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• W2056369163 abstract "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. 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. Many recent studies demonstrate that astrocytes play a significant modulatory role in synaptic physiology (1Araque A. Parpura V. Sanzgiri R.P. Haydon P.G. Trends Neurosci. 1999; 22: 208-215Abstract Full Text Full Text PDF PubMed Scopus (1814) Google Scholar, 2Haydon P.G. Nat. Rev. Neurosci. 2001; 2: 185-193Crossref PubMed Scopus (1168) Google Scholar, 3Newman E.A. Trends Neurosci. 2003; 26: 536-542Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 4Fellin T. Pascual O. Haydon P.G. Physiology. 2006; 21: 208-215Crossref PubMed Scopus (155) Google Scholar). Astrocytes respond to neurotransmitters, integrate different inputs, and signal back to neurons or forward information to neighboring or more distant astrocytes (2Haydon P.G. Nat. Rev. Neurosci. 2001; 2: 185-193Crossref PubMed Scopus (1168) Google Scholar). In response to stimulation they release several chemical substances (5Martin D.L. Glia. 1992; 5: 81-94Crossref PubMed Scopus (294) Google Scholar, 6Porter J.T. McCarthy K.D. Prog. Neurobiol. 1997; 51: 439-455Crossref PubMed Scopus (410) Google Scholar, 7Evanko D.S. Zhang Q. Zorec R. Haydon P.G. Glia. 2004; 47: 233-240Crossref PubMed Scopus (70) Google Scholar), termed gliotransmitters, which can interfere with the neuronal communicating pathways (8Kang J. Jiang L. Goldman S.A. Nedergaard M. Nat. Neurosci. 1998; 1: 683-692Crossref PubMed Scopus (701) Google Scholar, 9Fellin T. Pascual O. Gobbo S. Pozzan T. Haydon P.G. Carmignoto G. Neuron. 2004; 43: 729-743Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar, 10Pascual O. Casper K.B. Kubera C. Zhang J. Revilla-Sanchez R. Sul J.-Y. Takano H. Moss S.J. McCarthy K. Haydon P.G. Science. 2005; 310: 113-116Crossref PubMed Scopus (1017) Google Scholar). One major extracellular messenger important for coordinating the function of astrocytes, as well as for the cross-talk between them and other cell types, is ATP (11Lazarowski E.R. Boucher R.C. Harden T.K. Mol. Pharmacol. 2003; 64: 785-795Crossref PubMed Scopus (475) Google Scholar). Whereas several lines of evidence support the idea of ATP release from astrocytes (12Cotrina M.L. Lin J.H.-C. Alves-Rodrigues A. Liu S. Li J. Azmi-Ghadimi H. Kang J. Naus C.C.G. Nedergaard M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15735-15740Crossref PubMed Scopus (674) Google Scholar, 13Bal-Price A. Moneer Z. Brown G.C. Glia. 2002; 40: 312-323Crossref PubMed Scopus (181) Google Scholar, 14Coco S. Calegari F. Pravettoni E. Pozzi D. Taverna E. Rosa P. Matteoli M. Verderio C. J. Biol. Chem. 2003; 278: 1354-1362Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar), the release mechanisms are not completely understood. Some studies have described a connexin hemichannel-mediated release (12Cotrina M.L. Lin J.H.-C. Alves-Rodrigues A. Liu S. Li J. Azmi-Ghadimi H. Kang J. Naus C.C.G. Nedergaard M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15735-15740Crossref PubMed Scopus (674) Google Scholar, 15Arcuino G. Lin J.H.-C. Takano T. Liu C. Jiang L. Gao Q. Kang J. Nedergaard M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9840-9845Crossref PubMed Scopus (266) Google Scholar, 16Stout C.E. Constantin J.L. Naus C.C. Charles A.C. J. Biol. Chem. 2002; 277: 10482-10488Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar) in both resting and activated conditions. Other possible mechanisms, like volume-regulated anion channels (17Queiroz G. Gebicke-Haerter P.J. Schobert A. Starke K. von Kugelgen I. Neuroscience. 1997; 78: 1203-1208Crossref PubMed Scopus (118) Google Scholar, 18Anderson C.M. Bergher J.P. Swanson R.A. J. Neurochem. 