ATP regulates anion-channel mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms

ATP regulates anion channel-mediated organic osmolyte releasefrom cultured rat astrocytes via multiple Ca2�-sensitive mechanisms

Alexander A. Mongin1 and Harold K. Kimelberg2

1Center for Neuropharmacology and Neuroscience, Albany MedicalCollege, and 2Ordway Research Institute, Albany, New York

Submitted 8 July 2004; accepted in final form 8 September 2004

Mongin, Alexander A., and Harold K. Kimelberg. ATP regu-lates anion channel-mediated organic osmolyte release from culturedrat astrocytes via multiple Ca2�-sensitive mechanisms. Am J PhysiolCell Physiol 288: C204–C213, 2005. First published September 15,2004; doi:10.1152/ajpcell.00330.2004.—Ubiquitously expressed vol-ume-regulated anion channels (VRACs) are activated in response tocell swelling but may also show limited activity in nonswollen cells.VRACs are permeable to inorganic anions and small organic os-molytes, including the amino acids aspartate, glutamate, and taurine.Several recent reports have demonstrated that neurotransmitters orhormones, such as ATP and vasopressin, induce or strongly potentiateastrocytic whole cell Cl� currents and amino acid release, which areinhibited by VRAC blockers. In the present study, we explored theintracellular signaling mechanisms mediating the effects of ATP onD-[3H]aspartate release via the putative VRAC pathway in rat primaryastrocyte cultures. Cells were exposed to moderate (5%) or substantial(30%) reductions in medium osmolarity. ATP strongly potentiatedD-[3H]aspartate release in both moderately swollen and substantiallyswollen cells. These ATP effects were blocked (�80% inhibition) byintracellular Ca2� chelation with BAPTA-AM, calmodulin inhibitors,or a combination of the inhibitors of protein kinase C (PKC) andcalmodulin-dependent kinase II (CaMK II). In contrast, controlD-[3H]aspartate release activated by the substantial hyposmotic swell-ing showed little (�25% inhibition) sensitivity to the same pharma-cological agents. These data indicate that ATP regulates VRACactivity via two separate Ca2�-sensitive signaling cascades involvingPKC and CaMK II and that cell swelling per se activates VRACs viaa separate Ca2�/calmodulin-independent signaling mechanism. Ca2�-dependent organic osmolyte release via VRACs may contribute to thephysiological functions of these channels in the brain, includingastrocyte-to-neuron intercellular communication.

volume-regulated anion channels; protein kinase C; calcium/calmod-ulin-dependent kinase II; glutamate release; neuron-glia communication

THE OVERWHELMING MAJORITY of mammalian cells respond toswelling by the activation of volume-regulated potassium andanion channels, resulting in a net loss of osmolytes, causingregulatory volume decrease (32, 46). Ubiquitously expressedvolume-regulated anion channels (VRACs) are permeable to avariety of inorganic anions, small organic anions, and un-charged molecules, including the amino acids taurine, gluta-mate, and aspartate (30, 49, 51, 71). Although the molecularidentity of VRACs has not been established, there is goodevidence that, at least in some cell types, more than one type ofanion channel may contribute to swelling-activated Cl� andorganic osmolyte fluxes (2, 26, 31, 78). In addition to their rolein cell volume homeostasis, VRACs are also thought to par-

ticipate in a variety of other processes including cell prolifer-ation, apoptosis, and mechanosensitivity in endothelial andmuscle cells (13, 52).

In the brain, VRACs contribute to physiological and patho-logical amino acid release. In ischemia and other brain pathol-ogies, uncontrolled cell swelling, primarily seen in astrocytes,causes massive efflux of excitatory amino acids that is sensitiveto VRAC inhibitors (45, 57, 69), and such release has beenimplicated in ischemic brain damage (27, 29). Under physio-logical conditions, VRACs are functional in the supraoptic andparaventricular nuclei of the hypothalamus. In these brainareas, small changes in extracellular osmolarity tonically reg-ulate taurine release via a VRAC permeability pathway inspecialized subpopulations of astrocytes (9, 20). Extracellulartaurine via glycine receptors modulates the electric activity ofmagnocellular neurons, which secrete key hormones of bodywater homeostasis, vasopressin and oxytocin (21, 22).

A role for VRACs in nonpathological glutamate and aspar-tate release has not yet been demonstrated. Although astrocytesshow changes in their volume in response to neuronal stimu-lation in situ (1), the small degree of astrocytic swelling aloneseems insufficient to activate VRACs to the level of functionalsignificance. However, the findings of a few recent in vitrostudies (42, 44, 62) suggest that neurotransmitters and neuro-modulators that evoke intracellular Ca2� increases may induceamino acid release via a putative VRAC pathway in nonswol-len or moderately swollen cells. Such VRAC-mediated organicosmolyte release may contribute to Ca2�-dependent astrocyte-to-neuron signaling, which is currently thought to be mediatedby glutamate (6, 18). In the present study, we used D-[3H]as-partate release as a measure of VRAC activity to explore theintracellular signaling mechanisms mediating VRAC activa-tion and/or modulation by extracellular ATP in cells exposed tomoderate (a 5% reduction in medium osmolarity) and substan-tial (a 30% reduction in medium osmolarity) hyposmotic gra-dients. We employed moderate cell swelling to test for VRACactivity under conditions resembling those to which astrocytesare exposed upon physiological stimulation. Substantial cellswelling, on the other hand, allowed us to examine the mech-anisms of full VRAC activation as well as the effects of ATPon fully activated VRACs. Several reports (35, 36) havesuggested that depending on the degree of swelling, cells mayutilize different ion channels or signaling mechanisms of ionchannel activation to regulate their volume. Portions of the datareported in this article were presented in preliminary form (43).

