A plethora of presynaptic proteins associated with ATP-storing organelles in cultured astrocytes

Cultured astrocytes can release a variety of messenger substances via recep tor-mediated mechanisms, implicating their potential for regulated exocytos is and the participation of proteins of the SNARE complex. Here we demonstr ate the astrocytic expression and organellar association of a large variety of synaptic proteins (synaptobrevin II, synaptotagmin I, synaptophysin, ra b3a, synapsin I, SNAP-25, and syntaxin I) and also of the ubiquitous cellub revin. As revealed by immunoblotting the expression of synaptic proteins wa s highest within the first few days after plating. Synaptophysin and SNAP-2 5 showed the most significant decline with prolonged culture time. Rab3a an d synaptobrevin II were retained at a high level and synaptotagmin I, synap sin I, and syntaxin I at a lower level until 20 DIV. The immunoreaction for cellubrevin was low at the beginning and increased with prolonged culture time. As revealed by light microscopical immunocytochemistry the proteins a re expressed by GFAP-positive astrocytes and associated with organelles of varying size. Immunoelectron microscopical analysis allocates synaptobrevin II and synaptophysin to the membranes of vesicular organelles. Double labe ling experiments for pairs of synaptic proteins reveal that individual syna ptic proteins can be entirely colocalized or partly reside on different org anelles. Subcellular fractionation of astrocyte cultures by sucrose density gradient centrifugation after 2, 6, 13, and 20 DIV showed that the protein s sediment with ATP containing organelles of a broad density range. Our dat a suggest that messenger substances may be released from cultured astrocyte s via receptor-mediated, Ca2+-dependent exocytosis. (C) 1999 Wiley-Liss, In c.