title: Kinetics of calcium steps underlying calcium oscillations in melanotrope cells of Xenopus laevis.
abstract: 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.
authors.affiliation: Department of Cellular Animal Physiology, University of Nijmegen, The Netherlands. werkoop@sci.kun.nl
authors.forename: W J
authors.initials: WJ
authors.lastname: Koopman
authors.forename: W J
authors.initials: WJ
authors.lastname: Scheenen
authors.forename: E W
authors.initials: EW
authors.lastname: Roubos
authors.forename: B G
authors.initials: BG
authors.lastname: Jenks
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D016257:Fura-2; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D018613:Microscopy, Confocal; D010902:Pituitary Gland / Q000166:cytology* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D005456:Fluorescent Dyes; D002110:Caffeine; D013973:Thyrotropin-Releasing Hormone; D002118:Calcium; D016257:Fura-2