Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 µM) activated a small EAA release in 70-80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5-10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo.
cell swelling; anion channels; glutamate release; adenosine 5'-triphosphate release; neuron-glia communication
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INTRODUCTION |
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ASTROCYTES
ARE the main cell type in the mammalian brain showing
cell volume perturbations in response to pathological conditions or
neuronal stimulation (1, 31, 34). As in the majority of
other cells, swelling of cultured astrocytes activates specific volume
regulatory mechanisms involving K+ and Cl
efflux through volume-regulated cation and anion channels (51, 58, 69, 77). Volume-regulated anion channels (VRACs) are also
permeable toward a broad spectrum of small organic anions and neutral
molecules termed "organic osmolytes," which include amino acids,
polyols, and methylamines (reviewed in Refs. 35 and 73).
Organic osmolytes along with Cl
contribute to astrocytic
cell volume regulation in response to acute and chronic osmotic stress
(32, 53, 57, 59, 77). At least in some brain areas, glial
amino acid release via VRACs modulates neuronal activity (14, 24,
25). Recent data also suggest that VRACs constitute the major
pathway for the release of excitatory amino acids (EAAs) in several
central nervous system (CNS) pathologies (4, 33, 61, 71).
Although the biophysical properties of VRACs have been studied in detail, their molecular identities and the transducing mechanisms linking volume changes to their activation remain unknown. Changes in intracellular ionic strength (48, 78), calmodulin (38, 39, 43), tyrosine phosphorylation (12, 75), and p21Rho small GTPase (49, 74) have all been suggested as obligatory elements of volume signal transduction (reviewed in Refs. 36 and 45). Wang and coauthors (79) found that cell swelling induces the release of endogenous ATP in hepatoma cells, followed by autocrine activation of P2Y purinergic receptors and, subsequently, volume-dependent anion channels. Anion channel activation and volume regulation in these cells are eliminated by scavenging extracellular ATP or by the P2Y receptor inhibitors. Therefore, ATP may be a key autocrine factor mediating cell volume regulation.
ATP release is common in mammalian cells and may be induced by various stimuli, including changes in cell volume (23, 76), mechanical stress (20), or cAMP (70). Brain astrocytes possess all the elements constituting the hypothetical loop of autocrine ATP regulation of cell volume, i.e., ATP release machinery, P2Y receptors, and volume-dependent anion channels (11, 21, 32, 47). Moreover, astrocytic processes are exposed to ATP released from neurons during excitation and ATP is the messenger for intercellular communication in astrocytic networks as well as between neurons and astrocytes in culture (11, 18, 21). In the present paper we explored the role of ATP as an activator or modulator of VRACs and channel-mediated release of EAAs in primary astrocyte cultures. We looked for a possible role of ATP, or the product of its enzymatic degradation, adenosine, in regulation of EAA release under conditions of mild "physiological" swelling, resembling changes on physiological neuronal excitation. The results of this study have been presented and published in a preliminary form (44).
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MATERIALS AND METHODS |
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Materials.
D-[3H]aspartate (specific activity 18 Ci/mM)
was obtained from Du Pont-NEN Research Products (Boston, MA). Dispase
(neutral protease dispase grade II) was purchased from Boehringer
Mannheim (Indianapolis, IN). All cell culture reagents were from GIBCO (Grand Island, NY).
1'-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) and tetanus toxin were from Calbiochem (La Jolla, CA). Alloxazine, ,
-methylene-D-adenosine 5'-triphosphate
disodium salt (
,
-MeATP), and pyridoxal
phosphate-6-azophenyl-2'4'-disulfonic acid (PPADS) were from RBI
(Natick, MA). (S)-dihydroxyphenylglycine (DHPG) and reactive blue 2 were from Tocris Cookson (Ballwin, MO). Suramin, ATP two-sodium salt,
2-methylthioadenosine 5'-triphosphate (2-MeSATP), and other chemicals,
unless otherwise specified, were from Sigma (St. Louis, MO).
Cell cultures.
Confluent primary astrocyte cultures were prepared from the cerebral
cortex of newborn Sprague-Dawley rats as described by Frangakis and
Kimelberg (19), with minor modifications as described below. All animal procedures were performed according to the NIH guide
for animal care and approved by the institutional animal care
committee. The cerebral cortices were separated from meninges and basal
ganglia, and tissue was dissociated with the neutral protease dispase.
