Abstract
Glial subcellular re-sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia-specific proteins glial fibrillary acidic protein (GFAP) and S-100, but not the neuronal proteins 95-kDa postsynaptic density protein (PSD-95), microtubule-associated protein 2 (MAP-2) and beta-tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin-alphaM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1-5 microm) efficiently stimulated the release of tritium from gliosomes pre-labelled with [3H]d-aspartate and of endogenous glutamate in a Ca(2+)-dependent and bafilomycin A1-sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca(2+)-dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d-aspartate release in a concentration- (0.1-3 mm) and Ca(2+)-dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin-1, vesicular-associated membrane protein type 2 (VAMP-2), 23-kDa synaptosome-associated protein (SNAP-23) and 25-kDa synaptosome-associated protein (SNAP-25)] co-existing with GFAP immunoreactivity. Moreover, GFAP or VAMP-2 co-expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several approximately 30-nm non-clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate-accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.
Publication types
-
Comparative Study
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Animals
-
CD11b Antigen / metabolism
-
Calcium / pharmacology
-
Cells, Cultured
-
Cerebral Cortex / cytology*
-
D-Aspartic Acid / metabolism
-
Disks Large Homolog 4 Protein
-
Dose-Response Relationship, Drug
-
Enzyme Inhibitors / pharmacology
-
Exocytosis / physiology*
-
Fluorescent Antibody Technique / methods
-
Glial Fibrillary Acidic Protein / metabolism
-
Glutamic Acid / metabolism*
-
Intracellular Signaling Peptides and Proteins / metabolism
-
Ionomycin / pharmacology
-
Ionophores / pharmacology
-
L-Lactate Dehydrogenase / metabolism
-
Macrolides / pharmacology
-
Male
-
Membrane Proteins / metabolism
-
Microscopy, Confocal / methods
-
Microscopy, Electron, Transmission / methods
-
Microtubule-Associated Proteins / metabolism
-
Myelin Basic Protein / metabolism
-
Neuroglia / metabolism*
-
Neuroglia / ultrastructure
-
Propionates / metabolism
-
Rats
-
Rats, Sprague-Dawley
-
S100 Proteins / metabolism
-
Synaptosomal-Associated Protein 25 / metabolism
-
Synaptosomes / metabolism
-
Synaptosomes / ultrastructure
-
Time Factors
-
Tritium / metabolism
-
Tubulin / metabolism
-
Vesicle-Associated Membrane Protein 2 / metabolism
-
Vesicular Glutamate Transport Protein 1 / metabolism
-
Vesicular Transport Proteins / metabolism
Substances
-
CD11b Antigen
-
Disks Large Homolog 4 Protein
-
Dlg4 protein, rat
-
Enzyme Inhibitors
-
Glial Fibrillary Acidic Protein
-
Intracellular Signaling Peptides and Proteins
-
Ionophores
-
MAP2 protein, rat
-
Macrolides
-
Membrane Proteins
-
Microtubule-Associated Proteins
-
Myelin Basic Protein
-
Propionates
-
S100 Proteins
-
Snap23 protein, rat
-
Synaptosomal-Associated Protein 25
-
Tubulin
-
Vesicle-Associated Membrane Protein 2
-
Vesicular Glutamate Transport Protein 1
-
Vesicular Transport Proteins
-
sintenin
-
Tritium
-
Glutamic Acid
-
D-Aspartic Acid
-
Ionomycin
-
bafilomycin A1
-
L-Lactate Dehydrogenase
-
Calcium