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.
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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