Evidence for interactions between intracellular calcium stores during methylmercury-induced intracellular calcium dysregulation in rat cerebellar granule neurons

Acute exposure to methylmercury (MeHg) causes severe disruption of intracellular Ca2+ ([Ca2+](i)) regulation, which apparently contributes to neuronal death. Activation of the mitochondrial permeability transition pore (MTP) evidently contributes to this effect. We examined in more detail the contribution of mitochondrial Ca2+ ([Ca2+](m)) to elevations of [Ca2+](i) caused by acute exposure to a low concentration of MeHg in primary cultures of rat cerebellar granule neurons. In particular, we sought to determine whether interactions occurred between Ca-i(2+) pools in response to MeHg. Prior depletion of Ca2+ (m) using carbonyl cyanide m-chlorophenylhydrazone (CCCP) and oligomycin significantly decreased the amplitude of [Ca2+](i) release from intracellular stores, and delayed the onset of whole-cell [Ca2+](i) elevations, caused by 0.5 muM MeHg. CCCP alone hastened the MeHg-induced release of Ca2+ within the cell, whereas oligomycin alone delayed the MeHg-induced influx of extracellular Ca2+. In granule cells loaded with rhod-2 acetoxymethylester to measure changes in [Ca2+](m), MeHg exposure caused a biphasic increase in fluorescence. The initial increase in fluorescence occurred in the absence of extracellular Ca2+ and was abolished by mitochondrial depolarization. The secondary increase was associated with spreading of the dye from punctate staining to whole-cell distribution, and was delayed significantly by the MTP inhibitor cyclosporin A and the smooth endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin. We conclude that MeHg causes release of Ca2+ from the mitochondria through opening of the MTP, which contributes the bulk of the elevated [Ca2+](i) observed during MeHg neurotoxicity. Additionally, the Ca2+ that enters the mitochondria seems to originate in the smooth endoplasmic reticulum, providing a mechanism for the observed mitochondrial Ca2+ overload.