Britigan B E, Hamill D R
Department of Internal Medicine, Veterans Administration Medical Center, Iowa City, IA.
Free Radic Biol Med. 1990;8(5):459-70. doi: 10.1016/0891-5849(90)90059-r.
Previously we had utilized the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) to detect superoxide (.O2-) formation by human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan. When N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A were substituted as the neutrophil stimulus spin trap evidence of neutrophil free radical production was not detected. Consequently, the hypothesis that DMPO interfered with neutrophil stimulus response coupling was examined. DMPO exhibited a concentration-related inhibition of neutrophil .O2- secretion (ferricytochrome C reduction) following exposure to six different stimuli. The extent of inhibition was stimulus dependent--large (FMLP, concanavalin A), moderate (PMA, opsonized zymosan, A23187), and mild (arachidonic acid). Inhibition was reversible. Onset was nearly instantaneous and was observed even if DMPO was added after stimulus-induced .O2- formation was ongoing. DMPO had only minimal effect on .O2- production by a cell-free NADPH-oxidase membrane preparation. DMPO also inhibited the neutrophil degranulation response for elastase and lactoferrin but not vitamin B12 binding protein. DMPO-mediated inhibition of neutrophil function was not related to alteration in stimulus binding (FMLP or concanavalin A). DMPO had minimal impact on the stimulus-induced rise in intracellular calcium. However, the presence of DMPO resulted in a concentration-dependent depolarization of the resting neutrophil membrane and blunting of the depolarization response to each stimulus examined. These data are of importance to investigators applying spin-trapping techniques to phagocytic cells and suggest DMPO could be used as a tool for investigating neutrophil stimulus-response mechanisms.
此前我们曾利用自旋捕捉剂5,5-二甲基-1-吡咯啉-N-氧化物（DMPO）来检测佛波酯（PMA）或调理酵母聚糖刺激的人中性粒细胞中超氧阴离子（·O₂⁻）的形成。当用N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（FMLP）或伴刀豆球蛋白A替代作为中性粒细胞刺激物时，未检测到中性粒细胞产生自由基的自旋捕捉证据。因此，对DMPO干扰中性粒细胞刺激反应偶联这一假说进行了研究。暴露于六种不同刺激后，DMPO对中性粒细胞·O₂⁻分泌（高铁细胞色素C还原）呈现浓度依赖性抑制。抑制程度取决于刺激物——抑制作用大（FMLP、伴刀豆球蛋白A）、中等（PMA、调理酵母聚糖、A23187）、轻度（花生四烯酸）。抑制作用是可逆的。起效几乎是瞬间的，即使在刺激诱导的·O₂⁻形成已经开始后添加DMPO也能观察到抑制作用。DMPO对无细胞NADPH氧化酶膜制剂产生·O₂⁻的影响极小。DMPO还抑制中性粒细胞对弹性蛋白酶和乳铁蛋白的脱颗粒反应，但不抑制维生素B₁₂结合蛋白。DMPO介导的中性粒细胞功能抑制与刺激物结合（FMLP或伴刀豆球蛋白A）的改变无关。DMPO对刺激诱导的细胞内钙升高影响极小。然而，DMPO的存在导致静息中性粒细胞膜出现浓度依赖性去极化，并使对所检测的每种刺激的去极化反应减弱。这些数据对于将自旋捕捉技术应用于吞噬细胞的研究人员具有重要意义，并表明DMPO可作为研究中性粒细胞刺激反应机制的工具。
