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Originally published In Press as doi:10.1074/jbc.M409803200 on November 29, 2004

J. Biol. Chem., Vol. 280, Issue 5, 3548-3554, February 4, 2005
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Hydrogen Peroxide Potentiates Volume-sensitive Excitatory Amino Acid Release via a Mechanism Involving Ca2+/Calmodulin-dependent Protein Kinase II*{boxs}

Renée E. Haskew-Layton{ddagger}, Alexander A. Mongin{ddagger}§, and Harold K. Kimelberg¶

From the {ddagger}Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208 and Ordway Research Institute, Center for Medical Science, Albany, New York 12208

Received for publication, August 26, 2004 , and in revised form, November 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Excessive excitatory amino acid (EAA) release in cerebral ischemia is a major mechanism responsible for neuronal damage and death. A substantial fraction of ischemic EAA release occurs via volume-regulated anion channels (VRACs). Hydrogen peroxide (H2O2), which is abundantly produced during ischemia and reperfusion, activates a number of protein kinases critical for VRAC functioning and has recently been reported to activate VRACs. In the present study, we explored the effects of H2O2 on volume-dependent EAA release in cultured astrocytes, measured as the release of preloaded D-[3H]aspartate. 100–1,000 µM H2O2 enhanced swelling-induced EAA release by ~2.5–3-fold (EC50 {approx} 10 µM). The VRAC blockers ATP, phloretin, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) potently inhibited both control swelling-induced and the H2O2-potentiated release, suggesting a role for VRACs. The H2O2-induced component of EAA release was attenuated by the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) and completely eliminated by the calmodulin antagonists trifluoperazine and W-7 and the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93. Inhibitors of tyrosine kinases, protein kinase C, and the myosin light chain kinase were ineffective in blocking the H2O2 response. H2O2 treatment of swollen astrocytes, but not swelling alone, resulted in CaMKII activation that was inhibited by KN-93, as determined by a phospho-Thr286 CaMKII antibody. These data demonstrate that H2O2 strongly up-regulates astrocytic volume-sensitive EAA release via a CaMKII-dependent mechanism and in this way may potently promote pathological EAA release and brain damage in ischemia.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The pathological release of the excitatory amino acids (EAAs)1 glutamate and aspartate, followed by the subsequent activation of glutamate receptors, is considered an early obligatory event in the excitotoxicity cascade that leads to neuronal damage and death in cerebral ischemia (1, 2). Volume-regulated anion channels (VRACs) are thought to constitute a major pathway for EAA release, as suggested by the inhibition of pathological glutamate and aspartate release by several VRAC blockers in animal models of global and focal cerebral ischemia (3–6). VRACs are ubiquitously expressed, activated in response to cell swelling, and characterized by Eisenman type I anion selectivity (I– > NO –3 > Br– > Cl– > F–), moderate outward rectification, and inactivation at positive potentials (7–9). These anion channels are also permeable to a variety of small organic osmolytes including amino acids, polyols, and methylamines (7, 10, 11). Although the molecular identity of VRACs and the precise mechanisms of their regulation are not known, VRAC activation is probably provoked by pronounced pathological cell swelling, primarily seen in astrocytes in ischemia (12–14).

It is not known whether, in addition to cell swelling, other pathological factors play a role in VRAC-mediated EAA release. Several recent publications have demonstrated that hydrogen peroxide (H2O2) induces typical VRAC currents in the absence of cell swelling in HeLa and hepatoma cell lines (15, 16) and positively regulates swelling-activated organic osmolyte release in NIH3T3 fibroblasts (17). Because H2O2 and other reactive oxygen species are abundantly produced in ischemia and during reperfusion (18, 19), it is plausible that H2O2 causes the activation or positive modulation of excitatory amino acid release in the ischemic brain. In line with such a suggestion, reactive oxygen species positively modulate the activity of a number of protein kinases, such as tyrosine kinases, mitogen-activated protein kinases (MAPKs), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) (20–22). All of these kinases have been found to contribute to VRAC activation and/or modulation in a variety of cell types (9, 23–28). Robust changes in tyrosine phosphorylation and MAPK activity, as well as the translocation of PKC and CaMKII from the cytosol to the membrane fraction, have been found in ischemia (29–32). In the present work, we examined the potential role of H2O2 in mediating the activation or modulation of excitatory amino acid release via a putative VRAC pathway and explored intracellular signaling mechanisms involved in the H2O2 effects in primary astrocyte cultures.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—The neutral protease dispase was obtained from Roche Diagnostics, and all other cell culture reagents were from Invitrogen. D-[3H]aspartate was purchased from PerkinElmer Life and Analytical Sciences and Na2[51Cr]O4 from Amersham Biosciences. Hydrogen peroxide, ATP disodium salt, phloretin, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and trifluoperazine were purchased from Sigma-Aldrich. PP2, U0126, W-7, and W-5 were obtained from Tocris (Ellisville, MO). SU6656, tyrphostin A51, chelerythrine, Go6983, bisindolylmaleimide I, Ro32-0432, BAPTA-AM, ML-7, and KN-93 were purchased from Calbiochem.

