1993 Fiscal Year Final Research Report Summary
Nuclear Calcium Signals in Immune Responses
| Project/Area Number |
04454621
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Nagoya City University |
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Principal Investigator |
NAKANISHI Mamoru Nagoya City Univ.Professor Fac.Pharm.Sci., 薬学部, 教授 (90090472)
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| Co-Investigator(Kenkyū-buntansha) |
FURUNO Tadahide Nagoya City Univ.Instructor Fac.Pharm.Sci., 薬学部, 助手 (80254308)
TORIGOE Chikako Nagoya City Univ.Lectyure Fac.Pharm.Sci., 薬学部, 講師 (70237163)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Immune Response / Calcium ion / Nuclear signal / Confocal Microscope / T-cell / B-cell / Basophil / Image Analysis |
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| Research Abstract |
We have studied receptor-mediated calcium signals in antigen-specific B cells (trinitrophenol-specific B cell clone, TP67.21) and rat basophilic leukemia cells (RBL-2H3) using a confocal fluorescence microscope with an argon ion laser (488nm) and a He-Cd laser (325nm). Confocal fluorescence images of fluo-3-loaded B cells and RBL-2H3 cells, excited by an argon ion laser, became much brighter and more nonhomogeneous than those before antigen stimulation. Time-dependent fluorescence changes in intensities were abrupt and quite similar to the patterns of the intracellular calcium ion concentration [Ca^<2+>]_i observed by a conventional fluorescence microscope using fura-2. From the morphological patterns of the calcium images, the parts of the bright fluorescence seemed to belong to the nucleus in B cells (or RBL-2H3 cells). To confirm the above events we measured the confocal fluorescence images of the nucleus. From the fluorescence images of co-loaded Hoechst 33342 (a DNA-specific fluorescence probe), which excited by a He-Cd laser, the brighter parts of the fluo-3 fluorescence intensities were identified to the nucleus in B cells and RBL-2H3 cells. These nuclear calcium signals were also observed by the addition of thapsigargin, an inhibitor of the intracellular calcium pump. These results suggested the possibility that the increased intranuclear calcium ions may play a nuclear third messenger in B cells and RBL-2H3 cells. Then, we tried to determine whether the increased intranuclear calcium ions are transferred from the cytoplasm or they are released from the nuclear stores. We used Ba^<2+> and Mn^<2+> instead of Ca^<2+>, because Ba^<2+> and Mn^<2+> are known to enter via Ca^<2+> channels but they quench the fluo-3 fluorescence. The results showed that Ba^<2+> and Mn^<2+> entered into the nucleus as well as into the cytoplasm and quenched the fluo-3 fluorescence both in the nucleus and in the cytoplasm. This sug
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