Abstract
In the present study, we aimed to evaluate the pathways contributing to ATP release from mouse astrocytes during hypoosmotic stress. We first examined the expression of mRNAs for proteins constituting possible ATP-releasing pathways that have been suggested over the past several years. In RT-PCR analysis using both control and osmotically swollen astrocytes, amplification of cDNA fragments of expected size was seen for connexins (Cx32, Cx37, Cx43), pannexin 1 (Px1), the P2X7 receptor, MRP1 and MDR1, but not CFTR. Inhibitors of exocytotic vesicular release, gap junction hemi-channels, CFTR, MRP1, MDR1, the P2X7 receptor, and volume-sensitive outwardly rectifying chloride channels had no significant effects on the massive ATP release from astrocytes. In contrast, the hypotonicity-induced ATP release from astrocytes was most effectively inhibited by gadolinium (50 μM), an inhibitor of the maxi-anion channel, which has recently been shown to serve as a pathway for ATP release from several other cell types. Thus, we propose that the maxi-anion channel constitutes a major pathway for swelling-induced ATP release from cultured mouse astrocytes as well.
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Introduction
In the brain, astrocytes play active roles in cell-to-cell signaling by releasing gliotransmitters, such as glutamate and ATP, and thus forming neuron-glia and glia-glia networks 1, 2, 3, 4, 5. Astrocytic ATP release is involved in modulating a variety of neuronal activities, affecting excitability 6, synaptic transmission 7, 8, cell death 9, growth and survival 9, 10. ATP released from astrocytes also modulates the activities of astrocytes 11, 12 and microglia 13, 14, 15 through the stimulation of purinergic receptors.
In a variety of cell types, the release of ATP has been reported to be mediated by a number of non-lytic membrane transport pathways including exocytotic vesicular transport, connexin or pannexin hemichannels, the P2X7 receptor, ABC transporters such as MDR1 and MRP, as well as anion channels such as CFTR, volume-sensitive outwardly rectifying (VSOR) anion channels and maxi-anion channels 17, 18, 19: for Reviews.
Astrocytes have been shown to release ATP in a non-lytic manner in response to osmotic swelling 11, mechanical stimulation 8, 20, 21, 22, 23, deprivation of extracellular Ca2+ 22, 23, 24, and stimulation with glutamate 7, UTP 25, 26, noradrenaline 27 or NO 28. However, the precise pathway for ATP release remains controversial. In the present study, we aimed to identify the pathway for swelling-induced ATP release from mouse astrocytes in primary culture by performing RT-PCR analyses and pharmacological studies to test candidate pathways hitherto reported.
Results
Swelling-induced ATP release from mouse astrocytes
In normal Ringer solution, the basal release of ATP from mouse astrocytes was low and did not exceed 1.00 ± 0.13 nM over a 35-min incubation time (Figure 1, open squares). In contrast, when cell swelling was induced by exposure to a hypotonic solution (210 mosmol/kg-H2O), the extracellular ATP concentration rapidly reached a maximum level of approx. 2.5-3 nM within 15-25 min (Figure 1, open circles).
When the cells were incubated in solutions of different osmolality (ranging from 290 to 130 mosmol/kg-H2O) for 15 min, the release of ATP increased with decreasing medium osmolality (Figure 2). The osmolality dependence of ATP release had a sigmoidal shape with half-maximal ATP release observed at a medium osmolality of 228 ± 16 mosmol/kg-H2O.
Gene expression of molecules comprising candidate pathways for ATP release in mouse astrocytes
A number of possible ATP-releasing pathways have been suggested over the past several years. To determine which candidate pathway may be important in mouse astrocytes, we assayed the expression of candidate pathway molecule mRNAs by RT-PCR analysis. Under normotonic conditions, as shown in Figure 3A and 3B, RT-PCR yielded amplified cDNA fragments of expected size for connexins (Cx32, Cx37, Cx43), pannexin 1 (Px1), MRP1, P2X7 receptors (P2X7), MDR1a, and MDR1b. However, CFTR mRNA was not detected in mouse astrocytes but could be detected in mouse lung homogenates (MLHs), which were used as a positive control (Figure 3C). This profile of gene expression was not affected by osmotic swelling of the astrocytes induced by exposure to hypotonic solution (210 mosmol/kg-H2O) for 15 min (data not shown).
