Ca2+-dependent exocytosis of L-glutamate by alpha TC6, clonal mouse pancreatic alpha-cells

Pancreatic islet cells express receptors and transporters for L-glutamate a nd are thus believed to nse L-glutamate as an intercellular signaling molec ule. However, the mechanism by which L-glutamate appears in the islets is u nknown. In the present study, me investigated whether L-glutamate is secret ed through exocytosis by alpha TC6 cells (clonal mouse pancreatic or-cells) . An appreciable amount of L-glutamate mas released from cultured cells aft er the addition of KCl or A23181 in the presence of Ca2+ and 10 mmol/l gluc ose in the medium. The KCl-induced glutamate release was significantly redu ced when assayed in the absence of Ca2+ or when the cells were pretreated w ith EGTA-AM. The KCl-induced Ca2+-dependent glutamate release was inhibited similar to 40% by voltage-gated Ca2+ channel blockers, such as nifedipine at 20 mu mol/l. The degree of KCl-induced Ca2+-dependent glutamate release was correlated with an increase in intracellular [Ca2+], as monitored by fu ra-a fluorescence. Botulinum neurotoxin type E inhibited 55% of the KCl-ind uced Ca2+ dependent glutamate release, followed by specific cleavage of 25 kDa synaptosomal-associated protein. Furthermore, bafilomycin A,, a specifi c inhibitor of vacuolar Hf-ATPase, inhibited 40% of the KCl-induced Ca2+-de pendent glutamate release. Immunoelectronmicroscopy with antibodies against synaptophysin, a marker for neuronal synaptic vesicles and endocrine synap tic-like microvesicles, revealed a large number of synaptophysin-positive c lear vesicles in cells. Digitonin-permeabilized cells took up L-glutamate o nly in the presence of MgATP, which is sensitive to bafilomycin A(1) or 3,5 -di-tert-butyl-4-hydroxybenzglidene-malononitrile (a proton conductor) but insensitive to either oligomycin or vanadate. From these results, it was co ncluded that alpha TC6 cells accumulate L-glutamate in the synaptophysin-co ntaining 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 gl ucose, suggesting that low glucose treatment stimulates the release of glut amate. Our results are consistent with the idea that L-glutamate is secrete d by alpha -cells through Ca2+-dependent regulated exocytosis.