Cell Culture—Confluent primary astrocyte cultures were prepared according to the method of Frangakis and Kimelberg (33) with minor modifications as described elsewhere (34). In brief, the cerebral cortices of neonatal Sprague-Dawley rats were separated from the basal ganglia, followed by the removal of the meninges. Tissue was dissociated using the neutral protease Dispase. The cells were then plated on poly-D-lysine-coated glass coverslips or Petri dishes and grown for 3–4 weeks in minimal essential medium supplemented with 10% heat-inactivated horse serum and 50 units/ml penicillin plus 50 µg/ml streptomycin in a humidified 5% CO2/95% air atmosphere at 37 °C. Culture medium was replaced biweekly, and the penicillin and streptomycin were removed from the medium after 10 days of cultivation.

Excitatory Amino Acid and [51Cr] Release—Confluent astrocyte cultures grown on 18 x 18 mm glass coverslips were loaded overnight with D-[3H]aspartate (4 µCi/ml; final concentration, 270 nM) or Na2[51Cr]O4 (16 µCi/ml; final concentration, 340 nM) in minimal essential medium supplemented with 10% heat-inactivated horse serum. D-[3H]aspartate is a non-metabolized analogue of L-glutamate and L-aspartate, which is taken up by glutamate transporters in the same manner as L-glutamate. Before the start of each experiment, extracellular isotope was washed out with a basal HEPES-buffered medium (122 mM NaCl, 3.3 mM KCl, 0.4 mM MgSO4, 1.3 mM CaCl2, 1.2 mM KH2PO4, 10 mM D-glucose, and 25 mM HEPES, pH 7.4, 285 mOsM). The coverslips were then placed into a custom-made Lucite perfusion chamber, maintained in a 37 °C incubator throughout the experiments, and superfused at a flow rate of 1.2 ml/min with the basal HEPES-buffered medium or a hypo-osmotic HEPES-buffered medium (72 mM NaCl, 3.3 mM KCl, 0.4 mM MgSO4, 1.3 mM CaCl2, 1.2 mM KH2PO4, 10 mM D-glucose, and 25 mM HEPES, pH 7.4, 195 mOsM). Media osmolarities were checked using a freezing point µOsmette osmometer (Precision Systems, Natick, MA). The superfusate samples were collected each minute, and at the end of each experiment, the remaining intracellular isotope was extracted with 2% SDS + 8 mM EDTA. The radioactivity of each sample was counted for 3Hor 51Cr by a Tri-Carb 2900TR liquid scintillation counter (PerkinElmer Life and Analytical Sciences). Isotope release levels are presented as fractional release values for each time point, calculated by dividing the radioactivity released at each minute interval by the radioactivity remaining in the cells at that time point (includes isotope levels from cell lysis plus isotope levels in the remaining fractions).

Cell Volume Measurements—Relative cell volume changes were measured using the cell-permeable fluorescent probe calcein. Changes in the calcein signal positively correlate with changes in cell volume (35). Cells were loaded with 1 µM calcein-AM (Molecular Probes, Eugene, OR) for 15 min at room temperature. After washout of extracellular calcein, astrocytes plated on round coverslips were transferred into a perfusion chamber (Warner Instruments, Hamden, CT), and perfused at ~1 ml/min throughout the experiment with HEPES-buffered basal or hypo-osmotic media (see above for composition). All experiments were performed at room temperature. The changes in fluorescence intensity of a field of confluent cells were monitored with a 25x objective using a Zeiss LSM 510 confocal microscope (Zeiss, Heidelberg, Germany) at 488-nm excitation and long pass 505-nm emission. Images were scanned every 30 s. Relative changes in fluorescence intensity were calculated using Zeiss image analysis software.