Pharmacological sensitivity of swelling-induced ATP release from mouse astrocytes
We next tested the sensitivity of ATP release from swollen astrocytes to blockers of not only the candidate pathways examined in the above RT-PCR analysis but also other possible pathways including exocytotic vesicular transport as well as the VSOR anion channel and the maxi-anion channel, which have not yet been molecularly identified. When mouse astrocytes were exposed to hypotonic solution of 210 mosmol/kg-H2O for 15 min in the absence of any blockers, massive ATP release was observed as before (Figure 4A, first column). We then observed the effects of different blockers for candidate pathways by adding to the hypotonic solution at concentrations that were previously reported to be most effective. The data are summarized in Figure 4A (other columns). Swelling-induced ATP release was unaffected by the presence of glibenclamide (200 μM), a known blocker of the CFTR anion channel 29, a member of the ABC transporter superfamily. This is in accord with the RT-PCR data for CFTR (Figure 3C). Similarly, swelling-induced ATP release was not significantly affected by blockers, probenecid (1 mM) and verapamil (10 μM), which are known to block other ABC transporter proteins, MRP1 30, 31 and MDR1 32, respectively. Blockers of gap junction hemichannels also failed to affect swelling-induced ATP release; no significant effects were observed by adding 2 mM 1-octanol, a blocker of connexins 33, or 100 μM carbenoxolone, a blocker of both connexins and pannexins 34. A blocker of the P2X7 receptor 24, 35, brilliant blue G (1 μM), was also ineffective. When exocytotic vesicular transport was suppressed by adding an intracellular Ca2+ chelator, BAPTA-AM (50 μM), together with an inhibitor of vesicular transport 23, brefeldin A (BFA; 5 μM), ATP release from swollen astrocytes was not significantly affected. We then observed the effects of anion channel blockers. The ATP release was suppressed to an intermediate degree by NPPB (100 μM) and SITS (100 μM), both of which are known to moderately inhibit maxi-anion channels, but more strongly block VSOR anion channels in mouse astrocytes 36. In contrast, phloretin (100 μM), which is a blocker relatively specific to the VSOR anion channel 37, had no significant effect on swelling-induced ATP release. Also, DCPIB (10 μM), another blocker specific to the VSOR anion channel 38, failed to affect swelling-induced ATP release. We next examined the effect of Gd3+ (50 μM), which blocks, from the extracellular side, maxi-anion channels expressed in many cell types 17, 39, 40, 41 including mouse astrocytes 36. As reported previously 42, Gd3+itself suppressed the luciferin-luciferase reaction. As shown in Figure 4B, the slope of a calibration curve obtained by adding known amounts of ATP to normal Ringer solution in the presence of Gd3+ (triangles) was significantly smaller than that in the absence of Gd3+ (squares). However, the Gd3+ effect on the luciferin-luciferase reaction was completely eliminated by adding 600 μM EDTA to the luciferase solution (Figure 4B, circles). By supplementing the luciferin-luciferase reaction with EDTA, Gd3+ was found to be most effective in inhibiting hypotonicity-induced ATP release (Figure 4A, last column).
As shown in Figure 5, marked, moderate and little blocking effects of Gd3+, SITS and DCPIB, respectively, were observed on swelling-induced ATP release during the whole time range (up to 35 min) of observations.
From the above results, it is concluded that swelling-induced astrocytic ATP release does not involve vesicular transport, ABC transporters such as CFTR, MDR1 and MRP1, connexin or pannexin hemichannels, or swelling-activated VSOR anion channels. Instead, the evidence suggests that the Gd3+-sensitive maxi-anion channel serves as the pathway for ATP release from swollen mouse astrocytes.
