Abstract
BackgroundWe 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 Ca2+ levels in HEK293 cells with acquired NMDAR channels. Here, we evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca2+ levels in cultured murine neocortical neurons.Methodology/Principal FindingsIn neurons exposed to glutamate or NMDA, a significant increase was seen in mitochondrial Ca2+ 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 Ca2+ levels. In neurons overexpressing UCP2, exposure to ethanol resulted in significantly more effective inhibition of the NMDA-induced increase in mitochondrial free Ca2+ levels than in those without UCP2 overexpression, despite a similarly efficient increase in intracellular Ca2+ 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 Ca2+ 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.Conclusions/SignificanceEthanol could inhibit the interaction between UCP2 and NMDAR channels to prevent the mitochondrial Ca2+ incorporation and cell death after NMDAR activation in neurons.
Highlights
Evidence that mitochondria are a key mediator of cell death through apoptotic and/or necrotic processes [1,2] as well as an organelle essential for cellular respiration is accumulating in the literature
Mitochondrial Ca2+ Levels in Cultured Neurons Cortical neurons were cultured for 8 days, followed by loading of the mitochondrial Ca2+ dye Rhod-2 and subsequent exposure to
For evaluation of the concentration dependency, cultured neurons were exposed to a fixed concentration of NMDA, followed by cumulative addition of ethanol at final concentrations of 50 to 250 mM for determination of Rhod-2 fluorescence
Summary
Evidence that mitochondria are a key mediator of cell death through apoptotic and/or necrotic processes [1,2] as well as an organelle essential for cellular respiration is accumulating in the literature. Calcium entry is shown to more occur into mitochondria after activation of N-methyl-D-aspartate (NMDA) receptor (NMDAR). The mechanism seems to involve the mitochondrial calcium uniporter (mCU), which has been a conceptual carrier of cytoplasmic free Ca2+ ions into the matrix across inner membranes in mitochondria [12,13]. Uncoupling proteins (UCPs) are proposed to be a candidate carrier for the influx of cytoplasmic Ca2+ ions toward the regulation of matrix Ca2+ levels in mitochondria [14,15]. We have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity by Nmethyl-D-aspartate receptor (NMDAR) through a mechanism relevant to the increased mitochondrial Ca2+ levels in HEK293 cells with acquired NMDAR channels. We evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca2+ levels in cultured murine neocortical neurons
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