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Diabetes 50:1012-1020, 2001
© 2001 by the American Diabetes Association, Inc.

Ca2+-Dependent Exocytosis of L-Glutamate by {alpha}TC6, Clonal Mouse Pancreatic {alpha}-Cells

Hiroshi Yamada1, Masato Otsuka1, Mitsuko Hayashi1, Shuuichi Nakatsuka1, Kazuyuki Hamaguchi2, Akitsugu Yamamoto3,4, and Yoshinori Moriyama1,4

1 Department of Biochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama
2 Faculty of Medicine, Oita Medical University, Oita
3 Department of Physiology, Kansai Medical University, Moriguchi
4 CREST, Japan Science and Technology Corporation, Osaka, Japan

Pancreatic islet cells express receptors and transporters for L-glutamate and are thus believed to use L-glutamate as an intercellular signaling molecule. However, the mechanism by which L-glutamate appears in the islets is unknown. In the present study, we investigated whether L-glutamate is secreted through exocytosis by {alpha}TC6 cells (clonal mouse pancreatic {alpha}-cells). An appreciable amount of L-glutamate was released from cultured cells after the addition of KCl or A23187 in the presence of Ca2+ and 10 mmol/l glucose in the medium. The KCl-induced glutamate release was significantly reduced when assayed in the absence of Ca2+ or when the cells were pretreated with EGTA-AM. The KCl-induced Ca2+-dependent glutamate release was inhibited ~40% by voltage-gated Ca2+ channel blockers, such as nifedipine at 20 µmol/l. The degree of KCl-induced Ca2+-dependent glutamate release was correlated with an increase in intracellular [Ca2+], as monitored by fura-2 fluorescence. Botulinum neurotoxin type E inhibited 55% of the KCl-induced Ca2+-dependent glutamate release, followed by specific cleavage of 25 kDa synaptosomal-associated protein. Furthermore, bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, inhibited 40% of the KCl-induced Ca2+-dependent glutamate release. Immunoelectronmicroscopy with antibodies against synaptophysin, a marker for neuronal synaptic vesicles and endocrine synaptic-like microvesicles, revealed a large number of synaptophysin-positive clear vesicles in cells. Digitonin-permeabilized cells took up L-glutamate only in the presence of MgATP, which is sensitive to bafilomycin A1 or 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (a proton conductor) but insensitive to either oligomycin or vanadate. From these results, it was concluded that {alpha}TC6 cells accumulate L-glutamate in the synaptophysin-containing vesicles in an ATP-dependent manner and secrete it through a Ca2+-dependent exocytic mechanism. The Ca2+-dependent glutamate release was also triggered when cells were transferred in the medium containing 1 mmol/l glucose, suggesting that low glucose treatment stimulates the release of glutamate. Our results are consistent with the idea that L-glutamate is secreted by {alpha}-cells through Ca2+-dependent regulated exocytosis.


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