Ca2+-dependent exocytosis of L-glutamate by alphaTC6, clonal mouse pancreatic alpha-cells.
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 alphaTC6 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 approximately 40% by voltage-gated Ca2+ channel blockers, such as nifedipine at 20 micromol/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 alphaTC6 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.
Yamada
H
H
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Otsuka
M
M
Hayashi
M
M
Nakatsuka
S
S
Hamaguchi
K
K
Yamamoto
A
A
Moriyama
Y
Y
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001905:Botulinum Toxins / Q000494:pharmacology; D019556:COS Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020013:Calcium Signaling / Q000502:physiology; D002522:Chlorocebus aethiops; D002999:Clone Cells; D004110:Diltiazem / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007515:Islets of Langerhans / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D007700:Kinetics; D018942:Macrolides; D051379:Mice; D009543:Nifedipine / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D016708:Synaptophysin / Q000032:analysis / Q000378:metabolism; D013696:Temperature; D014638:Vanadates / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D018942:Macrolides; D009570:Nitriles; D009840:Oligomycins; D016708:Synaptophysin; D000001:Calcimycin; D018698:Glutamic Acid; D014638:Vanadates; C001784:SF 6847; D011189:Potassium Chloride; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; D001905:Botulinum Toxins; D004110:Diltiazem; D009543:Nifedipine; D002118:Calcium; C096326:botulinum toxin type E