Address for reprint requests and other correspondence: A. A. Mongin,Center for Neuropharmacology and Neuroscience, Albany Medical College,47 New Scotland Ave., MC-136, Albany, NY 12208 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Cell Physiol 288: C204–C213, 2005.First published September 15, 2004; doi:10.1152/ajpcell.00330.2004.

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MATERIALS AND METHODS

Astrocyte cultures. Confluent primary astrocyte cultures were pre-pared from cortices of newborn Sprague-Dawley rat pups as previ-ously described (15), with minor modifications. Pups were killed bydecapitation according to the procedure conforming to the PublicHealth Service Policy on Humane Care and Use of LaboratoryAnimals and approved by the Albany Medical College InstitutionalAnimal Care and Use Committee. The cerebral cortices were sepa-rated from meninges and basal ganglia, and tissue was dissociatedusing the neutral protease dispase and DNase I. Dissociated cells wereseeded on poly-D-lysine-coated 18 � 18-mm glass coverslips (Caro-lina Biological Supply, Burlington, NC) and grown for 2–4 wk inminimal essential medium supplemented with 10% heat-inactivatedhorse serum, 50 U/ml penicillin, and 50 �g/ml streptomycin at 37°Cin a humidified 5% CO2-95% air atmosphere. Culture medium wasreplaced twice weekly. After 10 days of cultivation, penicillin andstreptomycin were removed from the culture medium. Immunocyto-chemistry showed that �95% of the cells stained positively for theastrocytic marker, glial fibrillary acid protein.

Excitatory amino acid release. Release of excitatory amino acidswas measured as previously described (42) using D-[3H]aspartate, anonmetabolized analog of L-glutamate and L-aspartate, which is takenup by glutamate transporters in the same manner as L-glutamate.Astrocytes were loaded overnight with 4 �Ci/ml D-[3H]aspartate (finalconcentration 270 nM) in 2.5 ml of serum-containing minimumessential medium in an atmosphere of 5% CO2-95% room air at 37°C.In double-labeling experiments, cells were loaded with 4 �Ci/mlD-[3H]aspartate and 1 �Ci/ml [14C]taurine (final concentration 9.3�M). Before the start of the efflux measurements, the cells werewashed free of extracellular isotope and serum-containing medium inHEPES-buffered solution. The basal HEPES-buffered medium con-tained (in mM) 122 NaCl, 3.3 KCl, 0.4 MgSO4, 1.3 CaCl2, 1.2KH2PO4, 10 D-glucose, and 25 HEPES. pH was adjusted to 7.4 withNaOH (�15 mM). The coverslips were inserted into a Lucite perfu-sion chamber that had a depression precisely cut in the bottom toaccommodate the coverslip and a Teflon screw top, leaving a spaceabove the cells of �100–150 �m in height. The cells were superfusedat a constant flow rate of 1.2 ml/min in an incubator set at 37°C withHEPES-buffered media. Hyposmotic media were prepared by 5%dilution with H2O (a 5% decrease in medium osmolarity) or by 50mM reduction of [NaCl] (a 30% decrease in medium osmolarity). Theosmolarities of all buffers were checked using a freezing pointosmometer (�Osmomette; Precision Systems, Natick, MA) and weremeasured to be 288 � 2, 273.5 � 2, and 197 � 3 mosM for isosmotic,�5% hyposmotic, and �30% hyposmotic media, respectively. Super-fusate fractions were collected at 1-min intervals. At the end of eachexperiment, the isotope remaining in the cells was extracted with asolution containing 2% sodium dodecyl sulfate plus 8 mM EDTA.Ecoscint scintillation cocktail (4 ml; National Diagnostics, Atlanta,GA) was added, and each fraction was counted for [3H] or [3H]/[14C]in a Packard Tri-Carb 1900TR liquid scintillation analyzer (PackardInstrument, Meriden, CT). Percent fractional isotope release for eachtime point was calculated by dividing the radioactivity released ineach 1-min interval by the radioactivity left in the cells (the sum of allthe radioactive counts in the remaining fractions up to the beginningof the fraction being measured, plus the radioactivity left in the celldigest) using a custom computer program, as previously described (41).

Data analysis. Data are presented as the means � SE of 4–10experiments performed on at least two different astrocyte prepara-tions. Effects of all agonists and inhibitors of intracellular signalingwere always compared with the controls performed on the same dayand on the same culture preparation. In all cases we comparedmaximal amino acid release values measured during the second tothird minute of exposure to hyposmotic medium. The data wereanalyzed by one-way ANOVA followed by the post hoc Newman-Keuls test when multiple comparisons were made. Significance levels

of P � 0.05 were accepted as statistically different. Origin 7.5(OriginLab, Northampton, MA) and Statistica 6.1 (StatSoft, Tulsa,OK) were used for statistical analysis.

