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Sample records for calcium atpase type

  1. Vacuolar ATPase Regulates Surfactant Secretion in Rat Alveolar Type II Cells by Modulating Lamellar Body Calcium

    OpenAIRE

    Chintagari, Narendranath Reddy; Mishra, Amarjit; Su, Lijing; Wang, Yang; Ayalew, Sahlu; Hartson, Steven D; Liu, Lin

    2010-01-01

    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 alveol...

  2. 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.

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

    Science.gov (United States)

    Chintagari, Narendranath Reddy; Mishra, Amarjit; Su, Lijing; Wang, Yang; Ayalew, Sahlu; Hartson, Steven D; Liu, Lin

    2010-01-01

    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. PMID:20169059

  4. P-type ATPases.

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    Palmgren, Michael G; Nissen, Poul

    2011-01-01

    P-type ATPases form a large superfamily of cation and lipid pumps. They are remarkably simple with only a single catalytic subunit and carry out large domain motions during transport. The atomic structure of P-type ATPases in different conformations, together with ample mutagenesis evidence, has provided detailed insights into the pumping mechanism by these biological nanomachines. Phylogenetically, P-type ATPases are divided into five subfamilies, P1-P5. These subfamilies differ with respect to transported ligands and the way they are regulated. PMID:21351879

  5. Calcium-ATPases: Gene disorders and dysregulation in cancer.

    Science.gov (United States)

    Dang, Donna; Rao, Rajini

    2016-06-01

    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. PMID:26608610

  6. The evolutionary history of sarco(endoplasmic calcium ATPase (SERCA.

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    Ianina Altshuler

    Full Text Available Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na(+/K(+ transporters, H(+/K(+ transporters, and plasma membrane Ca(2+ pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endoplasmic reticulum calcium ATPase (SERCA, which maintains calcium homeostasis in the cell by actively pumping Ca(2+ into the sarco(endoplasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain.

  7. Revisiting the mechanisms of copper toxicity to rainbow trout: Time course, influence of calcium, unidirectional Na(+) fluxes, and branchial Na(+), K(+) ATPase and V-type H(+) ATPase activities.

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    Chowdhury, M Jasim; Girgis, Mina; Wood, Chris M

    2016-08-01

    In order to resolve uncertainties as to the mechanisms of toxic action of Cu and the protective effects of water [Ca], juvenile rainbow trout were acclimated to baseline soft water (SW, [Na(+)]=0.07, [Ca(2+)]=0.15, [Mg(2+)]=0.05mmolL(-1)) and then exposed to Cu with or without elevated [Ca] but at constant titratable alkalinity (0.27mmolL(-1)). The 96-h LC50 was 7-fold higher (63.8 versus 9.2μgCuL(-1); 1.00 versus 0.14μmolCuL(-1)) at [Ca]=3.0 versus 0.15mmolL(-1). Gill Cu burden increased with exposure concentration, and higher [Ca] attenuated this accumulation. At 24h, the gill Cu load (LA50≈0.58μgCug(-1); 9.13nmolCug(-1)) predictive of 50% mortality by 96h was independent of [Ca], in accord with Biotic Ligand Model (BLM) theory. Cu exposure induced net Na(+) losses (J(Na)net) by increasing unidirectional Na(+) efflux rates (J(Na)out) and inhibiting unidirectional Na(+) uptake rates (J(Na)in). The effect on J(Na)out was virtually immediate, whereas the effect on J(Na)in developed progressively over 24h and was associated with an inhibition of branchial Na(+), K(+) ATPase activity. The J(Na)in inhibition was eventually significant at a lower Cu threshold concentration (15μgCuL(-1)) than the J(Na)out stimulation (100μg Cu L(-1)). Elevated Ca protected against both effects, as well as against the inhibition of Na(+), K(+) ATPase activity. Branchial V-type H(+) ATPase activity was also inhibited by Cu exposure (100μgCuL(-1)), but only after 24h at high [Ca] (3.0mmolL(-1)). These novel results therefore reinforce the applicability of BLM theory to Cu, clarify that whether Na(+) influx or efflux is more sensitive depends on the duration of Cu exposure, show that elevated water [Ca], independent of alkalinity, is protective against both mechanisms of Cu toxicity, and identify V-type H(+)ATPase as a new Cu target for future investigation. PMID:27262060

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

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    Geisler Markus

    1998-01-01

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

  9. Evolution of Plant P-Type ATPases

    OpenAIRE

    Pedersen, Christian N. S.; Kristian B. Axelsen; Harper, Jeffrey F.; Palmgren, Michael G.

    2012-01-01

    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 tauri and Chlamydomonas reinhardtii, and the streptophytes Physcomitrella patens (a non-vascular moss), Selaginella moellendorffii (a primitive vascular plant), and Arabidopsis thaliana (a model flowering plant). Each organism contained sequences for all five...

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

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

    2013-09-01

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

  11. Cyclopiazonic acid, an inhibitor of calcium-dependent ATPases with antiviral activity against human respiratory syncytial virus.

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    Cui, Rui; Wang, Yizhuo; Wang, Liu; Li, Guiming; Lan, Ke; Altmeyer, Ralf; Zou, Gang

    2016-08-01

    Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in infants and young children worldwide, yet no vaccine or effective antiviral treatment is available. To search for new anti-RSV agents, we developed a cell-based assay that measures inhibition of RSV-induced cytopathic effect (CPE) and identified cyclopiazonic acid (CPA), an intracellular calcium ATPase inhibitor as a RSV inhibitor (EC50 values 4.13 μM) by screening of natural product library. CPA inhibited the replication of RSV strains belonging to both A and B subgroups and human parainfluenza virus type 3, but not Enterovirus 71. Mechanism of action study by time-of-addition assay and minigenome assay revealed that CPA acts at the step of virus genome replication and/or transcription. Moreover, two other calcium ATPase inhibitors (Thapsigargin and BHQ) and calcium ionophores (A23187 and ionomycin), but not calcium channel blockers (nifedipine, nimodipine, and tetrandrine), also had similar effect. These results indicate that an increase in intracellular calcium concentration is detrimental to RSV replication. Thus, our findings provide a new strategy for anti-RSV therapy via increasing intracellular calcium concentration. PMID:27210812

  12. V-type ATPase proton pump expression during enamel formation.

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    Sarkar, Juni; Wen, Xin; Simanian, Emil J; Paine, Michael L

    2016-01-01

    Several diseases such as proximal and distal renal tubular acidosis and osteoporosis are related to intracellular pH dysregulation resulting from mutations in genes coding for ion channels, including proteins comprising the proton-pumping V-type ATPase. V-type ATPase is a multi-subunit protein complex expressed in enamel forming cells. V-type ATPase plays a key role in enamel development, specifically lysosomal acidification, yet our understanding of the relationship between the endocytotic activities and dental health and disease is limited. The objective of this study is to better understand the ameloblast-associated pH regulatory networks essential for amelogenesis. Quantitative RT-PCR was performed on tissues from secretory-stage and maturation-stage enamel organs to determine which of the V-type ATPase subunits are most highly upregulated during maturation-stage amelogenesis: a time when ameloblast endocytotic activity is highest. Western blot analyses, using specific antibodies to four of the V-type ATPase subunits (Atp6v0d2, Atp6v1b2, Atp6v1c1 and Atp6v1e1), were then applied to validate much of the qPCR data. Immunohistochemistry using these same four antibodies was also performed to identify the spatiotemporal expression profiles of individual V-type ATPase subunits. Our data show that cytoplasmic V-type ATPase is significantly upregulated in enamel organ cells during maturation-stage when compared to secretory-stage. These data likely relate to the higher endocytotic activities, and the greater need for lysosomal acidification, during maturation-stage amelogenesis. It is also apparent from our immunolocalization data, using antibodies against two of the V-type ATPase subunits (Atp6v1c1 and Atp6v1e1), that significant expression is seen at the apical membrane of maturation-stage ameloblasts. Others have also identified this V-type ATPase expression profile at the apical membrane of maturation ameloblasts. Collectively, these data better define the

  13. Engineering a prototypic P-type ATPase Listeria Monocytogenes Ca(2+)-ATPase 1 for single-molecule FRET studies

    DEFF Research Database (Denmark)

    Dyla, Mateusz; Andersen, Jacob; Kjaergaard, Magnus;

    2016-01-01

    Approximately 30% of the ATP generated in the living cell is utilized by P-type ATPase primary active transporters to generate and maintain electrochemical gradients across biological membranes. P-type ATPases undergo large conformational changes during their functional cycle to couple ATP hydrol...

  14. Elucidating Functional Aspects of P-type ATPases

    DEFF Research Database (Denmark)

    Autzen, Henriette Elisabeth

    2015-01-01

    P-type ATPases are proteins that act to maintain ion homeostasis and electrochemical gradients through the translocation of cations across cell membranes. Underscoring their significance in humans, dysfunction of the ATPases can lead to crucial diseases. Dysfunction of the sarco(endo)plasmic reti......P-type ATPases are proteins that act to maintain ion homeostasis and electrochemical gradients through the translocation of cations across cell membranes. Underscoring their significance in humans, dysfunction of the ATPases can lead to crucial diseases. Dysfunction of the sarco...... cancer and pathogenic microbes. The goal of this Ph.D. dissertation was to functionally characterize SERCA1a and CopA from Legionella pneumophila (LpCopA) through a range of different methods within structural biology. Crystallographic studies of SERCA1a led to a newly determined crystal structure...... that the bacterial, anionic phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL), have an increased propensity to bind to certain areas of the transmembrane domain. Further studies are required to infer whether these observations support specific lipid-protein interactions and what their significance...

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  16. Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca2+-ATPase.

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    Huang, Yongqi; Li, Huifang; Bu, Yuxiang

    2009-10-01

    Calcium ATPase is a member of the P-type ATPase, and it pumps calcium ions from the cytoplasm into the reticulum against a concentration gradient. Several X-ray structures of different conformations have been solved in recent years, providing basis for elucidating the active transport mechanism of Ca2+ ions. In this work, molecular dynamics (MD) simulations were performed at atomic level to investigate the dynamical process of calcium ions moving from the outer mouth of the protein to their binding sites. Five initial locations of Ca2+ ions were considered, and the simulations lasted for 2 or 6 ns, respectively. Specific pathways leading to the binding sites and large structural rearrangements around binding sites caused by uptake of calcium ions were identified. A cooperative binding mechanism was observed from our simulation. Firstly, the first Ca2+ ion binds to site I, and then, the second Ca2+ ion approaches. The interactions between the second Ca2+ and the residues around site I disturb the binding state of site I and weaken its binding ability for the first bound Ca2+. Because of the electrostatic repulsion of the second Ca2+ and the electrostatic attraction of site II, the first bound Ca2+ shifts from site I to site II. Concertedly, the second Ca2+ binds to site I, forming a binding state with two Ca2+ ions, one at site I and the other at site II. Both of Glu908 and Asp800 coordinate with the two Ca2+ ions simultaneously during the concerted binding process, which is believed to be the hinge to achieve the concerted binding. In our simulations, four amino acid residues that serve as the channel to link the outer mouth and the binding sites during the binding process were recognized, namely Tyr837, Tyr763, Asn911, and Ser767. The analyses regarding the activity of the proteins via mutations of some key residues also supported our cooperative mechanism. PMID:19242958

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

    Institute of Scientific and Technical Information of China (English)

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

    2010-01-01

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

  18. Demethoxycurcumin is a potent inhibitor of P-type ATPases from diverse kingdoms of life

    DEFF Research Database (Denmark)

    Dao, Trong Tuan; Sehgal, Pankaj; Thanh Tung, Truong;

    2016-01-01

    the curcuminoids, demethoxycurcumin was the most potent inhibitor of all tested P-type ATPases from fungal (Pma1p; H+-ATPase), plant (AHA2; H+-ATPase) and animal (SERCA; Ca2+-ATPase) cells. All three curcuminoids acted as non-competitive antagonist to ATP and hence may bind to a highly conserved allosteric site......P-type ATPases catalyze the active transport of cations and phospholipids across biological membranes. Members of this large family are involved in a range of fundamental cellular processes. To date, a substantial number of P-type ATPase inhibitors have been characterized, some of which are used...... as drugs. In this work a library of natural compounds was screened and we first identified curcuminoids as plasma membrane H+-ATPases inhibitors in plant and fungal cells. We also found that some of the commercial curcumins contain several curcuminoids. Three of these were purified and, among...

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

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

    2014-02-01

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

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

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

    2010-02-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Amanda; Kathleen; Fakira; Stella; Elkabes

    2010-01-01

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

  2. Demethoxycurcumin Is A Potent Inhibitor of P-Type ATPases from Diverse Kingdoms of Life

    Science.gov (United States)

    Dao, Trong Tuan; Sehgal, Pankaj; Tung, Truong Thanh; Møller, Jesper Vuust; Nielsen, John; Palmgren, Michael; Christensen, Søren Brøgger

    2016-01-01

    P-type ATPases catalyze the active transport of cations and phospholipids across biological membranes. Members of this large family are involved in a range of fundamental cellular processes. To date, a substantial number of P-type ATPase inhibitors have been characterized, some of which are used as drugs. In this work a library of natural compounds was screened and we first identified curcuminoids as plasma membrane H+-ATPases inhibitors in plant and fungal cells. We also found that some of the commercial curcumins contain several curcuminoids. Three of these were purified and, among the curcuminoids, demethoxycurcumin was the most potent inhibitor of all tested P-type ATPases from fungal (Pma1p; H+-ATPase), plant (AHA2; H+-ATPase) and animal (SERCA; Ca2+-ATPase) cells. All three curcuminoids acted as non-competitive antagonist to ATP and hence may bind to a highly conserved allosteric site of these pumps. Future research on biological effects of commercial preparations of curcumin should consider the heterogeneity of the material. PMID:27644036

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-11-25

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

  4. Computational approaches for classification and prediction of P-type ATPase substrate specificity in Arabidopsis.

    Science.gov (United States)

    Zinati, Zahra; Alemzadeh, Abbas; KayvanJoo, Amir Hossein

    2016-01-01

    As an extended gamut of integral membrane (extrinsic) proteins, and based on their transporting specificities, P-type ATPases include five subfamilies in Arabidopsis, inter alia, P4ATPases (phospholipid-transporting ATPase), P3AATPases (plasma membrane H(+) pumps), P2A and P2BATPases (Ca(2+) pumps) and P1B ATPases (heavy metal pumps). Although, many different computational methods have been developed to predict substrate specificity of unknown proteins, further investigation needs to improve the efficiency and performance of the predicators. In this study, various attribute weighting and supervised clustering algorithms were employed to identify the main amino acid composition attributes, which can influence the substrate specificity of ATPase pumps, classify protein pumps and predict the substrate specificity of uncharacterized ATPase pumps. The results of this study indicate that both non-reduced coefficients pertaining to absorption and Cys extinction within 280 nm, the frequencies of hydrogen, Ala, Val, carbon, hydrophilic residues, the counts of Val, Asn, Ser, Arg, Phe, Tyr, hydrophilic residues, Phe-Phe, Ala-Ile, Phe-Leu, Val-Ala and length are specified as the most important amino acid attributes through applying the whole attribute weighting models. Here, learning algorithms engineered in a predictive machine (Naive Bays) is proposed to foresee the Q9LVV1 and O22180 substrate specificities (P-type ATPase like proteins) with 100 % prediction confidence. For the first time, our analysis demonstrated promising application of bioinformatics algorithms in classifying ATPases pumps. Moreover, we suggest the predictive systems that can assist towards the prediction of the substrate specificity of any new ATPase pumps with the maximum possible prediction confidence. PMID:27186030

  5. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    Energy Technology Data Exchange (ETDEWEB)

    Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

    2013-09-15

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.

  6. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    International Nuclear Information System (INIS)

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells

  7. Na+,K+-ATPase Na+ affinity in rat skeletal muscle fiber types

    DEFF Research Database (Denmark)

    Kristensen, Michael; Juel, Carsten

    2010-01-01

    Previous studies in expression systems have found different ion activation of the Na(+)/K(+)-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na(+),K(+)-ATPase activity, and the Na(+) affinity of Na......(+),K(+)-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na(+) affinity was higher (K(m) lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na......) and alpha(2)beta(2), respectively. The affinity differences and isoform distributions imply that the degree of activation of Na(+),K(+)-ATPase at physiological Na(+) concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions...

  8. Expression and characterization of P-type ATPases for structural studies

    OpenAIRE

    Chintalapati, Sivaram Chandra

    2007-01-01

    Two types of proteins transport ions across the membrane – ion channels and ion pumps. Ion pumps transport ions against their electrochemical gradient by co-transporting another ion or a substrate molecule through a concentration gradient or by coupling this process to an energy source like ATP. Those that couple ATP hydrolysis to ion transport are called ion motive ATPases and can be classified as ‘V’, ‘F’ and ‘P’ types. In this thesis, two sub-classes of P-type ATPases, PIIIA and PIB were s...

  9. Structure function relationship in P-type ATPases : a biophysical approach

    OpenAIRE

    Apell, Hans-Jürgen

    2003-01-01

    P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins the energy-providing ATP hydrolysis is coupled to ion-transport that builds up or maintains the electrochemical potential gradients of one or two ion species across the membrane. P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, and they are transporters of a broad variety of ions. So far, a crystal structure with atomic resolut...

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

    Directory of Open Access Journals (Sweden)

    Vida Praitis

    2013-05-01

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

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

    Science.gov (United States)

    Gourdon, Pontus; Liu, Xiang-Yu; Skjørringe, Tina; Morth, J Preben; Møller, Lisbeth Birk; Pedersen, Bjørn Panyella; Nissen, Poul

    2011-07-01

    Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu(+)-ATPase, in a copper-free form, as determined by X-ray crystallography at 3.2 Å resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  13. Copper-transporting P-type ATPases use a unique ion-release pathway

    DEFF Research Database (Denmark)

    Andersson, Magnus; Mattle, Daniel; Sitsel, Oleg;

    2014-01-01

    Heavy metals in cells are typically regulated by PIB-type ATPases. The first structure of the class, a Cu(+)-ATPase from Legionella pneumophila (LpCopA), outlined a copper transport pathway across the membrane, which was inferred to be occluded. Here we show by molecular dynamics simulations...... that extracellular water solvated the transmembrane (TM) domain, results indicative of a Cu(+)-release pathway. Furthermore, a new LpCopA crystal structure determined at 2.8-Å resolution, trapped in the preceding E2P state, delineated the same passage, and site-directed-mutagenesis activity assays support...

  14. Structure and mechanism of Zn2+-transporting P-type ATPases

    DEFF Research Database (Denmark)

    Wang, Kaituo; Sitsel, Oleg; Meloni, Gabriele;

    2014-01-01

    Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis. In prokaryotes and photosynthetic eukaryotes, Zn(2+)-transporting P-type ATPases of class IB (Znt...

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

    Institute of Scientific and Technical Information of China (English)

    宋营改

    2012-01-01

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

  16. A Cadmium-transporting P1B-type ATPase in Yeast Saccharomyces cerevisiae*

    OpenAIRE

    Adle, David J.; Sinani, Devis; Kim, Heejeong; Lee, Jaekwon

    2006-01-01

    Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P1B-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpe...

  17. Subunit topology in the V type ATPase and related enzymes

    NARCIS (Netherlands)

    Chaban, Yuriy

    2005-01-01

    During the last decades impressive progress has been made in understanding of the catalytic mechanism of F-type ATP synthase, which is the key enzyme in the energy metabolism of eukaryotes and most bacteria. This enzyme catalyzes the final step in the process of oxidative phosphorylation in bacteria

  18. Transient Expression of P-type ATPases in Tobacco Epidermal Cells.

    Science.gov (United States)

    Poulsen, Lisbeth R; Palmgren, Michael G; López-Marqués, Rosa L

    2016-01-01

    Transient expression in tobacco cells is a convenient method for several purposes such as analysis of protein-protein interactions and the subcellular localization of plant proteins. A suspension of Agrobacterium tumefaciens cells carrying the plasmid of interest is injected into the intracellular space between leaf epidermal cells, which results in DNA transfer from the bacteria to the plant and expression of the corresponding proteins. By injecting mixes of Agrobacterium strains, this system offers the possibility to co-express a number of target proteins simultaneously, thus allowing for example protein-protein interaction studies. In this chapter, we describe the procedure to transiently express P-type ATPases in tobacco epidermal cells, with focus on subcellular localization of the protein complexes formed by P4-ATPases and their β-subunits. PMID:26695049

  19. Reduced expression of plasma membrane calcium ATPase 2 and collapsin response mediator protein 1 promotes death of spinal cord neurons.

    Science.gov (United States)

    Kurnellas, M P; Li, H; Jain, M R; Giraud, S N; Nicot, A B; Ratnayake, A; Heary, R F; Elkabes, S

    2010-09-01

    The mechanisms underlying neuronal pathology and death in the spinal cord (SC) during inflammation remain elusive. We previously showed the important role of plasma membrane calcium ATPases (PMCAs) in the survival of SC neurons, in vitro. We also postulated that a decrease in PMCA2 expression could cause neuronal death during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The current studies were undertaken to define the specific contribution of PMCA2 to degeneration of SC neurons, the effectors downstream to PMCA2 mediating neuronal death and the triggers that reduce PMCA2 expression. We report that knockdown of PMCA2 in SC neurons decreases collapsin response mediator protein 1 (CRMP1) levels. This is followed by cell death. Silencing of CRMP1 expression also leads to neuronal loss. Kainic acid reduces both PMCA2 and CRMP1 levels and induces neuronal death. Administration of an alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor antagonist, at onset or peak of EAE, restores the decreased PMCA2 and CRMP1 levels to control values and ameliorates clinical deficits. Thus, our data link the reduction in PMCA2 expression with perturbations in the expression of CRMP1 and the ensuing death of SC neurons. This represents an additional mechanism underlying AMPA/kainate receptor-mediated excitotoxicity with relevance to neurodegeneration in EAE. PMID:20489728

  20. Phospholipase D2 mediates signaling by ATPase class I type 8B membrane 1[S

    OpenAIRE

    Chen, Frank; Ghosh, Ayantika; Shneider, Benjamin L.

    2013-01-01

    Functional defects in ATPase class I type 8B membrane 1 (ATP8B1 or familial intrahepatic cholestasis 1, FIC1) lead to cholestasis by mechanism(s) that are not fully understood. One proposed pathophysiology involves aberrant signaling to the bile acid sensor, the farnesoid X receptor (FXR), via protein kinase C ζ (PKCζ). The following cell line-based studies investigated whether phospholipase D2 may transduce a signal from FIC1 to FXR. PLD2 gain of function led to activation of the bile salt e...

  1. Association between erythrocyte Na+K+-ATPase activity and some blood lipids in type 1 diabetic patients from Lagos, Nigeria

    Directory of Open Access Journals (Sweden)

    Iwalokun Senapon O

    2007-10-01

    Full Text Available Abstract Background Altered levels of erythrocyte Na+K+-ATPase, atherogenic and anti-atherogenic lipid metabolites have been implicated in diabetic complications but their pattern of interactions remains poorly understood. This study evaluated this relationship in Nigerian patients with Type 1 diabetes mellitus. Methods A total of 34 consented Type 1 diabetic patients and age -matched 27 non-diabetic controls were enrolled. Fasting plasma levels of total cholesterol, triglycerides and HDL-cholesterol were determined spectrophotometrically and LDL-cholesterol estimated using Friedewald formula. Total protein content and Na+K+-ATPase activity were also determined spectrophotometrically from ghost erythrocyte membrane prepared by osmotic lysis. Results Results indicate significant (P +K+-ATPase activity in the Type 1 diabetic patients (0.38 ± 0.08 vs. 0.59 ± 0.07 uM Pi/mgprotein/h compared to the control but with greater reduction in the diabetic subgroup with poor glycemic control (n = 20 and in whom cases of hypercholesterolemia (8.8%, hypertriglyceridemia (2.9% and elevated LDL-cholesterol (5.9% each were found. Correlation analyses further revealed significant (P +K+-ATPase in this subgroup contrary to group with good glycemic control or non-diabetic subjects in which significant (P +K+-ATPase and HDL-C association were found (r = 0.427 - 0.489. The Na+K+-ATPase from the diabetic patients also exhibited increased sensitivity to digoxin and alterations in kinetic constants Vmax and Km determined by glycemic status of the patients. Conclusion It can be concluded that poor glycemic control evokes greater reduction in erythrocyte Na+K+-ATPase activity and promote enzyme-blood atherogenic lipid relationships in Type 1 diabetic Nigerian patients.

  2. Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

    International Nuclear Information System (INIS)

    P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca2+, Na+, K+ and H+), have been reported. They include reticulum and plasma-membrane Ca2+-ATPases, Na+/K+-ATPase and H+/K+-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg2+ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na+/K+-ATPase α1-isoform, H+/K+-ATPase α2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H+/K+-ATPase α2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

  3. A P-type ATPase importer that discriminates between essential and toxic transition metals.

    Science.gov (United States)

    Lewinson, Oded; Lee, Allen T; Rees, Douglas C

    2009-03-24

    Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q9I147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts.

  4. Transporters, chaperones, and P-type ATPases controlling grapevine copper homeostasis.

    Science.gov (United States)

    Leng, Xiangpeng; Mu, Qian; Wang, Xiaomin; Li, Xiaopeng; Zhu, Xudong; Shangguan, Lingfei; Fang, Jinggui

    2015-11-01

    With more copper and copper-containing compounds used as bactericides and fungicides in viticulture, copper homeostasis in grapevine (Vitis) has become one of the serious environmental crises with great risk. To better understand the regulation of Cu homeostasis in grapevine, grapevine seedlings cultured in vitro with different levels of Cu were utilized to investigate the tolerance mechanisms of grapevine responding to copper availability at physiological and molecular levels. The results indicated that Cu contents in roots and leaves arose with increasing levels of Cu application. With copper concentration increasing, malondialdehyde (MDA) content increased in roots and leaves and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased to protect the plant itself from damage. The expression patterns of 19 genes, encoding transporters, chaperones, and P-type ATPases involved in copper homeostasis in root and leaf of grapevine seedling under various levels of Cu(2+) were further analyzed. The expression patterns indicated that CTr1, CTr2, and CTr8 transporters were significantly upregulated in response both to Cu excess and deficiency. ZIP2 was downregulated in response to Cu excess and upregulated under Cu-deficient conditions, while ZIP4 had an opposite expression pattern under similar conditions. The expression of chaperones and P-type ATPases in response to Cu availability in grapevine were also briefly studied.

  5. In silico identification and characterization of the ion transport specificity for P-type ATPases in the Mycobacterium tuberculosis complex

    Directory of Open Access Journals (Sweden)

    Novoa-Aponte Lorena

    2012-10-01

    Full Text Available Abstract Background P-type ATPases hydrolyze ATP and release energy that is used in the transport of ions against electrochemical gradients across plasma membranes, making these proteins essential for cell viability. Currently, the distribution and function of these ion transporters in mycobacteria are poorly understood. Results In this study, probabilistic profiles were constructed based on hidden Markov models to identify and classify P-type ATPases in the Mycobacterium tuberculosis complex (MTBC according to the type of ion transported across the plasma membrane. Topology, hydrophobicity profiles and conserved motifs were analyzed to correlate amino acid sequences of P-type ATPases and ion transport specificity. Twelve candidate P-type ATPases annotated in the M. tuberculosis H37Rv proteome were identified in all members of the MTBC, and probabilistic profiles classified them into one of the following three groups: heavy metal cation transporters, alkaline and alkaline earth metal cation transporters, and the beta subunit of a prokaryotic potassium pump. Interestingly, counterparts of the non-catalytic beta subunits of Hydrogen/Potassium and Sodium/Potassium P-type ATPases were not found. Conclusions The high content of heavy metal transporters found in the MTBC suggests that they could play an important role in the ability of M. tuberculosis to survive inside macrophages, where tubercle bacilli face high levels of toxic metals. Finally, the results obtained in this work provide a starting point for experimental studies that may elucidate the ion specificity of the MTBC P-type ATPases and their role in mycobacterial infections.

  6. Proton Pumping and Slippage Dynamics of a Eukaryotic P-Type ATPase Studied at the Single-Molecule Level

    DEFF Research Database (Denmark)

    Veshaguri, Salome

    In all eukaryotes the plasma membrane potential and secondary transport systems are energized by P-type ATPases whose regulation however remains poorly understood. Here we monitored at the single-molecule level the activity of the prototypic proton pumping P-type ATPase Arabidopsis thaliana isoform......-intuitively increased the time spent pumping. Allosteric regulation by pH gradients affected the time spent pumping and the leakage probability but surprisingly not the intrinsic pumping rate. Interestingly, ATP dilution decreased the ATP hydrolysis rates in bulk while single molecule data revealed that intrinsic...

  7. Effect of Calcium on the Vanadium Extraction from High Calcium Type Stone Coal

    Institute of Scientific and Technical Information of China (English)

    BAO Shenxu; LIANG Liang; ZHANG Yimin; HAN Shihua; HU Yangjia

    2015-01-01

    The high calcium type stone coal from Hubei province was leached by water and dilute acid separately after being roasted with different dosage of NaCl. The water leaching rate of vanadium (WLRV) was low and only 26.8%of vanadium can be leached by water when 4%NaCl was added, but the acid leaching rate of vanadium (ALRV) was relatively high. Calcium in the high calcium type stone coal is greatly superfluous relative to vanadium, hence, the calcium reacts with vanadium to form Ca(VO3)2, Ca2V2O7 and Ca3(VO4)2 orderly during the stone coal roasting process and high temperature is beneficial to the reactions between calcium and vanadium, which was validated by simulated reactions between pure calcium carbonate and vanadium pentoxide. These calcium vanadates are all water insoluble but acid soluble and this causes the low WLRV and relatively high ALRV. After calcium removal by HCl, the WLRV is highly enhanced and reaches about 50%when only 2%NaCl was added. If the HCl content is too high, the stone coal is easily sintered and the formed glass structure can enwrap vanadium, which leads the WLRV to decline. Single water leaching process is not appropriate to extract vanadium from high calcium type stone coal.

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  9. Membrane Structure of CtrA3, a Copper-transporting P-type-ATPase from Aquifex aeolicus

    OpenAIRE

    Chintalapati, Sivaram; Kurdi, Rana Al; Terwisscha Van Scheltinga, Anke C; Kühlbrandt, Werner

    2008-01-01

    We have produced and characterized two new copper-transporting ATPases, CtrA2 and CtrA3 from Aquifex aeolicus, that belong to the family of heavy metal ion-transporting PIB-type ATPases. CtrA2 has a CPC metal-binding sequence in TM6 and a CxxC metal-binding N-terminal domain, while CtrA3 has a CPH metal-binding motif in TM6 and a histidine-rich N-terminal metal-binding domain. We have cloned both copper pumps, expressed them in Escherichia coli and characterized them functionally. CtrA2 is ac...

  10. Structural Basis for Metal Binding Specificity: the N-terminal Cadmium Binding Domain of the P1-type ATPase CadA

    OpenAIRE

    Banci, Lucia; Bertini, Ivano; Ciofi-Baffoni, Simone; Su, Xun-Cheng; Miras, Roger; Bal, Nathalie; Mintz, Elisabeth; Catty, Patrice; Shokes, Jacob E.; Scott, Robert A

    2005-01-01

    In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a βαββαβ fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sph...

  11. Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

    Science.gov (United States)

    Goldgof, Gregory M.; Durrant, Jacob D.; Ottilie, Sabine; Vigil, Edgar; Allen, Kenneth E.; Gunawan, Felicia; Kostylev, Maxim; Henderson, Kiersten A.; Yang, Jennifer; Schenken, Jake; LaMonte, Gregory M.; Manary, Micah J.; Murao, Ayako; Nachon, Marie; Stanhope, Rebecca; Prescott, Maximo; McNamara, Case W.; Slayman, Carolyn W.; Amaro, Rommie E.; Suzuki, Yo; Winzeler, Elizabeth A.

    2016-01-01

    The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity. PMID:27291296

  12. Rotary ATPases

    Science.gov (United States)

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

    2013-01-01

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    (+)-ATPase, in a copper-free form, as determined by X-ray crystallography at 3.2 Å resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative...... 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...

  14. Calcium signaling and T-type calcium channels in cancer cell cycling

    Institute of Scientific and Technical Information of China (English)

    James T Taylor; Xiang-Bin Zeng; Jonathan E Pottle; Kevin Lee; Alun R Wang; Stephenie G Yi; Jennifer A S Scruggs; Suresh S Sikka; Ming Li

    2008-01-01

    Regulation of intracellular calcium is an important signaling mechanism for cell proliferation in both normal and cancerous cells. In normal epithelial cells,free calcium concentration is essential for cells to enter and accomplish the S phase and the M phase of the cell cycle. In contrast, cancerous cells can pass these phases of the cell cycle with much lower cytoplasmic free calcium concentrations, indicating an alternative mechanism has developed for fulfilling the intracellular calcium requirement for an increased rate of DNA synthesis and mitosis of fast replicating cancerous cells. The detailed mechanism underlying the altered calcium loading pathway remains unclear;however, there is a growing body of evidence that suggests the T-type Ca2+ channel is abnormally expressed in cancerous cells and that blockade of these channels may reduce cell proliferation in addition to inducing apoptosis. Recent studies also show that the expression of T-type Ca2+ channels in breast cancer cells is proliferation state dependent, i.e. the channels are expressed at higher levels during the fast-replication period, and once the cells are in a non-proliferation state, expression of this channel isminimal. Therefore, selectively blocking calcium entry into cancerous cells may be a valuable approach for preventing tumor growth. Since T-type Ca2+ channels are not expressed in epithelial cells, selective T-type Ca2+ channel blockers may be useful in the treatment of certain types of cancers.

  15. H,K-ATPase type 2 contributes to salt-sensitive hypertension induced by K(+) restriction.

    Science.gov (United States)

    Walter, Christine; Tanfous, Mariem Ben; Igoudjil, Katia; Salhi, Amel; Escher, Geneviève; Crambert, Gilles

    2016-10-01

    In industrialized countries, a large part of the population is daily exposed to low K(+) intake, a situation correlated with the development of salt-sensitive hypertension. Among many processes, adaptation to K(+)-restriction involves the stimulation of H,K-ATPase type 2 (HKA2) in the kidney and colon and, in this study, we have investigated whether HKA2 also contributes to the determination of blood pressure (BP). By using wild-type (WT) and HKA2-null mice (HKA2 KO), we showed that after 4 days of K(+) restriction, WT remain normokalemic and normotensive (112 ± 3 mmHg) whereas HKA2 KO mice exhibit hypokalemia and hypotension (104 ± 2 mmHg). The decrease of BP in HKA2 KO is due to the absence of NaCl-cotransporter (NCC) stimulation, leading to renal loss of salt and decreased extracellular volume (by 20 %). These effects are likely related to the renal resistance to vasopressin observed in HKA2 KO that may be explained, in part by the increased production of prostaglandin E2 (PGE2). In WT, the stimulation of NCC induced by K(+)-restriction is responsible for the elevation in BP when salt intake increases, an effect blunted in HKA2-null mice. The presence of an activated HKA2 is therefore required to limit the decrease in plasma [K(+)] but also contributes to the development of salt-sensitive hypertension.

  16. A novel type of replicative enzyme harbouring ATPase, primase and DNA polymerase activity

    Science.gov (United States)

    Lipps, Georg; Röther, Susanne; Hart, Christina; Krauss, Gerhard

    2003-01-01

    Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life. Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E. The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity. Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner. Deoxynucleotides are preferentially used for the synthesis of primers ∼8 nucleotides long. The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length. The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase. ORF904 constitutes a new type of replication enzyme, which could have evolved indepen dently from the eubacterial and archaeal/eukaryal proteins of DNA replication. PMID:12743045

  17. Identification of small-molecule inhibitors of Yersinia pestis Type III secretion system YscN ATPase.

    Directory of Open Access Journals (Sweden)

    Wieslaw Swietnicki

    Full Text Available Yersinia pestis is a gram negative zoonotic pathogen responsible for causing bubonic and pneumonic plague in humans. The pathogen uses a type III secretion system (T3SS to deliver virulence factors directly from bacterium into host mammalian cells. The system contains a single ATPase, YscN, necessary for delivery of virulence factors. In this work, we show that deletion of the catalytic domain of the yscN gene in Y. pestis CO92 attenuated the strain over three million-fold in the Swiss-Webster mouse model of bubonic plague. The result validates the YscN protein as a therapeutic target for plague. The catalytic domain of the YscN protein was made using recombinant methods and its ATPase activity was characterized in vitro. To identify candidate therapeutics, we tested computationally selected small molecules for inhibition of YscN ATPase activity. The best inhibitors had measured IC(50 values below 20 µM in an in vitro ATPase assay and were also found to inhibit the homologous BsaS protein from Burkholderia mallei animal-like T3SS at similar concentrations. Moreover, the compounds fully inhibited YopE secretion by attenuated Y. pestis in a bacterial cell culture and mammalian cells at µM concentrations. The data demonstrate the feasibility of targeting and inhibiting a critical protein transport ATPase of a bacterial virulence system. It is likely the same strategy could be applied to many other common human pathogens using type III secretion system, including enteropathogenic E. coli, Shigella flexneri, Salmonella typhimurium, and Burkholderia mallei/pseudomallei species.

  18. PfeT, a P1B4 -type ATPase, effluxes ferrous iron and protects Bacillus subtilis against iron intoxication.

    Science.gov (United States)

    Guan, Guohua; Pinochet-Barros, Azul; Gaballa, Ahmed; Patel, Sarju J; Argüello, José M; Helmann, John D

    2015-11-01

    Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P1B4 -type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild-type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini-ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P1B4 -type ATPases previously implicated in bacterial pathogenesis. PMID:26261021

  19. Nitric oxide derived from L-arginine impairs cytoplasmic pH regulation by vacuolar-type H+ ATPases in peritoneal macrophages

    OpenAIRE

    1991-01-01

    The ability of macrophages (Mos) to function within an acidic environment has been shown to depend on cytoplasmic pH (pHi) regulation by vacuolar-type H+ ATPases. Mos metabolize L-arginine via an oxidative pathway that generates nitric oxide, nitrate, and nitrite. Since each of these products could potentially inhibit vacuolar-type H+ ATPases, we investigated the effect of L-arginine metabolism on Mo pHi regulation in thioglycolate-elicited murine peritoneal Mos. H+ ATPase- mediated pHi recov...

  20. T-Type Calcium Channel: A Privileged Gate for Calcium Entry and Control of Adrenal Steroidogenesis.

    Science.gov (United States)

    Rossier, Michel F

    2016-01-01

    Intracellular calcium plays a crucial role in modulating a variety of functions such as muscle contraction, hormone secretion, gene expression, or cell growth. Calcium signaling has been however shown to be more complex than initially thought. Indeed, it is confined within cell microdomains, and different calcium channels are associated with different functions, as shown by various channelopathies. Sporadic mutations on voltage-operated L-type calcium channels in adrenal glomerulosa cells have been shown recently to be the second most prevalent genetic abnormalities present in human aldosterone-producing adenoma. The observed modification of the threshold of activation of the mutated channels not only provides an explanation for this gain of function but also reminds us on the importance of maintaining adequate electrophysiological characteristics to make channels able to exert specific cellular functions. Indeed, the contribution to steroid production of the various calcium channels expressed in adrenocortical cells is not equal, and the reason has been investigated for a long time. Given the very negative resting potential of these cells, and the small membrane depolarization induced by their physiological agonists, low threshold T-type calcium channels are particularly well suited for responding under these conditions and conveying calcium into the cell, at the right place for controlling steroidogenesis. In contrast, high threshold L-type channels are normally activated by much stronger cell depolarizations. The fact that dihydropyridine calcium antagonists, specific for L-type channels, are poorly efficient for reducing aldosterone secretion either in vivo or in vitro, strongly supports the view that these two types of channels differently affect steroid biosynthesis. Whether a similar analysis is transposable to fasciculata cells and cortisol secretion is one of the questions addressed in the present review. No similar mutations on L-type or T-type channels

  1. T-type calcium channel: a privileged gate for calcium entry and control of adrenal steroidogenesis

    Directory of Open Access Journals (Sweden)

    Michel Florian Rossier

    2016-05-01

    Full Text Available Intracellular calcium plays a crucial role in modulating a variety of functions such as muscle contraction, hormone secretion, gene expression or cell growth. Calcium signaling has been however shown to be more complex than initially thought. Indeed, it is confined within cell microdomains and different calcium channels are associated with different functions, as shown by various channelopathies.Sporadic mutations on voltage-operated L-type calcium channels in adrenal glomerulosa cells have been shown recently to be the second most prevalent genetic abnormalities present in human aldosterone-producing adenoma. The observed modification of the threshold of activation of the mutated channels not only provides an explanation for this gain of function but reminds us on the importance of maintaining adequate electrophysiological characteristics to make channels able to exert specific cellular functions. Indeed, the contribution to steroid production of the various calcium channels expressed in adrenocortical cells is not equal and the reason has been investigated for a long time. Given the very negative resting potential of these cells, and the small membrane depolarization induced by their physiological agonists, low threshold T-type calcium channels are particularly well suited for responding under these conditions and conveying calcium into the cell, at the right place for controlling steroidogenesis. In contrast, high threshold L-type channels are normally activated by much stronger cell depolarizations. The fact that dihydropyridine calcium antagonists, specific for L-type channels, are poorly efficient for reducing aldosterone secretion either in vivo or in vitro, strongly supports the view that these two types of channels differently affect steroid biosynthesis.Whether a similar analysis is transposable to fasciculata cells and cortisol secretion is one of the questions addressed in the present review. No similar mutations on L-type or T-type

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    ECA3, a Golgi-localized P2A-type ATPase, plays a crucial role in manganese nutrition in Arabidopsis. Mills RF , Doherty ML , López-Marqués RL , Weimar T , Dupree P , Palmgren MG , Pittman JK , Williams LE . Calcium (Ca) and manganese (Mn) are essential nutrients required for normal plant growth and......, several independent T-DNA insertion mutant alleles were isolated. When grown on medium lacking Mn, eca3 mutants, but not eca2 mutants, displayed a striking difference from wild-type plants. After approximately 8 to 9 d on this medium, eca3 mutants became chlorotic, and root and shoot growth were strongly...... inhibited compared to wild-type plants. These severe deficiency symptoms were suppressed by low levels of Mn, indicating a crucial role for ECA3 in Mn nutrition in Arabidopsis. eca3 mutants were also more sensitive than wild-type plants and eca2 mutants on medium lacking Ca; however, the differences were...

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

    Science.gov (United States)

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-12-31

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

  5. Demethoxycurcumin is a potent inhibitorof P-type ATPases from diverse kingdoms of life

    DEFF Research Database (Denmark)

    Dao, Trung Tuan; Sehgal, Pankaj; Thanh Tung, Truong;

    2016-01-01

    as drugs. In this work a library of natural compounds was screened and we first identified curcuminoids as plasma membrane H+-ATPases inhibitors in plant and fungal cells.We also found that some of the commercial curcumins contain several curcuminoids. Three of these were purified and, among...... of these pumps. Future research on biological effects of commercial preparations of curcumin should consider the heterogeneity of the material....

  6. Crystallization of P-type ATPases by the High Lipid-Detergent (HiLiDe) Method

    DEFF Research Database (Denmark)

    Sitsel, Oleg; Wang, Kaituo; Liu, Xiangyu;

    2016-01-01

    Determining structures of membrane proteins remains a significant challenge. A technique utilizing high lipid-detergent concentrations ("HiLiDe") circumvents the major bottlenecks of current membrane protein crystallization methods. During HiLiDe, the protein-lipid-detergent ratio is varied...... in a controlled way in order to yield initial crystal hits, which may be subsequently optimized by variation of the crystallization conditions and/or utilizing secondary detergents. HiLiDe preserves the advantages of classical lipid-based methods, yet is compatible with both the vapor diffusion and batch...... crystallization techniques. The method has been applied with particular success to P-type ATPases....

  7. Crystallization of P-type ATPases by the High Lipid-Detergent (HiLiDe) Method.

    Science.gov (United States)

    Sitsel, Oleg; Wang, Kaituo; Liu, Xiangyu; Gourdon, Pontus

    2016-01-01

    Determining structures of membrane proteins remains a significant challenge. A technique utilizing high lipid-detergent concentrations ("HiLiDe") circumvents the major bottlenecks of current membrane protein crystallization methods. During HiLiDe, the protein-lipid-detergent ratio is varied in a controlled way in order to yield initial crystal hits, which may be subsequently optimized by variation of the crystallization conditions and/or utilizing secondary detergents. HiLiDe preserves the advantages of classical lipid-based methods, yet is compatible with both the vapor diffusion and batch crystallization techniques. The method has been applied with particular success to P-type ATPases.

  8. Antifungal Mechanism of Action of Lactoferrin: Identification of H+-ATPase (P3A-Type) as a New Apoptotic-Cell Membrane Receptor.

    Science.gov (United States)

    Andrés, María T; Acosta-Zaldívar, Maikel; Fierro, José F

    2016-07-01

    Human lactoferrin (hLf) is a protein of the innate immune system which induces an apoptotic-like process in yeast. Determination of the susceptibility to lactoferrin of several yeast species under different metabolic conditions, respiratory activity, cytoplasmic ATP levels, and external medium acidification mediated by glucose assays suggested plasma membrane Pma1p (P3A-type ATPase) as the hLf molecular target. The inhibition of plasma membrane ATPase activity by hLf and the identification of Pma1p as the hLf-binding membrane protein confirmed the previous physiological evidence. Consistent with this, cytoplasmic ATP levels progressively increased in hLf-treated Candida albicans cells. However, oligomycin, a specific inhibitor of the mitochondrial F-type ATPase proton pump (mtATPase), abrogated the antifungal activity of hLf, indicating a crucial role for mtATPase in the apoptotic process. We suggest that lactoferrin targeted plasma membrane Pma1p H(+)-ATPase, perturbing the cytoplasmic ion homeostasis (i.e., cytoplasmic H(+) accumulation and subsequent K(+) efflux) and inducing a lethal mitochondrial dysfunction. This initial event involved a normal mitochondrial ATP synthase activity responsible for both the ATP increment and subsequent hypothetical mitochondrial proton flooding process. We conclude that human lactoferrin inhibited Pma1p H(+)-ATPase, inducing an apoptotic-like process in metabolically active yeast. Involvement of mitochondrial H(+)-ATPase (nonreverted) was essential for the progress of this programmed cell death in which the ionic homeostasis perturbation seems to precede classical nonionic apoptotic events. PMID:27139463

  9. Cellular function and pathological role of ATP13A2 and related P-type transport ATPases in Parkinson’s disease and other neurological disorders

    Directory of Open Access Journals (Sweden)

    Sarah evan Veen

    2014-05-01

    Full Text Available Mutations in ATP13A2 lead to Kufor-Rakeb syndrome, a parkinsonism with dementia. ATP13A2 belongs to the P-type transport ATPases, a large family of primary active transporters that exert vital cellular functions. However, the cellular function and transported substrate of ATP13A2 remain unknown. To discuss the role of ATP13A2 in neurodegeneration, we first provide a short description of the architecture and transport mechanism of P-type transport ATPases. Then, we briefly highlight key P-type ATPases involved in neuronal disorders such as the copper transporters ATP7A (Menkes disease, ATP7B (Wilson disease, the Na+/K+-ATPases ATP1A2 (familial hemiplegic migraine and ATP1A3 (rapid-onset dystonia parkinsonism. Finally, we review the recent literature of ATP13A2 and discuss ATP13A2’s putative cellular function in the light of what is known concerning the functions of other, better-studied P-type ATPases. We critically review the available data concerning the role of ATP13A2 in heavy metal transport and propose a possible alternative hypothesis that ATP13A2 might be a flippase. As a flippase, ATP13A2 may transport an organic molecule, such as a lipid or a peptide, from one membrane leaflet to the other. A flippase might control local lipid dynamics during vesicle formation and membrane fusion events.

  10. P4-ATPases

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  11. Evolution of the P-type II ATPase gene family in the fungi and presence of structural genomic changes among isolates of Glomus intraradices

    Directory of Open Access Journals (Sweden)

    Sanders Ian R

    2006-03-01

    Full Text Available Abstract Background The P-type II ATPase gene family encodes proteins with an important role in adaptation of the cell to variation in external K+, Ca2+ and Na2+ concentrations. The presence of P-type II gene subfamilies that are specific for certain kingdoms has been reported but was sometimes contradicted by discovery of previously unknown homologous sequences in newly sequenced genomes. Members of this gene family have been sampled in all of the fungal phyla except the arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota, which are known to play a key-role in terrestrial ecosystems and to be genetically highly variable within populations. Here we used highly degenerate primers on AMF genomic DNA to increase the sampling of fungal P-Type II ATPases and to test previous predictions about their evolution. In parallel, homologous sequences of the P-type II ATPases have been used to determine the nature and amount of polymorphism that is present at these loci among isolates of Glomus intraradices harvested from the same field. Results In this study, four P-type II ATPase sub-families have been isolated from three AMF species. We show that, contrary to previous predictions, P-type IIC ATPases are present in all basal fungal taxa. Additionally, P-Type IIE ATPases should no longer be considered as exclusive to the Ascomycota and the Basidiomycota, since we also demonstrate their presence in the Zygomycota. Finally, a comparison of homologous sequences encoding P-type IID ATPases showed unexpectedly that indel mutations among coding regions, as well as specific gene duplications occur among AMF individuals within the same field. Conclusion On the basis of these results we suggest that the diversification of P-Type IIC and E ATPases followed the diversification of the extant fungal phyla with independent events of gene gains and losses. Consistent with recent findings on the human genome, but at a much smaller geographic scale, we provided evidence

  12. [Electrogenic activity of Na-K-ATPase and calcium ions in m. soleus fibers of rats and Mongolian gerbil during simulation of gravitational unloading].

    Science.gov (United States)

    Kravtsova, V V; Ogneva, I V; Altaeva, E G; Razgovorova, I A; Tiapkina, O V; Nikol'skiĭ, E E; Shenkman, B S; Krivoĭ, I I

    2010-01-01

    Some of the electrophysiological parameters of m. soleus of rat and Mongolian gerbil, and Ca ions content in fiber myoplasm were compared in different periods of gravitational unloading simulated by tail-suspension. No difference was found between the control animals as for membrane potential at rest, electrogenic activities of Na-K-ATPase and its isoforms, and input resistance of m. soleus fibers. At the same time, unlike rats, gerbils exhibited a substantial Ca decrease in myoplasm. From day one to 14 of gravitational unloading the pace of electrophysiological changes in gerbil's m. soleus was noticeably slower than of rat's, whereas Ca ions depositing in myoplasm was observed in both species already at the beginning ofsuspension. Analysis of the results suggests that adaptive changes in m. soleus of Mongolian gerbil and rat during simulated gravitational unloading are fundamentally different due to, probably, peculiar water-electrolyte metabolism, type of locomotion, and other factors which are still unclear. PMID:20799658

  13. Cellular function and pathological role of ATP13A2 and related P-type transport ATPases in Parkinson's disease and other neurological disorders

    DEFF Research Database (Denmark)

    van Veen, Sarah; Sørensen, Danny M.; Holemans, Tine;

    2014-01-01

    . To discuss the role of ATP13A2 in neurodegeneration, we first provide a short description of the architecture and transport mechanism of P-type transport ATPases. Then, we briefly highlight key P-type ATPases involved in neuronal disorders such as the copper transporters ATP7A (Menkes disease), ATP7B (Wilson...... disease), the Na+/K+-ATPases ATP1A2 (familial hemiplegic migraine) and ATP1A3 (rapid-onset dystonia parkinsonism). Finally, we review the recent literature of ATP13A2 and discuss ATP13A2's putative cellular function in the light of what is known concerning the functions of other, better-studied P...

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

    Institute of Scientific and Technical Information of China (English)

    WANG Xue; FAN Weimin; XIE Liping; ZHANG Rongqing

    2008-01-01

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

  15. Glucocorticoids specifically enhance L-type calcium current amplitude and affect calcium channel subunit expression in the mouse hippocampus.

    NARCIS (Netherlands)

    P.J.P. Chameau; Y.J. Qin; G. Smit; M. Joëls

    2007-01-01

    Previous studies have shown that corticosterone enhances whole cell calcium currents in CA1 pyramidal neurons, through a pathway involving binding of glucocorticoid receptor homodimers to the DNA. We examined whether glucocorticoids show selectivity for L- over N-type of calcium currents. Moreover,

  16. Glucocorticoids specifically enhance L-type calcium current amplitude and affect calcium channel subunit expression in the mouse hippocampus.

    Science.gov (United States)

    Chameau, Pascal; Qin, Yongjun; Spijker, Sabine; Smit, August Benjamin; Smit, Guus; Joëls, Marian

    2007-01-01

    Previous studies have shown that corticosterone enhances whole cell calcium currents in CA1 pyramidal neurons, through a pathway involving binding of glucocorticoid receptor homodimers to the DNA. We examined whether glucocorticoids show selectivity for L- over N-type of calcium currents. Moreover, we addressed the putative gene targets that eventually lead to the enhanced calcium currents. Electrophysiological recordings were performed in nucleated patches that allow excellent voltage control. Calcium currents in these patches almost exclusively involve N- and L-type channels. We found that L- but not N-type calcium currents were largely enhanced after treatment with a high dose of corticosterone sufficient to activate glucocorticoid receptors. Voltage dependency and kinetic properties of the currents were unaffected by the hormone. Nonstationary noise analysis suggests that the increased current is not caused by a larger unitary conductance, but rather to a doubling of the number of functional channels. Quantitative real-time PCR revealed that transcripts of the Ca(v)1 subunits encoding for the N- or L-type calcium channels are not upregulated in the mouse CA1 area; instead, a strong, direct, and consistent upregulation of the beta4 subunit was observed. This indicates that the corticosteroid-induced increase in number of L-type calcium channels is not caused by a simple transcriptional regulation of the pore-forming subunit of the channels.

  17. Δ²,³-ivermectin ethyl secoester, a conjugated ivermectin derivative with leishmanicidal activity but without inhibitory effect on mammalian P-type ATPases.

    Science.gov (United States)

    Noël, François; Pimenta, Paulo Henrique Cotrim; Dos Santos, Anderson Rouge; Tomaz, Erick Carlos Loureiro; Quintas, Luis Eduardo Menezes; Kaiser, Carlos Roland; Silva, Claudia Lucia Martins; Férézou, Jean-Pierre

    2011-01-01

    Looking at a new putative target for the large spectrum antiparasitic drug ivermectin, we recently showed that avermectin-derived drugs are active against promastigote and amastigote forms of Leishmania amazonensis at low micromolar concentrations. However, we then reported that at this concentration range ivermectin is also able to inhibit three important mammalian P-type ATPases so that unacceptable adverse effects could occur if this drug were used at such high doses therapeutically. The present work aimed to test the activity of ten ivermectin analogs on these rat ATPases in search of a compound with similar leishmanicidal activity but with no effect on the mammalian (host) ATPases at effective concentrations. We synthesized three new ivermectin analogs for testing on rat SERCA (1a and 1b), Na+, K+-ATPase (α₁ and α₂/α₃ isoforms) and H+/K+-ATPase activity, along with seven analogs already characterized for their leishmanicidal activity. Our main finding is that one of the prepared derivatives, Δ²,³-ivermectin ethyl secoester 8, is equipotent to ivermectin 1 for the in vitro leishmanicidal effects but is nearly without effect on the rat ATPases, indicating that it could have a better therapeutic index in vivo and could serve as a candidate for hit-to-lead progression. This conclusion is further supported by the fact that compound 8 produced only 6% (vs 77% for ivermectin) inhibition of the human kidney enzyme at 5 μM, a concentration corresponding to the IC₅₀ for the activity against L. amazonensis amastigotes. PMID:21088826

  18. Final Report for DE-FG02-04ER15626: P-type ATPases in Plants – Role of Lipid Flippases in Membrane Biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Harper, Jeffrey F. [Univ. of Nevada, Reno, NV (United States)

    2015-02-24

    The long-range goal of the research is to understand the structure and biological functions of different P-type ATPases (ion pumps) in plant cells, and to use that knowledge to enhance the production of bioenergy from plants, or plant-research inspired technologies. Ptype ATPases include ion pumps that specifically transport H+, Ca2+, Zn2+, Cu2+, K+, or Na+, as well as at least one unusual subfamily that appears to function as lipid flippases, flipping specific lipids from one side of a membrane bilayer to the other. As a group, P-type ATPases are thought to consume more than 1/3 of the cellular ATP in typical eukaryotic cells. Recent research in the Harper lab focused on understanding the biochemical and biological functions of P-type ATPases that flip lipids. These flippases belong to the P4 subfamily of P-type ATPases. The activity of lipid flippases is thought to induce membrane curvature and/or create an asymmetry in which certain lipid head groups are preferential exposed to one surface or the other. In Arabidopsis thaliana there are 12 members of this family referred to as Aminophospholipid ATPase (ALA) 1 to ALA12. Using genetic knockouts, the Harper lab has established that this unusual subfamily of P-type ATPases are critical for plants to cope with even modest changes in temperature (e.g., down to 15°C, or up to 30°C). In addition, members of one subclade are critical for cell expansion, and loss of function mutants result in severe dwarfism. Other members of this same sub-clade are critical for pollen tube growth, and loss of function mutants are sterile under conditions of hot days and cold nights. While the cellular processes that depend on lipid flippases are still unclear, the genetic analysis of loss of function mutants clearly show they are of fundamental importance to plant growth and response to the environment.

