Pancreatic islet cells express receptors and transporters for L-glutamate and are thus believed to use Lglutamate 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 ␣TC6 cells (clonal mouse pancreatic ␣-cells -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. Digitoninpermeabilized cells took up L-glutamate only in the presence of MgATP, which is sensitive to bafilomycin A 1 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 ␣TC6 cells accumulate L-glutamate in the synaptophysin-containing vesicles in an ATP-dependent manner and secrete it through a Ca 2؉ -dependent exocytic mechanism. The Ca 2؉ -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 ␣-cells through Ca 2؉ -dependent regulated exocytosis.

(7). In the pineal gland, it has been shown that D-aspartate is present in pinealocytes, endocrine cells for melatonin (8,9). Upon incubation of pinealocytes with exogenous D-aspartate, melatonin synthesis is strongly inhibited through the inhibition of N-acetyltransferase activity (9, 10). Thus, D-aspartate seems to be a modulator of melatonin synthesis. Furthermore, exogenous Daspartate stimulates the release of luteinizing hormone and growth hormone in the anterior pituitary (11).As chemical transmitters, D-serine and D-aspartate should be secreted from neuroendocrine cells. However, the mechanism by which these amino acids are secreted from neuroendocrine cells is less understood. D-Serine is released from astrocytes upon stimulation by glutamate (5). Because D-serine is present in the cytoplasm, reversed D-serine transport through a Na ϩ -dependent serine transporter at the plasma membrane was proposed (5). Similarly, D-aspartate is present in the cytoplasm of pinealocytes and is released from the cells (8, 9). Pinealocytes express the Na ϩ -dependent glutamate transporter, which recognizes D-aspartate as a substrate, and its inhibition by various antagonists decreases release of D-aspartate (9, 10), suggesting that the Na ϩ -dependent glutamate transporter is involved in the release of D-aspartate in pinealocytes.Here we present another type of mechanism of secretion of D-aspartate in neuroendocrine cells. A subset of rat pheochromocytoma PC12 cells contains an appreciable amount of Daspartate (12). We have extensively investigated the localization and release of D-aspartate in PC12 cells and found that PC12 cells store D-aspartate in secretory granules and secrete it through a Ca 2ϩ -dependent exocytotic mechanism. EXPERIMENTAL PROCEDURESCell Culture-PC12 cells were cultured in 20 ml of Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% fetal calf serum, 5% horse serum, 55 g/ml sodium pyruvate, 4.5 g/liter