Discussion
Under stimulation of receptors or exposure to stress, brain astrocytes affect neuronal brain functions by releasing a number of gliotransmitters, including glutamate and ATP. Ubiquitously expressed swelling-activated anion channels, called VSOR anion channels or volume-regulated anion channels (VRACs), are believed to serve as a pathway for swelling-induced release of the excitatory amino acids glutamate and aspartate from astrocytes 43, 44. In fact, Abdullaev et al. 45 have recently shown that swelling-induced aspartate efflux is mediated by Gd3+-insensitive VSOR anion channels in rat astrocytes. We have provided compelling evidence that not only the VSOR anion channel but also a Gd3+-sensitive maxi-anion channel represent major conductive pathways for the release of glutamate from mouse astrocytes challenged by hypotonic or ischemic stress 36. Recent pore sizing studies using a nonelectrolyte exclusion technique showed that the pore radius of the maxi-anion channel (∼1.3 nm) 46, but not of the VSOR anion channel (∼0.63 nm) 47, is large enough for the channel to be permeated not only by glutamate (cut-off radius ∼0.35 nm) but also by the anionic forms of ATP (0.58∼0.65 nm). Direct evidence has accumulated for the ATP conductivity of maxi-anion channels in a number of other cell types 39, 40, 41. It is therefore highly likely that swelling-induced ATP release from mouse astrocytes is also mediated by Gd3+-sensitive maxi-anion channels that are known to be activated by hypotonic and ischemic stresses in this cell type 36. The present study showed, in fact, that ATP release from osmotically swollen astrocytes was most effectively blocked by Gd3+.
Thus far, a large variety of molecules have been suggested to constitute the non-lytic and non-exocytotic pathways of ATP release. These include CFTR, MDR1 and MRP1 31 for rat astrocytes, the connexins 22 and P2X7 receptors 24 for mouse astrocytes, as well as the pannexins 19 for other cell types. The present RT-PCR study demonstrated molecular expression of connexins (Cx32, Cx37, Cx43), Px1, MDR1, MRP1 and the P2X7 receptor in both non-swollen and swollen mouse astrocytes. These data are in agreement with those from previous RT-PCR studies that examined MRP1 30, 48 and the P2X7 receptor 49 in mouse astrocytes in normotonic conditions, and Px1 50, MDR1 31, 45, 51 and MRP1 31, 48 in rat astrocytes, also in normotonic conditions. However, the present study failed to detect the CFTR transcript in mouse astrocytes, in contrast to a previous report 30. Previous pharmacological studies suggested that ATP release involves ABC transporters in rat cortical astrocytes 11, 31 and the P2X7 receptor in mouse spinal cord astrocytes 24. However, the present pharmacological study showed that swelling-induced ATP release from mouse cortical astrocytes was insensitive to blockers of ABC transporters (glibenclamide, probenecid, verapamil) and the P2X7 receptor (brilliant blue G). In addition, it was found that blockers of connexin and pannexin hemichannels (1-octanol, carbenoxolone) and VSOR anion channels (phloretin, DCPIB) did not significantly affect swelling-induced ATP release from mouse astrocytes.
It is evident that exocytotic release of secretory granules or vesicles is a non-lytic source of ATP in some secretory cell types 17. A recent single-vesicle imaging study has provided evidence for astrocyte exocytosis 52. On the basis of sensitivity to bafilomycin A1, tetanus neurotoxin, botulinum neurotoxin C and Ca2+ chelators, it was suggested that mechanostress- or NO-induced ATP release from rat astrocytes is mediated by exocytosis 23, 28. In the present study, however, intracellular Ca2+ chelation by pretreatment with a Ca2+ chelator, BAPTA-AM, and an inhibitor of vesicular transport, BFA, failed to inhibit swelling-induced ATP release from mouse astrocytes. Although the Ca2+ chelator and BFA may affect many processes, it appears that swelling-induced ATP release does not involve exocytosis which is an event essentially dependent on cytosolic Ca2+ and sensitive to BFA.
Since ATP exists in anionic forms at physiological pH, there is a possibility that some anion channel type (other than CFTR) may serve as the pathway for ATP release from mouse astrocytes. An involvement of the Ca2+-dependent anion channel may be ruled out because of insensitivity of the ATP release to cytosolic Ca2+ chelation. Its insensitivity to phloretin and DCPIB may similarly rule out an involvement of the VSOR anion channel. The most effective blocking agent for the ATP release from swollen astrocytes was Gd3+. Gd3+ is known to block not only maxi-anion channels 17, 36, 39, 40, 41 but also some cation channels such as mechano-gated cation channels 53 and several members of the TRP cation channel family 54 at similar concentrations, although it is not known how Gd3+ blocks both cation and anion channels. However, involvements of cation channels in release of anionic ATP would be implausible.
