Rylett_1993_Brain.Res_626_184

Membrane carriers display structural and functional asymmetry with a substrate binding site which can be oriented alternately, but not simultaneously, to the extracellular and intracellular environment. Hemicholinium-3 is an inhibitor of the high-affinity choline carrier in cholinergic nerve terminals which binds to the transporter at the outer membrane surface but is not taken up into the cell. In the present study, we investigated the decline in choline transport which occurs during the first few minutes cholinergic nerve terminals are incubated in physiological salt solutions. Following incubation of rat hippocampal synaptosomes with hemicholinium-3, samples were washed free of the inhibitor and high-affinity choline uptake was measured. Choline uptake into hemicholinium-treated nerve terminals was significantly greater than control (132 +/- 4%). This effect appeared not to be due to an increase in uptake of choline above initial values in the hemicholinium-treated synaptosomes, but to a decrease in choline carrier activity in control samples by more than 25% during the first few minutes of incubation. Addition of hemicholinium-3 to samples after the preincubation induced decrease in choline uptake, followed by a wash period to remove the inhibitor resulted in elevation of choline uptake levels to initial levels. The effect of hemicholinium-3 was concentration-dependent, requiring near saturating concentrations of the inhibitor to elicit the effect. Measurement of acetylcholine content of synaptosomes at different points during the incubation procedure revealed that there was a trend for transmitter levels to vary inversely compared to choline uptake activity, but the differences were not statistically significant during treatments when significant changes in transport activity were measured.