2004; 88: 246-256Crossref PubMed Scopus (204) Google Scholar) and ATP-binding cassette transporters (multidrug resistance P-glycoprotein (19Abraham E.H. Prat A.G. Gerweck L. Seneveratne T. Arceci R.J. Kramer R. Guidotti G. Cantiello H.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 312-316Crossref PubMed Scopus (365) Google Scholar), or cystic fibrosis transmembrane conductance regulator (20Schwiebert E.M. Egan M.E. Hwang T.H. Fulmer S.B. Allen S.S. Cutting G.R. Guggino W.B. Cell. 1995; 81: 1063-1073Abstract Full Text PDF PubMed Scopus (596) Google Scholar) have also been reported. On the contrary, only few studies have focused on the possibility of exocytotic, vesicular ATP release mechanism operating in astrocytes (13Bal-Price A. Moneer Z. Brown G.C. Glia. 2002; 40: 312-323Crossref PubMed Scopus (181) Google Scholar, 14Coco S. Calegari F. Pravettoni E. Pozzi D. Taverna E. Rosa P. Matteoli M. Verderio C. J. Biol. Chem. 2003; 278: 1354-1362Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar), even though it has been shown that astrocytes express the elements of the exocytotic apparatus (21Parpura V. Fang Y. Basarsky T.A. Jahn R. Haydon P.G. FEBS Lett. 1995; 377: 489-492Crossref PubMed Scopus (172) Google Scholar, 22Montana V. Ni Y. Sunjara V. Hua X. Parpura V. J. Neurosci. 2004; 24: 2633-2642Crossref PubMed Scopus (306) Google Scholar, 23Wilhelm A. Volknandt W. Langer D. Nolte C. Kettenmann H. Zimmermann H. Neurosci. Res. 2004; 48: 249-257Crossref PubMed Scopus (80) Google Scholar, 24Zhang Q. Pangršič T. Kreft M. Kržan M. Li N. Sul J.Y. Halassa M. Van Bockstaele E. Zorec R. Haydon P.G. J. Biol. Chem. 2004; 279: 12724-12733Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Furthermore, ATP has been found in secretory vesicles together with classical neurotransmitters (for instance, acetylcholine in neurons and noradrenaline in neurons and chromaffin cells (25Zimmermann H. Trends Neurosci. 1994; 17: 420-426Abstract Full Text PDF PubMed Scopus (424) Google Scholar). Therefore, it seems likely that ATP is released by the process of exocytosis from excitable cells (26Fabbro A. Skorinkin A. Grandolfo M. Nistri A. Giniatullin R. J. Physiol. 2004; 560: 505-517Crossref PubMed Scopus (35) Google Scholar, 27Grandolfo M. Nistri A. Neuroreport. 2005; 16: 381-385Crossref PubMed Scopus (2) Google Scholar). In our previous studies we have described a Ca2+-dependent exocytotic release mechanism of atrial natriuretic peptide (ANP) 3The abbreviations used are: ANPatrial natriuretic peptideEGFPenhanced green fluorescent proteinPBSphosphate-buffered salineBSAbovine serum albuminTIRFtotal internal reflection fluorescenceSTICsmall transient inward currents. and glutamate from cortical astrocytes (24Zhang Q. Pangršič T. Kreft M. Kržan M. Li N. Sul J.Y. Halassa M. Van Bockstaele E. Zorec R. Haydon P.G. J. Biol. Chem. 2004; 279: 12724-12733Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 28Kržan M. Stenovec M. Kreft M. Pangršič T. Grilc S. Haydon P.G. Zorec R. J. Neurosci. 2003; 23: 1580-1583Crossref PubMed Google Scholar, 29Kreft M. Stenovec M. Rupnik M. Grilc S. Kržan M. Potokar M. Pangršič T. Haydon P.G. Zorec R. Glia. 2004; 46: 437-445Crossref PubMed Scopus (149) Google Scholar). Before engaging in exocytosis, vesicles are transported through the cytoplasm of cells to the plasma membrane. In our recent study we have shown that transport of ANP-containing vesicles through the cytoplasm of astrocytes is supported by different types of the cytoskeleton (30Potokar M. Kreft M. Li L. Andersson D. Pangršič T. Chowdhury H.H. Pekny M. Zorec R. Traffic. 