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.

Glutamate receptors play the key role in excitatory synaptic transmission in the central nervous system (CNS). N-methyl-D-aspartate-activated glutamate receptors (NMDARs) are ion channels permeable...

Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+-independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 μM and 61.8 ± 8.9 μM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to postsynaptic responses mediated by GluN2A-containing NMDA receptors. On the other hand, HCY toxicity may be limited by desensitization typical for HCY-induced activation of GluN2B-containing extrasynaptic receptors. Our findings, therefore, provide an evidence for the physiological relevance of endogenous HCY, which may represent an effective endogenous modulator of the central excitatory neurotransmission.

To evaluate the possible role of the plasma membrane Na⁺/Ca²⁺-exchanger (NCX) in regulation of N-methyl-d-aspartate (NMDA) receptors (NMDARs), we studied effects of 2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate (KB-R7943; KBR) and lithium (inhibitors of NCX) on NMDA-elicited whole-cell currents using the patch-clamp technique on rat cortical neurons and human embryonic kidney 293T cells expressing recombinant NMDARs. KBR inhibited NMDAR currents in a voltage-independent manner with similar potency for receptors of GluN1/2A and GluN1/2B subunit compositions that excludes open-channel block and GluN2B-selective inhibition. The inhibition by KBR depended on glycine (Gly) concentration. At 30 μM NMDA, the KBR IC₅₀ values were 5.3 ± 0.1 and 41.2 ± 8.8 μM for 1 and 300 μM Gly, respectively. Simultaneous application of NMDA + KBR in the absence of Gly induced robust inward NMDAR currents that peaked and then rapidly decreased. KBR, therefore, is an agonist (EC₅₀ is 1.18 ± 0.16 µM) of the GluN1 subunit coagonist binding sites. The decrease of NMDA-elicited currents in the presence of KBR was abolished in Ca²⁺-free solution and was not observed in the presence of extracellular Ca²⁺ on 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-loaded neurons, suggesting that Ca²⁺ affects NMDARs from the cytosol. In agreement, the substitution of Li⁺ for extracellular Na⁺ caused a considerable decrease of NMDAR currents, which was not observed in the absence of extracellular Ca²⁺. Most likely, the accumulation of intracellular Ca²⁺ is caused by the inhibition of Ca²⁺ extrusion via NCX. Thus, KBR and Li⁺ provoke Ca²⁺-dependent receptor inactivation due to the disruption of Ca²⁺ extrusion by the NCX. The data reveal the role of NCX in regulation of Ca²⁺-dependent inactivation of NMDARs.

Although the tricyclic antidepressant amitriptyline (ATL) is widely used in the clinic, the mechanism underlying its high therapeutic efficacy against neuropathic pain remains unclear. NMDA receptors (NMDARs) represent a target for ATL and are involved in sensitization of neuropathic pain. Here we describe two actions of ATL on NMDARs: 1) enhancement of Ca²⁺-dependent desensitization and 2) trapping channel block. Inhibition of NMDARs by ATL was found to be dependent upon external Ca²⁺concentration ([Ca²⁺]) in a voltage-independent manner, with an IC₅₀of 0.72 μM in 4 mM [Ca²⁺]. The ATL IC₅₀value increased exponentially with decreasing [Ca²⁺], with ane-fold change observed per 0.69 mM decrease in [Ca²⁺]. Loading neurons with BAPTA abolished Ca²⁺-dependent inhibition, suggesting that Ca²⁺affects NMDARs from the cytosol. Since there is one known Ca²⁺-dependent process in gating of NMDARs, we conclude that ATL most likely promotes Ca²⁺-dependent desensitization. We also found ATL to be a trapping open-channel blocker of NMDARs with an IC₅₀of 220 µM at 0 mV. Ane-fold change in ATL IC₅₀was observed to occur with a voltage shift of 50 mV in 0.25 mM [Ca²⁺]. Thus, we disclose here a robust dependence of ATL potency on extracellular [Ca²⁺], and demonstrate that ATL bound in the NMDAR pore can be trapped by closure of the channel.