Na+,K+-ATPase functionally interacts with the plasma membrane Na+,Ca2+ exchanger to prevent Ca2+ overload and neuronal apoptosis in excitotoxic stress

J Pharmacol Exp Ther. 2012 Dec;343(3):596-607. doi: 10.1124/jpet.112.198341. Epub 2012 Aug 27.

Abstract

Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca(2+) 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 Ca(2+) 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(+),Ca(2+)-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 Ca(2+) extrusion from neurons via the Na(+),Ca(2+) exchanger. Because intracellular Ca(2+) accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca(2+) 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects*
  • Binding Sites
  • Calcium / metabolism*
  • Cell Culture Techniques
  • Cell Membrane / drug effects*
  • Cell Membrane / metabolism
  • Cell Membrane / pathology
  • Cell Survival / drug effects
  • Cells, Cultured
  • Cerebral Cortex / cytology
  • Cerebral Cortex / drug effects
  • Cerebral Cortex / metabolism
  • Dose-Response Relationship, Drug
  • Electrical Synapses
  • Excitatory Amino Acid Agonists / administration & dosage
  • Excitatory Amino Acid Agonists / pharmacology
  • Excitatory Postsynaptic Potentials / drug effects
  • Immunohistochemistry
  • Kainic Acid / administration & dosage
  • Kainic Acid / pharmacology
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • N-Methylaspartate / administration & dosage
  • N-Methylaspartate / pharmacology
  • Neurons / drug effects*
  • Neurons / metabolism
  • Neurons / pathology
  • Neuroprotective Agents / administration & dosage
  • Neuroprotective Agents / pharmacology
  • Ouabain / administration & dosage
  • Ouabain / pharmacology
  • Patch-Clamp Techniques
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Rats
  • Rats, Wistar
  • Sodium-Calcium Exchanger / metabolism*
  • Sodium-Potassium-Exchanging ATPase / metabolism*

Substances

  • Excitatory Amino Acid Agonists
  • Neuroprotective Agents
  • Proto-Oncogene Proteins c-bcl-2
  • Sodium-Calcium Exchanger
  • Ouabain
  • N-Methylaspartate
  • Sodium-Potassium-Exchanging ATPase
  • Kainic Acid
  • Calcium