title: Overestimation of NADH-driven vascular oxidase activity due to lucigenin artifacts.
abstract: Several limitations have recently been described for lucigenin, a probe frequently used to assess the activity of vascular NAD(P)H oxidase, a major superoxide source. The preferential reducing substrate of such oxidase remains unclear. We assessed whether lucigenin artifacts could affect detection of NAD(P)H oxidase activity. Initial chemiluminescence assays were performed with vascular rings or homogenates at 5, 50, or 250 microM concentrations. Results showed preferential signals with NADPH (vs. NADH) with 5 and 50 microM lucigenin, which were blocked by diphenylene iodonium (DPI), superoxide dismutase (SOD), or its cell-permeable mimetic MnTBAP. With 250 microM lucigenin, the relative signal with NADH became larger than with NADPH, and was poorly inhibited by all three antagonists above. All SOD/DPI-resistant signals were effectively blocked by the electron acceptor nitrobluetetrazolium. Spin trapping with DMPO showed an approximate doubling of DMPO-OH radical adduct signal upon addition of 5 microM lucigenin to homogenates incubated with either NADPH or NADH. With 50 or 250 microM lucigenin, much larger increases were observed with NADH, as opposed to NADPH. Furthermore, oxygen consumption measurements showed analogous results. In summary, our data suggest that: (i) Lucigenin redox-cycling is detectable in vascular tissue even at 5 microM concentrations, while at 250 microM redox-cycling becomes predominant and is markedly increased when NADH is the assayed substrate; and (ii) With 250 microM lucigenin, preferentially with NADH, signals are further overestimated by direct, oxidase-dependent, superoxide-independent two-electron transfer. Therefore, previous reports of preferential NADH affinity of the vascular oxidase may have been due to these artifacts.
authors.affiliation: Emergency Medicine Department, School of Medicine, University of São Paulo, São Paulo, Brazil.
authors.forename: Mariano
authors.initials: M
authors.lastname: Janiszewski
authors.forename: Heraldo P
authors.initials: HP
authors.lastname: Souza
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Liu
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Pedro
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
authors.forename: Francisco R M
authors.initials: FR
authors.lastname: Laurindo
mesh_terms: D000166:Acridines / Q000494:pharmacology*; D000818:Animals; D016477:Artifacts; D002339:Carotid Arteries / Q000201:enzymology*; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D007083:Iliac Artery / Q000201:enzymology*; D008163:Luminescent Measurements; D008297:Male; D009243:NAD / Q000494:pharmacology*; D009249:NADP / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D011817:Rabbits; D013113:Spin Labels; D014652:Vascular Diseases / Q000378:metabolism
chemical_list: D000166:Acridines; D003497:Cyclic N-Oxides; D013113:Spin Labels; D009243:NAD; C033472:10,10'-dimethyl-9,9'-biacridinium; D009249:NADP; C069977:2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy; D019255:NADPH Oxidases