D-aspartate is stored in secretory granules and released through a Ca2+-dependent pathway in a subset of rat pheochromocytoma PC12 cells

D-Aspartate in mammalian neuronal and neuroendocrine cells is suggested to play a regulatory role(s) in the neuroendocrine function. Although D-aspartate is known to be released from neuroendocrine cells, the mechanism underlying the release is less understood. Rat pheochromocytoma PC12 cells contain an appreciable amount of D-aspartate (257 +/- 31 pmol/10(7) cells). Indirect immunofluorescence microscopy with specific antibodies against D-aspartate indicated that the amino acid is present within a particulate structure, which is co-localized with dopamine and chromogranin A, markers for secretory granules, but not with synaptophysin, a marker for synaptic-like microvesicles. After sucrose density gradient centrifugation of the postnuclear particulate fraction, about 80% of the D-aspartate was recovered in the secretory granule fraction. Upon the addition of KCI, an appreciable amount of D-aspartate (about 40 pmol/10(7) cells at 10 min) was released from cultured cells on incubation in the presence of Ca2+ in the medium. The addition of A23187 also triggered D-aspartate release. Botulinum neurotoxin type E inhibited about 40% of KCl- and Ca2+-dependent D-aspartate release followed by specific cleavage of 25-kDa synaptosomal-associated protein. alpha -latrotoxin increased the intracellular [Ca2+] and caused the Ca2+-dependent D-aspartate release. Bafilomycin Al dissipated the intracellular acidic regions and inhibited 40% of the Ca2+-dependent D-aspartate release. These properties are similar to those of the exocytosis of dopamine. Furthermore, digitonin-permeabilized cells took up radiolabeled D-aspartate depending on MgATP, which is sensitive to bafilomycin Al or 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile. Taken together, these results strongly suggest that D-aspartate is stored in secretory granules and then secreted through a Ca2+-dependent exocytotic mechanism. Exocytosis of D-aspartate further supports the role(s) of D-aspartate as a chemical transmitter in neuroendocrine cells.