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 specia
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.
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Metadaten
Author:Flávia Figueiredo de Rezende Felipe, Kim-Kristin Prior, Oliver Löwe, Ilka Wittig, Valentina Strecker, Franziska Moll, Valeska Helfinger, Frank Schnütgen, Nina Susanne Kurrle, Frank Wempe, Maria Walter, Sven Zukunft, Bert Luck, Ingrid Fleming, Norbert Weißmann, Ralf Peter Louis Brandes, Katrin Schröder
URN:urn:nbn:de:hebis:30:3-455678
DOI:http://dx.doi.org/10.1016/j.freeradbiomed.2016.11.019
ISSN:1873-4596
ISSN:0891-5849
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=27863990
Parent Title (English):Free radical biology and medicine
Publisher:Elsevier
Place of publication:New York, NY [u. a.]
Document Type:Article
Language:English
Year of Completion:2016
Date of first Publication:2016/11/15
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2018/01/30
Tag:Chemiluminescence; Lucigenin; Membrane assays; NADPH oxidase; Nox; Reactive oxygen species; Superoxide
Volume:102
Pagenumber:10
First Page:57
Last Page:66
Note:
© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
HeBIS PPN:428712258
Institutes:Medizin
Dewey Decimal Classification:610 Medizin und Gesundheit
Sammlungen:Universitätspublikationen
Licence (German):License LogoCreative Commons - Namensnennung 4.0

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