Overstimation of NADH-driven vascular oxidase activity due to lucigenin artifacts

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 μM concentrations. Results showed preferential signals with NADPH (vs. NADH) with 5 and 50 μM lucigenin, which were blocked by diphenylene iodonium (DPI), superoxide dismutase (SOD), or its cell-permeable mimetic MnTBAP. With 250 μM 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 μM lucigenin to homogenates incubated with either NADPH or NADH. With 50 or 250 μM 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 μM concentrations, while at 250 μM redox-cycling becomes predominant and is markedly increased when NADH is the assayed substrate; and (ii) With 250 μM 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.