Rezende Flávia, Löwe Oliver, Helfinger Valeska, Prior Kim-Kristin, Walter Maria, Zukunft Sven, Fleming Ingrid, Weissmann Norbert, Brandes Ralf P, Schröder Katrin
1 Institute for Cardiovascular Physiology, Goethe-University , Frankfurt, Germany .
2 Institute for Vascular Signaling, Goethe-University , Frankfurt, Germany .
Antioxid Redox Signal. 2016 Mar 1;24(7):392-9. doi: 10.1089/ars.2015.6314. Epub 2015 Jun 30.
NADPH oxidases of the Nox family are considered important sources of cellular reactive oxygen species (ROS) production. This conclusion is, in part, based on the ability of NADPH to elicit a chemiluminescence signal in tissue/cell homogenates or membrane preparations in the presence of enhancers such as lucigenin, luminol, or L012. However, the ability of these particular assays to specifically detect Nox activity and Nox-derived ROS has not been proven. In this study, we demonstrate that combined knockout of the three main Nox enzymes of the mouse (Nox1-Nox2-Nox4 triple knockout) had no impact on NADPH-stimulated chemiluminescence signals in the aorta, heart, and kidney homogenates. In the NADPH-stimulated membrane assays, no effect of in vivo angiotensin II pretreatment or deletion of Nox enzymes was observed. In in vitro studies in HEK293 cells, the overexpression of Nox5 or Nox4 markedly increased ROS production in intact cells, whereas overexpression of Nox5 or Nox4 had no influence on the signal in membrane assays. In contrast, overexpression of nitric oxide synthase or cytochrome P450 enzymes resulted in an increased chemiluminescence signal in isolated membranes. On the basis of these observations, we propose the hypothesis that NADPH-stimulated chemiluminescence-based membrane assays, as currently used, do not reflect Nox activity.
Nox家族的NADPH氧化酶被认为是细胞活性氧（ROS）产生的重要来源。这一结论部分基于NADPH在诸如光泽精、鲁米诺或L012等增强剂存在的情况下，能够在组织/细胞匀浆或膜制剂中引发化学发光信号的能力。然而，这些特定检测方法特异性检测Nox活性和Nox衍生ROS的能力尚未得到证实。在本研究中，我们证明小鼠三种主要Nox酶的联合敲除（Nox1 - Nox2 - Nox4三联敲除）对主动脉、心脏和肾脏匀浆中NADPH刺激的化学发光信号没有影响。在NADPH刺激的膜检测中，未观察到体内血管紧张素II预处理或Nox酶缺失的影响。在HEK293细胞的体外研究中，Nox5或Nox4的过表达显著增加了完整细胞中的ROS产生，而Nox5或Nox4的过表达对膜检测中的信号没有影响。相反，一氧化氮合酶或细胞色素P450酶的过表达导致分离膜中的化学发光信号增加。基于这些观察结果，我们提出一个假设，即目前使用的基于NADPH刺激化学发光的膜检测方法不能反映Nox活性。
