Confocal fluorescence microscopy for studying thapsigargin-induced bivalent-cation entry into B cells.
We studied thapsigargin-induced bivalent-cation entry into antigen-specific B cells (TP67.21) with a confocal fluorescence microscope. Confocal fluorescence images of fluo-3-loaded B cells showed that thapsigargin-stimulated Ca2+ signals were transferred not only to the cytoplasm but also to the nucleus. In the absence of external Ca2+ ions, the free Ca2+ concentrations both in the cytosol and in the nucleus declined to basal levels by 5 min after addition of thapsigargin. However, subsequent addition of Ca2+ in the external medium made the fluo-3 (fura-2) fluorescence intensity rise, reflecting the fact that Ca2+ accumulated again in the nucleus as well as in the cytoplasm. Then, we added Ba2+ and Mn2+ instead of Ca2+, because Ba2+ and Mn2+ are known to enter via Ca2+ channels. The addition of Ba2+ and Mn2+ in the external medium quenched the fluo-3 fluorescence both in the nucleus and in the cytoplasm of B cells. This suggested the possibility that the increase in intranuclear Ca2+ after thapsigargin stimulation may come from the cytoplasm, not from the nuclear stores.
Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
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Y
Okamoto
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Furuno
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T
Hamano
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Nakanishi
D000814:Aniline Compounds; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D001464:Barium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014966:Xanthenes
D000814:Aniline Compounds; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D013729:Terpenes; D014966:Xanthenes; C059715:Fluo-3; D001464:Barium; D008345:Manganese; D019284:Thapsigargin; D002118:Calcium