There is a possibility that the data shown in Figure 1 represent underestimates of the actual amount of ATP released from mouse astrocytes, because the activity of ecto-ATPases was not inhibited in the present experiments. To test this possibility, the effects of potent ecto-ATPase blockers, such as suramin and DIDS 55, 56 as well as ARL 67156 57, could be examined. However, suramin was found to almost completely inhibit the luciferin-luciferase reaction (HT Liu and Y Okada, unpublished), and DIDS was reported to block the maxi-anion channel activity 58. Thus, we made preliminary experiments by applying ARL 67156 (100 μM) and found that this ecto-ATPase blocker markedly increased the extracellular concentration of ATP released from mouse astrocytes in response to a hypotonic challenge (210 mosmol/kg-H2O for 15 min) (HT Liu and Y Okada, unpublished). Even in the presence of ARL 67156, the hypotonicity-induced ATP release was found to be significantly suppressed by Gd3+ (HT Liu and Y Okada, unpublished). Although it is not known at present whether ARL 67156 affects any candidate pathways for ATP release, it appears that the Gd3+-sensitive maxi-anion channel serves as a major pathway for swelling-induced ATP release from mouse astrocytes even under the conditions where ecto-ATPases were inhibited.
Taken together, our results suggest that swelling-induced release of anionic ATP from mouse cortical astrocytes is mediated by Gd3+-sensitive maxi-anion channels. There is little information about the molecular nature of the maxi-anion channel. A mitochondrial porin (voltage-dependent anion channel (VDAC)) located in the plasma membrane has long been considered as the molecule underlying the maxi-anion channel activity 59, 60, 61, 62, based upon similarities in the biophysical properties of these two channels and the purported presence of VDAC protein in the plasma membrane. In our recent study 63, we have deleted all the three genes encoding the VDAC isoforms and demonstrated that the maxi-anion channel activity in VDAC-deficient mouse fibroblasts was unaltered. The lack of correlation between VDAC protein expression and maxi-anion channel activity strongly argues against the long-held hypothesis of plasmalemmal VDAC being the maxi-anion channel. Molecular identification of the maxi-anion channel awaits further investigation.
Brain astrocytes are known to exhibit swelling in response to ischemic and traumatic brain injury 44. Also, astrocytes are found to undergo swelling after stimulation with glutamate 64 as well as during hyperammonemia 65, 66 and lactacidosis 67. Therefore, it is conceivable that ATP release from swollen astrocytes plays some role in such pathological conditions.
Materials and methods
Solutions and chemicals
The normal Ringer solution contained (mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 Na-HEPES, 6 HEPES, and 5 glucose (pH 7.4, 290 mosmol/kg-H2O). Hypotonic solutions were prepared by mixing the normal Ringer solution with a HEPES-buffered solution containing (mM): 5 KCl, 2 CaCl2, 1 MgCl2, 5 Na-HEPES, 6 HEPES, and 5 glucose (pH 7.4, 38 mosmol/kg-H2O).
GdCl3 was stored as a 50-mM stock solution in water and added directly to the hypotonic solution immediately before each experiment. NPPB, glibenclamide, SITS, phloretin, BFA, carbenoxolone, 1-octanol, probenecid and brilliant blue G were purchased from Sigma-Aldrich (St. Louis, MO). Verapamil and BAPTA/AM were obtained from Nacalai Tesque (Kyoto, Japan) and DOJINDO (Kumamoto, Japan), respectively. The drugs were added to hypotonic solution immediately before use from stock solutions in DMSO. DMSO did not have any effect, when added alone at a concentration less than 0.1%. Osmolality of all solutions was measured using a freezing-point depression osmometer (OM802: Vogel, Kevelaer, Germany).
Cell culture and tissue preparation
The experimental protocol was approved in advance by the Ethics Review Committee for Animal Experimentation of the National Institute for Physiological Sciences. Astrocytes were obtained from 2–3-day old pups of Slc:ddy mice (Japan SLC, Inc., Hamamatsu, Japan) using the method described previously 36. The cells were cultured in 12- or 24-well plates, and, upon reaching confluency, were used for ATP measurements and RT-PCR assays. The cell number per well in 12- or 24-well plates was (2.35 ± 0.19) × 105 or (9.49 ± 1.03) × 104, respectively.