  19. Crystallization and preliminary X-ray analysis of Salmonella FliI, the ATPase component of the type III flagellar protein-export apparatus

    International Nuclear Information System (INIS)

    Crystals of an N-terminally truncated variant of the Salmonella flagellar ATPase FliI, which exports substrate proteins into the central channel of the growing flagellar structure by utilizing the energy of ATP hydrolysis, have been obtained and characterized by X-ray diffraction. Most of the structural components making up the bacterial flagellum are translocated through the central channel of the growing flagellar structure by the type III flagellar protein-export apparatus in an ATPase-driven manner and are assembled at the growing end. FliI is the ATPase that drives flagellar protein export using the energy of ATP hydrolysis. FliI forms an oligomeric ring structure in order to attain maximum ATPase activity. In this study, FliI(Δ1–18), an N-terminally truncated variant of FliI lacking the first 18 residues, was purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion technique with PEG 8000 as a precipitant. FliI(Δ1–18) crystals grew in the monoclinic space group P21, with unit-cell parameters a = 48, b = 73, c = 126 Å, β = 94°, and diffracted to 2.4 Å resolution. Anomalous difference Patterson maps of Os-derivative and Pt-derivative crystals showed significant peaks in their Harker sections, indicating that both derivatives are suitable for structure determination

  20. The cardiac L-type calcium channel distal carboxy terminus autoinhibition is regulated by calcium.

    Science.gov (United States)

    Crump, Shawn M; Andres, Douglas A; Sievert, Gail; Satin, Jonathan

    2013-02-01

    The L-type calcium channel (LTCC) provides trigger Ca(2+) for sarcoplasmic reticulum Ca-release, and LTCC function is influenced by interacting proteins including the LTCC distal COOH terminus (DCT) and calmodulin. DCT is proteolytically cleaved and reassociates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current (I(Ba,L)) in reconstituted channel complexes, yet the contribution of DCT to LTCC Ca(2+) current (I(Ca,L)) in cardiomyocyte systems is unexplored. This study tests the hypothesis that DCT attenuates cardiomyocyte I(Ca,L). We measured LTCC current and Ca(2+) transients with DCT coexpressed in murine cardiomyocytes. We also heterologously coexpressed DCT and Ca(V)1.2 constructs with truncations corresponding to the predicted proteolytic cleavage site, Ca(V)1.2Δ1801, and a shorter deletion corresponding to well-studied construct, Ca(V)1.2Δ1733. DCT inhibited I(Ba,L) in cardiomyocytes, and in human embryonic kidney (HEK) 293 cells expressing Ca(V)1.2Δ1801 and Ca(V)1.2Δ1733. Ca(2+)-CaM relieved DCT block in cardiomyocytes and HEK cells. The selective block of I(Ba,L) combined with Ca(2+)-CaM effects suggested that DCT-mediated blockade may be relieved under conditions of elevated Ca(2+). We therefore tested the hypothesis that DCT block is dynamic, increasing under relatively low Ca(2+), and show that DCT reduced diastolic Ca(2+) at low stimulation frequencies but spared high frequency Ca(2+) entry. DCT reduction of diastolic Ca(2+) and relief of block at high pacing frequencies and under conditions of supraphysiological bath Ca(2+) suggests that a physiological function of DCT is to increase the dynamic range of Ca(2+) transients in response to elevated pacing frequencies. Our data motivate the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    Eukaryotes encompass a remarkable variety of organisms and unresolved lineages. Different phylogenetic analyses have lead to conflicting conclusions as to the origin and associations between lineages and species. In this work, we investigated evolutionary relationship of a family of cation pumps ...... far, while P5B ATPases appear to be lost in three eukaryotic lineages; excavates, entamoebas and land plants. A lineage-specific gene expansion of up to four different P5B ATPases is seen in animals....

  2. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    Science.gov (United States)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  3. Surface Display of Metal Fixation Motifs of Bacterial P1-Type ATPases Specifically Promotes Biosorption of Pb2+ by Saccharomyces cerevisiae▿

    OpenAIRE

    Kotrba, Pavel; Ruml, Tomas

    2010-01-01

    Biosorption of metal ions may take place by different passive metal-sequestering processes such as ion exchange, complexation, physical entrapment, and inorganic microprecipitation or by a combination of these. To improve the biosorption capacity of the potential yeast biosorbent, short metal-binding NP peptides (harboring the CXXEE metal fixation motif of the bacterial Pb2+-transporting P1-type ATPases) were efficiently displayed and covalently anchored to the cell wall of Saccharomyces cere...

  4. ATPase Class I Type 8B Member 1 and Protein Kinase C-ζ Induce the Expression of the Canalicular Bile Salt Export Pump in Human Hepatocytes

    OpenAIRE

    Chen, Frank; Ellis, Ewa; Strom, Stephen C.; Shneider, Benjamin L.

    2010-01-01

    The exact molecular mechanism(s) of the disease that results from defects in the ATPase Class I Type 8B Member 1 gene remains controversial. Prior investigations of human ileum and in intestinal and ovarian cell lines have suggested that Familial Intrahepatic Cholestasis 1 (FIC1) activates the Farnesoid X-Receptor (FXR) via a pathway involving Protein Kinase C ζ (PKCζ). Translational investigations of human liver from individuals with FIC1 disease have been confounded by secondary affects of ...

  5. Prophylactic and therapeutic functions of T-type calcium blockers against noise-induced hearing loss

    OpenAIRE

    Shen, Haiyan; Zhang, BaoPing; Shin, June-Ho; Lei, Debin; Du, Yafei; Gao, Xiang; Wang, Qiuju; Ohlemiller, Kevin K.; Piccirillo, Jay; Bao, Jianxin

    2006-01-01

    Cochlear noise injury is the second most frequent cause of sensorineural hearing loss, after aging. Because calcium dysregulation is a widely recognized contributor to noise injury, we examined the potential of calcium channel blockers to reduce noise-induced hearing loss (NIHL) in mice. We focused on two T-type calcium blockers, trimethadione and ethosuximide, which are anti-epileptics approved by the Food and Drug Administration. Young C57BL/6 mice of either gender were divided into three g...

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

    Science.gov (United States)

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

    2005-08-01

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

  7. Deletion of N-type calcium channels alters ethanol reward and reduces ethanol consumption in mice

    OpenAIRE

    Newton, P. M.; Orr, C J; Wallace, M J; Kim, C.; Shin, H. S.; Messing, R O

    2004-01-01

    N-type calcium channels are modulated by acute and chronic ethanol exposure in vitro at concentrations known to affect humans, but it is not known whether N-type channels are important for behavioral responses to ethanol in vivo. Here, we show that in mice lacking functional N-type calcium channels, voluntary ethanol consumption is reduced and place preference is developed only at a low dose of ethanol. The hypnotic effects of ethanol are also substantially diminished, whereas ethanol-induced...

  8. Calcium in plant cells

    Directory of Open Access Journals (Sweden)

    V. V. Schwartau

    2014-04-01

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

  9. Hydrogen peroxide mediates oxidant-dependent stimulation of arterial smooth muscle L-type calcium channels.

    Science.gov (United States)

    Chaplin, Nathan L; Amberg, Gregory C

    2012-05-01

    Changes in calcium and redox homeostasis influence multiple cellular processes. Dysregulation of these signaling modalities is associated with pathology in cardiovascular, neuronal, endocrine, and other physiological systems. Calcium and oxidant signaling mechanisms are frequently inferred to be functionally related. To address and clarify this clinically relevant issue in the vasculature we tested the hypothesis that the ubiquitous reactive oxygen molecule hydrogen peroxide mediates oxidant-dependent stimulation of cerebral arterial smooth muscle L-type calcium channels. Using a combinatorial approach including intact arterial manipulations, electrophysiology, and total internal reflection fluorescence imaging, we found that application of physiological levels of hydrogen peroxide to isolated arterial smooth muscle cells increased localized calcium influx through L-type calcium channels. Similarly, oxidant-dependent stimulation of L-type calcium channels by the vasoconstrictor ANG II was abolished by intracellular application of catalase. Catalase also prevented ANG II from increasing localized subplasmalemmal sites of increased oxidation previously associated with colocalized calcium influx through L-type channels. Furthermore, catalase largely attenuated the contractile response of intact cerebral arterial segments to ANG II. In contrast, enhanced dismutation of superoxide to hydrogen peroxide with SOD had no effect on ANG II-dependent stimulation of L-type calcium channels. From these data we conclude that hydrogen peroxide is important for oxidant-dependent regulation of smooth muscle L-type calcium channels and arterial function. These data also support the emerging concept of hydrogen peroxide as a biologically relevant oxidant second messenger in multiple cell types with a diverse array of physiological functions.

  10. Phosphosite mapping of P-type plasma membrane H+-ATPase in homologous and heterologous environments

    DEFF Research Database (Denmark)

    Rudashevskaya, Elena; Ye, Juanying; Jensen, Ole Nørregaard;

    2012-01-01

    Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric...... analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H(+)-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any...... phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H(+)-ATPase to protein kinase...

  11. Current data with inulin-type fructans and calcium, targeting bone health in adults.

    Science.gov (United States)

    Coxam, Véronique

    2007-11-01

    In humans, there is increasing evidence that the colon can absorb nutritionally significant amounts of calcium, and this process may be susceptible to dietary manipulation by fermentable substrates, especially inulin-type fructans. Inulin-type fructans can modulate calcium absorption because they are resistant to hydrolysis by mammalian enzymes and are fermented in the large intestine to produce short-chain fatty acids, which in turn reduce luminal pH and modify calcium speciation, and hence solubility, or exert a direct effect on the mucosal transport pathway. Quite a few intervention studies showed an improvement of calcium absorption in adolescents or young adults by inulin-type fructans. In the same way, a positive effect has been reported in older women.

  12. T-type calcium channel expression in cultured human neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    Xianjie Wen; Shiyuan Xu; Lingling Wang; Hua Liang; Chengxiang Yang; Hanbing Wang; Hongzhen Liu

    2011-01-01

    Human neuroblastoma cells (SH-SY5Y) have similar structures and functions as neural cells and have been frequently used for cell culture studies of neural cell functions. Previous studies have revealed Land N-type calcium channels in SH-SY5Y cells. However, the distribution of the low -voltage activated calcium channel (namely called T-type calcium channel, including Cav3.1, Cav3.2, and Cav3.3) in SH-SY5Y cells remains poorly understood. The present study detected mRNA and protein expression of the T-type calcium channel (Cav3.1, Cav3.2, and Cav3.3) in cultured SH-SY5Y cells using real-time polymerase chain reaction (PCR) and western blot analysis. Results revealed mRNA and protein expression from all three T-type calcium channel subtypes in SH-SY5Y cells. Moreover,Cav3.1 was the predominant T-type calcium channel subtype in SH-SY5Y cells.

  13. Structural and functional characterization of the rotor rings from a Fusobacterium F-type and a Burkholderia N-type rotary ATPase

    OpenAIRE

    Schulz, Sarah

    2016-01-01

    Rotary adenosine triphosphate (ATP)ases are ubiquitous, membrane-bound enzyme complexes involved in biological energy conversion. The first subtype, the so-called F1Fo ATP synthase, predominantly functions as an ATP synthesizing machinery in most bacteria, mitochondria and chloroplasts. The vacuolar subtype of enzyme, the V1Vo ATPase, operates as an ATP driven ion pump in eukaryotic membranes. The subtype found in archaea and some bacteria is called A1Ao ATP (synth)ase and is capable of worki...

  14. Genetic association study of the P-type ATPase ATP13A2 in late-onset Parkinson's disease

    OpenAIRE

    Rakovic, A.; Stiller, B.; Djarmati, A; Flaquer, A; Freudenberg, J; Toliat, M.R.; Linnebank, M; Kostic, V; K. Lohmann; Paus, S; Nürnberg, P; Kubisch, C; Klein, C; Wüllner, U.; RAMIREZ, A

    2009-01-01

    A role of ATP13A2 in early-onset Parkinsonism (EOP) has been proposed. Conversely, the contribution of this ATPase to late-onset Parkinson's disease (PD) remains unexplored. We therefore conducted a case-control association study in this age-of-onset group with PD. The initial sample was of German origin and consisted of 220 patients with late-onset PD (mean age of onset 60.1 years) and 232 age-matched unrelated controls. Five single nucleotide polymorphisms (SNPs) covering ATP13A2 and its co...

  15. The Listeria monocytogenes Fur-regulated virulence protein FrvA is an Fe(II) efflux P1B4 -type ATPase.

    Science.gov (United States)

    Pi, Hualiang; Patel, Sarju J; Argüello, José M; Helmann, John D

    2016-06-01

    Listeria monocytogenes FrvA (Lmo0641) is critical for virulence in the mouse model and is an ortholog of the Bacillus subtilis Fur- and PerR-regulated Fe(II) efflux P1B4 -type ATPase PfeT. Previously, FrvA was suggested to protect against heme toxicity. Here, we demonstrate that an frvA mutant is sensitive to iron intoxication, but not to other metals. Expression of frvA is induced by high iron and this induction requires Fur. FrvA functions in vitro as a divalent cation specific ATPase most strongly activated by ferrous iron. When expressed in B. subtilis, FrvA increases resistance to iron both in wild-type and in a pfeT null strain. FrvA is a high affinity Fe(II) exporter and its induction imposes severe iron limitation in B. subtilis resulting in derepression of both Fur- and PerR-regulated genes. FrvA also recognizes Co(II) and Zn(II) as substrates and can complement B. subtilis strains defective in the endogenous export systems for these cations. Building on these results, we conclude that FrvA functions in the efflux of Fe(II), and not heme during listerial infection. PMID:26946370

  16. Cd(2+) extrusion by P-type Cd(2+)-ATPase of Staphylococcus aureus 17810R via energy-dependent Cd(2+)/H(+) exchange mechanism.

    Science.gov (United States)

    Tynecka, Zofia; Malm, Anna; Goś-Szcześniak, Zofia

    2016-08-01

    Cd(2+) is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd(2+)-resistant due to possession of cadA-coded Cd(2+) efflux system, recognized here as P-type Cd(2+)-ATPase. This Cd(2+) pump utilizing cellular energy-ATP, ∆μ H (+) (electrochemical proton potential) and respiratory protons, extrudes Cd(2+) from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd(2+) extrusion remains unknown. Here we propose that two Cd(2+) taken up by strain 17810R via Mn(2+) uniporter down membrane potential (∆ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high PiB) are trapped probably by high affinity sites in cytoplasmic domain of Cd(2+)-ATPase, forming SCdS. This stops Cd(2+) transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of Pi-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ∆ψ, extrude two trapped cytoplasmic Cd(2+), which move to low affinity sites, being then extruded into extracellular space via ∆ψ-dependent Cd(2+)/H(+) exchange. In 1 mM phosphate buffer (low PiB), external Cd(2+) competing with decreased number of Pi-dependent protons, binds to ψs of Cd(2+)-ATPase channel, enters cytoplasm through the channel down ∆ψ via Cd(2+)/Cd(2+) exchange and blocks dithiols in ODHC. However, Mg(2+) pretreatment preventing external Cd(2+) countertransport through the channel down ∆ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd(2+) via Cd(2+)/H(+) exchange, despite low PiB.

  17. T-type voltage-gated calcium channels regulate the tone of mouse efferent arterioles

    DEFF Research Database (Denmark)

    Poulsen, Christian B; Al-Mashhadi, Rozh H; Cribbs, Leanne L;

    2011-01-01

    Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation-contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar...... tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium....... Low concentrations of nickel, an agent that blocks Ca(v)3.2, had a similar effect. Thus, T-type voltage-gated calcium channels are functionally important for depolarization-induced vasoconstriction and subsequent dilatation in mouse cortical efferent arterioles.Kidney International advance online...

  18. Effect of dendroaspis natriuretic peptide (DNP) on L-type calcium channel current and its pathway.

    Science.gov (United States)

    Zhang, Shu-Ying; Cai, Zheng-Xu; Li, Ping; Cai, Chun-Yu; Qu, Cheng-Long; Guo, Hui-Shu

    2010-09-24

    Dendroaspis natriuretic peptide (DNP), a newly-described natriuretic peptide, relaxes gastrointestinal smooth muscle. L-type calcium channel currents play an important role in regulating smooth muscle contraction. The effect of DNP on L-type calcium channel currents in gastrointestinal tract is still unclear. This study was designed to investigate the effect of DNP on barium current (I(Ba)) through the L-type calcium channel in gastric antral myocytes of guinea pigs and cGMP-pathway mechanism. The whole-cell patch-clamp technique was used to record L-type calcium channel currents. The content of cGMP in guinea pig gastric antral smooth muscle and perfusion solution was measured using radioimmunoassay. DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in perfusion medium. DNP concentration-dependently inhibited I(Ba) in freshly isolated guinea pig gastric antral circular smooth muscle cells (SMCs) of guinea pigs. DNP-induced inhibition of I(Ba) was partially blocked by LY83583, an inhibitor of guanylate cyclase. KT5823, a cGMP-dependent protein kinase (PKG) inhibitor, almost completely blocked DNP-induced inhibition of I(Ba). However, DNP-induced inhibition of I(Ba) was potentiated by zaprinast, an inhibitor of cGMP-sensitive phosphodiesterase. Taken together, DNP inhibits L-type calcium channel currents via pGC-cGMP-PKG-dependent signal pathway in gastric antral myocytes of guinea pigs. PMID:20594955

  19. A novel series of pyrazolylpiperidine N-type calcium channel blockers.

    Science.gov (United States)

    Subasinghe, Nalin L; Wall, Mark J; Winters, Michael P; Qin, Ning; Lubin, Mary Lou; Finley, Michael F A; Brandt, Michael R; Neeper, Michael P; Schneider, Craig R; Colburn, Raymond W; Flores, Christopher M; Sui, Zhihua

    2012-06-15

    Selective blockers of the N-type calcium channel have proven to be effective in animal models of chronic pain. However, even though intrathecally delivered synthetic ω-conotoxin MVIIA from Conus magnus (ziconotide [Prialt®]) has been approved for the treatment of chronic pain in humans, its mode of delivery and narrow therapeutic window have limited its usefulness. Therefore, the identification of orally active, small-molecule N-type calcium channel blockers would represent a significant advancement in the treatment of chronic pain. A novel series of pyrazole-based N-type calcium channel blockers was identified by structural modification of a high-throughput screening hit and further optimized to improve potency and metabolic stability. In vivo efficacy in rat models of inflammatory and neuropathic pain was demonstrated by a representative compound from this series. PMID:22608964

  20. T-type calcium channels in neuropathic pain.

    Science.gov (United States)

    Bourinet, Emmanuel; Francois, Amaury; Laffray, Sophie

    2016-02-01

    Pain is a quite frequent complaint accompanying numerous pathologies. Among these pathological cases, numerous neuropathies are retrieved with identified etiologies (chemotherapies, diabetes, surgeries…) and also more diffuse syndromes such as fibromyalgia. More broadly, pain is one of the first consequences of most inherited diseases. Despite its importance for the quality of life, current pain management is limited to drugs that are either old or with a limited efficacy or that possess a bad risk benefit ratio. As no new pharmacological concept has led to new analgesics in the last decades, the discovery of new medications is needed, and to this aim, the identification of new druggable targets in pain transmission is a first step. Therefore, studies of ion channels in pain pathways are extremely active. This is particularly true with ion channels in peripheral sensory neurons in dorsal root ganglia known how to express unique sets of these channels. Moreover, both spinal and supraspinal levels are clearly important in pain modulation. Among these ion channels, we and others revealed the important role of low voltage-gated calcium channels in cellular excitability in different steps of the pain pathways. These channels, by being activated nearby resting membrane potential, have biophysical characteristics suited to facilitate action potential generation and rhythmicity. In this review, we will present the current knowledge on the role of these channels in the perception and modulation of pain. PMID:26785151

  1. Templating route for mesostructured calcium phosphates with carboxylic acid- and amine-type surfactants.

    Science.gov (United States)

    Ikawa, Nobuaki; Hori, Hideki; Kimura, Tatsuo; Oumi, Yasunori; Sano, Tsuneji

    2008-11-18

    Mesostructured calcium phosphates constructed by ionic frameworks were synthesized using carboxylic acid- and amine-type surfactants in mixed solvent systems of ethanol and water. A lamellar mesostructured calcium phosphate was prepared using palmitic acid as an anionic surfactant, as in the case using n-alkylamines. A wormhole-like mesostructured calcium phosphate can be obtained using dicarboxyl N-lauroyl- l-glutamic acid, whose headgroup is larger than that of palmitic acid. Similar mesostructured product was obtained using 4-dodecyldiethylenetriamine with a large headgroup containing two primary amine groups. Interactions of carboxyl and primary amino groups in the surfactant molecules with inorganic species are quite important for the formation of mesostructured calcium phosphates. The Ca/P molar ratio of mesostructured calcium phosphates was strongly affected by the molecular structure of surfactants containing carboxyl and primary amino groups. Ca-rich materials can be obtained using carboxylic acid-type surfactants (Ca/P approximately 1.7) rather than amine-type surfactants (Ca/P approximately 1.0). PMID:18947246

  2. Preparation and mechanical property of core-shell type chitosan/calcium phosphate composite fiber

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Atsushi [Japan Society for the Promotion of Science, Ikenohata1-1-1, Daitou-ku, Tokyo 110-0008 (Japan) and Creative Research Initiative ' Sousei' , Hokkaido University, Sapporo, Hokkaido 001-0021 (Japan)]. E-mail: MATSUDA.Atsushi@nims.go.jp; Ikoma, Toshiyuki [Biomaterials Research Center, National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044 (Japan); Kobayashi, Hisatoshi [Biomaterials Research Center, National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044 (Japan)]. E-mail: Kobayashi.Hisatoshi@nims.go.jp; Tanaka, Junzo [Creative Research Initiative ' Sousei' , Hokkaido University, Sapporo, Hokkaido 001-0021 (Japan); Biomaterials Research Center, National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044 (Japan)

    2004-12-01

    Core-shell type chitosan/calcium phosphate composite fibers were prepared by a facile wet spinning method; the chitosan aqueous solution with PO{sub 4} ions was dropped and coagulated in the ethanol/calcium hydroxide solutions at different mixed ratio. X-ray diffraction (XRD) patterns indicated that the crystal phases of calcium phosphates in the composite fibers were a low-crystalline hydroxyapatite (HAp; Ca{sub 10}(PO{sub 4}){sub 6}(OH){sub 2})or the low-crystalline hydroxyapatite/brushite mixture depended on the ratio of ethanol/calcium hydroxide solutions. The inorganic contents were ca. 60 wt.% by using the TG-DTA analysis. The energy-dispersive X-ray spectroscopy (EDS) analysis indicated that Ca and P atoms were mainly distributed on the outer layer of the composite fiber to grow calcium phosphate crystals; however, a little amount of P atom still remained at the inside of the fiber. This indicated that the composite fibers formed a unique core-shell structure with shell of calcium phosphate and core of chitosan. The mechanical property of the fibers was reinforced by the initial concentration of chitosan solution.

  3. The action of calcium channel blockers on recombinant L-type calcium channel alpha1-subunits.

    OpenAIRE

    Morel, Nicole; Buryi, V; Feron, Olivier; Gomez, J. P.; Christen, M O; Godfraind, Theophile

    1998-01-01

    1. CHO cells expressing the alpha(1C-a) subunit (cardiac isoform) and the alpha(1C-b) subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for alpha1C isoforms. 2. Inward current evoked by the transfected alpha1 subunit was recorded by the patch-clamp technique in the whole-cell configuration. 3. Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-1...

  4. Generation of slow wave type action potentials in the mouse small intestine involves a non-L-type calcium channel.

    Science.gov (United States)

    Malysz, J; Richardson, D; Farraway, L; Christen, M O; Huizinga, J D

    1995-10-01

    Intrinsic electrical activities in various isolated segments of the mouse small intestine were recorded (i) to characterize action potential generation and (ii) to obtain a profile on the ion channels involved in initiating the slow wave type action potentials (slow waves). Gradients in slow wave frequency, resting membrane potential, and occurrence of spiking activity were found, with the proximal intestine exhibiting the highest frequency, the most hyperpolarized cell membrane, and the greatest occurrence of spikes. The slow waves were only partially sensitive to L-type calcium channel blockers. Nifedipine, verapamil, and pinaverium bromide abolished spikes that occurred on the plateau phase of the slow waves in all tissues. The activity that remained in the presence of L-type calcium channel blockers, the upstroke potential, retained a similar amplitude to the original slow wave and was of identical frequency. The upstroke potential was not sensitive to a reduction in extracellular chloride or to the sodium channel blockers tetrodotoxin and mexiletine. Abolishment of the Na+ gradient by removal of 120 mM extracellular Na+ reduced the upstroke potential frequency by 13 - 18% and its amplitude by 50 - 70% in the ileum. The amplitude was similarly reduced by Ni2+ (up to 5 mM), and by flufenamic acid (100 mu M), a nonspecific cation and chloride channel blocker. Gadolinium, a nonspecific blocker of cation and stretch-activated channels, had no effect. Throughout these pharmacological manipulations, a robust oscillation remained at 5 - 10 mV. This oscillation likely reflects pacemaker activity. It was rapidly abolished by removal of extracellular calcium but not affected by L-type calcium channel blockers. In summary, the mouse small intestine has been established as a model for research into slow wave generation and electrical pacemaker activity. The upstroke part of the slow wave has two components, the pacemaker component involves a non-L-type calcium channel

  5. The genetic background affects the vascular response in T-type calcium channels 3.2 deficient mice

    DEFF Research Database (Denmark)

    Svenningsen, Per; Hansen, Pernille B L

    2016-01-01

    -type channels are the dominant Ca(2+) entry pathway in vascular smooth muscle cells, however, T-type calcium channels are also expressed in the cardiovascular system where they play a functional role in the regulation of both contraction and vasodilation in (Chen et al. 2003; Hansen et al. 2001). This article......Voltage-gated calcium channels (Cav ) are important regulators of vascular tone and are attractive targets for pharmacological treatment of hypertension. The clinical used calcium blockers are often not selective for one channel but affect several types of calcium channels (Hansen 2015). L...

  6. Thermal conductivity measurements on xonotlite-type calcium silicate by the transient hot-strip method

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The experimental results of the thermal conductivities of xonotlite-type calcium silicate insulation materials were presented at different temperatures and pressures.Two appropriative surroundings,i.e.an elevated temperature surrounding from ambient temperature to 1450 K and a vacuum surrounding from atmosphere pressure to 10-3 Pa,were designed for the transient hot-strip (THS) method.The thermal conduetivities of xonotlite-type calcium silicate with four densities from ambient temperature to 1000 K and 0.045 Pa to atmospheric pressure were measured.The results show that the thermal conductivity of xunotlite-type calcium silicate decreases apparently with the fall of density,and decreases apparently with the drop of pressure,and reaches the least value at about 100 Pa.The thermal conductivity of xonotlite-type calcium silicate increases almost linearly with T3,and increases more abundantly with low density than with high density.The thermal conductivity measurement uncertainty is estimated to be approximately 3% at ambient temperature,and 6% at 800 K.

  7. New Role of P/Q-type Voltage-gated Calcium Channels

    DEFF Research Database (Denmark)

    Hansen, Pernille B L

    2015-01-01

    Voltage-gated calcium channels are important for the depolarization-evoked contraction of vascular smooth muscle cells (SMCs), with L-type channels being the classical channel involved in this mechanism. However, it has been demonstrated that the CaV2.1 subunit, which encodes a neuronal isoform o...

  8. L-type Voltage-Gated Calcium Channels in Conditioned Fear: A Genetic and Pharmacological Analysis

    Science.gov (United States)

    McKinney, Brandon C.; Sze, Wilson; White, Jessica A.; Murphy, Geoffrey G.

    2008-01-01

    Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear…

  9. [Discovering L-type calcium channels inhibitors of antihypertensive drugs based on drug repositioning].

    Science.gov (United States)

    Liang, Ying-xi; He, Yu-su; Jiang, Lu-di; Yue, Qiao-xin; Cui, Shuai; Bin, Li; Ye, Xiao-tong; Zhang, Xiao-hua; Zhang, Yang-ling

    2015-09-01

    This study was amid to construct the pharmacophore model of L-type calcium channel antagonist in the application of screening Drugbank and TCMD. This paper repositions the approved drugs resulting from virtual screening and discusses the relocation-based drug discovery methods, screening antihypertensive drugs with L-type calcium channel function from TCMD. Qualitative hypotheses wre generated by HipHop separately on the basis of 12 compounds with antagonistic action on L-type calcium channel expressed in rabbit cardiac muscle. Datebase searching method was used to evaluate the generated hypotheses. The optimum hypothesis was used to search Drugbank and TCMD. This paper repositions the approved drugs and evaluates the antihypertensive effect of the chemical constituent of traditional Chinese medicine resulting from virtual screening by the matching score and literature. The results showed that optimum qualitative hypothesis is with six features, which were two hydrogen-bond acceptors, four hydrophobic groups, and the CAI value of 2.78. Screening Drugbank achieves 93 approved drugs. Screening TCMD achieves 285 chemical constituents of traditional Chinese medicine. It was concluded that the hypothesis is reliable and can be used to screen datebase. The approved drugs resulting from virtual screening, such as pravastatin, are potentially L-type calcium channels inhibitors. The chemical constituents of traditional Chinese medicine, such as Arctigenin III and Arctigenin are potentially antihypertensive drugs. It indicates that Drug Repositioning based on hypothesis is possible. PMID:26983215

  10. [Discovering L-type calcium channels inhibitors of antihypertensive drugs based on drug repositioning].

    Science.gov (United States)

    Liang, Ying-xi; He, Yu-su; Jiang, Lu-di; Yue, Qiao-xin; Cui, Shuai; Bin, Li; Ye, Xiao-tong; Zhang, Xiao-hua; Zhang, Yang-ling

    2015-09-01

    This study was amid to construct the pharmacophore model of L-type calcium channel antagonist in the application of screening Drugbank and TCMD. This paper repositions the approved drugs resulting from virtual screening and discusses the relocation-based drug discovery methods, screening antihypertensive drugs with L-type calcium channel function from TCMD. Qualitative hypotheses wre generated by HipHop separately on the basis of 12 compounds with antagonistic action on L-type calcium channel expressed in rabbit cardiac muscle. Datebase searching method was used to evaluate the generated hypotheses. The optimum hypothesis was used to search Drugbank and TCMD. This paper repositions the approved drugs and evaluates the antihypertensive effect of the chemical constituent of traditional Chinese medicine resulting from virtual screening by the matching score and literature. The results showed that optimum qualitative hypothesis is with six features, which were two hydrogen-bond acceptors, four hydrophobic groups, and the CAI value of 2.78. Screening Drugbank achieves 93 approved drugs. Screening TCMD achieves 285 chemical constituents of traditional Chinese medicine. It was concluded that the hypothesis is reliable and can be used to screen datebase. The approved drugs resulting from virtual screening, such as pravastatin, are potentially L-type calcium channels inhibitors. The chemical constituents of traditional Chinese medicine, such as Arctigenin III and Arctigenin are potentially antihypertensive drugs. It indicates that Drug Repositioning based on hypothesis is possible.

  11. Effect of propionyl-L-carnitine on L-type calcium channels in human heart sarcolemma

    International Nuclear Information System (INIS)

    Propionyl-L-carnitine (PC) protects perfused rat hearts against damage by ischemia-reperfusion. Activation of L-type calcium channel play a role on ischemia-reperfusion damage. Therefore, we studied the effect of PC on some properties of L-type calcium channels in an in vitro preparation from human myocardium sarcolemma (from patients with idiopathic dilated cardiomyopathy). Binding of the L-type calcium channel blockers isradipine [3H]-PN 200-110 (PN) to plasma membrane preparations revealed a single population of binding sites (total number: Bmax = 213 +/- 34 fM/mg protein and affinity: Kd = 152 +/- 19 nM; n = 6). The characteristics of these binding sites were evaluated in the presence and in the absence of Ca2+ and of calcium blockers (D-888, a verapamillike drug, and diltiazem). Incubation in a Ca2+-containing buffer increased the affinity of PN binding sites. Binding sites for PN were modulated by organic calcium channel blockers; in competition isotherms at 37 degree C, D-888 (desmethoxyverapamil) decreased the PN binding, whereas diltiazem increased it. These results strongly suggest that the site labelled by PN is the voltage-operated calcium channel of the human myocardium. The addition of PC (1 mM) to plasma membranes labelled with PN at 37 degree C decreased the affinity of the binding; this effect was counteracted by the addition of Ca2+ to the medium. This result was consistent with a competition between Ca2+ and PC. The effect of PC incubation at 4 degree C was the opposite; at this temperature PC increased the affinity of the binding sites and the effect was obscured by Ca2+

  12. The action of calcium channel blockers on recombinant L-type calcium channel α1-subunits

    Science.gov (United States)

    Morel, Nicole; Buryi, Vitali; Feron, Olivier; Gomez, Jean-Pierre; Christen, Marie-Odile; Godfraind, Théophile

    1998-01-01

    CHO cells expressing the α1C-a subunit (cardiac isoform) and the α1C-b subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for α1C isoforms.Inward current evoked by the transfected α1 subunit was recorded by the patch-clamp technique in the whole-cell configuration.Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of α1C-b-subunit than of α1C-a-subunit. This difference was more marked at a holding potential of −100 mV than at −50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms.Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on α1C-a than on α1C-b subunit at Vh of −100 mV and −50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages.[3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the α1C-b than for the α1C-a subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the α1C-a subunit than for the α1C-b subunit.These results indicate marked differences among Ca2+ channel blockers in their selectivity for the α1C-a and α1C-b subunits of the Ca2+ channel. PMID:9846638

  13. The action of calcium channel blockers on recombinant L-type calcium channel alpha1-subunits.

    Science.gov (United States)

    Morel, N; Buryi, V; Feron, O; Gomez, J P; Christen, M O; Godfraind, T

    1998-11-01

    1. CHO cells expressing the alpha(1C-a) subunit (cardiac isoform) and the alpha(1C-b) subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for alpha1C isoforms. 2. Inward current evoked by the transfected alpha1 subunit was recorded by the patch-clamp technique in the whole-cell configuration. 3. Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of alpha(1C-)b-subunit than of alpha(1C-a)-subunit. This difference was more marked at a holding potential of -100 mV than at -50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms. 4. Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on alpha(1C-a) than on alpha(1C-b) subunit at Vh of -100 mV and -50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages. 5. [3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the alpha(1C-b) than for the alpha(1C-a) subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the alpha(1C-a) subunit than for the alpha(1C-b) subunit. 6. These results indicate marked differences among Ca2+ channel blockers in their selectivity for the alpha(1C-a) and alpha(1C-b) subunits of the Ca2+ channel. PMID:9846638

  14. Recycling (?): Relict Spinels (?) in Type B Calcium-Aluminum-Rich Inclusions

    OpenAIRE

    Connolly, H. C.; Burnett, D. S.

    1998-01-01

    Type B calcium-aluminum-rich inclusions (CAIs) can be thought of as a type of chondrule despite obvious differences in size, composition, and texture. Nevertheless, igneous CAIs likely experienced similar thermal histories. A major constraint on the thermal history of chondrules is that they have been recycled, mainly supported by the presence relict grains. Although well known that igneous CAIs experienced at least two melting events (counting Wark-Lovering rims), the identification of re...

  15. Role of T-type calcium channels in myogenic tone of skeletal muscle resistance arteries

    DEFF Research Database (Denmark)

    VanBavel, Ed; Sorop, Oana; Andreasen, Ditte;

    2002-01-01

    T-type calcium channels may be involved in the maintenance of myogenic tone. We tested their role in isolated rat cremaster arterioles obtained after CO(2) anesthesia and decapitation. Total RNA was analyzed by RT-PCR and Southern blotting for calcium channel expression. We observed expression...... of voltage-operated calcium (Ca(V)) channels Ca(V)3.1 (T-type), Ca(V)3.2 (T-type), and Ca(V)1.2 (L-type) in cremaster arterioles (n = 3 rats). Amplification products were observed only in the presence of reverse transcriptase and cDNA. Concentration-response curves of the relatively specific L-type blocker...... verapamil and the relatively specific T-type blockers mibefradil and nickel were made on cannulated vessels with either myogenic tone (75 mmHg) or a similar level of constriction induced by 30 mM K(+) at 35 mmHg. Mibefradil and nickel were, respectively, 162-fold and 300-fold more potent in inhibiting...

  16. Experimental determination of control by the H+-ATPase in Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole; Westerhoff, H. V.

    1995-01-01

    coefficient by the H+-ATPase with respect to growth rate and catabolic fluxes was measured. Control on growth rate was absent at the wildtype concentration of H+-ATPase, independent of whether the substrate for growth was glucose or succinate. Control by the H+-ATPase on the catabolic fluxes, including...... respiration, was negative at the wild-type H+-ATPase level. Moreover, the turnover number of the individual H+-ATPase enzymes increased as the H+-ATPase concentration was lowered. The negative control by the H+-ATPase on catabolism may thus be involved in a homeostatic control of ATP synthesis and, to some...

  17. GABA(A) Increases Calcium in Subventricular Zone Astrocyte-Like Cells Through L- and T-Type Voltage-Gated Calcium Channels

    DEFF Research Database (Denmark)

    Young, Stephanie Z; Platel, Jean-Claude; Nielsen, Jakob V;

    2010-01-01

    induced Ca(2+) increases in 40-50% of SVZ astrocytes. GABA(A)-induced Ca(2+) increases were prevented with nifedipine and mibefradil, blockers of L- and T-type voltage-gated calcium channels (VGCC). The L-type Ca(2+) channel activator BayK 8644 increased the percentage of GABA(A)-responding astrocyte...

  18. Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

    Institute of Scientific and Technical Information of China (English)

    Min Chen; Yanru Wang; Tingting Hou; Huiliang Zhang; Aijuan Qu; Xianhua Wang

    2011-01-01

    Mitochondrial calcium plays a crucial role in mitochondriai metabolism,cell calcium handling,and cell death.However,some mechanisms concerning mitochondrial calcium regulation are still unknown,especially how mitochondrial calcium couples with cytosolic calcium.In this work,we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation.Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester,a mitochondrial membrane potential indicator.The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2.The apparent Kd of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes.Furthermore,we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria.In HeLa cells,we found that mitochondrial calcium ([Ca2+]mito)responded to the changes of cytosolic calcium ([Ca2+]cyto)induced by histamine or thapasigargin.Moreover,external Ca2+ (100 μmol/L) directly induced an increase of [Ca2+]mito in permeabilized HeLa cells.However,in rat cardiomyocytes [Ca2+]mito did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine.In permeabilized cardiomyocytes,600 nmol/L free Ca2+ repeatedly increased the fluorescent signals of mito-GCaMP2,which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria.These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding,...

  20. The emerging structure of vacuolar ATPases.

    Science.gov (United States)

    Drory, Omri; Nelson, Nathan

    2006-10-01

    Bioenergetics and physiology of primary pumps have been revitalized by new insights into the mechanism of energizing biomembranes. Structural information is becoming available, and the three-dimensional structure of F-ATPase is being resolved. The growing understanding of the fundamental mechanism of energy coupling may revolutionize our view of biological processes. The F- and V-ATPases (vacuolar-type ATPase) exhibit a common mechanical design in which nucleotide-binding on the catalytic sector, through a cycle of conformation changes, drives the transmembrane passage of protons by turning a membrane-embedded rotor. This motor can run in forward or reverse directions, hydrolyzing ATP as it pumps protons uphill or creating ATP as protons flow downhill. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as an ATP-dependent proton pump. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. V- and F-ATPases have similar structure and mechanism of action, and several of their subunits evolved from common ancestors. Electron microscopy studies of V-ATPase revealed its general structure at low resolution. Recently, several structures of V-ATPase subunits, solved by X-ray crystallography with atomic resolution, were published. This, together with electron microscopy low-resolution maps of the whole complex, and biochemistry cross-linking experiments, allows construction of a structural model for a part of the complex that may be used as a working hypothesis for future research. PMID:16990452

  1. Formaldehyde increases intracellular calcium concentration in primary cultured hippocampal neurons partly through NMDA receptors and T-type calcium channels

    Institute of Scientific and Technical Information of China (English)

    Ye-Nan Chi; Xu Zhang; Jie Cai; Feng-Yu Liu; Guo-Gang Xing; You Wan

    2012-01-01

    Objective Formaldehyde at high concentrations is a contributor to air pollution.It is also an endogenous metabolic product in cells,and when beyond physiological concentrations,has pathological effects on neurons.Formaldehyde induces mis-folding and aggregation of neuronal tau protein,hippocampal neuronal apoptosis,cognitive impairment and loss of memory functions,as well as excitation of peripheral nociceptive neurons in cancer pain models.Intracellular calcium ([Ca2+]i) is an important intracellular messenger,and plays a key role in many pathological processes.The present study aimed to investigate the effect of formaldehyde on [Ca2+]i and the possible involvement of N-methyl-D-aspartate receptors (NMDARs) and T-type Ca2+ channels on the cell membrane.Methods Using primary cultured hippocampal neurons as a model,changes of [Ca2+]i in the presence of formaldehyde at a low concentration were detected by confocal laser scanning microscopy.Results Formaldehyde at 1 mmol/L approximately doubled [Ca2+]i.(2R)-amino-5-phosphonopentanoate (AP5,25 μtmol/L,an NMDAR antagonist) and mibefradil (MIB,1 μtmol/L,a T-type Ca2+ channel blocker),given 5 min after formaldehyde perfusion,each partly inhibited the formaldehyde-induced increase of [Ca2+]i,and this inhibitory effect was reinforced by combined application of AP5 and MIB.When applied 3 min before formaldehyde perfusion,AP5 (even at 50 μmol/L) did not inhibit the formaldehyde-induced increase of [Ca2+]i,but MIB (1 μmol/L) significantly inhibited this increase by 70%.Conclusion These results suggest that formaldehyde at a low concentration increases [Ca2+]i in cultured hippocampal neurons; NMDARs and T-type Ca2+ channels may be involved in this process.

  2. Inhibition of Recombinant Human T-type Calcium Channels by Δ9-Tetrahydrocannabinol and Cannabidiol*

    OpenAIRE

    Ross, Hamish Redmond; Napier, Ian; Connor, Mark

    2008-01-01

    Δ9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most prevalent biologically active constituents of Cannabis sativa. THC is the prototypic cannabinoid CB1 receptor agonist and is psychoactive and analgesic. CBD is also analgesic, but it is not a CB1 receptor agonist. Low voltage-activated T-type calcium channels, encoded by the CaV3 gene family, regulate the excitability of many cells, including neurons involved in nociceptive processing. We examined the eff...

  3. Mechanisms underlying the effects of inulin-type fructans on the intestinal calcium absorption

    OpenAIRE

    Raschka, Ladislav

    2005-01-01

    Inulin-type fructans in a diet are selectively fermented by the large intestinal microflora which causes a multitude of effects that are considered as beneficial for human health and well-being. One of these well documented actions is an increased intestinal calcium absorption, similarly observed in experimental animals and in humans. Since the underlying mechanisms are not yet understood, various in vivo and in vitro experiments with rats were conducted to elucidate the molecular actions of ...

  4. Functional Importance of L- and P/Q-Type Voltage-Gated Calcium Channels in Human Renal Vasculature

    DEFF Research Database (Denmark)

    Hansen, Pernille B; Poulsen, Christian B; Walter, Steen;

    2011-01-01

    revealed signals for Ca(v) 2.1 and Ca(v) 3.1 associated with smooth muscle cells of preglomerular and postglomerular vessels. In human intrarenal arteries, depolarization with potassium induced a contraction inhibited by the L-type antagonist nifedipine, EC(50) 1.2×10(-8) mol/L. The T-type antagonist...... L- and P/Q-type channels are of functional importance for the depolarization-induced vasoconstriction. The contribution of P/Q-type channels to contraction in the human vasculature is a novel mechanism for the regulation of renal blood flow and suggests that clinical treatment with calcium blockers......Calcium channel blockers are widely used for treatment of hypertension, because they decrease peripheral vascular resistance through inhibition of voltage-gated calcium channels. Animal studies of renal vasculature have shown expression of several types of calcium channels that are involved in...

  5. Signal processing by T-type calcium channel interactions in the cerebellum

    Directory of Open Access Journals (Sweden)

    Jordan D.T. Engbers

    2013-11-01

    Full Text Available T-type calcium channels of the Cav3 family are unique among voltage-gated calcium channels due to their low activation voltage, rapid inactivation, and small single channel conductance. These special properties allow Cav3 calcium channels to regulate neuronal processing in the subthreshold voltage range. Here, we review two different subthreshold ion channel interactions involving Cav3 channels and explore the ability of these interactions to expand the functional roles of Cav3 channels. In cerebellar Purkinje cells, Cav3 and intermediate conductance calcium-activated potassium (IKCa channels form a novel complex which creates a low voltage-activated, transient outward current capable of suppressing temporal summation of excitatory postsynaptic potentials (EPSPs. In large diameter neurons of the deep cerebellar nuclei, Cav3-mediated calcium current (IT and hyperpolarization-activated cation current (IH are activated during trains of IPSPs. These currents have distinct, and yet synergistic, roles in the subthreshold domain with IT generating a rebound burst and IH controlling first spike latency and rebound spike precision. However, by shortening the membrane time constant the membrane returns towards resting value at a faster rate, allowing IH to increase the efficacy of IT, and increase the range of burst frequencies that can be generated. The net effect of Cav3 channels thus depends on the channels with which they are paired. When expressed in a complex with a KCa channel, Cav3 channels reduce excitability when processing excitatory inputs. If functionally coupled with an HCN channel, the depolarizing effect of Cav3 channels is accentuated, allowing for efficient inversion of inhibitory inputs to generate a rebound burst output. Therefore, signal processing relies not only on the activity of individual subtypes of channels but also on complex interactions between ion channels whether based on a physical complex or by indirect effects on

  6. Signal processing by T-type calcium channel interactions in the cerebellum.

    Science.gov (United States)

    Engbers, Jordan D T; Anderson, Dustin; Zamponi, Gerald W; Turner, Ray W

    2013-11-27

    T-type calcium channels of the Cav3 family are unique among voltage-gated calcium channels due to their low activation voltage, rapid inactivation, and small single channel conductance. These special properties allow Cav3 calcium channels to regulate neuronal processing in the subthreshold voltage range. Here, we review two different subthreshold ion channel interactions involving Cav3 channels and explore the ability of these interactions to expand the functional roles of Cav3 channels. In cerebellar Purkinje cells, Cav3 and intermediate conductance calcium-activated potassium (IKCa) channels form a novel complex which creates a low voltage-activated, transient outward current capable of suppressing temporal summation of excitatory postsynaptic potentials (EPSPs). In large diameter neurons of the deep cerebellar nuclei, Cav3-mediated calcium current (I T) and hyperpolarization-activated cation current (I H) are activated during trains of inhibitory postsynaptic potentials. These currents have distinct, and yet synergistic, roles in the subthreshold domain with I T generating a rebound burst and I H controlling first spike latency and rebound spike precision. However, by shortening the membrane time constant the membrane returns towards resting value at a faster rate, allowing I H to increase the efficacy of I T and increase the range of burst frequencies that can be generated. The net effect of Cav3 channels thus depends on the channels with which they are paired. When expressed in a complex with a KCa channel, Cav3 channels reduce excitability when processing excitatory inputs. If functionally coupled with an HCN channel, the depolarizing effect of Cav3 channels is accentuated, allowing for efficient inversion of inhibitory inputs to generate a rebound burst output. Therefore, signal processing relies not only on the activity of individual subtypes of channels but also on complex interactions between ion channels whether based on a physical complex or by indirect

  7. Signal processing by T-type calcium channel interactions in the cerebellum

    Science.gov (United States)

    Engbers, Jordan D. T.; Anderson, Dustin; Zamponi, Gerald W.; Turner, Ray W.

    2013-01-01

    T-type calcium channels of the Cav3 family are unique among voltage-gated calcium channels due to their low activation voltage, rapid inactivation, and small single channel conductance. These special properties allow Cav3 calcium channels to regulate neuronal processing in the subthreshold voltage range. Here, we review two different subthreshold ion channel interactions involving Cav3 channels and explore the ability of these interactions to expand the functional roles of Cav3 channels. In cerebellar Purkinje cells, Cav3 and intermediate conductance calcium-activated potassium (IKCa) channels form a novel complex which creates a low voltage-activated, transient outward current capable of suppressing temporal summation of excitatory postsynaptic potentials (EPSPs). In large diameter neurons of the deep cerebellar nuclei, Cav3-mediated calcium current (IT) and hyperpolarization-activated cation current (IH) are activated during trains of inhibitory postsynaptic potentials. These currents have distinct, and yet synergistic, roles in the subthreshold domain with IT generating a rebound burst and IH controlling first spike latency and rebound spike precision. However, by shortening the membrane time constant the membrane returns towards resting value at a faster rate, allowing IH to increase the efficacy of IT and increase the range of burst frequencies that can be generated. The net effect of Cav3 channels thus depends on the channels with which they are paired. When expressed in a complex with a KCa channel, Cav3 channels reduce excitability when processing excitatory inputs. If functionally coupled with an HCN channel, the depolarizing effect of Cav3 channels is accentuated, allowing for efficient inversion of inhibitory inputs to generate a rebound burst output. Therefore, signal processing relies not only on the activity of individual subtypes of channels but also on complex interactions between ion channels whether based on a physical complex or by indirect

  8. Hydrothermal Synthesis of Xonotlite-type Calcium Silicate Insulation Material Using Industrial Zirconium Waste Residue

    Institute of Scientific and Technical Information of China (English)

    JIANG Jinguo; CUI Chong; LIU Jinqiang; LIAO Wenli

    2011-01-01

    Xonotlite-type insulation material was prepared by hydrothermal synthesis technology using industrial zirconium waste residue in this paper, and the phase analysis together with the observation of micro-morphology were also carried out by XRD, SEM and TEM. The density and thermal conductivity were measured finally. The results indicate, chlorine ion impurity contained in zirconium waste residue can be removed effectively via water washed process, and the reactive activity of silicon dioxide is almost not affected,which make it be a good substitution of silicon material for the preparation of calcium silicate insulation material by hydrothermal synthesis technique. The density and thermal conductivity of xonotlite-type calcium silicate insulation material obtained by hydrothermal synthesis technique can reach 159 kg/m3, 0.049 W/(m·°C), respectively, meeting with National Standard well, when synthesis conditions are selected as follows: Ca/Si molar ratio equal to 1, synthesis temperature at 210 ℃ and kept for 8 hrs. It provides a new approach to realize lightweight and low thermal conductivity of calcium silicate insulation material.

  9. Evolving therapeutic indications for N-type calcium channel blockers: from chronic pain to alcohol abuse.

    Science.gov (United States)

    Belardetti, Francesco

    2010-05-01

    Clinical exploitation of the therapeutic potential of calcium channels has long been limited to L-type blockers for cardiovascular diseases. Recently, N-type blockers have been fully validated for the treatment of chronic pain, following approval of the intrathecally active ziconotide (Prialt(®)). This review describes the successful efforts to broaden the therapeutic scope of this mechanism to other major CNS indications, based on the discovery of N-type blockers orally active against pain. In animal models, the N-type blocker and pain-reducing NP078585 is efficacious against key elements of ethanol dependency, including self-administration and relapse. NP078585 moderately stimulates brain dopamine release without inducing reward or hyperlocomotion. N-type blockers may emerge as a novel class of 'dopamine stabilizers' for the treatment of drug dependency and other neuropsychiatric disorders without the side effects of current therapies. PMID:21426203

  10. Hydrogen sulfide inhibits L-type calcium currents depending upon the protein sulfhydryl state in rat cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Rongyuan Zhang

    Full Text Available Hydrogen sulfide (H(2S is a novel gasotransmitter that inhibits L-type calcium currents (I (Ca, L. However, the underlying molecular mechanisms are unclear. In particular, the targeting site in the L-type calcium channel where H(2S functions remains unknown. The study was designed to investigate if the sulfhydryl group could be the possible targeting site in the L-type calcium channel in rat cardiomyocytes. Cardiac function was measured in isolated perfused rat hearts. The L-type calcium currents were recorded by using a whole cell voltage clamp technique on the isolated cardiomyocytes. The L-type calcium channel containing free sulfhydryl groups in H9C2 cells were measured by using Western blot. The results showed that sodium hydrosulfide (NaHS, an H(2S donor produced a negative inotropic effect on cardiac function, which could be partly inhibited by the oxidant sulfhydryl modifier diamide (DM. H(2S donor inhibited the peak amplitude of I( Ca, L in a concentration-dependent manner. However, dithiothreitol (DTT, a reducing sulfhydryl modifier markedly reversed the H(2S donor-induced inhibition of I (Ca, L in cardiomyocytes. In contrast, in the presence of DM, H(2S donor could not alter cardiac function and L type calcium currents. After the isolated rat heart or the cardiomyocytes were treated with DTT, NaHS could markedly alter cardiac function and L-type calcium currents in cardiomyocytes. Furthermore, NaHS could decrease the functional free sulfhydryl group in the L-type Ca(2+ channel, which could be reversed by thiol reductant, either DTT or reduced glutathione. Therefore, our results suggest that H(2S might inhibit L-type calcium currents depending on the sulfhydryl group in rat cardiomyocytes.

  11. Young adolescents who respond to an inulin-type fructan substantially increase total absorbed calcium and daily calcium accretion to the skeleton

    Science.gov (United States)

    Calcium absorption and whole-body bone mineral content are greater in young adolescents who receive 8 g/d of Synergy, a mixture of inulin-type fructans (ITF), compared with those who received a maltodextrin control. Not all adolescents responded to this intervention, however. We evaluated 32 respond...

  12. Comparing the calcium bioavailability from two types of nano-sized enriched milk using in-vivo assay.

    Science.gov (United States)

    Erfanian, Arezoo; Rasti, Babak; Manap, Yazid

    2017-01-01

    Calcium bioavailability from two types of enriched (calcium citrate and calcium carbonate) milks homogenized to a nano-sized particle distribution has been studied among 48 female Sprauge-dawley rats. Skim milk powder was enriched with some essential nutrients (Inulin, DHA & EPA, vitamins B6, K1, and D3) as enhancers of calcium bioavailability according to recommended dietary allowances of the West European and North American. Ovariectomized and ovariectomized-osteoporosis rats were used as a menopause and menopause-osteoporosis model, respectively. Although, nano-sized enriched milk powders had the greatest calcium bioavailability among the groups, but bioavailability of nano-sized calcium carbonate-enriched-milk was significantly (P<0.05) better than nano-sized calcium citrate-enriched-milk. Moreover, the trends were similar for bone calcium, strength and morphology. Therefore, based on the current results the calcium carbonate nano-sized enriched milk could be an effective enriched milk powder in ovariectomized-osteoporosis and ovariectomized rats as a model of menopause-osteoporosis and menopause women. PMID:27507516

  13. Effects of collagen types II and X on the kinetics of crystallization of calcium phosphate in biomineralization

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The effects of the components of cartilages matrix on the process of endochondral ossification and the kinetics of crystal growth of calcium phosphate have been studied in the presence of type II or X collagen. During the experiments, type I collagen was added as the seed material. FT-IR analysis shows that calcium phosphate crystallized on the surface of type I collagen was mainly hydroxyapatite. Both type II and X collagens could reduce the growth rate of calcium phosphate crystals, and the effect of type X collagen is more obvious. The reaction was in the fourth order in the presence of type II collagen. The results showed that type II or X collagen had the ability to make Ca2+ accumulate in the process of endochondral ossification, but has little effect on crystal growth and the product of biomineralization.

