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

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 mu M) 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 mu M but not at 5 mu M 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 mu M increased vascular O-2(.-) production several fold while 5 mu M lucigenin did not stimulate O-2(.-) production. Inhibition of NO synthase by N-G-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 mu M 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. (C) 1999 Academic Press.