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