Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA315

Poster Communications

Cytochrome P450 reductase contributes to NADPH-dependent signal in membrane assays used for measuring Nox activity

P. Malacarne1, O. Löwe1, I. Wittig2, V. Helfinger1, F. Moll1, N. Kurrle3, N. Müller1, I. Fleming4, R. Brandes1, K. Schröder1, F. Rezende1

1. VRC, Institute für kardiovaskuläre Physiologie, Frankfurt, Hessen, Germany. 2. Functional Proteomics, Frankfurt am Main, Germany. 3. Institute for Molecular Hematology, Frankfurt am Main, Germany. 4. VRC, Institute for Vascular Signaling, Frankfurt am Main, Germany.


The molecular basis of cardiovascular diseases (CVD) is complex and oxidative stress is a strong candidate contributor towards its development. The Nox NADPH oxidase family of enzymes uses NADPH as substrate to produce ROS (reactive oxygen species) and have been considered important sources of oxidative stress occurring in atherosclerosis, hypertension and heart failure. Such conclusions were mainly based on NADPH-stimulated assays in membrane fractions which were thought to measure NADPH oxidase (Nox) activity. These assays were often used to overcome the limitations of the available ROS probes. However, the contribution of Nox enzymes to the highly detected signal was not validated at the time they emerged. We have generated a triple Nox knockout mouse (Nox1-Nox2-Nox4-/-) which exhibited attenuated endothelial dysfunction, hypertension and hypertrophic response to angiotensin II (AngII). However, the knockout of the three main Nox enzymes had no impact on chemiluminescence signals in aorta, heart and kidney homogenates. In HEK cell overexpressing Nox4 and Nox5 (as sources of H2O2 and superoxide, respectively) the high ROS production of intact cells could not be recapitulated in NADPH-stimulated membrane assays with lucigenin. Thus, the signal has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH-stimulated assays and mass spectrometry, cytochrome P450 reductase (POR) was identified as the possible source of the assay signal. Microsomes overexpressing POR, cytochromes b5 and P450 generated a NADPH-dependent signal in assays utilizing lucigenin, L-012 and dihydroethidium (DHE). Knockout of POR by CRISPR/Cas9 technology (POR-/-) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR knockout abolished the signal in NADPH-stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells (SMC) treated with angiotensin II showed an increased NADPH-dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. POR shares structural and biological similarities with Nox enzymes as both contain flavin domains and consume NADPH to produce ROS. Interestingly, loss of POR and Nox activity also had similar physiological consequences: Cell proliferation was reduced to a similar extent as was agonist-induced phosphorylation of p38MAP kinase. Thus, we show that POR contributes to NADPH-dependent signal in membrane assays and shares biological effects attributed originally to Nox enzymes.

Where applicable, experiments conform with Society ethical requirements