Na(+)-K(+)-ATPase inhibition and depolarization induce glutamate release via reverse Na(+)-dependent transport in spinal cord white matter.
Excitotoxic mechanisms involving alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)/kainate receptors play an important role in mediating cellular damage in spinal cord injury. However, the precise cellular mechanisms of glutamate release from non-synaptic white matter are not well understood. We examined how the collapse of transmembrane Na(+) and K(+) gradients induces reverse operation of Na(+)-dependent glutamate transporters, leading to glutamate efflux and injury to rat spinal dorsal columns in vitro. Compound action potentials were irreversibly reduced to 43% of control after ouabain/high K(+)/low Na(+) exposure (500 microM ouabain for 30 min to increase [Na(+)](i), followed by 1 h ouabain+high K(+) (129 mM)/low Na(+) (27 mM), to further reverse transmembrane ion gradients) followed by a 2 h wash. Ca(2+)-free perfusate was very protective (compound action potential amplitude recovered to 87% vs. 43%). The broad spectrum glutamate antagonist kynurenic acid (1 mM) or the selective AMPA antagonist GYKI52466 (30 microM) were partially protective (68% recovery). Inhibition of Na(+)-dependent glutamate transport with L-trans-pyrrolidine-2,4-dicarboxylic acid (1 mM) also provided significant protection (71% recovery), similar to that seen with glutamate receptor antagonists. Blocking reverse Na(+)-Ca(2+) exchange with KB-R7943 (10 microM) however, was ineffective in this paradigm (49% recovery). Semiquantitative glutamate immunohistochemistry revealed that levels of this amino acid were significantly depleted in axon cylinders and, to a lesser degree, in oligodendrocytes (but not in astrocytes) by ouabain/high K(+)/low Na(+), which was largely prevented by glutamate transport inhibition. Our data show that dorsal column white matter contains the necessary glutamate pools and release mechanisms to induce significant injury. When Na(+) and K(+) gradients are disrupted, even in the absence of reduced cellular energy reserves, reverse operation of Na(+)-dependent glutamate transport will release enough endogenous glutamate to activate AMPA receptors and cause substantial Ca(2+)-dependent injury. This mechanism likely plays an important role during ischemic and traumatic white matter injury, where collapse of transmembrane Na(+) and K(+) gradients occurs.
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Ottawa Health Research Institute, Division of Neuroscience, Ottawa Hospital, Civic Campus, University of Ottawa, 725 Parkdale Avenue, Ottawa, ON, Canada K1Y 4K9.
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D000818:Animals; D014151:Anti-Anxiety Agents / Q000494:pharmacology; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D001569:Benzodiazepines; D002118:Calcium / Q000378:metabolism; D003998:Dicarboxylic Acids / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D007736:Kynurenic Acid / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009413:Nerve Fibers, Myelinated / Q000378:metabolism*; D014179:Neurotransmitter Uptake Inhibitors / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D011759:Pyrrolidines / Q000494:pharmacology; D051381:Rats; D020318:Rats, Long-Evans; D018091:Receptors, AMPA / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism*; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; D014151:Anti-Anxiety Agents; D000889:Anti-Arrhythmia Agents; D003998:Dicarboxylic Acids; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D014179:Neurotransmitter Uptake Inhibitors; D011759:Pyrrolidines; D018091:Receptors, AMPA; C061950:GYKI 52466; D001569:Benzodiazepines; D018698:Glutamic Acid; D010042:Ouabain; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; C083486:pyrrolidine-2,4-dicarboxylic acid; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D013890:Thiourea; D007736:Kynurenic Acid; D011188:Potassium; D002118:Calcium