Rathore Rakesh, Zheng Yun-Min, Niu Chun-Feng, Liu Qing-Hua, Korde Amit, Ho Ye-Shih, Wang Yong-Xiao
Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208, USA.
Free Radic Biol Med. 2008 Nov 1;45(9):1223-31. doi: 10.1016/j.freeradbiomed.2008.06.012. Epub 2008 Jun 21.
The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells, including pulmonary artery smooth muscle cells (PASMCs), remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22(phox), p47(phox), and p67(phox) were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47(phox) protein to the plasma membrane in pulmonary, but not mesenteric, arteries. The Nox inhibitor apocynin and p47(phox) gene deletion attenuated the hypoxic increase in intracellular concentrations of reactive oxygen species and Ca(2+) (ROS and Ca(2+)), as well as contractions in mouse PASMCs, and abolished the hypoxic activation of Nox in pulmonary arteries. The conventional/novel protein kinase C (PKC) inhibitor chelerythrine, specific PKCepsilon translocation peptide inhibitor, and PKCepsilon gene deletion, but not the conventional PKC inhibitor GO6976, prevented the hypoxic increase in Nox activity in pulmonary arteries and ROS in PASMCs. The PKC activator phorbol 12-myristate 13-acetate could increase Nox activity in pulmonary and mesenteric arteries. Inhibition of mitochondrial ROS generation with rotenone or myxothiazol prevented hypoxic activation of Nox. Glutathione peroxidase-1 (Gpx1) gene overexpression to enhance H(2)O(2) removal significantly inhibited the hypoxic activation of Nox, whereas Gpx1 gene deletion had the opposite effect. Exogenous H(2)O(2) increased Nox activity in pulmonary and mesenteric arteries. These findings suggest that acute hypoxia may distinctively activate Nox to increase ROS through the mitochondrial ROS-PKCepsilon signaling axis, providing a positive feedback mechanism to contribute to the hypoxic increase in ROS and Ca(2+) as well as contraction in PASMCs.
NADPH氧化酶（Nox）在包括肺动脉平滑肌细胞（PASMCs）在内的缺氧感应细胞的缺氧反应中的重要性仍不确定。在本研究中，我们通过蛋白质印迹分析发现，主要的Nox亚基Nox1、Nox4、p22(phox)、p47(phox)和p67(phox)在小鼠肺动脉和体循环（肠系膜）动脉中表达相当。然而，急性缺氧显著增加了肺动脉而非肠系膜动脉中的Nox活性以及p47(phox)蛋白向质膜的转位。Nox抑制剂夹竹桃麻素和p47(phox)基因缺失减弱了小鼠PASMCs中活性氧和Ca(2+)（ROS和Ca(2+)）细胞内浓度的缺氧增加以及收缩，并消除了肺动脉中Nox的缺氧激活。传统/新型蛋白激酶C（PKC）抑制剂白屈菜红碱、特异性PKCepsilon转位肽抑制剂和PKCepsilon基因缺失，但不是传统PKC抑制剂GO6976，阻止了肺动脉中Nox活性和PASMCs中ROS的缺氧增加。PKC激活剂佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯可增加肺动脉和肠系膜动脉中的Nox活性。用鱼藤酮或粘噻唑抑制线粒体ROS生成可阻止Nox的缺氧激活。谷胱甘肽过氧化物酶 - 1（Gpx1）基因过表达以增强H(2)O(2)清除显著抑制了Nox的缺氧激活，而Gpx1基因缺失则有相反的效果。外源性H(2)O(2)增加了肺动脉和肠系膜动脉中的Nox活性。这些发现表明，急性缺氧可能通过线粒体ROS - PKCepsilon信号轴特异性激活Nox以增加ROS，提供一种正反馈机制，促进ROS和Ca(2+)的缺氧增加以及PASMCs中的收缩。
