Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca 2ϩ imaging analysis, we report that ouabain, a specific ligand of the Na ϩ ,K ϩ -ATPase cardiac glycoside binding site, can prevent glutamate receptor agonistinduced apoptosis in cultured rat cortical neurons. In our model of excitotoxicity, a 240-min exposure to 30 M N-methyl-Daspartate (NMDA) or kainate caused apoptosis in ϳ50% 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 2ϩ overload induced by excitotoxic insults. Loading neurons with 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid or inhibition of the plasma membrane Na ϩ ,Ca 2ϩ -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 2ϩ extrusion from neurons via the Na ϩ ,Ca 2ϩ exchanger. Because intracellular Ca 2ϩ accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca 2ϩ extrusion abolishes its development. These antiapoptotic effects are independent of Na ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase in neuroprotection.

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