  14. Deltamethrin Inhibits the Human T-type Voltage-Sensitive Calcium Channel (Cav3.2

    Directory of Open Access Journals (Sweden)

    Steven B. Symington

    2009-01-01

    Full Text Available The goal of this study was to determine the effect of deltamethrin, a pyrethroid insecticide, on CaV3.2, a human T-type voltage-sensitive calcium channel expressed in Xenopus laevis (X.laevis oocytes. Cav3.2 cDNA was transcribed into cRNA; the cRNA was then injected into X.laevis oocytes and electrophysiologically characterized using the two-electrode voltage clamp technique with Ba2+ as a charge carrier. Deltamethrin (10-7 M reduced peak current in a nonreversible manner compared to the untreated control, but had no effect on the voltagedependent activation and inactivation kinetics. These findings confirm that human CaV3.2 is a target for deltamethrin and quite possibly other pyrethroid insecticides. These studies provide insight into the molecular mechanisms of the effect that pyrethroids have on voltage-sensitive calcium channels in general. This information will allow a more complete understanding of the molecular and cellular nature of pyrethroid-induced toxicity and expand our knowledge of the structure-activity relationships of pyrethroids with regard to their action on voltage-sensitive calcium channels.

  15. Studies on the role of heavy-metal transporting P-type ATPase family genes on zinc (Zn) transport and accumulation in Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Bagavathiannan, M.V. [Manitoba Univ., Winnipeg, MB (Canada). Dept. of Plant Science; Broadley, M.R.; Donnelly, S.J.; Smith, R.J.; Mills, V. [Nottingham Univ., Loughborough, Leicestershire (United Kingdom). School of Biosciences, Plant Sciences Division

    2006-07-01

    Although zinc (Zn) is an essential plant mineral nutrient for normal crop growth, excess amounts can cause environmental contamination problems. Higher amounts of Zn are added into soils every year through effluents from tanning industries and other sources such as sewage treatment plants, metal inputs from rivers and the atmosphere. Studies have shown that specific metal tolerances exist at the cellular level in plants, indicating that specific adaptations to metal ions occur in cells as well as in the whole plant. This paper described the mechanisms that plants develop in order to tolerate heavy metals and showed that transporter genes play a key role in uptake and sequestration of heavy metals in plant systems. Since metal ion transporting genes are also involved in transport and homeostasis of heavy metals in plants, this study examined the role of the metal ion transporting gene family members in Zn transport and tolerance in plant systems. Among the metal ion transporting gene families, P-type ATPase gene family members are considered to be efficient in metal transport in the model plant Arabidopsis thaliana. They form a diverse superfamily of transporters which carry a range of essential and potentially toxic metals across cellular membranes. Genetic-screening experiments were performed in which 3 SALK lines with known disruption in the target gene were studied physiologically and at the molecular level to determine their role in heavy-metal transportation and accumulation. The study showed that one of the family lines may have altered Zn tolerance and uptake characteristics. Ongoing research continues to examine the characteristics of this line. 27 refs., 1 tab., 8 figs.

  16. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne;

    2003-01-01

    in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal...

  17. Action of erythropoietin in vitro on rabbit reticulocyte membrane Ca2+-ATPase activity.

    OpenAIRE

    Lawrence, W D; Davis, P J; Blas, S D

    1987-01-01

    The mechanism of action of erythropoietin is thought to require specific interaction with the target cell surface and involve alteration of cellular calcium metabolism. Using the rabbit reticulocyte membrane as a model of the immature red cell membrane, we investigated the effects of human recombinant erythropoietin on membrane Ca2+-ATPase (calcium pump) activity in vitro. Erythropoietin in a concentration range of 0.025 to 3.0 U/ml progressively decreased membrane Ca2+-ATPase activity by up ...

  18. Structural and functional studies of a Cu+-ATPase from Legionella pneumophila

    DEFF Research Database (Denmark)

    Mattle, Daniel

    During his studies, Daniel Mattle explored the copper(I) export mechanism of a P-type Cu+ ATPase from Legionella pneumophila – a homologue to the human Cu+ ATPases. Cu+ ATPases are responsible for the homeostatic control of the physiological relevant – but toxic – copper(I) cations. To assess...

  19. P/Q-type and T-type voltage-gated calcium channels are involved in the contraction of mammary and brain blood vessels from hypertensive patients

    DEFF Research Database (Denmark)

    Thuesen, A D; Lyngsø, K S; Rasmussen, L;

    2016-01-01

    AIM: Calcium channel blockers are widely used in cardiovascular diseases. Besides L-type channels, T- and P/Q-type calcium channels are involved in the contraction of human renal blood vessels. It was hypothesized that T- and P/Q-type channels are involved in the contraction of human brain...... and mammary blood vessels. METHODS: Internal mammary arteries from bypass surgery patients and cerebral arterioles from patients with brain tumours with and without hypertension were tested in a myograph and perfusion set-up. PCR and immunohistochemistry were performed on isolated blood vessels. RESULTS...... labelling of mammary and cerebral arteries revealed the presence of Cav 2.1 in endothelial and smooth muscle cells. Cav 3.1 was also detected in mammary arteries. CONCLUSION: P/Q- and T-type Cav are present in human internal mammary arteries and in cerebral penetrating arterioles. P/Q- and T-type calcium...

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  1. MAPK-mediated enhanced expression of vacuolar H(+)-ATPase confers the improved adaption to NaCl stress in a halotolerate peppermint (Mentha piperita L.).

    Science.gov (United States)

    Li, Zhe; Zhen, Zhen; Guo, Kai; Harvey, Paul; Li, Jishun; Yang, Hetong

    2016-03-01

    Vacuolar H(+)-ATPase (V-H(+)-ATPase) has been proved to be of importance in maintenance of ion homeostasis inside plant cells under NaCl stress. In this study, the expression levels and salt-tolerate function of V-H(+)-ATPase genes were investigated in the roots and leaves of a halotolerate peppermint (Mentha × piperita L.) Keyuan-1 treated with different concentrations of NaCl. Results showed that the expressions of V-H(+)-ATPase in the transcriptional, protein and activity levels were significantly enhanced in the halotolerate peppermint Keyuan-1 compared to the wild-type (WT) peppermint under 50, 100, and 150 mM NaCl treatment. Moreover, inhibition experiments exhibited that V-H(+)-ATPase activity played vital roles in the salt tolerance of peppermint Keyuan-1 to 150 mM NaCl stress through increasing the vacuolar H(+) pumping activity and Na(+) compartmentalization capacity. Furthermore, results of Western blots showed that the activity of a mitogen-activated protein kinase (MAPK) was significantly increased under different concentrations of NaCl in the halotolerate peppermint Keyuan-1, which was much higher than that of WT peppermint. Further experiments with inhibitors suggested that this MAPK protein was responsible for the enhanced expression of V-H(+)-ATPase in the halotolerate peppermint Keyuan-1. In response to NaCl stress, increase of cytoplasmic calcium concentration ([Ca(2+)]cyt) occurred upstream of MAPK activation in the halotolerate peppermint Keyuan-1. In all, these findings demonstrated that increased V-H(+)-ATPase activity was positively correlated with the enhanced salt tolerance in the halotolerate peppermint Keyuan-1, providing the theoretic basis for the further development and utilization of peppermint in saline areas.

  2. MAPK-mediated enhanced expression of vacuolar H(+)-ATPase confers the improved adaption to NaCl stress in a halotolerate peppermint (Mentha piperita L.).

    Science.gov (United States)

    Li, Zhe; Zhen, Zhen; Guo, Kai; Harvey, Paul; Li, Jishun; Yang, Hetong

    2016-03-01

    Vacuolar H(+)-ATPase (V-H(+)-ATPase) has been proved to be of importance in maintenance of ion homeostasis inside plant cells under NaCl stress. In this study, the expression levels and salt-tolerate function of V-H(+)-ATPase genes were investigated in the roots and leaves of a halotolerate peppermint (Mentha × piperita L.) Keyuan-1 treated with different concentrations of NaCl. Results showed that the expressions of V-H(+)-ATPase in the transcriptional, protein and activity levels were significantly enhanced in the halotolerate peppermint Keyuan-1 compared to the wild-type (WT) peppermint under 50, 100, and 150 mM NaCl treatment. Moreover, inhibition experiments exhibited that V-H(+)-ATPase activity played vital roles in the salt tolerance of peppermint Keyuan-1 to 150 mM NaCl stress through increasing the vacuolar H(+) pumping activity and Na(+) compartmentalization capacity. Furthermore, results of Western blots showed that the activity of a mitogen-activated protein kinase (MAPK) was significantly increased under different concentrations of NaCl in the halotolerate peppermint Keyuan-1, which was much higher than that of WT peppermint. Further experiments with inhibitors suggested that this MAPK protein was responsible for the enhanced expression of V-H(+)-ATPase in the halotolerate peppermint Keyuan-1. In response to NaCl stress, increase of cytoplasmic calcium concentration ([Ca(2+)]cyt) occurred upstream of MAPK activation in the halotolerate peppermint Keyuan-1. In all, these findings demonstrated that increased V-H(+)-ATPase activity was positively correlated with the enhanced salt tolerance in the halotolerate peppermint Keyuan-1, providing the theoretic basis for the further development and utilization of peppermint in saline areas. PMID:25999237

  3. Functional importance of T-type voltage-gated calcium channels in the cardiovascular and renal system

    DEFF Research Database (Denmark)

    Hansen, Pernille B L

    2015-01-01

    Over the years, it has been discussed whether T-type calcium channels Cav3 play a role in the cardiovascular and renal system. T-type channels have been reported to play an important role in renal hemodynamics, contractility of resistance vessels, and pacemaker activity in the heart. However, the...

  4. New Conotoxin SO-3 Targeting N-type Voltage-Sensitive Calcium Channels

    Directory of Open Access Journals (Sweden)

    Lei Wen

    2006-04-01

    Full Text Available Selective blockers of the N-type voltage-sensitive calcium (CaV channels are useful in the management of severe chronic pain. Here, the structure and function characteristics of a novel N-type CaV channel blocker, SO-3, are reviewed. SO-3 is a 25-amino acid conopeptide originally derived from the venom of Conus striatus, and contains the same 4-loop, 6-cysteine framework (C-C-CC-C-C as O-superfamily conotoxins. The synthetic SO-3 has high analgesic activity similar to ω-conotoxin MVIIA (MVIIA, a selective N-type CaV channel blocker approved in the USA and Europe for the alleviation of persistent pain states. In electrophysiological studies, SO-3 shows more selectivity towards the N-type CaV channels than MVIIA. The dissimilarity between SO-3 and MVIIA in the primary and tertiary structures is further discussed in an attempt to illustrate the difference in selectivity of SO-3 and MVIIA towards N-type CaV channels.

  5. Effect of resveratrol on L-type calcium current in rat ventricular myocytes

    Institute of Scientific and Technical Information of China (English)

    Li-ping ZHANG; Jing-xiang YIN; Zheng LIU; Yi ZHANG; Qing-shan WANG; Juan ZHAO

    2006-01-01

    Aim: To study the effect of resveratrol on L-type calcium current (ICa-L) in isolated rat ventricular myocytes and the mechanisms underlying these effects. Methods:ICa-L was examined in isolated single rat ventricular myocytes by using the whole cell patch-clamp recording technique. Results: Resveratrol (10-40 μmol/L) reduced the peak amplitude of ICa-L and shifted the current-voltage (I-V) curve upwards in a concentration-dependent manner. Resveratrol (10, 20, 40 μmol/L)decreased the peak amplitude of ICa-L from -14.2± 1.5 pA/pF to -10.5± 1.5 pA/pF (P<0.05), -7.5±2.4 pA/pF (P<0.01), and -5.2±1.2 pA/pF (P<0.01), respectively.Resveratrol (40 μmol/L) shifted the steady-state activation curve of ICa-L to the right and changed the half-activation potential (V0.5) from -19.4±0.4 mV to -15.4±1.9 mV (P<0.05). Resveratrol at a concentration of 40 μmol/L did not affect the steady-state inactivation curve of ICa-L, but did markedly shift the timedependent recovery curve of ICa-L to the right, and slow down the recovery of ICa-L from inactivation. Sodium orthovanadate (Na3VO4; 1 mmol/L), a potent inhibitor of tyrosine phosphatase, significantly inhibited the effects of resveratrol (P<0.01). Conclusion: Resveratrol inhibited ICa- L mainly by inhibiting the activation of L-type calcium channels and slowing down the recovery of L-type calcium channels from inactivation. This inhibitory effect of resveratrol was mediated by the inhibition of protein tyrosine kinase in rat ventricular myocytes.

  6. 脱硫废弃物对碱胁迫下水稻叶片钙分布、Ca2+-ATPase活性及抗氧化特征的影响%Effects of desulfurization waste on calcium distribution, Ca2+-ATPase activity, and antioxidant characteristics of rice leaf under alkali stress

    Institute of Scientific and Technical Information of China (English)

    毛桂莲; 许兴; 曾瑾; 岳自慧; 杨淑娟

    2012-01-01

    为了探讨脱硫废弃物提高水稻抗盐碱的作用机制,采用盆栽法,研究脱硫废弃物对碱胁迫下水稻幼苗叶片总钙含量、Ca2+分布、细胞膜Ca2+-ATPase活性及活性氧含量等的变化.结果表明:对照处理的细胞中钙颗粒零星分布于细胞壁和叶绿体中,添加脱硫废弃物和CaSO4处理的细胞质膜、细胞间隙、细胞壁和液泡中有大量的钙颗粒分布;随着脱硫废弃物和CaSO4添加量的增加,叶片总钙含量增加,质膜和液泡膜Ca2+-ATPase活性呈上升趋势,质膜透性、MDA含量和活性氧O2-产生速率呈下降趋势,SOD、POD等保护酶活性升高.添加脱硫废弃物在一定程度上能够减缓碱胁迫对水稻造成的细胞伤害,起主要作用的物质可能是其主要成分CaSO4.%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 Ca2+-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 CaS04 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, Ca2+ -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 CaS04.

  7. Na+*K+-ATPase activity of erythrocyte membrane in diabetic type 2 angiopathies%2型糖尿病性血管病和红细胞膜Na+*K+-ATP酶活性

    Institute of Scientific and Technical Information of China (English)

    何浩明; 黄慧建; 徐宁; 李小民; 田小平; 汪洪流

    2001-01-01

    目的:探讨2型糖尿病性血管病患者的红细胞膜Na+*K+-ATP酶活性的变化及其意义。方法:按Reilini制膜法测定55例2型糖尿病性血管病红细胞膜Na+*K+ -ATP酶活性,并与35名健康组作对照,并且将其结果与红细胞内Na+*K+浓度、空腹血糖、糖化血红蛋白等进行相关分析。结果:2型糖尿病血管病患者红细胞膜Na+*K+-ATP酶含量显著低于正常人(P<0.01),且与红细胞内Na+*K+浓度、空腹血糖、糖化血红蛋白等密切相关。结论:红细胞膜Na+*K+-ATP酶活性的下降可能参与糖尿病性血管病变的发生、发展过程。%Objective:To study on the change of Na+*K +-ATPase activity of erythrocyte membrane of patients with diabetic type 2 an giopathies.Methods:According to Reilini method the concentrations of erythrocyt e membrane Na+*K+-ATPase of 55 cases with diabetic type 2 angiopathies and 35 normal controls were measured.With density of Na+ and K+,levels of f asting blood glucose(FBG),glycosylated hemoglobin(GHb) were also detected.Results:Na+*K+-ATPase activity of erythrocyte membrane was sig nificantly decreased in diabetic type 2 patients(P<0.01).Conclusions:Decreased Na+*K+-ATPase activity in erythrocyte me mbrane may be one of factors that contributed to the occurrence and development of diabetic type 2 mellitus.

  8. [Role of vitamin D and calcium in obesity and type 2 diabetes].

    Science.gov (United States)

    Kuroda, Masashi; Sakaue, Hiroshi

    2016-03-01

    Obesity, induced by unhealthy lifestyle choices, could be involved in the development of chronic diseases like type 2 diabete. Obesity is largely due to the imbalance of energy intake and expenditure, therefore we have put more emphasis on the amount of macronutrients including carbohydrates, fats and proteins as dietary therapy for obesity and related-conditions. On the other hand, several studies revealed obese or diabetic patients were more likely to have micronutrient deficiencies such as vitamins and minerals. Besides the effects on bone metabolism, vitamin D and calcium might contribute to metabolic disorder accompanied by obesity. However, it has not been concluded supplementation of these two nutrients has a benefit in obese or diabetic individuals. Further studies are needed. PMID:26923970

  9. Effect of power and type of substrate on calcium-phosphate coating morphology and microhardness

    Energy Technology Data Exchange (ETDEWEB)

    Kulyashova, Ksenia, E-mail: kseniya@ispms.tsc.ru; Glushko, Yurii, E-mail: glushko@ispms.tsc.ru [Institute of Strength Physics and Materials Science SB RAS, Tomsk, 634055 (Russian Federation); Sharkeev, Yurii, E-mail: sharkeev@ispms.tsc.ru [Institute of Strength Physics and Materials Science SB RAS, Tomsk, 634055 (Russian Federation); National Research Tomsk Polytechnic University, Tomsk, 634050 (Russian Federation); Sainova, Aizhan, E-mail: aizhan-sainova@mail.ru [Institute of Strength Physics and Materials Science SB RAS, Tomsk, 634055 (Russian Federation); National Research Tomsk State University, Tomsk, 634050 (Russian Federation)

    2015-10-27

    As known, the influence of the different sputtering process parameters and type of substrate on structure of the deposited coating is important to identify, because these parameters are significantly affected on structure of coating. The studies of the morphology and microhardness of calcium-phosphate (CaP) coatings formed and obtained on the surface of titanium, zirconium, titanium and niobium alloy for different values of the power of radio frequency discharge are presented. The increase in the radio frequency (rf) magnetron discharge leads to the formation of a larger grain structure of the coating. The critical depths of indentation for coatings determining the value of their microhardness have been estimated. Mechanical properties of the composite material on the basis of the bioinert substrate metal and CaP coatings are superior to the properties of the separate components that make up this composite material.

  10. Effect of power and type of substrate on calcium-phosphate coating morphology and microhardness

    Science.gov (United States)

    Kulyashova, Ksenia; Sharkeev, Yurii; Glushko, Yurii; Sainova, Aizhan

    2015-10-01

    As known, the influence of the different sputtering process parameters and type of substrate on structure of the deposited coating is important to identify, because these parameters are significantly affected on structure of coating. The studies of the morphology and microhardness of calcium-phosphate (CaP) coatings formed and obtained on the surface of titanium, zirconium, titanium and niobium alloy for different values of the power of radio frequency discharge are presented. The increase in the radio frequency (rf) magnetron discharge leads to the formation of a larger grain structure of the coating. The critical depths of indentation for coatings determining the value of their microhardness have been estimated. Mechanical properties of the composite material on the basis of the bioinert substrate metal and CaP coatings are superior to the properties of the separate components that make up this composite material.

  11. Specific T-type calcium channel isoforms are associated with distinct burst phenotypes in deep cerebellar nuclear neurons

    OpenAIRE

    Molineux, Michael L.; McRory, John E.; McKay, Bruce E.; Hamid, Jawed; Mehaffey, W. Hamish; Rehak, Renata; Snutch, Terrance P; Gerald W Zamponi; Turner, Ray W

    2006-01-01

    T-type calcium channels are thought to transform neuronal output to a burst mode by generating low voltage-activated (LVA) calcium currents and rebound burst discharge. In this study we assess the expression pattern of the three different T-type channel isoforms (Cav3.1, Cav3.2, and Cav3.3) in cerebellar neurons and focus on their potential role in generating LVA spikes and rebound discharge in deep cerebellar nuclear (DCN) neurons. We detected expression of one or more Cav3 channel isoforms ...

  12. Antibodies to mammalian and plant V-ATPases cross react with the V-ATPase of insect cation-transporting plasma membranes.

    Science.gov (United States)

    Russell, V E; Klein, U; Reuveni, M; Spaeth, D D; Wolfersberger, M G; Harvey, W R

    1992-05-01

    In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources. PMID:1534830

  13. Regulation of vacuolar H(+)-ATPase in microglia by RANKL.

    Science.gov (United States)

    Serrano, Eric M; Ricofort, Ryan D; Zuo, Jian; Ochotny, Noelle; Manolson, Morris F; Holliday, L Shannon

    2009-11-01

    Vacuolar H(+)-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor kappaB-ligand (RANKL). We found that Receptor Activator of Nuclear Factor kappaB (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  14. Regulation of Vacuolar H+-ATPase in Microglia by RANKL

    Science.gov (United States)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian; Ochotny, Noelle; Manolson, Morris F.; Holliday, L. Shannon

    2009-01-01

    Vacuolar H+-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPase play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor κ B -ligand (RANKL). We found that Receptor Activator of Nuclear Factor κ B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia. PMID:19715671

  15. Regulation of vacuolar H+-ATPase in microglia by RANKL

    International Nuclear Information System (INIS)

    Vacuolar H+-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor κB-ligand (RANKL). We found that Receptor Activator of Nuclear Factor κB (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  16. Purification and Properties of an ATPase from Sulfolobus solfataricus

    Science.gov (United States)

    Hochstein, Lawrence I.; Stan-Lotter, Helga

    1992-01-01

    A sulfite-activated ATPase isolated from Sulfolobus solfataricus had a relative molecular mass of 370,000. It was composed of three subunits whose relative molecular masses were 63,000, 48,000, and 24,000. The enzyme was inhibited by the vacuolar ATPase inhibitors nitrate and N-ethylmaleimide; 4-chloro-7-nitrobenzo-furazan (NBD-Cl) was also inhibitory. N-Ethylmaleimide was predominately bound to the largest subunit while NBD-CL was bound to both subunits. ATPase activity was inhibited by low concentrations of p-chloromercuri-phenyl sulfonate and the inhibition was reversed by cysteine which suggested that thiol groups were essential for activity. While the ATPase from S. solfataricus shared several properties with the ATPase from S. acidocaldarius there were significant differences. The latter enzyme was activated by sulfate and chloride and was unaffected by N-ethylmaleimide, whereas the S. solfataricus ATPase was inhibited by these anions as well as N-ethyimaleimide. These differences as well as differences that occur in other vacuolar-like ATPases isolated from the methanogenic and the extremely halophilic bacteria suggest the existence of several types of archaeal ATPases, none of which have been demonstrated to synthesize ATP.

  17. Tissue Reaction to Different Types of Calcium Hydroxide Paste in Rat.

    Science.gov (United States)

    Zarei, Mina; Javidi, Maryam; Gharechahi, Maryam; Kateb, Moaied; Zare, Reza; Kelagari, Ziba Shirkhani

    2016-01-01

    The purpose of this study was to compare the biocompatibility of two types of calcium hydroxide paste in subcutaneous tissue in rat. Twenty-two Wistar rats were divided into 4 experimental (n=5 each) and one control (n=2) group. A polyethylene tube filled with either Dentsply or Sure-Paste was implanted in each rat in the experimental groups, while an empty polyethylene tube was used in the control group. After 15 or 60 days, the animals were sacrificed and histopathological examination carried out. Tissue reaction was assessed by inflammatory cell infiltration using a 4-point scoring system, ranging from 0 to 3. Data were analyzed with the Kruskal-Wallis, Wilcoxon, and McNemar tests. Both types of paste induced an inflammatory response at each time point, although the intensity varied. A significant reduction in the number of inflammatory cells was observed at 60 days. Dentsply appeared to induce a more marked inflammatory response at both time points, although the difference was not significant. These results suggest that both types of paste are biocompatible with subcutaneous tissue in rat. PMID:27320294

  18. Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D;

    2001-01-01

    The distribution of voltage-dependent calcium channels in kidney pre- and postglomerular resistance vessels was determined at the molecular and functional levels. Reverse transcription-polymerase chain reaction analysis of microdissected rat preglomerular vessels and cultured smooth muscle cells...... showed coexpression of mRNAs for T-type subunits (Ca(V)3.1, Ca(V)3.2) and for an L-type subunit (Ca(V)1.2). The same expression pattern was observed in juxtamedullary efferent arterioles and outer medullary vasa recta. No calcium channel messages were detected in cortical efferent arterioles. Ca(V)1.......2 protein was demonstrated by immunochemical labeling of rat preglomerular vasculature and juxtamedullary efferent arterioles and vasa recta. Cortical efferent arterioles were not immunopositive. Recordings of intracellular calcium concentration with digital fluorescence imaging microscopy showed a...

  19. The T-type calcium channel antagonist Z944 disrupts prepulse inhibition in both epileptic and non-epileptic rats.

    Science.gov (United States)

    Marks, Wendie N; Greba, Quentin; Cain, Stuart M; Snutch, Terrance P; Howland, John G

    2016-09-22

    The role of T-type calcium channels in brain diseases such as absence epilepsy and neuropathic pain has been studied extensively. However, less is known regarding the involvement of T-type channels in cognition and behavior. Prepulse inhibition (PPI) is a measure of sensorimotor gating which is a basic process whereby the brain filters incoming stimuli to enable appropriate responding in sensory rich environments. The regulation of PPI involves a network of limbic, cortical, striatal, pallidal and pontine brain areas, many of which show high levels of T-type calcium channel expression. Therefore, we tested the effects of blocking T-type calcium channels on PPI with the potent and selective T-type antagonist Z944 (0.3, 1, 3, 10mg/kg; i.p.) in adult Wistar rats and two related strains, the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and Non-Epileptic Control (NEC). PPI was tested using a protocol that varied prepulse intensity (3, 6, and 12dB above background) and prepulse-pulse interval (30, 50, 80, 140ms). Z944 decreased startle in the Wistar strain at the highest dose relative to lower doses. Z944 dose-dependently disrupted PPI in the Wistar and GAERS strains with the most potent effect observed with the higher doses. These findings suggest that T-type calcium channels contribute to normal patterns of brain activity that regulate PPI. Given that PPI is disrupted in psychiatric disorders, future experiments that test the specific brain regions involved in the regulation of PPI by T-type calcium channels may help inform therapeutic development for those suffering from sensorimotor gating impairments. PMID:27365170

  20. Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases.

    Science.gov (United States)

    Menezes-Rodrigues, Francisco Sandro; Pires-Oliveira, Marcelo; Duarte, Thiago; Paredes-Gamero, Edgar Julian; Chiavegatti, Tiago; Godinho, Rosely Oliveira

    2013-11-15

    Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses.

  1. The L-Type Calcium Channel Blocker Nifedipine Impairs Extinction, but Not Reduced Contingency Effects, in Mice

    Science.gov (United States)

    Jami, Shekib; Barad, Mark; Cain, Christopher K.; Godsil, Bill P.

    2005-01-01

    We recently reported that fear extinction, a form of inhibitory learning, is selectively blocked by systemic administration of L-type voltage-gated calcium channel (LVGCC) antagonists, including nifedipine, in mice. We here replicate this finding and examine three reduced contingency effects after vehicle or nifedipine (40 mg/kg) administration.…

  2. Phospholemman-dependent regulation of the cardiac Na/K-ATPase activity is modulated by inhibitor-1 sensitive type-1 phosphatase.

    Science.gov (United States)

    El-Armouche, Ali; Wittköpper, Katrin; Fuller, William; Howie, Jacqueline; Shattock, Michael J; Pavlovic, Davor

    2011-12-01

    Cardiac Na/K-ATPase (NKA) is regulated by its accessory protein phospholemman (PLM). Whereas kinase-induced PLM phosphorylation has been shown to mediate NKA stimulation, the role of endogenous phosphatases is presently unknown. We investigated the role of protein phosphatase-1 (PP-1) on PLM phosphorylation and NKA activity in rat cardiomyocytes and failing human hearts. Incubation of rat cardiomyocytes with the chemical PP-1/PP-2A inhibitor okadaic acid or the specific PP-1-inhibitor peptide (I-1ct) identified PLM phosphorylation at Ser-68 as the main substrate for PP-1. Moreover, myocytes adenovirally overexpressing PP-1 inhibitor-1 protein (I-1,Ad-I-1/eGFP) showed a 70% increase in PLM Ser-68 phosphorylation and 65% increase in NKA current, compared with enhanced green fluorescence protein (eGFP)-infected controls (Ad-eGFP), using Western blotting and voltage clamping, respectively. Notably, in left ventricular myocardium from patients with heart failure, PLM Ser-68 phosphorylation was ≈ 50% lower (n=7) than in nonfailing controls (n=7). We provide the first physiological and biochemical evidence that PLM phosphorylation and cardiac Na/K-ATPase activity are negatively regulated by PP-1 and that this regulatory mechanism could be counteracted by I-1. This novel mechanism is markedly perturbed in failing hearts favoring PLM dephosphorylation and NKA deactivation and thus may contribute to maladaptive hypertrophy and arrhythmogenesis via chronically higher intracellular Na and Ca concentrations.

  3. Huwentoxin-XVI, an analgesic, highly reversible mammalian N-type calcium channel antagonist from Chinese tarantula Ornithoctonus huwena.

    Science.gov (United States)

    Deng, Meichun; Luo, Xuan; Xiao, Yucheng; Sun, Zhenghua; Jiang, Liping; Liu, Zhonghua; Zeng, Xiongzhi; Chen, Hanchun; Tang, Jianhua; Zeng, Weimin; Songping Liang

    2014-04-01

    N-type calcium channels play important roles in the control of neurotransmission release and transmission of pain signals to the central nervous system. Their selective inhibitors are believed to be potential drugs for treating chronic pain. In this study, a novel neurotoxin named Huwentoxin-XVI (HWTX-XVI) specific for N-type calcium channels was purified and characterized from the venom of Chinese tarantula Ornithoctonus huwena. HWTX-XVI is composed of 39 amino acid residues including six cysteines that constitute three disulfide bridges. HWTX-XVI could almost completely block the twitch response of rat vas deferens to low-frequency electrical stimulation. Electrophysiological assay indicated that HWTX-XVI specifically inhibited N-type calcium channels in rat dorsal root ganglion cells (IC50 ∼60 nM). The inhibitory effect of HWTX-XVI on N-type calcium channel currents was dose-dependent and similar to that of CTx-GVIA and CTx-MVIIA. However, the three peptides exhibited markedly different degrees of reversibility after block. The toxin had no effect on voltage-gated T-type calcium channels, potassium channels or sodium channels. Intraperitoneal injection of the toxin HWTX-XVI to rats elicited significant analgesic responses to formalin-induced inflammation pain. Toxin treatment also changed withdrawal latency in hot plate tests. Intriguingly, we found that intramuscular injection of the toxin reduced mechanical allodynia induced by incisional injury in Von Frey test. Thus, our findings suggest that the analgesic potency of HWTX-XVI and its greater reversibility could contribute to the design of a novel potential analgesic agent with high potency and low side effects. PMID:24467846

  4. Calcium- and CaMKII-dependent chloride secretion induced by the microsomal Ca(2+)-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone in cystic fibrosis pancreatic epithelial cells.

    OpenAIRE

    Chao, A C; Kouyama, K; Heist, E K; Dong, Y. J.; Gardner, P

    1995-01-01

    Microsomal Ca(2+)-ATPase inhibitors such as thapsigargin (THG), cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ) have been shown to inhibit Ca2+ reuptake by the intracellular stores and increase cytosolic free Ca2+ ([Ca2+]i). DBHQ is a commercially available non-toxic synthetic compound chemically unrelated to THG and CPA. In this study, we tested the feasibility of utilizing DBHQ to improve Cl- secretion via the Ca(2+)-dependent pathway, in the cystic fibrosis (CF)-de...

  5. L—type calcium channel blockers inhibit the development but not the expression of sensitization to morphine in mice

    Institute of Scientific and Technical Information of China (English)

    ZhanQ; ZhenJW

    2002-01-01

    The relationship between opioid actions and L-type calcium channel blockers has been well documented.However,there is no report relevant to L-type calcium channel blockers and morphinesensitization,which is suggested to be an analog of behaviors that are the characteristics of drug addiction.Here the effects of three L-type calcium channel blockers,nimodipine,nifedipine and verapamil,on morphine-induced locomotor activity,the development and the expression of sensitization to morphine were studied systematically.The results showed that both nimodipine and verapamil attenuated,while nifedipine had only a tendency to decrease morphine-induced locomotor activity.All the three drugs inhibited the development of sensitization to morphine.However,none of them showed any effects on the expression of morphine sensitization.These results indicate that blocking L-tpye calcium channel attenuates the locomotor stimulating effects of morphine and inhibits the development but not the expression of morphine-sensitization.

  6. Activity-Dependent Calcium, Oxygen, and Vascular Responses in a Mouse Model of Familial Hemiplegic Migraine Type 1

    DEFF Research Database (Denmark)

    Khennouf, Lila; Gesslein, Bodil; Lind, Barbara Lykke;

    2016-01-01

    Objective: Familial hemiplegic migraine type 1 (FHM1) is a subtype of migraine with aura caused by a gain-of-function mutation in the pore-forming α1 subunit of CaV2.1 (P/Q-type) calcium channels. However, the mechanisms underlying how the disease is brought about and the prolonged aura remain...... explain impaired neurovascular responses in the mutant, and these alterations could contribute to brain frailty in FHM1 patients...

  7. Calcium efflux systems in stress signalling and adaptation in plants

    Directory of Open Access Journals (Sweden)

    Jayakumar eBose

    2011-12-01

    Full Text Available Transient cytosolic calcium ([Ca2+]cyt elevation is an ubiquitous denominator of the signalling network when plants are exposed to literally every known abiotic and biotic stress. These stress-induced [Ca2+]cyt elevations vary in magnitude, frequency and shape, depending on the severity of the stress as well the type of stress experienced. This creates a unique stress-specific calcium signature that is then decoded by signal transduction networks. While most published papers have been focused predominantly on the role of Ca2+ influx mechanisms in shaping [Ca2+]cyt signatures, restoration of the basal [Ca2+]cyt levels is impossible without both cytosolic Ca2+ buffering and efficient Ca2+ efflux mechanisms removing excess Ca2+ from cytosol, to reload Ca2+ stores and to terminate Ca2+ signalling. This is the topic of the current review. The molecular identity of two major types of Ca2+ efflux systems, Ca2+-ATPase pumps and Ca2+/H+ exchangers, is described, and their regulatory modes are analysed in detail. The spatial and temporal organisation of calcium signalling networks is described, and the importance of existence of intracellular calcium microdomains is discussed. Experimental evidence for the role of Ca2+ efflux systems in plant responses to a range of abiotic and biotic factors is summarised. Contribution of Ca2+-ATPase pumps and Ca2+/H+ exchangers in shaping [Ca2+]cyt signatures is then modelled by using a four-component model (plasma- and endo- membrane-based Ca2+-permeable channels and efflux systems taking into account the cytosolic Ca2+ buffering. It is concluded that physiologically relevant variations in the activity of Ca2+-ATPase pumps and Ca2+/H+ exchangers are sufficient to fully describe all the reported experimental evidence and determine the shape of [Ca2+]cyt signatures in response to environmental stimuli, emphasising the crucial role these active efflux systems play in plant adaptive responses to environment.

  8. A biomimetic strategy to form calcium phosphate crystals on type I collagen substrate

    International Nuclear Information System (INIS)

    Objective: The aim of this study is to induce mineralization of collagen by introducing phosphate groups onto type I collagen from eggshell membrane (ESM) by treating with sodium trimetaphosphate (STMP). This strategy is based on the hypothesis that phosphate groups introduced on collagen can mimic the nucleating role of phosphorylated non-collagenous proteins bound to collagen for inducing mineralization in natural hard tissue. Method: The collagen membrane was phosphorylated by treating it with a solution of STMP and saturated calcium hydroxide. The phosphorylated collagen was subsequently exposed to a mineralization solution and the pattern of mineralization on the surface of phosphorylated collagen substrate was analyzed. Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), field emission electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD) and microhardness test were used to characterize the collagen substrate and the pattern of minerals formed on the collagen surface. Results: The FTIR and EDX results indicated that the phosphate groups were incorporated onto the collagen surface by treatment with STMP. During the mineralization process, the plate-like mineral, octacalcium phosphate (OCP), which was initially formed on the surface of ESM, was later transformed into needle-like hydroxyapatite (HAP) as indicated by the SEM, FESEM, EDX and XRD findings. The microhardness test displayed significant increase in the Knoop hardness number of the mineralized collagen. Conclusions: Phosphate groups can be introduced onto type I collagen surface by treating it with STMP and such phosphorylated collagen can induce the mineralization of type I collagen.

  9. A biomimetic strategy to form calcium phosphate crystals on type I collagen substrate

    Energy Technology Data Exchange (ETDEWEB)

    Xu Zhang [Department of Restorative Dentistry, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road 119074, Singapore (Singapore); Neoh, Koon Gee [Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge 119260, Singapore (Singapore); Kishen, Anil, E-mail: anil.kishen@utoronto.ca [Discipline of Endodontics, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON (Canada)

    2010-07-20

    Objective: The aim of this study is to induce mineralization of collagen by introducing phosphate groups onto type I collagen from eggshell membrane (ESM) by treating with sodium trimetaphosphate (STMP). This strategy is based on the hypothesis that phosphate groups introduced on collagen can mimic the nucleating role of phosphorylated non-collagenous proteins bound to collagen for inducing mineralization in natural hard tissue. Method: The collagen membrane was phosphorylated by treating it with a solution of STMP and saturated calcium hydroxide. The phosphorylated collagen was subsequently exposed to a mineralization solution and the pattern of mineralization on the surface of phosphorylated collagen substrate was analyzed. Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), field emission electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD) and microhardness test were used to characterize the collagen substrate and the pattern of minerals formed on the collagen surface. Results: The FTIR and EDX results indicated that the phosphate groups were incorporated onto the collagen surface by treatment with STMP. During the mineralization process, the plate-like mineral, octacalcium phosphate (OCP), which was initially formed on the surface of ESM, was later transformed into needle-like hydroxyapatite (HAP) as indicated by the SEM, FESEM, EDX and XRD findings. The microhardness test displayed significant increase in the Knoop hardness number of the mineralized collagen. Conclusions: Phosphate groups can be introduced onto type I collagen surface by treating it with STMP and such phosphorylated collagen can induce the mineralization of type I collagen.

  10. Structural divergence between the two subgroups of P5 ATPases

    DEFF Research Database (Denmark)

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

    2010-01-01

    differences in the primary sequences between the two subgroups. P5A and P5B ATPases appear have a very different membrane topology from other P-type ATPases with two and one, respectively, additional transmembrane segments inserted in the N-terminal end. Based on conservation of residues in the transmembrane...... region, the two P5 subgroups most likely have different substrate specificities although these cannot be predicted from their sequences. Furthermore, sequence differences between P5A and P5B ATPases are identified in the catalytic domains that could influence key kinetic properties differentially...

  11. Discovery of novel tetrahydroisoquinoline derivatives as orally active N-type calcium channel blockers with high selectivity for hERG potassium channels.

    Science.gov (United States)

    Ogiyama, Takashi; Inoue, Makoto; Honda, Shugo; Yamada, Hiroyoshi; Watanabe, Toshihiro; Gotoh, Takayasu; Kiso, Tetsuo; Koakutsu, Akiko; Kakimoto, Shuichiro; Shishikura, Jun-ichi

    2014-12-15

    N-type calcium channels represent a promising target for the treatment of neuropathic pain. The selective N-type calcium channel blocker ziconotide ameliorates severe chronic pain but has a narrow therapeutic window and requires intrathecal administration. We identified tetrahydroisoquinoline derivative 1a as a novel potent N-type calcium channel blocker. However, this compound also exhibited potent inhibitory activity against hERG channels. Structural optimizations led to identification of (1S)-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone ((S)-1h), which exhibited high selectivity for hERG channels while retaining potency for N-type calcium channel inhibition. (S)-1h went on to demonstrate in vivo efficacy as an orally available N-type calcium channel blocker in a rat spinal nerve ligation model of neuropathic pain. PMID:25456079

  12. Plasmodium falciparum isolates from southern Ghana exhibit polymorphisms in the SERCA-type PfATPase6 though sensitive to artesunate in vitro

    Directory of Open Access Journals (Sweden)

    Ofori Michael F

    2011-07-01

    Full Text Available Abstract Background In 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative pfATPase6 and pftctp, pfmdr1, pfcrt genes in Ghanaian isolates, particularly at a time when there is no report on artemisinin resistance in malaria parasites from Ghana. The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes. Methods The prevalence of point mutations in the above Plasmodium falciparum genes were assessed from filter-paper blood blot samples by DNA sequencing. In vitro drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test. Results All successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of IC50 value for artesunate was 0.73 nM (95% CI, 0.38-1.08, amodiaquine 30.69 nM (95% CI, 14.18-47.20 and chloroquine 58.73 nM (95% CI, 38.08-79.38. Twenty point mutations were observed in pfATPase6 gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (p = 0.42. The pftctp gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the pfcrt gene was identified in about half of the isolates; this was consistent with chloroquine IC50 values (p = 0.001. Mutations were present in pfmdr1 gene but were not associated with artemisinin response (p = 1.00. Conclusion The pfATPase6 gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just

  13. Molecular basis of toxicity of N-type calcium channel inhibitor MVIIA.

    Science.gov (United States)

    Wang, Fei; Yan, Zhenzhen; Liu, Zhuguo; Wang, Sheng; Wu, Qiaoling; Yu, Shuo; Ding, Jiuping; Dai, Qiuyun

    2016-02-01

    MVIIA (ziconotide) is a specific inhibitor of N-type calcium channel, Cav2.2. It is derived from Cone snail and currently used for the treatment of severe chronic pains in patients unresponsive to opioid therapy. However, MVIIA produces severe side-effects, including dizziness, nystagmus, somnolence, abnormal gait, and ataxia, that limit its wider application. We previously identified a novel inhibitor of Cav2.2, ω-conopeptide SO-3, which possesses similar structure and analgesic activity to MVIIA's. To investigate the key residues for MVIIA toxicity, MVIIA/SO-3 hybrids and MVIIA variants carrying mutations in its loop 2 were synthesized. The substitution of MVIIA's loop 1 with the loop 1 of SO-3 resulted in significantly reduced Cav2.2 binding activity in vitro; the replacement of MVIIA loop 2 by the loop 2 of SO-3 not only enhanced the peptide/Cav2.2 binding but also decreased its toxicity on goldfish, attenuated mouse tremor symptom, spontaneous locomotor activity, and coordinated locomotion function. Further mutation analysis and molecular calculation revealed that the toxicity of MVIIA mainly arose from Met(12) in the loop 2, and this residue inserts into a hydrophobic hole (Ile(300), Phe(302) and Leu(305)) located between repeats II and III of Cav2.2. The combinative mutations of the loop 2 of MVIIA or other ω-conopeptides may be used for future development of more effective Cav2.2 inhibitors with lower side effects. PMID:26344359

  14. 5,6-EET potently inhibits T-type calcium channels

    DEFF Research Database (Denmark)

    Cazade, M.; Bidaud, I.; Hansen, Pernille B. Lærkegaard;

    2014-01-01

    T-type calcium channels (T-channels) are important actors in neuronal pacemaking, in heart rhythm, and in the control of the vascular tone. T-channels are regulated by several endogenous lipids including the primary eicosanoid arachidonic acid (AA), which display an important role in vasodilation...... via its metabolism leading to prostanoids, leukotrienes, and epoxyeicosatrienoic acids (EETs). However, the effects of these latter molecules on T-currents have not been investigated. Here, we describe the effects of the major cyclooxygenase, lipoxygenase, and cytochrome P450 epoxygenase products...... on the three human recombinant T-channels (Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3), as compared to those of AA. We identified the P450 epoxygenase product, 5,6-EET, as a potent physiological inhibitor of Ca(v)3 currents. The effects of 5,6-EET were observed at sub-micromolar concentrations (IC50 = 0.54 mu M...

  15. T-type Calcium Channel Regulation of Neural Tube Closure and EphrinA/EPHA Expression

    Directory of Open Access Journals (Sweden)

    Sarah Abdul-Wajid

    2015-10-01

    Full Text Available A major class of human birth defects arise from aberrations during neural tube closure (NTC. We report on a NTC signaling pathway requiring T-type calcium channels (TTCCs that is conserved between primitive chordates (Ciona and Xenopus. With loss of TTCCs, there is a failure to seal the anterior neural folds. Accompanying loss of TTCCs is an upregulation of EphrinA effectors. Ephrin signaling is known to be important in NTC, and ephrins can affect both cell adhesion and repulsion. In Ciona, ephrinA-d expression is downregulated at the end of neurulation, whereas, with loss of TTCC, ephrinA-d remains elevated. Accordingly, overexpression of ephrinA-d phenocopied TTCC loss of function, while overexpression of a dominant-negative Ephrin receptor was able to rescue NTC in a Ciona TTCC mutant. We hypothesize that signaling through TTCCs is necessary for proper anterior NTC through downregulation of ephrins, and possibly elimination of a repulsive signal.

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

    Science.gov (United States)

    Madias, Christopher; Garlitski, Ann C; Kalin, John; Link, Mark S

    2016-01-01

    Background. In a commotio cordis swine model, ventricular fibrillation (VF) can be induced by a ball blow to the chest believed secondary to activation of mechanosensitive ion channels. The purpose of the current study is to evaluate whether stretch induced activation of the L-type calcium channel may cause intracellular calcium overload and underlie the VF in commotio cordis. Method and Results. Anesthetized juvenile swine received 6 chest wall strikes with a 17.9 m/s lacrosse ball timed to the vulnerable period for VF induction. Animals were randomized to IV verapamil (n = 6) or placebo (n = 6). There was no difference in the observed frequency of VF between verapamil (19/26: 73%) and placebo (20/36: 56%) treated animals (p = 0.16). There was also no significant difference in the combined endpoint of VF or nonsustained VF (21/26: 81% in verapamil versus 24/36: 67% in controls, p = 0.22). Conclusions. In this experimental model of commotio cordis, verapamil did not prevent VF induction. Thus, in commotio cordis it is unlikely that stretch activation of the L-type calcium channel with resultant intracellular calcium overload plays a prominent role. PMID:26925288

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

    Directory of Open Access Journals (Sweden)

    Christopher Madias

    2016-01-01

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

  18. Conserved V-ATPase c subunit plays a role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

    Science.gov (United States)

    Zhou, Aimin; Bu, Yuanyuan; Takano, Tetsuo; Zhang, Xinxin; Liu, Shenkui

    2016-01-01

    In plant cells, the vacuolar-type H(+)-ATPases (V-ATPase) are localized in the tonoplast, Golgi, trans-Golgi network and endosome. However, little is known about how V-ATPase influences plant growth, particularly with regard to the V-ATPase c subunit (VHA-c). Here, we characterized the function of a VHA-c gene from Puccinellia tenuiflora (PutVHA-c) in plant growth. Compared to the wild-type, transgenic plants overexpressing PutVHA-c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V-ATPase activity. Consistently, the Arabidopsis atvha-c5 mutant shows reduced V-ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA-c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V-ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA-c-GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling-1 (AtRGS1). These findings suggest that the decrease in V-ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA-c plays an important role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

  19. Gene cloning and expression characteristics of vacuolar-type ATPase subunit B in Bombyx mori%家蚕V型ATP酶B亚基的克隆及表达特征

    Institute of Scientific and Technical Information of China (English)

    陈慧芳; 王鑫; 谢康; 李懿; 赵萍

    2016-01-01

    V型ATP酶(Vacuolar-type ATPase)是一种定位于细胞膜和细胞器膜上的氢离子转运酶.它利用ATP水解的能量将氢离子转运到液泡、囊泡或者胞外,从而维持细胞内正常的酸碱环境.V型ATP酶B亚基(V-ATPase B)作为ATP的催化位点,也有着非常重要的作用.为了探讨家蚕V-ATPase B(BmV-ATPase B)的功能,首先从家蚕五龄幼虫的中肠cDNA中克隆了Bm V-A TPase B基因并构建原核表达载体进行原核表达,获得了重组蛋白,经质谱鉴定正确后,通过镍柱亲和层析的方法纯化了该蛋白并制备了多克隆抗体;最后分析了该蛋白在家蚕丝腺中的表达特征并利用免疫荧光对其在丝腺中的表达位置进行了定位.结果显示Bm V-A TPase B基因序列全长1 473 bp,预测蛋白分子量55 kDa,预测等电点5.3.通过Western blotting对家蚕5龄第3天和上蔟第1天幼虫丝腺的不同区段进行BmV-ATPase B蛋白的表达特征分析,发现在两个时期该蛋白均在前部丝腺高量表达,而在中部丝腺和后部丝腺表达量相对较低.进一步对两个时期丝腺的不同区段进行免疫荧光定位,发现该蛋白在两个时期的前部丝腺、中部丝腺和后部丝腺均定位于细胞层.利用激光共聚焦显微镜对该蛋白进行进一步的定位,发现该蛋白主要在丝腺的细胞膜表达.研究结果明确了该蛋白在丝腺中的表达模式,为深入研究该蛋白在蚕丝纤维形成中的作用奠定了基础.

  20. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe;

    2011-01-01

    transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na......(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps....

  1. Pinaverium acts as L-type calcium channel blocker on smooth muscle of colon.

    Science.gov (United States)

    Malysz, J; Farraway, L A; Christen, M O; Huizinga, J D

    1997-08-01

    The effect of pinaverium was electrophysiologically characterized and compared with the established L-type calcium channel blockers diltiazem, D600, and nitrendipine on canine colonic circular smooth muscle. Effects were studied on the electrical activity of the smooth muscle cells, in particular the spontaneously occurring slow wave. In addition, effects were examined on spontaneous contraction patterns and contractile activities generated by stimulation of cholinergic nerves or directly by stimulating muscarinic receptors. Effects were also examined on excitation of NO-releasing intrinsic nerves. Pinaverium bromide affected the slow wave by selectively inhibiting the plateau potential that is associated with generation of contractile activity. Pinaverium, similar to diltiazem and D600, produced reductions in cholinergic responses as well as spontaneous contractions. The IC50 values for inhibition of cholinergic responses for pinaverium, diltiazem, and D600 were 1.0 x 10(-6), 4.1 x 10(-7), and 5.3 x 10(-7) M, respectively. The IC50 values for inhibition of spontaneous contractile activity for pinaverium, diltiazem, and D600 were 3.8 x 10(-6), 9.7 x 10(-7), and 8.0 x 10(-7) M, respectively. Increases in contractility by carbachol were abolished by pretreatment with either pinaverium or D600. In addition, neither pinaverium nor D600 had any effects on the inhibitory NO-mediated relaxations. These data provide a rationale for the use of pinaverium in the treatment of colonic motor disorders where excessive contraction has to be suppressed. PMID:9360010

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

    Institute of Scientific and Technical Information of China (English)

    Yang Yong-qing; Wang Xiao-feng

    2005-01-01

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

  3. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

    Science.gov (United States)

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

    2016-08-01

    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease.

  4. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation.

    Science.gov (United States)

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

    2016-01-01

    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease. PMID:27488468

  5. Analysis of Amino Acid Residues of Potential Importance for Phosphati-dylserine Specificity of P4-type ATPase ATP8A2

    DEFF Research Database (Denmark)

    Mogensen, Louise; Vestergaard, Anna Lindeløv; Mikkelsen, Stine;

    The asymmetric structure of the plasma membrane is maintained through internalization of phos-pholipids by the family of P4-ATPases by a poorly characterized mechanism. Studies in yeast point towards a non-classical pathway involving important residues of a two-gate mechanism [1]. Glycine-230...... 302 of ATP8A2 with alanine (N302A), tyrosine (N302Y) and serine (N302S). Furthermore, a triple mutant of ATP8A2 (Q95GQ96AN302S) was studied to reveal any cooperativity between the two gates, as observed in yeast [1]. The affinities of the mutants for phosphatidylserine and phosphatidylethanolamine...... and differences between ATP8A2 and the yeast flippases. The results supplement recent studies of the ATP8A2 flippase revealing a hydro-phobic gate that facilitates the transport along a water-filled pathway in the protein transmembrane domain[2]. 1. Baldridge, R.D. and T.R. Graham, Proceedings of the National...

  6. Towards defining the substrate of orphan P5A-ATPases

    DEFF Research Database (Denmark)

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

    2015-01-01

    Background P-type ATPases are ubiquitous ion and lipid pumps found in cellular membranes. P5A-ATPases constitute a poorly characterized subfamily of P-type ATPases present in all eukaryotic organisms but for which a transported substrate remains to be identified. Scope of review This review aims ...... significance Identification of the substrate of P5A-ATPases would throw light on an important general process in the ER that is still not fully understood. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins....

  7. Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes.

    Science.gov (United States)

    Wei, Jinhong; Sun, Junqing; Xu, Hao; Shi, Liang; Sun, Lijun; Zhang, Jianbao

    2015-03-01

    Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15 Hz, 50 Hz, 75 Hz and 100 Hz) and at a flux density of 2 mT. Intracellular calcium concentration ([Ca(2+)]i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10 mM) was carried out to verify the function of the cardiac Na(+)/Ca(2+) exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca(2+)-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca(2+)]i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes. PMID:24499289

  8. Accelerated inactivation of the L-type calcium current due to a mutation in CACNB2b underlies Brugada syndrome

    DEFF Research Database (Denmark)

    Cordeiro, Jonathan M; Marieb, Mark; Pfeiffer, Ryan;

    2009-01-01

    revealed brief episodes of very rapid ventricular tachycardia. He was also diagnosed with vasovagal syncope. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 15 ion channel genes were amplified and sequenced. The only mutation uncovered was a missense mutation (T11I) in CACNB2b......Recent studies have demonstrated an association between mutations in CACNA1c or CACNB2b and Brugada syndrome (BrS). Previously described mutations all caused a loss of function secondary to a reduction of peak calcium current (I(Ca)). We describe a novel CACNB2b mutation associated with Br...... significantly faster in mutant channels between 0 and + 20 mV. Action potential voltage clamp experiments showed that total charge was reduced by almost half compared to WT. We report the first BrS mutation in CaCNB2b resulting in accelerated inactivation of L-type calcium channel current. Our results suggest...

  9. Effects of Losartan on L-type Calcium Current in Hypertrophied RatMyocytes

    Institute of Scientific and Technical Information of China (English)

    FuLiying; LiYang; ChengLan; WangFang; XiaGuojin; YaoWeixing

    2001-01-01

    Objective To investigate the alterations of L-type calcium current (IcaL) in abdominal aorticligation-induced hypertrophied rat hearts and the effect of losartan on these alterations. METHODS Cardiachypertrophy was induced by abdominal aortic ligation in rats. To record IcaL, whole-cell patch-clamp technique wasused. RESULTS Membrane capacitance was larger in hypertrophied cells (148±29 pF) than in sham-operated cells(102±14 pF, P<0.01) and losartan-treated cells (118±27, P<0.01). The maximal peak IcaL Was increased from-835±124 pA in sham-operated cells to -1404+_417 pA in hypertrophied cells (P<0.01), the corresponding IcaL density was increased from -7.5±1.8 pA.pF1 to -10.5±2.2 pA.pF1 (P<0.01), while they were reduced to -956-2:170pF (P<0.01) and -8.2±1.6 pA.pF1 (P<0.05) respectively in losartan-treated cells. The membrane potential of halfmaximal activation of the hypertrophied cells (-20.6±1.0 mV) shifted to more negative potentials than sham-operatedcells (-15.6±1.6 mV, P<0.01) and lorsartan-treated cells (-17.4±1.0 mV, P<0.01). The slope of the activation curveof hypertrophied cells (5.7±0.4) was decreased slightly than sham-operated cells (6.4±0.5, P<0.05). The membranepotential of half maximal inactivation of hypertrophied cells (-27.6±1.9 mV) shifted to more positive potentials thansham-operated cells (-31.4±2.2 mV, P<0.05). The slope of inactivation curves were not different in the three groups.

  10. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance.