2006; 8: 12-20Crossref Scopus (135) Google Scholar). atrial natriuretic peptide enhanced green fluorescent protein phosphate-buffered saline bovine serum albumin total internal reflection fluorescence small transient inward currents. The aim of the present study was to characterize the nature and behavior of ATP-containing vesicles in astrocytes and to further explore the properties of exocytosis as a potential mechanism of ATP release from astrocytes. To this end, we used several approaches: first, we stained ATP vesicles with quinacrine (14Coco S. Calegari F. Pravettoni E. Pozzi D. Taverna E. Rosa P. Matteoli M. Verderio C. J. Biol. Chem. 2003; 278: 1354-1362Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 31Bodin P. Burnstock G. J. Cardiovasc. Pharmacol. 2001; 38: 900-908Crossref PubMed Scopus (235) Google Scholar) and examined their subcellular localization with immunocytochemistry. Second, using total internal reflection fluorescence and confocal microscopy, we analyzed vesicle mobility and their fusion with the plasma membrane after various stimulatory challenges. Third, to directly test the properties of ATP release from astrocytes, a sniffer cell approach was used. For this purpose, HEK-293T cells, co-transfected with the purinergic P2X3 receptor and enhanced green fluorescent protein (EGFP), were plated onto cultured cortical astrocytes; green fluorescent HEK-293T cells were then patch-clamped to record ATP-mediated membrane currents during stimulation of neighboring astrocytes. Our experimental evidence shows a high degree of co-localization of quinacrine-loaded vesicles and vesicles containing ANP, demonstrating a likely co-storage of ATP and ANP. We report the SNARE-dependent reduction in number of quinacrine-loaded vesicles after stimulation with ionomycin, indicating the involvement of exocytotic cargo release. The mobility of the remaining vesicles, observed in the cytoplasm, significantly decreased after stimulation of cells. Finally, in sniffer engineered HEK-293T cells plated on astrocytes we detected small transient inward currents (STICs), which likely report the quantal release of ATP from astrocytes and are reminiscent of postsynaptically detected ATP-mediated events (26Fabbro A. Skorinkin A. Grandolfo M. Nistri A. Giniatullin R. J. Physiol. 2004; 560: 505-517Crossref PubMed Scopus (35) Google Scholar). In contrast, a sustained release of ATP because of permeation of channels or transporters would result in sustained increase in inward current in HEK-293T cells, whereas transient currents similar to STICs can only be reproduced with very short (10 ms) pulses of ATP application (26Fabbro A. Skorinkin A. Grandolfo M. Nistri A. Giniatullin R. J. Physiol. 2004; 560: 505-517Crossref PubMed Scopus (35) Google Scholar). The frequency of STICs increased after astrocyte stimulation by glutamate and drastically decreased in the absence of calcium in the extracellular solution. Together, these data demonstrate that various stimuli trigger a Ca2+-dependent, most likely vesicular release of ATP from astrocytes. Primary Rat Cortical and Hippocampal Astrocyte Cultures—Primary cortical astrocyte cultures were prepared from the cerebral cortices of 2-3-day-old rats as described (32Pangršič T. Potokar M. Haydon P.G. Zorec R. Kreft M. J. Neurochem. 2006; 99: 514-523Crossref PubMed Scopus (63) Google Scholar). Briefly, cells were grown in high glucose Dulbecco's modified Eagle's medium, containing 10% fetal bovine serum, 1 mm pyruvate, 2 mm glutamine, and 25 μg/ml penicillin/streptomycin in 5% CO2/95% air. When cells reached confluence, they were shaken three times overnight at 200 rpm and, after each shaking, the medium was changed. The cells were then trypsinized. After reaching confluence again, they were subcultured onto 22-mm diameter poly-l-lysine-coated coverslips and used in the experiments during the following 3 days. Hippocampi of Wistar rat embryos (E18) were removed and dissociated mechanically in a Ca2+- and Mg2+-free balanced salt solution (CMF-BSS) at pH 7.4, containing 137 mm NaCl, 5.36 mm KCl, 0.27 mm Na2HPO4, 1.1 mm KH2PO4, 6.1 mm