SNARE protein-dependent glutamate release from astrocytes.
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.
Araque
A
A
Laboratory of Cellular Signaling, Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011, USA. araque@cajal.csic.es
Li
N
N
Doyle
R T
RT
Haydon
P G
PG
D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors; D027322:Amino Acid Transport System X-AG; D000818:Animals; D000831:Animals, Newborn; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D001905:Botulinum Toxins / Q000494:pharmacology; D019274:Botulinum Toxins, Type A; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D003998:Dicarboxylic Acids / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000502:physiology*; D007608:Kainic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D018942:Macrolides; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D011759:Pyrrolidines / Q000494:pharmacology; D051381:Rats; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D000900:Anti-Bacterial Agents; D003998:Dicarboxylic Acids; D004791:Enzyme Inhibitors; D018942:Macrolides; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011759:Pyrrolidines; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; C096323:rimabotulinumtoxinB; C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D018698:Glutamic Acid; C020889:dihydrokainic acid; C040929:bafilomycin A1; C083486:pyrrolidine-2,4-dicarboxylic acid; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D006180:Proton-Translocating ATPases; D007608:Kainic Acid; D002118:Calcium