Reagents. D-[3H]aspartate (specific activity 16.2 Ci/mmol) and[14C]taurine (specific activity 108.5 mCi/mmol) were purchased fromPerkinElmer Life Sciences (Boston, MA). Dispase (neutral proteasedispase grade II) was obtained from Roche Applied Science (India-napolis, IN). All cell culture reagents were obtained from GIBCO-Invitrogen (Grand Island, NY). Bisindolylmaleimide I (Go-6850),ML-7, KN-62, KN-93, and Ro-32-0432 were obtained from Calbio-chem (San Diego, CA). BAPTA-AM, chelerythrine chloride, triflu-operazine, and other chemicals, unless otherwise specified, were fromSigma (St. Louis, MO).

RESULTS

Characterization of ATP-stimulated D-[3H]aspartate releasepathway. In the present study, moderate cell swelling, inducedby a 5% reduction in medium osmolarity, produced little effecton D-[3H]aspartate release in cultured astrocytes (�20–40%increase above the basal levels; Fig. 1A). When 10 �M ATPwas applied in combination with moderate cell swelling, itcaused a transient 250–450% (variation between different cellcultures) increase in D-[3H]aspartate efflux rate compared withbasal release levels (Fig. 1A). In our previous study (44), wefound that the potentiation of amino acid release by 10 �MATP was close to maximal and not statistically different fromthe effects of 100 �M and 1 mM ATP. Even in those culturesshowing the highest ATP sensitivity, the maximal rate of theATP-induced D-[3H]aspartate release in moderately swollencells was much smaller than the release induced by the sub-stantial hyposmotic swelling (compare the release values inFig. 1B). In substantially swollen cells, 10 �M ATP addition-ally potentiated D-[3H]aspartate release approximately three-fold (Fig. 1B; P � 0.001). In our previous studies (42, 44), wefound that both swelling-activated D-[3H]aspartate release andATP-induced D-[3H]aspartate release were sensitive to thenonselective VRAC blockers DIDS (200 �M) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 �M) and tothe open-pore VRAC blocker extracellular ATP (10 mM). Inaddition, 100 �M phloretin, which at this concentration largelydiscriminates VRACs vs. Ca2�-sensitive and CFTR Cl� chan-nels (14), potently blocked the swelling-activated and theATP-induced D-[3H]aspartate release (42). We have repeatedour previous experiments with NPPB (42) in substantiallyswollen cells (30% reduction in medium osmolarity), in mod-erately swollen cells (5% reduction in medium osmolarity) inthe presence of 10 �M ATP, and in substantially swollen cellsin the presence of 10 �M ATP, and we found 69, 82, and 78%inhibition, respectively (n � 3, all P � 0.01 vs. respectivecontrols; data not shown). To test the amino acid selectivity ofthe transport pathway involved in the ATP-induced amino acidrelease in moderately swollen cells, we measured simulta-neously [14C]taurine and D-[3H]aspartate release (Fig. 1C).ATP increased the release of both isotope-labeled amino acids,with [14C]taurine showing larger stimulation compared withthe basal release levels.

Ca2� and calmodulin dependence of ATP-stimulatedD-[3H]aspartate release. P2Y receptor-mediated cellular re-sponses to ATP are primarily due to the activation of phos-pholipase C (PLC) and subsequent intracellular Ca2� releasefrom inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular

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Ca2� stores (58). Therefore, we tested the Ca2� sensitivity ofthe ATP-mediated D-[3H]aspartate release in both moderatelyand substantially swollen cells. Preloading astrocytes for 20min with the intracellular Ca2� chelator 10 �M BAPTA-AMstrongly suppressed basal D-[3H]aspartate release and inhibitedthe ATP-induced amino acid release in moderately swollenastrocytes by �75% (Fig. 2A; P � 0.001). In substantiallyswollen cells, the ATP-induced increase in D-[3H]aspartaterelease was suppressed by �90% (Fig. 2B). In contrast, thehyposmotic medium-stimulated release itself showed a lowsensitivity (�25% inhibition; P � 0.052) to intracellular Ca2�

chelation (Fig. 2B), which was similar to our previously pub-lished data for [3H]taurine release (41).

We further checked whether the Ca2�-dependent activationand/or modulation of VRACs in cultured astrocytes involves acalmodulin-dependent step. The calmodulin antagonists 20�M trifluoperazine (IC50 � 5.8 �M) and 50 �M chlorproma-zine (IC50 � 17 �M) essentially eliminated the ATP-inducedD-[3H]aspartate release in moderately swollen cells (Fig. 3A).Trifluoperazine (20 �M) also completely suppressed the ATP-induced increase in amino acid release in substantially swollencells (Fig. 3B). As in the case of BAPTA-AM, trifluoperazinewas ineffective in reducing control, swelling-induced D-[3H]as-partate release (Fig. 3B, P � 0.427). These data are verysimilar to our previously published findings for swelling-activated [3H]taurine release (41).

