KINETICS OF CALCIUM STEPS UNDERLYING CALCIUM OSCILLATIONS IN MELANOTROPE CELLS OF XENOPUS-LAEVIS

Melanotrope cells of Xenopus laevis display intracellular calcium osci llations which are generated at the plasma membrane and travel as a wa ve through the cytoplasm into the nucleus. An oscillation involves dis crete increases in intracellular Ca2+ ('steps'), followed by a relativ ely smooth return to the basal Ca2+ level, The aim of our investigatio n was to determine what role these steps play in shaping the Ca2+ sign al in melanotrope cells, by conducting a high resolution spatio-tempor al analysis of the kinetics of the Ca2+ steps. To this end Fura-red lo aded cells were analysed by confocal laser scanning microscopy using t he line scanning method to achieve 6 ms time resolution. Furthermore, the kinetics of the steps were analysed in 3 different intracellular a reas, to see if there are spatial differences in Ca2+ signalling kinet ics. The results showed that each calcium oscillation is built up by 3 -4 steps that were generated very quickly anti had approximately the s ame size. Following each Ca2+ step, there was a slow removal of calciu m before the next step boosted the overall level of Ca2+. Since the Ca 2+ steps were most pronounced directly beneath the plasma membrane, th ey appear to be generated in this region. The speed of the Ca2+ wave n ear the membrane exceeded 40 mu m/s, indicating an active mechanism fo r wave propagation. In deeper regions of the cell, the wave speed was much slower (about 8 mu m/s) and the size of each step was smaller, in dicating that regulation occurs within a narrower range of [Ca2+](i). Inside the nucleus, however, the calcium wave accelerated again (23 mu m/s). Treatment with TRH evoked a high amplitude Ca2+ transient and i ncreased the number of Ca2+ steps to 5 or 6. Each step had approximate ly the sane size as the steps of the pretreatment Ca2+ oscillations. C affeine treatment, which increased the frequency of the oscillations, had no effect on the number or the size of the Ca2+ steps, but it redu ced the time needed for each step to reach its maximum height. We sugg est 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 oscill ations. Both phenomena may play a crucial role in the encoding of info rmation transduced from an extracellular input to the intracellular ta rget.