bafilomycin-a1 and 3-hydroxyaspartic-acid

We investigated the cellular mechanisms underlying the Ca(2+)-dependent release of glutamate from cultured astrocytes isolated from rat hippocampus. Using Ca(2+) imaging and electrophysiological techniques, we analyzed the effects of disrupting astrocytic vesicle proteins on the ability of astrocytes to release glutamate and to cause neuronal electrophysiological responses, i.e., a slow inward current (SIC) and/or an increase in the frequency of miniature synaptic currents. We found that the Ca(2+)-dependent glutamate release from astrocytes is not caused by the reverse operation of glutamate transporters, because the astrocyte-induced glutamate-mediated responses in neurons were affected neither by inhibitors of glutamate transporters (beta-threo-hydroxyaspartate, dihydrokainate, and L-trans-pyrrolidine-2,4-dicarboxylate) nor by replacement of extracellular sodium with lithium. We show that Ca(2+)-dependent glutamate release from astrocytes requires an electrochemical gradient necessary for glutamate uptake in vesicles, because bafilomycin A(1), a vacuolar-type H(+)-ATPase inhibitor, reduced glutamate release from astrocytes. Injection of astrocytes with the light chain of the neurotoxin Botulinum B that selectively cleaves the vesicle-associated SNARE protein synaptobrevin inhibited the astrocyte-induced glutamate response in neurons. Therefore, the Ca(2+)-dependent glutamate release from astrocytes is a SNARE protein-dependent process that requires the presence of functional vesicle-associated proteins, suggesting that astrocytes store glutamate in vesicles and that it is released through an exocytotic pathway.

Topics: Amino Acid Transport System X-AG; Animals; Animals, Newborn; Anti-Bacterial Agents; Aspartic Acid; Astrocytes; ATP-Binding Cassette Transporters; Botulinum Toxins; Botulinum Toxins, Type A; Calcium; Cells, Cultured; Coculture Techniques; Dicarboxylic Acids; Enzyme Inhibitors; Glutamic Acid; Hippocampus; Kainic Acid; Macrolides; Membrane Potentials; Membrane Proteins; Nerve Tissue Proteins; Neurons; Proton-Translocating ATPases; Pyrrolidines; Rats; SNARE Proteins; Vesicular Transport Proteins