Abstract
The relationship between changes in mitochondrial membrane potential (Δψ
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 Δψ
m
and plasma membrane potential (Δψ
P
). Glutamate usually caused an immediate decrease in Δψ
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.