Haskew-Layton Renée E, Mongin Alexander A, Kimelberg Harold K
Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208, USA.
J Biol Chem. 2005 Feb 4;280(5):3548-54. doi: 10.1074/jbc.M409803200. Epub 2004 Nov 29.
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.
脑缺血时兴奋性氨基酸（EAA）过度释放是导致神经元损伤和死亡的主要机制。缺血时相当一部分EAA释放是通过容积调节性阴离子通道（VRACs）发生的。过氧化氢（H2O2）在缺血和再灌注期间大量产生，可激活许多对VRAC功能至关重要的蛋白激酶，最近有报道称其可激活VRACs。在本研究中，我们探讨了H2O2对培养星形胶质细胞中容积依赖性EAA释放的影响，以预加载的D-[3H]天冬氨酸的释放量来衡量。100 - 1000微摩尔的H2O2使肿胀诱导的EAA释放增加约2.5 - 3倍（半数有效浓度约为10微摩尔）。VRAC阻滞剂ATP、根皮素和5 - 硝基 - 2 -（3 - 苯丙基氨基）- 苯甲酸酯（NPPB）可有效抑制对照肿胀诱导的释放以及H2O2增强的释放，表明VRACs发挥了作用。Ca2+螯合剂1,2 - 双（2 - 氨基苯氧基）乙烷 - N,N,N',N' - 四乙酸 - 乙酰氧基甲酯（BAPTA - AM）可减弱H2O2诱导的EAA释放成分，而钙调蛋白拮抗剂三氟拉嗪和W - 7以及Ca2+/钙调蛋白依赖性蛋白激酶II（CaMKII）抑制剂KN - 93可完全消除该成分。酪氨酸激酶、蛋白激酶C和肌球蛋白轻链激酶的抑制剂在阻断H2O2反应方面无效。用磷酸化苏氨酸286 CaMKII抗体测定发现，H2O2处理肿胀的星形胶质细胞（而非单独肿胀）可导致CaMKII激活，而KN - 93可抑制该激活。这些数据表明，H2O2通过CaMKII依赖性机制强烈上调星形胶质细胞容积敏感性EAA释放，从而可能有力地促进缺血时病理性EAA释放和脑损伤。