Dissociated cells were seeded on poly-D-lysine-coated 18 × 18-mm glass coverslips (Carolina Biological Supply,
Burlington, NC) and grown for 3-4 wk in minimal essential medium
(MEM) supplemented with 10% heat-inactivated horse serum (HIHS), 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in a humidified
5% CO2-95% air atmosphere. Culture medium was replaced twice a
week. After 2 wk of cultivation, penicillin and streptomycin were
removed from the culture medium. Immunocytochemistry showed that 98%
of the cells stained positively for the astrocytic marker glial
fibrillary acid protein.
Excitatory amino acid efflux measurements.
Excitatory amino acid efflux measurements were performed as previously
described (43). Astrocytes grown on glass coverslips were
loaded overnight with D-[3H]aspartate (4 µCi/ml, final concentration 220 nM) in 2.5 ml of MEM containing 10%
HIHS in an incubator set for 5% CO2-95% air at 37°C.
Before the start of the efflux measurements, the cells were washed of
extracellular isotope and serum-containing medium in HEPES-buffered
solution. The basal HEPES-buffered medium contained (in mM) 122 NaCl,
3.3 KCl, 0.4 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 D-glucose, and 25 HEPES.
pH was adjusted to 7.4 with NaOH (~15 mM). The coverslips were
inserted into a Lucite perfusion chamber that had a depression
precisely cut in the bottom to accommodate the coverslip and a Teflon
screw top leaving a space above the cells of ~100 µm in height. The
cells were superfused at a flow rate of 1.0 ml/min in an incubator set
at 37°C with HEPES-buffered medium. In hypoosmotic media NaCl
concentration was reduced to 115 mM (7 mM NaCl, a 5% decrease in
medium osmolarity) or to 72 mM (
50 mM NaCl, a 35% decrease in medium
osmolarity). Hyperosmotic media were made by adding sucrose. The
osmolarities of all buffers were checked with a freezing point
osmometer (Advanced Instruments, Needham Heights, MA). Superfusate
fractions were collected at 1-min intervals. At the end of each
experiment, the isotope remaining in the cells was extracted with a
solution containing 1% sodium dodecyl sulfate plus 4 mM EDTA. Four
milliliters of Ecoscint scintillation cocktail (National Diagnostics,
Atlanta, GA) was added, and each fraction was counted for
3H in a Packard Tri-Carb 1900TR liquid scintillation
analyzer (Packard Instrument, Meriden, CT). Percent fractional isotope
release for each time point was calculated by dividing radioactivity
released in each 1-min interval by the radioactivity left in the cells (the sum of all the radioactive counts in the remaining fractions up to
the beginning of the fraction being measured plus the radioactivity left in the cell digest) with a custom computer program.
ATP assays. ATP assays were done with luciferin-luciferase reaction as described elsewhere (68). Cells in 12-well culture plates were washed several times with isosmotic medium and then exposed for 1-10 min to isosmotic or hypoosmotic medium (for compositions see Excitatory amino acid efflux measurements). Aliquots of extracellular medium (100 µl) were taken for extracellular ATP determination. Extracellular medium was then aspirated, cells were lysed with 0.1% Triton X-100 (1 ml), and 40-µl aliquots were used for intracellular ATP content measurements in the same well.
Statistical analysis. Data are presented as mean ± SE values of 3-10 experiments performed on at least two different astrocyte preparations. Effects of all agonists and antagonists were always compared with the controls performed on the same day and on the same culture preparation. The data were analyzed by one-way ANOVA followed by post hoc Newman-Keuls when multiple comparisons were made.
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RESULTS |
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Synergistic effect of moderate cell swelling and ATP on EAA release
in astrocyte cultures.
In 14 of 18 astrocyte cultures tested, 10 µM ATP under isosmotic
conditions induced transient stimulation of
D-[3H]aspartate release with maximal release
rates of 50-120% over basal release levels (Fig.
1). Four cultures did not respond to ATP
under isosmotic conditions (data not shown). Because nonpathological neuronal excitation can induce moderate swelling of astrocytes (1), likely due to uptake of released K+ and
glutamate (31), we tested ATP effects on EAA release in cells subjected to mild hypotonic stress. Five percent reduction of
extracellular medium osmolarity by itself led to a twenty to forty-five
percent increase in EAA release over the basal levels (Fig.
