title: Vesicular storage and secretion of L-glutamate from glucagon-like peptide 1-secreting clonal intestinal L cells.
abstract: 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.
authors.affiliation: Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Uehara
authors.forename: Sun-Kyung
authors.initials: SK
authors.lastname: Jung
authors.forename: Riyo
authors.initials: R
authors.lastname: Morimoto
authors.forename: Shigeo
authors.initials: S
authors.lastname: Arioka
authors.forename: Takaaki
authors.initials: T
authors.lastname: Miyaji
authors.forename: Narinobu
authors.initials: N
authors.lastname: Juge
authors.forename: Miki
authors.initials: M
authors.lastname: Hiasa
authors.forename: Kahori
authors.initials: K
authors.lastname: Shimizu
authors.forename: Akinori
authors.initials: A
authors.lastname: Ishimura
authors.forename: Masato
authors.initials: M
authors.lastname: Otsuka
authors.forename: Akitsugu
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Pierre
authors.initials: P
authors.lastname: Maechler
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Moriyama
mesh_terms: D000818:Animals; D002999:Clone Cells; D005089:Exocytosis; D052216:Glucagon-Like Peptide 1 / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism* / Q000493:pharmacokinetics; D007421:Intestine, Small / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D022142:Secretory Vesicles / Q000378:metabolism*; D015398:Signal Transduction; D050599:Vesicular Glutamate Transport Protein 2 / Q000378:metabolism
chemical_list: D050599:Vesicular Glutamate Transport Protein 2; D018698:Glutamic Acid; D052216:Glucagon-Like Peptide 1