Intracellular Calcium Measurements—Qualitative changes in [Ca2+]i levels were determined using the cell-permeable fluorescent [Ca2+]i indicator fluo-4-AM (Molecular Probes, Eugene, OR). Confluent cultured astrocytes, plated on round coverslips, were loaded for 1 h with 5 µM fluo-4-AM at room temperature. After the washout of extracellular fluo-4, the coverslips were placed in a perfusion chamber (Warner Instruments, Hamden, CT) and perfused with HEPES-buffered basal or hypo-osmotic medium (for media composition see above). The experiments were all performed at room temperature. The fluorescent images were scanned every 5 s with a 25x objective using a Zeiss LSM 510 confocal microscope (Zeiss, Heidelberg, Germany) at an excitation wavelength of 488 nm and a long pass 505 nm emission. The relative changes in fluo-4 intensity for a field of confluent cells were calculated using Zeiss image analysis software.

Western Blot Analysis—Autophosphorylated active CaMKII levels and total CaMKII expression were assessed by Western blot analysis using a phospho-Thr286 CaMKII antibody (Promega, Madison, WI) and a pan CaMKII antibody (gift of Dr. Harold A. Singer, Albany Medical College, Albany, NY), respectively. Confluent astrocyte cultures were subjected to various treatment conditions as specified under "Results" and subsequently lysed with 2% SDS plus 8 mM EDTA. The total protein content of the lysates was determined using a colorimetric BCA assay kit (Pierce). Whole cell lysates were diluted with a standard reducing buffer and separated on a 10% polyacrylamide gel followed by transfer to an Immobilon-P membrane (Millipore, Bedford, MA). The transfer membranes were blocked overnight with 10% nonfat milk in a Tris-buffered solution containing 0.05% Tween 20. Membranes were then incubated for 2 h at room temperature with either a phospho-Thr286 CaMKII polyclonal antibody (1:5,000 dilution) or a pan CaMKII polyclonal antibody (1:2,000 dilution). After five washes with Tris-buffered solution containing 0.05% Tween 20, membranes were incubated with horseradish peroxidase-conjugated secondary rabbit antibodies (1:10,000; Amersham Biosciences). The horseradish peroxidase signal was detected by autoradiography using a chemiluminescence ECL reagent (Amersham Biosciences) and SuperRX x-ray film (Fuji, Tokyo, Japan). The optical densities of the immunoreactive bands were analyzed using TotalLab Control Centre software (Nonlinear Dynamics, Durham, NC).

Statistical Analysis—The statistical significance of the data was determined with one-way analyses of variance, followed by post-hoc Newman-Keuls tests for multiple comparisons. Repeated-measures analyses of variance were also used where indicated. Origin 7.5 (OriginLab Corp, Northampton, MA) and Statistica 6.1 (StatSoft, Tulsa, OH) were used for statistical analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Hydrogen Peroxide Potentiates Swelling-sensitive EAA Release—We used D-[3H]aspartate as a tracer for EAA because it is not metabolized and is released through the same swelling-induced permeability pathways as L-[3H]glutamate (36). In astrocyte cultures, cell swelling induced by a 30% reduction in medium osmolarity (–90 mOsM) initiated a large release of preloaded D-[3H]aspartate (Fig. 1A). H2O2, given 10 min before and continuously during hypo-osmotic medium application, potentiated peak swelling-induced EAA release in a dose-dependent manner (Fig. 1B). The H2O2-effect was saturated at concentrations ≥100 µM (EC50 = 8.2 ± 4.0 µM, as determined by a dose-response curve fit, R2 = 0.98). 500 µM H2O2 was used throughout the study to ensure maximal activity. 100–1,000 µM H2O2 enhanced swelling-induced EAA release by ~2.5–3-fold (Fig. 1, A and B). In the absence of cell swelling, 500 µM H2O2 failed to initiate any rapid increases in EAA release, although it did induce a gradual upward shift in D-[3H]aspartate baseline values (Fig. 1A). After removal of H2O2, we typically observed a secondary increase in D-[3H]aspartate release compared with control baseline release, the degree of which varied between cell culture preparations. Because this secondary release was observed only after the removal of H2O2, we did not investigate its origin. To verify that the H2O2-induced potentiation of EAA release was not caused by additional cell swelling, we monitored relative cell volume changes using calcein fluorescence (35). Exposure to hypo-osmotic medium produced a ~4.5% increase in calcein fluorescence (Fig. 1C), which positively correlates to changes in cell volume (35). 500 µM H2O2 produced no additional changes in calcein fluorescence before and during hypo-osmotic medium exposure (Fig. 1C).



