Ago Tetsuro, Kitazono Takanari, Ooboshi Hiroaki, Iyama Teruaki, Han Youn Hee, Takada Junichi, Wakisaka Masanori, Ibayashi Setsuro, Utsumi Hideo, Iida Mitsuo
Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
Circulation. 2004 Jan 20;109(2):227-33. doi: 10.1161/01.CIR.0000105680.92873.70. Epub 2004 Jan 12.
Recent evidence has suggested that reactive oxygen species are important signaling molecules in vascular cells and play a pivotal role in the development of vascular diseases. The activity of NAD(P)H oxidase has been identified as the major source of reactive oxygen species in vascular endothelial cells. However, the precise molecular structure and the mechanism of activation of the oxidase have remained poorly understood.
Here, we investigated the molecular identities and the superoxide-producing activity of endothelial NAD(P)H oxidase. We found that Nox4, a homologue of gp91phox/Nox2, was abundantly expressed in endothelial cells. The expression of Nox4 in endothelial cells markedly exceeded that of other Nox proteins, including gp91phox/Nox2, and was affected by cell growth. Using electron spin resonance and chemiluminescence, we measured the superoxide production and found that the endothelial membranes had an NAD(P)H-dependent superoxide-producing activity comparable to that of the neutrophil membranes, whereas the activity was not enhanced by the 2 recombinant proteins p47phox and p67phox, in contrast to that of the neutrophil membranes. Downregulation of Nox4 by an antisense oligonucleotide reduced superoxide production in endothelial cells in vivo and in vitro.
These findings suggest that Nox4 may function as the major catalytic component of an endothelial NAD(P)H oxidase.
最近的证据表明，活性氧是血管细胞中的重要信号分子，在血管疾病的发展中起关键作用。NAD(P)H氧化酶的活性已被确定为血管内皮细胞中活性氧的主要来源。然而，氧化酶的确切分子结构和激活机制仍知之甚少。
在此，我们研究了内皮NAD(P)H氧化酶的分子特性和产生超氧化物的活性。我们发现，gp91phox/Nox2的同源物Nox4在内皮细胞中大量表达。内皮细胞中Nox4的表达明显超过其他Nox蛋白，包括gp91phox/Nox2，并且受细胞生长的影响。使用电子自旋共振和化学发光，我们测量了超氧化物的产生，发现内皮细胞膜具有与中性粒细胞膜相当的NAD(P)H依赖性超氧化物产生活性，而与中性粒细胞膜不同的是，该活性不会被两种重组蛋白p47phox和p67phox增强。反义寡核苷酸下调Nox4可降低体内和体外内皮细胞中的超氧化物产生。
这些发现表明，Nox4可能作为内皮NAD(P)H氧化酶的主要催化成分发挥作用。
