Kinetic Data on the Inhibition of High‐affinity Choline Transport into Rat Forebrain Synaptosomes by Choline‐like Compounds and Nitrogen Mustard Analogues

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Abstract
A series of choline analogues and nitrogen mustard derivatives were evaluated as inhibitors of high‐affinity transport of choline in rat forebrain synaptosomes. When synaptosomes were preincubated for 10 min with choline mustard aziridinium ion, monoethylcholine and monoethylcholine mustard aziridinium ion, the agents appeared to be equipotent as inhibitors of high‐affinity uptake (Ki=2.63, 3.15 and 2.72 μm, respectively). Acetylcholine mustard aziridinium ion was less potent than these compounds (Ki= 27.8 μm), but it was more potent than ethoxycholine and ethoxycholine mustard aziridinium ion (Ki= 500 and 403 μm) as a blocker of choline transport. From study with these compounds it was concluded that the high‐affinity choline transport mechanism shows specificity for hydroxylated compounds over those in which the same hydroxyl has been acetylated (10‐fold) and that the carbonyl oxygen of the acetylated analogues is important, as its removal (to form the ethylether derivative) decreased affinity another 20‐fold. The presence of an aziridinium ring on the quaternary nitrogen in place of two methyl groups did not affect the blocking of transport at 10 min of inhibitor preincubation and replacement of a methyl group on the nitrogen by an ethyl group did not alter affinity for the high‐affinity carrier. The aziridinium ring on the nitrogen of the mustard analogues was important, however, in determining the extent of reversibility of the binding of these agents to the carrier protein. Choline transport was not restored by washing synaptosomes that were incubated with choline mustard aziridinium ion or monoethylcholine mustard aziridinium ion, but was readily obtained in washed synaptosomes preincubated with monoethylcholine, hemicholinium‐3, or pyrrolcholine. The results indicate that the mustard analogues may be potent alkylators of the high‐affinity choline carrier and thus, useful agents in monitoring acetylcholine turnover in systems where the carrier is blocked.