Journal
GLIA
Volume 56, Issue 1, Pages 38-49Publisher
WILEY-LISS
DOI: 10.1002/glia.20590
Keywords
adenosine triphosphate; quinacrine; TIRF; plasma membrane; vesicle; astrocyte; Ca2+
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Astrocytes release a variety of transmitter molecules, which mediate communication between glial cells in the brain and modulate synaptic transmission. ATP is a major glia-derived transmitter, but the mechanisms and kinetics of ATP release from astrocytes remain largely unknown. Here, we combined epifluorescence and total internal reflection fluorescence microscopy to monitor individual quinacrine-loaded ATP-containing vesicles undergoing exocytosis in cultured astrocytes. In resting cells, vesicles exhibited three-dimensional motility, spontaneous docking and release at low rate. Extracellular ATP application induced a Ca2+ -dependent increase in the rate of exocytosis, which persisted for several minutes. Using UV flash photolysis of caged Ca2+, the threshold [Ca2+](i) for ATP exocytosis was found to be similar to 350 nM. Subthreshold [Ca2+](i) transients predominantly induced vesicle docking at plasma membrane without subsequent release. ATP exocytosis triggered either by purinergic stimulation or by Ca2+ uncaging occurred after a substantial delay ranging from tens to hundreds of seconds, with only similar to 4% of release occurring during the first 30 s. The time course of the cargo release from vesicles had two peaks centered on <= 10 s and 60 s. These results demonstrate that: (1) [Ca2+](i) elevations in cultured astrocytes trigger docking and release of ATP-containin vesicles; (2) vesicle docking and release have different Ca thresholds; (3) ATP exocytosis is delayed by several minutes and highly asynchronous; (4) two populations of ATP-containing vesicles with distinct (fast and slow) time course of cargo release exist in cultured astrocytes. (c) 2007 Wiley-Liss, Inc.
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