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 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.

GluN2A Subunit-Containing NMDA Receptors Are the Preferential Neuronal Targets of Homocysteine

“…Given the preferential contribution of GluN2A-containing NMDARs to the HCY effects, this amino acid is expected to be much less efficient neurotoxicant, than NMDA. In consistence to achieve the similar levels of apoptosis in primary culture of rat cortical neurons a 24-h exposure to HCY was required (Abushik et al, 2014), instead of a 4-h exposure to NMDA (Sibarov et al, 2012). Unlike NMDA-induced excitotoxicity is sensitive to NMDAR inhibitors (Church et al, 1988; Mironova et al, 2007), apoptosis caused by HCY is highly sensitive to the mGluR5 antagonist MTEP (Abushik et al, 2014).…”

Inhibition of Plasma Membrane Na/Ca-Exchanger by KB-R7943 or Lithium Reveals Its Role in Ca-Dependent N-methyl-d-aspartate Receptor Inactivation

“…For example, its IC 50 for fish NCXs is 1.9 and 3 mM for the reverse and forward modes of action, respectively (Abramochkin et al, 2013). In cultured cortical neurons, 10 mM KBR caused an immediate rise of intracellular Ca 21 , suggesting inhibition of NCX forward mode of transport (Sibarov et al, 2012).…”

KB-R7943 reduces 4-aminopyridine-induced epileptiform activity in adult rats after neuronal damage induced by neonatal monosodium glutamate treatment

“…Then changes in NCXs described above are only a part of the mechanisms implicated in the process by which MSG-treated rats are more susceptible to seizures induced by 4-AP [44]. Furthermore, because neural depolarization may induce inversion of NCXs and the inversion may increase firing probability [75, 76], changes induced by MSG treatment as increased average frequency of basal EEG activity in both studied cerebral regions (reported here for first time; Table 1), suggest that the probability of NCXs inversion could be potentiated after the treatment; however this suggestion should be proved.…”

“…On the other wise, in vitro approaches have demonstrated that low concentrations (<1 μM) of KB-R7943 predominantly block the reverse mode of NCX3 [81], avoiding the rise in [Ca 2+ ] i and reducing the frequency of excitatory postsynaptic currents, but high concentrations (≥10 μM) can block both the reverse and forward modes of the NCXs and promote increases in both [Ca 2+ ] i and the frequency of excitatory postsynaptic currents [76]. Because very few studies have applied KB-R7943 intracerebrally, we are not able to specifically propose how KB-R7943 is exerting its effects on the seizures.…”