Ionic and metabolic requirements for high-affinity choline uptake and acetylcholine synthesis in nerve terminals at a neuromuscular junction.

Abstract

Abstract: We have shown previously that in the chick ciliary nerve‐iris muscle preparation Na+‐dependent high‐affinity choline uptake was confined to the nerve terminals. In this paper the sodium‐dependent high‐affinity choline uptake (SDHACU), which is coupled to acetylcholine (ACh) synthesis, was further characterized by measuring uptake of [3H]choline and its conversion to [3hjach under a variety of ionic and metabolic perturbations. Mannitol equilibration with the extracellular space was found to occur in less than 1 min in this preparation. Na+‐dependent choline (Ch+) uptake was shown to be linear for 16 min and to reach an equilibrium before Na+‐independent Ch+ uptake, which continued to increase for 60 min. Elevated [K+]0 concentrations inhibited Ch+ uptake and ACh synthesis. Glycolytic and respiratory inhibitors also reduced both processes, as did ouabain and omission of [K+]0. Incubation conditions that reduce transmitter release had no effect on inhibition by high [K+]0. Reduction of SDHACU and sodium‐dependent ACh synthesis by depolarization with high [K+]0 or by inhibition of Na, K‐ATPase implies that the electrochemical gradients for Ch+ and Na+ are important in providing a driving force for high‐affinity Ch+ uptake. The inhibition by metabolic blockers suggests active transport, but the effects may be indirect, caused by reduced Na, K‐ATPase activity and alterations in membrane potential. While most metabolic inhibitors exerted parallel effects on both Ch+ uptake and ACh synthesis, in some cases Ch+ uptake was more strongly inhibited than ACh synthesis. This occurred in preparations incubated with high [K+]0 and ouabain. Na+‐dependent Ch+ uptake and ACh synthesis were found to be temperature‐dependent with a Q10 (20–30°) of 3.6 and 6.6, respectively and a Q10 (30–40°) of 1.3 and 1.0, respectively. Inhibition of acetylcholinesterase by paraoxon increases to 92% the proportion of the Ch+ taken up which is converted to ACh. ACh did not reduce Ch+ transport when present at 100 μM.