glucose. After centrifugation at 1,000 rpm (200 g) for 5 min, the pellet was resuspended in culture medium (pH 7.6) containing Neurobasal medium, penicillin 100 units/ml and streptomycin 100 μg/ml. The cells were plated at a density of 1.5·105 cells/cm2 on MatTek dishes pre-treated with poly-l-lysine. Cultures were maintained in 5% CO2/95% air at 37 °C, allowed to grow to confluence and used at 14-21 days in vitro. Medium was changed every 3-4 days. Astrocytes were identified on the basis of their flat morphology and close adhesion to the substrate. Immunocytochemistry—Prior to immunocytochemistry, cortical astrocytes were labeled with quinacrine dihydrochloride (1 μm; 15 min at room temperature). Thereafter, cells were rinsed with phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. Nonspecific staining was minimized by incubating cells in blocking buffer containing 3% bovine serum albumin (BSA) and 10% goat serum in PBS at 37 °C for 1 h. The cells were stained with primary and secondary antibodies, diluted into 3% BSA in PBS and incubated at 37 °C, and then treated with Light Antifade Kit (Molecular Probes, Invitrogen). A primary antibody against ANP (1:1000; Abcam, Cambridge, UK) and a secondary antibody against rabbit IgG (Alexa Fluor 546; 1:600; Molecular Probes, Invitrogen) were used (28Kržan M. Stenovec M. Kreft M. Pangršič T. Grilc S. Haydon P.G. Zorec R. J. Neurosci. 2003; 23: 1580-1583Crossref PubMed Google Scholar). Confocal Microscopy—Images of live cortical astrocytes loaded with quinacrine dihydrochloride for detection of vesicle mobility and images of immunolabeled cells were obtained with an inverted Zeiss LSM 510 confocal microscope (Jena, Germany). After incubation in quinacrine solution as described above, cells were rinsed, supplied with extracellular solution and observed with an oil immersion objective 63 ×/NA 1.4. In the study of the mobility of cell vesicles images were recorded every 2 s. For excitation of quinacrine 488 nm line of the Ar-Ion laser was used. The fluorescence signal was band-pass filtered at 505-530 nm. For cell stimulation, 2 μm ionomycin was applied by bath superperfusion. In immunocytochemical studies we used the 543 nm beam of the He-Ne laser to excite Alexa Fluor 546 goat anti-rabbit IgG antibody. The emission fluorescence was long-pass filtered at 560 nm. For excitation of quinacrine, the Ar-Ion laser was used, as described above. Total Internal Reflection Fluorescence (TIRF) Microscopy—TIRF-equipped CellR (Olympus Europe, Hamburg, Germany) was used to study vesicles distribution and dynamics near the plasma membrane (33Axelrod D. Methods Enzymol. 2003; 361: 1-33Crossref PubMed Scopus (226) Google Scholar). TIRF microscopy selectively reveals membrane-proximal fluorescent molecules through use of evanescent excitation light that decays exponentially in intensity along the direction perpendicular to the glass/liquid interface to which the cells adhere (33Axelrod D. Methods Enzymol. 2003; 361: 1-33Crossref PubMed Scopus (226) Google Scholar). The exponentially decaying excitation light causes fluorescently tagged vesicles to appear progressively brighter as they move toward the interface, while the vesicles located >150-200 nm from the interface are invisible in TIRF. Conversely, the intensity of wide-field epifluorescence image of the same vesicles is insensitive to their sub-micrometer translocations in the vertical direction. The ratio between the vesicle image brightness in TIRF and its epifluorescence brightness (34Kolikova J. Afzalov R. Giniatullina A. Surin A. Giniatullin R. Khiroug L. Brain Cell Biol. 2006; 35: 75-86Crossref PubMed Scopus (19) Google Scholar) was used to estimate the percentage of vesicles docked on the plasma membrane under resting conditions. To characterize the time course of stimulus-induced