Baseline cytoplasmic calcium levels for each sample are reported as relative (to the memantine-untreated sample) fluorescence values

Baseline cytoplasmic calcium levels for each sample are reported as relative (to the memantine-untreated sample) fluorescence values. and IV (decreased at high concentrations) Vmax activities. APV did not alter the effects of chronic memantine exposure on citrate synthase and complex IV. We detected a lower mitochondrial peroxide production rate with acute exposure, and an increased mitochondrial peroxide production rate with chronic exposure. Micromolar memantine concentrations affect mitochondria, some of these effects are directly mediated, and acute and chronic effects may differ. 1. Introduction Activation of NMDA receptor complexes elevates cytosolic calcium concentration [1, 2]. Cells can address increased cytosolic calcium in part by transferring these cations into PF-02575799 negatively charged mitochondrial matrices [3]. Mitochondrial calcium content may in turn influence mitochondrial function. Alterations in oxidative phosphorylation status, electron transport chain (ETC) function, and mitochondrial reactive oxygen species (ROS) production are potential consequences [4, 5]. Less functionally obvious mitochondria-NMDA receptor complex relationships exist. For example, a mitochondrial DNA (mtDNA) encoded protein, ND2, actually serves to anchor NMDA receptor complexes to a regulatory tyrosine kinase [6]. Thus, in addition to playing an essential role in excitotoxic cascades, mitochondria and NMDA receptor complexes structurally overlap. Memantine is a moderate-affinity NMDA receptor antagonist [7]. It can reside within NMDA receptor complex channels, and impedes calcium influx that might otherwise occur through these channels. It is widely used for the treatment of Alzheimer’s disease (AD), and clinical trials show over a six month period AD patients randomized to memantine show less symptom progression than placebo-randomized AD patients [8, 9]. While it is postulated memantine’s clinical effects arise from NMDA channel IL5RA antagonism, this hypothesis has been challenged [10]. Other mechanisms that could potentially mediate memantine’s clinical effects therefore require consideration. AD is associated with numerous PF-02575799 histologic and biochemical abnormalities. Mitochondrial dysfunction is observed in both degenerating and non-degenerating tissues of AD subjects [11, 12, 13]. Because of recognized inter-relationships between NMDA receptor complexes, cell calcium homeostasis, and mitochondria, we evaluated whether memantine affects mitochondrial function. We studied this under in vitro conditions using the NT2 teratocarcinoma cell line, a neuron-like tumor cell line that expresses critical parts of the NMDA receptor complex [14, 15]. We found memantine can influence mitochondrial function, and that at least part (if not all) of this occurs independent of NMDA channel antagonism. 2. Materials and Methods 2.1 Cell culture Aside from addition of memantine or DL-2 amino-5-phosphono-valeric acid lithium salt (APV) to cell medium, human teratocarcinoma Ntera/D1 (NT2) neuronal precursor cells were maintained as previously described [16]. To accomplish memantine exposures, memantine-HCl powder (molecular weight 215.76) obtained from Forest Research Institute (Jersey City, NJ) was dissolved in sterile water to generate 1000 stock solutions. These 1000 stock solutions were then diluted in Optimem (Gibco BRL, Gaithersburg, MD) to create media containing 5?60 uM memantine. This concentration range exceeds serum levels of memantine obtained with human usage (0.5?1.0 uM), but is in accordance with the concentration spectrum typically used for in vitro studies [17, 18, 19]. To accomplish APV exposures, APV (formula weight 203.1; Sigma, St. Louis) was dissolved in sterile water to generate a 1000, 50 mM stock solution. This PF-02575799 stock solution was then diluted in Optimem to create media containing 50 uM APV. For chronic exposure experiments, NT2 cells were maintained in media containing 0?60 uM memantine, with or without concomitant 50 uM APV. Cells were maintained in their designated medium for at least two weeks prior to any assays. Cells were harvested when flasks reached 90% confluency. We also routinely changed the culture medium one day prior to harvesting. Adherent cells.