Cytosolic calcium oscillations in astrocytes may regulate exocytotic release of glutamate.
To obtain insights into the spatiotemporal characteristics and mechanism of Ca(2+)-dependent glutamate release from astrocytes, we developed a new experimental approach using human embryonic kidney (HEK) 293 cells transfected with the NMDA receptor (NMDAR), which act as glutamate biosensors, plated on cultured astrocytes. We here show that oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) in astrocytes trigger synchronous and repetitive [Ca(2+)](i) elevations in sensor HEK cells, and that these elevations are sensitive to NMDAR inhibition. By whole-cell patch-clamp recordings, we demonstrate that the activation of NMDARs in HEK cells results in inward currents that often have extremely fast kinetics, comparable with those of glutamate-mediated NMDAR currents in postsynaptic neurons. We also show that the release of glutamate from stimulated astrocytes is drastically reduced by agents that are known to reduce neuronal exocytosis, i.e., tetanus toxin and bafilomycin A(1). We conclude that [Ca(2+)](i) oscillations represent a frequency-encoded signaling system that controls a pulsatile release of glutamate from astrocytes. The fast activation of NMDARs in the sensor cells and the dependence of glutamate release on the functional integrity of both synaptobrevin and vacuolar H(+) ATPase suggest that astrocytes are endowed with an exocytotic mechanism of glutamate release that resembles that of neurons.
Pasti
L
L
Department of Experimental Biomedical Sciences and Consiglio Nazionale delle Ricerche Center for the Study of Biomembranes, University of Padova, 35121 Padova, Italy.
Zonta
M
M
Pozzan
T
T
Vicini
S
S
Carmignoto
G
G
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003600:Cytosol / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism; D013744:Tetanus Toxin / Q000494:pharmacology; D014162:Transfection
D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D018091:Receptors, AMPA; D018094:Receptors, Metabotropic Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D013744:Tetanus Toxin; C035979:tetanospasmin; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D008666:Metalloendopeptidases