Pharmacological Characteristics of Choline Transport System in Mouse Cerebral Cortical Neurons in Primary Culture

Abstract

The characteristics of [³H]choline transport with high affinity were investigated using primary cultured neurons obtained from the mouse cerebral cortex. [³H]Choline uptake was saturable as a function of extracellular [³H]choline concentration. Analysis by Lineweaver-Burk plot revealed that [³H]choline was transported into neurons by a high affinity transport system with a K_m value of 19.8 ± 0.8μM and V_max value of 0.334 plusmn; 0.022 nmol/mg protein/min. This high affinity transport of [³H]choline was significantly inhibited by the withdrawal of sodium from the incubation medium, incubation at low temperature (4°C) and addition of metabolic inhibitors such as monoiodoacetate. These results indicate that the high affinity [³H]choline uptake in primary cultured neurons is sodium- and energy-dependent. Hemicholinium-3 also showed a competitive inhibition on the [³H]choline transport. Depolarization by high K⁺ induced an enhancement of the [³H]choline uptake in the presence of Ca²⁺. The crude synaptosomal fraction obtained from primary cultured neurons possessed approximately forty-fold higher synthesizing activity of [³H]acetylcholine from [³H]choline than that found in the homogenate preparation of cultured neurons. The present results strongly suggest that the primary cultured neurons used in this study possess a sodium- and energy-dependent high-affinity choline uptake system as well as a synthesizing system for acetylcholine. Possible usefulness of these neurons for investigating neuronal uptake of choline and its functional role in the biosynthesis of acetylcholine are also suggested.