Ca2+-dependent exocytosis of L-glutamate by αTC6, clonal mouse pancreatic α-cells

Hiroshi Yamada, Masato Otsuka, Mitsuko Hayashi, Shuuichi Nakatsuka, Kazuyuki Hamaguchi, Akitsugu Yamamoto, Yoshinori Moriyama

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

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 αTC6 cells (clonal mouse pancreatic α-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 αTC6 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 α-cells through Ca2+-dependent regulated exocytosis.

Original languageEnglish
Pages (from-to)1012-1020
Number of pages9
JournalDiabetes
Volume50
Issue number5
Publication statusPublished - 2001

Fingerprint

Exocytosis
Glutamic Acid
Synaptophysin
Islets of Langerhans
Glucose
Synaptosomal-Associated Protein 25
Adenosine Triphosphate
Vacuolar Proton-Translocating ATPases
Oligomycins
Digitonin
Vanadates
Synaptic Vesicles
Fura-2
Calcimycin
Nifedipine
Protons
Cultured Cells

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

Yamada, H., Otsuka, M., Hayashi, M., Nakatsuka, S., Hamaguchi, K., Yamamoto, A., & Moriyama, Y. (2001). Ca2+-dependent exocytosis of L-glutamate by αTC6, clonal mouse pancreatic α-cells. Diabetes, 50(5), 1012-1020.

Ca2+-dependent exocytosis of L-glutamate by αTC6, clonal mouse pancreatic α-cells. / Yamada, Hiroshi; Otsuka, Masato; Hayashi, Mitsuko; Nakatsuka, Shuuichi; Hamaguchi, Kazuyuki; Yamamoto, Akitsugu; Moriyama, Yoshinori.

In: Diabetes, Vol. 50, No. 5, 2001, p. 1012-1020.

Research output: Contribution to journalArticle

Yamada, H, Otsuka, M, Hayashi, M, Nakatsuka, S, Hamaguchi, K, Yamamoto, A & Moriyama, Y 2001, 'Ca2+-dependent exocytosis of L-glutamate by αTC6, clonal mouse pancreatic α-cells', Diabetes, vol. 50, no. 5, pp. 1012-1020.
Yamada H, Otsuka M, Hayashi M, Nakatsuka S, Hamaguchi K, Yamamoto A et al. Ca2+-dependent exocytosis of L-glutamate by αTC6, clonal mouse pancreatic α-cells. Diabetes. 2001;50(5):1012-1020.
Yamada, Hiroshi ; Otsuka, Masato ; Hayashi, Mitsuko ; Nakatsuka, Shuuichi ; Hamaguchi, Kazuyuki ; Yamamoto, Akitsugu ; Moriyama, Yoshinori. / Ca2+-dependent exocytosis of L-glutamate by αTC6, clonal mouse pancreatic α-cells. In: Diabetes. 2001 ; Vol. 50, No. 5. pp. 1012-1020.
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