Storage and Release of ATP from Astrocytes in Culture
2003; Elsevier BV; Volume: 278; Issue: 2 Linguagem: Inglês
10.1074/jbc.m209454200
ISSN1083-351X
AutoresSilvia Coco, Federico Calegari, Elena Pravettoni, Davide Pozzi, Elena Taverna, Patrizia Rosa, Michela Matteoli, Claudia Verderio,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoATP is released from astrocytes and is involved in the propagation of calcium waves among them. Neuronal ATP secretion is quantal and calcium-dependent, but it has been suggested that ATP release from astrocytes may not be vesicular. Here we report that, besides the described basal ATP release facilitated by exposure to calcium-free medium, astrocytes release purine under conditions of elevated calcium. The evoked release was not affected by the gap-junction blockers anandamide and flufenamic acid, thus excluding purine efflux through connexin hemichannels. Sucrose-gradient analysis revealed that a fraction of ATP is stored in secretory granules, where it is accumulated down an electrochemical proton gradient sensitive to the v-ATPase inhibitor bafilomycin A1. ATP release was partially sensitive to tetanus neurotoxin, whereas glutamate release from the same intoxicated astrocytes was almost completely impaired. Finally, the activation of metabotropic glutamate receptors, which strongly evokes glutamate release, was only slightly effective in promoting purine secretion. These data indicate that astrocytes concentrate ATP in granules and may release it via a regulated secretion pathway. They also suggest that ATP-storing vesicles may be distinct from glutamate-containing vesicles, thus opening up the possibility that their exocytosis is regulated differently. ATP is released from astrocytes and is involved in the propagation of calcium waves among them. Neuronal ATP secretion is quantal and calcium-dependent, but it has been suggested that ATP release from astrocytes may not be vesicular. Here we report that, besides the described basal ATP release facilitated by exposure to calcium-free medium, astrocytes release purine under conditions of elevated calcium. The evoked release was not affected by the gap-junction blockers anandamide and flufenamic acid, thus excluding purine efflux through connexin hemichannels. Sucrose-gradient analysis revealed that a fraction of ATP is stored in secretory granules, where it is accumulated down an electrochemical proton gradient sensitive to the v-ATPase inhibitor bafilomycin A1. ATP release was partially sensitive to tetanus neurotoxin, whereas glutamate release from the same intoxicated astrocytes was almost completely impaired. Finally, the activation of metabotropic glutamate receptors, which strongly evokes glutamate release, was only slightly effective in promoting purine secretion. These data indicate that astrocytes concentrate ATP in granules and may release it via a regulated secretion pathway. They also suggest that ATP-storing vesicles may be distinct from glutamate-containing vesicles, thus opening up the possibility that their exocytosis is regulated differently. secretogranin II phorbol 12-myristate 13-acetate Krebs-Ringer-Hepes 2-amino-5-phosphonovaleric acid 6-cyano-7-nitroquinoxaline-2,3-dione α-methyl-4-carboxyphenilglycine glial fibrillar acidic protein 1-aminocyclopentane-trans-1,3-olicarboxylic acid α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis (acetoxymethyl ester) Astrocytes propagate long-range calcium signals to neighboring cells and affect the activity of neurons by evoking calcium transients (1Nedergaard M. Science. 1994; 263: 1768-1771Crossref PubMed Scopus (836) Google Scholar, 2Parpura V. Basarsky T.A. Liu F. Jeftinija K. Jeftinija S. Haydon P.G. Nature. 1994; 369: 744-747Crossref PubMed Scopus (1401) Google Scholar, 3Hassinger T.D. 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Several lines of evidence now suggest that ATP is the major extracellular messenger for inter-astrocyte calcium-mediated communication (10Guthrie P.B. Knappenberg J. Segal M. Bennett M.V.L. Charles A.C. Kater S.B. J. Neurosci. 