Regulation of acetylcholine synthesis in the presence of hemicholinium mustard

High affinity choline uptake (HACU) is a critical element in the synthetic pathway for acetylcholine (ACh), and is known to demonstrate activity-dependent regulation in vivo and in vitro. However, little is known about this important sodium-dependent transport protein at the biochemical level, and about the nature of its interaction with the ACh synthetic enzyme ChAT. Hemicholinium mustard (HCM), an irreversibly binding analog of hemicholinium-3 (HC3), was used to create a preparation with HACU that is completely inhibited in order to investigate the immediate source of Ch for ACh synthesis. Rat brain synaptosomes were pre-incubated with HCM and washed before transport incubations of increasing length (0-6 min) were carried out. The contribution of endogenous and extracellular (tracer) Ch to the ACh level was measured at each time point using a gas chromatography mass spectrometry (GCMS) system that allows quantitative measurement of endogenous (unlabelled; [H-2(0)]) Ch as well as tracer (deuterium-labelled; [H-2(4)]) Ch. The hypothesis was that if an endogenous intraterminal Ch pool can be used for ACh synthesis, an increase in unlabelled ACh across time would be observed. In neither HCM-treated nor control synaptosomes was an increase observed in intraterminal (pellet) unlabelled ACh. To test the effects of high tissue demand, in other experiments synaptosomes were depolarized with addition of 40 mM KCI to the buffer after HCM treatment; again, no significant increase in intraterminal unlabelled ACh was observed across time. These experiments demonstrate that endogenous unlabelled Ch does not contribute to ACh synthesis, even when HACU is inactivated, and under conditions of high demand.