SPATIAL AND TEMPORAL ASPECTS OF CA2-LAEVIS MELANOTROPE CELLS( OSCILLATIONS IN XENOPUS)

Spatio-temporal aspects of Ca2+ signaling in melanotrope cells of Xeno pus laevis have been studied with confocal laser-scanning microscopy. In the whole-frame scanning mode, two major intracellular Ca2+ compart ments, the cytoplasm and the nucleus, were visualized. The basal [Ca2] in the nucleus appeared to be lower than that in the cytoplasm and C a2+ oscillations seemed to arise synchronously in both compartments. T he N-type channel blocker omega-conotoxin eliminated oscillations in b oth 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 i s not a continuous process but consists of 3 or 4 discrete steps. Each step can be seen as a Ca2+-wave starting at the cell membrane and goi ng through the cytoplasm at a speed of 33.3 +/- 4.3 mu m/s. Before the Ca2+-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 mu m/s. Treatment with the Ca2+-ATPase inhibitor thapsigargin (1 mu M) almost completel y eliminated the apparent difference in the basal [Ca2+] in the cytopl asm and the nucleus, reduced the delay of a Ca2+-wave before entering the nucleus to 79.8 +/- 8.7 ms, and diminished the nuclear wave speed to 35.0 +/- 4.9 mu m/s. These results indicate that a cytoplasmic thap sigargin-sensitive ATPase near the nuclear envelope is involved in buf fering Ca2+ before the Ca2+ wave enters the nucleus. After sensitizing IP3 receptors by thimerosal (10 mu M) the speed of the cytoplasmic Ca 2+-wave was increased to 70.3 +/- 3.6 mu m/s, suggesting that IP3 rece ptors may be involved in the propagation of the cytoplasmic Ca2+ wave. Our results indicate that in melanotropes the generation and propagat ion of Ca2+ oscillations is a complex event involving influx of Ca2+ t hrough N-type Ca2+ channels, propagation of the cytoplasmic Ca2+ wave through mobilization of intracellular stores and a regulated Ca2+ entr y into the nucleus. We propose that Ca2+-binding proteins may act as a Ca2+ store for propagation of the wave in the nucleus.