1999; 19: 520-528Crossref PubMed Google Scholar, 13Cotrina M.L. Lin J.H. Lopez-Garcia J.C. Naus C.C.G. Nedergaard M. J. Neurosci. 2000; 20: 2835-2844Crossref PubMed Google Scholar, 14Wang Z. Haydon P.G. Yeung E.S. Anal. Chem. 2000; 72: 2001-2007Crossref PubMed Scopus (177) Google Scholar). First, ATP is released from astrocytes during calcium wave propagation (10Guthrie P.B. Knappenberg J. Segal M. Bennett M.V.L. Charles A.C. Kater S.B. J. Neurosci. 1999; 19: 520-528Crossref PubMed Google Scholar, 14Wang Z. Haydon P.G. Yeung E.S. Anal. Chem. 2000; 72: 2001-2007Crossref PubMed Scopus (177) Google Scholar). Second, the propagation can be reduced or abolished by purinergic antagonists (10Guthrie P.B. Knappenberg J. Segal M. Bennett M.V.L. Charles A.C. Kater S.B. J. 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Because these findings could be consistent with vesicular storage and regulated purine secretion from astrocytes, we investigated the mechanisms of ATP storage and release from primary cultures of hippocampal astrocytes. Antibodies against rat SgII1 were raised in rabbits, purified by affinity chromatography, and characterized as previously described (25Calegari F. Coco S. Taverna E. Bassetti M. Verderio C. Corradi N. Matteoli M. Rosa P. J. Biol. Chem. 1999; 274: 22539-22547Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The monoclonal antibodies against GFAP came from Roche Molecular Biochemicals; the polyclonal antibodies against colony-stimulating factor-1 receptor from Santa Cruz Biotechnology (Santa Cruz, CA); and the polyclonal antibodies against synaptobrevin/VAMPII were from Synaptic System GmbH (Gottingen, Germany). Ribophorin and complex 3 were kindly provided by Prof D. Borgese (Milan, Italy). The secondary antibodies conjugated to fluorescein isothiocyanate, Texas Red, 10 nm gold particles, and peroxidase were obtained from Jackson Immunoresearch Laboratories (West Grove, PA); APV, CNQX, MCPG, t-ACPD, AMPA, and bafilomycin A1 were from Tocris Neuramin (Bristol, UK); quinacrine dihydrochloride, bradykinin, glutamate, ATP, PPADS, PMA, anandamide, apyrase (grade II) and flufenamic acid, BAPTA/AM were from Sigma. The ATP assay kit came from Molecular Probes Europe (Leiden, NL) and the lactate dehydrogenase kit from Sigma (Milano, Italy). Hippocampal mixed-glia cultures from embryonic rat pups (E18) were obtained using previously described methods (25Calegari F. Coco S. Taverna E. Bassetti M. Verderio C. Corradi N. Matteoli M. Rosa P. J. Biol. Chem. 1999; 274: 22539-22547Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Briefly, after dissection, the hippocampi were dissociated by treatment with trypsin (0.25% for 10 min at 37 °C) followed by fragmentation with a fire-polished Pasteur pipette. The dissociated cells were plated onto glass coverslips at a density of 0.5 × 106 cells/ml, and the cultures were grown in minimum essential medium (Invitrogen) supplemented with 20% fetal bovine serum (Euroclone Ltd, UK) and glucose at a final concentration of 5.5 g/l (glial medium). To obtain a pure astrocyte monolayer, any microglia cells were harvested by shaking 3-week-old cultures. The primary hippocampal neuron cultures were prepared from E18 embryos as previously described (29Verderio C. Coco S. Fumagalli G. Matteoli M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6449-6453Crossref PubMed Scopus (58) Google Scholar). The cultures were fixed for 25 min at room temperature with 4% paraformaldehyde in 0.12 mphosphate buffer containing 0.12 m sucrose. The fixed cells were detergent-permeabilized and labeled with primary antibodies followed by fluorochrome-conjugated secondary antibodies. The coverslips were mounted in 70% glycerol in phosphate buffer containing 1 mg/ml phenylendiamine. The images were acquired using a BioRad MRC-1024 confocal microscope equipped with