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

Free Radic Biol Med. 2017 Jan:102:57-66. doi: 10.1016/j.freeradbiomed.2016.11.019. Epub 2016 Nov 15.

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.

In conclusion: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays.

Keywords: Chemiluminescence; Lucigenin; Membrane assays; NADPH oxidase; Nox; Reactive oxygen species; Superoxide.

MeSH terms

  • Acridines / chemistry
  • Acridines / metabolism*
  • Angiotensin II / metabolism*
  • Animals
  • Cytochrome P-450 Enzyme System / genetics*
  • Cytochrome P-450 Enzyme System / metabolism
  • Cytochrome b Group / genetics*
  • HEK293 Cells
  • Humans
  • Luminescence
  • Membranes / chemistry
  • Membranes / metabolism
  • Mice
  • Mice, Knockout
  • Myocytes, Smooth Muscle / metabolism
  • NADP / metabolism
  • NADPH Oxidase 1 / genetics
  • NADPH Oxidase 2 / genetics
  • NADPH Oxidase 4 / genetics
  • NADPH Oxidases / genetics*
  • NADPH Oxidases / metabolism
  • NADPH-Ferrihemoprotein Reductase / genetics*
  • Oxidation-Reduction
  • Rats
  • Reactive Oxygen Species / metabolism

Substances

  • Acridines
  • Cytochrome b Group
  • Reactive Oxygen Species
  • Angiotensin II
  • 10,10'-dimethyl-9,9'-biacridinium
  • NADP
  • Cytochrome P-450 Enzyme System
  • NADPH-Ferrihemoprotein Reductase
  • Cybb protein, mouse
  • NADPH Oxidase 1
  • NADPH Oxidase 2
  • NADPH Oxidase 4
  • NADPH Oxidases
  • NOX1 protein, mouse
  • Nox4 protein, mouse
  • Cyba protein, mouse