D-Aspartate is stored in secretory granules and released through a Ca(2+)-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 KCl, 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 Ca(2+) in the medium. The addition of also triggered d-aspartate release. Botulinum neurotoxin type E inhibited about 40% of KCl- and Ca(2+)-dependent d-aspartate release followed by specific cleavage of 25-kDa synaptosomal-associated protein. alpha-Latrotoxin increased the intracellular [Ca(2+)] and caused the Ca(2+)-dependent d-aspartate release. Bafilomycin A1 dissipated the intracellular acidic regions and inhibited 40% of the Ca(2+)-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 A1 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 Ca(2+)-dependent exocytotic mechanism. Exocytosis of D-aspartate further supports the role(s) of D-aspartate as a chemical transmitter in neuroendocrine cells.
Nakatsuka
S
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
Hayashi
M
M
Muroyama
A
A
Otsuka
M
M
Kozaki
S
S
Yamada
H
H
Moriyama
Y
Y
D000310:Adrenal Gland Neoplasms / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D005089:Exocytosis; D016716:PC12 Cells; D010673:Pheochromocytoma / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*
D001224:Aspartic Acid; D002118:Calcium