1B). When 10 µM ATP was added to the hypoosmotic medium, it caused a larger transient potentiation of EAA release compared with
the ATP effect under basal conditions (Fig. 1). It should be stressed
that ATP-induced potentiation was found even in those cultures not
responding to ATP under basal conditions. The ATP-induced release
increased with a further decrease in medium osmolarity (Fig. 1). In
contrast, when medium osmolarity was increased by 5% and 10% (14 and
28 mM sucrose addition, respectively), this potently suppressed the
ATP-induced EAA release to near-basal levels (Fig. 1).
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Effects of P2 receptor agonists and antagonists on astrocytic EAA
release.
To explore what receptor subtype mediates the ATP effects under
conditions of moderate cell swelling, we used agonists and antagonists
for P2Y/P2U and P2X receptors. 2-MeSATP, a potent agonist for
P2Y1 and some P2X receptors, and UTP, which is active at
P2U receptors (group of UTP-sensitive P2Y receptors including P2Y2, P2Y4, and P2Y6), both
increased EAA release in moderately swollen cells (Fig.
4A). Besides several P2Y
receptor subtypes, cultured astrocytes also express two P2X receptors,
P2X1 and P2X7 (3, 41). These
receptors seem not to be involved in the ATP effects, because both the
P2X1/3 agonist ,
-MeATP (10 µM, Fig. 4A)
and the P2X7 antagonist KN-62 (1 µM, n = 3, P > 0.85; data not shown) were ineffective. The
P2Y1 antagonist reactive blue 2 (10 µM) completely
inhibited the effect of 2-MeSATP and partially suppressed the effects
of ATP and UTP (Fig. 4B). Although in the experiments
presented in Fig. 4B inhibition of the ATP effect by
reactive blue 2 was not statistically significant, this partial inhibition was reproduced in two other cultures. As an additional approach to blocking ATP receptors, we used a 10-min preexposure to ATP
to desensitize P2 receptors (64). Preincubation with ATP
completely suppressed further ATP-induced potentiation of EAA release
in moderately swollen cells (Fig. 4C). Adenosine and the
selective group I metabotropic glutamate receptor agonist DHPG, both
known to increase intracellular Ca2+ concentration in
cultured and acutely isolated astrocytes (6, 62), did not
affect EAA release in astrocyte cultures exposed to mild hypotonic
stress (Fig. 4A).
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Substantial cell swelling induces ATP release from cultured
astrocytes.
In several cell types, and particularly in hepatoma cells, swelling
induces endogenous ATP release, which then activates volume-dependent anion channels (79). We found a large release of
endogenous ATP, measured as accumulation of ATP in extracellular medium
under nonperfusion conditions, in confluent astrocyte cultures exposed to substantial hypoosmotic stress (35% reduction in medium osmolarity; Fig. 5A). During the first 2 min of hypotonic exposure, the extracellular ATP concentration
increased ~4.5 times, equivalent to the release of ~5% of the
total cell ATP content. After this first phase the rate of ATP release
was decreased despite the persistence of an osmotic gradient. The
hypoosmotic medium-induced ATP release was not associated with
significant changes in intracellular ATP content (Fig. 5B),
suggesting a compensatory increase in cellular ATP production. This
also rules out cell lysis as a reason for ATP release. In our previous
work (43), we also did not observe any significant cell
lysis in hypoosmotic media by monitoring the release of preloaded
51Cr. In parallel experiments performed on the same culture
preparation, a change of medium from isosmotic to isosmotic led to a
~70% increase in extracellular ATP content; this rise was 2.7 times
smaller compared with hypoosmotic conditions (Fig.
5A). These results indicate that mechanostimulation
of astrocytes during changes of medium is not the major cause for the
ATP release under hypoosmotic conditions.
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Effects of extracellular ATP scavenging and purinergic receptor
antagonists on EAA release activated by substantial cell swelling.
To check whether the autocrine release of endogenous ATP is the cause
for activation of astrocytic volume-dependent anion channels, as
reported by Wang et al. (79) for hepatoma cells, we added
apyrase to hydrolyze extracellular ATP and also several P2 receptor
inhibitors. Scavenging of extracellular ATP with apyrase (5 U/ml)
reduced swelling-activated D-[3H]aspartate
release by 20-30% (Fig.
