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Research ArticleNeuropharmacology

Na+,K+-ATPase Functionally Interacts with the Plasma Membrane Na+,Ca2+ Exchanger to Prevent Ca2+ Overload and Neuronal Apoptosis in Excitotoxic Stress

Dmitry A. Sibarov, Artemiy E. Bolshakov, Polina A. Abushik, Igor I. Krivoi and Sergei M. Antonov
Journal of Pharmacology and Experimental Therapeutics December 2012, 343 (3) 596-607; DOI: https://doi.org/10.1124/jpet.112.198341
Dmitry A. Sibarov
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia (D.A.S., A.E.B., P.A.A., S.M.A.); Department of General Physiology, St. Petersburg State University, St. Petersburg, Russia (I.I.K.); and Laboratory of Molecular Neurodegeneration, St. Petersburg State Polytechnic University, St. Petersburg, Russia (D.A.S., P.A.A., S.M.A.)
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Artemiy E. Bolshakov
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia (D.A.S., A.E.B., P.A.A., S.M.A.); Department of General Physiology, St. Petersburg State University, St. Petersburg, Russia (I.I.K.); and Laboratory of Molecular Neurodegeneration, St. Petersburg State Polytechnic University, St. Petersburg, Russia (D.A.S., P.A.A., S.M.A.)
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Polina A. Abushik
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia (D.A.S., A.E.B., P.A.A., S.M.A.); Department of General Physiology, St. Petersburg State University, St. Petersburg, Russia (I.I.K.); and Laboratory of Molecular Neurodegeneration, St. Petersburg State Polytechnic University, St. Petersburg, Russia (D.A.S., P.A.A., S.M.A.)
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Igor I. Krivoi
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia (D.A.S., A.E.B., P.A.A., S.M.A.); Department of General Physiology, St. Petersburg State University, St. Petersburg, Russia (I.I.K.); and Laboratory of Molecular Neurodegeneration, St. Petersburg State Polytechnic University, St. Petersburg, Russia (D.A.S., P.A.A., S.M.A.)
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Sergei M. Antonov
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia (D.A.S., A.E.B., P.A.A., S.M.A.); Department of General Physiology, St. Petersburg State University, St. Petersburg, Russia (I.I.K.); and Laboratory of Molecular Neurodegeneration, St. Petersburg State Polytechnic University, St. Petersburg, Russia (D.A.S., P.A.A., S.M.A.)
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Abstract

Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca2+ imaging analysis, we report that ouabain, a specific ligand of the Na+,K+-ATPase cardiac glycoside binding site, can prevent glutamate receptor agonist-induced apoptosis in cultured rat cortical neurons. In our model of excitotoxicity, a 240-min exposure to 30 μM N-methyl-d-aspartate (NMDA) or kainate caused apoptosis in ∼50% of neurons. These effects were accompanied by a significant decrease in the number of neurons that were immunopositive for the antiapoptotic peptide Bcl-2. Apoptotic injury was completely prevented when the agonists were applied together with 0.1 or 1 nM ouabain, resulting in a greater survival of neurons, and the percentage of neurons expressing Bcl-2 remained similar to those obtained without agonist treatments. In addition, subnanomolar concentrations of ouabain prevented the increase of spontaneous excitatory postsynaptic current's frequency and the intracellular Ca2+ overload induced by excitotoxic insults. Loading neurons with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or inhibition of the plasma membrane Na+,Ca2+-exchanger by 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) eliminated ouabain's effects on NMDA- or kainite-evoked enhancement of spontaneous synaptic activity. Our data suggest that during excitotoxic insults ouabain accelerates Ca2+ extrusion from neurons via the Na+,Ca2+ exchanger. Because intracellular Ca2+ accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca2+ extrusion abolishes its development. These antiapoptotic effects are independent of Na+,K+-ATPase ion transport function and are initiated by concentrations of ouabain that are within the range of an endogenous analog, suggesting a novel functional role for Na+,K+-ATPase in neuroprotection.

Footnotes

  • This work was supported by the Russian Foundation for Basic Research [Grants 08-04-00423, 11-04-00397 (to S.M.A.); 10-04-00970 (to I.I.K.)]; the Russian Federation Ministry of Education and Science [Contract 8476] (to IEPhB RAS); and Saint-Petersburg State University [Research Grant 1.37. 118.2011] (to I.I.K.).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    http://dx.doi.org/10.1124/jpet.112.198341.

  • ABBREVIATIONS:

    GluR
    ionotropic glutamate receptor
    AM
    acetoxymethyl ester
    ANOVA
    analysis of variance
    AWCE
    alternative to GluR ways of Ca2+ entry
    AMPAR
    2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptor
    AO
    acridine orange
    BAPTA
    1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
    [Ca2+]i
    intracellular calcium concentration
    CNS
    central nervous system
    EB
    ethidium bromide
    EPSC
    excitatory postsynaptic current
    sEPSC
    spontaneous EPSC
    FVA
    fluorescent viability assay
    I-V
    current-voltage
    KA
    kainic acid
    KAR
    KA receptor
    KB-R7943
    2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate
    MK-801
    (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine
    NCX
    Na+/Ca2+ exchanger
    NKA
    Na+,K+-ATPase
    NMDA
    N-methyl-d-aspartate
    NMDAR
    NMDA receptor
    Ouab
    ouabain molecule
    PBS
    phosphate-buffered saline.

  • Received July 11, 2012.
  • Accepted August 24, 2012.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 343 (3)
Journal of Pharmacology and Experimental Therapeutics
Vol. 343, Issue 3
1 Dec 2012
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Research ArticleNeuropharmacology

Ouabain and Na+,K+-ATPase Regulate Neuroprotection

Dmitry A. Sibarov, Artemiy E. Bolshakov, Polina A. Abushik, Igor I. Krivoi and Sergei M. Antonov
Journal of Pharmacology and Experimental Therapeutics December 1, 2012, 343 (3) 596-607; DOI: https://doi.org/10.1124/jpet.112.198341

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Research ArticleNeuropharmacology

Ouabain and Na+,K+-ATPase Regulate Neuroprotection

Dmitry A. Sibarov, Artemiy E. Bolshakov, Polina A. Abushik, Igor I. Krivoi and Sergei M. Antonov
Journal of Pharmacology and Experimental Therapeutics December 1, 2012, 343 (3) 596-607; DOI: https://doi.org/10.1124/jpet.112.198341
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