Effects of tyrosine kinase inhibitors on ATP-dependentD-[3H]aspartate release. In cultured astrocytes, hyposmoticmedium-induced cell swelling causes Ca2�-dependent activa-tion of tyrosine kinase cascades (67), and tyrosine kinasesignaling has been implicated in astrocytic VRAC activation ormodulation (7, 10, 17). Therefore, we tested for tyrosine kinaseinvolvement in the ATP-induced potentiation of amino acidrelease. In moderately swollen cells, both the receptor tyrosinekinase inhibitor tyrphostin A51 (20 �M; Fig. 4A) (P � 0.005)and an inhibitor of nonreceptor tyrosine kinases of the Srcfamily, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2; 10 �M) (n � 5, P � 0.023; data not

Fig. 1. A and B: comparison of ATP-induced D-[3H]aspartate release in moderately and substantially swollen cultured astrocytes. A: moderate cell swelling wasinduced by a 5% reduction in medium osmolarity (�14.5 mosM). ATP (10 �M) was applied simultaneously with hyposmotic medium (Hypo). For mediumcomposition, see MATERIALS AND METHODS. Data are means � SE of 7 experiments performed on 3 different cell culture preparations. B: substantial cell swellingwas induced by a 30% reduction in medium osmolarity (�90 mosM). ATP (10 �M) was applied simultaneously with hyposmotic medium. Open squares showATP-induced D-[3H]aspartate release values in moderately swollen cells for comparison. Data are means � SE of 5 experiments performed on 2 cell culturepreparations. When not indicated, SE bars were smaller than symbols. C: simultaneous measurements of ATP-induced D-[3H]aspartate and [14C]taurine releasefrom moderately swollen cultured astrocytes. Astrocytes were preloaded overnight with D-[3H]aspartate and [14C]taurine, perfused for 20 min with isosmoticmedium, and then exposed for 10 min to a 5% reduction in medium osmolarity plus 10 �M ATP. Data are means � SE of 5 experiments.

Fig. 2. ATP-induced organic osmolyte release in astrocytes is dependent onintracellular [Ca2�]. A: effect of intracellular Ca2� chelator BAPTA-AM onATP-induced D-[3H]aspartate release from moderately swollen astrocytes.Cells were preincubated with 10 �M BAPTA-AM for 20 min, followed by a5-min wash to remove extracellular BAPTA-AM. Astrocytes were then ex-posed to a 5% reduction in medium osmolarity plus 10 �M ATP. Data aremeans � SE of 5 experiments performed on 2 cell culture preparations. B:effect of BAPTA-AM on maximal values of ATP-induced D-[3H]aspartaterelease from substantially swollen astrocytes. Cells were pretreated with 10�M BAPTA-AM for 20 min, washed free of extracellular BAPTA-AM for 5min, and then exposed to a 30% reduction in medium osmolarity with orwithout 10 �M ATP. Data are means � SE of 6 experiments performed on 3cell culture preparations. **P � 0.01 vs. hyposmotic control. ##P � 0.01 vs.ATP-induced release.

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shown), reduced the ATP-induced D-[3H]aspartate efflux by50–60%. In substantially swollen cells, tyrphostin A51 did notaffect the ATP-induced increase in D-[3H]aspartate release(Fig. 4B, P � 0.551) or the release under control hyposmoticconditions (Fig. 4B, P � 0.144). Therefore, the ATP-inducedorganic osmolyte release seems differentially sensitive to ty-rosine kinase inhibition depending on the degree of cell swelling.

MLCK does not contribute to the ATP effect on organicosmolyte release. The myosin light chain kinase (MLCK) is acalmodulin-dependent enzyme that contributes to VRAC acti-vation in vascular endothelial cells (50). This enzyme is ex-pressed in astrocytes both in primary culture and in situ (12).Because we had found that the regulation of D-[3H]aspartaterelease by ATP is calmodulin dependent, we tested for thecontribution of MLCK in mediating the ATP effects. Hypos-motic medium-stimulated D-[3H]aspartate efflux was surpris-ingly potentiated by the MLCK inhibitor ML-7 (10 �M)(2-fold potentiation; Fig. 5, P � 0.001). Furthermore, ML-7was completely ineffective when cells were exposed to acombination of hyposmotic shock and ATP (Fig. 5, P �

0.565). Thus these data do not support a critical role for MLCKin ATP signaling.

Effects of CaMK II inhibitors on ATP- and swelling-inducedorganic osmolyte release. Another major Ca2�/calmodulin-sensitive enzyme, one that is expressed in astrocytes and hasbeen reported to regulate VRAC activity, is Ca2�/calmodulin-sensitive kinase II (CaMK II) (4, 5). To test for the CaMK IIinvolvement in the ATP-induced D-[3H]aspartate release, weused the selective inhibitors of this enzyme, KN-62 (IC50 �900 nM) and KN-93 (IC50 � 370 nM), at 5 and 10 �M. Insubstantially swollen cells, both inhibitors, when used at thehigher 10 �M concentration, suppressed D-[3H]aspartate re-lease by �40–50% either under control hyposmotic conditionsor when hyposmotic stress was coapplied with ATP (Fig. 6, Aand B). Although the absolute values of the ATP-inducedD-[3H]aspartate release were reduced by the KN-93 and KN-62compounds, relative values of VRAC activation by ATP re-mained similar in cells treated or not treated with CaMK IIinhibitors. Therefore, CaMK II activation seems insufficient to

Fig. 4. Differential sensitivity of ATP-induced organic osmolyte release to thetyrosine kinase inhibitor tyrphostin A51 in moderately (A) and substantiallyswollen astrocytes (B). A: cultured astrocytes were pretreated with 20 �Mtyrphostin A51 (TP-51) for 15 min before and during exposure to a 5%reduction in medium osmolarity plus 10 �M ATP. Data are means � SE of 5experiments performed on 2 different cell culture preparations. Control valuesof D-[3H]aspartate release from moderately swollen cells are presented in Fig.1A. B: cultured astrocytes were pretreated with 20 �M tyrphostin A51 for 15min before and during exposure to a 30% reduction in medium osmolarity withor without 10 �M ATP. Data are means � SE of 5 experiments performed on2 different cell culture preparations.

