Expression and modulation of an NADPH oxidase in mammalian astrocytes
Expression and modulation of an NADPH oxidase in mammalian astrocytes
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DOI：
10.1523/jneurosci.1632-05.2005
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发表时间：
2005-10-05
影响因子：
5.3
通讯作者：
Duchen, MR
中科院分区：
文献类型：
--
作者：
Abramov, AY;Jacobson, J;Duchen, MR
淀粉样β肽通过对NADPH氧化酶抑制剂[二苯基碘鎓（DPI）和夹竹桃苷]敏感的机制在海马星形胶质细胞中产生氧化应激。为了寻找原代海马星形胶质细胞中酶表达和功能的证据，我们通过Western印迹分析和免疫荧光以及与星形胶质细胞特异性标记物胶质细胞酸性蛋白在培养物和体内的共表达证实了吞噬细胞NADPH氧化酶亚基的表达。使用光泽精发光、二氢乙啶或二羧基荧光素荧光来测量活性氧（ROS）的产生的功能测定证明DPI和夹竹桃苷敏感的ROS产生响应于佛波酯PMA和应用离子霉素或P（2u）受体活化后升高的[Ca（2+）]（c）。蛋白激酶C（PKC）抑制剂staurosporine和hispidin可抑制PMA的刺激作用，但不抑制Ca（2+）。缺乏gp91phox的转基因小鼠没有反应。gp91phox和p67phox的表达增加，反应性星形胶质细胞，这表明增加的速率都休息和刺激的ROS生成。NADPH氧化酶活性受细胞内pH调节，细胞内碱化抑制，酸化增强。质子载体羰基氰化物对三氟甲氧基苯腙抑制基础ROS的产生，但显着增加PMA刺激的ROS的产生。这是独立的线粒体ROS的生产，因为它是不受线粒体去极化鱼藤酮和寡霉素。因此，NADPH氧化酶在星形胶质细胞中表达，并且是功能性的，由PKC和细胞内钙激活，由pHi调节，并且由星形胶质细胞激活上调。星形胶质细胞NADPH氧化酶可能在中枢神经系统的生理和病理中起重要作用。
Amyloid beta peptides generate oxidative stress in hippocampal astrocytes through a mechanism sensitive to inhibitors of the NADPH oxidase [diphenylene iodonium (DPI) and apocynin]. Seeking evidence for the expression and function of the enzyme in primary hippocampal astrocytes, we confirmed the expression of the subunits of the phagocyte NADPH oxidase by Western blot analysis and by immunofluorescence and coexpression with the astrocyte- specific marker glial fibrillary acidic protein both in cultures and in vivo. Functional assays using lucigenin luminescence, dihydroethidine, or dicarboxyfluorescein fluorescence to measure the production of reactive oxygen species (ROS) demonstrated DPI and apocynin-sensitive ROS generation in response to the phorbol ester PMA and to raised [Ca(2+)](c) after application of ionomycin or P(2u) receptor activation. Stimulation by PMA but not Ca(2+) was inhibited by the protein kinase C (PKC) inhibitors staurosporine and hispidin. Responses were absent in transgenic mice lacking gp91phox. Expression of gp91phox and p67phox was increased in reactive astrocytes, which showed increased rates of both resting and stimulated ROS generation. NADPH oxidase activity was modulated by intracellular pH, suppressed by intracellular alkalinization, and enhanced by acidification. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone suppressed basal ROS generation but markedly increased PMA-stimulated ROS generation. This was independent of mitochondrial ROS production, because it was unaffected by mitochondrial depolarization with rotenone and oligomycin. Thus, the NADPH oxidase is expressed in astrocytes and is functional, activated by PKC and intracellular calcium, modulated by pHi, and upregulated by astrocyte activation. The astrocytic NADPH oxidase is likely to play important roles in CNS physiology and pathology.