

    Full Text Available Elevation of extracellular Ca(2+ concentration induces intracellular Ca(2+ signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca(2+ pathways, but the direct mechanism responsible for the rise of intracellular Ca(2+ concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca(2+ signaling in frog parathyroid cells and show that Ca(2+-activated Cl(- channels are activated by intracellular Ca(2+ increase through an inositol 1,4,5-trisphophate (IP(3-independent pathway. High extracellular Ca(2+ induced an outwardly-rectifying conductance in a dose-dependent manner (EC(50 ∼6 mM. The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca(2+-induced and Ca(2+ dialysis-induced currents reversed at the equilibrium potential of Cl(- and were inhibited by niflumic acid (a specific blocker of Ca(2+-activated Cl(- channel. Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca(2+-induced current, suggesting the change of intracellular Cl(- concentration in a few minutes. Extracellular Ca(2+-induced currents displayed a moderate dependency on guanosine triphosphate (GTP. All blockers for phospholipase C, diacylglycerol (DAG lipase, monoacylglycerol (MAG lipase and lipoxygenase inhibited extracellular Ca(2+-induced current. IP(3 dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG, arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S-HPETE dialysis increased the conductance identical to extracellular Ca(2+-induced conductance. These results indicate that high extracellular Ca(2+ raises intracellular Ca(2+ concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl(- conductance.

  9. Effect of [10]-Gingerol on [Ca2+]i and Cell Death in Human Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Chung-Yi Chen


    Full Text Available The effect of [10]-gingerol on cytosol free Ca2+ concentration ([Ca2+]i and viability is large unknown. This study examines the early signaling effects of [10]-gingerol on human colorectal cancer cells. It was found that this compound caused a slow and sustained rise of [Ca2+]i in a concentration-dependent manner. [10]-Gingerol also induced a [Ca2+]i rise when extracellular Ca2+ was removed, but the magnitude was reduced by 38%. In a Ca2+-free medium, the [10]-gingerol-induced [Ca2+]i rise was partially abolished by depleting stored Ca2+ with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor. The elevation of [10]-gingerol-caused [Ca2+]i in a Ca2+-containing medium was not affected by modulation of protein kinase C activity. The [10]-gingerol-induced Ca2+ influx was insensitive to L-type Ca2+ channel blockers. At concentrations of 10-100 mM, [10]-gingerol killed cells in a concentration-dependent manner. These findings suggest that [10]-gingerol induces [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from non-L-type Ca2+ channels in SW480 cancer cells.

  10. The permeability transition pore as a Ca(2+) release channel: new answers to an old question. (United States)

    Bernardi, Paolo; von Stockum, Sophia


    Mitochondria possess a sophisticated array of Ca(2+) transport systems reflecting their key role in physiological Ca(2+) homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca(2+) uptake, the ruthenium red (RR)-sensitive mitochondrial Ca(2+) uniporter (MCU); and one main mechanism for Ca(2+) release, the RR-insensitive 3Na(+)-Ca(2+) antiporter. An additional mechanism for Ca(2+) release is provided by a Na(+) and RR-insensitive release mechanism, the putative 3H(+)-Ca(2+) antiporter. A potential kinetic imbalance is present, however, because the V(max) of the MCU is of the order of 1400nmol Ca(2+)mg(-1) proteinmin(-1) while the combined V(max) of the efflux pathways is about 20nmol Ca(2+)mg(-1) proteinmin(-1). This arrangement exposes mitochondria to the hazards of Ca(2+) overload when the rate of Ca(2+) uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca(2+)]. In this short review we discuss the hypothesis that transient opening of the Ca(2+)-dependent permeability transition pore may provide mitocondria with a fast Ca(2+) release channel preventing Ca(2+) overload. We also address the relevance of a mitochondrial Ca(2+) release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. The permeability transition pore as a Ca2+ release channel: New answers to an old question (United States)

    Bernardi, Paolo; von Stockum, Sophia


    Mitochondria possess a sophisticated array of Ca2+ transport systems reflecting their key role in physiological Ca2+ homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca2+ uptake, the ruthenium red (RR)-sensitive mitochondrial Ca2+ uniporter (MCU); and one main mechanism for Ca2+ release, the RR-insensitive 3Na+–Ca2+ antiporter. An additional mechanism for Ca2+ release is provided by a Na+ and RR-insensitive release mechanism, the putative 3H+–Ca2+ antiporter. A potential kinetic imbalance is present, however, because the Vmax of the MCU is of the order of 1400 nmol Ca2+ mg−1 protein min−1 while the combined Vmax of the efflux pathways is about 20 nmol Ca2+ mg−1 protein min−1. This arrangement exposes mitochondria to the hazards of Ca2+ overload when the rate of Ca2+ uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca2+]. In this short review we discuss the hypothesis that transient opening of the Ca2+-dependent permeability transition pore may provide mitocondria with a fast Ca2+ release channel preventing Ca2+ overload. We also address the relevance of a mitochondrial Ca2+ release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals. PMID:22513364

  12. Crystal chemistry of ordered rocksalt-type Ca2NF (United States)

    Al-Azzawi, Mohanad; Zeller, Matthias; Li, Dingqiang; Wagner, Timothy R.


    Ordered rocksalt-type Ca2NF has a cubic unit cell that is doubled along [100] relative to the rocksalt-type polymorph due to N/F ordering. Crystals for the present study were prepared from a pure, dry KCuF3 precursor by reaction with Ca metal under nitrogen, and analyzed via high resolution single crystal X-ray diffraction. From this analysis, as well as qualitative compositional analysis using energy dispersive spectroscopy, it was determined that interstitial fluoride ions previously interpreted as Frenkel defects are actually due to a non-stoichiometric defect. The refined composition is Ca2N0.925F1.23, and the ordered cubic phase has space group Fd 3 bar m (No. 227) with a = 10.0301(5) Å and Z = 16.

  13. Inhibiting the Ca2+ Influx Induced by Human CSF

    Directory of Open Access Journals (Sweden)

    Anna Drews


    Full Text Available One potential therapeutic strategy for Alzheimer’s disease (AD is to use antibodies that bind to small soluble protein aggregates to reduce their toxic effects. However, these therapies are rarely tested in human CSF before clinical trials because of the lack of sensitive methods that enable the measurement of aggregate-induced toxicity at low concentrations. We have developed highly sensitive single vesicle and single-cell-based assays that detect the Ca2+ influx caused by the CSF of individuals affected with AD and healthy controls, and we have found comparable effects for both types of samples. We also show that an extracellular chaperone clusterin; a nanobody specific to the amyloid-β peptide (Aβ; and bapineuzumab, a humanized monoclonal antibody raised against Aβ, could all reduce the Ca2+ influx caused by synthetic Aβ oligomers but are less effective in CSF. These assays could be used to characterize potential therapeutic agents in CSF before clinical trials.

