REGULATION OF RAT-BRAIN SYNAPTOSOMAL [H-3] HEMICHOLINIUM-3 BINDING AND [H-3] CHOLINE TRANSPORT SITES FOLLOWING EXPOSURE TO CHOLINE MUSTARD AZIRIDINIUM ION

Choline uptake by cholinergic nerve terminals is increased by depolari zation; the literature suggests that this results from either the appe arance of occult transporters or the increased activity of existing on es. The present experiments attempt to clarify the mechanism by which choline transport is regulated by testing if the preexposure of synapt osomes to choline mustard aziridinium ion prevents the stimulation-ind uced appearance of hemicholinium-3 binding sites and/or choline transp ort activity. Choline mustard inhibited irreversibly most of the ''gro und-state'' (basal) high-affinity choline transport but only 50% of '' ground-state'' hemicholinium-3 binding sites. Exposure of both striata l and hippocampal synaptosomes to the mustard, before stimulation, inh ibited K+-stimulated increases in choline transport and of [H-3]hemich olinium-3 binding. We conclude that the mechanism by which choline tra nsport is regulated involves the increased activity of a pool of trans port sites that are occluded to hemicholinium-3 but are available to c holine mustard aziridinium ion, and presumably to choline, before stim ulation. However, the concentration of mustard needed to inhibit the s timulation-induced increase of [H-3]hemicholinium-3 binding and cholin e transport was lower for striatal synaptosomes than for hippocampal s ynaptosomes. In the absence of extracellular Ca2+ or presence of high Mg2+ levels, the choline mustard did not prevent the appearance of ext ra striatal hemicholinium-3 binding sites. Also, high Mg2+ levels remo ved the ability of the mustard to inhibit K+-stimulated increases of e ither [H-3]hemicholinium-3 binding or choline transport by hippocampal synaptosomes. In contrast, the preexposure of hippocampal synaptosome s to the mustard in the presence of a calcium ionophore (A23187) reduc ed the concentration of inhibitor needed to prevent the activation of [H-3]hemicholinium-3 binding and choline uptake. Thus, we conclude tha t the ability of the choline mustard to alkylate the pool of choline t ransporters that are activated by stimulation appears dependent on the entry of extracellular Ca2+.