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

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Angiotensin II / metabolism
  • Animals
  • Cell Membrane / metabolism
  • Cytochrome P-450 Enzyme System / metabolism
  • Enzyme Activation
  • Kidney / metabolism
  • Membrane Glycoproteins / deficiency
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Mice
  • Mice, Knockout
  • Myocardium / metabolism
  • NADH, NADPH Oxidoreductases / deficiency
  • NADH, NADPH Oxidoreductases / genetics
  • NADH, NADPH Oxidoreductases / metabolism
  • NADP / metabolism
  • NADPH Oxidase 1
  • NADPH Oxidase 2
  • NADPH Oxidase 4
  • NADPH Oxidases / deficiency
  • NADPH Oxidases / genetics*
  • NADPH Oxidases / metabolism*
  • Nitric Oxide Synthase Type III / metabolism
  • Oxidation-Reduction
  • Phenotype
  • Reactive Oxygen Species
  • Signal Transduction


  • Membrane Glycoproteins
  • Reactive Oxygen Species
  • Angiotensin II
  • NADP
  • Cytochrome P-450 Enzyme System
  • Nitric Oxide Synthase Type III
  • NADH, NADPH Oxidoreductases
  • Cybb protein, mouse
  • NADPH Oxidase 1
  • NADPH Oxidase 2
  • NADPH Oxidase 4
  • NADPH Oxidases
  • NOX1 protein, mouse
  • Nox4 protein, mouse