TY  - JOUR
A1  - Rezende Felipe, Flávia Figueiredo de
A1  - Prior, Kim-Kristin
A1  - Löwe, Oliver
A1  - Wittig, Ilka
A1  - Strecker, Valentina
A1  - Moll, Franziska
A1  - Helfinger, Valeska
A1  - Schnütgen, Frank
A1  - Kurrle, Nina Susanne
A1  - Wempe, Frank
A1  - Walter, Maria
A1  - Zukunft, Sven
A1  - Luck, Bert
A1  - Fleming, Ingrid
A1  - Weißmann, Norbert
A1  - Brandes, Ralf
A1  - Schröder, Katrin
T1  - Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays
T2  - Free radical biology and medicine
N2  - 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.
KW  - NADPH oxidase
KW  - Nox
KW  - Lucigenin
KW  - Chemiluminescence
KW  - Superoxide
KW  - Reactive oxygen species
KW  - Membrane assays
Y1  - 2016
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/45567
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-455678
SN  - 1873-4596
SN  - 0891-5849
N1  - © 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/).
VL  - 102
SP  - 57
EP  - 66
PB  - Elsevier
CY  - New York, NY [u. a.]
ER  -