Glia re-sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate.
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.
Stigliani
Sara
S
Pharmacology and Toxicology Section, Department of Experimental Medicine, University of Genoa, Genoa, Italy.
Zappettini
Simona
S
Raiteri
Luca
L
Passalacqua
Mario
M
Melloni
Edon
E
Venturi
Consuelo
C
Tacchetti
Carlo
C
Diaspro
Alberto
A
Usai
Cesare
C
Bonanno
Giambattista
G
D000818:Animals; D039481:CD11b Antigen / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D026603:D-Aspartic Acid / Q000378:metabolism; D000076122:Disks Large Homolog 4 Protein; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology*; D005455:Fluorescent Antibody Technique / Q000379:methods; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D004676:Myelin Basic Protein / Q000378:metabolism; D009457:Neuroglia / Q000378:metabolism* / Q000648:ultrastructure; D011422:Propionates / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D009418:S100 Proteins / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism / Q000648:ultrastructure; D013997:Time Factors; D014316:Tritium / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism; D050598:Vesicular Glutamate Transport Protein 1 / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism
D039481:CD11b Antigen; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D047908:Intracellular Signaling Peptides and Proteins; D007476:Ionophores; C489787:MAP2 protein, rat; D018942:Macrolides; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D004676:Myelin Basic Protein; D011422:Propionates; D009418:S100 Proteins; C498858:Snap23 protein, rat; D050825:Synaptosomal-Associated Protein 25; D014404:Tubulin; D050988:Vesicle-Associated Membrane Protein 2; D050598:Vesicular Glutamate Transport Protein 1; D033921:Vesicular Transport Proteins; C499736:sintenin; D014316:Tritium; D018698:Glutamic Acid; D026603:D-Aspartic Acid; D015759:Ionomycin; C040929:bafilomycin A1; D007770:L-Lactate Dehydrogenase; D002118:Calcium