Mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells.
The relationship between changes in mitochondrial membrane potential (Deltapsi(m)) and the failure of cytoplasmic Ca(2+) homeostasis, delayed Ca(2+)deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetramethylrhodamine methyl ester (TMRM(+)) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of Deltapsi(m) and plasma membrane potential (Deltapsi(P)). Glutamate usually caused an immediate decrease in Deltapsi(m) of <10 mV, attributable to Ca(2+) accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca(2+) that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca(2+) ([Ca(2+)](c)) has risen to the set point at which mitochondria retain Ca(2+). In the presence of oligomycin no increase in [Ca(2+)](c) occurs during this depolarization. We conclude that transient Ca(2+) loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca(2+) extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.
Ward
M W
MW
Neurosciences Institute, Department of Pharmacology and Neuroscience, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
Rego
A C
AC
Frenguelli
B G
BG
Nicholls
D G
DG
D000200:Action Potentials / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000968:Antimycin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003594:Cytoplasmic Granules; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism*; D011189:Potassium Chloride / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017470:Receptors, Glutamate / Q000378:metabolism; D012235:Rhodamines; D014475:Uncoupling Agents / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D017470:Receptors, Glutamate; D012235:Rhodamines; D014475:Uncoupling Agents; C401833:tetramethylrhodamine methyl ester; D018698:Glutamic Acid; D000968:Antimycin A; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D002118:Calcium