The role of sulfhydryl groups and calcium in the mercuric chloride-induced inhibition of glutamate uptake in rat primary astrocyte cultures

Inhibition by mercuric chloride (MC) of the astrocytic uptake of the excitotoxic neurotransmitter L-glutamate (L-GLU) has been postulated to contribute to MC neurotoxicity. In the present study, we analyzed the ability of two sulfhydryl (SH)-protecting agents: a cell membrane non-penetrating compound-reduced glutathione (GSH), and the membrane permeable dithiothreitol (DTT), to reverse the inhibitory action of MC on the initial rate of uptake of radiolabelled GLU (100 μM) in primary cultures of rat astrocytes. MC at 5 μM concentration reduced the uptake to 46% of control when present in the incubation medium during the 5 min of actual uptake, and to 27% of control when astrocytes were preincubation with MC was partly relieved by the addition of 1 mM DTT during the actual 5 min uptake period. However, this inhibition could not be reversed by 1 mM GSH. Accordingly, it is postulated that the inhibitory effect exerted by MC on GLU uptake is associated with vulnerable SH groups located within, but not on the surface of the cell membrane. Neither 5 μM N-ethylmaleimide (NEM) nor 5 μM or 25 μM iodoacetate (IA) affected GLU uptake, indicating steric hindrance of the access of these two sulfhydryl reagents to the SH groups critical for the uptake. The effect of MC on GLU uptake was not altered by omission of Ca²⁺ or addition of a non-specific calcium channel blocker La³⁺ (10 μM), suggesting that the effect was not subsequent to Hg²⁺ entry into the cell through Ca²⁺ channels.