Abstract

Neurotoxic mechanisms of methylmercury (Met-Hg) on presynaptic nerve terminals were studied using the synaptosomes from the cerebral cortex of guinea pig as a model. Cytosolic free calcium [Ca++)c was determined using intrasynaptically trapped fluorescence indicator, fura-2; the plasma membrane potential (delta Up) by measuring the diffusion potential of 86Rb+ and the mitochondrial membrane potential was monitored using the safranine method. Synaptosomal respiration, glycolysis and concentrations of ATP and ADP in the presence and absence of Met-Hg also were quantified. Met-Hg increased synaptosomal [Ca++]c by two distinctive mechanisms. Moderate elevation of [Ca++]c by 127 nM was observed at 30 microM Met-Hg, at which concentration synaptosomal respiration was inhibited completely, leading to partial depolarization of mitochondria. A 3-fold activation of anaerobic glycolysis upon inhibition of respiration was insufficient to sustain terminal energy levels. The delta Up did not depolarize significantly from the resting potential of--67 mV. Thus, the rise in [Ca++]c was due to the energy failure of the synaptosomes, which has been caused by Met-Hg. With 100 microM Met-Hg, [Ca++]c increased extensively by 882 nM. Upon addition of 100 microM Met-Hg the delta Up depolarized instantly dropping 36 mV within 1 min. Synaptosomes were severely energy-deprived, because anaerobic glycolysis was inhibited by 90% from the aerobic level and mitochondrial membrane potential dropped below the limit that could be detected by the safranine method. The proportion of fura-2 signal quenching by Mn++ also increased, indicating that the plasma membrane had become leaky. Thus, at high concentrations of Met-Hg, the rise in [Ca++]c was ascribed to increased ionic permeability of the plasma membrane. The contribution of presynaptic energy failure by Met-Hg is discussed as a possible biochemical mechanism underlying the neurotoxicity of organic mercury.