Vol. 304, Issue 3, 949-958, March 2003

Evidence for Interactions between Intracellular Calcium Stores during Methylmercury-Induced Intracellular Calcium Dysregulation in Rat Cerebellar Granule Neurons

Tobi L. Limke, James K. L. Otero-Montañez and William D. Atchison

Department of Pharmacology and Toxicology, Institute for Environmental Toxicology, and Neuroscience Program, Michigan State University, East Lansing, Michigan

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⁺_i 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 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.