Fig. 3. ATP-induced organic osmolyte release is a calmodulin-dependentprocess. A: effect of calmodulin inhibitors trifluoperazine (TFP; 20 �M) andchlorpromazine (CLZ; 50 �M) on ATP-induced D-[3H]aspartate release frommoderately swollen cultured astrocytes. Cells were exposed to TFP and CLZfor 10 min before and during exposure to a 5% reduction in medium osmolarityplus 10 �M ATP. Data are means � SE of 5–7 experiments performed on 3cell culture preparations. **P � 0.01 vs. hyposmotic control. #P � 0.05;##P � 0.01 vs. ATP-induced release. B: effect of 20 �M TFP on ATP-inducedD-[3H]aspartate release from substantially swollen cells. Cells were preincu-bated with TFP for 10 min before and during exposure to a 30% reduction inmedium osmolarity with or without 10 �M ATP. Data are means � SE of 5experiments performed on 2 cell culture preparations. **P � 0.01 vs. hypos-motic control. ##P � 0.01 vs. ATP-induced release.



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explain the full ATP-induced potentiation of VRAC activity.We were unable to use higher concentrations of the KNcompounds because of their limited solubility. In moderatelyswollen cells, 10 �M KN-93 inhibited the ATP-inducedD-[3H]aspartate release by �50% (n � 5, P � 0.008; data notshown), similar to the data obtained in substantially swollencells.

Effects of PKC inhibitors on ATP-dependent modulation ofamino acid release. Astrocytes express a variety of proteinkinase C isoforms, several of which are activated by elevationsin intracellular [Ca2�] ([Ca2�]i) (70). PKC was recently im-plicated in the positive modulation of volume-dependent or-ganic osmolyte release by muscarinic receptors in neuroblas-toma cells (34). We therefore tested for PKC involvement inthe ATP-induced activation of D-[3H]aspartate release by usingseveral specific PKC inhibitors. In moderately swollen cells,both 1 �M bisindolylmaleimide I and 10 �M chelerythrinepotently inhibited the ATP-induced VRAC activation by 70–80% (Figs. 7A and 8A, respectively). In substantially swollencells, bisindolylmaleimide or chelerythrine strongly reducedD-[3H]aspartate release in the ATP-treated cells but showed noeffect (chelerythrine; P � 0.762) or little effect (bisindolylma-leimide I, 27% inhibition; P � 0.037) on the release induced bysubstantial hyposmotic swelling (Figs. 7B and 8B). Overall,these inhibitors strongly reduced (�70% inhibition) the ATP-induced increment in D-[3H]aspartate release, suggesting thatPKC is an important element of the ATP-induced VRACmodulation. In contrast, PKC is not obligatory or plays only aminor role in VRAC activation by hyposmotic swelling alone.Two other potent and selective PKC inhibitors, Ro-32-0432 (1�M) and Go-6983 (1 �M), were less effective against theATP-induced D-[3H]aspartate release in substantially swollencells (30–35% inhibition, n � 4, P � 0.05 for both inhibitors;data not shown) and showed no inhibition under control hy-posmotic conditions (n � 3–4, P 0.4 for both inhibitors; datanot shown).

Additive action of PKC and CaMK II inhibitors on ATP-induced amino acid release. Because the pharmacologicalinhibition of CaMK II or PKC produced strong but incompleteinhibition of the ATP effects on D-[3H]aspartate release, we

tested for the additive actions of these two signaling enzymes.A combination of the most effective PKC inhibitor, bisindolyl-maleimide I (1 �M), and the CaMK II inhibitor KN-93 (10�M) completely blocked the ATP-induced increase inD-[3H]aspartate release from moderately swollen (Fig. 9A; P �0.001) and substantially swollen (Fig. 9B; P � 0.001) astro-cytes. In sharp contrast to the ATP-regulated component,control hyposmotic medium-induced amino acid release wasonly insignificantly affected by the combination of PKC andCaMK II blockers (Fig. 9B; 30% inhibition, P � 0.335).

DISCUSSION

In the present study, we explored intracellular signalingmechanisms involved in the regulation of cell volume-depen-dent astrocytic organic osmolyte release by extracellular ATP.It was recently found that in cultured astrocytes and severalother cell types, receptor agonists stimulating the PLC/[Ca2�]i

signaling pathway, such as ATP, bradykinin, and vasopressin,activate VRAC-like Cl� currents and organic osmolyte release

Fig. 5. Myosin light chain kinase (MLCK) does not contribute to ATP-induced organic osmolyte release from substantially swollen astrocytes. Cellswere pretreated with the MLCK inhibitor ML-7 (10 �M) for 20 min before andduring exposure to a 30% reduction in medium osmolarity with or without 10�M ATP. Data are means � SE of 5–6 experiments performed on 2 cellculture preparations.

Fig. 6. Ca2�/calmodulin-dependent kinase II (CaMK II) modulates volume-dependent organic osmolyte release in cultured astrocytes. A: cells werepretreated with the CaMK II inhibitor KN-93 (10 �M) for 20 min before andduring exposure to a 30% reduction in medium osmolarity with or without 10�M ATP. Data are means � SE of 4–6 experiments performed on 2 differentcell culture preparations. B: summary of effects of the CaMK II inhibitorsKN-93 and KN-62 tested at 5 and 10 �M. Data are means � SE of 4–8experiments performed on 3 different cell culture preparations. Becausecontrol release values varied among cell cultures, all data were normalized tohyposmotic medium controls performed in the same culture. **P � 0.01 vs.hyposmotic control. #P � 0.05; ##P � 0.01 vs. ATP-induced release.