    Science.gov (United States)

    Schep, Daniel G; Zhao, Jianhua; Rubinstein, John L

    2016-03-22

    Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases.

  11. Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems.

    Science.gov (United States)

    Roelofs, Kevin G; Jones, Christopher J; Helman, Sarah R; Shang, Xiaoran; Orr, Mona W; Goodson, Jonathan R; Galperin, Michael Y; Yildiz, Fitnat H; Lee, Vincent T

    2015-10-01

    Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP.

  12. Distal Renal Tubular Acidosis and Calcium Nephrolithiasis

    Science.gov (United States)

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

    2008-09-01

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

  13. Structure of the plasminogen kringle 4 binding calcium-free form of the C-type lectin-like domain of tetranectin

    DEFF Research Database (Denmark)

    Nielbo, Steen; Thomsen, Jens K; Graversen, Jonas Heilskov;

    2004-01-01

    Tetranectin is a homotrimeric protein containing a C-type lectin-like domain. This domain (TN3) can bind calcium, but in the absence of calcium, the domain binds a number of kringle-type protein ligands. Two of the calcium-coordinating residues are also critical for binding plasminogen kringle 4 (K...... no such flexibility is observed in holoTN3. In the 20 best nuclear magnetic resonance structures of apoTN3, the residues critical for K4 binding span a large conformational space. Together with the relaxation data, this indicates that the K4-ligand-binding site in apoTN3 is not preformed....

  14. The α2Na+/K+-ATPase is critical for skeletal and heart muscle function in zebrafish

    DEFF Research Database (Denmark)

    Doganli, Canan; Kjaer-Sørensen, Kasper; Knoeckel, Christopher;

    2012-01-01

    The Na+/K+-ATPase generates ion gradients across the plasma membrane, essential for multiple cellular functions. In mammals, four different Na+/K+-ATPase α-subunit isoforms are associated with characteristic cell-type expression profiles and kinetics. We found the zebrafish α2Na+/K+-ATPase associ......The Na+/K+-ATPase generates ion gradients across the plasma membrane, essential for multiple cellular functions. In mammals, four different Na+/K+-ATPase α-subunit isoforms are associated with characteristic cell-type expression profiles and kinetics. We found the zebrafish α2Na...... identified up- and down-regulation of specific phenotype-related proteins, such as parvalbumin, CaM, GFAP and multiple kinases, thus highlighting a potential proteome change associated with the dynamics of α2Na+/K+-ATPase. Taken together, our findings display that zebrafish α2Na+/K+-ATPase is important...

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

    Science.gov (United States)

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

    2005-03-01

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

  16. Cdc50p Plays a Vital Role in the ATPase Reaction Cycle of the Putative Aminophospholipid Transporter Drs2p*♦

    OpenAIRE

    Lenoir, Guillaume; Williamson, Patrick; Puts, Catheleyne F.; Holthuis, Joost C.M.

    2009-01-01

    Members of the P4 subfamily of P-type ATPases are believed to catalyze transport of phospholipids across cellular bilayers. However, most P-type ATPases pump small cations or metal ions, and atomic structures revealed a transport mechanism that is conserved throughout the family. Hence, a challenging problem is to understand how this mechanism is adapted in P4-ATPases to flip phospholipids. P4-ATPases form heteromeric complexes with Cdc50 proteins. The primary role of these additional polypep...

  17. Antioxidant effect of T-type calcium channel blockers in gastric injury.

    Science.gov (United States)

    Bilici, Dilek; Banoğlu, Z Nur; Kiziltunç, Ahmet; Avci, Bahattin; Ciftçioğlu, Akif; Bilici, Sefa

    2002-04-01

    It is known that calcium ion has an important role in the cellular function. For this reason, calcium channel blockers may have a protective action against gastric injury which is induced by various stimuli. In this study, the influence of mibefradil on ethanol-induced gastric injury was investigated in rats. Mibefradil was given at a dose 50 mg/kg intraperitoneally 30 min before administration of 1 ml absolute ethanol given by gavage. We compared this effect of mibefradil with that of omeprazol. Ethanol-induced mucosal damage was evaluated using three different approaches: analysis of biochemical parameters and pathologic and macroscopic investigation. It was found that pretreatment with mibefradil significantly reduced ethanol-induced macroscopic, pathologic, and biochemical changes in the gastric mucosa. In conclusion, it is speculated that this findings may prove important in the development of new and improved therapies for the treatment and prevention of gastric ulcers in humans. PMID:11991620

  18. Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

    Science.gov (United States)

    Gu, Yuanxing; Qi, Baozhu; Zhou, Yingshan; Jiang, Xiaowu; Zhang, Xian; Li, Xiaoliang; Fang, Weihuan

    2016-01-01

    Porcine circovirus type 2 (PCV2) induces autophagy via the 5′ adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKβ) by increasing cytosolic Ca2+ via inositol 1,4,5-trisphosphate receptor (IP3R). Elevation of cytosolic calcium ion (Ca2+) did not seem to involve inositol 1,4,5-trisphosphate (IP3) release from phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide phospholipase C-gamma (PLC-γ). CaMKKβ then activated both AMPK and calcium/calmodulin-dependent protein kinase I (CaMKI). PCV2 employed CaMKI and Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1) as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca2+ both in PK-15 cells and PCV2-targeted primary cells from pigs. PMID:27213427

  19. Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

    Directory of Open Access Journals (Sweden)

    Yuanxing Gu

    2016-05-01

    Full Text Available Porcine circovirus type 2 (PCV2 induces autophagy via the 5′ adenosine monophosphate-activated protein kinase (AMPK/extracellular signal-regulated kinase (ERK/tuberous sclerosis complex 2 (TSC2/mammalian target of rapamycin (mTOR pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi, we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKβ by increasing cytosolic Ca2+ via inositol 1,4,5-trisphosphate receptor (IP3R. Elevation of cytosolic calcium ion (Ca2+ did not seem to involve inositol 1,4,5-trisphosphate (IP3 release from phosphatidylinositol 4,5-bisphosphate (PIP2 by phosphoinositide phospholipase C-gamma (PLC-γ. CaMKKβ then activated both AMPK and calcium/calmodulin-dependent protein kinase I (CaMKI. PCV2 employed CaMKI and Trp-Asp (WD repeat domain phosphoinositide-interacting protein 1 (WIPI1 as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca2+ both in PK-15 cells and PCV2-targeted primary cells from pigs.

  20. Calcium gluconate in phosphate buffered saline increases gene delivery with adenovirus type 5.

    Directory of Open Access Journals (Sweden)

    Marko T Ahonen

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

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

    Science.gov (United States)

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

    2010-01-01

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

  2. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

    OpenAIRE

    Nichols, Grant S.; DeBello, William M.

    2015-01-01

    Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB) provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII) in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII e...

  3. Calcium channel blocker overdose

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/002580.htm Calcium channel blocker overdose To use the sharing features on this page, please enable JavaScript. Calcium channel blockers are a type of medicine used ...

  4. Fenoprofen calcium overdose

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/002649.htm Fenoprofen calcium overdose To use the sharing features on this page, please enable JavaScript. Fenoprofen calcium is a type of medicine called a nonsteroidal ...

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

    Science.gov (United States)

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

    2015-01-01

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

  6. Role of T-type calcium current in identified D-hair mechanoreceptor neurons studied in vitro

    OpenAIRE

    Dubreuil, A.S.; Boukhaddaoui, H.; Desmadryl, G; Martinez-Salgado, C.; Moshourab, R.; Lewin, G. R.; Carroll, P.; Valmier, J; Scamps, F.

    2004-01-01

    Different subsets of dorsal root ganglion (DRG) mechanoreceptors transduce low- and high-intensity mechanical stimuli. It was shown recently that, in vivo, neurotrophin-4 (NT-4)-dependent D-hair mechanoreceptors specifically express a voltage-activated T-type calcium channel (Ca(v)3.2) that may be required for their mechanoreceptive function. Here we show that D-hair mechanoreceptors can be identified in vitro by a rosette-like morphology in the presence of NT-4 and that these rosette neurons...

  7. Tight coupling of Na+/K+-ATPase with glycolysis demonstrated in permeabilized rat cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Mervi Sepp

    Full Text Available The effective integrated organization of processes in cardiac cells is achieved, in part, by the functional compartmentation of energy transfer processes. Earlier, using permeabilized cardiomyocytes, we demonstrated the existence of tight coupling between some of cardiomyocyte ATPases and glycolysis in rat. In this work, we studied contribution of two membrane ATPases and whether they are coupled to glycolysis--sarcoplasmic reticulum Ca2+ ATPase (SERCA and plasmalemma Na+/K+-ATPase (NKA. While SERCA activity was minor in this preparation in the absence of calcium, major role of NKA was revealed accounting to ∼30% of the total ATPase activity which demonstrates that permeabilized cell preparation can be used to study this pump. To elucidate the contribution of NKA in the pool of ATPases, a series of kinetic measurements was performed in cells where NKA had been inhibited by 2 mM ouabain. In these cells, we recorded: ADP- and ATP-kinetics of respiration, competition for ADP between mitochondria and pyruvate kinase (PK, ADP-kinetics of endogenous PK, and ATP-kinetics of total ATPases. The experimental data was analyzed using a series of mathematical models with varying compartmentation levels. The results show that NKA is tightly coupled to glycolysis with undetectable flux of ATP between mitochondria and NKA. Such tight coupling of NKA to PK is in line with its increased importance in the pathological states of the heart when the substrate preference shifts to glucose.

  8. T-type calcium channels promote predictive homeostasis of input-output relations in thalamocortical neurons of lateral geniculate nucleus.

    Science.gov (United States)

    Hong, Su Z; Kim, Haram R; Fiorillo, Christopher D

    2014-01-01

    A general theory views the function of all neurons as prediction, and one component of this theory is that of "predictive homeostasis" or "prediction error." It is well established that sensory systems adapt so that neuronal output maintains sensitivity to sensory input, in accord with information theory. Predictive homeostasis applies the same principle at the cellular level, where the challenge is to maintain membrane excitability at the optimal homeostatic level so that spike generation is maximally sensitive to small gradations in synaptic drive. Negative feedback is a hallmark of homeostatic mechanisms, as exemplified by depolarization-activated potassium channels. In contrast, T-type calcium channels exhibit positive feedback that appears at odds with the theory. In thalamocortical neurons of lateral geniculate nucleus (LGN), T-type channels are capable of causing bursts of spikes with an all-or-none character in response to excitation from a hyperpolarized potential. This "burst mode" would partially uncouple visual input from spike output and reduce the information spikes convey about gradations in visual input. However, past observations of T-type-driven bursts may have resulted from unnaturally high membrane excitability. Here we have mimicked within rat brain slices the patterns of synaptic conductance that occur naturally during vision. In support of the theory of predictive homeostasis, we found that T-type channels restored excitability toward its homeostatic level during periods of hyperpolarization. Thus, activation of T-type channels allowed two retinal input spikes to cause one output spike on average, and we observed almost no instances in which output count exceeded input count (a "burst"). T-type calcium channels therefore help to maintain a single optimal mode of transmission rather than creating a second mode. More fundamentally our results support the general theory, which seeks to predict the properties of a neuron's ion channels and

  9. Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

    Science.gov (United States)

    Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  10. Calcium thorium phosphate (Whitlockite-type mineral). Synthesis and structure refinement

    International Nuclear Information System (INIS)

    The crystal structure of a new calcium thorium phosphate has been refined by the full-profile Rietveld method using X-ray powder diffraction data. The sample has been synthesized by the sol-gel technique. The phosphate has been identified by X-ray powder diffraction and IR spectroscopy. The refined composition is represented by the formula Ca10.26Th0.12(PO4)7. The CaOn and PO4 polyhedra are distorted compared to the corresponding polyhedra in the basic compound β-Ca3(PO4)2.

  11. The Timothy syndrome mutation differentially affects voltage- and calcium-dependent inactivation of CaV1.2 L-type calcium channels

    OpenAIRE

    Barrett, Curtis F.; Tsien, Richard W.

    2008-01-01

    Calcium entry into excitable cells is an important physiological signal, supported by and highly sensitive to the activity of voltage-gated Ca2+ channels. After membrane depolarization, Ca2+ channels first open but then undergo various forms of negative feedback regulation including voltage- and calcium-dependent inactivation (VDI and CDI, respectively). Inactivation of Ca2+ channel activity is perturbed in a rare yet devastating disorder known as Timothy syndrome (TS), whose features include...

  12. Synthesis, structural, magnetic and dielectric properties of zirconium copper doped M-type calcium strontium hexaferrites

    Energy Technology Data Exchange (ETDEWEB)

    Ashiq, Muhammad Naeem; Qureshi, Raheela Beenish [Institute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800 (Pakistan); Malana, Muhammad Aslam, E-mail: draslammalana@gmail.com [Institute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800 (Pakistan); Ehsan, Muhammad Fahad [National Center for Nanoscience and Technology (NCNST), 11 Beiyitiao, Zhongguawn, Beijing (China)

    2014-12-25

    Highlights: • Particle size of these samples is suitable for high density recording media. • The samples have coercivity suitable for longitudinal magnetic recording media. • The sample (x = 0.8) has dielectric constant useful for high frequency applications. - Abstract: Zirconium copper substituted calcium strontium hexagonal ferrites with composition Ca{sub 0.5}Sr{sub 0.5}Fe{sub 12−2x}Zr{sub x}Cu{sub x}O{sub 19} (x = 0.0, 0.2, 0.4, 0.6, 0.8) have been synthesized by the chemical co precipitation procedure. These compounds were characterized by X-ray diffraction, thermogravimetry and scanning electron microscopy. Dielectric and magnetic properties of these hexaferrites were also explored. XRD analysis confirmed the single hexagonal phase of all the compounds and the average crystallite size was between 37 and 47 nm. The dielectric parameters show relaxation behaviour at higher frequencies. The values of dielectric parameters increase with dopants. In the range of magnetic field studied, the saturation magnetization decreases as the dopant contents increase which may be due to the nonmagnetic character of the substituents. The coercivity of the Zr–Cu doped derivatives of strontium calcium hexaferrites is increased up to x = 0.2 and then decreased. The values of coercivity are above 600 Oe which make them suitable materials for use in industries in longitudinal magnetic recording media.

  13. Potentiation of Opioid-Induced Analgesia by L-Type Calcium Channel Blockers: Need for Clinical Trial in Cancer Pain

    Directory of Open Access Journals (Sweden)

    S Basu Ray

    2008-01-01

    Full Text Available Previous reports indicate that the analgesic effect of opioids is due to both closure of specific voltage-gated calcium channels (N- and P/Q-types and opening of G protein-coupled inwardly rectifying potassium channels (GIRKs in neurons concerned with transmission of pain. However, administration of opioids leads to unacceptable levels of side effects, particularly at high doses. Thus, current research is directed towards simultaneously targeting other voltage-gated calcium channels (VGCCs like the L-type VGCCs or even other cell signaling mechanisms, which would aug-ment opioid-mediated analgesic effect without a concurrent increase in the side effects. Unfortunately, the results of these studies are often conflicting considering the different experimental paradigms (variable drug selection and their doses and also the specific pain test used for studying analgesia adopted by researchers. The present review focuses on some of the interesting findings regarding the analgesic effect of Opioids + L-VGCC blockers and suggests that time has come for a clinical trial of this combination of drugs in the treatment of cancer pain.

  14. Archazolid and apicularen: Novel specific V-ATPase inhibitors

    Directory of Open Access Journals (Sweden)

    Zeeck Axel

    2005-08-01

    Full Text Available Abstract Background V-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic membranes, they energize many different transport processes. Since their malfunction is correlated with various diseases in humans, the elucidation of the properties of this enzyme for the development of selective inhibitors and drugs is one of the challenges in V-ATPase research. Results Archazolid A and B, two recently discovered cytotoxic macrolactones produced by the myxobacterium Archangium gephyra, and apicularen A and B, two novel benzolactone enamides produced by different species of the myxobacterium Chondromyces, exerted a similar inhibitory efficacy on a wide range of mammalian cell lines as the well established plecomacrolidic type V-ATPase inhibitors concanamycin and bafilomycin. Like the plecomacrolides both new macrolides also prevented the lysosomal acidification in cells and inhibited the V-ATPase purified from the midgut of the tobacco hornworm, Manduca sexta, with IC50 values of 20–60 nM. However, they did not influence the activity of mitochondrial F-ATPase or that of the Na+/K+-ATPase. To define the binding sites of these new inhibitors we used a semi-synthetic radioactively labelled derivative of concanamycin which exclusively binds to the membrane Vo subunit c. Whereas archazolid A prevented, like the plecomacrolides concanamycin A, bafilomycin A1 and B1, labelling of subunit c by the radioactive I-concanolide A, the benzolactone enamide apicularen A did not compete with the plecomacrolide derivative. Conclusion The myxobacterial antibiotics archazolid and apicularen are highly efficient and specific novel inhibitors of V-ATPases. While archazolid at least partly shares a common binding site with the plecomacrolides bafilomycin and concanamycin, apicularen adheres to an independent binding site.

  15. Diabetes alters intracellular calcium transients in cardiac endothelial cells.

    Directory of Open Access Journals (Sweden)

    Abdul Q Sheikh

    Full Text Available Diabetic cardiomyopathy (DCM is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca(2+](i homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca(2+](i homeostasis due to altered sarcoplasmic reticulum Ca(2+ ATPase (SERCA and sodium-calcium exchanger (NCX activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO, elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca(2+ regulatory mechanisms in cardiac endothelial cells (CECs remains unknown. The objective of this study was to determine the effect of diabetes on [Ca(2+](i homeostasis in CECs in the rat model (streptozotocin-induced of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca(2+](i transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca(2+ ATPase (PMCA and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca(2+](i sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.

  16. Development of an 111In-labeled dihydropyridine complex for L-type calcium channel imaging

    International Nuclear Information System (INIS)

    [111In]-DTPA-Amlodipine complex ([111In]-DTPA-AMLO) was prepared starting high purity [111In]indium chloride and conjugated DTPA-AMLO in 30 min at room temperature in acetate buffer in high radiochemical purity (>99 %, RTLC/HPLC; specific activity: 8-10 GBq/mmol). The log P, stability, biodistribution studies and imaging studies in untreated and amlodipine-pretreated rats were determined. The tracer is mostly washed out through kidneys as expected for a dihydropyridine compound. Blocking studies demonstrated high specific binding of the tracer in calcium channel-rich organs including intestine, heart and colon. SPECT images fully supported above results in normal and treated rats. (author)

  17. Thermal conductivity of the gadolinium calcium silicate apatites: Effect of different point defect types

    International Nuclear Information System (INIS)

    The apatite crystal structure of the gadolinium calcium silicates can accommodate a wide range of point defects, including oxygen and cation vacancies, as well as anti-site defects, depending on the Gd/Ca ratio. Compositions having only cation or oxygen vacancies were identified and the thermal diffusivity and conductivity were measured up to 1000 deg. C. All the compositions, including the stoichiometric composition, exhibit low thermal conductivities from room temperature to high temperature with the defect-containing compositions having even lower thermal conductivities. The high-temperature thermal conductivity, at temperatures below the onset of significant radiative heat transport, decreases with the inverse square root of the cation and anion vacancy concentration, consistent with simple defect scattering models. Based on the data, it is concluded that the oxygen vacancies are slightly more effective in reducing thermal conductivity.

  18. Splice variants of the CaV1.3 L-type calcium channel regulate dendritic spine morphology

    Science.gov (United States)

    Stanika, Ruslan; Campiglio, Marta; Pinggera, Alexandra; Lee, Amy; Striessnig, Jörg; Flucher, Bernhard E.; Obermair, Gerald J.

    2016-01-01

    Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson’s disease. The L-type calcium channel CaV1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length CaV1.3L and two C-terminally truncated splice variants (CaV1.342A and CaV1.343S) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged CaV1.3L revealed its localization at the base-, neck-, and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in CaV1.3L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with CaV1.3L and correlated with increased CaV1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of CaV1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of CaV1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders. PMID:27708393

  19. The beta-adrenergic blocker carvedilol restores L-type calcium current in a myocardial infarction model of rabbit

    Institute of Scientific and Technical Information of China (English)

    LI Xia; HUANG Cong-xin; JIANG Hong; CAO Feng; WANG Teng

    2005-01-01

    Background Carvedilol, an antagonist of α1- and β-adrenergic receptors, has shown efficacy in reducing all-cause death and arrhythmia death for ischemic heart disease and congestive heart failure in several large-scale trials. It has been found to prevent ventricular remodeling, and recently was reported to reverse down-regulation of Na+ channel in a chronic heart failure model. This study was conducted to investigate whether carvedilol could reverse the ion remodeling in a myocardial infarction model of rabbit.Methods After the procedure of coronary ligation, animals were randomized to placebo or carvedilol treatment (5 mg/kg). Action potentials, L-type calcium current (Ica L) and the effect of isoproterenol stimulation on Ica L were measured using whole-cell patch method. Evaluation of the expression of calcium channel subunits was carried out by RT-PCR and Western blot. Results The results indicate that mean peak Ica L densities (pA/pF) at +10 mV was reduced in postinfarction myocytes (5.33±0.45, n=25) compared to sham myocytes (6.52±0.21, n=20). Treatment of myocardial infarction rabbits with carvedilol could restore it partially (5.91±0.39, n=20, P<0.05). However, steady-state activation parameters were similar in three groups. With stimulation by isoproterenol (1 μmol/L) Ica L increased in all three groups, but the increase was smaller in postinfarction myocytes. mRNA levels of calcium channel subunit CaA1 gene was decreased but CaB2a, CaB2b and CaB3 mRNA levels did not change after MI. Corresponding change in CaA1 protein was also observed. Conclusions The results demonstrate that carvedilol restores Ica L density and reverse the downregulation of CaA1 postinfarction.

  20. Measuring In Vitro ATPase Activity for Enzymatic Characterization.

    Science.gov (United States)

    Rule, Chelsea S; Patrick, Marcella; Sandkvist, Maria

    2016-01-01

    Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary. PMID:27584824

  1. Peroxisome is a reservoir of intracellular calcium.

    Science.gov (United States)

    Raychaudhury, Bikramjit; Gupta, Shreedhara; Banerjee, Shouvik; Datta, Salil C

    2006-07-01

    We have examined fura 2-loaded purified peroxisomes under confocal microscope to prove that this mammalian organelle is a store of intracellular calcium pool. Presence of calcium channel and vanadate sensitive Ca(2+)-ATPase in the purified peroxisomal membrane has been demonstrated. We have further observed that machineries to maintain calcium pool in this mammalian organelle are impaired during infection caused by Leishmania donovani. Results reveal that peroxisomes have a merit to play a significant role in the metabolism of intracellular calcium. PMID:16713100

  2. Gentamicin Blocks the ACh-Induced BK Current in Guinea Pig Type II Vestibular Hair Cells by Competing with Ca2+ at the l-Type Calcium Channel

    Directory of Open Access Journals (Sweden)

    Hong Yu

    2014-04-01

    Full Text Available Type II vestibular hair cells (VHCs II contain big-conductance Ca2+-dependent K+ channels (BK and L-type calcium channels. Our previous studies in guinea pig VHCs II indicated that acetylcholine (ACh evoked the BK current by triggering the influx of Ca2+ ions through l-type Ca2+ channels, which was mediated by M2 muscarinic ACh receptor (mAChRs. Aminoglycoside antibiotics, such as gentamicin (GM, are known to have vestibulotoxicity, including damaging effects on the efferent nerve endings on VHCs II. This study used the whole-cell patch clamp technique to determine whether GM affects the vestibular efferent system at postsynaptic M2-mAChRs or the membrane ion channels. We found that GM could block the ACh-induced BK current and that inhibition was reversible, voltage-independent, and dose-dependent with an IC50 value of 36.3 ± 7.8 µM. Increasing the ACh concentration had little influence on GM blocking effect, but increasing the extracellular Ca2+ concentration ([Ca2+]o could antagonize it. Moreover, 50 µM GM potently blocked Ca2+ currents activated by (--Bay-K8644, but did not block BK currents induced by NS1619. These observations indicate that GM most likely blocks the M2 mAChR-mediated response by competing with Ca2+ at the l-type calcium channel. These results provide insights into the vestibulotoxicity of aminoglycoside antibiotics on mammalian VHCs II.

  3. Types of voltage—dependent calcium channels involved in high potassium depolarization—induced amylase secretion in the exocrine pancreatic tumour cell line AR4—2J

    Institute of Scientific and Technical Information of China (English)

    CUIZONGJIE

    1998-01-01

    In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium,Attached cells when stimulated with high potassium secreted large amount of amylase.High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation.High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel anatagonists with an order of potency as follows:nifedipine>ω-agatoxin IVA>ω-conotoxin GVIA.In contrast,the L-type calcium channel anatagonist nifedipine almost completely inhibited potassium-induced amylase secretion,whereas the N-type channel antagonist ω-conotoxin GVIA was without effect.The P-type channel antagonist ω-agatoxin IVA had a small inhibitory effect,but this inhibition was not significant at the level of amylase secretion.In conclusion,the AR4-2J cell line posesses different voltage-dependent calcium channels(L,P,N)with the L-type predominantly involved in depolarization induced amylase secretion.

  4. On the origin of rhythmic calcium transients in the ICC-MP of the mouse small intestine.

    Science.gov (United States)

    Lowie, Bobbi-Jo; Wang, Xuan-Yu; White, Elizabeth J; Huizinga, Jan D

    2011-11-01

    Interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are pacemaker cells of the small intestine, producing the characteristic omnipresent electrical slow waves, which orchestrate peristaltic motor activity and are associated with rhythmic intracellular calcium oscillations. Our objective was to elucidate the origins of the calcium transients. We hypothesized that calcium oscillations in the ICC-MP are primarily regulated by the sarcoplasmic reticulum (SR) calcium release system. With the use of calcium imaging, study of the effect of T-type calcium channel blocker mibefradil revealed that T-type channels did not play a major role in generating the calcium transients. 2-Aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate receptor (IP(3)R) inhibitor, and U73122, a phospholipase C inhibitor, both drastically decreased the frequency of calcium oscillations, suggesting a major role of IP(3) and IP(3)-induced calcium release from the SR. Immunohistochemistry proved the expression of IP(3)R type I (IP(3)R-I), but not type II (IP(3)R-II) and type III (IP(3)R-III) in ICC-MP, indicating the involvement of the IP(3)R-I subtype in calcium release from the SR. Cyclopiazonic acid, a SR/endoplasmic reticulum calcium ATPase pump inhibitor, strongly reduced or abolished calcium oscillations. The Na-Ca exchanger (NCX) in reverse mode is likely involved in refilling the SR because the NCX inhibitor KB-R7943 markedly reduced the frequency of calcium oscillations. Immunohistochemistry revealed 100% colocalization of NCX and c-Kit in ICC-MP. Testing a mitochondrial NCX inhibitor, we were unable to show an essential role for mitochondria in regulating calcium oscillations in the ICC-MP. In summary, ongoing IP(3) synthesis and IP(3)-induced calcium release from the SR, via the IP(3)R-I, are the major drivers of the calcium transients associated with ICC pacemaker activity. This suggests that a biochemical clock intrinsic to ICC determines the pacemaker

  5. Branchial Na+:K+:2Cl- cotransporter 1 and Na+/K+-ATPase α-subunit in a brackish water-type ionocyte of the euryhaline freshwater white-rimmed stingray, Himantura signifer

    Directory of Open Access Journals (Sweden)

    Yuen K Ip

    2013-12-01

    Full Text Available Himantura signifer is a freshwater stingray which inhabits rivers in Southeast Asia. It can survive in brackish water but not seawater. In brackish water, it becomes partially ureosmotic, but how it maintains its plasma hypoionic to the external medium is enigmatic because of the lack of a rectal gland. Here, we report for the first time the expression of Na+:K+:2Cl− cotransporter 1 (nkcc1 in the gills of freshwater H. signifer, and its moderate up-regulation (~2-fold in response to brackish water (salinity 20 acclimation. The absence of the Ste20-related proline-alanine-rich kinase and oxidation stress response kinase 1 interaction site from the N-terminus of H. signifer Nkcc1 suggested that it might not be effectively activated by stress kinases in response to salinity changes as in more euryhaline teleosts. The increased activity of Nkcc1 during salt excretion in brackish water would lead to an influx of Na+ into ionocytes, and the maintenance of intracellular Na+ homeostasis would need the cooperation of Na+/K+-ATPase (Nka. We demonstrated for the first time the expression of nkaα1, nkaα2 and nkaα3 in the gills of H. signifer, and the up-regulation of the mRNA expression of nkaα3 and the overall protein abundance of Nkaα in response to acclimation to brackish water. Immunofluorescence microscopy revealed the presence of a sub-type of ionocyte, co-expressing Nkcc1 and Nkaα, near the base of the secondary lamellae in the gills of H. signifer acclimated to brackish water, but this type of ionocyte was absent from the gills of fish kept in fresh water. Hence, there could be a change in the function of the gills of H. signifer from salt absorption to salt excretion during brackish water acclimation in the absence of a functioning rectal gland.

  6. Dietary calcium and 1,25-dihydroxyvitamin D3 regulate transcription of calcium transporter genes in calbindin-D9k knockout mice.

    Science.gov (United States)

    Ko, Sang-Hwan; Lee, Geun-Shik; Vo, Thuy T B; Jung, Eui-Man; Choi, Kyung-Chul; Cheung, Ki-Wha; Kim, Jae Wha; Park, Jong-Gil; Oh, Goo Taeg; Jeung, Eui-Bae

    2009-04-01

    The effect(s) of oral calcium and vitamin D(3) were examined on the expression of duodenal and renal active calcium transport genes, i.e., calbindin-D9k (CaBP-9k) and calbindin-D28k (CaBP-28k), transient receptor potential cation channels (TRPV5 and TRPV6), Na(+)/Ca(2+) exchanger 1 (NCX1) and plasma membrane calcium ATPase 1b (PMCA1b), in CaBP-9k KO mice. Wild-type (WT) and KO mice were provided with calcium and vitamin D(3)-deficient diets for 10 weeks. The deficient diet significantly decreased body weights compared with the normal diet groups. The serum calcium concentration of the WT mice was decreased by the deficient diet but was unchanged in the KO mice. The deficient diet significantly increased duodenal transcription of CaBP-9k and TRPV6 in the WT mice, but no alteration was observed in the KO mice. In the kidney, the deficient diet significantly increased renal transcripts of CaBP-9k, TRPV6, PMCA1b, CaBP-28k and TRPV5 in the WT mice but did not alter calcium-relating genes in the KO mice. Two potential mediators of calcium-processing genes, vitamin D receptor (VDR) and parathyroid hormone receptor (PTHR), have been suggested to be useful for elucidating these differential regulations in the calcium-related genes of the KO mice. Expression of VDR was not significantly affected by diet or the KO mutation. Renal PTHR mRNA levels were reduced by the diet, and reduced expression was also seen in the KO mice given the normal diet. Taken together, these results suggest that the active calcium transporting genes in KO mice may have resistance to the deficiency diet of calcium and vitamin D(3).

  7. A New Type of Biphasic Calcium Phosphate Cement as a Gentamicin Carrier for Osteomyelitis

    Directory of Open Access Journals (Sweden)

    Wen-Yu Su

    2013-01-01

    Full Text Available Osteomyelitis therapy is a long-term and inconvenient procedure for a patient. Antibiotic-loaded bone cements are both a complementary and alternative treatment option to intravenous antibiotic therapy for the treatment of osteomyelitis. In the current study, the biphasic calcium phosphate cement (CPC, called α-TCP/HAP (α-tricalcium phosphate/hydroxyapatite biphasic cement, was prepared as an antibiotics carrier for osteomyelitis. The developed biphasic cement with a microstructure of α-TCP surrounding the HAP has a fast setting time which will fulfill the clinical demand. The X-ray diffraction and Fourier transform infrared spectrometry analyses showed the final phase to be HAP, the basic bone mineral, after setting for a period of time. Scanning electron microscopy revealed a porous structure with particle sizes of a few micrometers. The addition of gentamicin in α-TCP/HAP would delay the transition of α-TCP but would not change the final-phase HAP. The gentamicin-loaded α-TCP/HAP supplies high doses of the antibiotic during the initial 24 hours when they are soaked in phosphate buffer solution (PBS. Thereafter, a slower drug release is produced, supplying minimum inhibitory concentration until the end of the experiment (30 days. Studies of growth inhibition of Staphylococcus aureus and Pseudomonas aeruginosa in culture indicated that gentamicin released after 30 days from α-TCP/HAP biphasic cement retained antibacterial activity.

  8. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

    Directory of Open Access Journals (Sweden)

    Grant S. Nichols

    2015-01-01

    Full Text Available Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX, the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is “off” in adults.

  9. Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

    Directory of Open Access Journals (Sweden)

    Yoichiro Fukao

    2016-01-01

    Full Text Available The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex, respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

  10. Calcium-dependent expression of transient receptor potential canonical type 3 channels in patients with chronic kidney disease

    DEFF Research Database (Denmark)

    Liu, Ying; Krueger, Katharina; Hovsepian, Anahit;

    2011-01-01

    It is unknown whether extracellular calcium may regulate the expression of transient receptor potential canonical type 3 (TRPC3) channels in patients with chronic kidney disease. Using quantitative in-cell Western assay we compared the expression of TRPC3 channel protein in monocytes from 20...... patients with chronic kidney disease and 19 age- and sex-matched healthy control subjects. TRPC3 channels were identified by immunoblotting using specific antibodies and TRPC3 protein was further confirmed by mass spectrometry. We observed a significant increase of TRPC3 channel protein expression...... in patients with chronic kidney disease compared to healthy control subjects (normalized expression, 0.42±0.06 vs. 0.19±0.03; p...

  11. Structure of Na+,K+-ATPase at 11-A resolution: comparison with Ca2+-ATPase in E1 and E2 states.

    OpenAIRE

    Rice, W J; Young, H S; Martin, D W; Sachs, J R; Stokes, D.L.

    2001-01-01

    Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the cry...

  12. An integrated multiassay approach to the discovery of small-molecule N-type voltage-gated calcium channel antagonists.

    Science.gov (United States)

    Finley, Michael F A; Lubin, Mary Lou; Neeper, Michael P; Beck, Edward; Liu, Yi; Flores, Christopher M; Qin, Ning

    2010-12-01

    Abstract The N-type voltage-gated calcium channel (Cav2.2) has been intensively explored as a target for novel, small-molecule analgesic drugs because of its distribution in the pain pathway and its role in nociceptive processing. For example, Cav2.2 is localized at presynaptic terminals of pain fibers in the dorsal horn, and it serves as a downstream effector of μ-opioid receptors. Most importantly, antagonism of the channel by the highly specific and potent Cav2.2 blocker ω-conotoxin MVIIA (ziconotide) produces clinical efficacy in the treatment of severe, intractable pain. To identify novel small-molecule Cav2.2 inhibitors, we developed new tools and screening methods critical to enhance the efficiency and probability of success. First, we established and characterized a new cell line stably expressing the three subunits of the Cav2.2, including an α-subunit splice variant that is uniquely expressed by dorsal root ganglion neurons. Second, using this cell line, we validated and employed a fluorescence-based calcium flux assay. Third, we developed a new "medium-throughput" electrophysiology assay using QPatch-HT to provide faster turnaround on high-content electrophysiology data that are critical for studying ion channel targets. Lastly, we used a therapeutically relevant, ex vivo spinal cord calcitonin gene-related peptide-release assay to confirm activities in the other assays. Using this approach we have identified compounds exhibiting single-digit nM IC₅₀ values and with a positive correlation across assay methods. This integrated approach provides a more comprehensive evaluation of small-molecule N-type inhibitors that may lead to improved therapeutic pharmacology. PMID:21050074

  13. Cardioprotective effect of an L/N-type calcium channel blocker in patients with hypertensive heart disease

    International Nuclear Information System (INIS)

    Left ventricular (LV) diastolic dysfunction is related to increased cardiac sympathetic activity. We investigated the effect of cilnidipine, an L/N-type calcium channel blocker, on LV diastolic function and cardiac sympathetic activity in patients with hypertensive heart disease (HHD) using radionuclide myocardial imaging. Thirty-two frame electrocardiography (ECG)-gated 99mTc-sestamibi (MIBI) myocardial single photon emission computed tomography (SPECT), and 123I-metaiodobenzylguanidine (MIBG) imaging were performed before and 6 months after drug administration in 32 outpatients with HHD. Sixteen of the patients were treated with cilnidipine and the other 16 were treated with nifedipine retard. The parameters for assessing LV diastolic function evaluated using ECG-gated 99mTc-MIBI SPECT were peak filling rate (PFR), first-third filling rate (1/3FR), and time to peak filling (TPF). Cardiac sympathetic activity was assessed as early and delayed heart to mediastinum (H/M) ratios and a washout rate (WR), using 123I-MIBG imaging. The PFR and 1/3FR significantly increased after 6 months of treatment with cilnidipine (p<0.05 for both), but did not with nifedipine retard. The H/M ratios significantly increased (p<0.05 for both) in conjunction with a decreased WR (p<0.05) in the cilnidipine group. Moreover, a significant positive correlation was seen between the rate of change in PFR and the rate of change in early and delayed H/M ratios in the cilnidipine group (p<0.05 for both). The same results were obtained for the relationship between the rate of change in 1/3FR and the rate of change in H/M ratios (p<0.05 for both). However, no such relationship was seen in the nifedipine group. These data indicate that cilnidipine seems to suppress cardiac sympathetic overactivity via blockade of N-type calcium channels and improves LV diastolic function in patients with HHD. (author)

  14. Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes

    OpenAIRE

    Srinivasan, Padma P.; Parajuli, Ashutosh; Price, Christopher; Wang, Liyun; Duncan, Randall L.; Kirn-Safran, Catherine B.

    2015-01-01

    Voltage-sensitive calcium channels (VSCC) regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1) and chondrocytes were treated with a selective T-VS...

  15. Cdc50p plays a vital role in the ATPase reaction cycle of the putative aminophospholipid transporter Drs2p

    NARCIS (Netherlands)

    Lenoir, G.F.; WIlliamson, P.L.; Puts, C.F.; Holthuis, J.C.M.

    2009-01-01

    Members of the P4 subfamily of P-type ATPases are believed to catalyze transport of phospholipids across cellular bilayers. However, most P-type ATPases pump small cations or metal ions, and atomic structures revealed a transport mechanism that is conserved throughout the family. Hence, a challengin

  16. Mechanism and significance of P4 ATPase-catalyzed lipid transport: lessons from a Na+/K+-pump

    NARCIS (Netherlands)

    Puts, C.F.; Holthuis, J.C.M.

    2009-01-01

    Members of the P4 subfamily of P-type ATPases are believed to catalyze phospholipid transport across membrane bilayers, a process influencing a host of cellular functions. Atomic structures and functional analysis of P-type ATPases that pump small cations and metal ions revealed a transport mechanis

  17. Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

    Energy Technology Data Exchange (ETDEWEB)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Ochotny, Noelle [Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Manolson, Morris F. [Faculty of Dentistry, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Holliday, L. Shannon, E-mail: sholliday@dental.ufl.edu [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610 (United States)

    2009-11-06

    Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  18. Connections between connexins, calcium, and cataracts in the lens.

    Science.gov (United States)

    Gao, Junyuan; Sun, Xiurong; Martinez-Wittinghan, Francisco J; Gong, Xiaohua; White, Thomas W; Mathias, Richard T

    2004-10-01

    There is a good deal of evidence that the lens generates an internal micro circulatory system, which brings metabolites, like glucose, and antioxidants, like ascorbate, into the lens along the extracellular spaces between cells. Calcium also ought to be carried into the lens by this system. If so, the only path for Ca2+ to get out of the lens is to move down its electrochemical gradient into fiber cells, and then move by electrodiffusion from cell to cell through gap junctions to surface cells, where Ca-ATPase activity and Na/Ca exchange can transport it back into the aqueous or vitreous humors. The purpose of the present study was to test this calcium circulation hypothesis by studying calcium homeostasis in connexin (Cx46) knockout and (Cx46 for Cx50) knockin mouse lenses, which have different degrees of gap junction coupling. To measure intracellular calcium, FURA2 was injected into fiber cells, and the gradient in calcium concentration from center to surface was mapped in each type of lens. In wild-type lenses the coupling conductance of the mature fibers was approximately 0.5 S/cm2 of cell to cell contact, and the best fit to the calcium concentration data varied from 700 nM in the center to 300 nM at the surface. In the knockin lenses, the coupling conductance was approximately 1.0 S/cm2 and calcium varied from approximately 500 nM at the center to 300 nM at the surface. Thus, when the coupling conductance doubled, the concentration gradient halved, as predicted by the model. In knockout lenses, the coupling conductance was zero, hence the efflux path was knocked out and calcium accumulated to approximately 2 microM in central fibers. Knockout lenses also had a dense central cataract that extended from the center to about half the radius. Others have previously shown that this cataract involves activation of a calcium-dependent protease, Lp82. We can now expand on this finding to provide a hypothesis on each step that leads to cataract formation: knockout of

  19. Cav1.4 L-Type Calcium Channels Contribute to Calpain Activation in Degenerating Photoreceptors of rd1 Mice.

    Directory of Open Access Journals (Sweden)

    Christian Schön

    Full Text Available Retinitis pigmentosa is an inherited blinding disorder characterized by progressive degeneration and loss of photoreceptors. The exact mechanism of degeneration and cell death of photoreceptors is not known, but is thought to involve disturbed Ca2+-signaling. Ca2+ can enter the photoreceptor cell via outer segment cyclic nucleotide-gated (CNG channels or synaptic Cav1.4 L-type voltage-gated calcium channels (VGCC. Previously, we have shown that genetic ablation of the Cngb1 gene encoding the B subunit of the rod CNG channel delays the fast progressing degeneration in the rd1 mutant mouse model of retinitis pigmentosa. In this study, we crossbred rd1 mice with the Cacna1f-deficient mouse lacking the Cav1.4 α1 subunit of the L-type VGCC. Longitudinal in vivo examinations of photoreceptor layer thickness by optical coherence tomography revealed a significant, but not sustained delay of retinal degeneration in Cacna1f x rd1 double mutant mice compared to rd1 mice. This was accompanied by a reduction of TUNEL positive cells in the early phase of rod degeneration. Remarkably, Cacna1f x rd1 double mutant mice displayed a strong decrease in the activation of the Ca2+-dependent protease calpain during photoreceptor loss. Our results show that genetic deletion of the synaptic Cav1.4 L-type VGCCs impairs calpain activation and leads to a short-term preservation of photoreceptors in the rd1 mouse.

  20. Plasma membrane ATPases

    DEFF Research Database (Denmark)

    Palmgren, Michael Broberg; Bækgaard, Lone; Lopez Marques, Rosa Laura;

    2011-01-01

    The plasma membrane separates the cellular contents from the surrounding environment. Nutrients must enter through the plasma membrane in order to reach the cell interior, and toxic metabolites and several ions leave the cell by traveling across the same barrier. Biological pumps in the plasma...... membrane include ABC transporters, vacuolar (V-type) H+ pumps, and P-type pumps. These pumps all utilize ATP as a fuel for energizing pumping. This review focuses on the physiological roles of plasma membrane P-type pumps, as they represent the major ATP hydrolytic activity in this membrane....

  1. Mechanism and significance of P4 ATPase-catalyzed lipid transport: lessons from a Na+/K+-pump.

    Science.gov (United States)

    Puts, Catheleyne F; Holthuis, Joost C M

    2009-07-01

    Members of the P(4) subfamily of P-type ATPases are believed to catalyze phospholipid transport across membrane bilayers, a process influencing a host of cellular functions. Atomic structures and functional analysis of P-type ATPases that pump small cations and metal ions revealed a transport mechanism that appears to be conserved throughout the family. A challenging problem is to understand how this mechanism is adapted in P(4) ATPases to flip phospholipids. P(4) ATPases form oligomeric complexes with members of the CDC50 protein family. While formation of these complexes is required for P(4) ATPase export from the endoplasmic reticulum, little is known about the functional role of the CDC50 subunits. The Na(+)/K(+)-ATPase and closely-related H(+)/K(+)-ATPase are the only other P-type pumps that are oligomeric, comprising mandatory beta-subunits that are strikingly reminiscent of CDC50 proteins. Besides serving a role in the functional maturation of the catalytic alpha-subunit, the beta-subunit also contributes specifically to intrinsic transport properties of the Na(+)/K(+) pump. As beta-subunits and CDC50 proteins likely adopted similar structures to accomplish analogous tasks, current knowledge of the Na(+)/K(+)-ATPase provides a useful guide for understanding the inner workings of the P(4) ATPase class of lipid pumps. PMID:19233312

  2. L-type voltage-operated calcium channels contribute to astrocyte activation In vitro.

    Science.gov (United States)

    Cheli, Veronica T; Santiago González, Diara A; Smith, Jessica; Spreuer, Vilma; Murphy, Geoffrey G; Paez, Pablo M

    2016-08-01

    We have found a significant upregulation of L-type voltage-operated Ca(++) channels (VOCCs) in reactive astrocytes. To test if VOCCs are centrally involved in triggering astrocyte reactivity, we used in vitro models of astrocyte activation in combination with pharmacological inhibitors, siRNAs and the Cre/lox system to reduce the activity of L-type VOCCs in primary cortical astrocytes. The endotoxin lipopolysaccharide (LPS) as well as high extracellular K(+) , glutamate, and ATP promote astrogliosis in vitro. L-type VOCC inhibitors drastically reduce the number of reactive cells, astrocyte hypertrophy, and cell proliferation after these treatments. Astrocytes transfected with siRNAs for the Cav1.2 subunit that conducts L-type Ca(++) currents as well as Cav1.2 knockout astrocytes showed reduce Ca(++) influx by ∼80% after plasma membrane depolarization. Importantly, Cav1.2 knock-down/out prevents astrocyte activation and proliferation induced by LPS. Similar results were found using the scratch wound assay. After injuring the astrocyte monolayer, cells extend processes toward the cell-free scratch region and subsequently migrate and populate the scratch. We found a significant increase in the activity of L-type VOCCs in reactive astrocytes located in the growing line in comparison to quiescent astrocytes situated away from the scratch. Moreover, the migration of astrocytes from the scratching line as well as the number of proliferating astrocytes was reduced in Cav1.2 knock-down/out cultures. In summary, our results suggest that Cav1.2 L-type VOCCs play a fundamental role in the induction and/or proliferation of reactive astrocytes, and indicate that the inhibition of these Ca(++) channels may be an effective way to prevent astrocyte activation. GLIA 2016. GLIA 2016;64:1396-1415. PMID:27247164

  3. Six types of multistability in a neuronal model based on slow calcium current.

    Directory of Open Access Journals (Sweden)

    Tatiana Malashchenko

    Full Text Available BACKGROUND: Multistability of oscillatory and silent regimes is a ubiquitous phenomenon exhibited by excitable systems such as neurons and cardiac cells. Multistability can play functional roles in short-term memory and maintaining posture. It seems to pose an evolutionary advantage for neurons which are part of multifunctional Central Pattern Generators to possess multistability. The mechanisms supporting multistability of bursting regimes are not well understood or classified. METHODOLOGY/PRINCIPAL FINDINGS: Our study is focused on determining the bio-physical mechanisms underlying different types of co-existence of the oscillatory and silent regimes observed in a neuronal model. We develop a low-dimensional model typifying the dynamics of a single leech heart interneuron. We carry out a bifurcation analysis of the model and show that it possesses six different types of multistability of dynamical regimes. These types are the co-existence of 1 bursting and silence, 2 tonic spiking and silence, 3 tonic spiking and subthreshold oscillations, 4 bursting and subthreshold oscillations, 5 bursting, subthreshold oscillations and silence, and 6 bursting and tonic spiking. These first five types of multistability occur due to the presence of a separating regime that is either a saddle periodic orbit or a saddle equilibrium. We found that the parameter range wherein multistability is observed is limited by the parameter values at which the separating regimes emerge and terminate. CONCLUSIONS: We developed a neuronal model which exhibits a rich variety of different types of multistability. We described a novel mechanism supporting the bistability of bursting and silence. This neuronal model provides a unique opportunity to study the dynamics of networks with neurons possessing different types of multistability.

  4. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe;

    2011-01-01

    transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na......(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps....

  5. Soft chemical synthesis and electrochemical properties of calcium ferrite-type LixMn2O4

    Science.gov (United States)

    Mamiya, Mikito; Tokiwa, Kazuyasu; Akimoto, Junji

    2016-04-01

    Calcium ferrite (CaFe2O4)-type LixMn2O4 was prepared via high-pressure and soft chemical synthesis method. The framework structure of CaFe2O4-type NaMn2O4 was synthesized from the stoichiometric mixture of Na2CO3 and MnO2 annealed by 1273 K for 1 h under 4.5 GPa. Na/Li ion-exchange of the CaFe2O4-type NaMn2O4 was carried out by soaking molten LiNO3 at 633 K for 12 h. The electrochemical properties of the ion-exchanged CaFe2O4-type LixMn2O4 were measured. The initial discharge profile in the voltage range from 4.0 to 1.0 V showed 458 mAh g-1 of the discharge capacity with two plateaus near 3.7 V and 2.7 V (vs. Li/Li+). The discharge capacity was decreased with increasing the cycle number. After 30 cycles, the capacity was decreased to 375 mAh g-1. When the range was set between 4.8 and 3.0 V, the discharge capacity was 113 mAh g-1 in initial, and 111 mAh g-1 after 50th cycle. The reference CaFe2O4-type LiMn2O4 was prepared via one-step high-pressure synthesis and compared the electrochemical properties with the ion-exchanged sample. The initial discharge capacity of the one-step synthesized one was 108 mAh g-1 at 1.0 V (vs. Li/Li+), which was 73% lower than the value of the ion-exchanged one.

  6. The Effect of Extrogenous Phosphocreatine on L-type Calcium Current in Ischemic Guinea Pig Ventricular Myocytes

    Institute of Scientific and Technical Information of China (English)

    Shi Xiangmin; Li Tiande; Yang Tingshu; Wang Yutang; Shan Zhaoliang

    2005-01-01

    Objectives Heart failure (HF)is one of the most common outcome for all kinds of heart diseases, the effects of energetic therapy on HF remains controversial, especially to ischemic HF. The aim of this study was to explore the effect of exogenous phosphocreatine with different concentration on L-type calcium(ICa-L) current in ischemic ventricular myocytes of guinea pig and to investigate its underlying electrophysiological mechanism for the treatment of ischemic HF. Methods Single ventricular myocytes were isolated enzymatically from left ventricle of guinea pig. Peak ICa-L current were recorded using patch clamp techniques in the whole-cell configuration when myocytes had been superfused with normal Tyrode solution, simple ischemic solution, ischemic solution containing phosphocreatine with different concentration for 10 minutes respectively. Results Peak ICa-L current density of myocytes superfused with simple simulated ischemic solution was remarkably inhibited by 80.6 ±5.2% compared with myocytes superfused with normal Tyrode solution(P<0.05). Ischemic solution containing phosphocreatine of 5, 10, 20, 30mmol/L inhibited Peak ICa-L current density by (53.8±6.7)%, (41.8±8.2)%, (38.1±7.4)%, (36.6±9.7)% respectively.There was no statistical significance among phosphocreation of 10, 20, 30 mmol / L. Conclusions Extrogenous phosphocreatine could reverse the inhibition of ICa-L current under ischemic condition,which could be the ionic basis for the treatment of ischemic heart failure. 0~10 mmol/L phosphocreatine exerted significant dose-effect relationship which no longer existed as concentration more than 10 mmol/L.It is supposed that phosphocreatine increased ICa-Lcurrent by many pathways rather than simple substrate for ATP synthesis.

  7. Cellular signaling underlying atrial tachycardia remodeling of L-type calcium current

    NARCIS (Netherlands)

    Qi, Xiao Yan; Yeh, Yung-Hsin; Xiao, Ling; Burstein, Brett; Maguy, Ange; Chartier, Denis; Villeneuve, Louis R.; Brundel, Bianca J. J. M.; Dobrev, Dobromir; Nattel, Stanley

    2008-01-01

    Atrial tachycardia (AT) downregulates L-type Ca2+ current (I-CaL) and causes atrial fibrillation -promoting electric remodeling. This study assessed potential underlying signal transduction. Cultured adult canine atrial cardiomyocytes were paced at 0, 1, or 3 Hz (P0, P1, P3) for up to 24 hours. Cell

  8. Mutation in the α2 isoform of Na,K-ATPase associated Familial Hemiplegic Migraine type 2 (FHM2) leads to elevated contractility and vasodilatation of cerebral arteries in mice

    DEFF Research Database (Denmark)

    Hangaard, Lise; Lykke-Hartmann, Karin; Xie, Zijian;

    is associated with few point mutations in the α2 isoform Na,K-ATPase. Mice bearing a mutation corresponding to the inherited mutation in FHM2 patients (G301R) were used in functional studies of middle cerebral arteries. Middle cerebral arteries from heterozygote G301R mice were not different in total α2 Na...

  9. The Ca2+/H+ antiporter TMEM165 expression, localization in the developing, lactating and involuting mammary gland parallels the secretory pathway Ca2+ATPase (SPCA1)

    Science.gov (United States)

    Plasma membrane Ca2+-ATPase 2 (PMCA2) knockout mice showed that ~ 60 % of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca2+-ATPase’s 1 and/or 2 (SP...

  10. High glucose enhances transient receptor potential channel canonical type 6-dependent calcium influx in human platelets via phosphatidylinositol 3-kinase-dependent pathway

    DEFF Research Database (Denmark)

    Liu, Daoyan; Maier, Alexandra; Scholze, Alexandra;

    2008-01-01

    Transient receptor potential canonical type 6 (TRPC6) channels mediating 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced calcium entry have been identified on human platelets. In the present study we tested the hypothesis that hyperglycemia increases the expression of TRPC6 channels....

  11. Like Extinction, Latent Inhibition of Conditioned Fear in Mice Is Blocked by Systemic Inhibition of L-Type Voltage-Gated Calcium Channels

    Science.gov (United States)

    Blouin, Ashley M.; Cain, Chris K.; Barad, Mike

    2004-01-01

    Having recently shown that extinction of conditioned fear depends on L-type voltage-gated calcium channels (LVGCCs), we have been seeking other protocols that require this unusual induction mechanism. We tested latent inhibition (LI) of fear, because LI resembles extinction except that cue exposures precede, rather than follow, cue-shock pairing.…

  12. The L-Type Voltage-Gated Calcium Channel Ca[subscript v]1.3 Mediates Consolidation, but Not Extinction, of Contextually Conditioned Fear in Mice

    Science.gov (United States)

    McKinney, Brandon C.; Murphy, Geoffrey G.

    2006-01-01

    Using pharmacological techniques, it has been demonstrated that both consolidation and extinction of Pavlovian fear conditioning are dependent to some extent upon L-type voltage-gated calcium channels (LVGCCs). Although these studies have successfully implicated LVGCCs in Pavlovian fear conditioning, they do not provide information about the…

  13. Plasma level of D-dimer accompanying different types of gynecologic surgery and effects of prophylactic subcutaneous injection of heparin calcium

    Directory of Open Access Journals (Sweden)

    Sakika Yanai

    2015-06-01

    Conclusions: Plasma levels of D-dimer on POD-1 were higher than those on the DPE in each type of gynecologic surgery. The D-dimer level remained high even on POD-6, and not changed by prophylactic subcutaneous injection of heparin calcium. [Int J Reprod Contracept Obstet Gynecol 2015; 4(3.000: 551-554

  14. The effects of the calcium-restricted diet of urolithiasis patients with absorptive hypercalciuria type II on risk factors for kidney stones and osteopenia

    NARCIS (Netherlands)

    Faassen, A. van; Ploeg, E.M.C. van der; Habets, H.M.L.; Meer, R. van der; Hermus, R.J.J.; Janknegt, R.A.

    1998-01-01

    The calcium (Ca)-restricted diet of urolithiasis patients with absorptive hypercalciuria type II may decrease Ca excretion but increase biochemical markers of risk for osteopenia. We randomly allocated 25 patients from six hospitals into an experimental group (Ca restriction to 500 mg/day, oxalate-r

  15. A combination of prebiotic short- and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young adolescents

    Science.gov (United States)

    BACKGROUND: Short-term studies in adolescents have generally shown an enhancement of calcium absorption by inulin-type fructans (prebiotics). Results have been inconsistent, however, and no studies have been conducted to determine whether this effect persists with long-term use. OBJECTIVE: The obje...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-02-28

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

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

  19. Cilnidipine, but not amlodipine, ameliorates osteoporosis in ovariectomized hypertensive rats through inhibition of the N-type calcium channel.

