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J. Biol. Chem., Vol. 282, Issue 39, 28749-28758, September 28, 2007

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Exocytotic Release of ATP from Cultured Astrocytes^*

Tina Pangri¹, Maja Potokar¹, Matja Stenovec, Marko Kreft, Elsa Fabbretti^¶, Andrea Nistri^¶, Evgeny Pryazhnikov^||, Leonard Khiroug^||, Rashid Giniatullin^¶, and Robert Zorec²

From the Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloka 4, SI-1000 Ljubljana, Slovenia, the Celica Biomedical Center, Proletarska cesta 4, SI-1000 Ljubljana, Slovenia, the ^||Neuroscience Center, University of Helsinki, PO Box 56 (Viikinkaari 4), FIN-00014 Helsinki, Finland, and the ^¶Neurobiology Sector, International School for Advanced Studies (SISSA), Via Beirut 2-4, 34014 Trieste, Italy

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 [Ca²⁺]_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 [Ca²⁺]_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 Ca²⁺-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X₃ 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 Ca²⁺. 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.

Received for publication, January 10, 2007 , and in revised form, July 11, 2007.

^* This work was supported in part by Grants Z3-7476-1683-06 of Slovenian Research Agency and P3 310, of The Ministry of Higher Education, Science and Technology of The Republic of Slovenia, BI-US/06-07-015, EC Contract DECG QLG3-CT-2001-02004, and by Center for International Mobility (CIMO), Finland, and the Academy of Finland. The work carried out at SISSA was supported by a FIRB grant (to A. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ These authors equally contributed to first authorship.

² To whom correspondence should be addressed. Tel.: 38615437020; Fax: 38615437036; E-mail: robert.zorec{at}mf.uni-lj.si.

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