Ishibashi Kaname, Okazaki Shigetoshi, Hiramatsu Mitsuo
Laboratory of Molecular Biophotonics, 5000 Hirakuchi, Hamamatsu 434-8555, Japan.
Biochem Biophys Res Commun. 2006 Jun 2;344(2):571-80. doi: 10.1016/j.bbrc.2006.02.173. Epub 2006 Mar 10.
We invented a simultaneous measuring instrument of fluorescence and chemiluminescence, realizing the analysis of chronological correlation between change in intracellular Ca2+ concentration ([Ca2+]i) and superoxide generation. A human monocytic cell line, THP-1, differentiated to be neutrophil-like cells generated superoxide with increase in intracellular Ca2+ concentration when stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) whereas PMA, phorbol ester-stimulated superoxide response occurred without change in [Ca2+]i. The cells treated with TMB-8, an intracellular Ca2+ antagonist, generated superoxide rapidly as well as transiently with transient [Ca2+]i elevation after stimulation with fMLP, whereas EGTA-treated cells generated superoxide slowly as well as persistently with transient [Ca2+]i elevation after the stimulation. These results suggest that the rapid and transient contents of superoxide generation are specific for Ca2+ influx from the extracellular domain. Verapamil, voltage-dependent Ca2+ channel blocker, dose-dependently inhibited fMLP-stimulated extracellular Ca2+ influx and superoxide generation without affecting PMA-stimulated superoxide generation. Other channel blockers tested, nifedipine and diltiazem, similarly inhibited these fMLP-stimulated responses. Numerical analysis of the values of the response curves elucidated that TMB-8 or the channel blocker reveals or eliminates the same contents of superoxide generation by the antagonism of intracellular Ca2+ release or extracellular Ca2+ influx, respectively. Taking these results together, the characteristic extracellular Ca2+ influx essential for superoxide generation was first revealed by the simultaneous measurement of superoxide generation and change in [Ca2+]i.
我们发明了一种荧光和化学发光同步测量仪器，实现了对细胞内钙离子浓度（[Ca2+]i）变化与超氧化物生成之间时间相关性的分析。一种人类单核细胞系THP-1分化为类中性粒细胞，在用甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）刺激时，随着细胞内钙离子浓度升高而产生超氧化物，而佛波酯PMA刺激产生的超氧化物反应在[Ca2+]i无变化的情况下发生。用细胞内钙离子拮抗剂TMB-8处理的细胞，在用fMLP刺激后，随着[Ca2+]i短暂升高，迅速且短暂地产生超氧化物，而用乙二醇双四乙酸（EGTA）处理的细胞在刺激后随着[Ca2+]i短暂升高，缓慢且持续地产生超氧化物。这些结果表明，超氧化物生成的快速和短暂含量是细胞外区域钙离子内流所特有的。电压依赖性钙离子通道阻滞剂维拉帕米剂量依赖性地抑制fMLP刺激的细胞外钙离子内流和超氧化物生成，而不影响PMA刺激的超氧化物生成。所测试的其他通道阻滞剂硝苯地平和地尔硫卓同样抑制这些fMLP刺激的反应。对反应曲线值的数值分析表明，TMB-8或通道阻滞剂分别通过拮抗细胞内钙离子释放或细胞外钙离子内流，揭示或消除了相同的超氧化物生成含量。综合这些结果，通过同时测量超氧化物生成和[Ca2+]i变化，首次揭示了超氧化物生成所必需的特征性细胞外钙离子内流。
