Excitotoxic injury to mitochondria isolated from cultured neurons

Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca²⁺ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca²⁺ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by ∼10%. The Ca ²⁺ load in mitochondria from glutamate-treated neurons was estimated to be 167 ± 19 nmol/mg protein. The glutamate-induced Ca²⁺ load was less than the maximal Ca²⁺ uptake capacity of the mitochondria determined in vitro (363 ± 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca²⁺ uptake capacity (686 ± 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca²⁺ was a smaller percentage of the maximal Ca²⁺ uptake capacity. Thus, this study indicated that Ca²⁺-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death.