6A), but this effect was not statistically significant. Efficiency of ATP scavenging by apyrase was
checked in the experiments with exogenously added 10 µM ATP, where
apyrase completely inhibited the ATP-induced potentiation of EAA
release (n = 3; data not shown). The broad-spectrum P2 receptor antagonists suramin (100 µM) and PPADS (50 µM)
(64) blocked the hypoosmotic medium-induced EAA release by
67% and 60%, respectively (Fig. 6, B and C). In
contrast to these agents, reactive blue 2, a more selective inhibitor
of the P2Y receptor subfamily (it also inhibits P2X2
receptors; Ref. 27), failed to block the swelling-induced
EAA release at concentrations of 10 and 50 µM (Fig. 6C).
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P2Y receptor-mediated modulation of EAA release in substantially
swollen astrocytes.
Although endogenous ATP release does not seem to be necessary for the
activation of astrocytic VRACs (see Fig. 6A), this
does not exclude the possibility of modulatory effects of ATP in
substantially swollen cells. Because we measure the volume-dependent
EAA release with a superfusion system, removal of endogenously released
ATP will likely diminish the activation of purinergic receptors,
masking potential modulatory effects of ATP on VRACs. We therefore
tested the effects of exogenous ATP, P2X, and P2Y receptor agonists
added to the superfusion medium on EAA release. At concentrations of 10 µM, ATP and the P2Y agonists UTP and 2-MeSATP increased
volume-dependent D-[3H]aspartate release
2.5-3.5 times with a potency in the order of UTP ATP
2-MeSATP (Figs. 7, A and
B). In contrast,
,
-MeATP (10 µM), an agonist
selective toward P2X1 and P2X3 receptors
(27), did not significantly affect the swelling-induced
EAA release (Fig. 7B). The ATP-induced increment in EAA
release was nearly completely suppressed by the P2Y antagonist reactive
blue 2 (Fig. 7A; for the lack of reactive blue 2 effect on
release induced by substantial cell swelling, see Fig. 6C).
Potentiating effects of other P2Y agonists were also suppressed by
reactive blue 2. As in the case of moderately swollen cells, the
2-MeSATP-induced increment in EAA release was completely inhibited by
reactive blue 2 (~95% inhibition, n = 3; data not
shown), but the UTP effect was only partially sensitive to reactive
blue 2 (~40% inhibition, n = 5; data not shown).
Desensitization of purinergic receptors by a 10-min preexposure to ATP
completely inhibited any subsequent ATP-induced increment in EAA
release in substantially swollen cells (Fig. 7C). It should
be noted, however, that although ATP receptor desensitization
eliminated the ATP effect, the volume-dependent component of EAA
release remained unchanged (compare with the control volume-dependent
release in Fig. 7C). These data further support the view
that activation of the P2Y receptors is not a necessary step in the
activation of anion channels but is rather involved in channel
modulation.
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Effect of adenosine on volume-dependent EAA release in
substantially swollen cells.
In brain tissue extracellular ATP is rapidly hydrolyzed to
adenosine by ecto-ATPases and ecto-5'-nucleotidase (80).
Astrocytes in situ and in culture express several types of adenosine
receptors (62, 63), and adenosine receptors have also been
implicated in the activation (8, 9) or modulation
(46) of volume-dependent anion channels in tracheal and
ciliary epithelial cells. We therefore tested the effects of adenosine
and the A2B adenosine receptor antagonist alloxazine, on
volume-dependent EAA release in substantially swollen cells. Adenosine
(100 µM) potentiated hypoosmotic medium-induced D-[3H]aspartate release by ~100% (Fig.
8). This was different from the lack of
adenosine effect in moderately swollen cells (Fig. 4A). In
substantially swollen cells, alloxazine (2 µM) suppressed the
adenosine effect by 70% but by itself did not affect control volume-dependent EAA release (Fig. 8).
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DISCUSSION |
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In this study we demonstrate that ATP works synergistically with
cell swelling in activating EAA release from primary astrocyte cultures. This release is likely mediated by volume-dependent anion
channels, which are permeable to Cl and small organic
anions and uncharged molecules (35, 73). The ATP effect
strongly depends on cell volume; even a small degree of cell shrinkage
completely eliminates the ATP-induced EAA release observed in 80%
astrocyte cultures not subjected to osmotic gradients. This is opposite
to the idea of a direct volume-independent activation of anion channels
by ATP in nonswollen cells and is important for understanding the
mechanisms of ATP actions in vivo. Our pharmacological data suggest
that ATP exerts its modulatory effects on EAA release from astrocytes
via activation of P2Y receptors.