LaserSharp 3.2 software. Electron microscopy was performed as previously described (25Calegari F. Coco S. Taverna E. Bassetti M. Verderio C. Corradi N. Matteoli M. Rosa P. J. Biol. Chem. 1999; 274: 22539-22547Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Quinacrine staining was performed by incubating living cultures for 30 min at 37 °C with Krebs-Ringer-Hepes (KRH: 125 mm NaCl, 5 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, 2 mmCaCl2, 6 mm glucose, 25 mmHepes/NaOH, pH 7.4) containing 5 × 10−7m quinacrine dihydrochloride. Quinacrine-fluorescent living astrocytes were examined with a Zeiss microscope equipped with epifluorescence and photographated using a TMAX 400 (Eastman Kodak Co.). After being grown on Petri dishes until near confluence, the astrocytes were scraped, washed, and resuspended 1:4 in homogenization buffer (10 mm Hepes-KOH, pH 7.4, 250 mm sucrose, 1 mm Mg acetate, 0.5 mm phenylmethylsulfonyl fluoride, 2 μg/ml pepstatin, 10 μg/ml aprotinin). The cells were homogenized using a cell cracker (European Molecular Biology Laboratory, Heidelberg, Germany) and centrifuged at 1,000 ×g for 10 min to prepare the post-nuclear supernatant. This supernatant was loaded onto a 0.4–1.8 M sucrose gradient and spun in a 41 SW rotor (Beckman Instruments, Inc., Palo Alto, CA) at 25,000 rpm for 18 h. Fractions (1 ml) were collected and analyzed by SDS-PAGE followed by Western blotting as previously described (25Calegari F. Coco S. Taverna E. Bassetti M. Verderio C. Corradi N. Matteoli M. Rosa P. J. Biol. Chem. 1999; 274: 22539-22547Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Briefly, after electrophoresis, the proteins were transferred to nitrocellulose filters which, after being incubated in blocking buffer (5% milk, 25 mm Tris-HCl, pH 7.5, 150 mm NaCl), were labeled with primary antibodies followed by the appropriate secondary antibodies conjugated to peroxidase diluted in blocking buffer containing 0.1–0.3% Tween 20. After extensive washing, the immunodecoration pattern was revealed using an enhanced chemiluminescence system (SuperSignal from Pierce, Rockford, IL) following the manufacturer' s protocol. The cultured cells were loaded for 60–90 min at 37 °C with 5 μm FURA-2 pentacetoxy-methylester in KRH, washed in the same solution, and transferred to the recording chamber of an inverted microscope (Axiovert 100; Zeiss, Oberkochen, Germany) equipped with a calcium imaging unit. For the assays, a modified CAM-230 dual-wavelength microfluorimeter (Jasco, Tokyo, Japan) was used as a light source. The experiments were performed using an Axon Imaging Workbench 2.2 equipped with a PCO SuperVGA SensiCam (Axon Instruments, Foster City, CA). The ratio values in discrete areas of interest were calculated from sequences of images to obtain temporal analyses. The images were acquired at 1–3 340/380 ratios/s. The experiments were performed in a static bath at room temperature (24–25 °C). The increases in calcium were quantified by measuring the peak and/or area of the response. The biological assay for glutamate detection was performed as previously described (29Verderio C. Coco S. Fumagalli G. Matteoli M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6449-6453Crossref PubMed Scopus (58) Google Scholar). Specifically, monolayers of astrocyte cultures in 60-mm Petri dishes were kept in 1 ml of KRH in the absence and then in the presence of PMA or t-ACPD for 10–30 min at 37 °C. Neuronal cultures loaded with FURA-2 were then exposed to the different aliquots of KRH. Immediately before challenging, the aliquot collected from unstimulated astrocytes was supplemented with the stimuli. To verify that the biological activity of the conditioned medium was caused by accumulated glutamate, a subset of recordings were made in the presence of glutamate receptor antagonists, APV and CNQX (29Verderio C. Coco S. Fumagalli G. Matteoli M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6449-6453Crossref PubMed Scopus (58) Google Scholar, 30Verderio C. Bruzzone S. Zocchi E. Fedele E. Schenk U., De Flora A. Matteoli M. J. Neurochem. 