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even in the absence of cell swelling, but they do so to a muchlarger extent in swollen cells (8, 42, 62, 75, 76). Exocrinerelease of endogenous ATP was initially postulated to be anecessary step of VRAC activation (76). However, subsequentstudies have shown that in the majority of cell types, endoge-nous ATP release is not necessary or sufficient for VRACactivation but, instead, positively modulates already activeVRACs (19, 25, 42, 60, 75). We demonstrated in the presentstudy that in cultured astrocytes, the ATP-induced, Ca2�-dependent regulation of organic osmolyte release via a VRAC-like pathway involves at least two Ca2�-dependent intracellu-lar signaling cascades that incorporate PKC and CaMK II. ThisATP-dependent mechanism of VRAC regulation is differentfrom VRAC activation by hyposmotic cell swelling, which islargely independent of intracellular Ca2� increases and cal-modulin.

What transport pathway mediates the ATP-stimulated aminoacid release? This question of what specific transport pathwaysare responsible for ATP-induced amino acid release was ini-tially addressed in our previous study (42), in which we foundthat ATP-induced D-[3H]aspartate release is inhibited by sev-eral structurally unrelated VRAC blockers and by small de-grees of cell shrinkage. However, our present findings ofsubstantial differences in the intracellular signaling mecha-nisms contributing to the regulation of D-[3H]aspartate releaseby hyposmotic cell swelling and by ATP naturally return us tothe possibility that hyposmotic cell swelling and ATP activatedifferent or multiple transport systems. Besides VRACs, whichare activated under hyposmotic conditions and are permeableto excitatory amino acids (3, 23, 28, 63), cultured astrocytesexpress several other transport pathways potentially contribut-ing to excitatory amino acid release. These pathways includeexcitatory amino acid transporters working in a reverse mode,the P2X7 ATP receptor channels, connexin hemichannels, and

Ca2�-dependent glutamate release through an exocytoticmechanism (11, 54, 55, 65, 77).

Both ATP-induced aspartate release and swelling-activatedaspartate release are potently inhibited by several VRACblockers that we have tested, including 100 �M NPPB, 200�M DIDS, 10 mM extracellular ATP, and 100 �M phloretin(42, 44). One previous study found that reversal of amino acidtransporters is insensitive to NPPB and extracellular ATP (64).P2X7 and connexin hemichannels are fully active only at lowextracellular Ca2� and Mg2� concentrations and show little orno sensitivity to typical VRAC blockers (11, 77). Therefore,they are also unlikely to be major contributors to hyposmoticand ATP-regulated D-aspartate release. The Ca2�-dependentvesicular glutamate release is less characterized in terms of itssensitivity to VRAC inhibitors. However, as shown by ourdata, the ATP-activated release pathway in moderately swollencells is permeable to both D-[3H]aspartate and [14C]taurine.Because in glial cells taurine is known to be localized in thecytoplasm and released through a VRAC route (9, 24), thisfinding seems to exclude a substantial contribution of thevesicular release pathway. Furthermore, the ATP-induced D-aspartate release is strongly potentiated by cell swelling andcompletely suppressed by cell shrinkage (42).

On the basis of the foregoing studies, we concluded thatunder our experimental conditions, ATP-induced excitatoryamino acid release in cultured astrocytes occurs predominantlyvia a VRAC-like channel, and we therefore use the termVRAC throughout this article. We cannot exclude the possi-bility, however, that other transport systems may provide aminor contribution to D-[3H]aspartate fluxes measured in ourexperiments or that more than one VRAC-like permeabilitypathway may exist in cultured astrocytes (26, 47).

Different roles for [Ca2�]i /calmodulin signaling in VRACregulation by ATP and cell swelling. Our data suggest that incultured astrocytes, VRAC regulation by ATP requires in-

Fig. 7. Effect of the protein kinase C (PKC) inhibitor bisindolylmaleimide I(BIM) on ATP-induced organic osmolyte release in moderately (A) andsubstantially swollen astrocytes (B). A: cells were pretreated with 1 �M BIMfor 20 min before and during exposure to a 5% reduction in medium osmolarityplus 10 �M ATP. Data are means � SE of 5 experiments performed on 2different cell culture preparations. **P � 0.01 vs. hyposmotic control. #P �0.05 vs. ATP-induced release. B: cells were pretreated with 1 �M BIM for 20min before and during exposure to a 30% reduction in medium osmolarity withor without 10 �M ATP. Data are means � SE of 5 experiments performed on2 different cell culture preparations. *P � 0.05; **P � 0.01 vs. hyposmoticcontrol. ##P � 0.05 vs. ATP-induced release.

Fig. 8. Effect of the PKC inhibitor chelerythrine on ATP-induced organicosmolyte release in moderately (A) and substantially swollen astrocytes (B). A:cells were pretreated with 10 �M chelerythrine (Chel.) for 20 min before andduring exposure to a 5% reduction in medium osmolarity plus 10 �M ATP.Data are means � SE of 4 experiments performed on 1 cell culture preparation.**P � 0.01 vs. basal release. #P � 0.05 vs. ATP-induced release. ND, notdone. B: cells were treated with 10 �M chelerythrine for 20 min before andduring exposure to a 30% reduction in medium osmolarity with or without 10�M ATP. Data are means � SE of 4 experiments performed on 1 cell culturepreparation. **P � 0.01 vs. hyposmotic control. ##P � 0.01 vs. ATP-inducedrelease.