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  		FIG. 1.
Hydrogen peroxide (H2O2) augments volume-sensitive excitatory amino acid release in cultured astrocytes. A, the effect of 500 µM H2O2 on volume-sensitive D-[3H]aspartate release. Cultured astrocytes were exposed for 10 min to hypo-osmotic medium (–90 mOsM) ({circ}, n = 9). 500 µM H2O2 was given 10 min before and during hypo-osmotic medium perfusion (•, n = 9). The results are compared with 20-min exposure to H2O2 in the absence of cell swelling ({triangledown}, n = 5). The data represent the means ± S.E. ***, p < 0.001, H2O2 versus all other groups. B, the dose-dependent the effect of H2O2 on volume-sensitive D-[3H]aspartate release. Cultured astrocytes were exposed for 10 min to hypo-osmotic medium (–90 mOsM) to induce cell swelling. H2O2 was given 10 min before and during hypo-osmotic medium perfusion. The data are the means ± S.E. of four to six experiments, representing peak swelling-induced D-[3H]aspartate release in the presence of 1–1,000 µM H2O2. C, H2O2 does not induce additional cell swelling. Calcein-loaded cultured astrocytes were exposed to hypo-osmotic medium for 10 min in the absence ({circ}, n = 7) or presence (•, n = 6) of 500 µM H2O2. The data represent the means ± S.E.

 
Hydrogen Peroxide-induced EAA Release Is Mediated by Volume-regulated Anion Channels—To confirm that the H2O2-induced enhancement of EAA release was not caused by cell lysis or detachment, we monitored the efflux of preloaded Na2[51Cr]O4. Radiolabeled chromate is taken inside cells where it binds to intracellular components, and its appearance in the superfusate corresponds mostly to cell lysis or detachment (37). Exposure to 500 µM H2O2 produced negligible increases in [51Cr] efflux levels compared with the robust release of D-[3H]aspartate, thus ruling out cell lysis or toxicity as a reason for H2O2-induced EAA release (Fig. 2A). 20-min exposure to 500 µM H2O2 produced no changes in astrocyte morphology (Supplemental Figure). To establish that the H2O2 enhancement of hypo-osmotic EAA release is mediated by VRACs, we used several VRAC blockers and compared their effects on EAA release in the presence or absence of H2O2. 10 mM ATP (which at this concentration acts as an open pore VRAC blocker (8)), 100 µM phloretin (which inhibits VRACs but not cystic fibrosis transmembrane conductance regulator channel or Ca2+-activated Cl– channels at the concentration used (38)), and the broad spectrum anion channel blocker 100 µM NPPB (8) all potently inhibited hypo-osmotic medium-induced EAA release both during control astrocyte swelling and in the presence of 500 µM H2O2 (Fig. 2B).



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  		FIG. 2.
The H2O2 effect on swelling-activated excitatory amino acid release is mediated by volume-regulated anion channels. A, H2O2 does not induce nonspecific 51Cr efflux levels from swollen astrocytes. Astrocytes were preloaded overnight with either Na2[51Cr]O4 ({circ}, n = 4) or D-[3H]aspartate (•, n = 3), and then treated with 500 µM H2O2 10 min before and during hypo-osmotic medium (–90 mOsM) exposure. Data represent the means ± S.E. B, the effect of anion channel blockers on peak swelling-induced D-[3H]aspartate release values in the presence and absence of H2O2. 500 µM H2O2 was given 10 min before and during 10-min hypo-osmotic medium (–90 mOsM) exposure. The anion channel blockers 10 mM ATP, 100 µM phloretin, or 100 µM NPPB were present during hypo-osmotic medium exposure in the absence or presence of H2O2. The data represent the means ± S.E. of the peak swelling-induced release values, in three to six experiments for each group. ***, p < 0.001 versus hypo-osmotic control; ###, p < 0.001 versus H2O2.

 
Intracellular Signaling Mechanisms Involved in the H2O2 Effect on EAA Release—H2O2 activates non-receptor tyrosine kinases of the src family, receptor tyrosine kinases, and, down-stream, enzymes of the MAPK cascade (22, 39), all of which have been found to contribute to VRAC activation (23–25). Therefore, we tested for the contribution of tyrosine and MAP kinases in mediating the H2O2 effect on swelling-induced EAA release. The selective src kinase inhibitors PP2 (10 µM) or SU6656 (5 µM) (40), the receptor tyrosine kinase inhibitor tyrphostin A51 (20 µM) (41), or the MEK inhibitor U0126 (10 µM) (42), did not block the H2O2-induced potentiation of EAA release (Fig. 3A).