  14. Crosstalk between mitochondrial and sarcoplasmic reticulum Ca2+ cycling modulates cardiac pacemaker cell automaticity.

    Directory of Open Access Journals (Sweden)

    Yael Yaniv

    Full Text Available Mitochondria dynamically buffer cytosolic Ca(2+ in cardiac ventricular cells and this affects the Ca(2+ load of the sarcoplasmic reticulum (SR. In sinoatrial-node cells (SANC the SR generates periodic local, subsarcolemmal Ca(2+ releases (LCRs that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+-Ca(2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP.To determine if mitochondrial Ca(2+ (Ca(2+ (m, cytosolic Ca(2+ (Ca(2+ (c-SR-Ca(2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.Inhibition of mitochondrial Ca(2+ influx into (Ru360 or Ca(2+ efflux from (CGP-37157 decreased [Ca(2+](m to 80 ± 8% control or increased [Ca(2+](m to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+ influx or efflux, the SR Ca(2+ load, and LCR size, duration, amplitude and period (imaged via confocal linescan significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+ signal were highly correlated with the change in the SR Ca(2+ load (r(2 = 0.97. Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control in response to changes in [Ca(2+](m were predicted by concurrent changes in LCR period (r(2 = 0.84.A change in SANC Ca(2+ (m flux translates into a change in the AP firing rate by effecting changes in Ca(2+ (c and SR Ca(2+ loading, which affects the characteristics of spontaneous SR Ca(2+ release.


    DEFF Research Database (Denmark)

    Andersen, A.; Cornett, Claus; Lauridsen, A.


    The importance of the sesquiterpene lactone thapsigargin as a tool for studying Ca2+ homeostasis has created a need for structure-activity relationship studies and consequently for procedures for selective transformations of the functional groups in the molecule. Methods for the selective inversi...... of configuration at C-3 and C-8, selective acetylation, and selective cleavage of the ester groups at 0-3, 0-8 and 0-10 are presented....

  16. Active transport of Ca2+ by an artificial photosynthetic membrane. (United States)

    Bennett, Ira M; Farfano, Hebe M Vanegas; Bogani, Federica; Primak, Alex; Liddell, Paul A; Otero, Luis; Sereno, Leonides; Silber, Juana J; Moore, Ana L; Moore, Thomas A; Gust, Devens


    Transport of calcium ions across membranes and against a thermodynamic gradient is essential to many biological processes, including muscle contraction, the citric acid cycle, glycogen metabolism, release of neurotransmitters, vision, biological signal transduction and immune response. Synthetic systems that transport metal ions across lipid or liquid membranes are well known, and in some cases light has been used to facilitate transport. Typically, a carrier molecule located in a symmetric membrane binds the ion from aqueous solution on one side and releases it on the other. The thermodynamic driving force is provided by an ion concentration difference between the two aqueous solutions, coupling to such a gradient in an auxiliary species, or photomodulation of the carrier by an asymmetric photon flux. Here we report a different approach, in which active transport is driven not by concentration gradients, but by light-induced electron transfer in a photoactive molecule that is asymmetrically disposed across a lipid bilayer. The system comprises a synthetic, light-driven transmembrane Ca2+ pump based on a redox-sensitive, lipophilic Ca2+-binding shuttle molecule whose function is powered by an intramembrane artificial photosynthetic reaction centre. The resulting structure transports calcium ions across the bilayer of a liposome to develop both a calcium ion concentration gradient and a membrane potential, expanding Mitchell's concept of a redox loop mechanism for protons to include divalent cations. Although the quantum yield is relatively low (approximately 1 per cent), the Ca2+ electrochemical potential developed is significant.

  17. Vasopressin inhibits LTP in the CA2 mouse hippocampal area.

    Directory of Open Access Journals (Sweden)

    Magda Chafai

    Full Text Available Growing evidence points to vasopressin (AVP as a social behavior regulator modulating various memory processes and involved in pathologies such as mood disorders, anxiety and depression. Accordingly, AVP antagonists are actually envisaged as putative treatments. However, the underlying mechanisms are poorly characterized, in particular the influence of AVP on cellular or synaptic activities in limbic brain areas involved in social behavior. In the present study, we investigated AVP action on the synapse between the entorhinal cortex and CA2 hippocampal pyramidal neurons, by using both field potential and whole-cell recordings in mice brain acute slices. Short application (1 min of AVP transiently reduced the synaptic response, only following induction of long-term potentiation (LTP by high frequency stimulation (HFS of afferent fibers. The basal synaptic response, measured in the absence of HFS, was not affected. The Schaffer collateral-CA1 synapse was not affected by AVP, even after LTP, while the Schaffer collateral-CA2 synapse was inhibited. Although investigated only recently, this CA2 hippocampal area appears to have a distinctive circuitry and a peculiar role in controlling episodic memory. Accordingly, AVP action on LTP-increased synaptic responses in this limbic structure may contribute to the role of this neuropeptide in controlling memory and social behavior.

  18. Possible involvement of transient receptor potential ankyrin 1 in Ca2+signaling via T-type Ca2+channel in mouse sensory neurons. (United States)

    Nishizawa, Yuki; Takahashi, Kenji; Oguma, Naoko; Tominaga, Makoto; Ohta, Toshio


    T-type Ca 2+ channels and TRPA1 are expressed in sensory neurons and both are associated with pain transmission, but their functional interaction is unclear. Here we demonstrate that pharmacological evidence of the functional relation between T-type Ca 2+ channels and TRPA1 in mouse sensory neurons. Low concentration of KCl at 15 mM (15K) evoked increases of intracellular Ca 2+ concentration ([Ca 2+ ] i ), which were suppressed by selective T-type Ca 2+ channel blockers. RT-PCR showed that mouse sensory neurons expressed all subtypes of T-type Ca 2+ channel. The magnitude of 15K-induced [Ca 2+ ] i increase was significantly larger in neurons sensitive to allylisothiocyanate (AITC, a TRPA1 agonist) than in those insensitive to it, and in TRPA1 -/- mouse sensory neurons. TRPA1 blockers diminished the [Ca 2+ ] i responses to 15K in neurons sensitive to AITC, but failed to inhibit 40 mM KCl-induced [Ca 2+ ] i increases even in AITC-sensitive neurons. TRPV1 blockers did not inhibit the 15K-induced [Ca 2+ ] i increase regardless of the sensitivity to capsaicin. [Ca 2+ ] i responses to TRPA1 agonist were enhanced by co-application with 15K. These pharmacological data suggest the possibility of functional interaction between T-type Ca 2+ channels and TRPA1 in sensory neurons. Since TRPA1 channel is activated by intracellular Ca 2+ , we hypothesize that Ca 2+ entered via T-type Ca 2+ channel activation may further stimulate TRPA1, resulting in an enhancement of nociceptive signaling. Thus, T-type Ca 2+ channel may be a potential target for TRPA1-related pain. © 2017 Wiley Periodicals, Inc.