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creases in [Ca2�]i and calmodulin. This is in contrast to VRACactivation by hyposmotic cell swelling, which is essentiallyCa2� and calmodulin independent. In hyposmotically swollenastrocytes, D-[3H]aspartate release was only modestly (�25%)inhibited by the calmodulin inhibitor trifluoperazine or bychelation of intracellular Ca2� with BAPTA-AM. In contrast,the same pharmacological agents nearly completely suppressedthe ATP-induced increases in D-[3H]aspartate release in bothmoderately and substantially swollen cells (see Figs. 2 and 3).These data are in line with previous findings of van der Wijket al. (75) on the Ca2�-dependent modulation of swelling-activated 125I� fluxes in human intestine 407 cells. In culturedcerebellar astrocytes, calmodulin antagonists and BAPTA-AMhave shown little effect on hyposmotic medium-induced[3H]taurine release but completely inhibited the positive mod-ulation of such release by the calcium ionophore ionomycin (5,48). A small component of swelling-activated organic os-molyte release, which is sensitive to Ca2� chelators and cal-modulin inhibitors, observed by us and others (8, 42, 75), maybe due to autocrine ATP release and subsequent VRAC mod-ulation.

In contrast to the Ca2�-independent VRAC activation byhyposmotic cell swelling observed in this study, Li et al. (33)

found a complete dependency of swelling-induced Cl� andtaurine currents on calmodulin and intracellular Ca2� (33) andstrong inhibition of Cl� currents by an intracellular applicationof anti-calmodulin antibodies (53). However, they used highconcentrations of calmodulin inhibitors (100 �M trifluopera-zine and 300 �M W-7), which we could not test in ourexperiments because of their pronounced nonspecific effects,and a combination of 1 mM extracellular EGTA and 20 mMintracellular BAPTA, which likely reduces [Ca2�]i below thepermissive levels required for VRAC functioning (66, 73).

ATP-dependent VRAC regulation involves several Ca2�-dependent protein kinase cascades. Several Ca2� and calmod-ulin-sensitive signaling enzymes have been reported to con-tribute to VRAC activation and/or modulation, three of which(MLCK, CaMK II, and PKC) are highly expressed in astro-cytes (4, 12, 70). MLCK contributes to the swelling-inducedVRAC activation in endothelial cells (50). However, in cul-tured astrocytes (data from the present work) and in NIH/3T3mouse fibroblasts (56), the MLCK inhibitor ML-7 potentiated(rather than inhibited) organic osmolyte release in hyposmoti-cally swollen cells and had no effect on the release in substan-tially swollen cells treated with ATP. Therefore, a positivemodulation of VRAC by the MLCK appears to be cell-type

Fig. 9. Additive action of the PKC inhibitor BIM and the CaMK II inhibitorKN-93 on ATP-induced organic osmolyte release from substantially (A) ormoderately swollen cultured astrocytes (B). A: cells were treated with acombination of 1 �M BIM and 10 �M KN-93 (BIM/KN) for 20 min beforeand during exposure to a 30% reduction of medium osmolarity with or without10 �M ATP. Data are means � SE of 5 experiments performed on 2 differentcell culture preparations. B: cells were treated with BIM/KN for 20 min beforeand during exposure to a 5% reduction of medium osmolarity plus 10 �MATP. Data are means � SE of 4 experiments performed on 2 different cellculture preparations.

Fig. 10. Hypothetical scheme of the intracellular signaling pathways contrib-uting to ATP-induced and volume-dependent activation of the putative vol-ume-regulated anion channel (VRAC) pathway based on data presented in thisstudy and in our preceding report (42). ATP and cell swelling regulate VRACsvia 2 separate mechanisms. VRAC regulation by ATP is dependent on cellswelling, since ATP is incapable of fully activating VRACs in nonswollen ormoderately swollen cells to the levels seen in cells exposed to a substantialhyposmotic swelling. 1) ATP activates P2Y receptors and, via a phospholipaseC (PLC)/inositol 1,4,5-trisphosphate signaling pathway, releases Ca2� fromintracellular stores. The increased intracellular Ca2� then activates PKC andcalmodulin (CM) as parallel pathways. Calmodulin, in turn, activates Ca2�/calmodulin-dependent kinase II (CaMK II). PKC and CaMK II cooperativelymodulate VRAC activity, directly or via associated regulatory protein(s)represented as “X”. 2) Cell swelling activates VRAC via a separate Ca2�/calmodulin-independent signaling pathway incorporating a hypothetical vol-ume sensor and regulatory protein(s) represented as “Y”. 3) Tyrosine kinases(TKs) are also activated by elevated intracellular Ca2� (67), but they modulateVRACs in moderately swollen cells only. Unlike PKC and CaMK II, TKs mayregulate the volume sensitivity of the cell volume sensor rather than the VRACitself (10). In substantially swollen cells, the volume signal becomes “satu-rated.” For further details, see DISCUSSION. Dashed lines represent signalingsteps that were not pharmacologically tested or identified in this study.



