Relationship between methylmercury-induced disruption of intracellular calcium and neuronal death

The mechanism by which the environmental neurotoxicant methylmercury (MeHg) causes elevations of intracellular Ca2+ ([Ca 2+]_i) and subsequent neuronal death was examined using single cell microfluorimetry of rat cerebellar granule neurons in primary culture. In granule cells loaded with fura-2 to monitor changes in [Ca 2+]_i, 0.2–1.0 μM MeHg causes early release of Ca2+ from at least one intracellular source; the possible sources examined were the smooth endoplasmic reticulum (SER) and the mitochondria. The non-specific muscarinic acetylcholine (ACh) receptor antagonist atropine delayed McHg-induced elevations of [Ca2+]_i, while down-regulation of the muscarinic receptors and the inositol-1,4,5-triphosphate (IP₃) receptor with 24 hr application of the muscarinic agonist bethanechol decreased the amplitude of the MeHg-induced release of Ca 2+_i by approximately 30–40%. Depletion of SER Ca 2+ stores with thapsigargin also reduced the amplitude of the MeHg-induced release of Ca2+_i by approximately 30–40%. Inhibition of Ca2+ release through the ryanodine receptors had minimal effect. Removal of mitochondrial Ca2+ (Ca 2+_m) content prior to MeHg exposure using carbonyl cyanide m-chlorophenylhydrazone (CCCP) and oligomycin decreased the amplitude of the MeHg-induced release of Ca2+_i by approximately 70%. Additionally, inhibition of the mitochondrial permeability transition pore (MTP) using cyclosporin A (CsA) delayed the increase in [Ca2+ ]_i. In granule cells loaded with tetramethylrhodamine ethyl ester (TMRE) to monitor changes in mitochondrial membrane potential, CsA delayed the irreversible loss of membrane potential caused by 0.5 μM MeHg. In granule cells loaded with rhod-2 to monitor changes in [Ca2+] _m, MeHg caused an early increase in [Ca2+]_m, followed several minutes later by release of dye from the mitochondria into the cytosol. The initial increase in [Ca2+]_m occurred independently of extracellular Ca2+, while the release of dye from the mitochondria was delayed by prior application of thapsigargin or CsA. Use of a calcein AM-ethidium homodimer cell viability assay revealed that increasing concentrations of MeHg (0.2–1.0 μM caused a corresponding increase in cell death at 24 hr post-exposure. Atropine and ryanodine did not protect against MeHg-induced cell death, while 24 hr BCh pretreatment significantly protected against cell killing. The BCh-mediated protection was reversed by atropine and the M3 muscarinic ACh receptor antagonist 4-diphenylacetoxyl-N-methylpiperidine methiodide (4-DAMP) but not by the M2 receptor antagonist methoctramine or the nicotinic ACh receptor antagonist dihydro-β-erythroidine hydrobromide (DHE). Thapsigargin itself was toxic, highlighting the sensitivity of granule cells to disruption of SER Ca2+. CsA also provided significant protection against cell death at 24 hr post-MeHg exposure. These results suggest that MeHg: (1) acts at M3 muscarinic ACh receptors to cause production of IP₃, which releases Ca2+ from the SER and (2) causes Ca2+ uptake into mitochondria which is then released into the cytosol via opening of the MTP. Additionally, these disruptions of [Ca2+]_i contribute to MeHg-induced neuronal death, and may underlie the specific neurotoxicity of MeHg within granule neurons of the cerebellar cortex.