Vesicular storage and secretion of L-glutamate from glucagon-like peptide 1-secreting clonal intestinal L cells
Vesicular storage and secretion of L-glutamate from glucagon-like peptide 1-secreting clonal intestinal L cells
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DOI：
10.1111/j.1471-4159.2005.03575.x
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发表时间：
2006-01-01
影响因子：
4.7
通讯作者：
Moriyama, Y
中科院分区：
文献类型：
--
作者：
Uehara, S;Jung, SK;Moriyama, Y
囊泡谷氨酸转运体（VGLUT）负责囊泡储存（L）-谷氨酸，并在CNS和一些神经内分泌细胞中谷氨酸介导的细胞间信号传递中起重要作用。肠L细胞是负责分泌胰高血糖素样肽1（GLP-1）的葡萄糖响应性神经内分泌细胞。我们已经表明，肠L细胞表达VGLUT 2，VGLUT亚型，这表明L细胞分泌（L）-谷氨酸。在本研究中，我们使用GLUTag小鼠克隆L细胞研究了这种可能性。RT-PCR和北方印迹分析显示VGLUT 1和VGLUT 2基因表达，但不表达VGLUT 3基因。蛋白质印迹分析揭示了VGLUT 2的免疫对应物，而未检测到VGLUT 1的免疫对应物。间接免疫荧光显微镜显示VGLUT 2免疫反应性在整个细胞中呈点状分布，与GLP-1共定位。双标记免疫电镜证实了VGLUT 2与含GLP-1的分泌颗粒的关联。膜组分表现出ATP依赖性的（L）-谷氨酸摄取，这是敏感的巴夫洛霉素A1（液泡质子ATP酶抑制剂）和伊文思蓝（VGLUT抑制剂），但不敏感的（D，L）-天冬氨酸。在用KCl去极化后，GLUTag细胞分泌可观量的（L）-谷氨酸和GLP-1。（D）-葡萄糖和甲基-α-（D）-吡喃葡萄糖苷（GLP-1胞吐的刺激物）也触发（L）-谷氨酸的分泌。L-谷氨酸的分泌部分依赖于Ca ~（2+），对巴弗洛霉素A_1敏感。这些结果表明，GLUTag细胞将（L）-谷氨酸储存在分泌颗粒中，并通过胞吐作用将其与GLP-1一起分泌。由于GLUTag细胞和肠L细胞表达红藻氨酸受体和质膜谷氨酸转运蛋白，这些结果支持肠L细胞附近的（L）-谷氨酸介导的细胞间信号传导的概念。
Vesicular glutamate transporter (VGLUT) is responsible for the vesicular storage of (L)-glutamate, and plays an essential role in glutamate-mediated intercellular signal transmission in the CNS and in some neuroendocrine cells. Intestinal L cells are the glucose-responsive neuroendocrine cells responsible for the secretion of glucagon-like peptide 1 (GLP-1). We have shown that intestinal L cells express VGLUT2, a VGLUT isoform, which suggests that L cells secrete (L)-glutamate. In the present study, we investigated this possibility using GLUTag mouse clonal L cells. RT-PCR and northern blot analyses revealed expression of the VGLUT1 and VGLUT2 genes, but not of the VGLUT3 gene. Western blot analysis revealed immunological counterparts for VGLUT2, whereas an immunological counterpart of VGLUT1 was not detected. Indirect immunofluorescence microscopy revealed a punctate distribution of VGLUT2 immunoreactivity throughout the cells, which co-localized with GLP-1. Double-labeling immunoelectronmicroscopy confirmed the association of VGLUT2 with GLP-1-containing secretory granules. The membrane fraction exhibited ATP-dependent (L)-glutamate uptake, which was sensitive to bafilomycin A1 (a vacuolar proton ATPase inhibitor) and Evans blue (a VGLUT inhibitor) but insensitive to (D,L)-aspartate. Upon depolarization with KCl, GLUTag cells secreted appreciable amounts of (L)-glutamate and GLP-1. (D)-Glucose and methyl-alpha-(D)-glucopyranoside, stimulators of exocytosis of GLP-1, also triggered the secretion of (L)-glutamate. The (L)-glutamate secretion was partially dependent on Ca2+ and sensitive to bafilomycin A1. These results demonstrated that GLUTag cells stored (L)-glutamate in secretory granules and secreted it with GLP-1 by exocytosis. As GLUTag cells and intestinal L cells express kainate receptors and plasma membrane glutamate transporters, these results support the concept of (L)-glutamate-mediated intercellular signaling in the vicinity of intestinal L cells.