Janiszewski Mariano, Souza Heraldo P, Liu Xiaoping, Pedro Marcelo A, Zweier Jay L, Laurindo Francisco R M
Emergency Medicine Department, School of Medicine, University of São Paulo, São Paulo, Brazil.
Free Radic Biol Med. 2002 Mar 1;32(5):446-53. doi: 10.1016/s0891-5849(01)00828-0.
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.
最近，人们描述了光泽精存在的几个局限性。光泽精是一种常用于评估血管NAD(P)H氧化酶活性的探针，而血管NAD(P)H氧化酶是超氧化物的主要来源。这种氧化酶的优先还原底物仍不清楚。我们评估了光泽精假象是否会影响NAD(P)H氧化酶活性的检测。最初的化学发光分析是在5、50或250微摩尔浓度下用血管环或匀浆进行的。结果显示，在5和50微摩尔光泽精存在下，NADPH（相对于NADH）产生优先信号，该信号被二苯碘鎓（DPI）、超氧化物歧化酶（SOD）或其细胞可渗透模拟物MnTBAP阻断。在250微摩尔光泽精存在下，NADH的相对信号变得比NADPH的更大，并且上述三种拮抗剂对其抑制作用较弱。所有抗SOD/DPI的信号均被电子受体硝基蓝四唑有效阻断。用DMPO进行自旋捕获显示，在向用NADPH或NADH孵育的匀浆中添加5微摩尔光泽精后，DMPO-OH自由基加合物信号大约增加了一倍。在50或250微摩尔光泽精存在下，与NADPH相比，NADH的增加幅度要大得多。此外，耗氧量测量也显示了类似的结果。总之，我们的数据表明：（i）即使在5微摩尔浓度下，光泽精的氧化还原循环在血管组织中也可检测到，而在250微摩尔时，氧化还原循环占主导地位，并且当NADH作为被检测底物时会显著增加；（ii）在250微摩尔光泽精存在下，特别是与NADH相关时，信号会因直接的、依赖氧化酶的、不依赖超氧化物的双电子转移而被进一步高估。因此，先前关于血管氧化酶对NADH有优先亲和力的报道可能是由于这些假象。