docking of individual quinacrine-loaded vesicles on the basal plasma membrane, time-lapse image series were generated by taking a pair of images (TIRF and epifluorescence) every 1-5 s. Vesicle docking was detected as an increase in its TIRF fluorescence with epifluorescence unchanged. Fusion of a vesicle and release of its content into extracellular space was detected as a signal loss both in TIRF and epifluorescence. Vesicle Tracking and Co-localization Analysis—Vesicle mobility was analyzed with Particle TR software (Celica, Ljubljana, Slovenia) as previously described (35Potokar M. Kreft M. Pangršič T. Zorec R. Biochem. Biophys. Res. Commun. 2005; 329: 678-683Crossref PubMed Scopus (73) Google Scholar). We calculated the following parameters of vesicle mobility: current time (time from the beginning of single vesicle tracking), step length (displacement of a vesicle in each time interval), track length (the total length of the analyzed vesicle pathway), maximal displacement (a measure of the net translocation of a vesicle) and the directionality index (maximal displacement/total track length) of vesicles as described previously (35Potokar M. Kreft M. Pangršič T. Zorec R. Biochem. Biophys. Res. Commun. 2005; 329: 678-683Crossref PubMed Scopus (73) Google Scholar, 36Wacker I. Kaether C. Kromer A. Migala A. Almers W. Gerdes H.H. J. Cell Sci. 1997; 110: 1453-1463Crossref PubMed Google Scholar). The analysis of vesicle mobility was performed for epochs of 30 s. The analysis of the co-localization degree between fluorescent pixels labeling different vesicles was performed with ColoC software (Celica, Ljubljana, Slovenia). Briefly, the software counts the number of green, red and co-localized pixels in each image. The degree of co-localization used in the analysis was expressed as the degree (%) of co-localized pixels in the cell in comparison to all green pixels in the cell, which are labeling ATP-containing vesicles. Overlaid green and red signals, indicating the co-localization between the ATP and peptide hormones, were observed as a yellow color in the images. Co-cultures of Astrocytes and Transfected HEK-293T Cells—Primary astrocyte cultures were prepared as mentioned above. Astrocytes were plated on poly-l-lysine-coated coverslips on the day of HEK-293T cell transfection. HEK-293T cells, obtained from the SISSA cell bank, were maintained in culture using the medium for astrocytes. Co-transfection of HEK-293T cells with plasmids encoding for mutated rat P2X3 receptor (provided by SISSA) and for GFP (EGFP-N1, Clontech, Takara Bio Europe, Saint-Germain-enLaye, France) was performed using the calcium phosphate transfection method (37Fabbretti E. Sokolova E. Masten L. D'Arco M. Fabbro A. Nistri A. Giniatullin R. J. Biol. Chem. 2004; 279: 53109-53115Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). For these experiments, we used the rP2X3 receptor mutated at D266A to exploit the fact that, because this mutant shows very reduced desensitization (37Fabbretti E. Sokolova E. Masten L. D'Arco M. Fabbro A. Nistri A. Giniatullin R. J. Biol. Chem. 2004; 279: 53109-53115Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), it greatly improves the detection of ATP-evoked current responses. HEK-293T cells were trypsinized 24 h after transfection and replated on top of astrocytes attached to coverslips. Apyrase (1 unit/ml) was added to coverslips to prevent excessive stimulation of transfected HEK-293T cells by ambient ATP. Co-cultures were used within the next 2 days after P2X3-expressing HEK-293T cell plating. The successfulness of co-transfection of P2X3 and EGFP was tested by immunofluorescence experiments. Paraformaldehyde-fixed co-trasfected HEK-293T cells were processed with anti-P2X3 receptor antibody (dilution 1:200, Alomone, Jerusalem, Israel) and an AlexaFluor 