  19. Sheep serum complement sensitisation of sheep erythrocyte-rabbit antibody complexes for haemolysis by guinea-pig complement plus EDTA or Mg2+-EGTA. (United States)

    Jonas, W; Stankiewicz, M


    Sheep erythrocyte (E)-rabbit antibody (A) complexes incubated with sheep serum diluted up to 1:5120 or 1:20480 and washed can be haemolysed by guinea-pig (g-p) serum (complement, C) containing EDTA or Mg2+-EGTA respectively as haemolytic finishing reagents. Sheep E carrying a high dose of rabbit A were necessary for this reaction, particularly with g-p C-EDTA. G-p serum (stored by freezing) was active as a haemolytic finishing reagent with both EDTA and Mg2+-EGTA. Reconstituted freeze-dried g-p serum (also stored by freezing) was haemolytically active with Mg2+-EGTA only. G-p serum preserved by Richardson's method did not function as a finishing reagent with EDTA or Mg2+-EGTA. A non-haemolytic prozone occurred with sheep E-rabbit A treated with dilutions of sheep serum or body fluid up to 1:160, particularly when g-p C (frozen)-EDTA was used as the finishing reagent. Sheep E-rabbit A were sensitized by serum, foetal lamb serum, pericardiac-, synovial- or ovarian follicle-fluids colostrum or milk for haemolysis by g-p C (frozen)-EDTA or -Mg2+-EGTA. With the C3 inhibitors cobra venom factor or salicylaldoxime, serum sensitisation of sheep E-rabbit A for haemolysis by g-p C (frozen)-EDTA or -Mg2+-EGTA was not blocked. Sensitisation by serum heated at 50 degrees C for 30 min (partial inactivation of C2) was incomplete. Inhibitors of C1 (antrypol, chelators of Ca2+ or heating serum at 56 degrees C for 30 min) partially or fully blocked sensitisation for haemolysis by both g-p C (frozen)-EDTA or -Mg2+-EGTA. These results show that at a minimum, components C1, C4 and C2 are present and functionally active in serum and some body fluids of sheep.

  20. Age-dependent alterations in Ca2+ homeostasis: role of TRPV5 and TRPV6

    NARCIS (Netherlands)

    Abel, M. van; Huybers, S.; Hoenderop, J.G.J.; Kemp, J.W.C.M. van der; Leeuwen, J.P.P.M. van; Bindels, R.J.M.


    Aging is associated with alterations in Ca2+ homeostasis, which predisposes elder people to hyperparathyroidism and osteoporosis. Intestinal Ca2+ absorption decreases with aging and, in particular, active transport of Ca2+ by the duodenum. In addition, there are age-related changes in renal Ca2+


    NARCIS (Netherlands)


    1 Bradykinin caused a transient reduction of about 25% in the cyclic AMP level in forskolin prestimulated DDT1 MF-2 smooth muscle cells (IC50: 36.4 +/- 4.9 nM) and a pronounced, sustained inhibition (40%) of the isoprenaline-stimulated cyclic AMP level (IC50: 37.5 +/- 1.1 nM). 2 The Ca2+ ionophore,

  2. Ca2+-independent and Ca2+/GTP-binding protein-controlled exocytosis in a plant cell


    Homann, Ulrike; Tester, Mark


    Exocytosis allows the release of secretory products and the delivery of new membrane material to the plasma membrane. So far, little is known about the underlying molecular mechanism and its control in plant cells. We have used the whole-cell patch-clamp technique to monitor changes in membrane capacitance to study exocytosis in barley aleurone protoplasts. To investigate the involvement of Ca2+ and GTP-binding proteins in exocytosis, protoplasts were dialyzed with very low (

  3. Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling. (United States)

    Kim, Jung Kuk; Choi, Jung Woong; Lim, Seyoung; Kwon, Ohman; Seo, Jeong Kon; Ryu, Sung Ho; Suh, Pann-Ghill


    Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca(2+) signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca(2+) source is enough for PLC-η1 activation. Furthermore, the IP(3) receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca(2+) mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca(2+) mobilization from the ER, and therefore amplifies GPCR-mediated signaling. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Effect of [10]-Gingerol on [Ca2+]i and Cell Death in Human Colorectal Cancer Cells


    Chung-Yi Chen; Yi-Wen Li; Soong-Yu Kuo


    The effect of [10]-gingerol on cytosol free Ca2+ concentration ([Ca2+]i) and viability is large unknown. This study examines the early signaling effects of [10]-gingerol on human colorectal cancer cells. It was found that this compound caused a slow and sustained rise of [Ca2+]i in a concentration-dependent manner. [10]-Gingerol also induced a [Ca2+]i rise when extracellular Ca2+ was removed, but the magnitude was reduced by 38%. In a Ca2+-free medium, the [10]-gingerol-induced [Ca2+]i rise w...