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  		FIG. 3.
Tyrosine kinases, MEK, and protein kinase C are not responsible for the H2O2 effect on excitatory amino acid release. A, the effects of the src inhibitors PP2 (10 µM), and SU6656 (5 µM), the receptor tyrosine kinase inhibitor tyrphostin A51 (20 µM; TP51), and the MEK inhibitor U0126 (10 µM) on the H2O2-induced modulation of EAA release. The experimental design was similar to that presented in Fig. 2A. PP2 and SU6656 were given 10 min before and during exposure to 500 µM H2O2. Tyrphostin A51 and U0126 were present during exposure to H2O2. Both the control H2O2 data (black bars) and the inhibitor data (open bars) are presented as the means ± S.E. of the maximal swelling-induced release values for four experiments in each group, normalized to average H2O2 control values performed on the same days. B, the effect of the protein kinase C inhibitors chelerythrine (5 µM), Go6983 (1 µM), bisindolylmaleimide I (1 µM; BIM), and Ro32-0432 (5 µM) on H2O2-induced D-[3H]aspartate release from swollen astrocytes. The experimental design was similar to that presented in Fig. 2A. The PKC inhibitors were present during the 500 µM H2O2 exposure. Both the control H2O2 data (black bars) and the inhibitor data (open bars) are presented as the means ± S.E. of the maximal swelling-induced release values for four to six experiments per group, normalized to average H2O2 control values performed on the same days. ***, p < 0.001 versus H2O2.

 
We also examined the potential role of PKC because this enzyme is also activated by H2O2 (43, 44) and has been shown to regulate VRAC activity (26, 45). The potent PKC inhibitors 1 µM Go6983, 1 µM bisindolylmaleimide I, and 5 µM Ro32-0432 (46–48) were ineffective in blocking the H2O2-enhanced EAA release during cell swelling (Fig. 3B). In contrast, the PKC inhibitor 5 µM chelerythrine completely blocked the H2O2-evoked enhancement in EAA release (Fig. 3B).

Two Ca2+/calmodulin-dependent enzymes known to be activated by H2O2, the myosin light chain kinase (MLCK), and CaMKII, have also been found to regulate VRACs (20, 27, 49, 50). We therefore explored whether changes in intracellular Ca2+ and the activation of Ca2+/calmodulin-dependent enzymes are involved in the H2O2 effect. Using the fluorescent Ca2+ indicator fluo-4-AM, we found that H2O2 pretreatment produces a steady rise in [Ca2+]i levels before the induction of cell swelling (Fig. 4, A and B). Hypo-osmotic medium-induced cell swelling itself caused a steep increase in [Ca2+]i followed by a gradual recovery toward baseline values (Fig. 4B). In the presence of 500 µM H2O2, swelling-induced [Ca2+]i release on average tended to be somewhat higher, but this effect was not statistically significant (Fig. 4B).



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  		FIG. 4.
H2O2 modulates swelling-induced EAA release via a Ca2+/calmodulin-dependent mechanism. A and B, the effect of H2O2 on intracellular calcium levels measured with the [Ca2+]i indicator fluo-4-AM. Astrocytes were perfused with basal and hypo-osmotic medium in the presence or absence of 500 µM H2O2. Representative images in A show changes in fluo-4 fluorescence in response to H2O2 and hypo-osmotic medium. B represents the average values ± S.E. of fluo-4 fluorescence intensities of four experiments in each group (error bars are shown only at 1-min intervals for clarity). All data are normalized to fluorescence intensities in the beginning of each experiment. *, p < 0.05 for the entire 5–15-min time interval as determined by repeated measures analysis of variance. C, the effect of Ca2+ chelation with BAPTA-AM on H2O2-induced D-[3H]aspartate release. Cultured astrocytes, not treated ({circ}, n = 6) or pretreated with 10 µM BAPTA-AM ({triangledown}, n = 6) (20-min pretreatment, followed by 10-min washout) were exposed for 10 min to hypo-osmotic medium. In corresponding experiments, cells were exposed to 500 µM H2O2, without (•, n = 7) or with 10 µM BAPTA-AM pretreatment ({blacktriangledown}, n = 7). Data represent the mean values ± S.E. ***, p < 0.001 H2O2 versus control; #, p = 0.05 H2O2 versus H2O2 + BAPTA-AM. D, the effect of the calmodulin inhibitors 20 µM trifluoperazine (TFP) and 20 µM W-7 on H2O2-potentiated D-[3H]aspartate release. 20 µM W-5 was used as a negative control for W-7. Astrocytes were treated with the calmodulin inhibitors, or W-5, for 10 min before and during 10-min exposure to hypo-osmotic medium (–90 mOsM) alone (left) or in the presence of 500 µM H2O2 given 10 min before and during cell swelling (right). The data represent the means ± S.E. of peak swelling-induced release values for four to seven experiments for each group. ***, p < 0.001 versus H2O2.

