Calcium signalling in individual T-cells measured by confocal microscopy.
Laser-scanning confocal microscopy was used in conjunction with a highly fluorescent Ca2+ indicator fluo-3 to visualize real-time alterations in the intracellular Ca2+ concentration ([Ca2+]i) in individual living Jurkat T-cells during the first minutes of activation by phytohaemagglutinin (PHA) at the physiological temperature (37 degrees C). With a delay of 30-120 s, PHA induced a strong [Ca2+]i peak in the micromolar range (1-3 microM). The rise in [Ca2+]i lasted for 1-2 minutes, and was followed by a sustained plateau of elevated [Ca2+]i in the 0.2-0.5 microM range. Some cells (10-20%) responded with additional asynchronous 0.5-1.5 microM peaks during the plateau phase. These oscillations continued for 10-20 minutes. The spans of the peaks ranged from 30 to 100 s, intervals between peaks varied from 60 to 300 s. It was shown that the initial [Ca2+]i peak was associated with Ca2+ mobilisation from internal sources, whereas the plateau was maintained by an influx of Ca2+ from external medium. In K(+)-rich medium or in the presence of quinine, a K+ channel blocker, no secondary response to PHA-activation characterised by an elevated plateau was observed. The data suggest that the Ca2+ influx was dependent on the membrane potential and/or the extracellular K(+)-concentration. Optical sectioning showed that the intracellular Ca2+ distributed almost homogeneously throughout the cell volume both in control and in PHA-stimulated cells including those exhibiting Ca2+ oscillations. This suggests that Ca2+ signals are localized not only in cytoplasm at the cell plasma membrane but can be also transferred directly into the nucleus.
Maltsev
V A
VA
Institute of Immunology, Ministry of Health of Russia, Moscow.
Wolff
B
B
Hess
J
J
Werner
G
G
D000814:Aniline Compounds; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D018613:Microscopy, Confocal; D010835:Phytohemagglutinins / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D010835:Phytohemagglutinins; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium