Hydrogen peroxide potentiates volume-sensitive excitatory amino acid release via a mechanism involving Ca2+/calmodulin-dependent protein kinase II.

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 microm H2O2 enhanced swelling-induced EAA release by approximately 2.5-3-fold (EC50 approximately 10 microM). 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.

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)(4)(5)(6). VRACs are ubiquitously expressed, activated in response to cell swelling, and characterized by Eisenman type I anion selectivity (I Ϫ Ͼ NO 3 Ϫ Ͼ Br Ϫ Ͼ Cl Ϫ Ͼ F Ϫ ), moderate outward rectification, and inactivation at positive potentials (7)(8)(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)(13)(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 (H 2 O 2 ) 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 H 2 O 2 and other reactive oxygen species are abundantly produced in ischemia and during reperfusion (18,19), it is plausible that H 2 O 2 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 Ca 2ϩ /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)(24)(25)(26)(27)(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 H 2 O 2 in mediating the activation or modulation of excitatory amino acid release via a putative VRAC pathway and explored intracellular signaling mechanisms involved in the H 2 O 2 effects in primary astrocyte cultures.
Excitatory Amino Acid and [ 51  The superfusate samples were collected each minute, and at the end of each experiment, the remaining intracellular isotope was extracted with 2% SDS ϩ 8 mM EDTA. The radioactivity of each sample was counted for 3 H or 51 Cr 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 M 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 ϳ1 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 25ϫ 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 [Ca 2ϩ ] i levels were determined using the cell-permeable fluorescent [Ca 2ϩ ] i indicator fluo-4-AM (Molecular Probes, Eugene, OR). Confluent cultured astrocytes, plated on round coverslips, were loaded for 1 h with 5 M 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 25ϫ 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 Trisbuffered 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.