  5. Sodium-calcium exchanger and R-type Ca(2+) channels mediate spontaneous [Ca(2+)]i oscillations in magnocellular neurones of the rat supraoptic nucleus. (United States)

    Kortus, Stepan; Srinivasan, Chinnapaiyan; Forostyak, Oksana; Zapotocky, Martin; Ueta, Yoichi; Sykova, Eva; Chvatal, Alexandr; Verkhratsky, Alexei; Dayanithi, Govindan


    Isolated supraoptic neurones generate spontaneous [Ca(2+)]i oscillations in isolated conditions. Here we report in depth analysis of the contribution of plasmalemmal ion channels (Ca(2+), Na(+)), Na(+)/Ca(2+) exchanger (NCX), intracellular Ca(2+) release channels (InsP3Rs and RyRs), Ca(2+) storage organelles, plasma membrane Ca(2+) pump and intracellular signal transduction cascades into spontaneous Ca(2+) activity. While removal of extracellular Ca(2+) or incubation with non-specific voltage-gated Ca(2+) channel (VGCC) blocker Cd(2+) suppressed the oscillations, neither Ni(2+) nor TTA-P2, the T-type VGCC blockers, had an effect. Inhibitors of VGCC nicardipine, ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA (for L-, N-, P and P/Q-type channels, respectively) did not affect [Ca(2+)]i oscillations. In contrast, a specific R-type VGCC blocker SNX-482 attenuated [Ca(2+)]i oscillations. Incubation with TTX had no effect, whereas removal of the extracellular Na(+) or application of an inhibitor of the reverse operation mode of Na(+)/Ca(2+) exchanger KB-R7943 blocked the oscillations. The mitochondrial uncoupler CCCP irreversibly blocked spontaneous [Ca(2+)]i activity. Exposure of neurones to Ca(2+) mobilisers (thapsigargin, cyclopiazonic acid, caffeine and ryanodine); 4-aminopyridine (A-type K(+) current blocker); phospholipase C and adenylyl cyclase pathways blockers U-73122, Rp-cAMP, SQ-22536 and H-89 had no effect. Oscillations were blocked by GABA, but not by glutamate, apamin or dynorphin. In conclusion, spontaneous oscillations in magnocellular neurones are mediated by a concerted action of R-type Ca(2+) channels and the NCX fluctuating between forward and reverse modes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. New molecular players facilitating Mg(2+) reabsorption in the distal convoluted tubule.

    NARCIS (Netherlands)

    Glaudemans, B.; Knoers, N.V.A.M.; Hoenderop, J.G.J.; Bindels, R.J.M.


    The renal distal convoluted tubule (DCT) has an essential role in maintaining systemic magnesium (Mg(2+)) concentration. The DCT is the final determinant of plasma Mg(2+) levels, as the more distal nephron segments are largely impermeable to Mg(2+). In the past decade, positional candidate

  7. In vitro aging promotes endoplasmic reticulum (ER)-mitochondria Ca2+cross talk and loss of store-operated Ca2+entry (SOCE) in rat hippocampal neurons. (United States)

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


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

  8. Lipid membranes loaded with Ca2+ and Zn2+ cations (United States)

    Kučerka, N.; Dushanov, E.; Kholmurodov, K. T.; Katsaras, J.; Uhríková, D.


    We have studied the interactions of calcium and zinc with the biomimetic membrane made of dipalmitoyl-phosphatidylcholine (DPPC). The small angle neutron experiment on oriented multilamellar samples showed clearly differences in the effects of the two cations. For both, a bilayer thickness increases due to divalent metal ion (Me2+) binding, reaching the maximum at stoichiometry Me2+:DPPC∼1:7 mol/mol. However, while the further increase in Ca2+ results in a bilayer thinning down to the level of pure DPPC, the Zn2+ binding indicates the behaviour of a typical isotherm, reaching a level of saturation.

  9. Differential Somatic Ca2+ Channel Profile in Midbrain Dopaminergic Neurons. (United States)

    Philippart, Fabian; Destreel, Geoffrey; Merino-Sepúlveda, Paulina; Henny, Pablo; Engel, Dominique; Seutin, Vincent


    Dopaminergic (DA) neurons located in the ventral midbrain continuously generate a slow endogenous pacemaker activity, the mechanism of which is still debated. It has been suggested that, in the substantia nigra pars compacta (SNc), the pacemaking relies more on Ca(2+) channels and that the density of L-type Ca(2+) channels is higher in these DA neurons than in those located in the ventral tegmental area (VTA). This might lead to a higher Ca(2+) load in SNc DA neurons and explain their higher susceptibility to degeneration. However, direct evidence for this hypothesis is lacking. We found that the L-type current and channel density are indeed higher in the somata of rat SNc DA neurons and that this current undergoes less inactivation in this region. Nonstationary fluctuation analysis measurements showed a much higher number of L-type channels in the soma of SNc DA neurons, as well as a smaller single-channel conductance, pointing to a possible different molecular identity of L-type channels in DA neurons from the two areas. A major consequence of this is that pacemaking and, even more so, bursting are associated with a larger Ca(2+) entry through L-type channels in SNc DA neurons than in their VTA counterparts. Our results establish a molecular and functional difference between two populations of midbrain DA neurons that may contribute to their differential sensitivity to neurodegeneration. Dopamine neurons from the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) are involved in various brain functions, such as movement initiation and goal directed behavior, respectively. This work shows that, although both neurons fire in a similar regular and slow pacemaker mode, this firing activity is supported by different calcium channel landscapes. Indeed, the L-type calcium current is larger in the soma of dopamine neurons of the SNc, leading to a higher charge transfer through L-type channels during pacemaking and bursting. Therefore, these neurons may

  10. Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure.

    Directory of Open Access Journals (Sweden)

    Mani Sadredini

    Full Text Available Abnormal cellular Ca2+ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial

  11. Na(+), K(+)-ATPase dysfunction causes cerebrovascular endothelial cell degeneration in rat prefrontal cortex slice cultures. (United States)

    Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Katsuki, Hiroshi


    Cerebrovascular endothelial cell dysfunction resulting in imbalance of cerebral blood flow contributes to the onset of psychiatric disorders such as depression, schizophrenia and bipolar disorder. Although decrease in Na(+), K(+)-ATPase activity has been reported in the patients with schizophrenia and bipolar disorder, the contribution of Na(+), K(+)-ATPase to endothelial cell dysfunction remains poorly understood. Here, by using rat neonatal prefrontal cortex slice cultures, we demonstrated that pharmacological inhibition of Na(+), K(+)-ATPase by ouabain induced endothelial cell injury. Treatment with ouabain significantly decreased immunoreactive area of rat endothelial cell antigen-1 (RECA-1), a marker of endothelial cells, in a time-dependent manner. Ouabain also decreased Bcl-2/Bax ratio and phosphorylation level of glycogen synthase kinase 3β (GSK3β) (Ser9), which were prevented by lithium carbonate. On the other hand, ouabain-induced endothelial cell injury was exacerbated by concomitant treatment with LY294002, an inhibitor of phosphoinositide 3- (PI3-) kinase. We also found that xestospongin C, an inhibitor of inositol triphosphate (IP3) receptor, but not SEA0400, an inhibitor of Na(+), Ca(2+) exchanger (NCX), protected endothelial cells from cytotoxicity of ouabain. These results suggest that cerebrovascular endothelial cell degeneration induced by Na(+), K(+)-ATPase inhibition resulting in Ca(2+) release from endoplasmic reticulum (ER) and activation of GSK3β signaling underlies pathogenesis of these psychiatric disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs.