 
To test for Ca2+ involvement in the H2O2 effect on EAA release, we pretreated cultured astrocytes for 20 min with the cell membrane-permeable Ca2+ chelator 10 µM BAPTA-AM, followed by a 10-min wash out. BAPTA-AM pretreatment partially reduced the H2O2-induced increment in EAA release but had no statistically significant effect on control swelling-induced EAA release (Fig. 4C). The calmodulin antagonists trifluoperazine (20 µM) and W-7 (20 µM) (51, 52) completely eliminated the H2O2-induced potentiation of swelling-induced D-[3H]aspartate release (Fig. 4D). On the other hand, the inactive structural analogue of W-7, W-5 (20 µM), was ineffective in reducing the H2O2 effect (Fig. 4D). In contrast to their effects in the presence of H2O2, trifluoperazine and W-7 did not reduce EAA release under control hypo-osmotic conditions (Fig. 4D).

To determine the specific Ca2+/calmodulin-dependent enzymes involved in the H2O2 effect, we used inhibitors of MLCK (ML-7 (40, 53)), and CaMKII (KN-93 (54)). Addition of 5 µM ML-7 during 500 µM H2O2 perfusion had no effect on the ability of H2O2 to enhance swelling-sensitive D-[3H]aspartate release (n = 4 per group, p = 0.243; data not shown). In contrast, the application of the CaMKII inhibitor 5 µM KN-93 completely abolished the effect of H2O2 but had no effect on control swelling-induced EAA release (Fig. 5A).



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  		FIG. 5.
H2O2 potentiates swelling-activated excitatory amino acid release via a CaMKII-dependent mechanism. A, the effect of the CaMKII inhibitor KN-93 on the H2O2-induced modulation of D-[3H]aspartate release. Astrocytes were exposed to hypo-osmotic medium in the absence ({circ}, n = 4) or presence of 5 µM KN-93 ({triangledown}, n = 5). In parallel experiments, cells were exposed to 500 µM H2O2 without (•, n = 4) or with ({blacktriangledown}, n = 5) 5 µM KN-93. The data represent the mean values ± S.E. ***, p < 0.001 H2O2 versus all other groups. B and C, the effect of H2O2 on astrocytic CaMKII activity, measured using a phospho-Thr286 CaMKII antibody. The treatment conditions were similar to those shown in A. Astrocytes were lysed at the time point corresponding to the 25th minute of the efflux experiments. B shows representative Western blots for phospho-Thr286 CaMKII and pan-CaMKII antibodies. Phosphorylation levels were compared with cells kept in basal medium for 25 min. The average optical densities ± S.E. of the immunoreactive bands from five different cell preparations are presented in C. *, p < 0.05, H2O2 versus all other groups.

 
We further tested the effects of H2O2 on CaMKII activity using a pospho-Thr286 CaMKII antibody recognizing the autophosphorylated active form of CaMKII (55). In astrocyte lysates, this antibody recognized a single protein band of ~52 kDa that corresponds to the predicted molecular mass of CaMKII. CaMKII expression and loading levels were not different between treatment groups, as verified using a pan CaMKII antibody. We found that H2O2 caused CaMKII activation in swollen astrocytes, which was blocked by 5 µM KN-93 (Fig. 5, B and C). Hypo-osmotic medium-induced swelling itself did not increase CaMKII activity above basal levels (Fig. 5, B and C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have found that hydrogen peroxide acts as a potent positive modulator of swelling-activated excitatory amino acid release in cultured astrocytes. When combined with hypo-osmotic swelling, H2O2 dose-dependently potentiated swelling-induced D-[3H]aspartate release by up to 3-fold (EC50{approx}10 µM). We found no morphological changes in astrocytes treated with 500 µM H2O2, which is in line with data showing that astrocytes are highly resistant to oxidative stress (56, 57). Furthermore, potentiation of swelling-induced D-[3H]aspartate release by H2O2 was not related to nonspecific changes in membrane permeability, monitored as the release of preloaded [51Cr]. H2O2 also did not enhance astrocyte swelling under hypo-osmotic conditions. Pharmacological analysis suggested that the H2O2-induced increase in EAA release is probably caused by the potentiation of VRAC activity in swollen cells. In our experiments, the VRAC blockers ATP (10 mM), phloretin (100 µM), and NPPB (100 µM) (8, 38) potently reduced EAA release in both the presence and absence of H2O2. Although there are currently no specific VRAC blockers available, the use of these three distinct inhibitors probably rules out alternative transport pathways that might also contribute to astrocytic EAA release. Such pathways might include other anion channels, a vesicular glutamate release pathway, connexin hemichannels, P2X7 receptor channels, and excitatory amino acid transporters working in the reverse mode (58–63).

