Exocytotic release of ATP from cultured astrocytes.
Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.
Pangršič
Tina
T
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, SI-1000 Ljubljana, Slovenia; Celica Biomedical Center, Proletarska cesta 4, SI-1000 Ljubljana, Slovenia.
Potokar
Maja
M
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, SI-1000 Ljubljana, Slovenia; Celica Biomedical Center, Proletarska cesta 4, SI-1000 Ljubljana, Slovenia.
Stenovec
Matjaž
M
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, SI-1000 Ljubljana, Slovenia; Celica Biomedical Center, Proletarska cesta 4, SI-1000 Ljubljana, Slovenia.
Kreft
Marko
M
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, SI-1000 Ljubljana, Slovenia; Celica Biomedical Center, Proletarska cesta 4, SI-1000 Ljubljana, Slovenia.
Fabbretti
Elsa
E
Neurobiology Sector, International School for Advanced Studies (SISSA), Via Beirut 2-4, 34014 Trieste, Italy.
Nistri
Andrea
A
Neurobiology Sector, International School for Advanced Studies (SISSA), Via Beirut 2-4, 34014 Trieste, Italy.
Pryazhnikov
Evgeny
E
Neuroscience Center, University of Helsinki, PO Box 56 (Viikinkaari 4), FIN-00014 Helsinki, Finland.
Khiroug
Leonard
L
Neuroscience Center, University of Helsinki, PO Box 56 (Viikinkaari 4), FIN-00014 Helsinki, Finland.
Giniatullin
Rashid
R
Neurobiology Sector, International School for Advanced Studies (SISSA), Via Beirut 2-4, 34014 Trieste, Italy.
Zorec
Robert
R
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, SI-1000 Ljubljana, Slovenia; Celica Biomedical Center, Proletarska cesta 4, SI-1000 Ljubljana, Slovenia. Electronic address: robert.zorec@mf.uni-lj.si.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D011796:Quinacrine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058477:Receptors, Purinergic P2X3; D022142:Secretory Vesicles / Q000378:metabolism*; D013569:Synapses / Q000502:physiology
D007476:Ionophores; C546407:P2RX3 protein, human; D018048:Receptors, Purinergic P2; D058477:Receptors, Purinergic P2X3; D018698:Glutamic Acid; D015759:Ionomycin; D000255:Adenosine Triphosphate; D011796:Quinacrine