Spatial and temporal aspects of Ca2+ oscillations in Xenopus laevis melanotrope cells

Spatio-temporal aspects of Ca2+ signaling in melanotrope cells of Xenopus laevis have been studied with confocal laser-scanning microscopy. In the whole-frame scanning mode, two major intracellular Ca2+ compartments, the cytoplasm and the nucleus, were visualized. The basal [Ca2+] in the nucleus appeared to be lower than that in the cytoplasm and Ca2+ oscillations seemed to arise synchronously in both compartments. The N-type channel blocker omega-conotoxin eliminated oscillations in both regions, indicating a strong coupling between the two compartments with respect to Ca2+ dynamics. Line-scanning mode, which gives higher time resolution, revealed that the rise phase of a Ca2+ oscillation is not a continuous process but consists of 3 or 4 discrete steps. Each step can be seen as a Ca(2+)-wave starting at the cell membrane and going through the cytoplasm at a speed of 33.3 +/- 4.3 microns/s. Before the Ca(2+)-wave enters the nucleus, a delay of 120.0 +/- 24.1 ms occurred. In the nucleus, the speed of a wave was 80.0 +/- 3.0 microns/s. Treatment with the Ca(2+)-ATPase inhibitor thapsigargin (1 MicroM) almost completely eliminated the apparent difference in the basal [Ca2+] in the cytoplasm and the nucleus, reduced the delay of a Ca(2+)-wave before entering the nucleus to 79.8 +/- 8.7 ms, and diminished the nuclear wave speed to 35.0 +/- 4.9 microns/s. These results indicate that a cytoplasmic thapsigargin-sensitive ATPase near the nuclear envelope is involved in buffering Ca2+ before the Ca2+ wave enters the nucleus. After sensitizing IP3 receptors by thimerosal (10 microM) the speed of the cytoplasmic Ca(2+)-wave was increased to 70.3 +/- 3.6 microns/s, suggesting that IP3 receptors may be involved in the propagation of the cytoplasmic Ca2+ wave. Our results indicate that in melanotropes the generation and propagation of Ca2+ oscillations is a complex event involving influx of Ca2+ through N-type Ca2+ channels, propagation of the cytoplasmic Ca2+ wave through mobilization of intracellular stores and a regulated Ca2+ entry into the nucleus. We propose that Ca(2+)-binding proteins may act as a Ca2+ store for propagation of the wave in the nucleus.