title: Selective inhibition by ethanol of mitochondrial calcium influx mediated by uncoupling protein-2 in relation to N-methyl-D-aspartate cytotoxicity in cultured neurons.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School, Kanazawa, Ishikawa, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Fukumori
authors.forename: Takeshi
authors.initials: T
authors.lastname: Takarada
authors.forename: Ryota
authors.initials: R
authors.lastname: Nakazato
authors.forename: Koichi
authors.initials: K
authors.lastname: Fujikawa
authors.forename: Miki
authors.initials: M
authors.lastname: Kou
authors.forename: Eiichi
authors.initials: E
authors.lastname: Hinoi
authors.forename: Yukio
authors.initials: Y
authors.lastname: Yoneda
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D000431:Ethanol / Q000494:pharmacology*; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D007473:Ion Channels / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D016202:N-Methylaspartate / Q000494:pharmacology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D000071246:Uncoupling Protein 2
chemical_list: D007473:Ion Channels; D024101:Mitochondrial Proteins; D016194:Receptors, N-Methyl-D-Aspartate; C000604389:UCP2 protein, human; C000604390:Ucp2 protein, mouse; D000071246:Uncoupling Protein 2; D000431:Ethanol; D018698:Glutamic Acid; D016202:N-Methylaspartate; D002118:Calcium