Tanigawa T, Kotake Y, Reinke L A
National Biomedical Center for Spin Trapping and Free Radicals, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Free Radic Res Commun. 1993;19(2):101-10. doi: 10.3109/10715769309056504.
Dahinden et al. reported that N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide release from polymorphonuclear leukocytes (PMNs) lasted more than 60 min when the cells were allowed to attach to a petri dish before induction. In contrast, it lasted only for 2.5 min when cells were in suspension (J. Clin. Invest. 72: 113-121, 1983). In spite of this report, the effect of cell adhesion has been ignored in most spin trapping studies of superoxide release from PMNs. This study shows that most PMNs in a quartz flat electron paramagnetic resonance (EPR) cuvette which was placed horizontally adhered to the wall within 3 min. In contrast, if the cuvette was placed vertically, only 20-30% of the cells became adherent in 30 min. We performed spin trapping studies using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap, and monitored the effect of cell adhesion on superoxide generation. When spin trapping was conducted on PMNs in suspension, the EPR signal of superoxide adduct (DMPO-OOH) was undetectable after stimulation with fMLP. However, PMNs which were allowed to adhere to the cuvette after stimulation generated superoxide for hours. Moreover, when PMNs were allowed to adhere prior to the stimulation, the magnitude of superoxide release was augmented three-to fourfold. Unlike fMLP, phorbol myristate acetate (PMA), which has been most commonly used in spin trapping studies, induced superoxide release which was not influenced by cell adhesion. We emphasize the importance of specifying the cell-adhesion-state in spin trapping studies.
达欣登等人报告称，当多形核白细胞（PMN）在诱导前附着于培养皿时，N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）诱导的超氧化物释放持续超过60分钟。相比之下，当细胞处于悬浮状态时，超氧化物释放仅持续2.5分钟（《临床研究杂志》72: 113 - 121, 1983）。尽管有此报告，但在大多数关于PMN超氧化物释放的自旋捕获研究中，细胞黏附的影响一直被忽视。本研究表明，水平放置的石英扁平电子顺磁共振（EPR）比色皿中的大多数PMN在3分钟内就会附着于壁上。相比之下，如果比色皿垂直放置，在30分钟内只有20 - 30%的细胞会黏附。我们使用5,5 - 二甲基吡咯啉 - N - 氧化物（DMPO）作为自旋捕获剂进行自旋捕获研究，并监测细胞黏附对超氧化物生成的影响。当对悬浮的PMN进行自旋捕获时，用fMLP刺激后超氧化物加合物（DMPO - OOH）的EPR信号无法检测到。然而，刺激后附着于比色皿的PMN会产生超氧化物达数小时之久。此外，当PMN在刺激前就被允许黏附时，超氧化物释放的幅度会增加三到四倍。与fMLP不同，在自旋捕获研究中最常用的佛波酯肉豆蔻酸酯（PMA）诱导的超氧化物释放不受细胞黏附的影响。我们强调在自旋捕获研究中明确细胞黏附状态的重要性。