2001; 78: 646-657Crossref PubMed Scopus (106) Google Scholar). Although embryonic hippocampal neurons lack t-ACPD receptors coupled to calcium mobilization, 2C. Verderio and M. Matteoli, unpublished data. the specific antagonist MCPG was always added to the conditioned medium upon t-ACPD stimulation. To test the tetanus neurotoxin (TeNT) sensitivity of glutamate release, the same astrocyte monolayer was challenged with PMA before and after 20-h incubation with the neurotoxin. Collected aliquots were frozen and then tested on the same FURA-2-loaded neurons. The endogenous glutamate concentration in the conditioned medium was determined by HPLC analysis coupled with fluorimetric detection as previously described (30Verderio C. Bruzzone S. Zocchi E. Fedele E. Schenk U., De Flora A. Matteoli M. J. Neurochem. 2001; 78: 646-657Crossref PubMed Scopus (106) Google Scholar). Off-site ATP bioassay aliquots of KRH (1 ml), conditioned as described above for glutamate detection, were split into two parts before testing on FURA-2-loaded astrocytes. One part was pretreated with apyrase (30 units/ml) for 15 min before testing. Before being exposed to ATP sensor cells, each aliquot was supplemented with a mixture of glutamate antagonists (APV 100 μm, CNQX 20 μm, MCPG 1 mm) and the appropriate stimulus when conditioned under control conditions. KRH conditioned under mechanical stimulation was collected from astrocyte monolayers shaken for 5 min on an orbital shaker (Stuart Scientific, UK). The same aliquots were tested for lactate dehydrogenase activity following the manufacturer's protocol. ATP levels in the superfusates of pure astrocyte monolayers were measured using a luciferin/luciferase assay (Molecular Probes, Leiden, NL) and a luminometer (Lumat, Berthold, LB9501) according to the manufacturer's instructions. Each sample was run in duplicate. Most of the samples were assayed within 5–10 min of collection; the others were frozen for subsequent ATP determination. ATP was detected on subcellular fractions by means of the same assay of equal aliquots of sucrose fractions that were boiled for 5 min before being frozen. To study the mechanisms of ATP release from hippocampal astrocytes, both “on line” (Fig. 1 A) and “off-site” (Fig. 1 D) biological assays were performed. The first method is based on the finding that microglia co-cultured with astrocytes may act as ATP reporter cells by selectively responding to the ATP released from adjacent astrocytes as [Ca2+]i increases (19Verderio C. Matteoli M. J. Immunol. 2001; 166: 6383-6391Crossref PubMed Scopus (200) Google Scholar). In this assay, FURA-2-loaded astrocyte-microglia co-cultures (Fig. 1 A) were digitally imaged in the presence of glutamate receptor antagonists (100 μm APV and 20 μmCNQX) to exclude the possible contributions of released glutamate ord-serine. A gentle touch of the astrocyte with a glass pipette (a widely used stimulus for ATP secretion: 10, 12, 13) (Fig. 1,A and B) generated an increase in [Ca2+]i in the stimulated cell, followed by a delayed [Ca2+]iresponse in neighboring astrocytes and microglial cells. Despite the efficient propagation of the calcium signal among astrocytes (19Verderio C. Matteoli M. J. Immunol. 2001; 166: 6383-6391Crossref PubMed Scopus (200) Google Scholar), the microglia [Ca2+]i responses were completely blocked or substantially inhibited when mechanically stimulated in the presence of the nonselective purinergic antagonist PPADS (50 μm) or the ATP-degrading enzyme apyrase (30 units/ml) (Fig. 1 C). No significant changes in mean astrocyte calcium responses were recorded in the presence of PPADS or apyrase (percent changes in Δ340/380 fluorescence ratio: PPADS, 74.5 ± 7.8, n = 5, p = 0.14; apyrase, 115.68 ± 21.5, n = 6, p = 0.43, data normalized to controls). These data indicate that ATP is the extracellular messenger responsible for microglial [Ca2+]i responses. Furthermore, a significant delay in the residual response was observed in the presence of the same blockers (a 514 ± 42% increase in the time to peak response in the presence of PPADS, and 420 ± 35% in the presence of apyrase, as compared with controls). Similar results were obtained when the mixed cultures were stimulated with 1 μm bradykinin (Fig. 1 C), which selectively increases [Ca2+]i in astrocytes (19Verderio C. Matteoli M. J. Immunol. 