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.
Footnotes
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This study was supported by National Institute of Environmental Health Sciences Grant ES03299. T.L.L. was supported by National Institute of Environmental Health Sciences Grant T32-ES07255. The Leica confocal laser scanning microscope was acquired with a grant from the Michigan Life Sciences Corridor Program of the Michigan Economic Development Corporation. This article is submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy (T.L.). Portions of this study were presented in abstract form [Toxicologist (2000) 49:79; Biophys J (2002) 82:122].
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DOI: 10.1124/jpet.102.042457
- Abbreviations:
- MeHg
- methylmercury
- [Ca2+]i
- intracellular free Ca2+concentration
- Ca2+e
- extracellular Ca2+
- MTP
- mitochondrial permeability transition pore
- [Ca2+]m
- mitochondrial matrix Ca2+
- SER
- smooth endoplasmic reticulum
- IP3
- inositol 1,4,5-triphosphate
- AM
- acetoxymethylester
- TMRE
- tetramethylrhodamine ethyl ester
- CsA
- cyclosporin A
- CCCP
- carbonyl cyanide m-chlorophenylhydrazone
- HBS
- HEPES-buffered saline solution
- ψm
- mitochondrial membrane potential
- Received July 30, 2002.
- Accepted October 14, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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