Imaging of Calcium Release in Inositol 1, 4, 5-trisphosphate-sensitive Internal Stores in Permeabilized HSY Cells Using Fluorescent Indicators

Abstract

We described methods for imaging the IP₃-induced Ca²⁺ release in Ca²⁺ storage organelles. IP₃-indnced changes in Ca²⁺ concentrations within Ca²⁺ stores ([Ca²⁺]_L in permeabilized HSY cells were monitored using the low affinity Ca²⁺ indicators, mag-fura-2 and mag-fura-red. The ratio images of mag-fura-2 were used to estimate the [Ca²⁺]_L, in the store. The apparent [Ca²⁺]_L, was 300-1000 μM at the cell periphery, whereas the [Ca²⁺]_L in the cytoplasm around the nucleus was 70-150 μM. The [Ca²⁺]_L throughout the cytoplasm was reduced by the application of 10 μM IP₃ to 30-70 μM, and could be largely recovered after removal of IP₃. The structure of IP₃-sensitive Ca²⁺ stores was investigated by confocal microscopy using mag-fura-red. An IP₃-induced increase in fluorescence was observed in the ER-like network and reticulum structures of the cytoplasm, and also in the nuclear envelope, suggesting that these organelles serve as IPs-sensitive Ca²⁺ stores. An analogous localization of the network and tubular elements of the ER was also demonstrated by electron microscopy. These observations suggest that these fluorescence techniques are useful to study the correlation between the distribution and function of Ca²⁺ stores.