The MLHs were prepared from 100 mg tissue samples of the lung dissected from 90-day old mice after homogenizing with a glass/Teflon homogenizer (Iuchi, Osaka, Japan) in 1 ml Sepasol RNA I reagent.
Luciferin-luciferase ATP assay
The bulk extracellular ATP concentration was measured by the luciferin-luciferase assay, as described previously 36, 39, using astrocytes cultured in 12- or 24-well plates. After the culture medium was totally replaced with isotonic Ringer solution (1 000 and 425μl for 12- and 24-well plates, respectively), cells were incubated at 37°C for 60 min. As a control sample for background ATP release, an aliquot (100 μl) of the extracellular solution was collected. An osmotic challenge was then applied by gently removing most of the remaining extracellular solution (875 and 300 μl for 12- and 24-well plates, respectively), adding the hypotonic solution (1 000 and 400μl for 12- and 24-well plates, respectively), and then placing the plates in an incubator at 37 °C. At specified time points, the plate was carefully rocked again to ensure homogeneity of the extracellular solution. For the luminometric ATP assay, samples (20 and 50 μl for 12- and 24-well plates, respectively) were collected from each well at specified time points. The ATP concentration was measured, under nearly normotonic conditions, by mixing 20 or 50 μl of sample solution with 530 or 500 μl normal Ringer solution, respectively, and 50 μl of luciferin-luciferase reagent. Ionic salt sensitivity of the luciferase reaction was negligible. When required, drugs were added to the hypotonic solution to give the final concentrations as indicated. We supplemented the luciferin-luciferase assay mixture with 600 μM EDTA when the samples contained Gd3+, because Gd3+ interfered with the luciferin-luciferase reaction (see Figure 4B), as reported previously 42. Other drugs employed in the present study had no significant effect on the luciferin-luciferase reaction.
RT-PCR analysis of mRNA expression
Total RNA was isolated from cultured mouse cortical astrocytes before and after 15-min exposure to hypotonic solution (210 mosmol/kg-H2O) or from homogenates of the mouse lung using Sepasol RNA I reagent (Nacalai Tesque) according to the manufacturer's instructions. First-strand cDNA was synthesized from the isolated RNA using reverse transcriptase (Roche Diagnostics GmbH, Mannheim, Germany) and oligo-dT primers (Invitrogen Corp., Carlsbad, CA). The synthesis of cDNA was performed according to the manufacturer's protocol. The sequences of primers for P2X7, MRP1 and CFTR were the same as reported previously 30, 49. All other gene-specific primers used for PCR were designed using Primer3 software (www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The sequences of primers for RT-PCR experiments are given in supplementary information, Table S1. PCR was carried out with Blend Taq (TOYOBO, Osaka, Japan) and a Gene Amp PCR System 9600 thermal cycler (Perkin-Elmer Life Sciences, Boston, MA). Cycling conditions were 4 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at the annealing temperature given for the individual primers (supplementary information, Table S1), and 1 min at 72 °C, and, finally, 10 min at 72 °C. The integrity of the isolated RNA and the reverse transcription reaction were examined using specific primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin. All PCR products were analyzed on a 2% agarose gel and had the sizes expected from the known cDNA sequences.
Data analysis
Data were analyzed with OriginPro 7.0 (MicroCal Software, Northampton, MA). Pooled data are given as means ± SEM of observations (n). Statistical differences of the data were evaluated by ANOVA and the paired or unpaired Student's t test where appropriate, and considered significant at P < 0.05.
(Supplementary Information is linked to the online version of the paper on the Cell Research website.)
References
Hansson E, Ronnback L . Glial neuronal signaling in the central nervous system. FASEB J 2003; 17:341–348.
Araque A, Perea G . Glial modulation of synaptic transmission in culture. Glia 2004; 47:241–248.
Fields RD, Burnstock G . Purinergic signalling in neuron-glia interactions. Nat Rev Neurosci 2006; 7:423–436.
Haydon PG, Carmignoto G . Astrocyte control of synaptic transmission and neurovascular coupling. Physiol Rev 2006; 86:1009–1031.