Extracellular ATP is a potent modulator of astrocytic
volume-dependent anion channels but is not required for their
activation.
ATP can be released from cells by a large variety of factors including
changes in cell volume, hypoxia, mechanical stress, cAMP, and receptor
stimulation (reviewed in Refs. 10 and 18). In the CNS and
peripheral nervous system (PNS), ATP is also coreleased synaptically
with other neurotransmitters (16, 30, 64). From this
perspective, the finding by Wang et al. (79) on the autocrine activation of anion channels by ATP with or without changes
in cell volume may have special importance in the brain. It is possible
that the ATP-induced glutamate release from astrocytes, which has been
proposed to mediate glial-neuronal communication (7, 18),
is due to activation of VRACs showing significant permeability toward
EAAs (26, 32). Similar to hepatoma cells, Darby et al.
(13) found both inhibition of volume-sensitive Cl currents by P2 antagonists or apyrase in swollen
cultured astrocytes and activation of Cl
currents by
exogenous ATP in nonswollen cells. More recently, Jeremic et al.
(28) observed stimulation of NPPB-sensitive endogenous glutamate and aspartate release in astrocyte cultures on application of
100 µM ATP.
Multiple purinergic receptors are involved in modulation of EAA
release.
Cultured astrocytes express at least two ionotropic P2X receptors,
P2X1 and P2X7 (3, 41), as well as
four subtypes of metabotropic ATP receptors, P2Y1,
P2Y2, P2Y4, and P2Y6 (29, 37). To date, no pharmacological tools allow us to reliably distinguish between each purinergic receptor subtype. Nonetheless, some
conclusions can be drawn based on the pharmacological data obtained in
the present study. A contribution of P2X1 and
P2X7 can be ruled out on the basis of the insensitivity to
the P2X1 agonist ,
-MeATP and the P2X7
antagonist KN-62, respectively. The most important observation seems to
be the ability of UTP to mimic the effects of ATP on EAA release. UTP
activates P2Y2 and P2Y4 and P2Y6
(collectively termed P2U) receptors, whereas it is completely inactive
at any of the P2X or P2Y1 receptors (27, 64).
Thus P2Y2, P2Y4, and/or P2Y6 are
good candidates for the effects observed in our study. P2Y2
is insensitive to reactive blue 2, whereas P2Y4 and
P2Y6 possess a low sensitivity to this compound
(IC50
20 µM; Ref. 27). Reactive blue
2 partially inhibited the ATP and UTP effects in moderately swollen
cells and was highly potent against ATP effects in substantially
swollen cells. 2-MeSATP, which discriminates P2Y1 from all
P2U receptors, potentiated the EAA release in both moderately and
substantially swollen cells, and these effects were highly sensitive to
reactive blue 2, consistent with the involvement of P2Y1
(27, 50). Overall these data suggest that both
P2Y1 and P2U-like receptors can equally contribute to
potentiation of EAA release. However, depending on the degree of cell
swelling the endogenous agonist ATP seems to act preferentially via P2U
(moderately swollen cells, low sensitivity to reactive blue 2) or
P2Y1 (substantially swollen cells, high
sensitivity to reactive blue 2). The potent effects of 2-MeSATP in
moderately swollen cells and UTP in substantially swollen cells
somewhat contradict this model. Potential explanations for this
discrepancy include a UTP/2-MeSATP-induced ATP release followed by a
cross-activation of other receptor subtypes, a phenomenon contributing
to the propagation of Ca2+ waves (21) and/or a
deviation of the pharmacological profiles of astrocytic P2Y receptors
from their cloned counterparts (37). In endothelial cells
hypoosmotic medium-induced ATP release accelerates volume regulation
via activation of P2Y receptors with pharmacological properties similar
to those found in the present study (72).
