Glutamate-induced intracellular acidification of cultured hippocampal neurons demonstrates altered energy metabolism resulting from Ca2+ loads.
1. Glutamate-evoked increases in intracellular free H+ concentration ([H+]i) were recorded from single rat hippocampal neurons grown in primary culture with carboxy SNARF-based dual emission microfluorimetry. The possibility that this acidification resulted from altered energy metabolism was investigated. 2. The response to 10 microM glutamate (delta pH = 0.41 +/- 0.14, mean +/- SD) was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist CGS19755 (10 microM) and required extracellular Ca2+. 3. Substituting the metabolic inhibitor 2-deoxyglucose for glucose in the extracellular buffer prevented glutamate-induced acidification. 4. Ba2+, which carries charge through Ca2+ channels, including the Ca2+ uniporter on the inner mitochondrial membrane, substituted for Ca2+ in mediating glutamate-induced cytoplasmic acidification. 5. Microinjection of ruthenium red, a compound that blocks mitochondrial Ca2+ sequestration, significantly inhibited glutamate-induced acidification. 6. The mitochondrial uncoupler, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP, 0.1 microM), mimicked and partially occluded the glutamate-induced [H+]i increase. 7. These findings indicate that glutamate-induced Ca2+ loads are sequestered by mitochondria, uncouple respiration, and produce metabolic acidosis. The glutamate-induced acidification is symptomatic of metabolic stress and may indicate that mitochondria play an important role in glutamate-induced neuronal death.
Wang
G J
GJ
Program in Neuroscience, University of Minnesota Medical School, Minneapolis 55455.
Randall
R D
RD
Thayer
S A
SA
D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000378:metabolism; D004734:Energy Metabolism / Q000187:drug effects*; D018698:Glutamic Acid / Q000494:pharmacology*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008845:Microinjections; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010875:Pipecolic Acids / Q000494:pharmacology; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000378:metabolism
D010875:Pipecolic Acids; D016194:Receptors, N-Methyl-D-Aspartate; D001464:Barium; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D018698:Glutamic Acid; C053672:selfotel; D003847:Deoxyglucose; D002118:Calcium