CALCIUM SIGNALING 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 mu M). 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 mu M range. Some cells (10-20%) responded with additional asynchronous 0.5-1.5 mu M 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.