Details

Autor(en) / Beteiligte

• 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

Titel

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

Ist Teil von

• Free radical biology & medicine, 2017-01, Vol.102, p.57-66

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2017

Quelle

MEDLINE

Beschreibungen/Notizen

• 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-dependent signal in SMC membranes.