Spatial and temporal aspects of Ca 2+ oscillations in Xenopus laevis melanotrope cells

Abstract

Spatio-temporal aspects of Ca 2+ signaling in melanotrope cells of Xenopus laevis have been studied with confocal laser-scanning microscopy. In the whole-frame scanning mode, two major intracellular Ca 2+ compartments, the cytoplasm and the nucleus, were visualized. The basal [Ca 2+] in the nucleus appeared to be lower than that in the cytoplasm and Ca 2+ oscillations seemed to arise synchronously in both compartments. The N-type channel blocker ω-conotoxin eliminated oscillations in both regions, indicating a strong coupling between the two compartments with respect to Ca 2+ dynamics. Line-scanning mode, which gives higher time resolution, revealed that the rise phase of a Ca 2+ 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 μm/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 μm/s. Treatment with the Ca 2+-ATPase inhibitor thapsigargin (1 μM) almost completely eliminated the apparent difference in the basal [Ca 2+] 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 μ/s. These results indicate that a cytoplasmic thapsigargin-sensitive ATPase near the nuclear envelope is involved in buffering Ca 2+ before the Ca 2+ wave enters the nucleus. After sensitizing IP 3 receptors by thimerosal (10 μM) the speed of the cytoplasmic Ca 2+-wave was increased to 70.3 ± 3.6 μ/s, suggesting that IP 3 receptors may be involved in the propagation of the cytoplasmic Ca 2+ wave. Our results indicate that in melanotropes the generation and propagation of Ca 2+ oscillations is a complex event involving influx of Ca 2+ through N-type Ca 2+ channels, propagation of the cytoplasmic Ca 2+ wave through mobilization of intracellular stores and a regulated Ca 2+ entry into the nucleus. We propose that Ca 2+-binding proteins may act as a Ca 2+ store for propagation of the wave in the nucleus.