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 Km value of 19.8 +/- 0.8 microM and Vmax value of 0.334 +/- 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 degrees 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.