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Review
. 2016 Mar 1;24(7):392-9.
doi: 10.1089/ars.2015.6314. Epub 2015 Jun 30.

Unchanged NADPH Oxidase Activity in Nox1-Nox2-Nox4 Triple Knockout Mice: What Do NADPH-Stimulated Chemiluminescence Assays Really Detect?

Flávia Rezende  1 , Oliver Löwe  1 , Valeska Helfinger  1 , Kim-Kristin Prior  1 , Maria Walter  1 , Sven Zukunft  2 , Ingrid Fleming  2 , Norbert Weissmann  3 , Ralf P Brandes  1 , Katrin Schröder  1
Affiliations
Review

Unchanged NADPH Oxidase Activity in Nox1-Nox2-Nox4 Triple Knockout Mice: What Do NADPH-Stimulated Chemiluminescence Assays Really Detect?

Flávia Rezende et al. Antioxid Redox Signal. .

Abstract

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.

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Comment in

  • Chemiluminescence and the Nox1-Nox2-Nox4 Triple Knockout.
    Pagano PJ, Griendling KK, Miller FJ, Laurindo FR, Touyz RM. Pagano PJ, et al. Antioxid Redox Signal. 2015 Nov 20;23(15):1246-7. doi: 10.1089/ars.2015.6382. Epub 2015 Jul 14. Antioxid Redox Signal. 2015. PMID: 26054248 No abstract available.
  • Response to Pagano et al.
    Rezende F, Löwe O, Helfinger V, Prior KK, Walter M, Zukunft S, Fleming I, Weissmann N, Brandes RP, Schröder K. Rezende F, et al. Antioxid Redox Signal. 2015 Nov 20;23(15):1247-9. doi: 10.1089/ars.2015.6396. Epub 2015 Jul 14. Antioxid Redox Signal. 2015. PMID: 26173053

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