    Science.gov (United States)

    Shimizu, Hideo; Nakagami, Hironori; Yasumasa, Natsuki; Mariana, Osako Kiomy; Kyutoku, Mariko; Koriyama, Hiroshi; Nakagami, Futoshi; Shimamura, Munehisa; Rakugi, Hiromi; Morishita, Ryuichi

    2012-01-01

    Both osteoporosis and high blood pressure are major diseases in aging populations. Recent studies demonstrated that some antihypertensive drugs reduced the risk of bone fracture in elderly patients. Although calcium channel blockers (CCB) are widely used as first-line antihypertensive agents, there is no evidence that they prevent osteoporosis. In this study, we investigated the effects of two types of CCB on bone metabolism: cilnidipine (L-/N-type CCB), which suppresses norepinephrine release from the sympathetic nerve, and amlodipine (L-type CCB). In ovariectomized female spontaneous hypertensive rats, administration of cilnidipine, but not amlodipine, resulted in a significant increase in the ratio of alkaline phosphatase to tartrate-resistant acid phosphatase (TRAP) and a decrease in the number of osteoclasts, as assessed by TRAP staining in the proximal tibia. Bone mineral density, moreover, was significantly higher in the cilnidipine group as compared with the amlodipine group and was associated with a significant decrease in a urinary collagen degradation product (deoxypyridinoline). The degree of prevention of osteoporosis by cilnidipine was similar to that of carvedilol (a β-blocker) because β-blockers reduce fracture risks though the inhibition of osteoclast activation. Interestingly, these effects cannot be attributed to the reduction of blood pressure because all three drugs significantly decreased blood pressure. In contrast, both cilnidipine and carvedilol, but not amlodipine, significantly decreased heart rate, indicating that both cilnidipine and carvedilol suppressed sympathetic nervous activity. Overall, our present data showed that cilnidipine (L-/N-type CCB) ameliorated osteoporosis in ovariectomized hypertensive rats. These pleiotropic effects of antihypertensive drugs such as cilnidipine and carvedilol might provide additional benefits in the treatment of hypertensive postmenopausal women.

  20. Inhibition of voltage-gated calcium currents in type II vestibular hair cells by cinnarizine.

    Science.gov (United States)

    Arab, Sonja F; Düwel, Philip; Jüngling, Eberhard; Westhofen, Martin; Lückhoff, Andreas

    2004-06-01

    Cinnarizine is pharmaceutically used in conditions with vestibular vertigo such as Meniere's disease. It is thought to act on extra-vestibular targets. We hypothesized that cinnarizine, as a blocker of L-type Ca2+ channels, may directly target vestibular hair cells where Ca2+ currents are important for the mechano-electrical transduction and transmitter release. Our aim was to clarify whether cinnarizine affected voltage-dependent Ca2+ currents in vestibular type II hair cells. Such cells were isolated from inner ears of guinea pigs by enzymatic and mechanical dissection from the gelatinous otolithic membrane and studied with the patch-clamp technique in conventional whole-cell mode. Ca2+ currents were elicited by depolarizing pulses in a solution containing 1.8 mM Ca2+ and 40 mM Ba2+. These currents resembled L-type currents (I(Ca,L)) with respect to their voltage-dependence and their inhibition by nifedipine and Cd2+ but did not show time-dependent inactivation. The currents were inhibited by cinnarizine in a concentration-dependent and reversible manner. The IC50 was 1.5 microM. A block exceeding 80% was achieved with 10 microM. The onset of current block was faster with higher concentrations but the reversibility after wash-out was less, suggesting accumulation in the membrane. We conclude that these direct actions of cinnarizine on hair cells should be considered as molecular mechanisms contributing to therapeutic effects of cinnarizine in vertigo. PMID:15138660

  1. An extended nomenclature for mammalian V-ATPase subunit genes and splice variants.

    Directory of Open Access Journals (Sweden)

    Kevin C Miranda

    Full Text Available The vacuolar-type H(+-ATPase (V-ATPase is a multisubunit proton pump that is involved in both intra- and extracellular acidification processes throughout the body. Multiple homologs and splice variants of V-ATPase subunits are thought to explain its varied spatial and temporal expression pattern in different cell types. Recently subunit nomenclature was standardized with a total of 22 subunit variants identified. However this standardization did not accommodate the existence of splice variants and is therefore incomplete. Thus, we propose here an extension of subunit nomenclature along with a literature and sequence database scan for additional V-ATPase subunits. An additional 17 variants were pulled from a literature search while 4 uncharacterized potential subunit variants were found in sequence databases. These findings have been integrated with the current V-ATPase knowledge base to create a new V-ATPase subunit catalogue. It is envisioned this catalogue will form a new platform on which future studies into tissue- and organelle-specific V-ATPase expression, localization and function can be based.

  2. Overproduction of PIB-Type ATPases

    DEFF Research Database (Denmark)

    Liu, Xiangyu; Sitsel, Oleg; Wang, Kaituo;

    2016-01-01

    Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction and purifi......Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction...

  3. Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells.

    Science.gov (United States)

    Chan, Chun-Yuan; Dominguez, Dennis; Parra, Karlett J

    2016-07-22

    Yeast 6-phosphofructo-1-kinase (PFK-1) has two subunits, Pfk1p and Pfk2p. Deletion of Pfk2p alters glucose-dependent V-ATPase reassembly and vacuolar acidification (Chan, C. Y., and Parra, K. J. (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J. Biol. Chem. 289, 19448-19457). This study capitalized on the mechanisms suppressing vacuolar H(+)-ATPase (V-ATPase) in pfk2Δ to gain new knowledge of the mechanisms underlying glucose-dependent V-ATPase regulation. Because V-ATPase is fully assembled in pfk2Δ, and glycolysis partially suppressed at steady state, we manipulated glycolysis and assessed its direct involvement on V-ATPase function. At steady state, the ratio of proton transport to ATP hydrolysis increased 24% after increasing the glucose concentration from 2% to 4% to enhance the glycolysis flow in pfk2Δ. Tighter coupling restored vacuolar pH when glucose was abundant and glycolysis operated below capacity. After readdition of glucose to glucose-deprived cells, glucose-dependent V1Vo reassembly was proportional to the glycolysis flow. Readdition of 2% glucose to pfk2Δ cells, which restored 62% of ethanol concentration, led to equivalent 60% V1Vo reassembly levels. Steady-state level of assembly (100% reassembly) was reached at 4% glucose when glycolysis reached a threshold in pfk2Δ (≥40% the wild-type flow). At 4% glucose, the level of Pfk1p co-immunoprecipitated with V-ATPase decreased 58% in pfk2Δ, suggesting that Pfk1p binding to V-ATPase may be inhibitory in the mutant. We concluded that V-ATPase activity at steady state and V-ATPase reassembly after readdition of glucose to glucose-deprived cells are controlled by the glycolysis flow. We propose a new mechanism by which glucose regulates V-ATPase catalytic activity that occurs at steady state without changing V1Vo assembly.

  4. L-type calcium channel blockers enhance 5-HTP-induced antinociception in mice

    Institute of Scientific and Technical Information of China (English)

    Jian-hui LIANG; Jun-xu LI; Xu-hua WANG; Bi CHEN; Ying LU; Pan ZHANG; Rong HAN; Xiang-feng YE

    2004-01-01

    AIM: To investigate the involvement of L-type Ca2+ channels in antinociceptive action induced by the 5-HT precursor,5-hydroxytryptophan (5-HTP). METHODS: Female Kunming mice were treated with either 5-HTP (20-80 mg/kg,ip) alone, or the combination of 5-HTP and fluoxetine (2-8 mg/kg, ip), pargyline (15-60 mg/kg, ip), nimodipine (2.5-10 mg/kg, ip), nifedipine (2.5-10 mg/kg, ip), verapamil (2.5-10 mg/kg, ip), CaC12 (5-20 mmol/L, icv), or EGTA (0.5-3 mmol/L, icv) prior to the hot-plate test (55 ℃, hind-paw licking latency). In addition, locomotor activity in mice treated with 5-HTP alone was measured using an ambulometer with five activity boxes. RESULTS: Ip injection of 5-HTP alone had no influence on the spontaneous locomotor activity, whereas dose-dependently increased the latency to licking hind-paw in the hot-plate test in mice. The inhibitory effects of 5-HTP on nociceptive response were significantly enhanced by fluoxetine in the mouse hot-plate test. At a sub-effective dose, pargyline could cause a leftward shift in the dose-response curve of 5-HTP-induced antinociception. Co-administration with 5-HTP and nimodipine, nifedipine, or verapamil obviously potentiated the antinociceptive effects elicited by 5-HTP.Interestingly, 5-HTP-induced antinociception was antagonized by CaC12 and enhanced by EGTA injected icv in the mouse hot-plate test. CONCLUSION: These findings suggest that systemic administration of 5-HTP may yield the antinociceptive effects, which are related to Ca2+ influx from extracellular fluid through L-type Ca2+ channels.

  5. Anti-proliferative actions of T-type calcium channel inhibition in Thy1 nephritis.

    Science.gov (United States)

    Cove-Smith, Andrea; Mulgrew, Christopher J; Rudyk, Olena; Dutt, Neelanjana; McLatchie, Linda M; Shattock, Michael J; Hendry, Bruce M

    2013-08-01

    Aberrant proliferation of mesangial cells (MCs) is a key finding in progressive glomerular disease. TH1177 is a small molecule that has been shown to inhibit low-voltage activated T-type Ca(2+) channels (TCCs). The current study investigates the effect of TH1177 on MC proliferation in vitro and in vivo. The effect of Ca(2+) channel inhibition on primary rat MC proliferation in vitro was studied using the microculture tetrazolium assay and by measuring bromodeoxyuridine incorporation. In vivo, rats with Thy1 nephritis were treated with TH1177 or vehicle. Glomerular injury and average glomerular cell number were determined in a blinded fashion. Immunostaining for Ki-67 and phosphorylated ERK were also performed. The expression of TCC isoforms in healthy and diseased tissue was investigated using quantitative real-time PCR. TCC blockade caused a significant reduction in rat MC proliferation in vitro, whereas L-type inhibition had no effect. Treatment of Thy1 nephritis with TH1177 significantly reduced glomerular injury (P < 0.005) and caused a 49% reduction in glomerular cell number (P < 0.005) compared to the placebo. TH1177 also reduced Ki-67-positive and pERK-positive cells per glomerulus by 52% (P < 0.01 and P < 0.005, respectively). These results demonstrate that TH1177 inhibits MC proliferation in vitro and in vivo, supporting the hypothesis that TCC inhibition may be a useful strategy for studying and modifying MC proliferative responses to injury. PMID:23746655

  6. Melanopsin Phototransduction Contributes to Light-Evoked Choroidal Expansion and Rod L-Type Calcium Channel Function In Vivo

    Science.gov (United States)

    Berkowitz, Bruce A.; Schmidt, Tiffany; Podolsky, Robert H.; Roberts, Robin

    2016-01-01

    Purpose In humans, rodents, and pigeons, the dark → light transition signals nonretinal brain tissue to increase choroidal thickness, a major control element of choroidal blood flow, and thus of photoreceptor and retinal pigment epithelium function. However, it is unclear which photopigments in the retina relay the light signal to the brain. Here, we test the hypothesis that melanopsin (Opn4)-regulated phototransduction modulates light-evoked choroidal thickness expansion in mice. Methods Two-month-old C57Bl/6 wild-type (B6), 4- to 5-month-old C57Bl/6/129S6 wild-type (B6 + S6), and 2-month-old melanopsin knockout (Opn4−/−) on a B6 + S6 background were studied. Retinal anatomy was evaluated in vivo by optical coherence tomography and MRI. Choroidal thickness in dark and light were measured by diffusion-weighted MRI. Rod cell L-type calcium channel (LTCC) function in dark and light (manganese-enhanced MRI [MEMRI]) was also measured. Results Opn4−/− mice did not show the light-evoked expansion of choroidal thickness observed in B6 and B6 + S6 controls. Additionally, Opn4−/− mice had lower than normal rod cell and inner retinal LTCC function in the dark but not in the light. These deficits were not due to structural abnormalities because retinal laminar architecture and thickness, and choroidal thickness in the Opn4−/− mice were similar to controls. Conclusions First time evidence is provided that melanopsin phototransduction contributes to dark → light control of murine choroidal thickness. The data also highlight a contribution in vivo of melanopsin phototransduction to rod cell and inner retinal depolarization in the dark. PMID:27727394

  7. Modulation pf pulmonary surfactant secretion from alveolar type II cells by cytoplasmic free calcium ([Ca2+]/sub i/)

    International Nuclear Information System (INIS)

    Ca2+ is regulator of a variety of cellular functions including exocytosis. TPA and terbutaline have been shown to stimulate surfactant secretion from alveolar type II cells. The authors examined changes in [Ca2+]/sub i/ and surfactant secretion by secretagogues in primary culture of alveolar type II cells. Cells were isolated from adult rats and were cultured for 24 h with 3H-choline to label phosphatidylcholine. Percent secretion was determined by counting the lipids of cells and medium; cytotoxicity was excluded by measuring lactate dehydrogenase as cells and medium. [Ca2+]/sub i/ was determined by measuring quin2 fluroescence of cells cultured on a glass coverslip. Ionomycin increased secretion as well as [Ca2+] in dose dependent manner at the concentration from 25 to 400 nM. Ionomycin (50 nM) increased terbutaline-induced secretion in a synergistic manner but only increased TPA-induced secretion in an additive manner. Terbutaline mobilized [Ca2+]/sub i/ from intracellular stores and increased [Ca2+]/sub i/ by 20% from a basal level of 140 nM. TPA itself did not change [Ca2+]/sub i/ but inhibited the effect of terbutaline on [Ca2+]/sub i/. Loading of quin2 in the absence of extracellular calcium lowered [Ca2+]/sub i/ from 143 nM to 31 nM. Lowering [Ca2+]/sub i/ inhibited TPA- or terbutaline-induced secretion by 22% and 40% respectively. These results indicate that [Ca2+]/sub i/ effects cAMp-induced secretion more than protein kinase C-mediated secretion in alveolar type II cells

  8. Modulation pf pulmonary surfactant secretion from alveolar type II cells by cytoplasmic free calcium ((Ca/sup 2 +/)/sub i/)

    Energy Technology Data Exchange (ETDEWEB)

    Sano, K.; Voelker, D.R.; Mason, R.J.

    1986-05-01

    Ca/sup 2 +/ is regulator of a variety of cellular functions including exocytosis. TPA and terbutaline have been shown to stimulate surfactant secretion from alveolar type II cells. The authors examined changes in (Ca/sup 2 +/)/sub i/ and surfactant secretion by secretagogues in primary culture of alveolar type II cells. Cells were isolated from adult rats and were cultured for 24 h with /sup 3/H-choline to label phosphatidylcholine. Percent secretion was determined by counting the lipids of cells and medium; cytotoxicity was excluded by measuring lactate dehydrogenase as cells and medium. (Ca/sup 2 +/)/sub i/ was determined by measuring quin2 fluroescence of cells cultured on a glass coverslip. Ionomycin increased secretion as well as (Ca/sup 2 +/) in dose dependent manner at the concentration from 25 to 400 nM. Ionomycin (50 nM) increased terbutaline-induced secretion in a synergistic manner but only increased TPA-induced secretion in an additive manner. Terbutaline mobilized (Ca/sup 2 +/)/sub i/ from intracellular stores and increased (Ca/sup 2 +/)/sub i/ by 20% from a basal level of 140 nM. TPA itself did not change (Ca/sup 2 +/)/sub i/ but inhibited the effect of terbutaline on (Ca/sup 2 +/)/sub i/. Loading of quin2 in the absence of extracellular calcium lowered (Ca/sup 2 +/)/sub i/ from 143 nM to 31 nM. Lowering (Ca/sup 2 +/)/sub i/ inhibited TPA- or terbutaline-induced secretion by 22% and 40% respectively. These results indicate that (Ca/sup 2 +/)/sub i/ effects cAMp-induced secretion more than protein kinase C-mediated secretion in alveolar type II cells.

  9. Altered expression and insulin-induced trafficking of Na+-K+-ATPase in rat skeletal muscle

    DEFF Research Database (Denmark)

    Galuska, Dana; Kotova, Olga; Barres, Romain;

    2009-01-01

    Skeletal muscle Na(+)-K(+)-ATPase plays a central role in the clearance of K(+) from the extracellular fluid, therefore maintaining blood [K(+)]. Na(+)-K(+)-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise...... function precede the development of skeletal muscle insulin resistance. Disturbances in skeletal muscle Na(+)-K(+)-ATPase regulation, particularly the alpha(2)-subunit, may contribute to impaired ion homeostasis in insulin-resistant states such as obesity and type 2 diabetes....

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

    Science.gov (United States)

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

    2016-04-01

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

  11. N-type calcium channel/syntaxin/SNAP-25 complex probed by antibodies to II-III intracellular loop of the α1B subunit

    International Nuclear Information System (INIS)

    Neuronal voltage-dependent calcium channels are integral components of cellular excitation and neurosecretion. In addition to mediating the entry of calcium across the plasma membrane, both N-type and P/Q-type voltage-dependent calcium channels have been shown to form stable complexes with synaptic vesicle and presynaptic membrane proteins, indicating a structural role for the voltage-dependent calcium channels in secretion. Recently, detailed structural analyses of N-type calcium channels have identified residues amino acids 718-963 as the site in the rat α1B subunit that mediates binding to syntaxin, synaptosome-associated protein of 25andpuncsp; omitted000 mol. wt and synaptotagmin [Sheng et al. (1996) Nature 379, 451-454]. The purpose of this study was to employ site-directed antibodies to target domains within and outside of the interaction site on the rat α1B to probe potential binding sites for syntaxin/SNAP-25/synaptotagmin.Our results demonstrate that both antibodies employed in this study have access to their epitopes on the α1B as evidenced by equivalent immunoprecipitation of native [125I]omega-conotoxin GVIA-labeled α1B protein from CHAPS-solubilized preparations. The N-type voltage-dependent calcium channel immunoprecipitated by Ab CW14, the antibody directed to a domain outside of the synprint site, is associated with syntaxin and SNAP-25 with the recovery of these proteins, increasing in parallel to the recovery of α1B. However, when we used the antibody raised to an epitope within the synprint site (Ab CW8) to immunoprecipitate N-type calcium channels, the α1B was depleted of more than 65% of syntaxin and 80% of SNAP-25 when compared to the recovery of these proteins using Ab CW14. This is the first report of a defined epitope on the α1B subunit II-III loop (amino acids 863-875) whose perturbation by a site-directed antibody influences the dissociation of SNAP-25 and syntaxin. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights

  12. Calcium signaling in pancreatic β-cells in health and in Type 2 diabetes.

    Science.gov (United States)

    Gilon, Patrick; Chae, Hee-Young; Rutter, Guy A; Ravier, Magalie A

    2014-11-01

    Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]c) play a crucial role in the control of insulin secretion from the electrically excitable pancreatic β-cell. Secretion is controlled by the finely tuned balance between Ca(2+) influx (mainly through voltage-dependent Ca(2+) channels, but also through voltage-independent Ca(2+) channels like store-operated channels) and efflux pathways. Changes in [Ca(2+)]c directly affect [Ca(2+)] in various organelles including the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, secretory granules and lysosomes, as imaged using recombinant targeted probes. Because most of these organelles have specific Ca(2+) influx and efflux pathways, they mutually influence free [Ca(2+)] in the others. In this article, we review the mechanisms of control of [Ca(2+)] in various compartments and particularly the cytosol, the endoplasmic reticulum ([Ca(2+)]ER), acidic stores and mitochondrial matrix ([Ca(2+)]mito), focusing chiefly on the most important physiological stimulus of β-cells, glucose. We also briefly review some alterations of β-cell Ca(2+) homeostasis in Type 2 diabetes.

  13. Specific inhibition of long-lasting, L-type calcium channels by synthetic parathyroid hormone

    International Nuclear Information System (INIS)

    The effect of an active synthetic N-terminal fragment of bovine parathyroid hormone (bPTH), bPTH-(1-34), on Ca2+ channels was studied in mouse neuroblastoma cells (N1E-115). With the whole-cell variation of the patch-clamp technique, T (transient) and L (long-lasting) types of Ca2+ currents were identified. Pharmacological characterization showed that the L current was amplified by the Ca2+ channel stimulator BAY K-8644, but the T current was unaffected. The administration of bPTH-(1-34) produced dose-related inhibition of the L current, which could be reversed by BAY K-8644. The peptide had no effect on the T current. In addition, use of the fluorescent indicator fura-2 showed that bPTH-(1-34) inhibited the KCl-stimulated increase in intracellular free Ca2+ in neuroblastoma cells with L channels but not in cells with T channels. An inactivated (oxidized) preparation of bPTH-(1-34) failed to affect the L current. High-affinity binding of labeled PTH analog to these neuroblastoma cells was also demonstrated. In addition, bPTH-(1-34) inhibited the L current in cultured vascular smooth muscle cells from rat tail artery. These data indicate that, in some tissues PTH can act as an endogenous blocker of Ca2+ entry

  14. High-Velocity Features of Calcium and Silicon in the Spectra of Type Ia Supernovae

    CERN Document Server

    Silverman, Jeffrey M; Marion, G H; Wheeler, J Craig; Barna, Barnabas; Szalai, Tamas; Mulligan, Brian; Filippenko, Alexei V

    2015-01-01

    "High-velocity features" (HVFs) are spectral features in Type Ia supernovae (SNe Ia) that have minima indicating significantly higher (by greater than about 6000 km/s) velocities than typical "photospheric-velocity features" (PVFs). The PVFs are absorption features with minima indicating typical photospheric (i.e., bulk ejecta) velocities (usually ~9000-15,000 km/s near B-band maximum brightness). In this work we undertake the most in-depth study of HVFs ever performed. The dataset used herein consists of 445 low-resolution optical and near-infrared (NIR) spectra (at epochs up to 5 d past maximum brightness) of 210 low-redshift SNe Ia that follow the "Phillips relation." A series of Gaussian functions is fit to the data in order to characterise possible HVFs of Ca II H&K, Si II {\\lambda}6355, and the Ca II NIR triplet. The temporal evolution of the velocities and strengths of the PVFs and HVFs of these three spectral features is investigated, as are possible correlations with other SN Ia observables. We f...

  15. Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2012-01-01

    ATP7A and ATP7B are copper-transporting P-1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and C

  16. Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase.

    Science.gov (United States)

    Tani, Motohiro; Toume, Moeko

    2015-12-01

    In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group and five types of ceramide; however, the physiological significance of the structural diversity is not fully understood. Here, we report that deletion of vacuolar H+-ATPase (V-ATPase) in yeast causes dramatic alteration of the complex sphingolipid composition, which includes decreases in hydroxylation at the C-4 position of long-chain bases and the C-2 position of fatty acids in the ceramide moiety, decreases in inositol phosphorylceramide (IPC) levels, and increases in mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)2C] levels. V-ATPase-deleted cells exhibited slow growth at pH 7.2, whereas the increase in MIPC levels was significantly enhanced when V-ATPase-deleted cells were incubated at pH 7.2. The protein expression levels of MIPC and M(IP)2C synthases were significantly increased in V-ATPase-deleted cells incubated at pH 7.2. Loss of MIPC synthesis or an increase in the hydroxylation level of the ceramide moiety of sphingolipids on overexpression of Scs7 and Sur2 sphingolipid hydroxylases enhanced the growth defect of V-ATPase-deleted cells at pH 7.2. On the contrary, the growth rate of V-ATPase-deleted cells was moderately increased on the deletion of SCS7 and SUR2. In addition, supersensitivities to Ca2+, Zn2+ and H2O2, which are typical phenotypes of V-ATPase-deleted cells, were enhanced by the loss of MIPC synthesis. These results indicate the possibility that alteration of the complex sphingolipid composition is an adaptation mechanism for a defect of V-ATPase.

  17. Calcium-Driven Folding of RTX Domain β-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts.

    Science.gov (United States)

    Bumba, Ladislav; Masin, Jiri; Macek, Pavel; Wald, Tomas; Motlova, Lucia; Bibova, Ilona; Klimova, Nela; Bednarova, Lucie; Veverka, Vaclav; Kachala, Michael; Svergun, Dmitri I; Barinka, Cyril; Sebo, Peter

    2016-04-01

    Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens.

  18. Hyperpolarization-activated cation and T-type calcium ion channel expression in porcine and human renal pacemaker tissues.

    Science.gov (United States)

    Hurtado, Romulo; Smith, Carl S

    2016-05-01

    Renal pacemaker activity triggers peristaltic upper urinary tract contractions that propel waste from the kidney to the bladder, a process prone to congenital defects that are the leading cause of pediatric kidney failure. Recently, studies have discovered that hyperpolarization-activated cation (HCN) and T-type calcium (TTC) channel conductances underlie murine renal pacemaker activity, setting the origin and frequency and coordinating upper urinary tract peristalsis. Here, we determined whether this ion channel expression is conserved in the porcine and human urinary tracts, which share a distinct multicalyceal anatomy with multiple pacemaker sites. Double chromagenic immunohistochemistry revealed that HCN isoform 3 is highly expressed at the porcine minor calyces, the renal pacemaker tissues, whereas the kidney and urinary tract smooth muscle lacked this HCN expression. Immunofluorescent staining demonstrated that HCN(+) cells are integrated within the porcine calyx smooth muscle, and that they co-express TTC channel isoform Cav3.2. In humans, the anatomic structure of the minor calyx pacemaker was assayed via hematoxylin and eosin analyses, and enabled the visualization of the calyx smooth muscle surrounding adjacent papillae. Strikingly, immunofluorescence revealed that HCN3(+) /Cav3.2(+) cells are also localized to the human minor calyx smooth muscle. Collectively, these data have elucidated a conserved molecular signature of HCN and TTC channel expression in porcine and human calyx pacemaker tissues. These findings provide evidence for the mechanisms that can drive renal pacemaker activity in the multi-calyceal urinary tract, and potential causes of obstructive uropathies. PMID:26805464

  19. Characterisation of marrubenol, a diterpene extracted from Marrubium vulgare, as an L-type calcium channel blocker.

    Science.gov (United States)

    El-Bardai, Sanae; Wibo, Maurice; Hamaide, Marie-Christine; Lyoussi, Badiaa; Quetin-Leclercq, Joelle; Morel, Nicole

    2003-12-01

    1. The objective of the present study was to investigate the mechanism of the relaxant activity of marrubenol, a diterpenoid extracted from Marrubium vulgare. In rat aorta, marrubenol was a more potent inhibitor of the contraction evoked by 100 mM KCl (IC50: 11.8+/-0.3 microM, maximum relaxation: 93+/-0.6%) than of the contraction evoked by noradrenaline (maximum relaxation: 30+/-1.5%). 2. In fura-2-loaded aorta, marrubenol simultaneously inhibited the Ca2+ signal and the contraction evoked by 100 mM KCl, and decreased the quenching rate of fura-2 fluorescence by Mn2+. 3. Patch-clamp data obtained in aortic smooth muscle cells (A7r5) indicated that marrubenol inhibited Ba2+ inward current in a voltage-dependent manner (KD: 8+/-2 and 40+/-6 microM at holding potentials of -50 and -100 mV, respectively). 4. These results showed that marrubenol inhibits smooth muscle contraction by blocking L-type calcium channels. PMID:14597602

  20. Characterisation of marrubenol, a diterpene extracted from Marrubium vulgare, as an L-type calcium channel blocker.

    Science.gov (United States)

    El-Bardai, Sanae; Wibo, Maurice; Hamaide, Marie-Christine; Lyoussi, Badiaa; Quetin-Leclercq, Joelle; Morel, Nicole

    2003-12-01

    1. The objective of the present study was to investigate the mechanism of the relaxant activity of marrubenol, a diterpenoid extracted from Marrubium vulgare. In rat aorta, marrubenol was a more potent inhibitor of the contraction evoked by 100 mM KCl (IC50: 11.8+/-0.3 microM, maximum relaxation: 93+/-0.6%) than of the contraction evoked by noradrenaline (maximum relaxation: 30+/-1.5%). 2. In fura-2-loaded aorta, marrubenol simultaneously inhibited the Ca2+ signal and the contraction evoked by 100 mM KCl, and decreased the quenching rate of fura-2 fluorescence by Mn2+. 3. Patch-clamp data obtained in aortic smooth muscle cells (A7r5) indicated that marrubenol inhibited Ba2+ inward current in a voltage-dependent manner (KD: 8+/-2 and 40+/-6 microM at holding potentials of -50 and -100 mV, respectively). 4. These results showed that marrubenol inhibits smooth muscle contraction by blocking L-type calcium channels.

  1. The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

    Directory of Open Access Journals (Sweden)

    Niv Bachnoff

    2011-01-01

    Full Text Available A PKA consensus phosphorylation site S1928 at the α11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α11.2 or α11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s at the C-tail of α11.2, the pore forming subunit of CaV1.2.

  2. Calcium - urine

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003603.htm Calcium - urine To use the sharing features on this ... enable JavaScript. This test measures the amount of calcium in urine. All cells need calcium in order ...

  3. Calcium supplements

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007477.htm Calcium supplements To use the sharing features on this page, please enable JavaScript. WHO SHOULD TAKE CALCIUM SUPPLEMENTS? Calcium is an important mineral for the ...

  4. Plasma membrane calcium pumps and their emerging roles in cancer

    Institute of Scientific and Technical Information of China (English)

    Sarah; J; Roberts-Thomson; Merril; C; Curry; Gregory; R; Monteith

    2010-01-01

    Alterations in calcium signaling and/or the expression of calcium pumps and channels are an increasingly recognized property of some cancer cells.Alterations in the expression of plasma membrane calcium ATPase(PMCA) isoforms have been reported in a variety of cancer types,including those of breast and colon,with some studies of cancer cell line differentiation identifying specific PMCA isoforms,which may be altered in some cancers.Some studies have also begun to assess levels of PMCA isoforms in clinical tumor samples and to address mechanisms of altered PMCA expression in cancers.Both increases and decreases in PMCA expression have been reported in different cancer types and in many cases these alterations are isoform specific.In this review,we provide an overview of studies investigating the expression of PMCA in cancer and discuss how both the overexpression and reduced expression of a PMCA isoform in a cancer cell could bestow a growth advantage,through augmenting responses to proliferative stimuli or reducing sensitivity to apoptosis.

  5. 钙型卤水净化与真空制盐新工艺途径%Calcium type brine purification and vacuum salt making process approach

    Institute of Scientific and Technical Information of China (English)

    阙勇

    2015-01-01

    Through the brine purification, this paper can provide a new thought for calcium type bittern as raw materials in salt making enterprises by converting the mode of calcium type brine sodium to sulfate type brine. From the view of its production, the saving of steam, water and energy effect is obvious.%通过卤水净化,把钙型卤水转换成芒硝型卤水的生产方式,可以为以钙型卤水为原料的制盐企业提供一条新思路。从其生产情况来看,节汽、节水和节电效果比较明显。

  6. Comparative effect of angiotensin II type I receptor blockers and calcium channel blockers on laboratory parameters in hypertensive patients with type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Nishida Yayoi

    2012-05-01

    Full Text Available Abstract Background Both angiotensin II type I receptor blockers (ARBs and calcium channel blockers (CCBs are widely used antihypertensive drugs. Many clinical studies have demonstrated and compared the organ-protection effects and adverse events of these drugs. However, few large-scale studies have focused on the effect of these drugs as monotherapy on laboratory parameters. We evaluated and compared the effects of ARB and CCB monotherapy on clinical laboratory parameters in patients with concomitant hypertension and type 2 diabetes mellitus. Methods We used data from the Clinical Data Warehouse of Nihon University School of Medicine obtained between Nov 1, 2004 and July 31, 2011, to identify cohorts of new ARB users (n = 601 and propensity-score matched new CCB users (n = 601, with concomitant mild to moderate hypertension and type 2 diabetes mellitus. We used a multivariate-adjusted regression model to adjust for differences between ARB and CCB users, and compared laboratory parameters including serum levels of triglyceride (TG, total cholesterol (TC, non-fasting blood glucose, hemoglobin A1c (HbA1c, sodium, potassium, creatinine, alanine aminotransferase (ALT, aspartate aminotransferase (AST, gamma-glutamyltransferase (GGT, hemoglobin and hematocrit, and white blood cell (WBC, red blood cell (RBC and platelet (PLT counts up to 12 months after the start of ARB or CCB monotherapy. Results We found a significant reduction of serum TC, HbA1c, hemoglobin and hematocrit and RBC count and a significant increase of serum potassium in ARB users, and a reduction of serum TC and hemoglobin in CCB users, from the baseline period to the exposure period. The reductions of RBC count, hemoglobin and hematocrit in ARB users were significantly greater than those in CCB users. The increase of serum potassium in ARB users was significantly greater than that in CCB users. Conclusions Our study suggested that hematological adverse effects and

  7. Calcium fluxes in Hoplosternum littorale (tamoatá exposed to different types of Amazonian waters

    Directory of Open Access Journals (Sweden)

    Bernardo Baldisserotto

    2009-09-01

    Full Text Available Fishes that live in the Amazonian environment may be exposed to several kinds of waters: "black waters", containing high dissolved organic carbon and acidic pH, "white waters", with ten fold higher Ca2+ concentrations than black waters and neutral pH, and "clear waters", with two fold higher Ca2+ concentrations than black waters and also neutral pH. Therefore, the aim of the present study was to analyze Ca2+ fluxes in the facultative air-breather Hoplosternum littorale (tamoatá exposed to different Amazonian waters. Fishes were acclimated in well water (similar to clear water and later placed in individual chambers for Ca2+ fluxes measurements. After 4 h, water from the chambers was replaced by a different type of water. Transfer of tamoatás to ion-poor black or acidic black water resulted in net Ca2+ loss only in the first 2 h of experiment. However, transfer from black or acidic black water to white water led to only net Ca2+ influxes. The results obtained allowed us to conclude that transfer of tamoatás to ion-poor waters (black and acidic black water led to transient net Ca2+ loss, while the amount of Ca2+ in the ion-rich white water seems adequate to prevent Ca2+ loss after transfer. Therefore, transfer of tamoatás between these Amazonian waters does not seem to result in serious Ca2+ disturbance.Os peixes que vivem na Amazônia são expostos a vários tipos de água: águas pretas, contendo grande quantidade de carbono orgânico dissolvido, águas brancas, com concentração de Ca2+ dez vezes maior que as águas pretas e pH neutro, e águas claras, com concentração de Ca2+ duas vezes maior que as águas pretas e pH também neutro. Dessa forma, o objetivo deste trabalho foi analisar o fluxo de Ca2+ no peixe de respiração aérea facultativa Hoplosternum littorale (tamoatá exposto a diferentes tipos de águas amazônicas. Os peixes foram aclimatados em água de poço artesiano (semelhante à água clara e depois colocados

  8. The transport mechanism of bacterial Cu+-ATPases: distinct efflux rates adapted to different function

    OpenAIRE

    Raimunda, Daniel; González-Guerrero, Manuel; Leeber, Blaise W.; Argüello, José M.

    2011-01-01

    Cu+-ATPases play a key role in bacterial Cu+ homeostasis by participating in Cu+ detoxification and cuproprotein assembly. Characterization of Archaeoglobus fulgidus CopA, a model protein within the subfamily of P1B-1 type ATPases, has provided structural and mechanistic details on this group of transporters. Atomic resolution structures of cytoplasmic regulatory metal binding domains (MBDs) and catalytic actuator, phosphorylation, and nucleotide binding domains are available. These, in combi...

  9. Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs*

    OpenAIRE

    Winther, Anne-Marie L.; Liu, Huizhen; Sonntag, Yonathan; Olesen, Claus; le Maire, Marc; Soehoel, Helmer; Olsen, Carl-Erik; Christensen, S. Brøgger; Nissen, Poul; Møller, Jesper V.

    2010-01-01

    Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca2+-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed b...

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Muscarinic cholinergic regulation of L-type calcium channel in heart of embryonic mice at different developmental stages

    Institute of Scientific and Technical Information of China (English)

    Hua-minLIANG; MingTANG; Chang-jinLIU; Hong-yanLUO; Yuan-longSONG; Xin-wuHU; Jiao-yaXI; Lin-linGAO; BinNIE; Su-yunLI; Ling-lingLAI; JuergenHESCHELER

    2004-01-01

    AIM: To investigate the muscarinic regulation of L-type calcium current (ICa-L) during development. METHODS:The whole cell patch-clamp technique was used to record ICa-L in mice embryonic cardiomyocytes at different stages (the early developmental stage, EDS; the intermediate developmental stage, IDS; and the late developmental stage, LDS). Carbachol (CCh) was used to stimulate M-receptor in the embryonic cardiomyocytes of mice.RESULTS: The expression of lCa.L density did not change in different developmental stages (P>0.05). There was no difference in the sensitivity of ICa-L to CCh during development (P>0.05). This inhibitory action of CCh was mediated by inhibition of cyclic AMP since 8-bromo-cAMP completely reversed the muscarinic inhibitory action. IBMX, a non-selective inhibitor of phosphodiesterase (PDE), reversed the inhibitory action of M-receptor on ICa-L current by 71.2 %±9.2% (n=8) and 11.3%±2.5% (n=9) in EDS and LDS respectively. However forskolin, an agonist of adenylyl cyclase (AC), reversed the action of CCh by 14.5%±3.5% (n=5) and 82.7%± 10.4% (n=7) in EDS and LDS respectively. CONCLUSION: The inhibitory action of CCh on lca.L current was mediated in different pathways: in EDS, the inhibitory action of M-receptor on ICa-L channel mainly depended on the stimulation of PDE. However, in LDS, the regulation by M-receptor on lCa.L channel mainly depended on the inactivation of AC.

  12. Circadian profiles in the embryonic chick heart: L-type voltage-gated calcium channels and signaling pathways.

    Science.gov (United States)

    Ko, Michael L; Shi, Liheng; Grushin, Kirill; Nigussie, Fikru; Ko, Gladys Y-P

    2010-10-01

    Circadian clocks exist in the heart tissue and modulate multiple physiological events, from cardiac metabolism to contractile function and expression of circadian oscillator and metabolic-related genes. Ample evidence has demonstrated that there are endogenous circadian oscillators in adult mammalian cardiomyocytes. However, mammalian embryos cannot be entrained independently to light-dark (LD) cycles in vivo without any maternal influence, but circadian genes are well expressed and able to oscillate in embryonic stages. The authors took advantage of using chick embryos that are independent of maternal influences to investigate whether embryonic hearts could be entrained under LD cycles in ovo. The authors found circadian regulation of L-type voltage-gated calcium channels (L-VGCCs), the ion channels responsible for the production of cardiac muscle contraction in embryonic chick hearts. The mRNA levels and protein expression of VGCCα1C and VGCCα1D are under circadian control, and the average L-VGCC current density is significantly larger when cardiomyocytes are recorded during the night than day. The phosphorylation states of several kinases involved in insulin signaling and cardiac metabolism, including extracellular signal-regulated kinase (Erk), stress-activated protein kinase (p38), protein kinase B (Akt), and glycogen synthase kinase-3β (GSK-3β), are also under circadian control. Both Erk and p38 have been implicated in regulating cardiac contractility and in the development of various pathological states, such as cardiac hypertrophy and heart failure. Even though both Erk and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways participate in complex cellular processes regarding physiological or pathological states of cardiomyocytes, the circadian oscillators in the heart regulate these pathways independently, and both pathways contribute to the circadian regulation of L-VGCCs.

  13. G(o) transduces GABAB-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons.

    Science.gov (United States)

    Menon-Johansson, A S; Berrow, N; Dolphin, A C

    1993-11-01

    High-voltage-activated (HVA) calcium channel currents (IBa) were recorded from acutely replated cultured dorsal root ganglion (DRG) neurons. IBa was irreversibly inhibited by 56.9 +/- 2.7% by 1 microM omega-conotoxin-GVIA (omega-CTx-GVIA), whereas the 1,4-dihydropyridine antagonist nicardipine was ineffective. The selective gamma-aminobutyric acidB (GABAB) agonist, (-)-baclofen (50 microM), inhibited the HVA IBa by 30.7 +/- 5.4%. Prior application of omega-CTx-GVIA completely occluded inhibition of the HVA IBa by (-)-baclofen, indicating that in this preparation (-)-baclofen inhibits N-type current. To investigate which G protein subtype was involved, cells were replated in the presence of anti-G protein antisera. Under these conditions the antibodies were shown to enter the cells through transient pores created during the replating procedure. Replating DRGs in the presence of anti-G(o) antiserum, raised against the C-terminal decapeptide of the G alpha o subunit, reduced (-)-baclofen inhibition of the HVA IBa, whereas replating DRGs in the presence of the anti-Gi antiserum did not. Using anti-G alpha o antisera (1:2000) and confocal laser microscopy, G alpha o localisation was investigated in both unreplated and replated neurons. G alpha o immunoreactivity was observed at the plasma membrane, neurites, attachment plaques and perinuclear region, and was particularly pronounced at points of cell-to-cell contact. The plasma membrane G alpha o immunoreactivity was completely blocked by preincubation with the immunising G alpha o undecapeptide (1 microgram.ml-1) for 1 h at 37 degrees C. A similar treatment also blocked recognition of G alpha o in brain membranes on immunoblots.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8309795

  14. Chronic deficit in nitric oxide elicits oxidative stress and augments T-type calcium-channel contribution to vascular tone of rodent arteries and arterioles

    DEFF Research Database (Denmark)

    Howitt, Lauren; Kuo, Ivana Y; Ellis, Anthie;

    2013-01-01

    AIMS: As cardiovascular disease is characterized by reduced nitric oxide bioavailability, our aim was to determine the impact of this change on the mechanism underlying vascular tone of pressurized arteries in vitro and in vivo. METHODS AND RESULTS: We used pressurized cerebral and mesenteric......, by regulating the bioavailability of reactive oxygen species produced by NADPH oxidase. Our data provide evidence for a novel causal link between nitric oxide deficit, oxidative stress, and T-type calcium channel function....

  15. Combined Effect of a Microporous Layer and Type I Collagen Coating on a Biphasic Calcium Phosphate Scaffold for Bone Tissue Engineering

    OpenAIRE

    Mun-Hwan Lee; Changkook You; Kyo-Han Kim

    2015-01-01

    In this study, type I collagen was coated onto unmodified and modified microporous biphasic calcium phosphate (BCP) scaffolds. Surface characterization using a scanning electron microscope (SEM) and a surface goniometer confirmed the modification of the BCP coating. The quantity of the collagen coating was investigated using Sirius Red staining, and quantitative assessment of the collagen coating showed no significant differences between the two groups. MG63 cells were used to evaluate cell p...

  16. Rotary ATPases: models, machine elements and technical specifications.

    Science.gov (United States)

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

    2013-01-01

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

  17. A spider toxin, ω-agatoxin IV A, binds to fixed as well as living tissues: cytochemical visualization of P/Q-type calcium channels.

    Science.gov (United States)

    Nakanishi, Setsuko

    2016-08-01

    ω-Agatoxin IV A, a peptidyl toxin from Agelenopsis aperta venom, selectively binds to voltage-gated P/Q-type calcium channels. ω-Agatoxin IV A has been used as a selective tool in pharmacological and electrophysiological studies. Visualization of P/Q-type calcium channels has previously been accomplished using biotin-conjugated ω-Agatoxin IV A in freshly prepared mouse cerebellar and hippocampal slices (Nakanishi et al, J. Neurosci. Res., 41: , 532, 1995). Here biotinylated ω-agatoxin IV A was applied to transcardially fixed brain slices prepared with various fixatives. ω-Agatoxin IV A did not bind to fixed tissues from P/Q-type calcium channel knockout mice, confirming that binding to normal, fixed tissues was not an artifact. Using transmission electron microscopy, locations of biotinylated ω-agatoxin IV A binding sites visualized with gold-conjugated streptavidin showed a similar pattern to those visualized with antibody. The ability of biotinylated ω-agatoxin IV A to bind to fixed tissue provides a new cytochemical technique to study molecular architecture of synapses. PMID:27095701

  18. Regulation of PKC mediated signaling by calcium during visceral leishmaniasis.

    Science.gov (United States)

    Roy, Nivedita; Chakraborty, Supriya; Paul Chowdhury, Bidisha; Banerjee, Sayantan; Halder, Kuntal; Majumder, Saikat; Majumdar, Subrata; Sen, Parimal C

    2014-01-01

    Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca2+ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis. PMID:25329062

  19. Regulation of PKC mediated signaling by calcium during visceral leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Nivedita Roy

    Full Text Available Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca2+ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA and Sarco/endoplasmic reticulum calcium ATPase (SERCA which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis.

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

    Science.gov (United States)

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

    2006-08-18

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

  1. Urinary calcium to creatinine ratio: a potential marker of secondary hyperparathyroidism in patients with vitamin D-dependent rickets type 1A.

    Science.gov (United States)

    Miyai, Kentaro; Onishi, Toshikazu; Kashimada, Kenichi; Hasegawa, Yukihiro

    2015-01-01

    Patients with vitamin D-dependent rickets type 1A (VDDR1A) are usually treated with alfacalcidol, an analog of vitamin D. Around puberty, an increased dose of alfacalcidol is recommended for these patients to avoid hypocalcemia and secondary hyperparathyroidism. However, no indicators of secondary hyperparathyroidism except for PTH are presently known. The aim of this study is to evaluate whether urinary calcium to creatinine ratio (U-Ca/Cr) is useful as a biomarker of secondary hyperparathyroidism in VDDR1A patients in order to determine the proper dose of alfacalcidol. Two brothers with VDDR1A were recruited who had null mutations of CYP27B1 which encodes 1-alpha-hydroxylase of vitamin D. We investigated the relationship between U-Ca/Cr and intact-PTH around puberty when the brothers showed hypocalcemia with secondary hyperparathyroidism. The results were compared to those of five patients with vitamin D deficiency (VDD). As a result, high intact-PTH levels were observed when U-Ca/Cr decreased to less than 0.1 (mg/mg) in both VDDR1A brothers. This relationship was also observed in the VDD patients. However, it is necessary to take into account body calcium status, either in depletion or in excess, to accurately evaluate the relationship between U-Ca/Cr and secondary hyperparathyroidism. First, low U-Ca/Cr was detected in situations with calcium depletion without hyperparathyroidism in the VDDR1A patients. Second, high U-Ca/Cr with hyperparathyroidism could be detected theoretically in a condition of excess calcium supply. In conclusion, a U-Ca/Cr ratio of less than 0.1 (mg/mg) in VDDR1A patients is useful to accurately evaluate calcium depletion and secondary hyperparathyroidism.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-01-27

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

  3. Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Zhi-ying LIN; Li-min CHEN; Jing ZHANG; Xiao-dong PAN; Yuan-gui ZHU; Qin-yong YE; Hua-pin HUANG; Xiao-chun CHEN

    2012-01-01

    Aim:To investigate the effect of ginsenoside Rb1 on voltage-gated calcium currents in cultured rat hippocampal neurons and the modulatory mechanism.Methods:Cultured hippocampal neurons were prepared from Sprague Dawley rat embryos.Whole-cell configuration of the patchclamp technique was used to record the voltage-gated calcium currents (VGCCs)from the hippocampal neurons,and the effect of Rb1 was examined.Results:Rb1 (2-100 μmol/L)inhibited VGCCs in a concentration-dependent manner,and the current was mostly recovered upon wash-out.The specific L-type Ca2+ channel inhibitor nifedipine (10 μmol/L)occluded Rb1-induced inhibition on VGCCs.Neither the selective N-type Ca2+ channel blocker ω-conotoxin-GVlA (1 μmoVL),nor the selective P/Q-type Ca2+ channel blocker ωo-agatoxin IVA (30 nmol/L)diminished Rb1-sensitive VGCCs.Rb1 induced a leftward shift of the steady-state inactivation curve of Ica to a negative potential without affecting its activation kinetics or reversal potential in the I-V curve.The inhibitory effect of Rb1 was neither abolished by the adenylyl cyclase activator forskolin (10 μmol/L),nor by the PKA inhibitor H-89 (10 μmol/L).Conclusion:Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels,without affecting the N-type or P/Q-type Ca2+ channels in hippocampal neurons,cAMP-PKA signaling pathway is not involved in this effect.

  4. The Role of L- and T-Type Calcium Channels in Local and Remote Calcium Responses in Rat Mesenteric Terminal Arterioles

    DEFF Research Database (Denmark)

    Braunstein, Thomas Hartig; Inoue, Ryuji; Cribbs, Leanne;

    2009-01-01

    Background/Aims: The roles of intercellular communication and T-type versus L-type voltage-dependent Ca(2+) channels (VDCCs) in conducted vasoconstriction to local KCl-induced depolarization were investigated in mesenteric arterioles. Methods: Ratiometric Ca(2+) imaging (R) using Fura-PE3 with...... local (DeltaR = 0.54) and remote (DeltaR = 0.17 at 500 mum) increases in intracellular Ca(2+). Remote Ca(2+) responses were inhibited by the gap junction uncouplers carbenoxolone and palmitoleic acid. Ca(V)1.2, Ca(V)3.1 and Ca(V)3.2 channels were immunolocalized in vascular smooth muscle cells and Ca...... arterioles (at 200-300 mum) using micro-application of VDCC blockers. Conclusion: Both L- and T-type channels mediate Ca(2+) entry during conducted vasoconstriction to local KCl in mesenteric arterioles. However, these channels do not participate in the conduction process per se....

  5. Kinase-Mediated Regulation of P4-ATPases

    DEFF Research Database (Denmark)

    Frøsig, Merethe Mørch

    Abstract Kinase-Mediated Regulation of P4-ATPases Understanding kinase-mediated regulation and designing novel tools to study regulatory proteins of P4-ATPases P4-ATPases play a critical role in the biogenesis of transport vesicles in the secretory and endocytic pathways, and P4-ATPase activity...

  6. Elevated NT-proBNP and coronary calcium score in relation to coronary artery disease in asymptomatic type 2 diabetic patients with elevated urinary albumin excretion rate

    DEFF Research Database (Denmark)

    Reinhard, Henrik; Hansen, Peter R; Persson, Frederik;

    2011-01-01

    Elevated plasma N-terminal (NT)-proBNP levels and coronary calcium score (CCS) not only predicts myocardial ischaemia and coronary artery stenosis but also adverse cardiovascular events and mortality in type 2 diabetic patients with an increased urinary albumin excretion rate (UAER), whereas low...... levels are associated with low frequency of coronary artery disease (CAD) and good prognosis. The underlying causes of poor prognosis in patients with elevated NT-proBNP are not known; thus, we investigated the role of putative asymptomatic CAD in type 2 diabetic patients with UAER >30 mg/24 h and...... elevated P-NT-proBNP and/or CCS....

  7. Elevated NT-proBNP and coronary calcium score in relation to coronary artery disease in asymptomatic type 2 diabetic patients with elevated urinary albumin excretion rate

    DEFF Research Database (Denmark)

    Reinhard, Henrik; Hansen, Peter R; Persson, Frederik;

    2011-01-01

    Elevated plasma N-terminal (NT)-proBNP levels and coronary calcium score (CCS) not only predicts myocardial ischaemia and coronary artery stenosis but also adverse cardiovascular events and mortality in type 2 diabetic patients with an increased urinary albumin excretion rate (UAER), whereas low...... levels are associated with low frequency of coronary artery disease (CAD) and good prognosis. The underlying causes of poor prognosis in patients with elevated NT-proBNP are not known; thus, we investigated the role of putative asymptomatic CAD in type 2 diabetic patients with UAER >30 mg/24 h...... and elevated P-NT-proBNP and/or CCS....

  8. The basidiomycete Ustilago maydis has two plasma membrane H⁺-ATPases related to fungi and plants.

    Science.gov (United States)

    Robles-Martínez, Leobarda; Pardo, Juan Pablo; Miranda, Manuel; Mendez, Tavis L; Matus-Ortega, Macario Genaro; Mendoza-Hernández, Guillermo; Guerra-Sánchez, Guadalupe

    2013-10-01

    The fungal and plant plasma membrane H⁺-ATPases play critical roles in the physiology of yeast, plant and protozoa cells. We identified two genes encoding two plasma membrane H⁺-ATPases in the basidiomycete Ustilago maydis, one protein with higher identity to fungal (um02581) and the other to plant (um01205) H⁺-ATPases. Proton pumping activity was 5-fold higher when cells were grown in minimal medium with ethanol compared to cells cultured in rich YPD medium, but total vanadate-sensitive ATPase activity was the same in both conditions. In contrast, the activity in cells cultured in minimal medium with glucose was 2-fold higher than in YPD or ethanol, implicating mechanisms for the regulation of the plasma membrane ATPase activity in U. maydis. Analysis of gene expression of the H⁺-ATPases from cells grown under different conditions, showed that the transcript expression of um01205 (plant-type) was higher than that of um02581 (fungal-type). The translation of the two proteins was confirmed by mass spectrometry analysis. Unlike baker's yeast and plant H⁺-ATPases, where the activity is increased by a short incubation with glucose or sucrose, respectively, U. maydis H⁺-ATPase activity did not change in response to these sugars. Sequence analysis of the two U. maydis H⁺-ATPases revealed the lack of canonical threonine and serine residues which are targets of protein kinases in Saccharomyces cerevisiae and Arabidopsis thaliana plasma membrane H⁺-ATPases, suggesting that phosphorylation of the U. maydis enzymes occurs at different amino acid residues.

  9. The role of Na(+), K(+)-ATPase in the hypoxic vasoconstriction in isolated rat basilar artery.

    Science.gov (United States)

    Shen, Haitao; Liang, Peng; Qiu, Suhua; Zhang, Bo; Wang, Yongli; Lv, Ping

    2016-06-01

    Hypoxia-induced cerebrovascular dysfunction is a key factor in the occurrence and the development of cerebral ischemia. Na(+), K(+)-ATPase affects the regulation of intracellular Ca(2+) concentration and plays an important role in vascular smooth muscle function. However, the potential role of Na(+), K(+)-ATPase in hypoxia-induced cerebrovascular dysfunction is unknown. In this study, we found that the KCl-induced contraction under hypoxia in rat endothelium-intact basilar arteries is similar to that of denuded arteries, suggesting that hypoxia may cause smooth muscle cell (SMC)-dependent vasoconstriction in the basilar artery. The Na(+), K(+)-ATPase activity of the isolated basilar artery with or without endothelium significantly reduced with prolonged hypoxia. Blocking the Na(+)-Ca(2+) exchanger with Ni(2+) (10(-3)M) or the L-type Ca(2+) channel with nimodipine (10(-8)M) dramatically attenuated KCl-induced contraction under hypoxia. Furthermore, prolonged hypoxia significantly reduced Na(+), K(+)-ATPase activity and increased [Ca(2+)]i in cultured rat basilar artery SMCs. Hypoxia reduced the protein and mRNA expression of the α2 isoform of Na(+), K(+)-ATPase in SMCs in vitro. We used a low concentration of the Na(+), K(+)-ATPase inhibitor ouabain, which possesses a high affinity for the α2 isoform. The contractile response in the rat basilar artery under hypoxia was partly inhibited by ouabain pretreatment. The decreased Na(+), K(+)-ATPase activity in isolated basilar artery and the increased [Ca(2+)]i in SMCs induced by hypoxia were partly inhibited by pretreatment with a low concentration of ouabain. These results suggest that hypoxia may educe Na(+), K(+)-ATPase activity in SMCs through the α2 isoform contributing to vasoconstriction in the rat basilar artery.

  10. Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

    Energy Technology Data Exchange (ETDEWEB)

    Miles, Andrew J. [Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX (United Kingdom); Fedosova, Natalya U. [Department of Biomedicine, Aarhus University, DK-8000 Aarhus (Denmark); Hoffmann, Søren V. [ISA, Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus (Denmark); Wallace, B.A. [Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX (United Kingdom); Esmann, Mikael, E-mail: me@biophys.au.dk [Department of Biomedicine, Aarhus University, DK-8000 Aarhus (Denmark)

    2013-05-31

    Highlights: •Ouabain binding to pig and shark Na,K-ATPase enhances thermal stability. •Ouabain stabilises both membrane-bound and solubilised Na,K-ATPase. •Synchrotron radiation circular dichroism is used for structure determination. •Secondary structure in general is not affected by ouabain binding. •Stabilisation is due to re-arrangement of tertiary structure. -- Abstract: Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.