Fellin T, Pascual O, Haydon PG . Astrocytes coordinate synaptic networks: balanced excitation and inhibition. Physiology (Bethesda) 2006: 21:208–215.
Newman EA . Glial cell inhibition of neurons by release of ATP. J Neurosci 2003; 23:1659–1666.
Zhang JM, Wang HK, Ye CQ, et al. ATP released by astrocytes mediates glutamatergic activity-dependent heterosynaptic suppression. Neuron 2003; 40:971–982.
Koizumi S, Fujishita K, Tsuda M, Shigemoto-Mogami Y, Inoue K . Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures. Proc Natl Acad Sci USA 2003; 100:11023–11028.
Volonte C, Amadio S, Cavaliere F, D'Ambrosi N, Vacca F, Bernardi G . Extracellular ATP and neurodegeneration. Curr Drug Targets CNS Neurol Disord 2003; 2:403–412.
Franke H, Illes P . Involvement of P2 receptors in the growth and survival of neurons in the CNS. Pharmacol Ther 2006; 109:297–324.
Darby M, Kuzmiski JB, Panenka W, Feighan D, MacVicar BA . ATP released from astrocytes during swelling activates chloride channels. J Neurophysiol 2003; 89:1870–1877.
Shinozaki Y, Koizumi S, Ishida S, Sawada J, Ohno Y, Inoue K . Cytoprotection against oxidative stress-induced damage of astrocytes by extracellular ATP via P2Y1 receptors. Glia 2005; 49:288–300.
Inoue K . Microglial activation by purines and pyrimidines. Glia 2002; 40:156–163.
McLarnon JG . Purinergic mediated changes in Ca2+ mobilization and functional responses in microglia: effects of low levels of ATP. J Neurosci Res 2005; 81:349–356.
Davalos D, Grutzendler J, Yang G, et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 2005; 8:752–758.
Xiang Z, Chen M, Ping J, et al. Microglial morphology and its transformation after challenge by extracellular ATP in vitro. J Neurosci Res 2006; 83:91–101.
Sabirov RZ, Okada Y . ATP release via anion channels. Purinergic Signalling 2005; 1:311–328.
Schwiebert EM, Zsembery A . Extracellular ATP as a signaling molecule for epithelial cells. Biochim Biophys Acta 2003; 1615:7–32.
Barbe MT, Monyer H, Bruzzone R . Cell-cell communication beyond connexins: the pannexin channels. Physiology (Bethesda) 2006; 21:103–114.
Harden TK, Lazarowski ER . Release of ATP and UTP from astrocytoma cells. Prog Brain Res 1999; 120:135–143.
Wang Z, Haydon PG, Yeung ES . Direct observation of calcium-independent intercellular ATP signaling in astrocytes. Anal Chem 2000; 72:2001–2007.
Stout CE, Costantin JL, Naus CC, Charles AC . Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J Biol Chem 2002; 277:10482–10488.
Coco S, Calegari F, Pravettoni E, et al. Storage and release of ATP from astrocytes in culture. J Biol Chem 2003; 278:1354–1362.
Suadicani SO, Brosnan CF, Scemes E . P2X7 receptors mediate ATP release and amplification of astrocytic intercellular Ca2+ signaling. J Neurosci 2006; 2:1378–1385.
Cotrina ML, Lin JH, Alves-Rodrigues A, et al. Connexins regulate calcium signaling by controlling ATP release. Proc Natl Acad Sci USA 1998; 95:15735–15740.
Liu GJ, Werry EL, Bennett MR . Secretion of ATP from Schwann cells in response to uridine triphosphate. Eur J Neurosci 2005; 21:151–160.
Gordon GR, Baimoukhametova DV, Hewitt SA, Rajapaksha WR, Fisher TE, Bains JS . Norepinephrine triggers release of glial ATP to increase postsynaptic efficacy. Nat Neurosci 2005; 8:1078–1086.
Bal-Price A, Moneer Z, Brown GC . Nitric oxide induces rapid, calcium-dependent release of vesicular glutamate and ATP from cultured rat astrocytes. Glia 2002; 40:312–323.
Sheppard DN, Welsh MJ . Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents. J Gen Physiol 1992; 100:573–591.
