Induction of Ca super(2+) oscillations by selective, U73122-mediated, depletion of inositol-trisphosphate-sensitive Ca super(2+) stores in rabbit pancreatic acinar cells

The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca super(2+) sequestering and releasing properties of internal Ca super(2+) stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca super(2+) uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P sub(3)]-sensitive, but not the Ins(1,4,5)P sub(3)-insensitive, Ca super(2+) store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca super(2+) indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca super(2+) concentration ([Ca super(2+)] sub(i,av)), which was largely independent of external Ca super(2+). Addition of U73122 to cell suspensions prestimulated with either cholecystokinin octapeptide or JMV-180 revealed an inverse relationship in size between the U73122- and the agonist-evoked [Ca super(2+)] sub(i,av) transient. Moreover, thapsigargin-induced inhibition of intracellular Ca super(2+)-ATPase activity resulted in a [Ca super(2+)] sub(i,av) transient, the size of which was not different following maximal prestimulation with either U73122 or agonist. These observations suggest that U73122 selectively affects the Ins(1,4,5)P sub(3)- casu quo agonist-sensitive internal Ca super(2+) store, whereas thapsigargin affects both the Ins(1,4,5)P sub(3)-sensitive and -insensitive Ca super(2+) store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca super(2+)] sub(i). The U73122-evoked oscillations were abolished in the absence of external Ca super(2+). The ability of U73122 to generate external Ca super(2+)-dependent Ca super(2+) oscillations suggests that depletion of the agonist-sensitive store leads to an increase in Ca super(2+) permeability of the plasma membrane and that the Ins(1,4,5)P sub(3)-insensitive Ca super(2+) pool is necessary for the Ca super(2+) oscillations.