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-induced 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 IP3-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.