Methylmercury uptake in rat primary astrocyte cultures: the role of the neutral amino acid transport system

The significance of the dense labeling pattern of methylmercury (MeHg) over astrocytes in areas of damaged cortex remains obscure, and the extent to which individual neurons are altered by MeHg accumulation in astrocytes is unknown. As a first step in understanding the relationship between the astrocyte and the mechanisms of MeHg's neurotoxicity, studies were directed at how MeHg is transported into cultured astrocytes. Uptake of [²⁰³Hg]MeHg in primary astrocyte cultures from neonatal rat cerebral cortex following incubations with MeHgCl conformed to a simple diffusion process. Uptake of [²⁰³Hg]MeHg by astrocytes exhibited the kinetic criteria of a specific transport system when added to the media as the l-cysteine conjugate. Saturation kinetics, substrate specificity and inhibition, and trans-stimulation were demonstrated in the presence of this SH-containing amino acid. Cysteine-mediated uptake of MeHg was inhibited by the coadministration of l-methionine, and 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid. 2-Methylaminoisobutyric acid was ineffective in inhibiting the uptake of the MeHg-cysteine conjugate. Preloading of the astrocytes with glutamate was moderately effective in trans-stimulating the uptake of MeHg-cysteine conjugates, while in the absence of cysteine, uptake of [²⁰³Hg]MeHg was unchanged. These results indicate the presence in astrocytes of a neutral amino acid carrier transport System L, capable of selectively mediating cysteine-MeHg uptake. The substrate specificity and high affinity of this transport system resemble the properties of the System L neutral amino acid transport across the blood-brain barrier in the rat. Cellular uptake of MeHg-cysteine conjugates was not inhibited by preincubation of astrocytes with 100 μM N-ethylmaleimide or NaF. Hence, endocytotic or pinocytotic mechanisms, and shuttling of MeHg via sequential sulfhydryl membrane ligand exchange do not appear to operate in the transport of MeHg into the astrocyte.