Simultaneous measurement of superoxide generation and intracellular Ca2+ concentration reveals the effect of extracellular Ca2+ on rapid and transient contents of superoxide generation in differentiated THP-1 cells.
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.
Ishibashi
Kaname
K
Laboratory of Molecular Biophotonics, 5000 Hirakuchi, Hamamatsu 434-8555, Japan. kaname.ishibashi@ims.jti.co.jp
Okazaki
Shigetoshi
S
Hiramatsu
Mitsuo
M
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D045604:Extracellular Fluid; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D007700:Kinetics; D008657:Metabolic Clearance Rate; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
D013481:Superoxides; D002118:Calcium