  11. The role of individual domains and the significance of shedding of ATP6AP2/(prorenin receptor in vacuolar H(+-ATPase biogenesis.

    Directory of Open Access Journals (Sweden)

    Kenichiro Kinouchi

    Full Text Available The ATPase 6 accessory protein 2 (ATP6AP2/(prorenin receptor (PRR is essential for the biogenesis of active vacuolar H(+-ATPase (V-ATPase. Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD and transmembrane domain (TM of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M and causes X-linked mental retardation Hedera type (MRXSH and X-linked parkinsonism with spasticity (XPDS in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

  12. The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left-right asymmetry in zebrafish.

    Science.gov (United States)

    Gokey, Jason J; Dasgupta, Agnik; Amack, Jeffrey D

    2015-11-01

    Asymmetric fluid flows generated by motile cilia in a transient 'organ of asymmetry' are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H(+)-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer's vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures-neuromasts and olfactory placodes-suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry.

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

    Science.gov (United States)

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae. PMID:16350755

  14. V-ATPase as an effective therapeutic target for sarcomas

    International Nuclear Information System (INIS)

    Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment. - Highlights: • Osteosarcoma, rhabdomyosarcoma, and chondrosarcoma survive in acidic microenvironment. • At acidic extracellular pH, sarcoma survival is dependent on V-ATPase expression. • Esomeprazole administration induce a significant dose-dependent toxicity

  15. V-ATPase as an effective therapeutic target for sarcomas

    Energy Technology Data Exchange (ETDEWEB)

    Perut, Francesca, E-mail: francesca.perut@ior.it [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Avnet, Sofia; Fotia, Caterina; Baglìo, Serena Rubina; Salerno, Manuela [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Hosogi, Shigekuni [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kusuzaki, Katsuyuki [Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Baldini, Nicola [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna (Italy)

    2014-01-01

    Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment. - Highlights: • Osteosarcoma, rhabdomyosarcoma, and chondrosarcoma survive in acidic microenvironment. • At acidic extracellular pH, sarcoma survival is dependent on V-ATPase expression. • Esomeprazole administration induce a significant dose-dependent toxicity.

  16. Release of transforming growth factor beta 1 and platelet derived growth factor type AB from canine platelet gels obtained by the tube method and activated with calcium salts

    Directory of Open Access Journals (Sweden)

    RF Silva

    2013-01-01

    Full Text Available The objectives of this study were: 1 to measure the concentrations of transforming growth factor beta 1 (TGF-β1 and platelet-derived growth factor type AB (PDGF-AB in plasma and platelet gel (PG activated with calcium salts (gluconate or chloride in dogs, and 2 to determine correlations between cell results and growth factors (GF concentrations. Blood samples were collected from fourteen Brazilian Fila dogs. EDTA was used to obtain whole blood and plasma while ACD-A solution was used to prepare platelet concentrates (PC. Calcium salts were added to PC to induce their gelification. Platelet and leukocyte count was performed before PC activation. The concentration of growth factors in PG supernatants and plasma was determined by ELISA. Statistically significant differences (P < 0.01 between platelet and leukocyte count were observed when comparing whole blood and PC. No statistically significant differences were found between the concentrations of TGF-β1 and PDGF-AB in PC and plasma according to the calcium salt used for the activation of PC. The TGF-β1 concentration was highly correlated with the number of platelets concentrated in the PC. This methodology was useful for producing PG with therapeutic potential for canine regenerative medicine.

  17. The effect of two types chewing gum containing casein phosphopeptide-amorphous calcium phosphate and xylitol on salivary Streptococcus mutans

    OpenAIRE

    Shila Emamieh; Yosra Khaterizadeh; Hossein Goudarzi; Amir Ghasemi; Alireza Akbarzadeh Baghban; Hasan Torabzadeh

    2015-01-01

    Aim: The aim was to evaluate the effect of sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and xylitol on salivary Streptococcus mutans. Materials and Methods: A total of 60 dental students of 20-25 years old, who volunteered after checking their health condition and signing an informed consent, were randomly allocated to receive one of the following interventions: (A) Chewing gum containing CPP-ACP; (B) containing xylitol. Subjects within the ...

  18. N- and L-Type Voltage-Gated Calcium Channels Mediate Fast Calcium Transients in Axonal Shafts of Mouse Peripheral Nerve.

    Science.gov (United States)

    Barzan, Ruxandra; Pfeiffer, Friederike; Kukley, Maria

    2016-01-01

    In the peripheral nervous system (PNS) a vast number of axons are accommodated within fiber bundles that constitute peripheral nerves. A major function of peripheral axons is to propagate action potentials along their length, and hence they are equipped with Na(+) and K(+) channels, which ensure successful generation, conduction and termination of each action potential. However little is known about Ca(2+) ion channels expressed along peripheral axons and their possible functional significance. The goal of the present study was to test whether voltage-gated Ca(2+) channels (VGCCs) are present along peripheral nerve axons in situ and mediate rapid activity-dependent Ca(2+) elevations under physiological circumstances. To address this question we used mouse sciatic nerve slices, Ca(2+) indicator Oregon Green BAPTA-1, and 2-photon Ca(2+) imaging in fast line scan mode (500 Hz). We report that transient increases in intra-axonal Ca(2+) concentration take place along peripheral nerve axons in situ when axons are stimulated electrically with single pulses. Furthermore, we show for the first time that Ca(2+) transients in peripheral nerves are fast, i.e., occur in a millisecond time-domain. Combining Ca(2+) imaging and pharmacology with specific blockers of different VGCCs subtypes we demonstrate that Ca(2+) transients in peripheral nerves are mediated mainly by N-type and L-type VGCCs. Discovery of fast Ca(2+) entry into the axonal shafts through VGCCs in peripheral nerves suggests that Ca(2+) may be involved in regulation of action potential propagation and/or properties in this system, or mediate neurotransmitter release along peripheral axons as it occurs in the optic nerve and white matter of the central nervous system (CNS).

  19. Effects of low-dose ionising radiation on pituitary adenoma: is there a role for L-type calcium channel?

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Marcella Araugio; Santos, Raquel Gouvea dos [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia]. E-mail: santosr@cdtn.br

    2005-10-15

    Pituitary adenomas constitute about 6-18% of brain tumours in adults. Activation of voltage gated calcium currents can account for growth hormone over secretion in some GH-secreting pituitary adenomas that produce an acromegaly appearance and increase mortality. Ca{sup 2+} ions, as mediators of intracellular signalling, are crucial for the development of apoptosis. However, the role of [Ca{sup 2+}] in the development of apoptosis is ambiguous. In this study, the effects of low-dose ionising gamma radiation ({sup 60} Co) on rat pituitary adenoma cells survival and proliferation and the role of calcium channels on the apoptosis radio-induced were evaluated. Doses as low as 3 Gy were found to inhibit GH3 cell proliferation. Even though there was a significant number of live cells,168 hours following irradiation, they were not able to proliferate. The results indicate that the blockade of extracellular calcium influx through these channels does not interfere in the radiation-induced apoptosis in GH3 cells. (author)

  20. L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

    Science.gov (United States)

    Maddala, Rupalatha; Nagendran, Tharkika; de Ridder, Gustaaf G; Schey, Kevin L; Rao, Ponugoti Vasantha

    2013-01-01

    Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial

  1. L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

    Directory of Open Access Journals (Sweden)

    Rupalatha Maddala

    Full Text Available Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations

  2. A pivotal role of vacuolar H+-ATPase in regulation of lipid production in Phaeodactylum tricornutum

    Science.gov (United States)

    Zhang, Huiying; Zeng, Rensen; Chen, Daoyi; Liu, Jian

    2016-01-01

    Microalgal lipids have been considered as a promising source for biodiesel production. Alkaline pH can induce neutral lipid accumulation in microalgae cells. However, whether and how proton pumps, especially vacuolar H+-ATPase (V-ATPase), function in these processes is not well known. In this study, we treated Phaeodactylum tricornutum with V-ATPase specific inhibitor bafilomycin A1 (BFA1) to determine its role in lipid production. Firstly, V-ATPase activity was increased in the latter phase of microalgae growth. BFA1 treatment decreased the cell density and lipid contents. Further analysis showed that BFA1 treatment reduced the number and size of oil bodies. GC-MS analysis showed that lipid components were not affected by BFA1 treatment. Intracellular pH was decreased and nitrogen depletion was delayed after BFA1 treatment. RNA-Seq analysis showed that expression of genes involved in calcium signaling, sulfur metabolism, cell cycle, glycolysis, pentose phosphate pathway, porphyrin, chlorophyll metabolism and lipid catabolic metabolism were upregulated, while expression of genes involved in ion transmembrane transport, ubiquitin mediated proteolysis, SNARE interactions in vesicular transport, fatty acid biosynthesis were downregulated under BFA1 treatment. Our findings provided insights into the molecular mechanisms underlying lipid accumulation and the key genes involved in lipid metabolism in Phaeodactylum tricornutum in response to BFA1. PMID:27499168

  3. A pivotal role of vacuolar H(+)-ATPase in regulation of lipid production in Phaeodactylum tricornutum.

    Science.gov (United States)

    Zhang, Huiying; Zeng, Rensen; Chen, Daoyi; Liu, Jian

    2016-01-01

    Microalgal lipids have been considered as a promising source for biodiesel production. Alkaline pH can induce neutral lipid accumulation in microalgae cells. However, whether and how proton pumps, especially vacuolar H(+)-ATPase (V-ATPase), function in these processes is not well known. In this study, we treated Phaeodactylum tricornutum with V-ATPase specific inhibitor bafilomycin A1 (BFA1) to determine its role in lipid production. Firstly, V-ATPase activity was increased in the latter phase of microalgae growth. BFA1 treatment decreased the cell density and lipid contents. Further analysis showed that BFA1 treatment reduced the number and size of oil bodies. GC-MS analysis showed that lipid components were not affected by BFA1 treatment. Intracellular pH was decreased and nitrogen depletion was delayed after BFA1 treatment. RNA-Seq analysis showed that expression of genes involved in calcium signaling, sulfur metabolism, cell cycle, glycolysis, pentose phosphate pathway, porphyrin, chlorophyll metabolism and lipid catabolic metabolism were upregulated, while expression of genes involved in ion transmembrane transport, ubiquitin mediated proteolysis, SNARE interactions in vesicular transport, fatty acid biosynthesis were downregulated under BFA1 treatment. Our findings provided insights into the molecular mechanisms underlying lipid accumulation and the key genes involved in lipid metabolism in Phaeodactylum tricornutum in response to BFA1. PMID:27499168

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

    Institute of Scientific and Technical Information of China (English)

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

    2010-01-01

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

  5. Cdc50p plays a vital role in the ATPase reaction cycle of the putative aminophospholipid transporter Drs2p.

    Science.gov (United States)

    Lenoir, Guillaume; Williamson, Patrick; Puts, Catheleyne F; Holthuis, Joost C M

    2009-07-01

    Members of the P(4) subfamily of P-type ATPases are believed to catalyze transport of phospholipids across cellular bilayers. However, most P-type ATPases pump small cations or metal ions, and atomic structures revealed a transport mechanism that is conserved throughout the family. Hence, a challenging problem is to understand how this mechanism is adapted in P(4)-ATPases to flip phospholipids. P(4)-ATPases form heteromeric complexes with Cdc50 proteins. The primary role of these additional polypeptides is unknown. Here, we show that the affinity of yeast P(4)-ATPase Drs2p for its Cdc50-binding partner fluctuates during the transport cycle, with the strongest interaction occurring at a point where the enzyme is loaded with phospholipid ligand. We also find that specific interactions with Cdc50p are required to render the ATPase competent for phosphorylation at the catalytically important aspartate residue. Our data indicate that Cdc50 proteins are integral components of the P(4)-ATPase transport machinery. Thus, acquisition of these subunits may have been a crucial step in the evolution of flippases from a family of cation pumps. PMID:19411703

  6. The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid subunits

    Science.gov (United States)

    Hilario, E.; Gogarten, J. P.

    1998-01-01

    Changes in the primary and quarternary structure of vacuolar and archaeal type ATPases that accompany the prokaryote-to-eukaryote transition are analyzed. The gene encoding the vacuolar-type proteolipid of the V-ATPase from Giardia lamblia is reported. Giardia has a typical vacuolar ATPase as observed from the common motifs shared between its proteolipid subunit and other eukaryotic vacuolar ATPases, suggesting that the former enzyme works as a hydrolase in this primitive eukaryote. The phylogenetic analyses of the V-ATPase catalytic subunit and the front and back halves of the proteolipid subunit placed Giardia as the deepest branch within the eukaryotes. Our phylogenetic analysis indicated that at least two independent duplication and fusion events gave rise to the larger proteolipid type found in eukaryotes and in Methanococcus. The spatial distribution of the conserved residues among the vacuolar-type proteolipids suggest a zipper-type interaction among the transmembrane helices and surrounding subunits of the V-ATPase complex. Important residues involved in the function of the F-ATP synthase proteolipid have been replaced during evolution in the V-proteolipid, but in some cases retained in the archaeal A-ATPase. Their possible implication in the evolution of V/F/A-ATPases is discussed.

  7. Dietary Calcium and Dairy Modulation of Oxidative Stress and Mortality in aP2-Agouti and Wild-type Mice

    OpenAIRE

    Antje Bruckbauer; Zemel, Michael B

    2009-01-01

    Oxidative and inflammatory stress have been implicated as major contributors to the aging process. Dietary Ca reduced both factors in short-term interventions, while milk exerted a greater effect than supplemental Ca. In this work, we examined the effects of life-long supplemental and dairy calcium on lifespan and life-span related biomarkers in aP2-agouti transgenic (model of diet-induced obesity) and wild-type mice fed obesigenic diets until their death. These data demonstrate that dairy Ca...

  8. To evaluate the levels of glycated hemoglobin, serum calcium, magnesium, phosphate, uric acid and microalbuminuria in patients with newly diagnosed type 2 diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Qazi Najeeb

    2014-08-01

    Conclusion: There is decrease in serum calcium, magnesium and phosphate levels, all these plays an important role in the regulation of glucose level in the blood. Hence oral supplementation of all these ions other than diet is recommended. Increased serum uric acid and microalbuminuria was seen with reduced glucose tolerance hence early estimation of both the parameters should be done while monitoring case of Type-2 diabetes and thus will help to decrease the incidence of renal complications. [Int J Res Med Sci 2014; 2(4.000: 1462-1465

  9. Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed

    2008-01-01

    . Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na+ cycle from an Na+/K+ cycle in the pump. The Na+ cycle possibly involves transport through the recently characterized Na+-specific site...

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

    Science.gov (United States)

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

    2016-08-01

    V-ATPases are ATP-driven proton pumps that function within both intracellular compartments and the plasma membrane in a wide array of normal physiological and pathophysiological processes. V-ATPases are composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that transports protons. Regulated assembly of the V-ATPase represents an important mechanism of regulating V-ATPase activity in response to a number of environmental cues. Our laboratory has demonstrated that glucose-dependent assembly of the V-ATPase complex in yeast is controlled by the Ras/cAMP/PKA pathway. By contrast, increased assembly of the V-ATPase during dendritic cell maturation involves the PI-3 kinase and mTORC1 pathways. Recently, we have shown that amino acids regulate V-ATPase assembly in mammalian cells, possibly as a means to maintain adequate levels of amino acids upon nutrient starvation. V-ATPases have also been implicated in cancer cell survival and invasion. V-ATPases are targeted to different cellular membranes by isoforms of subunit a, with a3 targeting V-ATPases to the plasma membrane of osteoclasts. We have shown that highly invasive human breast cancer cell lines express higher levels of the a3 isoform than poorly invasive lines and that knockdown of a3 reduces both expression of V-ATPases at the plasma membrane and in vitro invasion of breast tumor cells. Moreover, overexpression of a3 in a non-invasive breast epithelial line increases both plasma membrane V-ATPases and in vitro invasion. Finally, specific ablation of plasma membrane V-ATPases in highly invasive human breast cancer cells using either an antibody or small molecule approach inhibits both in vitro invasion and migration. These results suggest that plasma membrane and a3-containing V-ATPases represent a novel and important target in the development of therapeutics to limit breast cancer metastasis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics

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

    Science.gov (United States)

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

    2016-08-01

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

  12. Clusterin (Apolipoprotein J), a Molecular Chaperone That Facilitates Degradation of the Copper-ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2011-01-01

    The copper-transporting P1B-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mut

  13. C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells

    DEFF Research Database (Denmark)

    Galuska, Dana; Pirkmajer, Sergej; Barres, Romain;

    2011-01-01

    Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in...

  14. Secondary structure of the intact H+,K+ -ATPase and of its membrane-embedded region. An attenuated total reflection infrared spectroscopy, circular dichroism and Raman spectroscopy study

    NARCIS (Netherlands)

    Raussens, V.; Jongh, H. de; Pézolet, M.; Ruysschaert, J.-M.; Goormaghtigh, E.

    1998-01-01

    Models of P-type ATPase predict that membrane-embedded fragments represent about 20% of the protein and adopt an all-α-helical structure. While this prediction was confirmed for the Ca2+ -ATPase [Corbalan-Garcia, S., Teruel, J., Villalain, J. and Gomez-Fernandez, J. (1994) Biochemistry 33, 8247-8254

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

    Science.gov (United States)

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

    2014-05-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

    Objective Overexpression of SERCA2a could improve cardiac function in human and experimental heart failure(HF)models.We observed the proteomics changes post SERCA2a overexpression in a pacing induced HF model in dogs.Methods Beagles were divided into four groups:control group,HF group(230 beats/min for 4 weeks),HF+EGFP group(myocardial injection of 1 × 1012 v.g recombinant adeno-associated virus carrying enhanced green fluorescent protein gene,rAAV2/1-EGFP)and HF+ SERCA2a group ( myocardial injection of 1 × 1012 v.g recombinant adeno-associated virus carrying SERCA2a gene,rAAV2/1-SERCA2a).Thirty days after gene transduction,left ventficular systolic and diastolic functions were measured by echoeardiography and invasive hemodynamics in all animals.By use of 2-dimensional gel electrophoresis(2-DE),-500 distinct protein spots were detected in myocardium of all animals.Protein spots observed to be altered between failing and SERCA2a overexpressed hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis.Results At 30 day after gene transfer,HF signs were significantly reduced,cardiac function[LVSP:(214.72±31.74)mm Hg(1 mm Hg=0.133 kPa)vs.(139.32±36.79)mm Hg,+dp/dtmax:(6779.43±217.58)mm Hg/s vs.(2746.85±931.23)mm Hg/s and -dp/dtmax:(-4341.42±322.02)mm Hg/s vs.(r-2531.14 ±616.15)mm Hg/s,LVEDP:(21.86±6.95)mm Hg vs.(59.78±6.92)mm Hg]significantly improved in HF+SERCA2a dogs than those in HF+ EGFP group(all P<0.05)and parameters were comparable between HF+SERCA2a and control groups.We identified alterations in the expression level of more than 10 proteins in myocardium.These protein changes were observed mainly in two subcellular compartments:the cardiac contractile apparatus and metabolism/energetics.Conclusion These results showed that overexpression of SERCA2a could improve cardiac function accompanied with numerous alterations in protein expressions involved in calcium

  17. Altered Calcium Handling in Reperfusion Injury.

    Science.gov (United States)

    Bompotis, Georgios C; Deftereos, Spyridon; Angelidis, Christos; Choidis, Efthymios; Panagopoulou, Vasiliki; Kaoukis, Andreas; Vassilikos, Vassilios P; Cleman, Michael W; Giannopoulos, Georgios

    2016-01-01

    Coronary Heart Disease (CHD) is the major mortality cause in the Western Hemisphere. Reinstituting blood flow in the acutely occluded coronary vessel became the standard intervention to prevent Myocardial Infarct (MI) progression. Ever since their conception, thrombolysis, Percutaneous Coronary Intervention (PCI) and Coronary Artery Bypass Grafting (CABG) have been at the forefront of CHD treatment, limiting MI size. However, it quickly became apparent that after a period of ischemia, reperfusion itself sets off a cascade of events leading to cell injury. It seems that cellular changes in the ischemic period, prime the cell for a loss of homeostasis once blood flow returns. Loss of calcium (Ca(2+)) regulation has been found to be a main culprit in both ischemia and reperfusion. Indeed, sarcoplasmic Ca(2+) overload during reperfusion is related to hypercontracture, proteolysis and mitochondrial failure--the so-called Reperfusion Injury (RI). Ca(2+) channels of the sarcolemma (SL) (L-Type Ca((2+)) Channels, Sodium / Calcium Exchanger) initiate Ca(2+) flux and those of the Sarcoplasmic Reticulum (SR) (Ca(2+) ATPase, Ca(2+) release channel) sustain the rise in intracellular Ca(2+) concentration. Ensuing interplay between Ca(2+), SR, mitochondria, myofilaments and proteolytic cascades i.e. calpain activation, results in cell injury. Novel insight about this interplay and details about the extent by which each of these players contributes to the RI, may allow scientists to devise and design proper interventions that ultimately reduce RI in clinical practice. The present article reviews the literature about key subcellular players participating in the sustained rise of cardiac myocyte cytosolic Ca(2+) during ischemia and reperfusion.

  18. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Guo, Yan; Cuin, Tracey A.;

    2007-01-01

    Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report that an ...... an increase in the concentration of cytosolic-free calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM Hþ-ATPase regulation....

  19. Statin, Calcium Channel Blocker and Beta Blocker Therapy May Decrease the Incidence of Tuberculosis Infection in Elderly Taiwanese Patients with Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Mei-Yueh Lee

    2015-05-01

    Full Text Available Background: It is well known that diabetes mellitus impairs immunity and therefore is an independent risk factor for tuberculosis. However, the influence of associated metabolic factors, such as hypertension, dyslipidemia and gout has yet to be confirmed. This study aimed to investigate whether the strong association between tuberculosis and diabetes mellitus is independent from the influence of hypertension and dyslipidemia, and its treatment in elderly Taiwanese patients. Methods: A total of 27,958 patients aged more than 65 years were identified from the National Health Insurance Research Database (NIHRD in 1997 and were followed from 1998 to 2009. The demographic characteristics between the patients with and without diabetes were analyzed using the χ2 test. A total of 13,981 patients with type 2 diabetes were included in this study. Cox proportional hazard regression models were used to determine the independent effects of diabetes on the risk of tuberculosis. Results: After adjusting for age, sex, other co-morbidities and medications, calcium channel blocker, beta blocker and statin users had a lower independent association, with risk ratios of 0.76 (95% CI, 0.58–0.98, 0.72 (95% CI, 0.58–0.91 and 0.76 (95% CI, 0.60–0.97, respectively. Conclusion: Calcium channel blocker, beta blocker and statin therapy may decrease the incidence of tuberculosis infection in elderly Taiwanese patients with type 2 diabetes.

  20. Newinsightsintostore-independentCa21entry:secretory pathwaycalciumATPase2innormalphysiologyandcancer

    Institute of Scientific and Technical Information of China (English)

    Ming-Ye Feng; Rajini Rao

    2013-01-01

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

  1. DNA binding to SMC ATPases-trapped for release.

    Science.gov (United States)

    Schüler, Herwig; Sjögren, Camilla

    2016-04-01

    The SMC/Rad50/RecN proteins are universal DNA‐associated ABC‐type ATPases with crucial functions in genome maintenance. New insights into Rad50-DNA complex structure and cohesin regulation inspire a speculative look at the entire superfamily. Identification of a continuous DNA binding site across the Rad50 dimer interface (Liu et al, 2016; Seifert et al, 2016) suggests a similar site in cohesin. The localization of this site hints a DNA-activated mechanism for cohesin removal from chromosomes.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-04-09

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

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

    Science.gov (United States)

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

    2005-01-01

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

  5. Combined Effect of a Microporous Layer and Type I Collagen Coating on a Biphasic Calcium Phosphate Scaffold for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Mun-Hwan Lee

    2015-03-01

    Full Text Available In this study, type I collagen was coated onto unmodified and modified microporous biphasic calcium phosphate (BCP scaffolds. Surface characterization using a scanning electron microscope (SEM and a surface goniometer confirmed the modification of the BCP coating. The quantity of the collagen coating was investigated using Sirius Red staining, and quantitative assessment of the collagen coating showed no significant differences between the two groups. MG63 cells were used to evaluate cell proliferation and ALP activity on the modified BCP scaffolds. The modified microporous surfaces showed low contact angles and large surface areas, which enhanced cell spreading and proliferation. Coating of the BCP scaffolds with type I collagen led to enhanced cell-material interactions and improved MG63 functions, such as spreading, proliferation, and differentiation. The micropore/collagen-coated scaffold showed the highest rate of cell response. These results indicate that a combination of micropores and collagen enhances cellular function on bioengineered bone allograft tissue.

  6. Dietary Calcium and Dairy Modulation of Oxidative Stress and Mortality in aP2-Agouti and Wild-type Mice

    Directory of Open Access Journals (Sweden)

    Antje Bruckbauer

    2009-08-01

    Full Text Available Oxidative and inflammatory stress have been implicated as major contributors to the aging process. Dietary Ca reduced both factors in short-term interventions, while milk exerted a greater effect than supplemental Ca. In this work, we examined the effects of life-long supplemental and dairy calcium on lifespan and life-span related biomarkers in aP2-agouti transgenic (model of diet-induced obesity and wild-type mice fed obesigenic diets until their death. These data demonstrate that dairy Ca exerts sustained effects resulting in attenuated adiposity, protection against age-related muscle loss and reduction of oxidative and inflammatory stress in both mouse strains. Although these effects did not alter maximum lifespan, they did suppress early mortality in wild-type mice, but not in aP2-agouti transgenic mice.

  7. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

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

  8. [3H]TA-3090, a selective benzothiazepine-type calcium channel receptor antagonist: In vitro characterization

    International Nuclear Information System (INIS)

    Binding of the new benzothiazepine calcium channel blocker, (+)-(2S,3S)-3-acetoxy-8-chloro-5-(2-(dimethylamino)ethyl)-2,3-dihydro-2- (4- methoxyphenyl)-1,5-benzothiazepine-4-(5H)-one maleate, [3H]TA-3090, was characterized and its specificity for rat myocardial benzothiazepine receptors described. Scatchard plots and nonlinear regression analysis of specific [3H]TA-3090 binding best fit a one-site binding model (Kd = 8.8 +/- 2.7 nM, Bmax = 132 +/- 38 fmol/mg protein). Kinetically derived affinity constants were in close agreement (Kd = 7.86 nM) with those obtained from analysis of equilibrium binding data. In comparison, under identical conditions [3H]diltiazem exhibited a Kd of 38 nM and Bmax, 106 fmol/mg protein. Specific binding was saturable, reversible and stereoselective (d-cis-TA-3090 Ki = 14 nM; 1-cis-TA-3090 Ki = 2700 nM). Competitions for [3H]TA-3090 binding were conducted with nifedipine, propranolol, prazosin, quinuclidinyl benzilate, verapamil and yohimbine. Only the calcium channel blockers nifedipine and verapamil inhibited specific [3H]TA-3090 binding. Nifedipine could maximally inhibit only 52% of specifically bound [3H]TA-3090 at 10 microM. In contrast, however, 10 microM verapamil completely inhibited specific radioligand binding (Ki = 93 +/- 28 nM) but with six times less efficacy than TA-3090. Thus, these data demonstrate that [3H]TA-3090 is a potent radioligand selective for the benzothiazepine binding site and is consistent with the hypothesis that [3H]TA-3090 interacts with a myocardial benzothiazepine receptor site

  9. Effect of ionizing radiation on catalytic properties of Ca2+-ATP-ase from sarcoplasmic reticulum of skeletal muscle

    International Nuclear Information System (INIS)

    It was studied kinetic and thermodynamic characteristics of Ca2+-ATP-ase of rat skeletal muscle (membranes of sarcoplasmic reticulum) after irradiation in doses 0,5, 4,0 and 8,0 Gy. It was shown that external gamma-irradiation at different doses changed kinetic and thermodynamic characteristics of the enzyme of sarcoplasmic reticulum membranes of skeletal muscle. These alterations probably correlate with disbalance of hormonal regulation of intracellular calcium metabolism and changes in membrane structure and functions

  10. Effect of ionizing radiation on catalytic properties of Ca2+-ATPase from sarcoplasmic reticulum of skeletal muscle

    International Nuclear Information System (INIS)

    It was studied kinetic and thermodynamic characteristics of Ca2+-ATPase of rat skeletal muscle (membranes of sarcoplasmic reticulum) after irradiation in doses 0,5, 4,0 and 8,0 Gy. It was shown that external gamma-irradiation at different doses changed kinetic and thermodynamic characteristics of the enzyme of sarcoplasmic reticulum membranes of skeletal muscle. These alterations probably correlate with dis balance of hormonal regulation of intracellular calcium metabolism and changes in membrane structure and functions

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  12. Mutations in the Gene Encoding the Calcium-Permeable Ion Channel TRPV4 Produce Spondylometaphyseal Dysplasia, Kozlowski Type and Metatropic Dysplasia

    Science.gov (United States)

    Krakow, Deborah; Vriens, Joris; Camacho, Natalia; Luong, Phi; Deixler, Hannah; Funari, Tara L.; Bacino, Carlos A.; Irons, Mira B.; Holm, Ingrid A.; Sadler, Laurie; Okenfuss, Ericka B.; Janssens, Annelies; Voets, Thomas; Rimoin, David L.; Lachman, Ralph S.; Nilius, Bernd; Cohn, Daniel H.

    2009-01-01

    The spondylometaphyseal dysplasias (SMDs) are a group of short-stature disorders distinguished by abnormalities in the vertebrae and the metaphyses of the tubular bones. SMD Kozlowski type (SMDK) is a well-defined autosomal-dominant SMD characterized by significant scoliosis and mild metaphyseal abnormalities in the pelvis. The vertebrae exhibit platyspondyly and overfaced pedicles similar to autosomal-dominant brachyolmia, which can result from heterozygosity for activating mutations in the gene encoding TRPV4, a calcium-permeable ion channel. Mutation analysis in six out of six patients with SMDK demonstrated heterozygosity for missense mutations in TRPV4, and one mutation, predicting a R594H substitution, was recurrent in four patients. Similar to autosomal-dominant brachyolmia, the mutations altered basal calcium channel activity in vitro. Metatropic dysplasia is another SMD that has been proposed to have both clinical and genetic heterogeneity. Patients with the nonlethal form of metatropic dysplasia present with a progressive scoliosis, widespread metaphyseal involvement of the appendicular skeleton, and carpal ossification delay. Because of some similar radiographic features between SMDK and metatropic dysplasia, TRPV4 was tested as a disease gene for nonlethal metatropic dysplasia. In two sporadic cases, heterozygosity for de novo missense mutations in TRPV4 was found. The findings demonstrate that mutations in TRPV4 produce a phenotypic spectrum of skeletal dysplasias from the mild autosomal-dominant brachyolmia to SMDK to autosomal-dominant metatropic dysplasia, suggesting that these disorders should be grouped into a new bone dysplasia family. PMID:19232556

  13. Preventing effect of L-type calcium channel blockade on electrophysiological alterations in dentate gyrus granule cells induced by entorhinal amyloid pathology.

    Directory of Open Access Journals (Sweden)

    Hamid Gholami Pourbadie

    Full Text Available The entorhinal cortex (EC is one of the earliest affected brain regions in Alzheimer's disease (AD. EC-amyloid pathology induces synaptic failure in the dentate gyrus (DG with resultant behavioral impairment, but there is little known about its impact on neuronal properties in the DG. It is believed that calcium dyshomeostasis plays a pivotal role in the etiology of AD. Here, the effect of the EC amyloid pathogenesis on cellular properties of DG granule cells and also possible neuroprotective role of L-type calcium channel blockers (CCBs, nimodipine and isradipine, were investigated. The amyloid beta (Aβ 1-42 was injected bilaterally into the EC of male rats and one week later, electrophysiological properties of DG granule cells were assessed. Voltage clamp recording revealed appearance of giant sIPSC in combination with a decrease in sEPSC frequency which was partially reversed by CCBs in granule cells from Aβ treated rats. EC amyloid pathogenesis induced a significant reduction of input resistance (Rin accompanied by a profound decreased excitability in the DG granule cells. However, daily administration of CCBs, isradipine or nimodipine (i.c.v. for 6 days, almost preserved the normal excitability against Aβ. In conclusion, lower tendency to fire AP along with reduced Rin suggest that DG granule cells might undergo an alteration in the membrane ion channel activities which finally lead to the behavioral deficits observed in animal models and patients with early-stage Alzheimer's disease.

  14. Differential rescue of spatial memory deficits in aged rats by L-type voltage-dependent calcium channel and ryanodine receptor antagonism.

    Science.gov (United States)

    Hopp, S C; D'Angelo, H M; Royer, S E; Kaercher, R M; Adzovic, L; Wenk, G L

    2014-11-01

    Age-associated memory impairments may result as a consequence of neuroinflammatory induction of intracellular calcium (Ca(+2)) dysregulation. Altered L-type voltage-dependent calcium channel (L-VDCC) and ryanodine receptor (RyR) activity may underlie age-associated learning and memory impairments. Various neuroinflammatory markers are associated with increased activity of both L-VDCCs and RyRs, and increased neuroinflammation is associated with normal aging. In vitro, pharmacological blockade of L-VDCCs and RyRs has been shown to be anti-inflammatory. Here, we examined whether pharmacological blockade of L-VDCCs or RyRs with the drugs nimodipine and dantrolene, respectively, could improve spatial memory and reduce age-associated increases in microglia activation. Dantrolene and nimodipine differentially attenuated age-associated spatial memory deficits but were not anti-inflammatory in vivo. Furthermore, RyR gene expression was inversely correlated with spatial memory, highlighting the central role of Ca(+2) dysregulation in age-associated memory deficits.

  15. Macrophage activation by a vanadyl-aspirin complex is dependent on L-type calcium channel and the generation of nitric oxide

    International Nuclear Information System (INIS)

    Bone homeostasis is the result of a tight balance between bone resorption and bone formation where macrophage activation is believed to contribute to bone resorption. We have previously shown that a vanadyl(IV)-aspirin complex (VOAspi) regulates cell proliferation and differentiation of osteoblasts in culture. In this study, we assessed VOAspi and VO effects and their possible mechanism of action on a mouse macrophage cell line RAW 264.7. Both vanadium compounds inhibited cell proliferation in a dose-dependent manner. Nifedipine completely reversed the VOAspi-induced macrophage cytotoxicity, while it could not block the effect of VO. VOAspi also stimulated nitric oxide (NO) production, the oxidation of dihydrorhodamine 123 (DHR-123) and enhanced the expression of both constitutive and inducible isoforms of nitric oxide syntases (NOS). All these effects were abolished by nifedipine. Althogether our finding give evidence that VOAspi-induced macrophage cytotoxicity is dependent on L-type calcium channel and the generation of NO though the induction of eNOS and iNOS. Contrary, the parent compound VO exerted a cytotoxic effect by mechanisms independent of a calcium entry and the NO/NOS activation

  16. Control analysis of the dependence of Escherichia coli physiology on the H(+)-ATPase.

    OpenAIRE

    Jensen, P. R.; Michelsen, O; Westerhoff, H.V.

    1993-01-01

    The H(+)-ATPase plays a central role in Escherichia coli free-energy transduction and hence in E. coli physiology. We here investigate the extent to which this enzyme also controls the growth rate, growth yield, and respiratory rate of E. coli. We modulate the expression of the atp operon and determine the effect on said properties. When quantified in terms of control coefficients, we find that, in the wild-type cell growing on glucose in minimal medium, this key enzyme (H(+)-ATPase) exerts v...

  17. Regulation of plant plasma membrane H+- and Ca2+-ATPases by terminal domains

    DEFF Research Database (Denmark)

    Bækgaard, Lone; Fuglsang, Anja Thoe; Palmgren, Michael Gjedde

    2005-01-01

    In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H(+)-and Ca(2+)-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulatory...... mechanisms of these pumps. In plant plasma membrane H(+)- and Ca(2+)-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed....

  18. Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes.

    Directory of Open Access Journals (Sweden)

    Padma P Srinivasan

    Full Text Available Voltage-sensitive calcium channels (VSCC regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1 and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration.

  19. Arrhythmogenicity of the hypertrophied and senescent heart and relationship to membrane proteins involved in the altered calcium handling.

    Science.gov (United States)

    Carré, F; Rannou, F; Sainte Beuve, C; Chevalier, B; Moalic, J M; Swynghedauw, B; Charlemagne, D

    1993-10-01

    The high incidence of arrhythmias in human left ventricular hypertrophy has been well established but the mechanisms of arrhythmias are not well defined. In attempt to clarify these mechanisms, we tried to determine if a relationship might exist in the hypertrophied or senescent hearts between the incidence of arrhythmias and alterations in the gene expression of the main membrane proteins involved in the regulation of calcium movements. Holter monitoring was used in young and senescent rats where hypertrophy had been induced by aortic stenosis and hyperthyroidism (young rats) or by DOCA-salt treatment (senescent rats). Different types of spontaneous arrhythmias were detected. In the aortic stenosis group, the heart rate and the number of supraventricular premature beats were increased significantly, whereas the number of ventricular premature beats was increased in some animals but not in all. In senescent rats, the numbers of ventricular and supraventricular premature beats and the incidence of atrioventricular block were very high. At the cellular level, the density of calcium channels from the sarcolemma and of the alpha 1 subunit of the Na+/K(+)-ATPase were unchanged in the hypertrophied and senescent hearts but most of the proteins involved in the regulation of calcium movements (calcium release channel and Ca(2+)-ATPase from the sarcoplasmic reticulum, Na+/Ca2+ exchange, and beta adrenergic and muscarinic receptors from the sarcolemma) have a decreased density or activity. These changes might account for the slowing of the maximum shortening velocity and the impaired contractility of the hypertrophied and senescent hearts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8275524

  20. Additive effects of cilnidipine, an L-/N-type calcium channel blocker, and an angiotensin II receptor blocker on reducing cardiorenal damage in Otsuka Long-Evans Tokushima Fatty rats with type 2 diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Mori Y

    2014-06-01

    Full Text Available Yutaka Mori,1,2 Shizuka Aritomi,3 Kazumi Niinuma,3 Tarou Nakamura,3 Kenichi Matsuura,1 Junichi Yokoyama,1 Kazunori Utsunomiya1 1Division of Diabetes and Endocrinology, Department of Internal Medicine, The Jikei University School of Medicine, Minato-ku, Japan; 2Department of Clinical Research, National Hospital Organization, Utsunomiya National Hospital, Utsunomiya, Japan; 3Research Center, Ajinomoto Pharmaceuticals Co, Ltd, Kanagawa, Japan Abstract: Cilnidipine (Cil, which is an L-/N-type calcium channel blocker (CCB, has been known to provide renal protection by decreasing the activity of the sympathetic nervous system (SNS and the renin–angiotensin system. In this study, we compared the effects of the combination of Cil and amlodipine (Aml, which is an L-type CCB, with an angiotensin (Ang II receptor blocker on diabetic cardiorenal damage in spontaneously type 2 diabetic rats. Seventeen-week-old Otsuka Long-Evans Tokushima Fatty rats were randomly assigned to receive Cil, Aml, valsartan (Val, Cil + Val, Aml + Val, or a vehicle (eight rats per group for 22 weeks. Antihypertensive potencies were nearly equal among the CCB monotherapy groups and the combination therapy groups. The lowering of blood pressure by either treatment did not significantly affect the glycemic variables. However, exacerbations of renal and heart failure were significantly suppressed in rats administered Cil or Val, and additional suppression was observed in those administered Cil + Val. Although Val increased the renin–Ang system, Aml + Val treatment resulted in additional increases in these parameters, while Cil + Val did not show such effects. Furthermore, Cil increased the ratio of Ang-(1–7 to Ang-I, despite the fact that Val and Aml + Val decreased the Ang-(1–7 levels. These actions of Cil + Val might be due to their synergistic inhibitory effect on the activity of the SNS, and on aldosterone secretion through N-type calcium channel antagonism and Ang II

  1. Neurological disease mutations compromise a C-terminal ion pathway in the Na(+)/K(+)-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Khandelia, Himanshu; Morth, J Preben;

    2010-01-01

    The Na(+)/K(+)-ATPase pumps three sodium ions out of and two potassium ions into the cell for each ATP molecule that is split, thereby generating the chemical and electrical gradients across the plasma membrane that are essential in, for example, signalling, secondary transport and volume...... regulation in animal cells. Crystal structures of the potassium-bound form of the pump revealed an intimate docking of the alpha-subunit carboxy terminus at the transmembrane domain. Here we show that this element is a key regulator of a previously unrecognized ion pathway. Current models of P-type ATPases...... operate with a single ion conduit through the pump, but our data suggest an additional pathway in the Na(+)/K(+)-ATPase between the ion-binding sites and the cytoplasm. The C-terminal pathway allows a cytoplasmic proton to enter and stabilize site III when empty in the potassium-bound state, and when...

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca2+-ATPase. A comparison of the configurations of bound......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 catalytic site as a planar VO3− in complex with water and Mg2+ in a dephosphorylation transition-state-like conformation. Validating bound VO3− by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl− site near the dephosphorylation site. Crystallization...

  3. Distribution of Na,K-ATPase α subunits in rat vestibular sensory epithelia

    NARCIS (Netherlands)

    Schuth, Olga; McLean, Will J; Eatock, Ruth Anne; Pyott, Sonja J

    2014-01-01

    The afferent encoding of vestibular stimuli depends on molecular mechanisms that regulate membrane potential, concentration gradients, and ion and neurotransmitter clearance at both afferent and efferent relays. In many cell types, the Na,K-ATPase (NKA) is essential for establishing hyperpolarized m

  4. Calredoxin represents a novel type of calcium-dependent sensor-responder connected to redox regulation in the chloroplast.

    Science.gov (United States)

    Hochmal, Ana Karina; Zinzius, Karen; Charoenwattanasatien, Ratana; Gäbelein, Philipp; Mutoh, Risa; Tanaka, Hideaki; Schulze, Stefan; Liu, Gai; Scholz, Martin; Nordhues, André; Offenborn, Jan Niklas; Petroutsos, Dimitris; Finazzi, Giovanni; Fufezan, Christian; Huang, Kaiyao; Kurisu, Genji; Hippler, Michael

    2016-01-01

    Calcium (Ca(2+)) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca(2+)- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca(2+)-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca(2+)-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca(2+)-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca(2+)-dependent 'sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation. PMID:27297041

  5. Effects of the 1, 4-dihydropyridine L-type calcium channel blocker benidipine on bone marrow stromal cells.

    Science.gov (United States)

    Ma, Zhong-ping; Liao, Jia-cheng; Zhao, Chang; Cai, Dao-zhang

    2015-08-01

    Osteoporosis (OP) often increases the risk of bone fracture and other complications and is a major clinical problem. Previous studies have found that high blood pressure is associated with bone formation abnormalities, resulting in increased calcium loss. We have investigated the effect of the antihypertensive drug benidipine on bone marrow stromal cell (BMSC) differentiation into osteoblasts and bone formation under osteoporotic conditions. We used a combination of in vitro and in vivo approaches to test the hypothesis that benidipine promotes murine BMSC differentiation into osteoblasts. Alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), β-catenin, and low-density lipoprotein receptor-related protein 5 (LRP5) protein expression was evaluated in primary femoral BMSCs from C57/BL6 mice cultured under osteogenic conditions for 2 weeks to examine the effects of benidipine. An ovariectomized (OVX) mouse model was used to investigate the effect of benidipine treatment for 3 months in vivo. We found that ALP, OCN, and RUNX2 expression was up-regulated and WNT/β-catenin signaling was enhanced in vitro and in vivo. In OVX mice that were intragastrically administered benidipine, bone parameters (trabecular thickness, bone mineral density, and trabecular number) in the distal femoral metaphysis were significantly increased compared with control OVX mice. Consistently, benidipine promoted BMSC differentiation into osteoblasts and protected against bone loss in OVX mice. Therefore, benidipine might be a suitable candidate for the treatment of patients with postmenopausal osteoporosis and hypertension.

  6. Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity.

    Science.gov (United States)

    Fatehi, Mohammad; Carter, Chris R J; Youssef, Nermeen; Hunter, Beth E; Holt, Andrew; Light, Peter E

    2015-01-01

    ATP-sensitive K(+) (KATP) channels play an important role in insulin secretion. KATP channels possess intrinsic MgATPase activity that is important in regulating channel activity in response to metabolic changes, although the precise structural determinants are not clearly understood. Furthermore, the sulfonylurea receptor 1 (SUR1) S1369A diabetes risk variant increases MgATPase activity, but the molecular mechanisms remain to be determined. Therefore, we hypothesized that residue-residue interactions between 1369 and 1372, predicted from in silico modelling, influence MgATPase activity, as well as sensitivity to the clinically used drug diazoxide that is known to increase MgATPase activity. We employed a point mutagenic approach with patch-clamp and direct biochemical assays to determine interaction between residues 1369 and 1372. Mutations in residues 1369 and 1372 predicted to decrease the residue interaction elicited a significant increase in MgATPase activity, whereas mutations predicted to possess similar residue interactions to wild-type (WT) channels elicited no alterations in MgATPase activity. In contrast, mutations that were predicted to increase residue interactions resulted in significant decreases in MgATPase activity. We also determined that a single S1369K substitution in SUR1 caused MgATPase activity and diazoxide pharmacological profiles to resemble those of channels containing the SUR2A subunit isoform. Our results provide evidence, at the single residue level, for a molecular mechanism that may underlie the association of the S1369A variant with type 2 diabetes. We also show a single amino acid difference can account for the markedly different diazoxide sensitivities between channels containing either the SUR1 or SUR2A subunit isoforms. PMID:26181369

  7. Obstacle Effects on One-Dimensional Translocation of ATPase

    Institute of Scientific and Technical Information of China (English)

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

    2002-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2016-01-01

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

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

    Science.gov (United States)

    Chen, Hong-Lin; Zhao, Jing; Zhang, Guan-Jun; Kou, Jun-Ping; Yu, Bo-Yang

    2016-06-01

    Myosin II plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin II ATPase activity based on molybdenum blue method, using a known myosin II ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate (ATP) and calcium chloride, pH, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin II ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin II ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin II ATPase inhibitors in vitro. PMID:27473959

  10. Calcium Phosphate Biomaterials: An Update

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Current calcium phosphate (CaP) biomaterials for bone repair, substitution, augmentation and regeneration include hydroxyapatite ( HA ) from synthetic or biologic origin, beta-tricalcium phosphate ( β-TCP ) , biphasic calcium phosphate (BCP), and are available as granules, porous blocks, components of composites (CaP/polymer) cements, and as coatings on orthopedic and dental implants. Experimental calcium phosphate biomaterials include CO3- and F-substituted apatites, Mg-and Zn-substituted β-TCP, calcium phosphate glasses. This paper is a brief review of the different types of CaP biomaterials and their properties such as bioactivity, osteoconductivity, osteoinductivity.

  11. Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

    Directory of Open Access Journals (Sweden)

    Flora H.F. D'Angelis

    2014-09-01

    Full Text Available This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50 and alkaline incubation (pH=10.50, at a temperature of 37ºC, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue, type IIA (oxidative-glycolytic, intermediate blue and type IIX (glycolytic, dark blue. There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.

  12. The Vacuolar H+-ATPase B1 Subunit Polymorphism p.E161K Associates with Impaired Urinary Acidification in Recurrent Stone Formers.

    Science.gov (United States)

    Dhayat, Nasser A; Schaller, Andre; Albano, Giuseppe; Poindexter, John; Griffith, Carolyn; Pasch, Andreas; Gallati, Sabina; Vogt, Bruno; Moe, Orson W; Fuster, Daniel G

    2016-05-01

    Mutations in the vacuolar-type H(+)-ATPase B1 subunit gene ATP6V1B1 cause autosomal-recessive distal renal tubular acidosis (dRTA). We previously identified a single-nucleotide polymorphism (SNP) in the human B1 subunit (c.481G>A; p.E161K) that causes greatly diminished pump function in vitro To investigate the effect of this SNP on urinary acidification, we conducted a genotype-phenotype analysis of recurrent stone formers in the Dallas and Bern kidney stone registries. Of 555 patients examined, 32 (5.8%) were heterozygous for the p.E161K SNP, and the remaining 523 (94.2%) carried two wild-type alleles. After adjustment for sex, age, body mass index, and dietary acid and alkali intake, p.E161K SNP carriers had a nonsignificant tendency to higher urinary pH on a random diet (6.31 versus 6.09; P=0.09). Under an instructed low-Ca and low-Na diet, urinary pH was higher in p.E161K SNP carriers (6.56 versus 6.01; PKidney stones of p.E161K carriers were more likely to contain calcium phosphate than stones of wild-type patients. In acute NH4Cl loading, p.E161K carriers displayed a higher trough urinary pH (5.34 versus 4.89; P=0.01) than wild-type patients. Overall, 14.6% of wild-type patients and 52.4% of p.E161K carriers were unable to acidify their urine below pH 5.3 and thus, can be considered to have incomplete dRTA. In summary, our data indicate that recurrent stone formers with the vacuolar H(+)-ATPase B1 subunit p.E161K SNP exhibit a urinary acidification deficit with an increased prevalence of calcium phosphate-containing kidney stones. The burden of E161K heterozygosity may be a forme fruste of dRTA. PMID:26453614

  13. Calcium Carbonate

    Science.gov (United States)

    ... before being swallowed; do not swallow them whole. Drink a full glass of water after taking either the regular or chewable tablets or capsules. Some liquid forms of calcium carbonate must be shaken well before use.Do not ...

  14. Calcium Calculator

    Science.gov (United States)

    ... Latvia - Lebanon - Libya - Lithuania - Luxembourg - Macedonia, Republic of - Malaysia - Malta - Mexico - Moldova - Morocco - Netherlands - New Zealand - Nigeria - ... and Statistics Popular content Calcium content of common foods What is Osteoporosis? The Board Introduction to Bone ...

  15. Calcium Electroporation

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; Gibot, Laure; Madi, Moinecha;

    2015-01-01

    BACKGROUND: Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill...... efficacy-and normal cell sensitivity. METHODS: Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29), a bladder transitional cell carcinoma (SW780......), and a breast adenocarcinoma (MDA-MB231), as well as on primary normal human dermal fibroblasts (HDF-n). RESULTS: The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p

  16. Age-dependent impact of CaV 3.2 T-type calcium channel deletion on myogenic tone and flow-mediated vasodilatation in small arteries

    DEFF Research Database (Denmark)

    Mikkelsen, Miriam F; Björling, Karl; Jensen, Lars Jørn

    2016-01-01

    , structural remodeling, and mRNA + protein expression in small mesenteric arteries from CaV 3.2 knock-out vs. wild-type mice at young vs. mature adult age. In young mice, only, deletion of CaV 3.2 led to enhanced myogenic response and ∼50 % reduction of flow-mediated vasodilatation. Ni(2+) had both CaV 3.......2-dependent and -independent effects. No changes in mRNA expression of several important K(+) and Ca(2+) channel genes were induced by CaV 3.2 knock-out. However, the expression of the other T-type channel isoform (CaV 3.1) was reduced at the mRNA and protein level in mature adult compared to young WT...... arteries. Our study shows important roles of the CaV 3.2 T-type calcium channels in myogenic tone and flow-mediated vasodilation that disappear with aging. Since increased arterial tone is a risk factor for cardiovascular disease we conclude that CaV 3.2 channels, by modulating pressure- and flow...

  17. Versatile roles of V-ATPases accessory subunit Ac45 in osteoclast formation and function.

    Directory of Open Access Journals (Sweden)

    An Qin

    Full Text Available Vacuolar-type H(+-ATPases (V-ATPases are macromolecular proton pumps that acidify intracellular cargos and deliver protons across the plasma membrane of a variety of specialized cells, including bone-resorbing osteoclasts. Extracellular acidification is crucial for osteoclastic bone resorption, a process that initiates the dissolution of mineralized bone matrix. While the importance of V-ATPases in osteoclastic resorptive function is well-defined, whether V-ATPases facilitate additional aspects of osteoclast function and/or formation remains largely obscure. Here we report that the V-ATPase accessory subunit Ac45 participates in both osteoclast formation and function. Using a siRNA-based approach, we show that targeted suppression of Ac45 impairs intracellular acidification and endocytosis, both are prerequisite for osteoclastic bone resorptive function in vitro. Interestingly, we find that knockdown of Ac45 also attenuates osteoclastogenesis owing to a reduced fusion capacity of osteoclastic precursor cells. Finally, in an effort to gain more detailed insights into the functional role of Ac45 in osteoclasts, we attempted to generate osteoclast-specific Ac45 conditional knockout mice using a Cathepsin K-Cre-LoxP system. Surprisingly, however, insertion of the neomycin cassette in the Ac45-Flox(Neo mice resulted in marked disturbances in CNS development and ensuing embryonic lethality thus precluding functional assessment of Ac45 in osteoclasts and peripheral bone tissues. Based on these unexpected findings we propose that, in addition to its canonical function in V-ATPase-mediated acidification, Ac45 plays versatile roles during osteoclast formation and function.

  18. Analysis of the Inhibitory Effect of Gypenoside on Na+,K+-ATPase in Rats' Heart and Brain and Its Kinetics

    Institute of Scientific and Technical Information of China (English)

    HAN Xiao-yan; WEI Hong-bo; ZHANG Fu-cheng

    2007-01-01

    ObjectiYe: To study the effects of gypenoside (Gyp) on the activity of microsomal Na+,K+-ATPase in rat's heart and brain in vitro. Methods: The microsomal Na+, K+-ATPase was prepared from rat's heart and brain by differential centrifugation. The activity of microsomal Na+, K+-ATPase was assayed by colorimetric technique. Enzyme kinetic analysis method was used to analyze the effect of Gyp on the microsomal Na+, K+-ATPase of rats. Results: Gyp reversibly inhibited the brain and heart's microsomal Na+, K+-ATPase in a concentration-dependent manner, and showed a more potent effect on enzyme in the brain. The IC50 of Gyp for the heart and brain were 58.79± 8.05 mg/L and 52.07 ±6.25 mg/L, respectively. The inhibition was enhanced by lowering the Na+, or K+ concentrations or increasing the ATP concentration. Enzyme kinetic studies indicated that the inhibitory effect of Gyp on the enzyme is like that of competitive antagonist of Na+, the counter-competitive inhibitor for the substrate ATP, and the mixed-type inhibitor for K+. Conclusion: Gyp displays its cardiotonic and central inhibitory effects by way of inhibiting heart and brain's microsomal Na+, K+-ATPase activities in rats.

  19. Addition of subunit γ, K⁺ ions, and lipid restores the thermal stability of solubilized Na,K-ATPase.

    Science.gov (United States)

    Yoneda, Juliana Sakamoto; Rigos, Carolina Fortes; Ciancaglini, Pietro

    2013-02-15

    Differential scanning calorimetry (DSC) was applied to ascertain the effect caused by K⁺, Na⁺, ATP, detergent, DPPC, DPPE, and subunit γ on the thermostability of Na,K-ATPase. The enthalpy variation (ΔH) for the thermal denaturation of the membrane-bound is twice the ΔH value obtained for solubilized Na,K-ATPase. Denaturation occurs in five steps for membrane-bound against three steps for the solubilized enzyme, therefore a multi-step unfolding process. In the presence of Na⁺, the melting temperature is 61.6°C, and the ΔH is lower as compared with the ΔH obtained in the presence or in the absence of K⁺. Addition of ATP does not alter the transition temperatures significantly, but the shape of the curve is modified. Subunit γ probably stabilizes Na,K-ATPase in the beginning of thermal unfolding, and different amounts of detergents in the solubilized sample change the protein stability. Reconstitution of Na,K-ATPase into a liposome shows that lipids exert a protector effect. These results reveal differences on the thermostability depending on the conformation of Na,K-ATPase. They are relevant because it allows a comparison with future studies, e.g. how the composition of the membrane interferes on the stability of Na, K-ATPase, elucidating the importance of the lipid type contained in cell membrane.

