Chang S, Girod R, Morimoto T, O'Donoghue M, Popov S
Department of Physiology and Biophysics, University of Illinois at Chicago 60612, USA.
Biophys J. 1998 Sep;75(3):1354-64. doi: 10.1016/S0006-3495(98)74053-6.
Fibroblasts in cell culture were loaded with exogenous neurotransmitter acetylcholine (ACh). ACh secretion from loaded cells was detected by whole-cell patch clamp recordings from Xenopus myocytes manipulated into contact with ACh-loaded cells. Two different approaches were used for ACh loading. In the first approach, fibroblasts were incubated in the culture medium containing ACh. Recordings from myocytes revealed fast inward currents that resemble miniature endplate currents found at neuromuscular synapses. The currents observed in recordings from myocytes were due to exocytosis of ACh-containing vesicles. Although exogenous ACh penetrated through the plasma membrane of fibroblasts during incubation and was present in the cytoplasm at detectable levels, cytoplasmic ACh did not contribute to the quantal ACh secretion. In the second approach, exogenous ACh was loaded into the cytoplasm of fibroblasts by microinjection. Under these experimental conditions, fibroblasts also exhibited spontaneous quantal ACh secretion. Analysis of the exocytotic events in fibroblasts following two different protocols of ACh loading revealed that the vesicular compartments responsible for uptake of exogenous ACh are associated with the endocytic recycling pathway. Extrapolation of our results to neuronal cells suggest that in cholinergic neurons, in addition to genuine synaptic vesicles, ACh can be secreted by the vesicles participating in endosomal membrane recycling.
将细胞培养中的成纤维细胞装载外源性神经递质乙酰胆碱（ACh）。通过对与装载ACh的细胞接触的非洲爪蟾肌细胞进行全细胞膜片钳记录来检测装载细胞分泌的ACh。采用了两种不同的方法进行ACh装载。在第一种方法中，将成纤维细胞在含有ACh的培养基中孵育。对肌细胞的记录显示出快速内向电流，类似于在神经肌肉突触处发现的微小终板电流。在肌细胞记录中观察到的电流是由于含ACh囊泡的胞吐作用。虽然在孵育过程中外源性ACh穿透成纤维细胞的质膜并以可检测水平存在于细胞质中，但细胞质中的ACh对量子化ACh分泌没有贡献。在第二种方法中，通过显微注射将外源性ACh装载到成纤维细胞的细胞质中。在这些实验条件下，成纤维细胞也表现出自发性量子化ACh分泌。对两种不同ACh装载方案后成纤维细胞胞吐事件的分析表明，负责摄取外源性ACh的囊泡区室与内吞再循环途径有关。将我们的结果外推到神经元细胞表明，在胆碱能神经元中，除了真正的突触囊泡外，ACh还可以由参与内体膜再循环的囊泡分泌。