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specific and not relevant to the ATP-stimulated organic os-molyte release in astrocytes.

The second candidate enzyme, CaMK II, likely modulatesVRAC activity in astrocytes. In our experiments, the CaMK IIinhibitors KN-62 (10 �M) and KN-93 (10 �M) attenuated theVRAC-mediated D-[3H]aspartate release by �40–50% underall conditions tested. Although the absolute values of theATP-induced D-[3H]aspartate release were reduced, the rela-tive degree of VRAC stimulation by ATP remained similar inthe cells treated or untreated with the CaMK II inhibitors.Therefore, CaMK II does not appear to be crucial for theATP-dependent VRAC modulation. In contrast to our data,Cardin et al. (5) found that in cultured cerebellar astrocytes, 10�M KN-93 completely inhibits the Ca2�-dependent upregula-tion of [3H]taurine release by ionomycin but does not affectcontrol hyposmotic medium-stimulated amino acid release (5),pointing to a pivotal role for the CaMK II in VRAC modulationin ionomycin-treated cells.

Two PKC inhibitors, chelerythrine and bisindolylmaleimideI, potently suppressed the ATP-induced D-[3H]aspartate releasein our experiments while minimally affecting D-[3H]aspartaterelease induced by substantial hyposmotic swelling. Thus PKCappears to be a critical element of the ATP-induced VRACregulation in astrocytes but does not contribute to VRACactivation by hyposmotic swelling. However, PKC involve-ment is not consistent with our data presented in Fig. 3,showing strong calmodulin dependence of the ATP effect,because Ca2�-sensitive members of the PKC family are di-rectly regulated by [Ca2�]i and do not require calmodulinactivation. This contradiction may be explained by a calmod-ulin-dependent regulation of PLC, an upstream enzyme of thePKC signaling cascade (37, 68). Similar Ca2�- and PKC-dependent modulation of anion channel-mediated organic os-molyte efflux has been found in neuroblastoma cells (34). PKCcontributes to VRAC activation or positive modulation in some(34, 39, 59, 61), but not all (40, 79), cell types.

Although PKC inhibitors strongly attenuated the ATP-in-duced D-[3H]aspartate release, inhibition was incomplete. Wetherefore tested for the additive action of PKC and CaMK II. Acombination of the inhibitors of both enzymes caused completesuppression of the ATP-induced VRAC regulation in moder-ately and substantially swollen cells. This finding implies thatboth PKC and CaMK II contribute to the ATP effects, withPKC playing the dominant role. Cooperative action of PKCand CaMK II has been found to be critical for the regulation ofdelayed rectifier potassium channels by angiotensin II inCATH.a cells (72), activation of phospholipase D by musca-rinic acetylcholine receptors in a heterologous expression sys-tem (38), and ATP-dependent activation of ERK-1/2 in smoothmuscle cells (16).

Tyrosine kinases differently regulate VRAC activity depend-ing on degree of cell swelling. Both the PKC and the CaMK IIinhibitors, when applied in combination or alone, blocked theATP-induced VRAC activation in astrocytes independently ofthe degree of cell swelling. In contrast, tyrosine kinase inhib-itors showed strong inhibition of the ATP-induced D-[3H]as-partate release in moderately swollen cells but were ineffectivein substantially swollen cells with or without ATP. Thustyrosine kinase actions are limited to smaller degrees of cellswelling and are not essential for VRAC activation in substan-tially swollen cells. A similar trend for stronger inhibition of

the volume-dependent organic osmolyte fluxes by tyrosinekinase blockers in “less swollen” compared with “more swol-len” cells has been reported in epithelial cells, isolated brainsupraoptic nuclei, and cultured cerebellar astrocytes (5, 10, 74).One suggested explanation of this phenomenon is that tyrosinekinases modulate volume sensitivity of the hypothetical vol-ume sensor rather than directly regulate the VRAC function(10). “Saturation” of the volume signal in substantially swollencells may override the modulatory contribution of tyrosinekinases (74). Because combined inhibition of PKC and CaMKII essentially abolishes the ATP-induced amino acid release inmoderately swollen cells, tyrosine kinase signaling may workin the same signaling cascade, upstream or downstream ofPKC/CaMK II.

In summary, on the basis of the present data and ourprevious findings (42), we suggest that extracellular ATPregulates organic osmolyte release via a VRAC-like transportpathway through activation of P2Y receptors and at least twoCa2�/calmodulin-dependent intracellular signaling cascadesthat incorporate PKC and Ca2�/calmodulin-dependent kinaseII (Fig. 10). In contrast, substantial hyposmotic cell swellingactivates VRACs through a separate Ca2�/calmodulin-inde-pendent signaling cascade that was not identified in this study.The ATP-dependent positive modulation of VRACs likelyaccelerates the regulatory volume decrease process in substan-tially swollen cells and may induce functionally significantVRAC activity in nonswollen and moderately swollen cells. Inthe brain, the ATP-induced release of excitatory amino acidsvia a VRAC-like permeability pathway from nonswollen ormoderately swollen astrocytes may contribute to intercellularglutamate signaling.

ACKNOWLEDGMENTS

We thank Renee E. Haskew-Layton for critical reading of and helpfulcomments on the manuscript.

GRANTS

This work was supported in part by National Institutes of Health Grants R01NS-35205 (to H. K. Kimelberg) and F05 TW-05329 (to A. A. Mongin).

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ATP regulates anion-channel mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms