title: Na+,K+-ATPase functionally interacts with the plasma membrane Na+,Ca2+ exchanger to prevent Ca2+ overload and neuronal apoptosis in excitotoxic stress.
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.
authors.affiliation: Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Sibarov
authors.forename: Artemiy E
authors.initials: AE
authors.lastname: Bolshakov
authors.forename: Polina A
authors.initials: PA
authors.lastname: Abushik
authors.forename: Igor I
authors.initials: II
authors.lastname: Krivoi
authors.forename: Sergei M
authors.initials: SM
authors.lastname: Antonov
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D054351:Electrical Synapses; D018690:Excitatory Amino Acid Agonists / Q000008:administration & dosage / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D007150:Immunohistochemistry; D007608:Kainic Acid / Q000008:administration & dosage / Q000494:pharmacology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016202:N-Methylaspartate / Q000008:administration & dosage / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018696:Neuroprotective Agents / Q000008:administration & dosage / Q000494:pharmacology; D010042:Ouabain / Q000008:administration & dosage / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D018690:Excitatory Amino Acid Agonists; D018696:Neuroprotective Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D019831:Sodium-Calcium Exchanger; D010042:Ouabain; D016202:N-Methylaspartate; D000254:Sodium-Potassium-Exchanging ATPase; D007608:Kainic Acid; D002118:Calcium