2001; 166: 6383-6391Crossref PubMed Scopus (200) Google Scholar). The alternative bioassay for the study of ATP secretion was based on off-site measurements of released purine. Superfusates, conditioned by differently treated pure astrocyte monolayers, were added to FURA-2-loaded astrocytes as ATP sensor cells in the presence of glutamate receptor antagonists (Fig. 1 D). Fig. 1 Eshows the [Ca2+]i responses induced by the superfusates collected under static bath conditions (a) or during mechanical stimulation (b). The [Ca2+]i responses were completely prevented when the conditioned medium was treated with apyrase for 10–15 min (Fig. 1 F), thus indicating that ATP was the involved bioactive compound. Analysis of lactate dehydrogenase release revealed no significant difference between the extracellular media collected under static bath conditions or during mechanical stimulation (lactate dehydrogenase activity: control, 4.87 ± 0.69 units/liter; mechanically stimulated, 6.27 ± 1.2 units/liter, n = 4, p = 0.37; Triton X-100-treated, 247 ± 11.3,n = 4, p < 0.001), thus excluding ATP leakage caused by shear damage. The lack of cell damage was also confirmed by the exclusion of Trypan Blue from the mechanically stimulated astrocytes (data not shown). We used the two bioassays to obtain insights into the mechanisms that control ATP release from primary astrocytes on different kinds of stimulation. To investigate the possible calcium dependence of ATP release, astrocytes were mechanically stimulated after 45 min treatment with the intracellular calcium chelator BAPTA/AM (10 μm). Purine release was largely calcium-dependent, as the response in ATP reporter cells was significantly attenuated when the medium was collected from BAPTA-treated astrocytes (off-site bioassay, Fig. 1 E, c). BAPTA treatment significantly reduced the calcium response to below baseline levels (Fig. 1 F). Furthermore, ATP release was significantly increased by treatment with the potent secretagogues PMA (100 nm) (Fig. 1 G) or with the glutamate receptor agonists AMPA (100 μm) and t-ACPD (100 μm) (Fig. 1 H), which have been previously shown to stimulate glutamate secretion when simultaneously applied to astrocytes (31Bezzi P. Carmignoto G. Pasti L. Vesce S. Rossi D. Rizzini B.L. Pozzan T. Volterra A. Nature. 1998; 391: 281-285Crossref PubMed Scopus (989) Google Scholar). On the basis of a standard dose-response curve of [Ca2+]i response amplitude to different concentrations of exogenous ATP, the actual ATP concentration in the collected medium was estimated to be 130–290 nmunder static bath and 550–700 nm after mechanical stimulation or secretagogue treatment. Determination of the ATP in the extracellular medium using the sensitive luciferin-luciferase bioluminescence assay revealed a 4.5 ± 0.4-fold (n = 3) increase in ATP release after PMA stimulation. We then evaluated whether connexin hemichannels mediate ATP efflux from primary astrocytes upon stimulation. To test this hypothesis directly, we measured stimulated ATP release in the presence of the gap-junction blockers anandamide, which effectively uncouples astrocytes (11Guan X. Cravatt B.F. Ehring G.R. Hall J.E. Boger D.L. Lerner R.A. Gilula N. J. Cell Biol. 1997; 139: 1785-1792Crossref PubMed Scopus (216) Google Scholar) or flufenamic acid, which has been recently used as connexin hemichannel blocker (21Stout C.E. Costantin J.L. Naus C.C. Charles A.C. J. Biol. Chem. 2002; 277: 10482-10488Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar, 32Zhang Y. McBride D.W., Jr. Hamill O.P. J. Physiol. (Lond.). 