D-Aspartate is Stored in Secretory Granules and Released through a Ca 2+-dependent Pathway in a Subset of Rat Pheochromocytoma PC12 Cells

Shuuichi Nakatsuka, Mitsuko Hayashi, Akiko Muroyama, Masato Otsuka, Shunji Kozaki, Hiroshi Yamada, Yoshinori Moriyama

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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 languageEnglish
Pages (from-to)26589-26596
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number28
DOIs
Publication statusPublished - Jul 13 2001

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D-Aspartic Acid
PC12 Cells
Secretory Vesicles
Pheochromocytoma
Rats
Neuroendocrine Cells
Exocytosis
Dopamine
Synaptosomal-Associated Protein 25
Digitonin
Chromogranin A
Synaptophysin
Density Gradient Centrifugation
Centrifugation
Calcimycin
Indirect Fluorescent Antibody Technique
Fluorescence Microscopy

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 journalArticle

Nakatsuka, Shuuichi ; Hayashi, Mitsuko ; Muroyama, Akiko ; Otsuka, Masato ; Kozaki, Shunji ; Yamada, Hiroshi ; Moriyama, Yoshinori. / D-Aspartate is Stored in Secretory Granules and Released through a Ca 2+-dependent Pathway in a Subset of Rat Pheochromocytoma PC12 Cells. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 28. pp. 26589-26596.
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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.",
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