Kinetics of calcium steps underlying calcium oscillations in melanotrope cells of Xenopus laevis.

Melanotrope cells of Xenopus laevis display intracellular calcium oscillations which are generated at the plasma membrane and travel as a wave through the cytoplasm into the nucleus. An oscillation involves discrete increases in intracellular Ca2+ ('steps'), followed by a relatively smooth return to the basal Ca2+ level. The aim of our investigation was to determine what role these steps play in shaping the Ca2+ signal in melanotrope cells, by conducting a high resolution spatio-temporal analysis of the kinetics of the Ca2+ steps. To this end Fura-red loaded cells were analysed by confocal laser scanning microscopy using the line scanning method to achieve 6 ms time resolution. Furthermore, the kinetics of the steps were analysed in 3 different intracellular areas, to see if there are spatial differences in Ca2+ signalling kinetics. The results showed that each calcium oscillation is built up by 3-4 steps that were generated very quickly and had approximately the same size. Following each Ca2+ step, there was a slow removal of calcium before the next step boosted the overall level of Ca2+. Since the Ca2+ steps were most pronounced directly beneath the plasma membrane, they appear to be generated in this region. The speed of the Ca2+ wave near the membrane exceeded 40 microns/s, indicating an active mechanism for wave propagation. In deeper regions of the cell, the wave speed was much slower (about 8 microns/s) and the size of each step was smaller, indicating that regulation occurs within a narrower range of [Ca2+]i. Inside the nucleus, however, the calcium wave accelerated again (23 microns/s). Treatment with TRH evoked a high amplitude Ca2+ transient and increased the number of Ca2+ steps to 5 or 6. Each step had approximately the same size as the steps of the pretreatment Ca2+ oscillations. Caffeine treatment, which increased the frequency of the oscillations, had no effect on the number or the size of the Ca2+ steps, but it reduced the time needed for each step to reach its maximum height. We suggest a possible 'building block' function for the Ca2+ steps, whereby a cell generates more steps to achieve a high oscillation amplitude or accelerates the speed of the steps to increase the frequency of oscillations. Both phenomena may play a crucial role in the encoding of information transduced from an extracellular input to the intracellular target.