Monitoring of Ca2+ release from intracellular stores in permeabilized rat parotid acinar cells using the fluorescent indicators Mag-fura-2 and calcium green C18.

The operation of intracellular Ca2+ stores in saponin-permeabilized rat parotid acinar cells was studied by monitoring the Ca2+ concentration within organelles loaded with the low affinity Ca2+ indicator Mag-fura-2. Inositol 1, 4, 5-trisphosphate (InsP3) caused a decrease in the Mag-fura-2 ratio in a dose-dependent manner, and this effect was reversed by a removal of InsP3 or by an addition of the InsP3 receptor antagonist heparin. The changes in Mag-fura-2 ratio indicate the Ca2+ release from InsP3-sensitive Ca2+ stores and Ca2+ re-uptake into the stores in permeabilized acinar cells. The decrease in Mag-fura-2 ratio induced by InsP3 was observed at all regions of the acinar cells, suggesting that the InsP3-sensitive Ca2+ stores are located throughout the cells. The InsP3-induced Ca2+ release was also monitored using the membrane-bound Ca2+ indicator Calcium Green C18 which is sensitive to the changes in Ca2+ concentration immediately adjacent to the membrane of intracellular Ca2+ stores. InsP3 caused a large increase in the Calcium Green C18 fluorescence reflecting Ca2+ release from the stores. The Ca2+ pump inhibitor thapsigargin (ThG) itself had little or no effect on the Mag-fura-2 ratio or Calcium Green C18 fluorescence, but combined application of ThG with a low concentration of InsP3 evoked a significant decrease in the Mag-fura-2 ratio. This result supports the hypothesis that the ThG-induced Ca2+ release is due to InsP3-sensitive Ca2+ release which is mediated by the resting levels of InsP3. Further, none of cyclic ADP-ribose, caffeine or ryanodine changed the Mag-fura-2 ratio and Calcium Green C18 fluorescence, leading to the assumption that the ryanodine-sensitive Ca2+ stores are minor in rat parotid acinar cells.