REGULATION OF ACETYLCHOLINE SYNTHESIS IN THE PRESENCE OF HEMICHOLINIUM MUSTARD

High affinity choline uptake (HACU) is a critical element in the synth etic pathway for acetylcholine (ACh), and is known to demonstrate acti vity-dependent regulation in vivo and in vitro. However, little is kno wn about this important sodium-dependent transport protein at the bioc hemical level, and about the nature of its interaction with the ACh sy nthetic enzyme ChAT. Hemicholinium mustard (HCM), an irreversibly bind ing analog of hemicholinium-3 (HC3), was used to create a preparation with HACU that is completely inhibited in order to investigate the imm ediate source of Ch for ACh synthesis. Rat brain synaptosomes were pre -incubated with HCM and washed before transport incubations of increas ing length (0-6 min) were carried out. The contribution of endogenous and extracellular (tracer) Ch to the ACh level was measured at each ti me point using a gas chromatography mass spectrometry (GCMS) system th at allows quantitative measurement of endogenous (unlabelled; [H-2(0)] ) Ch as well as tracer (deuterium-labelled; [H-2(4)]) Ch. The hypothes is was that if an endogenous intraterminal Ch pool can be used for ACh synthesis, an increase in unlabelled ACh across time would be observe d. In neither HCM-treated nor control synaptosomes was an increase obs erved in intraterminal (pellet) unlabelled ACh. To test the effects of high tissue demand, in other experiments synaptosomes were depolarize d 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.