Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays

Measuring NADPH oxidase (Nox)-derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitiv...

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Published in:Free radical biology & medicine 2017-01, Vol.102, p.57-66
Main Authors: Rezende, Flávia, Prior, Kim-Kristin, Löwe, Oliver, Wittig, Ilka, Strecker, Valentina, Moll, Franziska, Helfinger, Valeska, Schnütgen, Frank, Kurrle, Nina, Wempe, Frank, Walter, Maria, Zukunft, Sven, Luck, Bert, Fleming, Ingrid, Weissmann, Norbert, Brandes, Ralf P., Schröder, Katrin
Format: Article
Language:English
Subjects:
Acridines - chemistry
Acridines - metabolism
Angiotensin II - metabolism
Animals
Chemiluminescence
Cytochrome b Group - genetics
Cytochrome P-450 Enzyme System - genetics
Cytochrome P-450 Enzyme System - metabolism
HEK293 Cells
Humans
Lucigenin
Luminescence
Membrane assays
Membranes - chemistry
Membranes - metabolism
Mice
Mice, Knockout
Myocytes, Smooth Muscle - metabolism
NADP - metabolism
NADPH oxidase
NADPH Oxidase 1 - genetics
NADPH Oxidase 2 - genetics
NADPH Oxidase 4 - genetics
NADPH Oxidases - genetics
NADPH Oxidases - metabolism
NADPH-Ferrihemoprotein Reductase - genetics
Nox
Oxidation-Reduction
Rats
Reactive oxygen species
Reactive Oxygen Species - metabolism
Superoxide
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Summary:Measuring NADPH oxidase (Nox)-derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH-stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1-Nox2-Nox4-/-) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH-stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH-stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH-stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH-dependent signal in assays utilizing lucigenin, L-012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR-/-) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR-/- abolished the signal in NADPH-stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells 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. In conclusion: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays. [Display omitted] •Nox activity of intact cells could not be recapitulated in membranes treated with NADPH.•Proteomics of membranes show P450 reductase as source of NADPH-dependent signal.•Microsomes overexpressing Cytochrome P450 system produce a NADPH-dependent signal.•Knockout of P450 reductase (CRISPR/Cas9) abolished lucigenin signal in HEK cell membranes.•Knockout of POR but not p22phox abolishes the basal and Angiotensin II-stimulated NADPH-dep
ISSN:0891-5849
1873-4596
DOI:10.1016/j.freeradbiomed.2016.11.019