[show abstract][hide abstract] ABSTRACT: We have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity by N-methyl-D-aspartate receptor (NMDAR) through a mechanism relevant to the increased mitochondrial Ca(2+) levels in HEK293 cells with acquired NMDAR channels. Here, we evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca(2+) levels in cultured murine neocortical neurons.
In neurons exposed to glutamate or NMDA, a significant increase was seen in mitochondrial Ca(2+) levels determined by Rhod-2 at concentrations of 0.1 to 100 µM. Further addition of 250 mM ethanol significantly inhibited the increase by glutamate and NMDA in Rhod-2 fluorescence, while similarly potent inhibition of the NMDA-induced increase was seen after exposure to ethanol at 50 to 250 mM in cultured neurons. Lentiviral overexpression of UCP2 significantly accelerated the increase by NMDA in Rhod-2 fluorescence in neurons, without affecting Fluo-3 fluorescence for intracellular Ca(2+) levels. In neurons overexpressing UCP2, exposure to ethanol resulted in significantly more effective inhibition of the NMDA-induced increase in mitochondrial free Ca(2+) levels than in those without UCP2 overexpression, despite a similarly efficient increase in intracellular Ca(2+) levels irrespective of UCP2 overexpression. Overexpression of UCP2 significantly increased the number of dead cells in a manner prevented by ethanol in neurons exposed to glutamate. In HEK293 cells with NMDAR containing GluN2B subunit, more efficient inhibition was similarly induced by ethanol at 50 and 250 mM on the NMDA-induced increase in mitochondrial Ca(2+) levels than in those with GluN2A subunit. Decreased protein levels of GluN2B, but not GluN2A, subunit were seen in immunoprecipitates with UCP2 from neurons with brief exposure to ethanol at concentrations over 50 mM.
Ethanol could inhibit the interaction between UCP2 and NMDAR channels to prevent the mitochondrial Ca(2+) incorporation and cell death after NMDAR activation in neurons.
PLoS ONE 01/2013; 8(7):e69718. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Necrotic damage leads to a massive leakage from injured cells of different intracellular constituents such as glutamate (Glu) and ATP, which are believed to play a role in the neuronal survival in the brain. In this study, we evaluated pharmacological properties of ATP, which is shown to be an endogenous inhibitor of N-methyl-D-aspartate (NMDA) receptors, on the neurotoxicity relevant to mitochondrial membrane potential disruption in cultured rat hippocampal neurons. Exposure to Glu or NMDA significantly inhibited cellular viability determined 24 and 48 h later, while simultaneous addition of 1 mM ATP significantly ameliorated the decreased viability in neurons exposed to Glu and NMDA, but not in those exposed to other cytotoxins. Both Glu and NMDA markedly increased intracellular free Ca(2+) levels in a manner sensitive to blockade by the exposure to ATP, but not by that to adenosine. Exposure to ATP significantly delayed the rate of mitochondrial membrane potential disruption induced by Glu and NMDA. These results suggest that extracellular ATP would play a role as an endogenous antagonist endowed to protect rat hippocampal neurons from the excitotoxicity mediated by NMDA receptors in association with the delayed mitochondrial membrane potential disruption after the liberation from adjacent cells under necrotic death.
Journal of Pharmacological Sciences 04/2012; 119(1):20-9. · 2.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have previously shown the possible involvement of mitochondrial membrane potential disruption in the mechanisms underlying the neurotoxicity seen after activation of N-methyl-d-aspartate (NMDA) receptors (NMDAR) in primary cultured rat hippocampal neurons. In this study, we attempted to demonstrate a pivotal role of mitochondrial uncoupling protein-2 (UCP2) as a determinant of the NMDA neurotoxicity by using acquired NMDAR channels artificially orchestrated in HEK293 cells. In cells with overexpression of UCP2, immunoreactive UCP2 was exclusively detected at intracellular locations stained with the mitochondrial marker MitoTracker. In cells with acquired NMDAR channels, exposure to either NMDA or the calcium ionophore A23187 similarly led to a significant increase in cytosolic Ca(2+) levels determined by Fluo-3 imaging irrespective of the overexpression of UCP2. By contrast, NMDA, but not A23187, was significantly more effective in increasing mitochondrial Ca(2+) levels determined by Rhod-2 fluorescence imaging in cells transfected with NMDAR subunit and UCP2 expression vectors than in those without UCP2 overexpression. Overexpression of UCP2 significantly increased the number of cells stained with propidium iodide in cultures with acquired NMDAR channels, but failed to significantly affect that in cells exposed to A23187. Immunocytochemical and immunoprecipitation analyses similarly revealed the possible interaction between GluN1 subunit and UCP2 in HEK293 cells with acquired NMDAR channels and UCP2 overexpression. These results suggest that UCP2 could play a role as a determinant of the neurotoxicity mediated by NMDAR through a mechanism related to the unidentified interaction with the essential GluN1 subunit toward modulation of mitochondrial Ca(2+) levels in neurons.
Neurochemistry International 04/2012; 61(4):498-505. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neural progenitor is a generic term used for undifferentiated cell populations of neural stem, neuronal progenitor and glial progenitor cells with abilities for proliferation and differentiation. We have shown functional expression of ionotropic N-methyl-D-aspartate (NMDA) and gamma-aminobutyrate type-A receptors endowed to positively and negatively regulate subsequent neuronal differentiation in undifferentiated neural progenitors, respectively. In this study, we attempted to evaluate the possible functional expression of nicotinic acetylcholine receptor (nAChR) by undifferentiated neural progenitors prepared from neocortex of embryonic rodent brains.
Reverse transcription polymerase chain reaction analysis revealed mRNA expression of particular nAChR subunits in undifferentiated rat and mouse progenitors prepared before and after the culture with epidermal growth factor under floating conditions. Sustained exposure to nicotine significantly inhibited the formation of neurospheres composed of clustered proliferating cells and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction activity at a concentration range of 1 µM to 1 mM without affecting cell survival. In these rodent progenitors previously exposed to nicotine, marked promotion was invariably seen for subsequent differentiation into cells immunoreactive for a neuronal marker protein following the culture of dispersed cells under adherent conditions. Both effects of nicotine were significantly prevented by the heteromeric α4β2 nAChR subtype antagonists dihydro-β-erythroidine and 4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine, but not by the homomeric α7 nAChR subtype antagonist methyllycaconitine, in murine progenitors. Sustained exposure to nicotine preferentially increased the expression of Math1 among different basic helix-loop-helix proneural genes examined. In undifferentiated progenitors from embryonic mice defective of NMDA receptor subunit-1, nicotine was still effective in significantly inhibiting the proliferation.
Functional α4β2 nAChR subtype would be constitutively expressed to play a role in the mechanism underlying the determination of proliferation and subsequent differentiation fate into a neuronal lineage in association with preferential promotion of Math1 expression in undifferentiated neural progenitors of developing rodent neocortex independently of NMDA receptor activation.
PLoS ONE 01/2012; 7(10):e46177. · 3.73 Impact Factor