d-Aspartate Is Stored in Secretory Granules and Released through a Ca2+-dependent Pathway in a Subset of Rat Pheochromocytoma PC12 Cells

Abstract

(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

Secretory Granule-mediated Co-secretion ofl-Glutamate and Glucagon Triggers Glutamatergic Signal Transmission in Islets of Langerhans

“…Recently, we showed that D-aspartate is accumulated in secretory granules and secreted from PC12 cells (48). Thus, co-secretion of excitatory amino acids with hormones might be a common feature in endocrine signal transmission.…”

Exocytosis of D-Aspartate from INS-1E Clonal .BETA. Cells

“…5,10,11) In brief, cultured cells (1ϫ10 7 cells) were washed three times with a Ringer's solution comprising 10 mM HEPES, pH 7.4, 0.2% BSA, 4.7 mM KCl and 2. Ringer containing 50 mM KCl was used.…”

“…The antibodies did not cross react with L-aspartate or L-glutamate, or other related amino acids, the details being given previously. 11) Monoclonal antibodies against insulin were obtained from Cymbus Biotechnology Ltd.…”

“…Immunohistochemical Analysis Immunohistochemical analysis was performed as described 11,13) with a slight modification. Cells were incubated in phosphate-buffered saline (PBS) containing 1% BSA and 2% goat serum and 0.2% saponin to permeabilize the cellular and organelle membranes for 15 min.…”

“…10) Pheochromocytoma PC12 cells also synthesize D-aspartate de novo, store it in dopamine-containing secretory granules, and secrete it through exocytosis. 11) In Leydig cells, D-aspartate is present in the cytoplasm and stimulates testosterone synthesis through modulation of steroidogenesis. 3,12) In pituitary cells, D-aspartate is metabolically converted to N-methyl-D-aspartate (NMDA), which in turn stimulates the secretion of hypothalamic releasing hormones.…”