Minich T, Riemer J, Schulz JB, Wielinga P, Wijnholds J, Dringen R . The multidrug resistance protein 1 (Mrp1), but not Mrp5, mediates export of glutathione and glutathione disulfide from brain astrocytes. J Neurochem 2006; 97:373–384.
Ballerini P, Di Iorio P, Ciccarelli R, et al. Glial cells express multiple ATP binding cassette proteins which are involved in ATP release. NeuroReport 2002; 13:1789–1792.
Roman RM, Lomri N, Braunstein G, et al. Evidence for multidrug resistance-1 P-glycoprotein-dependent regulation of cellular ATP permeability. J Membr Biol 2001; 183:165–173.
Ye ZC, Wyeth MS, Baltan-Tekkok S, Ransom BR . Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J Neurosci 2003; 23:3588–3596.
Bruzzone R, Barbe MT, Jakob NJ, Monyer H . Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in Xenopus oocytes. J Neurochem 2005; 92:1033–1043.
Anderson CM, Nedergaard M . Emerging challenges of assigning P2X7 receptor function and immunoreactivity in neurons. Trends Neurosci 2006; 29:257–262.
Liu HT, Tashmukhamedov BA, Inoue H, Okada Y, Sabirov RZ . Roles of two types of anion channels in glutamate release from mouse astrocytes under ischemic or osmotic stress. Glia 2006; 54:343–357.
Fan HT, Morishima S, Kida H, Okada Y . Phloretin differentially inhibits volume-sensitive and cAMP-activated, but not Ca-activated, Cl- channels. Br J Pharmacol 2001; 133:1096–1106.
Decher N, Lang HJ, Nilius B, Bruggemann A, Busch AE, Steinmeyer K . DCPIB is a novel selective blocker of ICl,swell and prevents swelling-induced shortening of guinea-pig atrial action potential duration. Br J Pharmacol 2001; 134:1467–1479.
Dutta AK, Sabirov RZ, Uramoto H, Okada Y . Role of ATP-conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischaemic or hypoxic conditions. J Physiol 2004; 559:799–812.
Sabirov RZ, Dutta AK, Okada Y . Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release. J Gen Physiol 2001; 118:251–266.
Bell PD, Lapointe JY, Sabirov RZ, et al. Macula densa cell signaling involves ATP release through a maxi anion channel. Proc Natl Acad Sci USA 2003; 100:4322–4327.
Boudreault F, Grygorcayk R . Cell swelling-induced ATP release and gadolinium sensitive channels. Am J Physiol 2002; 282:C219–C226.
Kimelberg HK . Water homeostasis in the brain: basic concepts. Neuroscience 2004; 129:851–860.
Kimelberg HK . Astrocytic swelling in cerebral ischemia as a possible cause of injury and target for therapy. Glia 2005; 50:389–397.
Abdullaev IF, Rudkouskaya A, Schools GP, Kimelberg HK, Mongin AA . Pharmacological comparison of swelling-activated excitatory amino acid release and Cl− currents in cultured rat astrocytes. J Physiol 2006; 572:677–689.
Sabirov RZ, Okada Y . Wide nanoscopic pore of maxi-anion channel suits its function as an ATP-conductive pathway. Biophys J 2004; 87:1672–1685.
Ternovsky VI, Okada Y, Sabirov RZ . Sizing the pore of the volume-sensitive anion channel by differential polymer partitioning. FEBS Lett 2004; 576:433–436.
Hirrlinger J, Moeller H, Kirchhoff F, Dringen R . Expression of multidrug resistance proteins (Mrps) in astrocytes of the mouse brain: a single cell RT-PCR study. Neurochem Res 2005; 30:1237–1244.
Duan S, Anderson CM, Keung EC, Chen Y, Chen Y, Swanson RA . P2X7 receptor-mediated release of excitatory amino acids from astrocytes. J Neurosci 2003; 23:1320–1328.
Lai CP, Bechberger JF, Thompson RJ, MacVicar BA, Bruzzone R, Naus CC . Tumor-suppressive effects of pannexin 1 in C6 glioma cells. Cancer Res 2007; 67:1545–1554.
