Bayraktutan U, Draper N, Lang D, Shah A M
Department of Cardiology, University of Wales College of Medicine, Cardiff, UK.
Cardiovasc Res. 1998 Apr;38(1):256-62. doi: 10.1016/s0008-6363(98)00003-0.
The production of reactive oxygen species (e.g., superoxide) by endothelial cells is relevant to tissue injury during ischemia-reperfusion, and may also play a role in intracellular signaling pathways. However, the molecular identities of the enzymes responsible for endothelial superoxide production are poorly defined, although xanthine oxidase, NADH/NADPH oxidoreductases and nitric oxide synthase are among proteins suggested to contribute. Recent studies suggest that an NADH/NADPH oxidase similar to that found in neutrophils is an important source of superoxide in vascular smooth muscle.
We investigated whether a phagocyte-type NADH/NADPH oxidase complex is present in rat cultured coronary microvascular endothelial cells. The expression of NADPH oxidase components was studied by RT-PCR and Western blot analyses, while functional activity was assessed by measurement of superoxide production by lucigenin-enhanced chemiluminescence.
The major component of the phagocyte-type NADH/NADPH oxidase complex, a cytochrome b558 heterodimer, was shown to be present both at mRNA and protein levels, using oligonucleotide primers designed from published neutrophil and vascular smooth muscle sequences and anti-neutrophil antibodies respectively. Functional activity of the enzyme was also confirmed by NADPH-evoked superoxide production in cell homogenates, which was inhibited either by the superoxide chelator Tiron or by diphenyleneiodonium, an inhibitor of the oxidase.
A functional phagocyte-type NADPH oxidase is expressed in coronary microvascular endothelial cells, where it may contribute to the physiological and/or pathophysiological effects of reactive oxygen species. These data, together with reports of the presence of a similar oxidase in other non-phagocytic cell types, suggest that this enzyme complex is widely expressed in many tissues where it may subserve signaling and other functions.
内皮细胞产生活性氧（如超氧化物）与缺血再灌注期间的组织损伤相关，并且可能在细胞内信号通路中发挥作用。然而，尽管黄嘌呤氧化酶、NADH/NADPH氧化还原酶和一氧化氮合酶被认为是可能起作用的蛋白质，但负责内皮细胞超氧化物产生的酶的分子身份仍不清楚。最近的研究表明，一种与中性粒细胞中发现的类似的NADH/NADPH氧化酶是血管平滑肌中超氧化物的重要来源。
我们研究了大鼠培养的冠状动脉微血管内皮细胞中是否存在吞噬细胞型NADH/NADPH氧化酶复合物。通过RT-PCR和蛋白质印迹分析研究NADPH氧化酶成分的表达，同时通过荧光素增强化学发光法测量超氧化物产生来评估功能活性。
使用分别根据已发表的中性粒细胞和血管平滑肌序列设计的寡核苷酸引物以及抗中性粒细胞抗体，显示吞噬细胞型NADH/NADPH氧化酶复合物的主要成分，即细胞色素b558异二聚体，在mRNA和蛋白质水平均存在。细胞匀浆中NADPH诱导的超氧化物产生也证实了该酶的功能活性，超氧化物螯合剂替诺隆或氧化酶抑制剂二苯碘鎓均可抑制该产生。
功能性吞噬细胞型NADPH氧化酶在冠状动脉微血管内皮细胞中表达，在其中它可能有助于活性氧的生理和/或病理生理作用。这些数据，连同其他非吞噬细胞类型中存在类似氧化酶的报道，表明这种酶复合物在许多组织中广泛表达，在其中它可能发挥信号传导和其他功能。