    Directory of Open Access Journals (Sweden)

    Yong-Qiang Zhang


    Full Text Available Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H(+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma(- phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca(2+ and H(+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca(2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

  13. Insulin regulation of beta-cell function involves a feedback loop on SERCA gene expression, Ca(2+) homeostasis, and insulin expression and secretion. (United States)

    Xu, G G; Gao, Z Y; Borge, P D; Jegier, P A; Young, R A; Wolf, B A


    The insulin receptor signaling pathway is present in beta-cells and is believed to be important in beta-cell function. We show here that insulin directly regulates beta-cell function in isolated rodent islets. Long-term insulin treatment caused a sustained increase in [Ca(2+)](i) and enhanced glucose-stimulated insulin secretion in rat islets, but failed to increase insulin content. Chronic activation of insulin receptor signaling by IRS-1 overexpression in the beta-cell inhibited gene expression of SERCA3, an endoplasmic reticulum Ca(2+)-ATPase. Insulin gene transcription was stimulated by insulin receptor signaling and insulin mimetic compound (L-783 281) in a glucose- and Grb2-dependent manner. Thus, beta-cell SERCA3 is a target for insulin regulation, which implies that beta-cell Ca(2+) homeostasis is regulated in an autocrine feedback loop by insulin. This study identifies a novel regulatory pathway of insulin secretion at the molecular level with two main components: (1) regulation of intracellular Ca(2+) homeostasis via SERCA3 and (2) regulation of insulin gene expression.

  14. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration. (United States)

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard


    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC, the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

  15. Microstructural Analysis of AM50/Mg2Si Cast Magnesium Composites

    Directory of Open Access Journals (Sweden)

    M.A. Malik


    Full Text Available AM50/Mg2Si composites containing 5.7 wt. % and 9.9 wt. %. of Mg2Si reinforcing phase were prepared successfully by casting method.The microstructure of the cast AM50/Mg2Si magnesium matrix composites was investigated by light microscopy and X-ray diffractometry (XRD. The microstructure of these composites was characterized by the presence of α-phase (a solid solution of aluminium in magnesium, Mg17Al12 (γ-phase, Al8Mn5 and Mg2Si. It was demonstrated that the Mg2Si phase was formed mainly as primary dendrites and eutectic.

  16. Microstructural Analysis of AM50/Mg2Si Cast Magnesium Composites

    Directory of Open Access Journals (Sweden)

    Malik M.A.


    Full Text Available AM50/Mg2Si composites containing 5.7 wt. % and 9.9 wt. %. of Mg2Si reinforcing phase were prepared successfully by casting method. The microstructure of the cast AM50/Mg2Si magnesium matrix composites was investigated by light microscopy and X-ray diffractometry (XRD. The microstructure of these composites was characterized by the presence of α-phase (a solid solution of aluminium in magnesium, Mg17Al12 (γ-phase, Al8Mn5 and Mg2Si. It was demonstrated that the Mg2Si phase was formed mainly as primary dendrites and eutectic.

  17. Molecular basis for interaction of Na+/K+-ATPase with other transporters in membrane microdomains of vascular smooth muscle cells

    DEFF Research Database (Denmark)

    Hansen, Anne Kirstine; Matchkov, Vladimir; Bouzinova, Elena


    small arteries and in the SMC cell line A7r5. Confocal microscopy and conventional patch clamp were used in functional studies. The Na+/K+-ATPase subunits in SMCs were found to be α1 and α2. As indicated by loss of mechanical synchronization and synchronization of Ca2+ transients between SMCs...... and by direct measurements of electrical coupling between SMCs, ouabain effectively uncouples SMCs in micromolar concentrations (1-10 µM). Since rodent α1 Na+/K+-ATPase subunits are ouabain-resistant, we conclude that α2 Na+/K+-ATPase subunits is involved in regulation of the intercellular communications via...

  18. Ubiquitous [Na+]i/[K+]i-Sensitive Transcriptome in Mammalian Cells: Evidence for Ca2+i-Independent Excitation-Transcription Coupling (United States)

    Koltsova, Svetlana V.; Trushina, Yulia; Haloui, Mounsif; Akimova, Olga A.; Tremblay, Johanne; Hamet, Pavel; Orlov, Sergei N.


    Stimulus-dependent elevation of intracellular Ca2+ ([Ca2+]i) affects the expression of numerous genes – a phenomenon known as excitation-transcription coupling. Recently, we found that increases in [Na+]i trigger c-Fos expression via a novel Ca2+i-independent pathway. In the present study, we identified ubiquitous and tissue-specific [Na+]i/[K+]i-sensitive transcriptomes by comparative analysis of differentially expressed genes in vascular smooth muscle cells from rat aorta (RVSMC), the human adenocarcinoma cell line HeLa, and human umbilical vein endothelial cells (HUVEC). To augment [Na+]i and reduce [K+]i, cells were treated for 3 hrs with the Na+,K+-ATPase inhibitor ouabain or placed for the same time in the K+-free medium. Employing Affymetrix-based technology, we detected changes in expression levels of 684, 737 and 1839 transcripts in HeLa, HUVEC and RVSMC, respectively, that were highly correlated between two treatments (p0.62). Among these Na+i/K+i-sensitive genes, 80 transcripts were common for all three types of cells. To establish if changes in gene expression are dependent on increases in [Ca2+]i, we performed identical experiments in Ca2+-free media supplemented with extracellular and intracellular Ca2+ chelators. Surprisingly, this procedure elevated rather than decreased the number of ubiquitous and cell-type specific Na+i/K+i-sensitive genes. Among the ubiquitous Na+i/K+i-sensitive genes whose expression was regulated independently of the presence of Ca2+ chelators by more than 3-fold, we discovered several transcription factors (Fos, Jun, Hes1, Nfkbia), interleukin-6, protein phosphatase 1 regulatory subunit, dual specificity phosphatase (Dusp8), prostaglandin-endoperoxide synthase 2, cyclin L1, whereas expression of metallopeptidase Adamts1, adrenomedulin, Dups1, Dusp10 and Dusp16 was detected exclusively in Ca2+-depleted cells. Overall, our findings indicate that Ca2+i-independent mechanisms of excitation-transcription coupling are involved in

  19. The cardioprotective effect of rosmarinic acid on acute myocardial infarction and genes involved in Ca2+ homeostasis. (United States)