1998; 508: 763-776Crossref Scopus (75) Google Scholar). As shown in figure 2A, no significant reduction in [Ca2+]i response was observed when the astrocytes were mechanically stimulated after 10–30 min incubation with 100 μm anandamide (on-line bioassay). Accordingly, [Ca2+]i responses in ATP reporter cells to the medium conditioned by a PMA-stimulated astrocyte monolayer were unaffected by the presence of the gap-junction blocker (off-site bioassay) (Fig. 2 B). To ensure that the doses of anandamide used in this study were appropriate to block gap-junction communication, FURA-2-loaded astrocytes were mechanically stimulated in the presence of the purinergic antagonist PPADS and the ATP-degrading enzyme apyrase with or without anandamide. When the extracellular pathway was inhibited by the purinergic blockers, anandamide completely prevented calcium signal propagation, thus indicating an efficient block of the gap-junction-mediated communication (wave propagation radius: PPADS, apyrase, without anandamide: 145.9 ± 11 μm, n = 4; PPADS, apyrase, with anandamide: 12.1 ± 6.4 μm, n = 5,p < 0.01). Furthermore, as blocking of gap-junctions may not be indicative of inhibition of connexin hemichannels, we tested whether anandamide blocks NAD+ influx down a concentration gradient, which is known to occur through connexin hemichannels (33Bruzzone S Guida L. Zocchi E. Franco L. De Flora A. FASEB J. 2001; 15: 10-12Crossref PubMed Scopus (394) Google Scholar). An 86 ± 13% reduction of NAD+ influx was caused by a 15-min preincubation with 100 μm anandamide. These data indicate that anandamide, which is effective in blocking nucleotide fluxes through connexin hemichannels, does not significantly impair ATP release evoked by astrocyte stimulation. Similarly, no significant reduction in ATP release, monitored as [Ca2+]i response in microglial cells (on-line bioassay), was observed when the astrocytes were mechanically stimulated after 10–30 min incubation with 50 μm flufenamic acid (data not shown). It has been reported in the literature that exposure of astrocytes to calcium-free medium facilitates ATP release and that this treatment promotes opening of connexin hemichannels (15Cotrina M.L. Lin J.H. Alves-Rodrigues A. Liu S., Li, J. Azmi-Ghadimi H. Naus C.C. Nerdergard M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15735-15740Crossref PubMed Scopus (664) Google Scholar, 21Stout C.E. Costantin J.L. Naus C.C. Charles A.C. J. Biol. Chem. 2002; 277: 10482-10488Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar). Treatment of hippocampal astrocytes with calcium-free medium significantly increased basal ATP release, as detected by using the sensitive luciferin-luciferase assay (196 ± 15% increase upon controls). ATP release was significantly reduced (30 ± 1.8% reduction) in cultures incubated with the gap-junction inhibitor anandamide. Our data indicated the existence of a calcium-dependent, gap-junction blocker-insensitive release of ATP in primary cultures of hippocampal astrocytes, suggesting a vesicular purine storage. In line with this hypothesis, labeling of astrocytes with quinacrine fluorescence dye, which is known to stain high levels of ATP bound to peptides in large granular vesicles (24Bodin P. Burnstock G. J. Cardiovasc. Pharmacol. 2001; 38: 900-908Crossref PubMed Scopus (234) Google Scholar, 34Belai A. Burnstock G. Neuroreport. 2000; 11: 5-8Crossref PubMed Scopus (27) Google Scholar) revealed the existence of an ATP-containing population of vesicular organelles, prominently localized in the perinuclear region of the astrocytes (Fig. 3 A). This vesicular staining was reminiscent of the localization of secretogranin II (SgII), a well established marker of dense-core vesicles (35Rosa P. Hille A. Lee R.W. Zanini A., De Camilli P. Huttner W
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