Abstract
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/107 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/107 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. α-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 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 Ca2+-dependent exocytotic mechanism. Exocytosis of D-aspartate further supports the role(s) of D-aspartate as a chemical transmitter in neuroendocrine cells.
| Original language | English |
|---|---|
| Pages (from-to) | 26589-26596 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 276 |
| Issue number | 28 |
| DOIs | |
| Publication status | Published - Jul 13 2001 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
D-Aspartate is Stored in Secretory Granules and Released through a Ca 2+-dependent Pathway in a Subset of Rat Pheochromocytoma PC12 Cells. / Nakatsuka, Shuuichi; Hayashi, Mitsuko; Muroyama, Akiko; Otsuka, Masato; Kozaki, Shunji; Yamada, Hiroshi; Moriyama, Yoshinori.
In: Journal of Biological Chemistry, Vol. 276, No. 28, 13.07.2001, p. 26589-26596.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - D-Aspartate is Stored in Secretory Granules and Released through a Ca 2+-dependent Pathway in a Subset of Rat Pheochromocytoma PC12 Cells
AU - Nakatsuka, Shuuichi
AU - Hayashi, Mitsuko
AU - Muroyama, Akiko
AU - Otsuka, Masato
AU - Kozaki, Shunji
AU - Yamada, Hiroshi
AU - Moriyama, Yoshinori
PY - 2001/7/13
Y1 - 2001/7/13
N2 - 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/107 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/107 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. α-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 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 Ca2+-dependent exocytotic mechanism. Exocytosis of D-aspartate further supports the role(s) of D-aspartate as a chemical transmitter in neuroendocrine cells.
AB - 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/107 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/107 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. α-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 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 Ca2+-dependent exocytotic mechanism. Exocytosis of D-aspartate further supports the role(s) of D-aspartate as a chemical transmitter in neuroendocrine cells.
UR - http://www.scopus.com/inward/record.url?scp=0035854808&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035854808&partnerID=8YFLogxK
U2 - 10.1074/jbc.M011754200
DO - 10.1074/jbc.M011754200
M3 - Article
C2 - 11333256
AN - SCOPUS:0035854808
VL - 276
SP - 26589
EP - 26596
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 28
ER -