Anion channels or Ca2+-dependent vesicular release? Recently much attention has been paid to the phenomenon of astrocytic Ca2+-dependent glutamate release, which shows many similarities to the vesicular glutamate release in neuronal cells (2, 5, 22, 54). Cultured astrocytes express many proteins constituting the apparatus of exocytosis (55) and respond to physiological elevations of cytoplasmic Ca2+ levels with endogenous glutamate release (56). Therefore, one should consider whether the effects of ATP on EAA release are due to stimulation of astrocytic Ca2+-dependent vesicular-like glutamate release, especially in nonswollen and moderately swollen cells. Several facts are inconsistent with such a hypothesis. First, the ATP-induced EAA release in moderately swollen cells shows a high sensitivity to anion channel blockers, which is similar to the volume-dependent release induced by substantial hypoosmotic shock. An extensive line of experimental evidence suggests that at least the latter release is attributed to activation of VRACs (26, 57, 59, 66, 73). Second, ATP effects are strictly dependent on medium osmolarity and are completely inhibited by moderate cell shrinkage. Third, ATP-induced EAA release in moderately swollen cells was completely insensitive to 24-h pretreatment with tetanus toxin, in contrast to the astrocytic vesicular-like glutamate release (60). Together, the data on ATP-induced modulation of astrocytic EAA release can be most parsimoniously explained by the modulation of VRACs. This process may be complementary or alternative to the vesicular-like astrocytic glutamate release described elsewhere (2, 5, 22, 54).
Possible physiological and pathological significance of ATP-induced EAA release. Volume-dependent anion channels contribute to EAA release and likely to neural tissue damage under various pathological conditions associated with astrocyte swelling (4, 33, 61, 71). As seen from our data in substantially swollen cells, ATP release from damaged or swollen neural cells may significantly increase pathological VRAC-mediated EAA release. Because activation of the VRACs is dependent on intracellular ATP, their contribution to EAA release in the infarction core is less likely (26, 52, 68). However, in the penumbra, intracellular ATP content remains as high as 70% of the normal tissue levels (40). Therefore, modulation of swelling-activated EAA release by released extracellular ATP and adenosine may persist in the penumbra over the total duration of ischemic episode and during reperfusion.
In contrast to pathological conditions, very little is known to date regarding the normal physiological functions of VRACs in the brain. In supraoptic and paraventricular nuclei of the hypothalamus, taurine is concentrated in glial cells and tonically released via VRACs in response to small changes in extracellular osmolarity (14). This release modulates electric activity and vasopressin release in magnocellular neurons, thereby contributing to hormonal control of body fluid homeostasis (24, 25). Similarly, VRAC-mediated glutamate release may contribute to regulation of neuronal activity in other brain areas. However, on the basis of in vitro studies we know that volume-dependent glutamate release requires significant changes in cell volume above a threshold level (67), which seems unlikely in the normal CNS. Our data strongly suggest that ATP may be a factor, which provides potent activation of VRACs under physiological conditions. Any degree of astrocytic swelling due to increases in extracellular K+ and/or glutamate concentration should drastically enhance the efficiency of an ATP signal, offering an additional level of regulation of astrocytic EAA release. Severalfold stimulation of basal astrocytic glutamate release is unlikely enough to activate low-affinity non-NMDA glutamate receptors. However, the anion channel-mediated glutamate release in astrocytic processes surrounding glutamatergic synapses may be sufficient to modulate neuronal NMDA receptor activity and contribute to the processes of synaptic plasticity such as long-term potentiation. Interestingly, ATP receptors have a certain specificity in this regard because 100 µM adenosine or 200 µM DHPG, two agents causing the phospholipase C/inositol 1,4,5-trisphosphate-mediated Ca2+ increases in cultured and acutely isolated astrocytes (6, 62), were unable to upregulate EAA release in moderately swollen cells. In summary, the novel findings of our study are that ATP-induced stimulation of astrocytic EAA release is strictly dependent on cell volume and can be strongly upregulated within physiological ranges of cell swelling. The volume dependence, high sensitivity to anion channel blockers, and insensitivity to long-term pretreatment with tetanus toxin are most consistent with EAA release via volume-regulated anion channels. Furthermore, pharmacological analysis suggests that ATP exerts its action via activation of multiple P2Y receptor subtypes (both P2Y1 and P2U-like). ![]() |
ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. N. Hussy for numerous helpful suggestions on the manuscript, C. J. Charniga for expert technical assistance, and R. E. Haskew for critical reading of the manuscript.
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FOOTNOTES |
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This work was supported by the National Institutes of Health (Grant R01-NS-35205 to H. K. Kimelberg and Fogarty International Center Grant F05 TW-05329 to A. A. Mongin).
Address for reprint requests and other correspondence: H. K. Kimelberg, Center for Neuropharmacology and Neuroscience, Albany Medical College, MC-60, 47 New Scotland Ave., Albany, NY 12208 (E-mail: kimelbh{at}mail.amc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 18, 2002;10.1152/ajpcell.00438.2001
Received 11 September 2001; accepted in final form 9 April 2002.
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