Hydrogen Peroxide Potentiates Swelling-sensitive EAA Release-We used D-[ 3 H]aspartate as a tracer for EAA because it
is not metabolized and is released through the same swellinginduced permeability pathways as L-[ 3 H]glutamate (36). In astrocyte cultures, cell swelling induced by a 30% reduction in medium osmolarity (Ϫ90 mOsM) initiated a large release of preloaded D-[ 3 H]aspartate (Fig. 1A). H 2 O 2 , 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 H 2 O 2 -effect was saturated at concentrations Ն100 M (EC 50 ϭ 8.2 Ϯ 4.0 M, as determined by a dose-response curve fit, R 2 ϭ 0.98). 500 M H 2 O 2 was used throughout the study to ensure maximal activity. 100 -1,000 M H 2 O 2 enhanced swelling-induced EAA release by ϳ2.5-3fold (Fig. 1, A and B). In the absence of cell swelling, 500 M H 2 O 2 failed to initiate any rapid increases in EAA release, although it did induce a gradual upward shift in D-[ 3 H]aspartate baseline values (Fig. 1A). After removal of H 2 O 2 , we typically observed a secondary increase in D-[ 3 H]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 H 2 O 2 , we did not investigate its origin. To verify that the H 2 O 2 -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 ϳ4.5% increase in calcein fluorescence (Fig. 1C), which positively correlates to changes in cell volume (35). 500 M H 2 O 2 produced no additional changes in calcein fluorescence before and during hypo-osmotic medium exposure (Fig. 1C).
Hydrogen  (8)), 100 M phloretin (which inhibits VRACs but not cystic fibrosis transmembrane conductance regulator channel or Ca 2ϩ -activated Cl Ϫ channels at the concentration used (38)), and the broad spectrum anion channel blocker 100 M NPPB (8) all potently inhibited hypo-osmotic medium-induced EAA release both during control astrocyte swelling and in the presence of 500 M H 2 O 2 (Fig. 2B).
Intracellular Signaling Mechanisms Involved in the H 2 O 2 Effect on EAA Release-H 2 O 2 activates non-receptor tyrosine kinases of the src family, receptor tyrosine kinases, and, downstream, enzymes of the MAPK cascade (22,39), all of which have been found to contribute to VRAC activation (23)(24)(25). Therefore, we tested for the contribution of tyrosine and MAP kinases in mediating the H 2 O 2 effect on swelling-induced EAA release. The selective src kinase inhibitors PP2 (10 M) or SU6656 (5 M) (40), the receptor tyrosine kinase inhibitor tyrphostin A51 (20 M) (41), or the MEK inhibitor U0126 (10 M) (42), did not block the H 2 O 2 -induced potentiation of EAA release (Fig. 3A).
We also examined the potential role of PKC because this enzyme is also activated by H 2 O 2 (43,44) and has been shown to regulate VRAC activity (26,45). The potent PKC inhibitors 1 M Go6983, 1 M bisindolylmaleimide I, and 5 M Ro32-0432 (46 -48) were ineffective in blocking the H 2 O 2 -enhanced EAA release during cell swelling (Fig. 3B). In contrast, the PKC inhibitor 5 M chelerythrine completely blocked the H 2 O 2evoked enhancement in EAA release (Fig. 3B).
Two Ca 2ϩ /calmodulin-dependent enzymes known to be activated by H 2 O 2 , 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 Ca 2ϩ and the activation of Ca 2ϩ /calmodulin-dependent enzymes are involved in the H 2 O 2 effect. Using the fluorescent Ca 2ϩ indicator fluo-4-AM, we found that H 2 O 2 pretreatment produces a steady rise in [Ca 2ϩ ] 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 [Ca 2ϩ ] i followed by a gradual recovery toward baseline values (Fig. 4B). In the presence of 500 M H 2 O 2 , swelling-induced [Ca 2ϩ ] i release on average tended to be somewhat higher, but this effect was not statistically significant (Fig. 4B).
To test for Ca 2ϩ involvement in the H 2 O 2 effect on EAA release, we pretreated cultured astrocytes for 20 min with the cell membrane-permeable Ca 2ϩ chelator 10 M BAPTA-AM, followed by a 10-min wash out. BAPTA-AM pretreatment partially reduced the H 2 O 2 -induced increment in EAA release but had no statistically significant effect on control swelling-induced EAA release (Fig. 4C) (Fig. 4D). On the other hand, the inactive structural analogue of W-7, W-5 (20 M), was ineffective in reducing the H 2 O 2 effect (Fig. 4D). In contrast to their effects in the presence of H 2 O 2 , trifluoperazine and W-7 did not reduce EAA release under control hypo-osmotic conditions (Fig. 4D).
To determine the specific Ca 2ϩ /calmodulin-dependent enzymes involved in the H 2 O 2 effect, we used inhibitors of MLCK (ML-7 (40, 53)), and CaMKII (KN-93 (54)). Addition of 5 M ML-7 during 500 M H 2 O 2 perfusion had no effect on the ability of H 2 O 2 to enhance swelling-sensitive D-[ 3 H]aspartate release (n ϭ 4 per group, p ϭ 0.243; data not shown). In contrast, the application of the CaMKII inhibitor 5 M KN-93 completely abolished the effect of H 2 O 2 but had no effect on control swelling-induced EAA release (Fig. 5A).
We further tested the effects of H 2 O 2 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 ϳ52 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 H 2 O 2 caused CaMKII activation in swollen astrocytes, which was blocked by 5 M 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 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, H 2 O 2 dose-dependently po- though 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 H 2 O 2 induces a gradual increase in Cl Ϫ conductance in non-swollen cells, which exhibits the typical current/voltage relationship and pharmacological properties of VRACs (15,16). Although H 2 O 2 strongly up-regulated volume-sensitive EAA release in our experiments, in the absence of cell swelling, even 20-min H 2 O 2 treatment produced only a slight upward shift in the baseline release rate that was more pronounced after the removal of H 2 O 2 . Such a difference between our efflux experiments and the studies showing H 2 O 2 -induced Cl Ϫ 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 H 2 O 2 induces the positive modulation of taurine release only in swollen NIH 3T3 fibroblasts.
Although H 2 O 2 -dependent VRAC activation and modulation of organic osmolyte release has been described in several cell lines (15)(16)(17), the intracellular signaling events responsible for such effects have not been determined. It is well known that H 2 O 2 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 H 2 O 2 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-[ 3 H]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 H 2 O 2 effect. PKC was also a potential candidate, in that it has been found to be activated by H 2 O 2 (43,44) and is also critical for the activation   (27, 70 -73).
To establish the Ca 2ϩ /calmodulin-dependent mechanism responsible for the H 2 O 2 effect, we looked into the involvement of two Ca 2ϩ /calmodulin-dependent enzymes, MLCK and CaMKII. The MLCK inhibitor ML-7 was ineffective in blocking the H 2 O 2 -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 Ca 2ϩ /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 H 2 O 2 effect without altering control volumesensitive EAA release. These data suggest that CaMKII is a critical component of the H 2 O 2 -dependent potentiation of swelling-sensitive EAA release. Similar to our findings, Cardin et al. have demonstrated that the Ca 2ϩ 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 H 2 O 2 stimulates CaMKII activity in swollen astrocytes. However, hypo-osmotic medium alone induced no changes in CaMKII activity despite similar increases in [Ca 2ϩ ] i levels in swollen astrocytes in the absence or presence of H 2 O 2 . One possible explanation is that pretreatment with H 2 O 2 and its associated [Ca 2ϩ ] i elevations may promote calmodulin trapping, which further enhances CaMKII activity in response to subsequent [Ca 2ϩ ] 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)(4)(5)(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)(13)(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 H 2 O 2 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.