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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^*

Renée E. Haskew-Layton, Alexander A. Mongin¶, and Harold K. Kimelberg¶

From the Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208 and ^¶Ordway Research Institute, Center for Medical Science, Albany, New York 12208

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 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.

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

^* 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.

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

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.

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