%0 Journal Article %A Dmitry A. Sibarov %A Artemiy E. Bolshakov %A Polina A. Abushik %A Igor I. Krivoi %A Sergei M. Antonov %T Na+,K+-ATPase Functionally Interacts with the Plasma Membrane Na+,Ca2+ Exchanger to Prevent Ca2+ Overload and Neuronal Apoptosis in Excitotoxic Stress %D 2012 %R 10.1124/jpet.112.198341 %J Journal of Pharmacology and Experimental Therapeutics %P 596-607 %V 343 %N 3 %X 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. %U https://jpet.aspetjournals.org/content/jpet/343/3/596.full.pdf