  20. Influence of calcium oxalate crystal accumulation on the calcium content of seeds from Medicago truncatula.

    Science.gov (United States)

    Nakata, Paul A

    2012-04-01

    Crystals of calcium oxalate often form in cells adjacent to the vascular bundles in the tissues along the xylem stream. This spatial crystal pattern suggests a role for calcium oxalate formation in regulating calcium transport and partitioning to edible organs such as seeds. To investigate this potential role, microscopic and biochemical comparisons were conducted on the different tissues of Medicago truncatula wild-type and the calcium oxalate defective (cod) 5 which lacks the ability to accumulate prismatic crystals in the cells adjacent to the vascular bundles. Calcium measurements showed that cod5 seeds had more calcium and cod5 pods contained less calcium than the corresponding wild-type tissues. Roots, stems, and leaves from cod5 and wild-type had similar calcium content. Although cod5 was devoid of prismatic crystals, cod5 pods were observed to form druse crystals of calcium oxalate not found in wild-type pods. Taken together these findings suggest a functional role for calcium oxalate formation in regulating calcium transport to the seeds. Regulating calcium uptake at the roots also appeared to be another point of control in determining seed calcium content. Overall, regulating the long distance transport and partitioning of calcium to the seeds appears to be a complex process with multiple points of control. PMID:22325887

  1. Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: Mapping of residues that when altered give rise to an activated enzyme

    DEFF Research Database (Denmark)

    Axelsen, K.B.; Venema, K.; Jah, T.;

    1999-01-01

    The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants, The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme....... Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H......+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP...

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

    Directory of Open Access Journals (Sweden)

    D.V. Vassallo

    2008-09-01

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

  3. A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes

    International Nuclear Information System (INIS)

    Rationale: Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca2+ channels and their renowned antioxidant properties. Methods: We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca2+ channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca2+ channel-blocking activity and antioxidant properties. The Ca2+ channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. Results: Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca2+ channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca2+ channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. Conclusions: Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring. - Highlights: • Dihydropyridine (DHP) molecules are widely used in cardiovascular disease. • DHPs block Ca2+ entry through LTCC—some DHPs have antioxidant activity as well. • We synthesized 6 new DHPs and tested their Ca2+ blocking and antioxidant activities. • 3-Aryl meta-hydroxyl substitution strongly increases their Ca2+ blocking activity. • 3-Aryl meta-hydroxyl substitution did not affect the antioxidant properties

  4. S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism.

    Science.gov (United States)

    Liu, Taiming; Schroeder, Hobe J; Zhang, Meijuan; Wilson, Sean M; Terry, Michael H; Longo, Lawrence D; Power, Gordon G; Blood, Arlin B

    2016-08-31

    S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

  5. S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism.

    Science.gov (United States)

    Liu, Taiming; Schroeder, Hobe J; Zhang, Meijuan; Wilson, Sean M; Terry, Michael H; Longo, Lawrence D; Power, Gordon G; Blood, Arlin B

    2016-08-31

    S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway. PMID:27235767

  6. Fingerprinting differential active site constraints of ATPases

    OpenAIRE

    Hacker, Stephan M.; Hardt, Norman; Buntru, Alexander; Pagliarini, Dana; Möckel, Martin; Mayer, Thomas U; Scheffner, Martin; Hauck, Christof R.; Marx, Andreas

    2013-01-01

    The free energy provided by adenosine triphosphate (ATP) hydrolysis is central to many cellular processes and, therefore, the number of enzymes utilizing ATP as a substrate is almost innumerable. Modified analogues of ATP are a valuable means to understand the biological function of ATPases. Although these enzymes have evolved towards binding to ATP, large differences in active site architectures were found. In order to systematically access the specific active site constraints of different A...

  7. Identification of a Region of the Polypeptide Chain of Na,K-ATPase α-Subunit Interacting with 67-kDa Melittin-Like Protein.

    Science.gov (United States)

    Kamanina, Yu V; Klimanova, E A; Dergousova, E A; Petrushanko, I Yu; Lopina, O D

    2016-03-01

    It was shown earlier that a 67-kDa protein purified from mouse kidney using polyclonal antibodies against melittin (a peptide from bee venom) interacted with Na,K-ATPase from rabbit kidney. In this study, a 43-kDa proteolytic fragment of Na,K-ATPase α-subunit interacting with the 67-kDa melittin-like protein was found. The α-subunit was hydrolyzed by trypsin in the presence of 0.5 mM ouabain (E2-conformation of Na,K-ATPase). A proteolytic fragment interacting with the 67-kDa melittin-like protein that was identified by mass-spectrometry is a region of the cytoplasmic domain of Na,K-ATPase α-subunit located between amino acid residues 591 and 775. The fragment includes a conservative DPPRA motif that occurs in many P-type ATPases. It was shown earlier that this motif of H,K-ATPase from gastric mucosa binds to melittin. We suggest that namely this motif of P-type ATPases is able to interact with proteins containing melittin-like modules. PMID:27262194

  8. A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Galvis-Pareja, David [Advanced Center for Chronic Diseases (ACCDiS), Facultad de Ciencias Químicas y Farmacéuticas and Facultad Medicina, Universidad de Chile, Santiago (Chile); Centro Estudios Moleculares de la Célula (CEMC), Facultad de Ciencias Químicas y Farmacéuticas and Facultad Medicina, Universidad de Chile, Santiago (Chile); Zapata-Torres, Gerald [Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago (Chile); Hidalgo, Jorge [Centro Estudios Moleculares de la Célula (CEMC), Facultad de Ciencias Químicas y Farmacéuticas and Facultad Medicina, Universidad de Chile, Santiago (Chile); Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago (Chile); Ayala, Pedro [Centro Estudios Moleculares de la Célula (CEMC), Facultad de Ciencias Químicas y Farmacéuticas and Facultad Medicina, Universidad de Chile, Santiago (Chile); and others

    2014-08-15

    Rationale: Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca{sup 2+} channels and their renowned antioxidant properties. Methods: We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca{sup 2+} channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca{sup 2+} channel-blocking activity and antioxidant properties. The Ca{sup 2+} channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. Results: Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca{sup 2+} channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca{sup 2+} channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. Conclusions: Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring. - Highlights: • Dihydropyridine (DHP) molecules are widely used in cardiovascular disease. • DHPs block Ca{sup 2+} entry through LTCC—some DHPs have antioxidant activity as well. • We synthesized 6 new DHPs and tested their Ca{sup 2+} blocking and antioxidant activities. • 3-Aryl meta-hydroxyl substitution strongly increases their Ca{sup 2+} blocking activity. • 3-Aryl meta-hydroxyl substitution did not affect the antioxidant properties.

  9. Spexin Enhances Bowel Movement through Activating L-type Voltage-dependent Calcium Channel via Galanin Receptor 2 in Mice

    Science.gov (United States)

    Lin, Cheng-yuan; Zhang, Man; Huang, Tao; Yang, Li-ling; Fu, Hai-bo; Zhao, Ling; Zhong, Linda LD; Mu, Huai-xue; Shi, Xiao-ke; Leung, Christina FP; Fan, Bao-min; Jiang, Miao; Lu, Ai-ping; Zhu, Li-xin; Bian, Zhao-xiang

    2015-01-01

    A novel neuropeptide spexin was found to be broadly expressed in various endocrine and nervous tissues while little is known about its functions. This study investigated the role of spexin in bowel movement and the underlying mechanisms. In functional constipation (FC) patients, serum spexin levels were significantly decreased. Consistently, in starved mice, the mRNA of spexin was significantly decreased in intestine and colon. Spexin injection increased the velocity of carbon powder propulsion in small intestine and decreased the glass beads expulsion time in distal colon in mice. Further, spexin dose-dependently stimulated the intestinal/colonic smooth muscle contraction. Galanin receptor 2 (GALR2) antagonist M871, but not Galanin receptor 3 (GALR3) antagonist SNAP37899, effectively suppressed the stimulatory effects of spexin on intestinal/colonic smooth muscle contraction, which could be eliminated by extracellular [Ca2+] removal and L-type voltage-dependentCa2+ channel (VDCC) inhibitor nifedipine. Besides, spexin dramatically increased the [Ca2+]i in isolated colonic smooth muscle cells. These data indicate that spexin can act on GALR2 receptor to regulate bowel motility by activating L-type VDCC. Our findings provide evidence for important physiological roles of spexin in GI functions. Selective action on spexin pathway might have therapeutic effects on GI diseases with motility disorders. PMID:26160593

  10. Role of platelet plasma membrane Ca2+-ATPase in health and disease

    Institute of Scientific and Technical Information of China (English)

    William; L; Dean

    2010-01-01

    Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.On the other hand,hyperactive platelets lead to heart attack and stroke.Calcium is a major second messenger in platelet activation,and elevated intracellular calcium leads to hyperactive platelets.Elevated platelet calcium has been documented in hypertension and diabetes;both conditions increase the likelihood of heart attack and stroke. Thus,proper regulation of calcium metabolism in the platelet is extremely important.Plasma membrane Ca2+-ATPase(PMCA)is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux.In keeping with the important role of calcium in platelet function,PMCA is a highly regulated transporter.In human platelets,PMCA is activated by Ca2+/calmodulin,by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide.It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis.In addition,the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation.Rapid regulation by phosphorylation results in changes in the rate of platelet activation,whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction.In hypertension and diabetes,PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation.Clearly,a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.

  11. Vacuolar-ATPase (V-ATPase) Mediates Progesterone-Induced Uterine Fluid Acidification in Rats.

    Science.gov (United States)

    Karim, Kamarulzaman; Giribabu, Nelli; Muniandy, Sekaran; Salleh, Naguib

    2016-04-01

    We hypothesized that progesterone-induced decrease in uterine fluid pH involves V-ATPase. In this study, expression and functional activity of V-ATPase in uterus were investigated under progesterone influence. Ovariectomized adult female rats received subcutaneous injection of estradiol-17β (1 µg/kg/day) or progesterone (20 mg/kg/day) for 3 days or 3 days estradiol-17β followed by 3 days vehicle, progesterone, or estradiol-17β plus progesterone. Mifepristone, a progesterone receptor blocker, was concomitantly given to the rats which received progesterone. A day after last injection, rate of uterine fluid secretion, its HCO3 (-) concentration, and pH were determined via in vivo uterine perfusion in rats under anesthesia. V-ATPase inhibitor, bafilomycin, was introduced into the perfusion buffer, and changes in these parameters were observed. Expression of V-ATPase A1 and B1/2 proteins and mRNAs in uterus were quantified by Western blotting and real-time PCR, respectively. Distribution of these proteins was observed by immunohistochemistry. Our findings showed that under progesterone influence, uterine fluid secretion rate, HCO3 (-) concentration, and pH were significantly reduced. Administration of bafilomycin did not cause significant changes in fluid secretion rate; however, HCO3 (-) concentration and pH were significantly elevated. In parallel with these changes, expression of V-ATPase A1 and B1/2 proteins and mRNAs were significantly increased with these proteins highly distributed in uterine luminal and glandular epithelia. In conclusion, increased expression and functional activity of V-ATPase were most likely responsible for the decreased in uterine fluid pH observed under progesterone influence. PMID:26403527

  12. Vacuolar-ATPase (V-ATPase) Mediates Progesterone-Induced Uterine Fluid Acidification in Rats.

    Science.gov (United States)

    Karim, Kamarulzaman; Giribabu, Nelli; Muniandy, Sekaran; Salleh, Naguib

    2016-04-01

    We hypothesized that progesterone-induced decrease in uterine fluid pH involves V-ATPase. In this study, expression and functional activity of V-ATPase in uterus were investigated under progesterone influence. Ovariectomized adult female rats received subcutaneous injection of estradiol-17β (1 µg/kg/day) or progesterone (20 mg/kg/day) for 3 days or 3 days estradiol-17β followed by 3 days vehicle, progesterone, or estradiol-17β plus progesterone. Mifepristone, a progesterone receptor blocker, was concomitantly given to the rats which received progesterone. A day after last injection, rate of uterine fluid secretion, its HCO3 (-) concentration, and pH were determined via in vivo uterine perfusion in rats under anesthesia. V-ATPase inhibitor, bafilomycin, was introduced into the perfusion buffer, and changes in these parameters were observed. Expression of V-ATPase A1 and B1/2 proteins and mRNAs in uterus were quantified by Western blotting and real-time PCR, respectively. Distribution of these proteins was observed by immunohistochemistry. Our findings showed that under progesterone influence, uterine fluid secretion rate, HCO3 (-) concentration, and pH were significantly reduced. Administration of bafilomycin did not cause significant changes in fluid secretion rate; however, HCO3 (-) concentration and pH were significantly elevated. In parallel with these changes, expression of V-ATPase A1 and B1/2 proteins and mRNAs were significantly increased with these proteins highly distributed in uterine luminal and glandular epithelia. In conclusion, increased expression and functional activity of V-ATPase were most likely responsible for the decreased in uterine fluid pH observed under progesterone influence.

  13. Structural and magnetic Properties of TbZn-substituted calcium barium M-type nano-structured hexa-ferrites

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Hasan M. [Department of Physics, Bahauddin Zakariya University, Multan 60800 (Pakistan); Department of Electronics, University of York, York YO10 5DD (United Kingdom); Islam, M.U., E-mail: dr.misbahulislam@bzu.edu.pk [Department of Physics, Bahauddin Zakariya University, Multan 60800 (Pakistan); Xu, Yongbing [Department of Electronics, University of York, York YO10 5DD (United Kingdom); Nanjing–York International Centre of Spintronics and Nano-Engineering, Nanjing University, Nanjing 210093 (China); Asif Iqbal, M. [Department of Physics, Bahauddin Zakariya University, Multan 60800 (Pakistan); National University of Science and Technology, College of E and ME, Islamabad (Pakistan); Ali, Irshad [Department of Physics, Bahauddin Zakariya University, Multan 60800 (Pakistan)

    2014-03-15

    Highlights: • Tb–Zn substituted Ca{sub 0.5}Ba{sub 0.5}Fe{sub 12}O{sub 19} samples exhibit single magnetoplumbite phase. • Lattice parameters a and c have increasing values. • Coercivity can be tuned at lower substitution level • Crystallites size was found in the range 18–25 nm by TEM and by Scherrer formula. • These hexa-ferrites are suitable for microwave devices and magnetic recording media. -- Abstract: Effect of TbZn substitution on the structural and magnetic properties of Ca{sub 0.5}Ba{sub 0.5−x}Tb{sub x}Zn{sub y}Fe{sub 12−y}O{sub 19}, (x = 0.00–0.10; y = 0.00–1.00) ferrites prepared by sol–gel auto combustion is reported. The synthesized samples were characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, transmission electron microscopy and Vibrating Sample magnetometery. The X-ray diffraction analysis confirmed single phase M-type hexa-ferrite structure. The lattice parameters were found to increase as TbZn contents increases, which is attributed to the ionic sizes of the implicated cations. The TbZn seems to be completely soluble in the lattice. The results of scanning electron microscopy and transmission electron microscopy shows that the grain size decreases with increase of TbZn substitution. The coercivity values (1277–2025 Oe) of all samples lies in the range of M-type hexa-ferrite and indicate that an increase of anisotropy was achieved by substitution of TbZn, while the size of nanoparticles was drastically reduced between 18 and 25 nm. The increased anisotropy and fine particle size are useful for many applications, such as improving signal noise ratio of recording devices.

  14. Growth differentiation factor-15 promotes glutamate release in medial prefrontal cortex of mice through upregulation of T-type calcium channels

    Science.gov (United States)

    Liu, Dong-Dong; Lu, Jun-Mei; Zhao, Qian-Ru; Hu, Changlong; Mei, Yan-Ai

    2016-01-01

    Growth differentiation factor-15 (GDF-15) has been implicated in ischemic brain injury and synapse development, but its involvement in modulating neuronal excitability and synaptic transmission remain poorly understood. In this study, we investigated the effects of GDF-15 on non-evoked miniature excitatory post-synaptic currents (mEPSCs) and neurotransmitter release in the medial prefrontal cortex (mPFC) in mice. Incubation of mPFC slices with GDF-15 for 60 min significantly increased the frequency of mEPSCs without effect on their amplitude. GDF-15 also significantly elevated presynaptic glutamate release, as shown by HPLC. These effects were blocked by dual TGF-β type I receptor (TβRI) and TGF-β type II receptor (TβRII) antagonists, but not by a TβRI antagonist alone. Meanwhile, GDF-15 enhanced pERK level, and inhibition of MAPK/ERK activity attenuated the GDF-15-induced increases in mEPSC and glutamate release. Blocking T-type calcium channels reduced the GDF-15 induced up-regulation of synaptic transmission. Membrane-protein extraction and use of an intracellular protein-transport inhibitor showed that GDF-15 promoted CaV3.1 and CaV3.3 α-subunit expression by trafficking to the membrane. These results confirm previous findings in cerebellar granule neurons, in which GDF-15 induces its neurobiological effects via TβRII and activation of the ERK pathway, providing novel insights into the mechanism of GDF-15 function in cortical neurons. PMID:27353765

  15. Phosphorylation of the Na+,K+-ATPase and the H+,K+-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Morth, Jens Preben; Jensen, Jan Egebjerg;

    2010-01-01

    Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na(+),K(+)- and H(+),K(+)-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two...... pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties...... as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations....

  16. Golgi-associated LC3 lipidation requires V-ATPase in noncanonical autophagy.

    Science.gov (United States)

    Gao, Ying; Liu, Yajun; Hong, Liang; Yang, Zuolong; Cai, Xinran; Chen, Xiaoyun; Fu, Yuanyuan; Lin, Yujie; Wen, Weijie; Li, Sitong; Liu, Xingguo; Huang, Heqing; Vogt, Andreas; Liu, Peiqing; Yin, Xiao-Ming; Li, Min

    2016-01-01

    Autophagy is an evolutionarily conserved catabolic process by which cells degrade intracellular proteins and organelles in the lysosomes. Canonical autophagy requires all autophagy proteins (ATGs), whereas noncanonical autophagy is activated by diverse agents in which some of the essential autophagy proteins are dispensable. How noncanonical autophagy is induced and/or inhibited is still largely unclear. In this study, we demonstrated that AMDE-1, a recently identified chemical that can induce canonical autophagy, was able to elicit noncanonical autophagy that is independent of the ULK1 (unc-51-like kinase 1) complex and the Beclin1 complex. AMDE-1-induced noncanonical autophagy could be specifically suppressed by various V-ATPase (vacuolar-type H(+)-ATPase) inhibitors, but not by disturbance of the lysosome function or the intracellular ion redistribution. Similar findings were applicable to a diverse group of stimuli that can induce noncanonical autophagy in a FIP200-independent manner. AMDE-1-induced LC3 lipidation was colocalized with the Golgi complex, and was inhibited by the disturbance of Golgi complex. The integrity of the Golgi complex was also required for multiple other agents to stimulate noncanonical LC3 lipidation. These results suggest that the Golgi complex may serve as a membrane platform for noncanonical autophagy where V-ATPase is a key player. V-ATPase inhibitors could be useful tools for studying noncanonical autophagy. PMID:27512951

  17. Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity.

    Science.gov (United States)

    de Almeida-Amaral, Elmo Eduardo; Caruso-Neves, Celso; Pires, Vanessa Maria Pereira; Meyer-Fernandes, José Roberto

    2008-02-01

    We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite. PMID:17825292

  18. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

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

    Institute of Scientific and Technical Information of China (English)

    陈立华; 于嘉; 刘丽旭; 曹美鸿

    2002-01-01

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

  20. Negative regulation of gamma-aminobutyric acid type A receptor on free calcium ion levels following facial nerve injury

    Institute of Scientific and Technical Information of China (English)

    Fugao Zhu; Dawei Sun; Yanqing Wang; Rui Zhou; Junfeng Wen; Xiuming Wan; Yanjun Wang; Banghua Liu

    2010-01-01

    Previous studies have demonstrated that muscarinic, and nicotinic receptors increase free Ca2+ levels in the facial nerve nucleus via various channels following facial nerve injury. However, intracellular Ca2+ overload can trigger either necrotic or apoptotic cell death. Gamma-aminobutyric acid (GABA), an important inhibitory neurotransmitter in the central nervous system, exists in the facial nerve nucleus. It is assumed that GABA negatively regulates free Ca2+ levels in the facial nerve nucleus. The present study investigated GABA type A (GABAA) receptor expression in the facial nerve nucleus in a rat model of facial nerve injury using immunohistochemistry and laser confocal microscopy, as well as the regulatory effects of GABAA receptor on nicotinic receptor response following facial nerve injury. Subunits α1, α3, α5, β1, β2, δ, and γ3 of GABAA receptors were expressed in the facial nerve nucleus following facial nerve injury. In addition, GABAA receptor expression significantly inhibited the increase in nicotinic receptor-mediated free Ca2+ levels in the facial nerve nucleus following facial nerve injury in a concentration-dependent fashion. These results suggest that GABAA receptors exhibit negative effects on nicotinic receptor responses following facial nerve injury.

  1. The Silicon and Calcium High-Velocity Features in Type Ia Supernovae from Early to Maximum Phases

    CERN Document Server

    Zhao, Xulin; Maeda, Keiichi; Sai, Hanna; Zhang, Tianmeng; Zhang, Jujia; Huang, Fang; Rui, Liming; Zhou, Qi; Mo, Jun

    2015-01-01

    The high-velocity features (HVFs) in optical spectra of type Ia supernovae (SNe Ia) are examined with a large sample including very early-time spectra (e.g., t < -7 days). Multiple Gaussian fits are applied to examine the HVFs and their evolutions, using constraints on expansion velocities for the same species (i.e., SiII 5972 and SiII 6355). We find that strong HVFs tend to appear in SNe Ia with smaller decline rates (e.g., dm15(B)<1.4 mag), clarifying that the finding by Childress et al. (2014) for the Ca-HVFs in near-maximum-light spectra applies both to the Si-HVFs and Ca-HVFs in the earlier phase. The Si-HVFs seem to be more common in fast-expanding SNe Ia, which is different from the earlier result that the Ca-HVFs are associated with SNe Ia having slower SiII 6355 velocities at maximum light (i.e., Vsi). This difference can be due to that the HVFs in fast-expanding SNe Ia usually disappear more rapidly and are easily blended with the photospheric components when approaching the maximum light. Mor...

  2. Calcium and bones

    Science.gov (United States)

    Bone strength and calcium ... calcium (as well as phosphorus) to make healthy bones. Bones are the main storage site of calcium in ... your body does not absorb enough calcium, your bones can get weak or will not grow properly. ...

  3. Calcium carbonate overdose

    Science.gov (United States)

    Tums overdose; Calcium overdose ... Calcium carbonate can be dangerous in large amounts. ... Some products that contain calcium carbonate are certain: ... and mineral supplements Other products may also contain calcium ...

  4. Get Enough Calcium

    Science.gov (United States)

    ... Calcium Print This Topic En español Get Enough Calcium Browse Sections The Basics Overview Foods and Vitamins ... 2 of 4 sections Take Action! Take Action: Calcium Sources Protect your bones – get plenty of calcium ...

  5. Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.

    Science.gov (United States)

    Márkus, Nóra M; Hasel, Philip; Qiu, Jing; Bell, Karen F S; Heron, Samuel; Kind, Peter C; Dando, Owen; Simpson, T Ian; Hardingham, Giles E

    2016-01-01

    Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms.

  6. Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.

    Directory of Open Access Journals (Sweden)

    Nóra M Márkus

    Full Text Available Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs, however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes, differing neuronal subtype (CA3 vs. CA1 hippocampus and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms.

  7. Preparation and preliminary biological evaluation of radiogallium-labeled DTPA-amlodipine complex for possible L-type calcium channel imaging

    International Nuclear Information System (INIS)

    A DTPA-conjugated amlodipine analog (DTPA-AMLO) 3, was prepared for possible voltage gated calcium channel imaging after radiolabeling with Ga-67. [67Ga]-DTPA-AMLO complex was prepared starting [67Ga]gallium chloride and DTPA-AMLO in 60-90 min at 50-60 C in phosphate buffer. The partition co-efficient and stability of the tracer was determined in final solution (25 C) and presence of human serum (37 C) up to 24 h. The biodistribution of the labeled compound in wild-type rats were determined up to 72 h using organ counting and SPECT. The radiolabled complex was prepared in high radiochemical purity (>96%, RTLC and >98% HPLC) and significant specific activity (7-10 GBq/mmol). The log P for the complex was calculated as -0.594, consistent with a water soluble complex. The tracer is mostly washed out through kidneys which were in full compliance with the amlodipine metabolism and imaging studies demonstrated the same behavior. The tracer uptake in organs with smooth muscles was observed in stomach, colon as well as intestine.

  8. Nonlinear Color--Metallicity Relations of Globular Clusters. VI. On Calcium II Triplet Based Metallicities of Globular Clusters in Early-type Galaxies

    CERN Document Server

    Chung, Chul; Lee, Sang-Yoon; Lee, Young-Wook

    2016-01-01

    The metallicity distribution function of globular clusters (GCs) in galaxies is a key to understanding galactic formation and evolution. The calcium II triplet (CaT) index has recently become a popular metal abundance indicator thanks to its sensitivity to GC metallicity. Here we revisit and assess the reliability of CaT as a metallicity indicator using our new stellar population synthesis simulations based on empirical, high-resolution fluxes. The model shows that the CaT strength of old ($>$ 10 Gyr) GCs is proportional to ${\\rm [Fe/H]}$ below $-0.5$. In the modest metal-rich regime, however, CaT does not increase anymore with ${\\rm [Fe/H]}$ due to the little contribution from coolest red giant stars to the CaT absorption. The nonlinear nature of the color--$CaT$ relation is confirmed by the observations of GCs in nearby early-type galaxies. This indicates that the CaT should be used carefully when deriving metallicities of metal-rich stellar populations. Our results offer an explanation for the observed sha...

  9. Preparation and preliminary biological evaluation of radiogallium-labeled DTPA-amlodipine complex for possible L-type calcium channel imaging

    Energy Technology Data Exchange (ETDEWEB)

    Firuzyar, Tahereh; Shafiee-Ardestani, Mehdi; Khalaj, Ali [Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of). Faculty of Pharmacy; Jalilian, Amir R.; Fazaeli, Yousef; Aboudzadeh, Mohammad Reza [Nuclear Science and Technology Research Institute (NSTRI), Tehran (Iran, Islamic Republic of). Radiopharmacy Research Group

    2014-07-01

    A DTPA-conjugated amlodipine analog (DTPA-AMLO) 3, was prepared for possible voltage gated calcium channel imaging after radiolabeling with Ga-67. [{sup 67}Ga]-DTPA-AMLO complex was prepared starting [{sup 67}Ga]gallium chloride and DTPA-AMLO in 60-90 min at 50-60 C in phosphate buffer. The partition co-efficient and stability of the tracer was determined in final solution (25 C) and presence of human serum (37 C) up to 24 h. The biodistribution of the labeled compound in wild-type rats were determined up to 72 h using organ counting and SPECT. The radiolabled complex was prepared in high radiochemical purity (>96%, RTLC and >98% HPLC) and significant specific activity (7-10 GBq/mmol). The log P for the complex was calculated as -0.594, consistent with a water soluble complex. The tracer is mostly washed out through kidneys which were in full compliance with the amlodipine metabolism and imaging studies demonstrated the same behavior. The tracer uptake in organs with smooth muscles was observed in stomach, colon as well as intestine.

  10. Inhibition of Glycolate Oxidase With Dicer-substrate siRNA Reduces Calcium Oxalate Deposition in a Mouse Model of Primary Hyperoxaluria Type 1.

    Science.gov (United States)

    Dutta, Chaitali; Avitahl-Curtis, Nicole; Pursell, Natalie; Larsson Cohen, Marita; Holmes, Benjamin; Diwanji, Rohan; Zhou, Wei; Apponi, Luciano; Koser, Martin; Ying, Bo; Chen, Dongyu; Shui, Xue; Saxena, Utsav; Cyr, Wendy A; Shah, Anee; Nazef, Naim; Wang, Weimin; Abrams, Marc; Dudek, Henryk; Salido, Eduardo; Brown, Bob D; Lai, Chengjung

    2016-04-01

    Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, metabolic disorder caused by mutations of alanine-glyoxylate aminotransferase (AGT), a key hepatic enzyme in the detoxification of glyoxylate arising from multiple normal metabolic pathways to glycine. Accumulation of glyoxylate, a precursor of oxalate, leads to the overproduction of oxalate in the liver, which accumulates to high levels in kidneys and urine. Crystalization of calcium oxalate (CaOx) in the kidney ultimately results in renal failure. Currently, the only treatment effective in reduction of oxalate production in patients who do not respond to high-dose vitamin B6 therapy is a combined liver/kidney transplant. We explored an alternative approach to prevent glyoxylate production using Dicer-substrate small interfering RNAs (DsiRNAs) targeting hydroxyacid oxidase 1 (HAO1) mRNA which encodes glycolate oxidase (GO), to reduce the hepatic conversion of glycolate to glyoxylate. This approach efficiently reduces GO mRNA and protein in the livers of mice and nonhuman primates. Reduction of hepatic GO leads to normalization of urine oxalate levels and reduces CaOx deposition in a preclinical mouse model of PH1. Our results support the use of DsiRNA to reduce liver GO levels as a potential therapeutic approach to treat PH1. PMID:26758691

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

    Science.gov (United States)

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

    1996-05-01

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

  12. Enhanced valine production in Corynebacterium glutamicum with defective H+-ATPase and C-terminal truncated acetohydroxyacid synthase.

    Science.gov (United States)

    Wada, Masaru; Hijikata, Nowaki; Aoki, Ryo; Takesue, Nobuchika; Yokota, Atsushi

    2008-11-01

    We have reported increased glutamate production by a mutant of Corynebacterium glutamicum ATCC14067 (strain F172-8) with reduced H(+)-ATPase activity under biotin-limiting culture conditions (Aoki et al. Biosci. Biotechnol. Biochem., 69, 1466-1472 (2005)). In the present study, we examined valine production by an H(+)-ATPase-defective mutant of C. glutamicum. Using the double-crossover chromosome replacement technique, we constructed a newly defined H(+)-ATPase-defective mutant from ATCC13032. After transforming the new strain (A-1) with a C-terminal truncation of acetohydroxyacid synthase gene (ilvBN), valine production increased from 21.7 mM for the wild-type strain to 46.7 mM for the A-1 in shaking flask cultures with 555 mM glucose. Increased production of the valine intermediate acetoin was also observed in A-1, and was reduced by inserting acetohydroxyacid isomeroreductase gene (ilvC) into the ilvBN plasmid. After transformation with this new construct, valine production increased from 38.3 mM for the wild-type strain to 95.7 mM for A-1 strain. To the best of our knowledge, this is the first report indicating that an H(+)-ATPase-defective mutant of C. glutamicum is capable of valine production. Our combined results with glutamate and valine suggest that the H(+)-ATPase defect is also effective in the fermentative production of other practical compounds.

  13. The L-type calcium channel Cav1.3 is required for proper hippocampal neurogenesis and cognitive functions.

    Science.gov (United States)

    Marschallinger, Julia; Sah, Anupam; Schmuckermair, Claudia; Unger, Michael; Rotheneichner, Peter; Kharitonova, Maria; Waclawiczek, Alexander; Gerner, Philipp; Jaksch-Bogensperger, Heidi; Berger, Stefan; Striessnig, Jörg; Singewald, Nicolas; Couillard-Despres, Sebastien; Aigner, Ludwig

    2015-12-01

    L-type voltage gated Ca(2+) channels (LTCCs) are widely expressed within different brain regions including the hippocampus. The isoforms Cav1.2 and Cav1.3 have been shown to be involved in hippocampus-dependent learning and memory, cognitive functions that require proper hippocampal neurogenesis. In vitro, functional LTCCs are expressed on neuronal progenitor cells, where they promote neuronal differentiation. Expression of LTCCs on neural stem and progenitor cells within the neurogenic regions in the adult brain in vivo has not been examined so far, and a contribution of the individual isoforms Cav1.2 and Cav1.3 to adult neurogenesis remained to be clarified. To reveal the role of these channels we first evaluated the expression patterns of Cav1.2 and Cav1.3 in the hippocampal dentate gyrus and the subventricular zone (SVZ) in adult (2- and 3-month old) and middle-aged (15-month old) mice on mRNA and protein levels. We performed immunohistological analysis of hippocampal neurogenesis in adult and middle-aged Cav1.3(-/-) mice and finally addressed the importance of Cav1.3 for hippocampal function by evaluating spatial memory and depression-like behavior in adult Cav1.3(-/-) mice. Our results showed Cav1.2 and Cav1.3 expression at different stages of neuronal differentiation. While Cav1.2 was primarily restricted to mature NeuN(+) granular neurons, Cav1.3 was expressed in Nestin(+) neural stem cells and in mature NeuN(+) granular neurons. Adult and middle-aged Cav1.3(-/-) mice showed severe impairments in dentate gyrus neurogenesis, with significantly smaller dentate gyrus volume, reduced survival of newly generated cells, and reduced neuronal differentiation. Further, Cav1.3(-/-) mice showed impairment in the hippocampus dependent object location memory test, implicating Cav1.3 as an essential element for hippocampus-associated cognitive functions. Thus, modulation of LTCC activities may have a crucial impact on neurogenic responses and cognition, which should be

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  15. Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

    Energy Technology Data Exchange (ETDEWEB)

    Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.

    1986-01-01

    The H translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ approx. = 70,000 and approx. = 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-( UC)ethylmaleimide and 7-chloro-4-nitro( UC)benzo-2-oxa-1,3-diazole, labeled the M/sub r/ approx. = 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-( UC)dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ approx. = 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10V, 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ approx. = 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F0F1 ATPases.

  16. Effects of percutaneous midband pulse current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats

    Directory of Open Access Journals (Sweden)

    Yi-zong ZHAI

    2015-06-01

    Full Text Available Objective To explore the effects of percutaneous impulsive current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats, in order to investigate the effect of exercise-induced fatigue. Methods Seventy-two 8-week old male Wistar rats were randomly divided into 4 groups (18 each: control group (group A, fatigue group (group B, stimulation before fatigue group (group C and stimulation after fatigue group (group D. Exhaustion of animals in B, C and D groups were reproduced by prolonged swimming. Current stimulation (1024Hz, 10mA, current cycle 1sec for 20 minutes was given to the rats of group C before swimming, and to those in group D after exhaustion. At the weekend of 1st, 3rd and 5th week after modeling, the rats were sacrificed in batches from each group (6 each. The activities of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase were determined by spectrophotometry, and Bradfood protein quantification was employed to quantitate the protein in rats' hepatic mitochondria. Results No significant difference was found in swimming-exhaustion time among 3 groups at the first weekend (P>0.05, while the swimming-exhaustion time was significantly prolonged at the 3rd and 5th weekends in group D than in group B and C (P0.05, while the enzyme activities were obviously lower at the 3rd and 5th weekend in group B than that in groups A, C and D (P<0.05, and they were also lower in group C than that in group D (P<0.05. Conclusions Exercise-induced fatigue can lower the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase. Percutaneous pulsive current stimulating hepatic region of exercise-induced fatigued rats may improve the enzyme activity, reduce the concentration of free calcium and calcium overload in mitochondria, stimulate the oxidative phosphorylation, accelerate the rate of respiratory chain, promote exercise endurance and score, and

  17. A plasma membrane H+-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana

    OpenAIRE

    Ivan R Baxter; Young, Jeffery C.; Armstrong, Gordon; Foster, Nathan; Bogenschutz, Naomi; Cordova, Tatiana; Peer, Wendy Ann; Hazen, Samuel P.; Murphy, Angus S.; Harper, Jeffrey F.

    2005-01-01

    The plasma membrane in plant cells is energized with an electrical potential and proton gradient generated through the action of H+ pumps belonging to the P-type ATPase superfamily. The Arabidopsis genome encodes 11 plasma membrane H+ pumps. Auto-inhibited H+-ATPase isoform 10 (AHA10) is expressed primarily in developing seeds. Here we show that four independent gene disruptions of AHA10 result in seed coats with a transparent testa (tt) phenotype (light-colored seeds). A quantitative analysi...

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

    Science.gov (United States)

    Nedukha, Olena

    2016-07-01

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

  19. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Bækgaard, Lone;

    2010-01-01

    of calcium-bound calmodulin (Ca(2+)-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The......Plasma-membrane Ca(2+)-ATPases (PMCAs) are calcium pumps that expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. They are of major physiological importance, with different isoforms being essential, for example...... crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 A, beta = 113.2 degrees. A complete data set was collected to 3.0 A resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin....

  20. Trypsin-induced ATPase activity in potato mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Jung, D.W.; Laties, G.G.

    1976-04-01

    Potato mitochondria (Solanum tuberosum var. Russet Burbank), which readily phosphorylate ADP in oxidative phosphorylation, show low levels of ATPase activity which is stimulated neither by Mg/sup 2 +/, 2,4-dinitrophenol, incubation with respiratory substrates, nor disruption by sonication or treatment with Triton X-100, individually or in concert. Treatment of disrupted potato mitochondria with trypsin stimulates Mg/sup 2 +/-dependent, oligomycin-sensitive ATPase activity 10- to 15-fold, suggesting the presence of an ATPase inhibitor protein. Trypsin-induced ATPase activity was unaffected by uncoupler. Oligomycin-sensitive ATPase activity decreases as exposure to trypsin is increased. Incubation at alkaline pH or heating at 60/sup 0/C for 2 minutes also activates ATPase of sonicated potato mitochondria. Disruption of cauliflower (Brassica oleracea), red sweet potato (Ipomoea batatas), and carrot (Daucus carota) mitochondria increases ATPase activity, which is further enhanced by treatment with trypsin. The significance of the tight association of the inhibitor protein and ATPase in potato mitochondria is not clear.

  1. Calcium paradox and calcium entry blockers

    NARCIS (Netherlands)

    Ruigrok, T.J.C.; Slade, A.M.; Nayler, W.G.; Meijler, F.L.

    1984-01-01

    Reperfusion of isolated hearts with calcium-containing solution after a short period of calcium-free perfusion results in irreversible cell damage (calcium paradox). This phenomenon is characterized by an excessive influx of calcium into the cells, the rapid onset of myocardial contracture, exhausti

  2. V-ATPase-mediated phagosomal acidification is impaired by Streptococcus pyogenes through Mga-regulated surface proteins.

    Science.gov (United States)

    Nordenfelt, Pontus; Grinstein, Sergio; Björck, Lars; Tapper, Hans

    2012-11-01

    Streptococcus pyogenes, a significant bacterial pathogen in humans, interferes with the membrane traffic of human neutrophils and survives following phagocytosis. The mechanism(s) behind this property is not known, but in contrast to wild-type bacteria, mutant bacteria lacking virulence factors regulated by the transcriptional regulator Mga, are phagocytosed and killed. In the present work we investigated whether differences in phagosomal acidification may contribute to this difference. Phagosomal pH in neutrophil-differentiated HL-60 cells was studied by fluorescence ratio imaging, and phagosomes containing wild-type S. pyogenes bacteria of the M1 serotype exhibited little or no acidification, whereas Mga mutant bacteria were found in more acidic phagosomes. With phagosomes containing these bacteria, proton delivery was inhibited by adding folimycin, a vacuolar-type adenosine triphosphatase (V-ATPase) inhibitor. This inhibitor had no effect on phagosomes containing wild-type bacteria, indicating either inactivation or removal of V-ATPases by the bacteria. Analysis of isolated bacteria-containing phagosomes confirmed the latter scenario and showed a more efficient delivery of V-ATPases to phagosomes containing Mga mutant bacteria. The results demonstrate that V-ATPase-mediated phagosomal proton delivery is reduced during phagocytosis of wild-type S. pyogenes, leading to impaired acidification, and that surface proteins of the mga regulon are responsible for this effect. PMID:22981599

  3. Role of tumour necrosis factor-a in the regulation of T-type calcium channel current in HL-1 cells.

    Science.gov (United States)

    Rao, Fang; Xue, Yu-Mei; Wei, Wei; Yang, Hui; Liu, Fang-Zhou; Chen, Shao-Xian; Kuang, Su-Juan; Zhu, Jie-Ning; Wu, Shu-Lin; Deng, Chun-Yu

    2016-07-01

    Increasing evidence indicates that inflammation contributes to the initiation and perpetuation of atrial fibrillation (AF). Although tumour necrosis factor (TNF)-α levels are increased in patients with AF, the role of TNF-α in the pathogenesis of AF remains unclear. Besides L-type Ca(2+) currents (IC a,L ), T-type Ca(2+) currents (IC a,T ) also plays an important role in the pathogenesis of AF. This study was designed to use the whole-cell voltage-clamp technique and biochemical assays to explore if TNF-α is involved in the pathogenesis of AF through regulating IC a,T in atrial myocytes. It was found that compared with sinus rhythm (SR) controls, T-type calcium channel (TCC) subunit mRNA levels were decreased, while TNF-α expression levels were increased, in human atrial tissue from patients with AF. In murine atrial myocyte HL-1 cells, after culturing for 24 h, 12.5, 25 and 50 ng/mL TNF-α significantly reduced the protein expression levels of the TCC α1G subunit in a concentration-dependent manner. The peak current was reduced by the application of 12.5 or 25 ng/mL TNF-α in a concentration-dependent manner (from -15.08 ± 1.11 pA/pF in controls to -11.89 ± 0.83 pA/pF and -8.54 ± 1.55 pA/pF in 12.5 or 25 ng/mL TNF-α group respectively). TNF-α application also inhibited voltage-dependent inactivation of IC a,T, shifted the inactivation curve to the left. These results suggest that TNF-α is involved in the pathogenesis of AF, probably via decreasing IC a,T current density in atrium-derived myocytes through impaired channel function and down-regulation of channel protein expression. This pathway thus represents a potential pathogenic mechanism in AF. PMID:27119319

  4. Z944, a Novel Selective T-Type Calcium Channel Antagonist Delays the Progression of Seizures in the Amygdala Kindling Model.

    Directory of Open Access Journals (Sweden)

    Pablo Miguel Casillas-Espinosa

    Full Text Available Temporal lobe epilepsy (TLE is the most common form of drug resistant epilepsy. Current treatment is symptomatic, suppressing seizures, but has no disease modifying effect on epileptogenesis. We examined the effects of Z944, a potent T-type calcium channel antagonist, as an anti-seizure agent and against the progression of kindling in the amygdala kindling model of TLE. The anti-seizure efficacy of Z944 (5mg/kg, 10mg/kg, 30mg/kg and 100mg/kg was assessed in fully kindled rats (5 class V seizures as compared to vehicle, ethosuximide (ETX, 100mg/kg and carbamazepine (30mg/kg. Each animal received the seven treatments in a randomised manner. Seizure class and duration elicited by six post-drug stimulations was determined. To investigate for effects in delaying the progression of kindling, naive animals received Z944 (30mg/kg, ETX (100mg/kg or vehicle 30-minutes prior to each kindling stimulation up to a maximum of 30 stimulations, with seizure class and duration recorded after each stimulation. At the completion of drug treatment, CaV3.1, CaV3.2 and CaV3.3 mRNA expression levels were assessed in the hippocampus and amygdala using qPCR. Z944 was not effective at suppressing seizures in fully kindled rats compared to vehicle. Animals receiving Z944 required significantly more stimulations to evoke a class III (p<0.05, IV (p<0.01 or V (p<0.0001 seizure, and to reach a fully kindled state (p<0.01, than animals receiving vehicle. There was no significant difference in the mRNA expression of the T-type Ca2+ channels in the hippocampus or amygdala. Our results show that selectively targeting T-type Ca2+ channels with Z944 inhibits the progression of amygdala kindling. This could be a potential for a new therapeutic intervention to mitigate the development and progression of epilepsy.

  5. Isolation and characterization of a specific endogenous Na/sup +/, K/sup +/-ATPase inhibitor from bovine adrenal

    Energy Technology Data Exchange (ETDEWEB)

    Tamura, M.; Lam, T.T.; Inagami, T.

    1988-06-14

    In order to identify a specific endogenous Na/sup +/,K/sup +/-ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na/sup +/,K/sup +/-ATPase; (ii) competitive displacing activity against (/sup 3/H)ouabain binding to the enzyme; (iii) inhibitory activity for /sup 86/Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C/sub 18/ open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na/sup +/,K/sup +/-ATPase. These results strongly suggest that this water-soluble nonpeptidic Na/sup +/,K/sup +/-ATPase inhibitor may be a specific endogenous regulator for the ATPase.

  6. Rice Phospholipase Dα is Involved in Salt Tolerance by the Mediation of H+-ATPase Activity and Transcription

    Institute of Scientific and Technical Information of China (English)

    Peng Shen; Rong Wang; Wen Jing; Wenhua Zhang

    2011-01-01

    Phospholipase Dα (PLDα) is involved in plant response to salt stress, but the mechanisms remain unclear.We investigated rice PLDα (OsPLDα) localization and its effect on tonoplast (TP) and plasma membrane (PM) H+-ATPase activity and transcription in response to NaCl. When rice suspension-cultured cells were treated with 100 mM NaCI, PLDα activity in cell extracts showed a transient activation with a threefold increase at 1 h. The amount of OsPLDα protein decreased slightly in the cytosolic fractions, whereas it increased significantly in the TP after NaCI treatment. OsPLDα1 knockdown cells were developed using RNA interference (RNAi) methods. The increase in TP and PM H+-ATPase activity induced by NaCl was significantly inhibited in OsPLDα1-RNAi cells. Knockdown of OsPLDα1 prevented the NaCl-induced increase in the transcript level of OsVHA-A (encodes TP H+-ATPase) and OSA2 (encodes PM H+-ATPase),as well as OsNHX1 (encodes TP Na+/H+ antiporter). The cells died more in OsPLDα1-RNAi mutant than in wild type when they were treated with NaCl. These results suggest that OsPLDα is involved in salt tolerance in rice through the mediation of H+-ATPase activity and transcription.

  7. Different Na+/K+-ATPase signal pathways was involved in the increase of [Ca2+]i induced by strophanthidin in normal and failing isolated guinea pig ventricular myocytes

    Institute of Scientific and Technical Information of China (English)

    Ya-juan QI; Su-wen SU; Jun-xia LI; Ji-he LI; Fang GUO; Yong-li WANG

    2008-01-01

    Aim: To determine whether different Na+/K+-ATPase signal transduction pathways have positive inotropic effects on normal ventricular myocytes (NC) and failing ventricular myocytes (FC), and are involved in an increase of [Ca2+]i induced by strophanthidin (Str). Methods: A guinea pig model of congestive heart failure was made by constricting descending aorta. The left ventricular myocytes were enzymatically isolated. The effects of 25 μmol/L Str with different signal-transducing inhibitors on contractility and the calcium transient of NC or FC from guinea pigs were simultaneously assessed and compared with those in the 25 μmol/L Str-only group by a video-based, motion-edge detection system. Results: Str at 1, 10, and 25 μmol/L in NC and Str at 0.1, 1, 10, and 25 μmol/L) in FC elevated the calcium transient amplitude and increased the positive inotropic effects in a concentration-dependent manner, respectively. At the same concentration, the effects of Str were more potent in FC than in NC. In FC, both the mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) signal transduction pathway of Na+/K+-ATPase were involved in the increase of the calcium transient induced by Str, but only activation of the MAPK pathway increased the calcium transient in NC. However, only the ROS pathway was involved in positive inotropic effects both in NC and FC. Conclusion: The present study suggests that Na+/K+-ATPase signaling pathways involved in the inotropic effects of Str in NC and FC are consistent, and Na+/K+-ATPase signaling pathways involved in the increase of [Ca2+]i by Str in NC and FC are different.

  8. Z944, a Novel Selective T-Type Calcium Channel Antagonist Delays the Progression of Seizures in the Amygdala Kindling Model.

    Science.gov (United States)

    Casillas-Espinosa, Pablo Miguel; Hicks, Ashleigh; Jeffreys, Amy; Snutch, Terrance P; O'Brien, Terence J; Powell, Kim L

    2015-01-01

    Temporal lobe epilepsy (TLE) is the most common form of drug resistant epilepsy. Current treatment is symptomatic, suppressing seizures, but has no disease modifying effect on epileptogenesis. We examined the effects of Z944, a potent T-type calcium channel antagonist, as an anti-seizure agent and against the progression of kindling in the amygdala kindling model of TLE. The anti-seizure efficacy of Z944 (5mg/kg, 10mg/kg, 30mg/kg and 100mg/kg) was assessed in fully kindled rats (5 class V seizures) as compared to vehicle, ethosuximide (ETX, 100mg/kg) and carbamazepine (30mg/kg). Each animal received the seven treatments in a randomised manner. Seizure class and duration elicited by six post-drug stimulations was determined. To investigate for effects in delaying the progression of kindling, naive animals received Z944 (30mg/kg), ETX (100mg/kg) or vehicle 30-minutes prior to each kindling stimulation up to a maximum of 30 stimulations, with seizure class and duration recorded after each stimulation. At the completion of drug treatment, CaV3.1, CaV3.2 and CaV3.3 mRNA expression levels were assessed in the hippocampus and amygdala using qPCR. Z944 was not effective at suppressing seizures in fully kindled rats compared to vehicle. Animals receiving Z944 required significantly more stimulations to evoke a class III (pkindled state (pkindling. This could be a potential for a new therapeutic intervention to mitigate the development and progression of epilepsy.

  9. Anion-sensitive regions of L-type CaV1.2 calcium channels expressed in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Norbert Babai

    Full Text Available L-type calcium currents (I(Ca are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of I(Ca and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca(2+ channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from approximately 75%-80% to approximately 50% by omitting beta subunits but unaffected by omitting alpha(2delta subunits. Similarly, gluconate inhibition was reduced to approximately 50% by deleting an alpha1 subunit N-terminal region of 15 residues critical for beta subunit interactions regulating open probability. Omitting beta subunits with this mutant alpha1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different beta subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from approximately 75%-80% to approximately 50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to approximately 60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to approximately 25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving beta subunit interactions with the N terminus and a short C terminal region.

  10. Sevoflurane postconditioning alleviates action potential duration shortening and L-type calcium current suppression induced by ischemia/reperfusion injury in rat epicardial myocytes

    Institute of Scientific and Technical Information of China (English)

    GONG Jun-song; YAO Yun-tai; FANG Neng-xin; HUANG Jian; LI Li-huan

    2012-01-01

    Background It has been proved that sevoflurane postconditioning (SpostC) could protect the heart against myocardial ischemia/reperfusion injury,however,there has been few research focused on the electrophysiological effects of SpostC.The objective of the study was to investigate the effects of SpostC on action potential duration (APD) and L-type calcium current (ICa,L) in isolated cardiomyocytes.Methods Langendorff perfused SD rat hearts were randomly assigned to one of the time control (TC),ischemia/reperfusion (I/R,25 minutes of ischemia followed by 30 minutes of reperfusion),and SpostC (postconditioned with 3% sevoflurane) groups.At the end of reperfusion,epicardial myocytes were dissociated enzymatically for patch clamp studies.Results Sevoflurane directly prolonged APD and decreased peak Ica,L densities in epicardial myocytes of the TC group (P<0.05).I/R injury shortened APD and decreased peak Ica,L densities in epicardial myocytes of the I/R group (P<0.05).SpostC prolonged APD and increased peak Ica,L densities in epicardial myocytes exposed to I/R injury (P<0.05).SpostC decreased intracellular reactive oxygen species (ROS) levels,reduced the incidence of ventricular tachycardia and ventricular fibrillation,and decreased reperfusion arrhythmia scores compared with the I/R group (all P<0.05).Conclusions SpostC attenuates APDshortening and ICa,L suppression induced by I/R injury.The regulation of APD and Ica,L by SpostC might be related with intracellular ROS modulation,which contributes to the alleviation of reperfusion ventricular arrhythmia.Chin Med J 2012;125(19):3485-3491

  11. Association between calcium sensing receptor gene polymorphisms and chronic pancreatitis in a US population: Role of serine protease inhibitor Kazal 1type and alcohol

    Institute of Scientific and Technical Information of China (English)

    Venkata Muddana; David C Whitcomb; Janette Lamb; Julia B Greer; Beth Elinoff; Robert H Hawes; Peter B cotton; Michelle A Anderson; Randall E Brand; Adam Slivka

    2008-01-01

    AIM: To test the hypothesis that calcium sensing receptor (CASR) polymorphisms are associated with chronic pancreatitis (CP), and to determine whether serine protease inhibitor Kazal 1type (SPfNK1) N34S or alcohol are necessary co-factors in its etiology.METHODS: Initially, 115 subjects with pancreatitis and 66 controls were evaluated, of whom 57 patients and 21 controls were predetermined to carry the high-risk SP/NK1 N34S polymorphism. We sequenced CASR gene exons 2, 3, 4, 5 and 7, areas containing the majority of reported polymorphisms and novel mutations. Based on the initial results, we added 223 patients and 239 controls to analyze three common nonsynonymous single nucleotide polymorphisrns (SNPs) in exon 7 (A986S, R990G, and Q1011E).RESULTS: The CASR exon 7 R990G polymorphism was significantly associated with CP (OR, 2.01; 95% CI, 1.12-3.59; P = 0.015). The association between CASR R990G and CP was stronger in subjects who reported moderate or heavy alcohol consumption (OR,3.12; 95% CI, 1.14-9.13; P = 0.018). There was no association between the various CASR genotypes and SPINK1 N34S in pancreatitis. None of the novel CASR polymorphisms reported from Germany and India was detected.CONCLUSION: Our United States-based study confirmed an association of CASR and CP and for the first time demonstrated that CASR R990G is a significant risk factor for CP. We also conclude that the risk of CP with CASR R990G is increased in subjects with moderate to heavy alcohol consumption.

  12. Effects of octreotide on expression of L-type voltage-operated calcium channels and on intracellular Ca2+ in activated hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    丁惠国; 王宝恩; 贾继东; 夏华向; 王振宇; 赵春惠; 徐燕琳

    2004-01-01

    Background The contractility of hepatic stellate cells (HSCs) may play an important role in the pathogenesis of cirrhosis with portal hypertension. The aim of this study was to research the effects of octreotide, an analogue of somatostatin, on intracellular Ca2+ and on the expression of L-type voltage-operated calcium channels (L-VOCCs) in activated HSCs, and to try to survey the use of octreotide in treatment and prevention of cirrhosis with portal hypertension complications. Methods HSC-T6, an activated HSCs line, was plated on small glass coverslips in 35-mm culture dishes at a density of 1×105/ml, and incubated in DMEM media for 24 hours. After the cells were loaded with Fluo-3/AM, intracellular Ca2+ was measured by Laser Scanning Confocal Microscopy (LSCM). The dynamic changes in activated HSCs of intracellular Ca2+, stimulated by octreotide, endothelin-1, and KCl, respectively, were also determined by LSCM. Each experiment was repeated six times. L-VOCC expression in HSCs was estimated by immunocytochemistry. Results After octreotide stimulation, a signifcant decrease in the intracellular Ca2+ of activated HSCs was observed. However, octreotide did not inhibit the increases in intracellular Ca2+ after stimulation by KCl and endothelin-1. Moreover, octreotide did not significantly affect L-VOCC expression. These results suggest that neither L-VOCC nor endothelin-1 receptors in activated HSCs are inhibited by octreotide. Conclusions Octreotide may decrease portal hypertension and intrahepatic vascular tension by inhibiting activated HSCs contractility through decreases in intracellular Ca2+. The somatostatin receptors in activated HSCs may be inhibited by octreotide.

  13. GsAPK, an ABA-activated and calcium-independent SnRK2-type kinase from G. soja, mediates the regulation of plant tolerance to salinity and ABA stress.

    Directory of Open Access Journals (Sweden)

    Liang Yang

    Full Text Available Plant Snf1 (sucrose non-fermenting-1 related protein kinase (SnRK, a subfamily of serine/threonine kinases, has been implicated as a crucial upstream regulator of ABA and osmotic signaling as in many other signaling cascades. In this paper, we have isolated a novel plant specific ABA activated calcium independent protein kinase (GsAPK from a highly salt tolerant plant, Glycine soja (50109, which is a member of the SnRK2 family. Subcellular localization studies using GFP fusion protein indicated that GsAPK is localized in the plasma membrane. We found that autophosphorylation and Myelin Basis Protein phosphorylation activity of GsAPK is only activated by ABA and the kinase activity also was observed when calcium was replaced by EGTA, suggesting its independence of calcium in enzyme activity. We also found that cold, salinity, drought, and ABA stress alter GsAPK gene transcripts and heterogonous overexpression of GsAPK in Arabidopsis alters plant tolerance to high salinity and ABA stress. In summary, we demonstrated that GsAPK is a Glycine soja ABA activated calcium independent SnRK-type kinase presumably involved in ABA mediated stress signal transduction.