Decleves X, Regina A, Laplanche JL, et al. Functional expression of P-glycoprotein and multidrug resistance-associated protein (Mrp1) in primary cultures of rat astrocytes. J Neurosci Res 2000; 60:594–601.
Bowser DN, Khakh BS . Two forms of single-vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy. Proc Natl Acad Sci USA 2007; 104:4212–4217.
Hamill OP, McBride DW Jr . The pharmacology of mechanogated membrane ion channels. Pharmacol Rev 1996; 48:231–252.
Moran MM, Xu H, Clapham DE . TRP ion channels in the nervous system. Curr Opin Neurobiol 2004; 14:362–369.
Collopy-Junior I, Kneipp LF, da Silva FC, et al. Characterization of an ecto-ATPase activity in Fonsecaea pedrosoi. Arch Microbiol 2006; 185:355–362.
Fonseca FV, Fonseca de Souza AL, Mariano AC, Entringer PF, Gondim KC, Meyer-Fernandes JR . Trypanosoma rangeli: characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity. Exp Parasitol 2006; 112:76–84.
Levesque SA, Lavoie EG, Lecka J, Bigonnesse F, Sevigny J . Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse ectonucleotidases. Br J Pharmacol 2007; 152:141–150.
Guibert B, Dermietzel R, Siemen S . Large conductance channel in plasma membranes of strocytic cells is functionally related to mitochondrial VDAC-channel. Intern J Biochem Cell Biol 1998; 30:379–391.
Dermietzel R, Hwang TK, Buettner R, et al. Cloning and in situ localization of a brain-derived porin that constitutes a large-conductance anion channel in astrocytic plasma membranes. Proc Natl Acad Sci USA 1994; 91:499–503.
Buettner R, Papoutsoglou G, Scemes E, Spray DC, Dermietzel R . Evidence for secretory pathway localization of a voltage-dependent anion channel isoform. Proc Natl Acad Sci USA 2000; 97:3201–3206.
Bahamonde MI, Fernandez-Fernandez JM, Guix FX, Vazquez E, Valverde MA . Plasma membrane voltage-dependent anion channel mediates antiestrogen-activated maxi Cl− currents in C1300 neuroblastoma cells. J Biol Chem 2003; 278:33284–33289.
Elinder F, Akanda N, Tofighi R, et al. Opening of plasma membrane voltage-dependent anion channels (VDAC) precedes caspase activation in neuronal apoptosis induced by toxic stimuli. Cell Death Differ 2005; 12:1134–1140.
Sabirov RZ, Sheiko T, Liu HT, Deng D, Okada Y, Craigen WJ . Genetic demonstration that the plasma membrane maxianion channel and voltage-dependent anion channels are unrelated proteins. J Biol Chem 2006; 281:1897–1904.
Schneider GH, Baethmann A, Kempski O . Mechanisms of glial swelling induced by glutamate. Can J Physiol Pharmacol 1992; 70:S334–S343.
Takahashi H, Koehler RC, Brusilow SW, Traystman RJ . Inhibition of brain glutamine accumulation prevents cerebral edema in hyperammonemic rats. Am J Physiol Heart Circ Physiol 1991; 261:H825–H829.
Willard-Mack CL, Koehler RC, Hirata T, et al. Inhibition of glutamine synthetase reduces ammonia-induced astrocyte swelling in rat. Neuroscience 1996; 71:589–599.
Izumi Y, Kirby-Sharkey CO, Benz AM, et al. Swelling of Muller cells induced by AP3 and glutamate transport substrates in rat retina. Glia 1996; 17:285–293.
Acknowledgements
This work was supported by The Grants-in-Aid for Scientific Research from JSPS (to H-TL and RZS) and from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to YO). We thank our colleagues EL Lee for manuscript reviewing, K Shigemoto and M Ohara for technical assistance, and T Okayasu for secretarial assistance.
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Supplementary information Table S1
Specificity, sequences, size and annealing temperature of primers designed for RT-PCR (PDF 25 kb)
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Liu, HT., Toychiev, A., Takahashi, N. et al. Maxi-anion channel as a candidate pathway for osmosensitive ATP release from mouse astrocytes in primary culture. Cell Res 18, 558–565 (2008). https://doi.org/10.1038/cr.2008.49
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DOI: https://doi.org/10.1038/cr.2008.49
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