Evidence that Ca2+-waves in Xenopus melanotropes depend on calcium-induced calcium release: a fluorescence correlation microscopy and linescanning study.

The neuroendocrine melanotrope cell displays Ca2+ oscillations that are build up by several discrete Ca2+ rises ('steps'). Each step is linked to Ca2+-entry across the plasma membrane via voltage-operated calcium channels and associated with a fast Ca2+-wave travelling from the plasma membrane to the central parts of the cell. Previously, linescanning with confocal laser scanning microscopy (CLSM) supported that these waves have high speeds (between 30 and 80 microm/s), which is considered indicative of the involvement of a calcium-induced calcium release (CICR) mechanism in fast-wave propagation. However, to firmly establish the presence of a CICR mechanism one must rule out the possibility that the Ca2+ signal is artifactually accelerated by the presence of a highly mobile Ca2+ probe and also eliminate imaging artifacts inherent to single wavelength imaging. In the present study both problems are addressed. Mobility and intracellular distribution of a generally used Ca2+ probe, Oregon-green 488 BAPTA-1 (O-green-1), were established using fluorescence correlation microscopy. We then used the ratio signal of co-loaded O-green-1 and Fura-Red to quantify the relative [Ca2+]i during linescanning. It was found that O-green-1 displays different diffusion times when regions near the plasma membrane and in the center of the cell are compared. However, the calculated diffusion constant of the probe was too low to account for the observed high speed of the Ca2+ wave. In conclusion, we established the authenticity of the high speed of Ca2+-waves in Xenopus melanotropes, providing evidence for the involvement of a CICR mechanism in wave propagation.