Uptake and metabolism of [3H]choline mustard by cholinergic nerve terminals from rat brain.

The objective of this study was to measure the uptake and metabolism of [3H]choline mustard aziridinium ion in rat brain synaptosomes. In previous investigations, we showed that this compound binds irreversibly to the choline carrier thereby inhibiting choline transport into nerve terminals; it also acts as both a substrate and inhibitor of the acetylcholine biosynthetic enzyme choline acetyltransferase. We now report that [3H]choline mustard aziridinium ion was transported into purified rat brain synaptosomes by a hemicholinium-sensitive mechanism, but at only a fraction of the rate of uptake of [3H]choline. Following 5 min incubation with the nerve terminal preparation, uptake of [3H]choline mustard aziridinium ion was 20% of that of [3H]choline transport, but this fell to 10% of [3H]choline accumulation at 30 min incubation. Apparent Michaelis constants derived from double reciprocal plots of velocity of transport versus substrate concentration revealed that the apparent affinity constants (Km) of the high-affinity choline carrier for [3H]choline mustard aziridinium ion and [3H]choline were not different (1.44 +/- 0.15 and 2.14 +/- 0.80 microM for choline and choline mustard aziridinium ion, respectively). Increasing the incubation time from 5 to 30 min, during which time a proportion of the high-affinity choline carriers were irreversibly inactivated by choline mustard aziridinium ion, did not alter the binding affinity for this compound. The maximum velocity of transport (Vmax) for the two compounds were significantly different with the maximum uptake of [3H]choline mustard aziridinium ion being 19.5% of that for choline at 5 min incubation, and falling to only 10.6% of the maximum rate of choline transport by 30 min incubation. [3H]Choline mustard aziridinium ion transported into synaptosomes on the high-affinity choline carrier was metabolized, with 27% being recovered as [3H]acetylcholine mustard aziridinium ion, 27% as [3H]phosphorylcholine mustard aziridinium ion, 7% as unmetabolized [3H]choline mustard aziridinium ion and 16% recovered as an unidentified metabolite. In parallel samples, [3H]choline taken up into synaptosomes was recovered as [3H]acetylcholine (71%) and unmetabolized [3H]choline (18%) with no net production of [3H]phosphorylcholine. Acetylation of [3H]choline mustard aziridinium ion amounted to only 7.6% of [3H]acetylcholine synthesized under the same conditions. These results show clearly that choline mustard aziridinium ion was accumulated into the cholinergic nerve terminals by the high-affinity choline carrier, but the amount was small relative to the uptake of choline and probably restricted by progressive inactivation of the transporters through covalent bond formation.