Uehara, Shunsuke, Jung, Sun-Kyung, Morimoto, Riyo, Arioka, Shigeo, Miyaji, Takaaki, Juge, Narinobu, Hiasa, Miki, Shimizu, Kahori, Ishimura, Akinori, Otsuka, Masato, Yamamoto, Akitsugu, Maechler, Pierre, and Moriyama, Yoshinori
Vesicular glutamate transporter (VGLUT) is responsible for the vesicular storage ofl-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 secretel-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-dependentl-glutamate uptake, which was sensitive to bafilomycin A1 (a vacuolar proton ATPase inhibitor) and Evans blue (a VGLUT inhibitor) but insensitive tod,l-aspartate. Upon depolarization with KCl, GLUTag cells secreted appreciable amounts ofl-glutamate and GLP-1.d-Glucose and methyl-α-d-glucopyranoside, stimulators of exocytosis of GLP-1, also triggered the secretion ofl-glutamate. Thel-glutamate secretion was partially dependent on Ca2+ and sensitive to bafilomycin A1. These results demonstrated that GLUTag cells storedl-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 ofl-glutamate-mediated intercellular signaling in the vicinity of intestinal L cells. [ABSTRACT FROM AUTHOR]