594-conjugated anti-rabbit secondary antibody (dilution 1:500, Molecular Probes, Invitrogen). In addition, cell nuclei were stained with DAPI. If not otherwise mentioned, all chemicals were obtained from Sigma. Electrophysiological Recordings and Analysis—Coverslips with co-cultures of astrocytes and HEK-293T cells were bathed in extracellular medium containing (in mm): NaCl 152, KCl 5, MgCl2 1, CaCl2 2, Na HEPES 10, d-glucose 10, pH adjusted to 7.4 with NaOH. Patch-clamp pipettes had a resistance of 2-5 MΩ and were filled with (in mm): CsCl 130, MgCl2 1, HEPES 20, Na2ATP 3, pH adjusted to 7.2 with CsOH. All recordings were made at room temperature. Whole cell patch-clamp recordings were performed with SWAM IIB (Celica, Ljubljana, Slovenia). Green fluorescent P2X3-expressing HEK-293T cells lying on top of or beside astrocytes were voltage clamped at -60 mV. Whole cell currents were acquired at 10 kHz, filtered at 1 kHz, and digitized by WinWCP software (Strathclyde University, Glasgow, UK). Off-line data analysis was performed using software subroutines written in MATLAB (MathWorks Inc.) with additional digital filtering as required. Slow drifts/fluctuations in the signal were digitally subtracted. We measured peak amplitude, rise time (20-80% of the peak amplitude) and half-decay time (to 50% of the peak amplitude) of transient current events and their apparent inter-event interval. To stimulate ATP release from astrocytes 100-300 μm glutamate was added to the extracellular solution. All data are given as mean ± S.E.; n denotes the number of individual cells assessed in patch-clamp studies or the number of events detected. Statistical differences were determined by two-tailed unpaired Student's t test and considered significant at p < 0.05. Quinacrine Loading into ATP Vesicles in Astrocytes—To visualize ATP-containing vesicles in astrocytes we incubated cultures in 1 μm quinacrine dihydrochloride (14Coco S. Calegari F. Pravettoni E. Pozzi D. Taverna E. Rosa P. Matteoli M. Verderio C. J. Biol. Chem. 2003; 278: 1354-1362Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 31Bodin P. Burnstock G. J. Cardiovasc. Pharmacol. 2001; 38: 900-908Crossref PubMed Scopus (235) Google Scholar). Fig. 1, displaying one section of a rat astrocyte, shows the presence of large numbers of ATP vesicles (∼80 vesicles/cell), which are randomly distributed over the cell profile. To identify the nature of ATP-containing vesicles, treated cells were immunolabeled with antibodies against ANP (Fig. 1). The double staining revealed a high degree of co-localization between two dyes (green for quinacrine, see Fig. 1A, and red for anti-ANP, see Fig. 1B), observed as yellow fluorescence (Fig. 1C). The average degree of quinacrine and ANP co-localization between containing vesicles, expressed as the ratio between all co-localized pixels and all green pixels in the cell, was 39.48 ± 6.59% (n = 12; n represents the number of analyzed cells). These results show that ATP was substantially co-stored in peptidergic vesicles with ANP in astrocytes. The Mobility of Quinacrine-loaded Vesicles Decreased after Stimulation—The total number of tracked quinacrine-stained vesicles was 817 in non-stimulated cells (n = 10), while in ionomycin-stimulated (2 μm, 1 min) cells (n = 6) only 109 vesicles could be tracked after bath application of the Ca2+ ionophore. When ionomycin was omitted from the bath, the total image fluorescence (supplemental Fig. S1A, filled bars) and the number of quinacrine-stained vesicles per cell did not change significantly over time (supplemental Fig. S1B, filled bars). On the contrary, when cells were stimulated by 2 μm ionomycin, the total image fluorescence decreased from 100% to 93.8 ± 2.3% (mean ± S.E., n = 6) within 1 min (supplemental Fig. S1, open bars) and the number of