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.