dizocilpine-maleate and dihydrorhodamine-123

Mitochondrial uptake of Ca(2+) has recently been found to play an important role in glutamate-induced neurotoxicity (GNT) as well as in the activation of Ca(2+)-dependent molecules, such as calmodulin and neuronal nitric oxide synthase (nNOS), in the cytoplasm. Prolonged exposure to glutamate injures motor neurons predominantly through the activation of Ca(2+)/calmodulin-nNOS, as previously reported, and is, in part, associated with the pathogenesis of amyotrophic lateral sclerosis (ALS). In the present study, we investigated how mitochondrial uptake of Ca(2+) is involved in GNT in spinal motor neurons. Acute excitotoxicity induced by exposure to 0.5 mM glutamate for 5 min was found in both motor and nonmotor neurons in cultured spinal cords from rat embryos and was dependent on extracellular Ca(2+) and on N-methyl-D-aspartate (NMDA) receptor activation. Mitochondrial uncouplers markedly blocked acute excitotoxicity, and membrane-permeable superoxide dismutase mimics attenuated acute excitotoxicity induced by glutamate and NMDA but not by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainate. Fluorimetric analysis showed that mitochondrial Ca(2+) was elevated promptly with subsequent accumulation of reactive oxygen species (ROS) in the mitochondria. An NMDA receptor antagonist and a mitochondrial uncoupler eliminated the increase in fluorescence of mitochondrial Ca(2+) and ROS indicators. These data indicate that acute excitotoxicity in spinal neurons is mediated by mitochondrial Ca(2+) overload and ROS generation through the activation of NMDA receptors. This mechanism is different from that of chronic GNT.

Topics: 2,4-Dinitrophenol; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Cyclosporine; Dibucaine; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluoresceins; Fluorescent Dyes; Glutamic Acid; Heterocyclic Compounds, 3-Ring; Imidazoles; Kainic Acid; Mitochondria; Motor Neuron Disease; Motor Neurons; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Neurotoxins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Rhodamines; Spinal Cord; Superoxide Dismutase; Uncoupling Agents