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 b-tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin-αM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1–5 µM) efficiently stimulated the release of tritium from gliosomes pre-labelled with [3H]Daspartate and of endogenous glutamate in a Ca2+-dependent and bafilomycin A1-sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca2+-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 Ca2+-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 ~30-nm non-clustered vesicles were present in the gliosomes 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.