  14. GsAPK, an ABA-activated and calcium-independent SnRK2-type kinase from G. soja, mediates the regulation of plant tolerance to salinity and ABA stress.

    Science.gov (United States)

    Yang, Liang; Ji, Wei; Gao, Peng; Li, Yong; Cai, Hua; Bai, Xi; Chen, Qin; Zhu, Yanming

    2012-01-01

    Plant Snf1 (sucrose non-fermenting-1) related protein kinase (SnRK), a subfamily of serine/threonine kinases, has been implicated as a crucial upstream regulator of ABA and osmotic signaling as in many other signaling cascades. In this paper, we have isolated a novel plant specific ABA activated calcium independent protein kinase (GsAPK) from a highly salt tolerant plant, Glycine soja (50109), which is a member of the SnRK2 family. Subcellular localization studies using GFP fusion protein indicated that GsAPK is localized in the plasma membrane. We found that autophosphorylation and Myelin Basis Protein phosphorylation activity of GsAPK is only activated by ABA and the kinase activity also was observed when calcium was replaced by EGTA, suggesting its independence of calcium in enzyme activity. We also found that cold, salinity, drought, and ABA stress alter GsAPK gene transcripts and heterogonous overexpression of GsAPK in Arabidopsis alters plant tolerance to high salinity and ABA stress. In summary, we demonstrated that GsAPK is a Glycine soja ABA activated calcium independent SnRK-type kinase presumably involved in ABA mediated stress signal transduction.

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

    Directory of Open Access Journals (Sweden)

    Rosa L López-Marqués

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

  16. IgG from Amyotrophic Lateral Sclerosis Patients Increases Current Through P-Type Calcium Channels in Mammalian Cerebellar Purkinje Cells and in Isolated Channel Protein in Lipid Bilayer

    Science.gov (United States)

    Llinas, R.; Sugimori, M.; Cherksey, B. D.; Smith, R. Glenn; Delbono, O.; Stefani, E.; Appel, S.

    1993-12-01

    The effect of the IgG from amyotrophic lateral sclerosis (ALS) patients was tested on the voltage-dependent barium currents (IBa) in mammalian dissociated Purkinje cells and in isolated P-type calcium channels in lipid bilayers. Whole cell clamp of Purkinje cells demonstrates that ALS IgG increases the amplitude of IBa without modifying their voltage kinetics. This increased IBa could be blocked by a purified nonpeptide toxin from Agelenopsis aperta venom (purified funnel-web spider toxin) or by a synthetic polyamine analog (synthetic funnel-web spider toxin) and by a peptide toxin from the same spider venom, ω-Aga-IVA. Similar results were obtained on single-channel recordings from purified P channel protein. The addition of ALS IgG increased single-channel IBa open time without affecting slope conductance. The results described above were not seen with normal human IgG nor with boiled ALS IgG. It is concluded that ALS IgG enhances inward current through P-type calcium channels. Since P-type Ca2+ channels are present in motoneuron axon terminals, we propose that the enhanced calcium current triggered by ALS IgG may contribute to neuronal damage in ALS.

  17. Regulatory interplay of Cockayne syndrome B ATPase and stress-response gene ATF3 following genotoxic stress

    DEFF Research Database (Denmark)

    Kristensen, Hans-Ulrik Svejstrup; Epanchintsev, Alexey; Rauschendorf, Marc-Alexander;

    2013-01-01

    Cockayne syndrome type B ATPase (CSB) belongs to the SwItch/Sucrose nonfermentable family. Its mutations are linked to Cockayne syndrome phenotypes and classically are thought to be caused by defects in transcription-coupled repair, a subtype of DNA repair. Here we show that after UV-C irradiatio...

  18. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    Science.gov (United States)

    Ledoux, Sarah; Guthrie, Christine

    2016-06-01

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. PMID:27072132

  19. Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

    Directory of Open Access Journals (Sweden)

    Bartlett Marilyn S

    2001-06-01

    Full Text Available Abstract Background Pneumocystis carinii causes pneumonia in immunocompromised patients with a high morbidity and mortality rate, but the interaction between this organism and the host cell is not well understood. The purpose of this research was to study the response of host cells to P. carinii infection on a molecular level. Results The technique of mRNA differential display was used to detect genes whose expression may be affected by P. carinii infection. The nucleotide sequence of one differentially displayed DNA fragment was found to be identical to that of the rat mitochondrial ATPase 6 gene, which is a subunit of the F0F1-ATP synthase complex. A four-fold increase in expression of this gene was verified by Northern blot analysis of total RNA extracted from P. carinii-infected rat lung versus that from mock-infected rat lung. Localization of the cells containing ATPase 6 mRNA was accomplished by in situ hybridization. In sections of non-infected rat lung, these cells were found lining the distal parts of the respiratory tree and in apical areas of the alveoli. Histological location of these cells suggested that they were Clara cells and type II pneumocytes. This hypothesis was confirmed by co-localizing the mRNAs for ATPase 6 and surfactant protein B (SP-B to the same cells by two-color fluorescent in situ hybridization. Conclusions The ATPase 6 gene is over expressed during P. carinii infection, and type II pneumocytes and Clara cells are the cell types responsible for this over-expression.

  20. Factors to consider in the selection of a calcium supplement.

    OpenAIRE

    Shangraw, R F

    1989-01-01

    Calcium supplements are widely used, yet many questions remain as to the absorption of various calcium salts. Because the solubility of many calcium salts is dependent upon pH, the type of salt used, the condition of the patient, and the time of administration should be considered. Studies show that many calcium supplements on the market today do not meet standards of quality established in the "U.S. Pharmacopeia" (USP). Consumers must be discerning about the products they purchase. Calcium s...

  1. The reduction of EPSC amplitude in CA1 pyramidal neurons by the peroxynitrite donor SIN-1 requires Ca2+ influx via postsynaptic non-L-type voltage gated calcium channels.

    Science.gov (United States)

    Zhaowei, Liu; Yongling, Xie; Jiajia, Yang; Zhuo, Yang

    2014-02-01

    The peroxynitrite free radical (ONOO(-)) modulation of miniature excitatory postsynaptic currents (mEPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) was investigated in rat CA1 pyramidal neurons using the whole-cell patch clamp technique. SIN-1(3-morpholino-sydnonimine), which can lead the simultaneous generation of superoxide anion and nitric oxide, and then form the highly reactive species ONOO(-), induced dose-dependent inhibition in amplitudes of both mEPSCs and sEPSCs. The SIN-1 action on mEPSC amplitude was completely blocked by U0126, a selective MEK inhibitor, suggesting that MEK contributed to the action of ONOO(-) on mEPSCs. The effect of SIN-1 was completely occluded either in the presence of the calcium chelator EGTA or the non-selective calcium channel antagonist Cd(2+). Furthermore, the application of nifedipine (20 μM), the L-type calcium channel blocker, had no effect on the ONOO(-)-induced decrease in mEPSC amplitude, excluding a role for L-type voltage-gated Ca(2+) channels in this process. SIN-1 inhibited the frequency of sEPSCs but had no effect on mEPSC frequency, which suggested a presynaptic action potential-dependent the action of ONOO(-) at CA1 pyramidal neuron synapses. The best-known glutamatergic input to CA1 pyramidal neurons is via Schaffer collaterals from CA3 area. However, no changes were observed in slices treated with SIN-1 on the spontaneous firing rates of CA3 pyramidal neurons. These findings suggested that SIN-1 inhibited glutamatergic synaptic transmission of CA1 pyramidal neurons by a postsynaptic non-L-type voltage gated calcium channel-dependent mechanism.

  2. Dissolution of the inorganic phase of bone leading to release of calcium regulates osteoclast survival

    DEFF Research Database (Denmark)

    Nielsen, Rasmus H; Karsdal, Morten A; Sørensen, Mette G;

    2007-01-01

    Osteoclasts are the sole cells possessing the ability to resorb calcified bone matrix. This occurs via secretion of hydrochloric acid mediated by the V-ATPase and the chloride channel ClC-7. Loss of acidification leads to osteopetrosis characterized by ablation of bone resorption and increased...... osteoclast numbers, indicating increased life span of the osteoclasts. To investigate the role of the inorganic phase of bone with respect to osteoclast life span, we used the V-ATPase inhibitor bafilomycin and the calcium uptake antagonist ryanodine on human osteoclasts cultured on calcified and decalcified...... bone slices. Bafilomycin inhibited bone resorption and increased osteoclast survival on calcified but not decalcified bones. Ryanodine attenuated calcium uptake and thereby augmented osteoclast survival on calcified bones. In summary, we found that acidification leading to calcium release from bone...

  3. Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

    OpenAIRE

    Mahmmoud, Yasser A.

    2008-01-01

    Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K+. Studies with purified membranes revealed that CPZ reduced Na+-dependent phosphorylation by interference with Na+ binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K+. Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, ...

  4. Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase.

    Science.gov (United States)

    Mahmmoud, Yasser A

    2008-02-01

    Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K(+). Studies with purified membranes revealed that CPZ reduced Na(+)-dependent phosphorylation by interference with Na(+) binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K(+). Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive (22)Na(+) influx into K(+)-loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump. Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na(+) cycle from an Na(+)/K(+) cycle in the pump. The Na(+) cycle possibly involves transport through the recently characterized Na(+)-specific site. A shift to such an uncoupled mode is believed to produce pumps mediating uncoupled Na(+) efflux by modifying the transport stoichiometry of single pump units. PMID:18230728

  5. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Directory of Open Access Journals (Sweden)

    Qingxi Yue

    Full Text Available Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the

  6. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Science.gov (United States)

    Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

    2016-01-01

    Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase

  7. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

    Science.gov (United States)

    Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  8. Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.

    Science.gov (United States)

    Trampari, Eleftheria; Stevenson, Clare E M; Little, Richard H; Wilhelm, Thomas; Lawson, David M; Malone, Jacob G

    2015-10-01

    The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.

  9. The transport mechanism of bacterial Cu+-ATPases: distinct efflux rates adapted to different function.

    Science.gov (United States)

    Raimunda, Daniel; González-Guerrero, Manuel; Leeber, Blaise W; Argüello, José M

    2011-06-01

    Cu(+)-ATPases play a key role in bacterial Cu(+) homeostasis by participating in Cu(+) detoxification and cuproprotein assembly. Characterization of Archaeoglobus fulgidus CopA, a model protein within the subfamily of P(1B-1) type ATPases, has provided structural and mechanistic details on this group of transporters. Atomic resolution structures of cytoplasmic regulatory metal binding domains (MBDs) and catalytic actuator, phosphorylation, and nucleotide binding domains are available. These, in combination with whole protein structures resulting from cryo-electron microscopy analyses, have enabled the initial modeling of these transporters. Invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites (TM-MBSs). These bind Cu(+) with high affinity in a trigonal planar geometry. The cytoplasmic Cu(+) chaperone CopZ transfers the metal directly to the TM-MBSs; however, loading both of the TM-MBSs requires binding of nucleotides to the enzyme. In agreement with the classical transport mechanism of P-type ATPases, occupancy of both transmembrane sites by cytoplasmic Cu(+) is a requirement for enzyme phosphorylation and subsequent transport into the periplasmic or extracellular milieus. Recent transport studies have shown that all Cu(+)-ATPases drive cytoplasmic Cu(+) efflux, albeit with quite different transport rates in tune with their various physiological roles. Archetypical Cu(+)-efflux pumps responsible for Cu(+) tolerance, like the Escherichia coli CopA, have turnover rates ten times higher than those involved in cuproprotein assembly (or alternative functions). This explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high copper environments. PMID:21210186

  10. Radioprotector modifying influence upon the ion transport ATPase activities

    International Nuclear Information System (INIS)

    The effects of aminothiol and biogenic amine radioprotectors (β-mercaptoethylamine, AET, serotonin, dopamine, histamine) on the basic ion transport enzymes, such as Na, K-ATP ase and Mg, Ca-ATPase activities were investigated in the tissues of numerous organs, with different radiosensitivity in the wistar rats. Experimental results showed that intraperitoneal injection of the used radioprotectors caused preliminary inhibition of the Na, K-ATPase activity in tissues from organs with different radioresistance, but had no influence on the Mg, Ca-ATPase activity in membranes of erythrocytes and rat brain cells. (2 tabs.)

  11. Calcium source (image)

    Science.gov (United States)

    Getting enough calcium to keep bones from thinning throughout a person's life may be made more difficult if that person has ... as a tendency toward kidney stones, for avoiding calcium-rich food sources. Calcium deficiency also effects the ...

  12. Calcium hydroxide poisoning

    Science.gov (United States)

    Hydrate - calcium; Lime milk; Slaked lime ... Calcium hydroxide ... These products contain calcium hydroxide: Cement Limewater Many industrial solvents and cleaners (hundreds to thousands of construction products, flooring strippers, brick cleaners, cement ...

  13. Calcium and bones (image)

    Science.gov (United States)

    Calcium is one of the most important minerals for the growth, maintenance, and reproduction of the human ... body, are continually being re-formed and incorporate calcium into their structure. Calcium is essential for the ...

  14. Coronary Calcium Scan

    Science.gov (United States)

    ... the NHLBI on Twitter. What Is a Coronary Calcium Scan? A coronary calcium scan is a test ... you have calcifications in your coronary arteries. Coronary Calcium Scan Figure A shows the position of the ...

  15. Calcium Pyrophosphate Deposition (CPPD)

    Science.gov (United States)

    ... Patient / Caregiver Diseases & Conditions Calcium Pyrophosphate Deposition (CPPD) Calcium Pyrophosphate Deposition (CPPD) Fast Facts The risk of ... young people, too. Proper diagnosis depends on detecting calcium pyrophosphate crystals in the fluid of an affected ...

  16. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    International Nuclear Information System (INIS)

    Highlights: ► We detect the functional Ca2+ currents and mRNA expression of VDCCL in rMSCs. ► Blockage of VDCCL exert antiproliferative and apoptosis-inducing effects on rMSCs. ► Inhibiting VDCCL can suppress the ability of rMSCs to differentiate into osteoblasts. ► α1C of VDCCL may be a primary functional subunit in VDCCL-regulating rMSCs. -- Abstract: L-type voltage-dependent Ca2+ channels (VDCCL) play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. They have been confirmed to contribute to the functional activities of osteoblasts. Recently, VDCCL expression was reported in mesenchymal stem cells (MSCs), but the role of VDCCL in MSCs is still undetermined. The aim of this study was to determine whether VDCCL may be regarded as a new regulator in the proliferation and osteogenic differentiation of rat MSC (rMSCs). In this study, we examined functional Ca2+ currents (ICa) and mRNA expression of VDCCL in rMSCs, and then suppressed VDCCL using nifedipine (Nif), a VDCCL blocker, to investigate its role in rMSCs. The proliferation and osteogenic differentiation of MSCs were analyzed by MTT, flow cytometry, alkaline phosphatase (ALP), Alizarin Red S staining, RT-PCR, and real-time PCR assays. We found that Nif exerts antiproliferative and apoptosis-inducing effects on rMSCs. ALP activity and mineralized nodules were significantly decreased after Nif treatment. Moreover, the mRNA levels of the osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2), were also down-regulated. In addition, we transfected α1C-siRNA into the cells to further confirm the role of VDCCL in rMSCs, and a similar effect on osteogenesis was found. These results suggest that VDCCL plays a crucial role in the proliferation and osteogenic differentiation of rMSCs.

  17. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wen, Li [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Wang, Yu [Department of Oncology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); Wang, Huan [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Kong, Lingmin [Department of Fundamental Medicine, Cell Engineering Research Centre, Fourth Military Medical University, Xi' an 710032 (China); Zhang, Liang [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Chen, Xin [Department of General Dentistry, The 174th Hospital of Chinese PLA, Xiamen 361003 (China); Ding, Yin, E-mail: dingyin@fmmu.edu.cn [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer We detect the functional Ca{sup 2+} currents and mRNA expression of VDCC{sub L} in rMSCs. Black-Right-Pointing-Pointer Blockage of VDCC{sub L} exert antiproliferative and apoptosis-inducing effects on rMSCs. Black-Right-Pointing-Pointer Inhibiting VDCC{sub L} can suppress the ability of rMSCs to differentiate into osteoblasts. Black-Right-Pointing-Pointer {alpha}1C of VDCC{sub L} may be a primary functional subunit in VDCC{sub L}-regulating rMSCs. -- Abstract: L-type voltage-dependent Ca{sup 2+} channels (VDCC{sub L}) play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. They have been confirmed to contribute to the functional activities of osteoblasts. Recently, VDCC{sub L} expression was reported in mesenchymal stem cells (MSCs), but the role of VDCC{sub L} in MSCs is still undetermined. The aim of this study was to determine whether VDCC{sub L} may be regarded as a new regulator in the proliferation and osteogenic differentiation of rat MSC (rMSCs). In this study, we examined functional Ca{sup 2+} currents (I{sub Ca}) and mRNA expression of VDCC{sub L} in rMSCs, and then suppressed VDCC{sub L} using nifedipine (Nif), a VDCC{sub L} blocker, to investigate its role in rMSCs. The proliferation and osteogenic differentiation of MSCs were analyzed by MTT, flow cytometry, alkaline phosphatase (ALP), Alizarin Red S staining, RT-PCR, and real-time PCR assays. We found that Nif exerts antiproliferative and apoptosis-inducing effects on rMSCs. ALP activity and mineralized nodules were significantly decreased after Nif treatment. Moreover, the mRNA levels of the osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2), were also down-regulated. In addition, we transfected {alpha}1C-siRNA into the cells to further confirm the role of VDCC{sub L} in rMSCs, and a similar effect on osteogenesis was found. These

  18. Electroconvulsive stimulations prevent chronic stress-induced increases in L-type calcium channel mRNAs in the hippocampus and basolateral amygdala

    DEFF Research Database (Denmark)

    Maigaard, Katrine; Pedersen, Ida Hageman; Jørgensen, Anders;

    2012-01-01

    Although affective disorders have high prevalence, morbidity and mortality, we do not fully understand disease etiopathology, nor have we determined the exact mechanisms by which treatment works. Recent research indicates that intracellular calcium ion dysfunction might be involved. Here we use...

  19. Detailed search for protein kinase(s) involved in plasma membrane H+-ATPase activity regulation of yeast cells.

    Science.gov (United States)

    Pereira, Renata R; Castanheira, Diogo; Teixeira, Janaina A; Bouillet, Leoneide E M; Ribeiro, Erica M C; Trópia, Maria M J; Alvarez, Florencia; Correa, Lygia F M; Mota, Bruno E F; Conceição, Luis Eduardo F R; Castro, Ieso M; Brandão, Rogelio L

    2015-03-01

    This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.

  20. Characterization of the flexibility of the peripheral stalk of prokaryotic rotary A-ATPases by atomistic simulations.

    Science.gov (United States)

    Papachristos, Kostas; Muench, Stephen P; Paci, Emanuele

    2016-09-01

    Rotary ATPases are involved in numerous physiological processes, with the three distinct types (F/A/V-ATPases) sharing functional properties and structural features. The basic mechanism involves the counter rotation of two motors, a soluble ATP hydrolyzing/synthesizing domain and a membrane-embedded ion pump connected through a central rotor axle and a stator complex. Within the A/V-ATPase family conformational flexibility of the EG stators has been shown to accommodate catalytic cycling and is considered to be important to function. For the A-ATPase three EG structures have been reported, thought to represent conformational states of the stator during different stages of rotary catalysis. Here we use long, detailed atomistic simulations to show that those structures are conformers explored through thermal fluctuations, but do not represent highly populated states of the EG stator in solution. We show that the coiled coil tail domain has a high persistence length (∼100 nm), but retains the ability to adapt to different conformational states through the presence of two hinge regions. Moreover, the stator network of the related V-ATPase has been suggested to adapt to subunit interactions in the collar region in addition to the nucleotide occupancy of the catalytic domain. The MD simulations reported here, reinforce this observation showing that the EG stators have enough flexibility to adapt to significantly different structural re-arrangements and accommodate structural changes in the catalytic domain whilst resisting the large torque generated by catalytic cycling. These results are important to understand the role the stators play in the rotary-ATPase mechanism. Proteins 2016; 84:1203-1212. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:27177595

  1. Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase for anti-fungal therapy

    Directory of Open Access Journals (Sweden)

    Summer R. Hayek

    2014-01-01

    Full Text Available Vacuolar proton-translocating ATPase (V-ATPase is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae (S. cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans (C. albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens.

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

    Science.gov (United States)

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

    2012-11-01

    intracellular calcium occurs by the entry of extracellular calcium ions through VGCCs which are sensitive to neomycin. N-type and P-type VGCCs are potential candidates because they are observed in osteoblasts and they are sensitive to neomycin. The calcium channels identified in this study provide new insight into mechanisms underlying the targeted repair process which is essential to bone adaptation.

  3. Dietary calcium attenuates platelet aggregation and intracellular Ca2+ mobilization in spontaneously hypertensive rats

    Science.gov (United States)

    Otsuka, K.; Watanabe, M.; Yue, Q.; McCarron, D. A.; Hatton, D.

    1997-01-01

    Spontaneously hypertensive rats (SHR) are known to be blood pressure sensitive to dietary calcium. The effects of dietary calcium on platelet aggregation and intracellular Ca2+ mobilization were assessed by turbidimetric methods and fura-2 methods, respectively, in washed platelets of SHR. Ca2+ ATPase activity was examined in aortic membrane fractions. Six weeks of dietary calcium supplementation attenuated the increase of systolic blood pressure (SBP 199 +/- 16 v 170 +/- 9 mm Hg, P ionomycin-induced intracellular calcium ([Ca2+]i) peak in the absence of external Ca2+, which reflects [Ca2+]i storage size, and thrombin-evoked [Ca2+]i release from [Ca2+]i storage were decreased by 2.0% Ca diet (472 +/- 55 v 370 +/- 23 nmol/L, P ionomycin-induced [Ca2+]i (r = 0.591, P = .0415), respectively. However, there was no significant effect of dietary calcium on Ca2+-ATPase activity in aortic membranes. These results suggest that dietary calcium supplementation had a beneficial effect on platelets of SHR by attenuating [Ca2+]i mobilization from [Ca2+]i storage. The hypotensive effect of dietary calcium might be associated with attenuated [Ca2+]i mobilization in SHR.

  4. Effects of a high intake of unsaturated and saturated oils on intestinal transference of calcium and calcium mobilization from bone in an ovariectomized rat model of osteoporosis.

    Science.gov (United States)

    Chanda, S; Islam, N; Ghosh, T K; Mitra, C

    1999-06-01

    Intestinal transference of calcium and rate of bone turnover were evaluated in ovariectomized rats fed for 15 days with a high amount (30%) of lipid enriched with monounsaturated (groundnut oil), polyunsaturated (sunflower oil) and saturated (coconut oil) fatty acids. The results were compared with those for sham-operated control and ovariectomized groups fed a normal diet (7% groundnut oil). Irrespective of the saturation and unsaturation characteristics, all lipids (edible oils) used in our study considerably decreased the rate of in situ intestinal transference of calcium. Likewise, the activities of intestinal mucosal enzymes, alkaline phosphatase (AP) and calcium ATPase (Ca2+-ATPase) were decreased significantly in all the segments of the small intestine in a descending gradient. Significant changes in bone turnover and bone calcium (Ca) mobilization were confirmed in these animals by marked alterations in plasma AP activity, urinary calcium and phosphate excretion and calcium to creatinine (Ca:creatinine) ratio. Lipid supplementation (30%) in such ovariectomized rats using groundnut oil (monounsaturated), sunflower oil (polyunsaturated) or coconut oil (saturated) for 15 days further enhanced all of the above observed parameters. These results suggest that the intake of high amounts of lipids with different unsaturation and saturation characteristics may be an important factor in determining bone loss in ovariectomized rats.

  5. Evolution of Copper Transporting ATPases in Eukaryotic Organisms

    OpenAIRE

    Gupta, Arnab; Lutsenko, Svetlana

    2012-01-01

    Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases i...

  6. Maternal inheritance in polyploid fish inferred from mitochondrial ATPase genes analysis

    Institute of Scientific and Technical Information of China (English)

    Jinpeng Yan; Xinhong Guo; Shaojun Liu; Jun Xiao; Zhen Liu; Yubao Chen; Yun Liu

    2009-01-01

    The sequences of the ATPase8/6 genes for the triploid, tetraploid and pentaploid hybrids as well as for their male parent blunt snout bream were determined. In order to examine mitochondrial maternal inheritance, the sequences were subjected to a comparative sequence analysis with the homologous sequences of red crucian carp, their female parent, and zebrafish as the outgroup. Base compo-sition and variation as well as the divergences based on nucleotide sequences and deduced amino acid sequences were calculated. Phy-logenetic trees were also constructed with maximum parsimony (MP), minimum evolution (ME), neighbor joining (NJ) and the unweighted pair group method with arithmetic mean (UPGMA) algorithms in MEGA 3.1. The results showed that most nucleotide sub-stitutions occurred at the third codon position of the two genes and thus represented synonymous mutations. The nucleotide sequence divergences of the ATPase8/6 genes ranged from 0.0% to 21.6% among ingroup samples (three types of polyploids and their parents), and 27.0-28.2% between their ingroup and the outgroup samples. All the polyploids were considerably closer in sequence relationship to the female parent red crucian carp (0.0-3.3%) compared to their male parent blunt snout bream (21.0-21.6%). The phylogenetic trees also showed a similar result. In conclusion, the mitochondrial ATPase8/6 genes of artificial polyploid fish stringently indicated maternal inheritance. Our results also suggested that the ATPase8/6 genes are valuable genetic markers to track genealogies and variations in the progenies of the hybrids.

  7. Calcium channel as a potential anticancer agent.

    Science.gov (United States)

    Kriazhev, L

    2009-11-01

    Anticancer treatment in modern clinical practices includes chemotherapy and radiation therapy with or without surgical interventions. Efficiency of both methods varies greatly depending on cancer types and stages. Besides, chemo- and radiotherapy are toxic and damaging that causes serious side effects. This fact prompts the search for alternative methods of antitumor therapy. It is well known that prolonged or high increase of intracellular calcium concentration inevitably leads to the cell death via apoptosis or necrosis. However, stimulation of cell calcium level by chemical agents is hardly achievable because cells have very sophisticated machinery for maintaining intracellular calcium in physiological ranges. This obstacle can be overridden, nevertheless. It was found that calcium channels in so called calcium cells in land snails are directly regulated by extracellular calcium concentration. The higher the concentration the higher the calcium intake is through the channels. Bearing in mind that extracellular/intracellular calcium concentration ratio in human beings is 10,000-12,000 fold the insertion of the channel into cancer cells would lead to fast and uncontrollable by the cells calcium intake and cell death. Proteins composing the channel may be extracted from plasma membrane of calcium cells and sequenced by mass-spectrometry or N-terminal sequencing. Either proteins or corresponding genes could be used for targeted delivery into cancer cells.

  8. Redox Modulation of Cellular Signaling and Metabolism Through Reversible Oxidation of Methionine Sensors in Calcium Regulatory Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bigelow, Diana J.; Squier, Thomas C.

    2005-01-17

    response to {beta}-adrenergic signaling in the heart. The important role of the Ca-ATPase in determining the properties of the intracellular calcium transient in muscle highlights the potential role of phospholamban oxidation in cellular stress response. We suggest that under acute conditions, such as inflammation or ischemia, these types of mechanisms ensure minimal nonspecific cellular damage, allowing for rapid restoration of cellular function through repair of oxidized methionines by methionine sulfoxide reductase and degradation pathways after restoration of normal cellular redox conditions.

  9. Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

    DEFF Research Database (Denmark)

    Timmermann, D B; Lund, T M; Belhage, B;

    2001-01-01

    in cytosolic calcium concentration. The results of this investigation demonstrate that pharmacologically distinct types of voltage dependent calcium channels are differentially localized in cell bodies, neurites and nerve terminals of mouse cortical neurons but that the Q-type calcium channel appears......The physiological significance and subcellular distribution of voltage dependent calcium channels was defined using calcium channel blockers to inhibit potassium induced rises in cytosolic calcium concentration in cultured mouse neocortical neurons. The cytosolic calcium concentration was measured...... using the fluorescent calcium chelator fura-2. The types of calcium channels present at the synaptic terminal were determined by the inhibitory action of calcium channel blockers on potassium-induced [3H]GABA release in the same cell preparation. L-, N-, P-, Q- and R-/T-type voltage dependent calcium...

  10. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

    Science.gov (United States)

    Sysoeva, Tatyana A; Yennawar, Neela; Allaire, Marc; Nixon, B Tracy

    2013-12-01

    One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators. PMID:24316836

  11. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ54-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP–BeFx

    International Nuclear Information System (INIS)

    This study reports the crystallization of a new nucleotide state of the ATPase domain of a bacterial transcription activator NtrC1 from the hyperthermophilic bacterium Aquifex aeolicus. Wild-type NtrC1 ATPase domain was crystallized in the presence of the ATP analog ADP–BeFx–Mg and the crystals diffracted anisotropically to at best 3.2, 5.2 and 3.2 Å resolution in the a*, b* and c* directions, respectively. One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ70 RNA polymerase (RNAP), σ54 RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP–BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators

  12. Comparative Features of Copper ATPases ATP7A and ATP7B Heterologously Expressed in COS-1 Cells

    OpenAIRE

    Liu, Yueyong; Pilankatta, Rajendra; Hatori, Yuta; Lewis, David; Inesi, Giuseppe

    2010-01-01

    ATP7A and ATP7B are P-type ATPases required for copper homeostasis and involved in the etiology of Menkes and Wilson diseases. We used heterologous expression of ATP7A or ATP7B in COS-1 cells infected with adenovirus vectors to characterize differential features pertinent to each protein expressed in the same mammalian cell type, rather than to extrinsic factors related to different cells sustaining expression. Electrophoretic analysis of the expressed protein, before and after purification, ...

  13. Calcium and Vitamin D

    Science.gov (United States)

    ... Home › Patients › Treatment › Calcium/Vitamin D Calcium/Vitamin D Getting enough calcium and vitamin D is essential ... counter medications and calcium supplements. What is Vitamin D and What Does it Do? Vitamin D plays ...

  14. Robustness of the rotary catalysis mechanism of F1-ATPase.

    Science.gov (United States)

    Watanabe, Rikiya; Matsukage, Yuki; Yukawa, Ayako; Tabata, Kazuhito V; Noji, Hiroyuki

    2014-07-11

    F1-ATPase (F1) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F1 as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the γ-phosphate of ATP (general base); and an arginine directly contacting the γ-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F1 is far more robust than previously thought.

  15. 蛇床子素对L-和N-型钙通道的影响%Effect of Osthole on L- and N- type Calcium Channels

    Institute of Scientific and Technical Information of China (English)

    袁春华; 李勃兴; 孙丽荣; 朱心红

    2011-01-01

    Objective: To determine the effects of osthole on voltage-dependent calcium channel subtypes. Methods: tsA-201 cells were transfected with the calcium channel subtypes of Cavl.2, Cavl.3, Cav2.2e [37a],and Cav2.2e [37b]. The calcium currents were recorded in tsA-201 cells using whole cell patch clamp technique. The effects of osthole on calcium currents were investigated. Results: Osthole could inhibit Cavl.2 and Cavl.3 currents in a concentration - dependent manner. The half-effective concentration of the inhibition was 162.1 μmol -L-1, and 56.2 μmo\\ -L-1, respectively. In addition, Osthole reduced the Cav2.2e [37a],and Cav2.2e[37b] currents by 38 %, and 61 %, respectively, at the concentration of 300 μmol-L -1. The inhibition was fast and reversible. Osthole decreased the current amplitudes at all tested potentials, but showed no significant influence on the activation kinetics of the above calcium channels subtypes. Conclusions: Osthole concentration - dependency inhibited calcium currents and displayed different affinity to the calcium channel subtypes.%比较蛇床子素对不同钙通道亚型的作用差异.方法:首先在tsA201细胞上瞬时转染Cavl.2,Cav1.3,Cav2.2e[37a],和Cav2.2e[37b]通道,然后采用全细胞膜片钳技术,记录tsA201细胞上的钙电流,并观察蛇床子素对各种钙通道亚型的影响.结果:蛇床子素可以浓度依赖性抑制Cav1.2和Cav1.3电流,抑制的半有效浓度分别为162.1 μmol·L1和56.2 μmol·L-1.此外,蛇床子素对Cav2.2通道也有一定的抑制作用,在300μmol·L-1的浓度下,抑制38%的Cav2.2e[37a]电流和61%的Cav2.2e[37b]电流.蛇床子素对钙电流的抑制是快速可逆的.蛇床子素在各个测试电位水平均能抑制上述四种钙通道电流,但不改变电流的激活阈值和最大峰值电流的激活电压.结论:蛇床子素以浓度依赖的方式抑制多种钙通道亚型并表现出不同的亲和力.

  16. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

    Science.gov (United States)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly ( P P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

  17. Rotating with the brakes on and other unresolved features of the vacuolar ATPase

    Science.gov (United States)

    Rawson, Shaun; Harrison, Michael A.; Muench, Stephen P.

    2016-01-01

    The rotary ATPase family comprises the ATP synthase (F-ATPase), vacuolar ATPase (V-ATPase) and archaeal ATPase (A-ATPase). These either predominantly utilize a proton gradient for ATP synthesis or use ATP to produce a proton gradient, driving secondary transport and acidifying organelles. With advances in EM has come a significant increase in our understanding of the rotary ATPase family. Following the sub nm resolution reconstructions of both the F- and V-ATPases, the secondary structure organization of the elusive subunit a has now been resolved, revealing a novel helical arrangement. Despite these significant developments in our understanding of the rotary ATPases, there are still a number of unresolved questions about the mechanism, regulation and overall architecture, which this mini-review aims to highlight and discuss. PMID:27284051

  18. Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

    DEFF Research Database (Denmark)

    Timmermann, D B; Lund, Trine Meldgaard; Belhage, B;

    2001-01-01

    The physiological significance and subcellular distribution of voltage dependent calcium channels was defined using calcium channel blockers to inhibit potassium induced rises in cytosolic calcium concentration in cultured mouse neocortical neurons. The cytosolic calcium concentration was measured...... using the fluorescent calcium chelator fura-2. The types of calcium channels present at the synaptic terminal were determined by the inhibitory action of calcium channel blockers on potassium-induced [3H]GABA release in the same cell preparation. L-, N-, P-, Q- and R-/T-type voltage dependent calcium...... most important voltage dependent calcium channel in all parts of the neuron. After treatment with thapsigargin the increase in cytosolic calcium was halved, indicating that calcium release from thapsigargin sensitive intracellular calcium stores is an important component of the potassium induced rise...

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

    Science.gov (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

    2013-01-01

    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.

  20. Lysophosphatidylcholines containing polyunsaturated fatty acids were found as Na+,K+-ATPase inhibitors in acutely volume-expanded hog

    International Nuclear Information System (INIS)

    Na+,K+-ATPase inhibitors activities against the specific binding of ouabain to Na+,K+-ATPase and 86Rb uptake into hog erythrocytes have been purified from the plasma of acutely saline-infused hog. The purifications were performed by a combination of Amberlite XAD-2 adsorption chromatography and four steps of high-performance liquid chromatography with four different types of columns. Fast atom bombardment (FAB) mass and proton NMR spectrometric studies identified the purified substances as γ-arachidoyl- [LPCA(γ), 34%], β-arachidoyl- [LPCA(β), 4%], γ-linoleoyl- (LPCL, 33%), and γ-oleoyl- (LPCO, 25%) lysophosphatidylcholine, expressed in molar ratio in the plasma. Small amounts of γ-docosapentaenoyl-, γ-eicosatrienoyl-, and γpalmitoyllysophosphatidylcholine were also detected by both FAB mass and 1H NMR spectrometric studies. The inhibition of Na+,K+-ATPase activity due to these compounds was always more sensitive than that of both ouabain-binding and 86Rb uptake activities. The ouabain-displacing activity in plasma due to these compounds increased with time during saline infusion. The maximal plasma level was approximately 10 times higher than that in the preinfusion plasma sample. Although these results suggest that γ-acyl-LPC's with long-chain polyunsaturated fatty acids are not simple competitive inhibitors to Na+,K+-ATPase, these compounds could be implicated in the pathogenesis of the circulation abnormality through the modulation of membrane enzyme

  1. Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii.

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    Peter Rohloff

    Full Text Available Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+/H(+ countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.

  2. Hydrogen peroxide-mediated oxidative stress disrupts calcium binding on calmodulin: More evidence for oxidative stress in vitiligo

    International Nuclear Information System (INIS)

    Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10-3 M H2O2. One consequence of this severe oxidative stress is an altered calcium homeostasis in epidermal keratinocytes and melanocytes. Here, we show decreased epidermal calmodulin expression in acute vitiligo. Since 10-3M H2O2 oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H2O2 utilising 45calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activities were followed in full skin biopsies from lesional skin of patients with acute vitiligo (n = 6) and healthy controls (n = 6). The results yielded a 4-fold decrease of ATPase activities in the patients. Computer simulation of native and oxidised calmodulin confirmed the loss of all four calcium ions from their specific EF-hand domains. Taken together H2O2-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed L-phenylalanine-uptake in the epidermis of acute vitiligo

  3. C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells.

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    Dana Galuska

    Full Text Available BACKGROUND: Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC in control and hyperglycemic conditions. METHODOLOGY/PRINCIPAL FINDINGS: HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86Rb(+ uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM. DNA binding activity was determined by electrical mobility shift assay (EMSA. Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α(1-subunit protein expression, accompanied with increase in (86Rb(+ uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6, concomitant with Na,K-ATPase α(1-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(1-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing. CONCLUSIONS/SIGNIFICANCE: Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription

  4. Calcium signals can freely cross the nuclear envelope in hippocampal neurons: somatic calcium increases generate nuclear calcium transients

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    Bading Hilmar

    2007-07-01

    Full Text Available Abstract Background In hippocampal neurons, nuclear calcium signaling is important for learning- and neuronal survival-associated gene expression. However, it is unknown whether calcium signals generated by neuronal activity at the cell membrane and propagated to the soma can unrestrictedly cross the nuclear envelope to invade the nucleus. The nuclear envelope, which allows ion transit via the nuclear pore complex, may represent a barrier for calcium and has been suggested to insulate the nucleus from activity-induced cytoplasmic calcium transients in some cell types. Results Using laser-assisted uncaging of caged calcium compounds in defined sub-cellular domains, we show here that the nuclear compartment border does not represent a barrier for calcium signals in hippocampal neurons. Although passive diffusion of molecules between the cytosol and the nucleoplasm may be modulated through changes in conformational state of the nuclear pore complex, we found no evidence for a gating mechanism for calcium movement across the nuclear border. Conclusion Thus, the nuclear envelope does not spatially restrict calcium transients to the somatic cytosol but allows calcium signals to freely enter the cell nucleus to trigger genomic events.

  5. 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.

    Science.gov (United States)

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

    2013-09-17

    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.

  6. Role of electrolytes disturbances and Na(+)-K(+)-ATPase in cisplatin - induced renal toxicity and effects of ethanolic extract of Cichorium intybus.

    Science.gov (United States)

    Noori, Shafaq; Mahboob, Tabassum

    2012-10-01

    Cisplatin is known by its toxicity by disturbing electrolytes homeostasis. Thus we aimed to find out the role of herbal plant Cichorium intybus on Cisplatin - induced toxicity. 24 male Albino Wistar rats were randomly divided into 4 groups: Group I is termed as untreated control; Group II is Cisplatin control and received 3 mg/ kg b.w.; i.p.; Group III received C. intybus ethanolic extract at a dose of 500 mg/kg b.w. orally for 10 consecutive days and Group IV is Cisplatin + C. intybus pretreated group. C. intybus is given 30 minutes prior to Cisplatin. Cisplatin - induced electrolytes disturbances is indicated by increase Intra - erythrocyte sodium content, decreased plasma magnesium, calcium and Intra-erythrocyte Na(+)-K(+)-ATPase which implicates the renal toxicity. At a dose of 500 mg/kg b.w. of C. Intybus pretreatment showed partial counter action on the electrolytes imbalances and Na(+)-K(+)-ATPase activity. PMID:23010005

  7. The molecular, temporal and region-specific requirements of the beta isoform of Calcium/Calmodulin-dependent protein kinase type 2 (CAMK2B) in mouse locomotion.

    Science.gov (United States)

    Kool, Martijn J; van de Bree, Jolet E; Bodde, Hanna E; Elgersma, Ype; van Woerden, Geeske M

    2016-01-01

    Genetic approaches using temporal and brain region-specific restricted gene deletions have provided a wealth of insight in the brain regions and temporal aspects underlying spatial and associative learning. However, for locomotion such extensive studies are still scarce. Previous studies demonstrated that Camk2b(-/-) mice, which lack the β isoform of Calcium/Calmodulin-dependent protein kinase 2 (CAMK2B), show very severe locomotion deficits. However, where these locomotion deficits originate is unknown. Here we made use of novel Camk2b mutants (Camk2b(f/f) and Camk2b(T287A)), to explore the molecular, temporal and brain region-specific requirements of CAMK2B for locomotion. At the molecular level we found that normal locomotion requires Calcium/Calmodulin mediated activation of CAMK2B, but CAMK2B autonomous activity is largely dispensable. At a systems level, we found that global deletion of Camk2b in the adult mouse causes only mild locomotion deficits, suggesting that the severe locomotion deficits of Camk2b(-/-) mice are largely of developmental origin. However, early onset deletion of Camk2b in cerebellum, striatum or forebrain did not recapitulate the locomotion deficits, suggesting that these deficits cannot be attributed to a single brain area. Taken together, these results provide the first insights into the molecular, temporal and region-specific role of CAMK2B in locomotion. PMID:27244486

  8. A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases.

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    Gianluca Bartolommei

    Full Text Available The detection of small amounts (nanomoles of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases, that produce inorganic phosphate by cleavage of the γ-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III oxide tartrate (originally employed for phosphate detection in environmental analysis to allow its use with phosphatase enzymes. In particular, the method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening.

  9. Cadmium, ATPase-P, yeast. From transport to toxicity

    International Nuclear Information System (INIS)

    Two projects has been developed during my PhD. One consisting in the functional study of CadA, the Cd2+-ATPase from Listeria monocytogenes, the other one was focused on the toxicity of cadmium and the associated response of the yeast Saccharomyces cerevisiae. This two studies used a a phenotype of sensitivity to cadmium induced by CadA expression in yeast. This phenotype was used as a screening tool to identify essential amino acids of Cd transport by CadA and to study cadmium toxicity and the corresponding yeast cellular response. CadA actively transports Cd using ATP hydrolysis as energy source. Directed mutagenesis of the membranous polar, sulphur and charged amino-acids revealed that Cd transport pathway implied four transmembrane segments (Tm) and more precisely the cysteine C354, C356 and proline P355 of the CPC motif located in Tm6, aspartate D692 in Tm8, glutamate E164 in Tm4 and methionine M149 in Tm5. From our studies, 2 Cd ions would be translocated for each hydrolysis ATP. Expression of CadA in the yeast Saccharomyces cerevisiae induces an hypersensitivity to Cd. A wild type cell can grow up to 100 μm cadmium whereas CadA expressing yeast cannot grow with 1 μm cadmium in the culture medium. This cadmium sensitivity was due to the localisation of CadA in the endoplasmic reticulum membrane. Transport of cadmium in this compartment produces an accumulation of mis-folded proteins that induces the Unfolded Protein Response (UPR). As UPR also occurs in a wild type yeast exposed to low Cd concentration, one can point out endoplasmic reticulum as a extremely sensitive cellular compartment. UPR also appears as an early response to Cd as it happens far before any visible signs of toxicity. (author)

  10. Altered calcium signaling following traumatic brain injury

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    John Thomas Weber

    2012-04-01

    Full Text Available Cell death and dysfunction after traumatic brain injury (TBI is caused by a primary phase, related to direct mechanical disruption of the brain, and a secondary phase which consists of delayed events initiated at the time of the physical insult. Arguably, the calcium ion contributes greatly to the delayed cell damage and death after TBI. A large, sustained influx of calcium into cells can initiate cell death signaling cascades, through activation of several degradative enzymes, such as proteases and endonucleases. However, a sustained level of intracellular free calcium is not necessarily lethal, but the specific route of calcium entry may couple calcium directly to cell death pathways. Other sources of calcium, such as intracellular calcium stores, can also contribute to cell damage. In addition, calcium-mediated signal transduction pathways in neurons may be perturbed following injury. These latter types of alterations may contribute to abnormal physiology in neurons that do not necessarily die after a traumatic episode. This review provides an overview of experimental evidence that has led to our current understanding of the role of calcium signaling in death and dysfunction following TBI.

  11. V-ATPase-dependent luminal acidification is required for endocytic recycling of a yeast cell wall stress sensor, Wsc1p

    Energy Technology Data Exchange (ETDEWEB)

    Ueno, Kazuma; Saito, Mayu; Nagashima, Makiko; Kojima, Ai; Nishinoaki, Show [Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Toshima, Junko Y., E-mail: yama_jun@aoni.waseda.jp [Faculty of Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480 (Japan); Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Toshima, Jiro, E-mail: jtosiscb@rs.noda.tus.ac.jp [Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan)

    2014-01-10

    Highlights: •A targeted genome screen identified 5 gene groups affecting Wsc1p recycling. •V-ATPase-dependent luminal acidification is required for Wsc1p recycling. •Activity of V-ATPase might be required for cargo recognition by the retromer complex. -- Abstract: Wsc1p is a major cell wall sensor protein localized at the polarized cell surface. The localization of Wsc1p is maintained by endocytosis and recycling from endosomes back to the cell surface, but changes to the vacuole when cells are subjected to heat stress. Exploiting this unique property of Wsc1p, we screened for yeast single-gene deletion mutants exhibiting defects in Wsc1p trafficking. By expressing 3GFP-tagged Wsc1p in mutants with deleted genes whose function is related to intracellular trafficking, we identified 5 gene groups affecting Wsc1p trafficking, impaired respectively in endocytic internalization, multivesicular body sorting, the GARP complex, endosomal maturation/vacuolar fusion, and V-ATPase. Interestingly, deletion of the VPH1 gene, encoding the V{sub o} subunit of vacuolar-type H{sup +}-ATPase (V-ATPase), led to mis-localization of Wsc1p from the plasma membrane to the vacuole. In addition, disruption of other V-ATPase subunits (vma mutants) also caused defects of Wsc1p trafficking and vacuolar acidification similar to those seen in the vph1Δ mutant. Moreover, we found that deletion of the VPS26 gene, encoding a subunit of the retromer complex, also caused a defect in Wsc1p recycling and mis-localization of Wsc1p to the vacuole. These findings clarified the previously unidentified Wsc1p recycling pathway and requirement of V-ATPase-dependent luminal acidification for Wsc1p recycling.

  12. REM sleep deprivation-induced noradrenaline stimulates neuronal and inhibits glial Na-K ATPase in rat brain: in vivo and in vitro studies.

    Science.gov (United States)

    Baskey, Ganesh; Singh, Abhishek; Sharma, Rakhi; Mallick, Birendra Nath

    2009-01-01

    Increased noradrenaline, induced by rapid eye movement (REM) sleep deprivation, stimulates Na-K ATPase activity in the rat brain. The brain contains neurons as well as glia and both possess Na-K ATPase, however, it was not known if REM sleep deprivation affects the enzyme in both types of cells identically. Rats were REM sleep deprived by the flowerpot method and free moving, large platform and recovery controls were carried out. Na-K ATPase activity was measured in membranes prepared from whole brain as well as from neuronal and glial fractions separated from REM sleep-deprived and control rats. The effects of noradrenaline (NA) in different fractions were studied with or without in vivo i.p. treatment of prazosin, an alpha1-adrenpceptor antagonist, as well as in vitro membranes prepared from neurons and glia separated from normal rat brain. Further, to confirm the findings, membranes were prepared from neuro2a and C6 cell lines treated with NA in the presence and absence of prazosin and Na-K ATPase activity was estimated. The results showed that neuron and neuro2a as well as glia and C6 possess comparable Na-K ATPase activity. After REM sleep deprivation the neuronal Na-K ATPase activity increased, while the glial enzyme activity decreased and these changes were mediated by NA acting on alpha1-adrenoceptor; comparable results were obtained by treating the neuro2a and C6 cell lines with NA. The opposite actions of NA on neuronal and glial Na-K ATPase activity probably help maintain neuronal homeostasis.

  13. Na+/K+-ATPase E960 and phospholemman F28 are critical for their functional interaction

    OpenAIRE

    Khafaga, Mounir; Bossuyt, Julie; Mamikonian, Luiza; Li, Joseph C.; Lee, Linda L.; Yarov-Yarovoy, Vladimir; Despa, Sanda; Bers, Donald M.

    2012-01-01

    Na+-K+-ATPase (NKA) establishes the transmembrane [Na+] gradient in cells. In heart, phospholemman (PLM) inhibits NKA activity by reducing its apparent Na+ affinity, an effect that is relieved by PLM phosphorylation. The NKA crystal structure suggests regions of PLM–NKA interaction, but the sites important for functional effects in live cells are not known. We tested wild type (WT) and CFP–NKA-α1 point mutants (alanine substitution at F956, E960, L964, and F967) for fluorescence resonance ene...

  14. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ54-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP–BeFx

    OpenAIRE

    Sysoeva, Tatyana A.; Yennawar, Neela; Allaire, Marc; Nixon, B. Tracy

    2013-01-01

    This study reports the crystallization of a new nucleotide state of the ATPase domain of a bacterial transcription activator NtrC1 from the hyperthermophilic bacterium Aquifex aeolicus. Wild-type NtrC1 ATPase domain was crystallized in the presence of the ATP analog ADP–BeFx–Mg and the crystals diffracted anisotropically to at best 3.2, 5.2 and 3.2 Å resolution in the a*, b* and c* directions, respectively.

  15. Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane.

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    Leilismara Sousa

    Full Text Available Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1, iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 μM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5% than in women and was associated with an increase (446% in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS and an increase (327% in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 μM FeCl3 for 24 h showed an increase (132% in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels.

  16. Role of the Na+/K+-ATPase in regulating the membrane potential in rat peritoneal mast cells.

    Science.gov (United States)

    Friis, U G; Praetorius, H A; Knudsen, T; Johansen, T

    1997-10-01

    1. The aim of this study was to investigate the effect of the Na+/K+-ATPase on the membrane potential of peritoneal mast cells isolated from male Sprague-Dawley SPF-rats. 2. Experiments were performed at 22-26 degrees C in the tight-seal whole-cell configuration of the patch-clamp technique by use of Sylgard-coated patch pipettes (3-6 M[omega]). High-resolution membrane currents were recorded with an EPC-9 patch-clamp amplifier controlled by the 'E9SCREEN' software. In addition, a charting programme on another computer synchronously recorded at low resolution (2 Hz) membrane potential and holding current (low-pass filtered at 500 Hz). 3. Na+/K+-ATPase activity was measured as the ouabain-sensitive change in the zero-current potential. The zero-current potential in rat peritoneal mast cells measured 2 min after obtaining whole-cell configuration amounted to 1.7 +/- 2.5 mV (n = 21). Ouabain (5 mM), a Na+/K+-ATPase-inhibitor, had only a very minor effect upon the membrane potential under resting conditions (n = 3). 4. When mast cells were superfused with nominal calcium-free external solution, the cells hyperpolarized (delta mV: 20.2 +/- 3.8 mV (n = 5)). In addition, when the mast cells were preincubated in nominal calcium-free external solution for 12 +/- 1.6 min before whole-cell configuration, the membrane potential amounted to -53.7 +/- 9.8 mV (n = 8). A subsequent superfusion with ouabain (5 mM) depolarized the membrane potential (ouabain-sensitive hyperpolarization (delta mV): 23.0 +/- 8.4 mV (n = 8)). 5. A high intracellular concentration of Na+ ([Na+]i) (26.6 mM) also resulted in hyperpolarization (delta mV: 20.2 +/- 9.1 mV (n = 7)), but only when ATP was present. A subsequent superfusion with ouabain (5 mM) repolarized these cells to -1.2 +/- 14 mV (ouabain-sensitive hyperpolarization (delta mV): 19.7 +/- 7.7 mV (n = 7)). 6. The size of the [Na+]i-dependent hyperpolarization was dose-dependent. Low [Na+]i (1 mM) had no effect on membrane potential and these

  17. Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

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    Kazi Md Kamrul Huda

    Full Text Available BACKGROUND: Plasma membrane Ca(2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+ from the cell, hence regulating Ca(2+ level within cells. Though plant Ca(2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. RESULTS: The 1478 bp promoter sequence of rice plasma membrane Ca(2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. CONCLUSIONS: The rice plasma membrane Ca(2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the

  18. Characterization of ATPase Activity of Recombinant Human Pif1

    Institute of Scientific and Technical Information of China (English)

    Yu HUANG; Deng-Hong ZHANG; Jin-Qiu ZHOU

    2006-01-01

    Saccharomyces cerevisiae Pif1p helicase is the founding member of the Pif1 subfamily that is conserved from yeast to human. The potential human homolog of the yeast PIF1 gene has been cloned from the cDNA library of the Hek293 cell line. Here, we described a purification procedure of glutathione Stransferase (GST)-fused N terminal truncated human Pif1 protein (hPif1△N) from yeast and characterized the enzymatic kinetics of its ATP hydrolysis activity. The ATPase activity of human Pif1 is dependent on divalent cation, such as Mg2+, Ca2+ and single-stranded DNA. Km for ATP for the ATPase activity is approximately 200 μM. As the ATPase activity is essential for hPif1's helicase activity, these results will facilitate the further investigation on hPif1.

  19. Ganglioside GM3 modulates conformation of reconstituted Ca2+ -ATPase

    Institute of Scientific and Technical Information of China (English)

    王丽华; 杨小毅; 屠亚平; 催肇春; 杨福愉

    1997-01-01

    Using steady-state fluorescence and nanosecond time-resolved fluorescence techniques, the Ca 2+-ATPase conformational changes induced by ganglioside GM3 were studied with different quenchers. The results showed that GM3 could significantly increase the lifetime of intrinsic fluorescence of Ca2 + -ATPase reconstituted into proteoliposomes, and could also weaken the intrinsic fluorescence quenching by KI or hypocrellin B, HB. Further-more, by using quenching kinetic analysis of the time-resolved fluorescence, in the presence of GM3, the quenching constant (Ksv) and quenching efficiency were significantly lowered. The obtained results suggest that the oligosaccha-ride chain and the ceramide moieties of the GM3 molecule could interact with its counterparts of the Ca2+ -ATPase re-spectively, thus change the conformation of the hydrophobic domain of the enzyme, making the tryptophan residues in different regions shift towards the hydrophilic-hydrophobic interface, and hence shorten the distance between the hy

  20. Ultracytochemical Localization and Functional Analysis of ATPase During the Endosperm Development in Oryza sativa L.

    Institute of Scientific and Technical Information of China (English)

    WEI Cun-xu; LAN Sheng-yin; XU Zhen-xiu

    2003-01-01

    Ultracytochemical localization of ATPase during development of rice endosperm was performed using a lead phosphate precipitation technique. The results indicated that, at the coenocyte and ceilularization stages, active ATPase was mainly distributed in an embryo sac wall, nucleus, and plasma membrane. At the early stage of development and differentiation, active ATPase was observed in the plasma membrane. At the grain filling stage, ATPase was highly active in the plasma membrane, intercellular space, and plasmodesmata in aleurone, moderately active on the plasma membrane in subaleurone. In starchy endosperm, ATPase was localized in the plasma membrane and degenerated nucleus. ATPase activity also appeared around vacuole and protein body in endosperm cell. The relationships between the ultracytochemical localization of ATPase and its function during the development of rice endosperm were discussed. Overall, ATPase was involved in the process of nutrition absorption and protein synthesis.