Kishi M, Ohkuma S, Ma F H, Kuriyama K
Department of Pharmacology, Kyoto Prefectural University of Medicine, Japan.
Jpn J Pharmacol. 1991 Feb;55(2):223-32. doi: 10.1254/jjp.55.223.
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.
利用从小鼠大脑皮层获得的原代培养神经元，研究了具有高亲和力的[³H]胆碱转运的特性。[³H]胆碱摄取作为细胞外[³H]胆碱浓度的函数是可饱和的。通过Lineweaver - Burk图分析表明，[³H]胆碱通过高亲和力转运系统转运到神经元中，其Km值为19.8±0.8微摩尔，Vmax值为0.334±0.022纳摩尔/毫克蛋白质/分钟。这种[³H]胆碱的高亲和力转运受到孵育培养基中钠的去除、低温（4℃）孵育以及添加代谢抑制剂如碘乙酸的显著抑制。这些结果表明，原代培养神经元中高亲和力的[³H]胆碱摄取是钠和能量依赖性的。半胱氨酸-3对[³H]胆碱转运也表现出竞争性抑制作用。在存在Ca²⁺的情况下，高K⁺去极化诱导[³H]胆碱摄取增强。从原代培养神经元获得的粗突触体部分从[³H]胆碱合成[³H]乙酰胆碱的活性比培养神经元匀浆制剂中高约40倍。目前的结果强烈表明，本研究中使用的原代培养神经元具有钠和能量依赖性的高亲和力胆碱摄取系统以及乙酰胆碱合成系统。还表明了这些神经元在研究胆碱的神经元摄取及其在乙酰胆碱生物合成中的功能作用方面可能的用途。