    Javidanpour, Somayeh; Dianat, Mahin; Badavi, Mohammad; Mard, Seyyed Ali


    Acute myocardial infarction (AMI) is a common cause of hospitalisation and high mortality due to lethal arrhythmias. Sarcoplasmic reticulum Ca2+ ATPase (SERCA2) and ryanodine receptor (RyR2) regulate the cytosolic Ca2+ ion concentration. Rosmarinic acid (RA) is one of the most common caffeic esters in Rosmarinus officinalis. The present study was conducted to test the hypothesis whether RA can protect cardiac function against AMI and arrhythmias induced by isoproterenol through the regulatory effect of SERCA2 and RyR2 gene expression. To this aim, male Sprague-Dawley rats were allocated into in vivo and ex vivo studies and received RA (10, 15, and 30 mg/kg; 14 days). AMI was induced by two consecutive subcutaneous injections of 100 mg/kg isoproterenol. Blood pressure (BP), heart rate, electrocardiography (ECG) parameters, plasma levels of cardiac biomarkers, and antioxidative enzymes were evaluated (in vivo study). Cardiac functions were measured in isolated hearts using Langendorff set up. Gene expressions of SERCA2 and RyR2 were measured in left ventricular heart. Isoproterenol administration showed a significant decline in BP, QRS voltage, activities of antioxidant enzymes, cardiac function, and gene expressions of SERCA2 and RyR2. The results also indicated a significant increase in heart rate, ST-elevation, cardiac biomarkers, and antioxidant enzymes. RA at 30 mg/kg dosage showed the best effect on the improvement of the mentioned factors. This study suggests that RA has potent cardioprotective effects against AMI and arrhythmia, which may be due to its ability to enhance expression of plasma antioxidant enzymes and genes involved in Ca2+ homeostasis SERCA2 and RyR2. The protective role of RA is also possibly related to its antiadrenergic effects.

  20. A novel biphenolic ligand for selective Mg2+and Zn2+ions sensing followed by colorimetric, spectroscopic and cell imaging methods. (United States)

    Maheswari, Palanisamy Uma; Renuga, Duraisamy; Jeeva Kumari, H L; Ruckmani, Kandasamy


    The (E)-2-((2-hydrohy-5-methylphenylimino) methyl) phenol ligand was synthesized. The receptor was characterized by IR, 1 H and 13 C NMR and CHN analysis. The ligand exhibits colorimetric and fluorometric sensing of Zn 2+ and Mg 2+ ions in semi-aqueous medium (DMSO-H2O). The receptor was tested with series of transition metal ions (Cr 2+ , Fe 2+ , Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ ) and heavy metal ions (Sn 2+ , Pd 2+ , Ce 2+ , Hg 2+ , Cd 2+ ) and the essential human body elements like Ca 2+ , Mg 2+ , Na + and K + ions. The naked eye colorimetric sensing was absorbed only for Zn 2+ and Mg 2+ . Both ions (ZnCl 2 and MgCl 2 in H 2 O), when added to the colorless solutions of the receptor of about 1 equivalence in incremental additions turn the solution into bright turmeric yellow. All other ions remain inactive, in colorimetric sensing. Further the Zn 2+ and Mg 2+ ions were probed by absorption and emission spectroscopy through incremental addition of respective metal ions. The in-situ deprotonation of the ligand on both Mg 2+ and Zn 2+ ions binding was confirmed by 1 H NMR titration studies. The imino nitrogen of the receptor is not coordinated to the metal ions. The Job's plot studies reveal the 1:2 binding ratio of metal ions to the receptor. The high fold fluorescence output on metal ions binding was positively used to sense the Zn 2+ and Mg 2+ ions, separately and together in HeLa cancer cells through cell imaging. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Cch1p mediates Ca2+ influx to protect Saccharomyces cerevisiae against eugenol toxicity. (United States)

    Roberts, Stephen K; McAinsh, Martin; Widdicks, Lisa


    Eugenol has antifungal activity and is recognised as having therapeutic potential. However, little is known of the cellular basis of its antifungal activity and a better understanding of eugenol tolerance should lead to better exploitation of eugenol in antifungal therapies. The model yeast, Saccharomyces cerevisiae, expressing apoaequorin was used to show that eugenol induces cytosolic Ca(2+) elevations. We investigated the eugenol Ca(2+) signature in further detail and show that exponentially growing cells exhibit Ca(2+) elevation resulting exclusively from the influx of Ca(2+) across the plasma membrane whereas in stationary growth phase cells Ca(2+) influx from intracellular and extracellular sources contribute to the eugenol-induced Ca(2+) elevation. Ca(2+) channel deletion yeast mutants were used to identify the pathways mediating Ca(2+) influx; intracellular Ca(2+) release was mediated by the vacuolar Ca(2+) channel, Yvc1p, whereas the Ca(2+) influx across the plasma membrane could be resolved into Cch1p-dependent and Cch1p-independent pathways. We show that the growth of yeast devoid the plasma membrane Ca(2+) channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca(2+) elevations. Taken together, these results indicate that a cch1p-mediated Ca(2+) influx is part of an intracellular signal which protects against eugenol toxicity. This study provides fresh insight into the mechanisms employed by fungi to tolerate eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies.

  2. Cch1p Mediates Ca2+ Influx to Protect Saccharomyces cerevisiae against Eugenol Toxicity (United States)

    Roberts, Stephen K.; McAinsh, Martin; Widdicks, Lisa


    Eugenol has antifungal activity and is recognised as having therapeutic potential. However, little is known of the cellular basis of its antifungal activity and a better understanding of eugenol tolerance should lead to better exploitation of eugenol in antifungal therapies. The model yeast, Saccharomyces cerevisiae, expressing apoaequorin was used to show that eugenol induces cytosolic Ca2+ elevations. We investigated the eugenol Ca2+ signature in further detail and show that exponentially growing cells exhibit Ca2+ elevation resulting exclusively from the influx of Ca2+ across the plasma membrane whereas in stationary growth phase cells Ca2+ influx from intracellular and extracellular sources contribute to the eugenol-induced Ca2+ elevation. Ca2+ channel deletion yeast mutants were used to identify the pathways mediating Ca2+ influx; intracellular Ca2+ release was mediated by the vacuolar Ca2+ channel, Yvc1p, whereas the Ca2+ influx across the plasma membrane could be resolved into Cch1p-dependent and Cch1p-independent pathways. We show that the growth of yeast devoid the plasma membrane Ca2+ channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca2+ elevations. Taken together, these results indicate that a cch1p-mediated Ca2+ influx is part of an intracellular signal which protects against eugenol toxicity. This study provides fresh insight into the mechanisms employed by fungi to tolerate eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies. PMID:23028482

