MONITORING OF CA2-CELLS USING THE FLUORESCENT INDICATORS MAG-FURA-2 AND CALCIUM GREEN C-18( RELEASE FROM INTRACELLULAR STORES IN PERMEABILIZED RAT PAROTID ACINAR)

The operation of intracellular Ca2+ stores in saponin-permeabilized ra t parotid acinar cells was studied by monitoring the Ca2+ concentratio n within organelles loaded with the low affinity Ca2+ indicator Mag-fu ra 2. Inositol 1, 4, 5-trisphosphate (InsP(3)) caused a decrease in th e Mag-fura-a ratio in a dose-dependent manner, and this effect was rev ersed by a removal of InsP(3) or by an addition of the InsP(3) recepto r antagonist heparin. The changes in Mag-fura-2 ratio indicate the Ca2 + release from InsP(3)-sensitive Ca2+ stores and Ca2+ re-uptake into t he stores in permeabilized acinar cells. The decrease in Mag-fura-2 ra tio induced by InsP(3) was observed at all regions of the acinar cells , suggesting that the InsP(3)-sensitive Ca2+ stores are located throug hout the cells. The InsP(3)-induced Ca2+ release was also monitored us ing the membrane-bound Ca2+ indicator Calcium Green C-18 which is sens itive to the changes in Ca2+ concentration immediately adjacent to the membrane of intracellular Ca2+ stores. InsP(3) caused a large increas e in the Calcium Green C-18 fluorescence reflecting Ca2+ release from the stores. The Ca2+ pump inhibitor thapsigargin (ThG) itself had litt le or no effect on the Mag-fura-2 ratio or Calcium Green C-18 fluoresc ence, but combined application of ThG with a low concentration of InsP (3) evoked a significant decrease in the Mag-fura-2 ratio. This result supports the hypothesis that the ThG-induced Ca2+ release is due to I nsP(3)-sensitive Ca2+ release which is mediated by the resting levels of InsP(3). Further, none of cyclic ADP-ribose, caffeine or ryanodine changed the Mag-fura-2 ratio and Calcium Green C-18 fluorescence, lead ing to the assumption that the ryanodine sensitive Ca2+ stores are min or in rat parotid acinar cells. (C) 1997 Academic Press.