quinacrine-stained vesicles decreased from 110 ± 13 to 93 ± 10 vesicles per cell (from 100% to 84.6 ± 3.0%) (p < 0.05, paired Student's t test). Following 2 min of incubation in the presence of ionomycin, the total image fluorescence decreased to 87.4 ± 3.7% and the number of quinacrine-stained vesicles decreased to 84 ± 10 vesicles per cell (the relative number of vesicles was reduced to 76.6 ± 5.2%), (supplemental Fig. S1, open bars), which is consistent with the Ca2+-dependent, exocytotic fluorescent cargo release from quinacrine-stained vesicles in stimulated astrocytes. To further test that these results reflect exocytotic cargo release from quinacrine-stained vesicles as considered previously (10Pascual O. Casper K.B. Kubera C. Zhang J. Revilla-Sanchez R. Sul J.-Y. Takano H. Moss S.J. McCarthy K. Haydon P.G. Science. 2005; 310: 113-116Crossref PubMed Scopus (1017) Google Scholar, 14Coco S. Calegari F. Pravettoni E. Pozzi D. Taverna E. Rosa P. Matteoli M. Verderio C. J. Biol. Chem. 2003; 278: 1354-1362Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar), astrocytes were transfected with a construct to express the dominant-negative SNARE domain peptide, which blocks the formation of the SNARE complex (24Zhang Q. Pangršič T. Kreft M. Kržan M. Li N. Sul J.Y. Halassa M. Van Bockstaele E. Zorec R. Haydon P.G. J. Biol. Chem. 2004; 279: 12724-12733Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Transfected cells displaying cytosolic EGFP fluorescence were stained with quinacrine and stimulated by 2 μm ionomycin to trigger Ca2+-dependent exocytosis. In transfected cells, the number of quinacrine-stained vesicles per cell was not reduced significantly (p > 0.05, paired Student's t test) following ionomycin stimulation (supplemental Fig. S1B, gray bars) clearly indicating that the decrease in quinacrine-stained vesicles is a SNARE-dependent process. To estimate the mobility these vesicles were tracked for 30 s in resting conditions and from 30 to 60 s after the addition of 2 μm ionomycin. Vesicles in resting conditions predominantly displayed non-directional mobility as only a few vesicles had almost rectilinear directional mobility (Fig. 2A). Directional and non-directional mobility was determined as described (35Potokar M. Kreft M. Pangršič T. Zorec R. Biochem. Biophys. Res. Commun. 2005; 329: 678-683Crossref PubMed Scopus (73) Google Scholar). Ionomycin application greatly affected both types of vesicle mobility. In non-stimulated cells, the maximal displacement of directional vesicles (nd) was 2.49 ± 0.38 μm (nd = 35) and the track length was 3.46 ± 0.37 μm. Following ionomycin application, none of tracked vesicles displayed directional mobility within 30-60 s after stimulation (note the absence of rectilinear tracks in Fig. 2B). However, ionomycin also significantly affected the mobility of non-directional vesicles. The maximal displacement of non-directional vesicles was significantly reduced from 0.37 ± 0.01 μm (nn = 782); (n represents the number of vesicles) in non-stimulated cells to 0.30 ± 0.01 μm in ionomycin-stimulated cells, (n = 109; p < 0.001). Similarly, the mean vesicle track length was significantly shorter in stimulated (1.14 ± 0.04 μm; n = 109) than in non-stimulated cells (1.30 ± 0.02 μm; p < 0.001). Directionality index (see “Experimental Procedures”) decreased from 0.66 ± 0.03 (nd) and 0.34 ± 0.001 (nn) to 0.28 ± 0.001 in stimulated cells. Following the elevation of free cytosolic [Ca2+] by ionomycin, vesicles are like" @default.
• W2056369163 created "2016-06-24" @default.
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• W2056369163 date "2007-09-01" @default.
• W2056369163 modified "2023-10-02" @default.
• W2056369163 title "Exocytotic Release of ATP from Cultured Astrocytes" @default.
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