Molecular characterization of a superoxide-generating NAD(P)H oxidase in the ventilatory muscles
Molecular characterization of a superoxide-generating NAD(P)H oxidase in the ventilatory muscles
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DOI：
10.1164/ajrccm.165.3.2103028
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发表时间：
2002-02-01
影响因子：
24.7
通讯作者：
Hussain, SNA
中科院分区：
文献类型：
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作者：
Javesghani, D;Magder, SA;Hussain, SNA
骨骼肌中活性氧簇(ROS)的分子来源尚不清楚。我们推测非吞噬细胞NAD(P)H氧化酶可能是肌肉纤维中ROS的来源之一。因此，我们研究了大鼠骨骼肌中非吞噬细胞NAD(P)H氧化酶的存在、结构及其对ROS产生的贡献。用荧光素增强化学发光法和NADH消耗率检测ROS生成和NAD(P)H氧化酶活性，用逆转录聚合酶链式反应和免疫印迹法检测酶的组成。正常大鼠肌条基础O-2(-)产生量平均为每10分钟1.4nmol/mg，加入NADH后，其基础O-2(-)产生量增加至接近每10分钟18nmol/mg。肌肉O-2(-)的产生和NADH的消耗可被TIron、超氧化物歧化酶、罗布宁和二苯基碘抑制，但不被环氧合酶、黄嘌呤氧化酶、一氧化氮合酶(NOS)和线粒体酶的抑制剂所抑制。我们检测了正常大鼠肌肉中p22(Phox)、gp91(Phox)、p47(Phox)和p67(Phox)亚单位的mRNA和蛋白。这些亚单位位于肌膜附近。脓毒症诱导的大鼠肌肉中O-2(-)的产生增加了一倍，而肌肉NADPH氧化物亚单位的表达没有重大变化。在内毒素处理的肌肉中，而在对照肌肉中，O-2(-)的产生通过抑制NOS而显著增加。我们的结论是，正常骨骼肌纤维中存在一个具有结构性活性的NAD(P)H氧化酶复合体，并参与ROS的产生。在脓毒症大鼠中，这种产生增加，但可测量的O-2(-)减少增加NO产生。
The molecular sources of reactive oxygen species (ROS) in skeletal muscles are not well understood. We hypothesized that nonphagocyte NAD(P)H oxidase could be a source of ROS in muscle fibers. We thus investigated the existence, structure, and contribution of nonphagocyte NAD(P)H oxidase to ROS production in rat skeletal muscles. ROS production and NAD(P)H oxidase activity were evaluated by lucigenin-enhanced chemiluminescence and NADH consumption rate, whereas enzyme composition was monitored by reverse transcription-polymerase chain reaction and immunoblotting. Basal O-2(-) production in muscle strips from normal rats averaged 1.4 nmol/mg per 10 min and increased to similar to 18 nmol/mg per 10 min in the presence of NADH. Muscle O-2(-) production and NADH consumption were inhibited by Tiron, superoxide dismutase, apocynin, and diphenyleneiodonium but not by inhibitors of cyclo-oxygenases, xanthine oxidase, nitric oxide synthases (NOS), and mitochondrial enzymes. We detected mRNA and proteins of p22(phox), gp91(phox), p47(phox), and p67(phox) subunits in normal rat muscles. These subunits were localized in close proximity to the sarcolemma. Induction of sepsis in rats doubled muscle O-2(-) production with no major changes in muscle NADPH oxide subunit expression. In lipopolysaccharide-treated but not in control muscles, O-2(-) production was increased significantly by NOS inhibition. We conclude that a constitutively active NAD(P)H oxidase enzyme complex exists in normal skeletal muscle fibers and contributes to ROS production. In septic rats, this production is increased but measurable O-2(-) is reduced by enhanced NO production.