S. Nakatsuka et al., D-aspartate is stored in secretory granules and released through a Ca2+-dependent pathway in a subset of rat pheochromocytoma PC12 cells, J BIOL CHEM, 276(28), 2001, pp. 26589-26596
D-Aspartate in mammalian neuronal and neuroendocrine cells is suggested to
play a regulatory role(s) in the neuroendocrine function. Although D-aspart
ate is known to be released from neuroendocrine cells, the mechanism underl
ying the release is less understood. Rat pheochromocytoma PC12 cells contai
n an appreciable amount of D-aspartate (257 +/- 31 pmol/10(7) cells). Indir
ect immunofluorescence microscopy with specific antibodies against D-aspart
ate indicated that the amino acid is present within a particulate structure
, which is co-localized with dopamine and chromogranin A, markers for secre
tory granules, but not with synaptophysin, a marker for synaptic-like micro
vesicles. After sucrose density gradient centrifugation of the postnuclear
particulate fraction, about 80% of the D-aspartate was recovered in the sec
retory 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 cultur
ed cells on incubation in the presence of Ca2+ in the medium. The addition
of A23187 also triggered D-aspartate release. Botulinum neurotoxin type E i
nhibited about 40% of KCl- and Ca2+-dependent D-aspartate release followed
by specific cleavage of 25-kDa synaptosomal-associated protein. alpha -latr
otoxin increased the intracellular [Ca2+] and caused the Ca2+-dependent D-a
spartate release. Bafilomycin Al dissipated the intracellular acidic region
s and inhibited 40% of the Ca2+-dependent D-aspartate release. These proper
ties are similar to those of the exocytosis of dopamine. Furthermore, digit
onin-permeabilized cells took up radiolabeled D-aspartate depending on MgAT
P, which is sensitive to bafilomycin Al or 3,5-di-tert-butyl-4-hydroxybenzy
lidene-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 cel
ls.