L-Glutamate is the major excitatory neurotransmitter in the central nervous system and plays important roles in many neuronal processes such as fast synaptic transmission and neuronal plasticity (1, 2). To use L-glutamate as an intercellular signaling molecule, neuronal cells develop the glutamatergic system. Thus, L-glutamate is accumulated in synaptic vesicles through vesicular glutamate transporters (VGLUTs), 1 and is secreted through exocytosis. The released L-glutamate binds to the receptor so as to transmit signals intercellularly. The excess amount of L-glutamate in synaptic cleft is sequestrated through plasma membrane-type glutamate transporter. Recent evidence has indicated that peripheral non-neuronal tissues also possess the glutamatergic system and use L-glutamate as an intercellular transmitter (3). The islet of Langerhans, a pancreatic miniature organ for the hormones regulating the blood glucose level, is composed of four major types of endocrine cells, i.e. insulin-secreting ␤ cells, glucagon-secreting ␣ cells, somatostatin-secreting ␦ cells, and pancreatic polypeptide-secreting F cells. These islet cells expresses functional glutamate receptors and plasma membrane-type glutamate transporter (4 -11), suggesting that L-glutamate functions as an intercellular transmitter in islet. In fact, L-glutamate has been shown to affect secretion of insulin or glucagon from islet cells, isolated islets, or perfused pancreas (4 -11). However, the role of L-glutamate as an intercellular chemical transmitter in the islets has been long controversial, mainly because critical issues, i.e. where, when, and how L-glutamate appears in the islets and what happens upon stimulation of glutamate receptors in the islets, remain unresolved.Recent findings indicate that brain-specific Na ϩ -dependent inorganic phosphate cotransporter (12) and its isoform, differentiation-associated Na ϩ -dependent inorganic phosphate cotransporter (13), function as VGLUTs and are thus abbreviated as VGLUT1 and VGLUT2, respectively (14 -21). These VGLUTs seem to be potential probes for the site of L-glutamate release in peripheral tissues as well as the central nervous system since these transporters are essential for L-glutamate signal output. We have shown that VGLUT2 is expressed in ␣TC6 cells, clonal ␣ cells, and islet ␣ cells, but not in ␤ or ␦ cells (18). These results are consistent with the occurrence of Ca 2ϩ -dependent exocytosis of L-glutamate from ␣TC6 cells (22) and suggest that ␣ cells are the sites of L-glutamate signal appearance.During course of the study, we noticed that the expression and subcellular localization of VGLUTs are of extraordinary

In islets of Langerhans, L-glutamate is stored in glucagon-containing secretory granules of ␣-cells and cosecreted with glucagon under low-glucose conditions. The L-glutamate triggers secretion of ␥-aminobutyric acid (GABA) from ␤-cells, which in turn inhibits glucagon secretion from ␣-cells through the GABAA receptor. In the present study, we tested the working hypothesis that L-glutamate functions as an autocrine/paracrine modulator and inhibits glucagon secretion through a glutamate receptor (

The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of ␥-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (⌬) (positive inside) but not ⌬pH. VIAAT-mediated GABA uptake had an absolute requirement for Cl ؊ and actually accompa-

Many metabolic factors affect the secretion of insulin from ␤-cells and glucagon from ␣-cells of the islets of Langerhans to regulate blood glucose. Somatostatin from ␦-cells, considered a local inhibitor of islet function, reduces insulin and glucagon secretion by activating somatostatin receptors in islet cells. Somatostatin secretion from ␦-cells is increased by high glucose via glucose metabolism in a similar way to insulin secretion from ␤-cells. However, it is unknown how low glucose triggers somatostatin secretion. Because L-glutamate is cosecreted with glucagon from ␣-cells under low-glucose conditions and acts as a primary intercellular messenger, we hypothesized that glutamate signaling triggers the secretion of somatostatin. In this study, we showed that ␦-cells express GluR4c-flip, a newly identified splicing variant of GluR4, an (RS)-␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptor of rat. After treatment with L-glutamate, AMPA, or kainate, secretion of somatostatin from isolated islets was significantly stimulated under low-glucose conditions. The glutamatedependent somatostatin secretion was Ca 2؉ dependent and blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. Somatostatin in turn inhibited the secretion of L-glutamate and glucagon from ␣-cells. These results indicate that L-glutamate triggers somatostatin secretion from ␦-cells by way of the GluR4c-flip receptor under lowglucose conditions. The released somatostatin may complete the feedback inhibition of ␣-cells. Thus, ␣-and ␦-cells may communicate with each other through Lglutamate and somatostatin signaling.

(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