Induction of Ca2+ oscillations by selective, U73122-mediated, depletion of inositol-trisphosphate-sensitive Ca2+ stores in rabbit pancreatic acinar cells.

The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca2+ sequestering and releasing properties of internal Ca2+ stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca2+ uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P3]-sensitive, but not the Ins(1,4,5)P3-insensitive, Ca2+ store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca2+ indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca2+ concentration ([Ca2+]i,av), which was largely independent of external Ca2+. 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 [Ca2+]i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca(2+)-ATPase activity resulted in a [Ca2+]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)P3- casu quo agonist-sensitive internal Ca2+ store, whereas thapsigargin affects both the Ins(1,4,5)P3-sensitive and -insensitive Ca2+ store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca2+]i. The U73122-evoked oscillations were abolished in the absence of external Ca2+. The ability of U73122 to generate external Ca(2+)-dependent Ca2+ oscillations suggests that depletion of the agonist-sensitive store leads to an increase in Ca2+ permeability of the plasma membrane and that the Ins(1,4,5)P3-insensitive Ca2+ pool is necessary for the Ca2+ oscillations.