VRACs, which are typically activated in response to cell swelling, are ubiquitously expressed and permeable toward small organic molecules in astrocytes and other cell types (10, 36, 64, 65). Two recent publications have shown that H2O2 induces a gradual increase in Cl– conductance in non-swollen cells, which exhibits the typical current/voltage relationship and pharmacological properties of VRACs (15, 16). Although H2O2 strongly up-regulated volume-sensitive EAA release in our experiments, in the absence of cell swelling, even 20-min H2O2 treatment produced only a slight upward shift in the baseline release rate that was more pronounced after the removal of H2O2. Such a difference between our efflux experiments and the studies showing H2O2-induced Cl– current activation in non-swollen cells may be related to cell type differences or a modified intracellular milieu resulting from cell dialysis in electrophysiological experiments. Lambert (17) has found that H2O2 induces the positive modulation of taurine release only in swollen NIH 3T3 fibroblasts.

Although H2O2-dependent VRAC activation and modulation of organic osmolyte release has been described in several cell lines (15–17), the intracellular signaling events responsible for such effects have not been determined. It is well known that H2O2 stimulates numerous intracellular signaling pathways, including tyrosine kinases, MAPKs, PKC, MLCK, and CaMKII, and inhibits tyrosine phosphatases (20–22, 49, 66). Because the same protein kinases have also been found to regulate VRAC activity, we hypothesized that H2O2 exerts its actions on astrocytic amino acid release via alterations in VRAC-associated intracellular signaling pathways. In our previous work, we found that the reactive nitrogen species peroxynitrite potentiates swelling-activated D-[3H]aspartate release via a tyrosine kinase-dependent mechanism (67). Therefore, we tested for the involvement of tyrosine kinases but found that none of the inhibitors of non-receptor tyrosine kinases (PP2 and SU6656) or receptor tyrosine kinases (tyrphostin A51) or the MEK inhibitor (U0126) prevented the H2O2 effect. PKC was also a potential candidate, in that it has been found to be activated by H2O2 (43, 44) and is also critical for the activation or modulation of VRACs (26, 45) and volume-dependent organic osmolyte release (50). However, we found that several potent PKC inhibitors (Go6983, bisindolylmaleimide I, and Ro32-0432) were ineffective in attenuating the H2O2-response. The PKC inhibitor chelerythrine blocked the H2O2-effect, but because the other PKC blockers did not replicate this effect, it is unlikely to be related to PKC inhibition.

H2O2 also elevates [Ca2+]i in a variety of cell lines (22, 68). Therefore, we investigated the possible involvement of Ca2+ and calmodulin in mediating the H2O2 effect on amino acid release. Because cell swelling itself is known to stimulate [Ca2+]i increases in a majority of cell types, including cultured astrocytes (69), we sought to determine whether the presence of H2O2 during astrocytic swelling would further potentiate the [Ca2+]i signal. In our experiments, H2O2 initiated a significant gradual [Ca2+]i rise before the onset of hypo-osmotic swelling. Under hypo-osmotic conditions, H2O2 modestly potentiated swelling-induced [Ca2+]i increases, but this effect was not statistically significant. Nonetheless, the Ca2+ chelator BAPTA-AM partially blocked the H2O2-induced EAA release. In line with the BAPTA-AM data, the calmodulin antagonists trifluoperazine and W-7 completely eliminated the H2O2 effect. Partial inhibition of the H2O2-induced potentiation of EAA release by 10 µM BAPTA-AM, compared with full inhibition by the calmodulin antagonists, may be caused by insufficient intracellular Ca2+ chelation. We attempted to use a higher BAPTA-AM concentration, but it caused potent nonspecific EAA release on its own. In contrast to their effects on the H2O2-response, BAPTA-AM and the calmodulin blockers did not significantly affect control swelling-induced D-[3H]aspartate release. These observations are consistent with a number of publications indicating that although swelling-induced [Ca2+]i increases are not necessary for VRAC activation, additional elevations in [Ca2+]i by the Ca2+ ionophore ionomycin and [Ca2+]i-releasing agonists potently modulate VRAC activity (27, 70–73).

