title: NMDA-induced calcium loads recycle across the mitochondrial inner membrane of hippocampal neurons in culture.
abstract: Mitochondria sequester N-methyl-D-aspartate (NMDA)-induced Ca(2+) loads and regulate the shape of intracellular Ca(2+) concentration ([Ca(2+)](i)) responses in neurons. When isolated mitochondria are exposed to high [Ca(2+)](,) Ca(2+) enters the matrix via the uniporter and returns to the cytosol by Na(+)/Ca(2+) exchange. Released Ca(2+) may re-enter the mitochondrion recycling across the inner membrane dissipating respiratory energy. Ca(2+) recycling, the continuous uptake and release of Ca(2+) by mitochondria, has not been described in intact neurons. Here we used single-cell microfluorimetry to measure [Ca(2+)](i) and mitochondrially targeted aequorin to measure matrix Ca(2+) concentration ([Ca(2+)](mt)) to determine whether Ca(2+) recycles across the mitochondrial inner membrane in intact neurons following treatment with NMDA. We used ruthenium red and CGP 37157 to block uptake via the uniporter and release via Na(+)/Ca(2+) exchange, respectively. As predicted by the Ca(2+) recycling hypothesis, blocking the uniporter immediately following challenge with 200 microM NMDA produced a rapid and transient increase in cytosolic Ca(2+) without a corresponding increase in matrix Ca(2+). Blocking mitochondrial Ca(2+) release produced the opposite effect, depressing cytosolic Ca(2+) levels and prolonging the time for matrix Ca(2+) levels to recover. The Ca(2+) recycling hypothesis uniquely predicts these reciprocal changes in the Ca(2+) levels between the two compartments. Ca(2+) recycling was not detected following treatment with 20 microM NMDA. Thus Ca(2+) recycling across the inner membrane was more pronounced following treatment with a high relative to a low concentration of NMDA, consistent with a role in Ca(2+)-dependent neurotoxicity.
authors.affiliation: Department of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455-0217, USA.
authors.forename: Guang Jian
authors.initials: GJ
authors.lastname: Wang
authors.forename: Stanley A
authors.initials: SA
authors.lastname: Thayer
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D002998:Clonazepam / Q000031:analogs & derivatives* / Q000494:pharmacology; D004396:Coloring Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D005260:Female; D006624:Hippocampus / Q000166:cytology; D008928:Mitochondria / Q000378:metabolism*; D016202:N-Methylaspartate / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D012430:Ruthenium Red / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D013841:Thiazepines / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D004396:Coloring Agents; D018690:Excitatory Amino Acid Agonists; D019831:Sodium-Calcium Exchanger; D013841:Thiazepines; D014475:Uncoupling Agents; D012430:Ruthenium Red; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002998:Clonazepam; D016202:N-Methylaspartate; C079032:CGP 37157; D012964:Sodium; D002118:Calcium