COMPARTMENTALIZATION OF CA2- IMPLICATIONS FOR THE QUANTAL BEHAVIOR OFCA2+ RELEASE( SIGNALING AND CA2+ POOLS IN PANCREATIC ACINI )

Streptolysin O-permeabilized pancreatic acini were used to study compa rtmentalization of Ca2+ signaling and Ca2+ pools. In these cells, the inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ channels could be ac tivated by a number of agonists (carbachol, cholecystokinin, or bombes in) or by activation of the entire cellular phospholipase C pool with GTP gamma S. Surprisingly, each of the antagonists interacting with ac inar cells inactivated the channels after stimulation with GTP gamma S . In addition, when permeabilized cells were stimulated with more than one agonist, any antagonist to the specific agonists employed inactiv ated the channels. The aberrant behavior of the antagonists in permeab le cells was not related to a loss of specificity since (a) when added before GTP gamma S, the antagonists had no effect on Ca2+ release and (b) when cells were stimulated with a single agonist, the antagonists prevented only the effect of their specific agonist. The differential behavior of the antagonists in intact and permeable cells suggests a compartmentalization of Ca2+ signaling into separate, agonist-specific units that is modified by cell permeabilization. Further evidence for compartmentalization of signaling was obtained by showing that the pa rtial agonist (the CCK octapeptide analogue JMV-180) can access and re lease only 50% of the cholecystokinin or IP3-mobilizabIe Ca2+ pool in intact and permeable cells. Kinetic measurements revealed a multiphasi c time course of agonist-evoked Ca2+ release in permeable cells. At hi gh agonist concentrations, all phases were fast and merged into an app arent single event of Ca2+ release. The phases were separated by three independent protocols: reduction in agonist concentrations, addition of heparin, or addition of guanosine-5'-O-(thio)diphosphate. Since all protocols that caused phase separation reduce IP3-mediated Ca2+ relea se, these findings demonstrate heterogeneity in the affinity for IP3 o f channels present in compartmentalized Ca2+ pools of the same cells. Compartmentalization of signaling and the heterogeneity in the affinit y for IP3 resulted in a quantal agonist-evoked Ca2+ release. The overa ll findings are discussed in the context of an integrated model of com partmentalization of signaling complexes, Ca2+ pools, and IP3-activate d Ca2+ channels.