Abstract

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 Ca²⁺_ipools in response to MeHg. Prior depletion of Ca²⁺_m using carbonyl cyanidem-chlorophenylhydrazone (CCCP) and oligomycin significantly decreased the amplitude of [Ca²⁺]_i release from intracellular stores, and delayed the onset of whole-cell [Ca²⁺]_ielevations, caused by 0.5 μM 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.