To establish the Ca2+/calmodulin-dependent mechanism responsible for the H2O2 effect, we looked into the involvement of two Ca2+/calmodulin-dependent enzymes, MLCK and CaMKII. The MLCK inhibitor ML-7 was ineffective in blocking the H2O2-response, ruling out MLCK involvement. Although MLCK is a potent positive modulator of VRAC activity in endothelial cells (74), in astrocytes and other cell types, this Ca2+/calmodulin-dependent enzyme seems not to contribute to the activation of VRACs and volume-dependent organic osmolyte release (75, 76).

In contrast to ML-7, the CaMKII inhibitor KN-93 completely eliminated the H2O2 effect without altering control volume-sensitive EAA release. These data suggest that CaMKII is a critical component of the H2O2-dependent potentiation of swelling-sensitive EAA release. Similar to our findings, Cardin et al. have demonstrated that the Ca2+ ionophore ionomycin enhances volume-sensitive taurine release in cerebellar astrocyte cultures through a CaMKII-dependent mechanism (27). In the same study, CaMKII inhibition was ineffective in blocking control swelling-induced release in the absence of ionomycin (27). Using a phospho-Thr286 antibody recognizing the autophosphorylated active form of CaMKII (55), we confirmed that H2O2 stimulates CaMKII activity in swollen astrocytes. However, hypo-osmotic medium alone induced no changes in CaMKII activity despite similar increases in [Ca2+]i levels in swollen astrocytes in the absence or presence of H2O2. One possible explanation is that pretreatment with H2O2 and its associated [Ca2+]i elevations may promote calmodulin trapping, which further enhances CaMKII activity in response to subsequent [Ca2+]i increases, as has been demonstrated using purified CaMKII (77, 78).

VRACs probably play a major role in mediating excessive EAA release during cerebral ischemia (3–6). The potent VRAC blocker tamoxifen reduces infarct size after transient and global ischemia, although the exact mechanism of tamoxifen protection remains to be elucidated (79, 80). Astrocytes are a plausible site for VRAC activation because, unlike neurons, they are highly susceptible to ischemia-induced cell swelling (12–14). However, swelling may not be the only factor responsible for pathological VRAC regulation. The massive release of neurotransmitters and neuromodulators from depolarized and damaged neuronal cells, along with the excessive production of reactive nitrogen and oxygen species, causes alterations in intracellular signaling (18, 22). In particular, severalfold increases in tyrosine phosphorylation and MAPK activity, as well as the robust translocation of PKC and CaMKII from the cytosol to the membrane fraction, have been found in animal ischemia models (29–32). Our in vitro data demonstrate that H2O2 strongly up-regulates astrocytic VRAC activity via a CaMKII-dependent mechanism and in this way may potently promote pathological excitatory amino acid release in the ischemic brain.


   FOOTNOTES
 
* This work was supported by NINDS, National Institutes of Health Grants R01-NS35205 (to H.K.K.) and F31-NS46961 (to R. E. H.-L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains a Supplemental Figure. Back

§ To whom correspondence should be addressed: 47 New Scotland Ave. (MC-136), Albany, NY 12208. Tel.: 518-262-5098; Fax: 518-262-6178; E-mail: mongina{at}maill.amc.edu.

1 The abbreviations used are: EAA, excitatory amino acid; VRAC, volume-regulated anion channel; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; CaMKII, Ca2+/calmodulin-dependent protein kinase II; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; BA-PTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; AM, acetoxymethyl ester; MLCK, myosin light chain kinase. Back


   ACKNOWLEDGMENTS
 
We thank C. J. Charniga and Z. Li for technical assistance, Dr. G. P. Schools for help with confocal microscopy experiments, Dr. P. J. Feustel for helpful discussions, and Dr. H. A. Singer for providing us with pan CaMKII antibodies.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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