  3. Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release. (United States)

    Haustein, Moritz; Hannes, Tobias; Trieschmann, Jan; Verhaegh, Rabea; Köster, Annette; Hescheler, Jürgen; Brockmeier, Konrad; Adelmann, Roland; Khalil, Markus


    Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 μM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, pmyocardium (n=5, pmyocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

  4. A sequential vesicle pool model with a single release sensor and a ca(2+)-dependent priming catalyst effectively explains ca(2+)-dependent properties of neurosecretion

    DEFF Research Database (Denmark)

    Walter, Alexander M; da Silva Pinheiro, Paulo César; Verhage, Matthijs


    Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca(2+) dependence, but also upstream steps depend on Ca(2+). After deletion of the Ca(2+) sensor for fast release - synaptot......Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca(2+) dependence, but also upstream steps depend on Ca(2+). After deletion of the Ca(2+) sensor for fast release...... identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca(2+)-dependent action: a Ca(2+)-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1...... that the elusive 'alternative Ca(2+) sensor' for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca(2+)-dependent properties of secretion without assuming parallel pools or sensors....

  5. Ca2+-binding protein-1 facilitates and forms a postsynaptic complex with Cav1.2 (L-type) Ca2+ channels. (United States)

    Zhou, Hong; Kim, Seong-Ah; Kirk, Elizabeth A; Tippens, Alyssa L; Sun, Hong; Haeseleer, Françoise; Lee, Amy


    Ca2+-binding protein-1 (CaBP1) is a Ca2+-binding protein that is closely related to calmodulin (CaM) and localized in somatodendritic regions of principal neurons throughout the brain, but how CaBP1 participates in postsynaptic Ca2+ signaling is not known. Here, we describe a novel role for CaBP1 in the regulation of Ca2+ influx through Ca(v)1.2 (L-type) Ca2+ channels. CaBP1 interacts directly with the alpha1 subunit of Ca(v)1.2 at sites that also bind CaM. CaBP1 binding to one of these sites, the IQ domain, is Ca2+ dependent and competitive with CaM binding. The physiological significance of this interaction is supported by the association of Ca(v)1.2 and CaBP1 in postsynaptic density fractions purified from rat brain. Moreover, in double-label immunofluorescence experiments, CaBP1 and Ca(v)1.2 colocalize in numerous cell bodies and dendrites of neurons, particularly in pyramidal cells in the CA3 region of the hippocampus and in the dorsal cortex. In electrophysiological recordings of cells transfected with Ca(v)1.2, CaBP1 greatly prolonged Ca2+ currents, prevented Ca2+-dependent inactivation, and caused Ca2+-dependent facilitation of currents evoked by step depolarizations and repetitive stimuli. These effects contrast with those of CaM, which promoted strong Ca2+-dependent inactivation of Ca(v)1.2 with these same voltage protocols. Our findings reveal how Ca2+-binding proteins, such as CaM and CaBP1, differentially adjust Ca2+ influx through Ca(v)1.2 channels, which may specify diverse modes of Ca2+ signaling in neurons.

  6. Role of Na+/Ca2+ Exchangers in Therapy Resistance of Medulloblastoma Cells. (United States)

    Pelzl, Lisann; Hosseinzadeh, Zohreh; Al-Maghout, Tamer; Singh, Yogesh; Sahu, Itishri; Bissinger, Rosi; Schmidt, Sebastian; Alkahtani, Saad; Stournaras, Christos; Toulany, Mahmoud; Lang, Florian


    Alterations of cytosolic Ca2+-activity ([Ca2+]i) are decisive in the regulation of tumor cell proliferation, migration and survival. Transport processes participating in the regulation of [Ca2+]i include Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether medulloblastoma cells express Na+/Ca2+-exchangers, whether expression differs between therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells, and whether Na+/Ca2+-exchangers participate in the regulation of cell survival. In therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells transcript levels were estimated by RT-PCR, protein abundance by Western blotting, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/ Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free by Na+ free and Ca2+ containing (2 mM) extracellular perfusate as well as cell death from PI -staining and annexin-V binding in flow cytometry. The transcript levels of NCX3, NCKX2, and NCKX5, protein abundance of NCX3, slope and peak of Δ[Ca2+]i as well as Ica were significantly lower in therapy sensitive D283 than in therapy resistant UW228-3 medulloblastoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i, and augmented the ionizing radiation-induced apoptosis but did not significantly modify clonogenicity of medulloblastoma cells. Apoptosis was further enhanced by NCX3 silencing. Na+/Ca2+-exchanger activity significantly counteracts apoptosis but does not significantly affect clonogenicity after radiation of medulloblastoma cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

  7. Wear Behavior of Al-Mg2Si Cast In-situ Composite: Effect of Mg2Si Different Volume Fractions (United States)

    Ghiasinejad, J.; Emamy, M.; Ghorbani, M. R.; Malekan, A.


    Al-Mg2Si in situ composites are great candidates for automobile brake discs due to their low density, reasonably high young's modulus and low thermal expansion coefficient. Thus, understanding wear properties of this composite is of a great importance. In this study wear behavior of an in-situ Al-Mg2Si composite, prepared from a simple casting route, has been investigated using a pin-on-disc configuration concerning the effect of Mg2Si volume fractions, 15, 20 and 25% respectively. It was found that the weight loss increases with increase in reinforce volume fraction which can be due to a coarse morphology of primary Mg2Si particles. It was found that the variations of weight loss with sliding distance comprise different regimes of which the mechanisms are discussed.

  8. Development and dissipation of Ca(2+) gradients in adrenal chromaffin cells. (United States)

    Marengo, F D; Monck, J R


    We used pulsed laser imaging to measure the development and dissipation of Ca(2+) gradients evoked by the activation of voltage-sensitive Ca(2+) channels in adrenal chromaffin cells. Ca(2+) gradients appeared rapidly (buffer capacity on the order of 1000 and appropriate affinity and kinetics, approximated the size of the Ca(2+) increases and rate of dissipation of the measured gradients. Finally, simulations without exogenous buffer suggest that the Ca(2+) signal due to Ca(2+) channel activation is restricted by the endogenous buffer to a space less than 1 microm from the cell membrane. PMID:11023887

  9. Arbuscular mycorrhizae improve low temperature tolerance in cucumber via alterations in H2O2 accumulation and ATPase activity. (United States)

    Liu, Airong; Chen, Shuangchen; Chang, Rui; Liu, Dilin; Chen, Haoran; Ahammed, Golam Jalal; Lin, Xiaomin; He, Chaoxing


    The combined effects of arbuscular mycorrhizal fungi (AMF) and low temperature (LT) on cucumber plants were investigated with respect to biomass production, H2O2 accumulation, NADPH oxidase, ATPase activity and related gene expression. Mycorrhizal colonization ratio was gradually increased after AMF-inoculation. However, LT significantly decreased mycorrhizal colonization ability and mycorrhizal dependency. Regardless of temperature, the total fresh and dry mass, and root activity of AMF-inoculated plants were significantly higher than that of the non-AMF control. The H2O2 accumulation in AMF-inoculated roots was decreased by 42.44% compared with the control under LT. H2O2 predominantly accumulated on the cell walls of apoplast but was hardly detectable in the cytosol or organelles of roots. Again, NADPH oxidase activity involved in H2O2 production was significantly reduced by AMF inoculation under LT. AMF-inoculation remarkably increased the activities of P-type H(+)-ATPase, P-Ca(2+)-ATPase, V-type H(+)-ATPase, total ATPase activity, ATP concentration and plasma membrane protein content in the roots under LT. Additionally, ATP concentration and expression of plasma membrane ATPase genes were increased by AMF-inoculation. These results indicate that NADPH oxidase and ATPase might play an important role in AMF-mediated tolerance to chilling stress, thereby maintaining a lower H2O2 accumulation in the roots of cucumber.

  10. ERK activation by Ca2+ ionophores depends on Ca2+ entry in lymphocytes but not in platelets, and does not conduct membrane scrambling. (United States)

    Arachiche, A; Badirou, I; Dachary-Prigent, J; Garcin, I; Geldwerth-Feniger, D; Kerbiriou-Nabias, D


    Rapid Ca2+-dependent phospholipid (PL) reorganization (scrambling) at the plasma membrane is a mechanism common to hematopoietic cells exposing procoagulant phosphatidylserine (PS). The aim of this research was to determine whether activation of the extracellular signal-regulated kinase (ERK) pathway was required for PL scrambling, based on a single report analyzing both responses induced by Ca2+ ionophores in megakaryoblastic HEL cells. Ca2+ ionophore-stimulated ERK phosphorylation was induced in platelets without external Ca2+, whereas exogenous Ca2+ entry was crucial for ERK activation in Jurkat T cells. In both cells, membrane scrambling only occurred following Ca2+ entry and was not blocked by inhibiting ERK phosphorylation. Furthermore, ERK proteins are strongly phosphorylated in transformed B lymphoblastic cell lines, which do not expose PS in their resting state. Overall, the data demonstrated that ERK activation and membrane scrambling are independent mechanisms.

  11. Supralinear dendritic Ca2+ signalling in young developing CA1 pyramidal cells (United States)

    Pohle, Jörg; Bischofberger, Josef


    Although Ca2+ is critically important in activity-dependent neuronal development, not much is known about the regulation of dendritic Ca2+ signals in developing neurons. Here, we used ratiometric Ca2+ imaging to investigate dendritic Ca2+ signalling in rat hippocampal pyramidal cells during the first 1–4 weeks of postnatal development. We show that active dendritic backpropagation of Nav channel-dependent action potentials (APs) evoked already large dendritic Ca2+ transients in animals aged 1 week with amplitudes of ∼150 nm, similar to the amplitudes of ∼160 nM seen in animals aged 4 weeks. Although the AP-evoked dendritic Ca2+ load increased about four times during the first 4 weeks, the peak amplitude of free Ca2+ concentration was balanced by a four-fold increase in Ca2+ buffer capacity κs (∼70 vs. ∼280). Furthermore, Ca2+ extrusion rates increased with postnatal development, leading to a slower decay time course (∼0.2 s vs. ∼0.1 s) and more effective temporal summation of Ca2+ signals in young cells. Most importantly, during prolonged theta-burst stimulation dendritic Ca2+ signals were up to three times larger in cells at 1 week than at 4 weeks of age and much larger than predicted by linear summation, which is attributable to an activity-dependent slow-down of Ca2+ extrusion. As Ca2+ influx is four-fold smaller in young cells, the larger Ca2+ signals are generated using four times less ATP consumption. Taken together, the data suggest that active backpropagations regulate dendritic Ca2+ signals during early postnatal development. Remarkably, during prolonged AP firing, Ca2+ signals are several times larger in young than in mature cells as a result of activity-dependent regulation of Ca2+ extrusion rates. PMID:25239458

  12. Variable luminal sarcoplasmic reticulum Ca(2+) buffer capacity in smooth muscle cells. (United States)

    Dagnino-Acosta, Adán; Guerrero-Hernández, Agustín


    Sarcoplasmic reticulum contains the internal Ca(2+) store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca(2+) level is the same all throughout the SR; however, whether the Ca(2+) buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca(2+) buffer capacity of the SR by comparing the reduction in SR Ca(2+) levels with the corresponding increase in [Ca(2+)](i) during activation of either IP(3)Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca(2+) buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca(2+) buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca(2+) buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca(2+) replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca(2+) influx, indicate the relevance of luminal SR Ca(2+) buffer capacity in the [Ca(2+)](i) response. To further study the importance of luminal SR Ca(2+) buffer capacity in the release process we used low levels of heparin to partially inhibit IP(3)Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca(2+) levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca(2+)-binding proteins in the high-capacity, low-affinity conformation, particularly for IP(3)R-mediated Ca(2+) release.

  13. Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Graves Bridget M


    Full Text Available Abstract The phosphoinositide 3-kinases (PI3K/Akt dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca2+]i, Ca2+ transients and membrane Ca2+ current, ICa, in cultured murine HL-1 cardiomyocytes. LY294002 (1–20 μM, a specific PI3K inhibitor, dramatically decreased HL-1 [Ca2+]i, Ca2+ transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM; β (TGX-221; 100 nM and γ (AS-252424; 100 nM, to determine the contribution of specific isoforms to HL-1 [Ca2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca2+ transients. Triciribine (1–20 μM, which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca2+ transients and ICa. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca2+]i required for excitation-contraction coupling in cardiomyoctyes.

  14. Stimulation-induced Ca(2+) influx at nodes of Ranvier in mouse peripheral mo