CALCIUM SIGNALING IN INDIVIDUAL T-CELLS MEASURED BY CONFOCAL MICROSCOPY

Laser-scanning confocal microscopy was used in conjunction with a high ly fluorescent Ca2+ indicator fluo-3 to visualize real-time alteration s in the intracellular Ca2+ concentration ([Ca2+](i)) in individual li ving Jurkat T-cells during the first minutes of activation by phytohae magglutinin (PHA) at the physiological temperature (37 degrees C). Wit h a delay of 30-120 s, PHA induced a strong [Ca2+](i) peak in the micr omolar 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 th e 0.2-0.5 mu M range. Some cells (10-20%) responded with additional as ynchronous 0.5-1.5 mu M peaks during the plateau phase. These oscillat ions continued for 10-20 minutes. The spans of the peaks ranged from 3 0 to 100 s, intervals between peaks varied from 60 to 300 s. It was sh own that the initial [Ca2+](i) peak was associated with Ca2+ mobilisat ion from internal sources, whereas the plateau was maintained by an in flux of Ca2+ from external medium. In K+-rich medium or in the presenc e of quinine, a K+ channel blocker, no secondary response to PHA-activ ation characterised by an elevated plateau was observed. The data sugg est that the Ca2+ influx was dependent on the membrane potential and/o r the extracellular K+-concentration. Optical sectioning showed that t he intracellular Ca2+ distributed almost homogeneously throughout the cell volume both in control and in PHA-stimulated cells including thos e exhibiting Ca2+ oscillations. This suggests that Ca2+ signals are lo calized not only in cytoplasm at the cell plasma membrane but can be a lso transferred directly into the nucleus.