Validation of lucigenin as a chemiluminescent probe to monitor vascular superoxide as well as basal vascular nitric oxide production

Biochem Biophys Res Commun. 1999 Jan 19;254(2):319-24. doi: 10.1006/bbrc.1998.9942.


Lucigenin has been widely used as a chemiluminescent substrate to monitor vascular superoxide (O*-2) formation. The validity of lucigenin for detection of O*-2 has been questioned because O*-2 is generated by lucigenin itself. It has been shown that the concentration of lucigenin is a critical parameter affecting the validity of this assay. In the present studies we evaluated a reduced concentration of lucigenin (5 microM) as a tool to quantify O*-2 production in vascular tissue. Lucigenin-induced effects on endothelial function were assessed by isometric tension recording of isolated aortic rings suspended in organ baths. The effects of lucigenin on O*-2 production were studied using spin trapping and electron spin resonance spectroscopy. Lucigenin at 250 microM but not at 5 microM caused a significant attenuation of endothelium-dependent relaxations to acetylcholine, which was prevented by pretreatment with superoxide dismutase. Spin-trapping studies revealed that lucigenin at 250 microM increased vascular O*-2 production several fold while 5 microM lucigenin did not stimulate O*-2 production. Inhibition of NO synthase by NG-momomethyl-l-arginine as well as the removal of the endothelium almost doubled lucigenin-derived chemiluminescence (LDCL), indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. Thus, lucigenin at a concentration of 5 microM seems to be a sensitive and valid probe for assessing O*-2 in vascular tissue. It can also be used as an indirect probe to estimate basal vascular NO release.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / pharmacology
  • Acridines* / pharmacology
  • Animals
  • Aorta / drug effects
  • Aorta / physiology
  • Aorta / physiopathology
  • Electron Spin Resonance Spectroscopy / methods
  • Endothelium, Vascular / physiology*
  • Endothelium, Vascular / physiopathology
  • Hypercholesterolemia / physiopathology*
  • In Vitro Techniques
  • Luminescent Measurements
  • Muscle, Smooth, Vascular / drug effects
  • Muscle, Smooth, Vascular / physiology*
  • Muscle, Smooth, Vascular / physiopathology
  • Nitric Oxide / analysis
  • Nitric Oxide / metabolism*
  • Pentetic Acid / pharmacology
  • Rabbits
  • Reproducibility of Results
  • Scintillation Counting / methods
  • Superoxide Dismutase
  • Superoxides / analysis
  • Superoxides / metabolism*
  • Vasodilation / drug effects
  • Vasodilation / physiology*
  • omega-N-Methylarginine / pharmacology


  • Acridines
  • Superoxides
  • 10,10'-dimethyl-9,9'-biacridinium
  • omega-N-Methylarginine
  • Nitric Oxide
  • Pentetic Acid
  • Superoxide Dismutase
  • Acetylcholine