title: Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays.
abstract: 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.
  
  
  
  the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays.
authors.affiliation: Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany.
authors.forename: Flávia
authors.initials: F
authors.lastname: Rezende
authors.affiliation: Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany.
authors.forename: Kim-Kristin
authors.initials: KK
authors.lastname: Prior
authors.affiliation: Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Löwe
authors.affiliation: Functional Proteomics, SFB 815 Core Unit, Goethe-Universität, Frankfurt, Germany.
authors.forename: Ilka
authors.initials: I
authors.lastname: Wittig
authors.affiliation: Functional Proteomics, SFB 815 Core Unit, Goethe-Universität, Frankfurt, Germany.
authors.forename: Valentina
authors.initials: V
authors.lastname: Strecker
authors.affiliation: Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Moll
authors.affiliation: Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany.
authors.forename: Valeska
authors.initials: V
authors.lastname: Helfinger
authors.affiliation: Institute for Molecular Hematology, Goethe-University, Frankfurt, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Schnütgen
authors.affiliation: Institute for Molecular Hematology, Goethe-University, Frankfurt, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Kurrle
authors.affiliation: Institute for Molecular Hematology, Goethe-University, Frankfurt, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Wempe
authors.affiliation: Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Walter
authors.affiliation: Institute for Vascular Signaling, Goethe-University, Frankfurt, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Zukunft
authors.affiliation: Institute for Vascular Signaling, Goethe-University, Frankfurt, Germany.
authors.forename: Bert
authors.initials: B
authors.lastname: Luck
authors.affiliation: Institute for Vascular Signaling, Goethe-University, Frankfurt, Germany.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Fleming
authors.affiliation: University of Giessen, Lung Center, Giessen, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Weissmann
authors.affiliation: Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany. Electronic address: brandes@vrc.uni-franfurt.de.
authors.forename: Ralf P
authors.initials: RP
authors.lastname: Brandes
authors.affiliation: Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany. Electronic address: schroeder@vrc.uni-frankfurt.de.
authors.forename: Katrin
authors.initials: K
authors.lastname: Schröder
mesh_terms: D000166:Acridines / Q000737:chemistry / Q000378:metabolism*; D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D003573:Cytochrome b Group / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D049449:Luminescence; D008566:Membranes / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D000074624:NADPH Oxidase 1 / Q000235:genetics; D000074662:NADPH Oxidase 2 / Q000235:genetics; D000074663:NADPH Oxidase 4 / Q000235:genetics; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics*; D010084:Oxidation-Reduction; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000166:Acridines; D003573:Cytochrome b Group; D017382:Reactive Oxygen Species; D000804:Angiotensin II; C033472:10,10'-dimethyl-9,9'-biacridinium; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C488842:Cybb protein, mouse; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C000614711:NOX1 protein, mouse; C422943:Nox4 protein, mouse; C515614:Cyba protein, mouse
keywords: Chemiluminescence; Lucigenin; Membrane assays; NADPH oxidase; Nox; Reactive oxygen species; Superoxide