Nuclear Calcium Signals in Immune Responses

我们用Ar离子激光(488 Nm)和He-Cd激光(325 Nm)共聚焦荧光显微镜研究了抗原特异性B细胞(三硝基苯酚B细胞克隆，TP67.21)和大鼠嗜碱性白血病细胞(RBL-2H3)受体介导的钙信号。经Ar+激光激发的B细胞和RBL-2H3细胞的共聚焦荧光图像比抗原刺激前变得更加明亮和不均匀。荧光强度随时间的变化是突然的，与用Fura-2常规荧光显微镜观察到的细胞内钙离子浓度[Ca^&lt；2+&gt；]_i非常相似。从钙离子图像的形态特征来看，B细胞(或RBL-2H3细胞)的部分明亮荧光似乎属于细胞核。为了证实上述情况，我们测量了细胞核的共聚焦荧光图像。在He-Cd激光激发下，共负载的Hoechst 33342(一种DNA特异性荧光探针)的荧光图像显示，在B细胞和Rbl-2H3细胞中，Fuo-3的荧光强度较亮的部分位于细胞核。细胞内钙泵抑制剂thapsigargin的加入也观察到了这些核钙信号。上述结果提示，B细胞和RBL-2H3细胞内钙离子的增加可能是B细胞和RBL-2H3细胞的第三信使。然后，我们试图确定增加的核内钙离子是从细胞质转移出来的，还是从核库释放出来的。我们使用Ba^lt；2+和Mn^lt；2+>；而不是Ca^&lt；2+>，因为已知Ba^&lt；2+和Mn^&lt；2+&gt；是通过Ca^&lt；2+&gt；通道进入的，但它们会猝灭Fluo-3荧光。结果表明，Ba2+-lt；2+-gt；和Mn2+-lt；2+-gt；既进入细胞核又进入细胞质，猝灭了胞核和细胞质中的Fluo-3荧光。这条建议

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