High-affinity cholecystokinin type A receptor/cytosolic phospholipase A(2)pathways mediate Ca2+ oscillations via a positive feedback regulation by calmodulin kinase in pancreatic acini

In rat pancreatic acini, we previously demonstrated that depending on the a gonist used, activation of cholecystokinin type A (CCKA) receptor (CCK-AR) results in the differential involvement of the cytosolic phospholipase A(2) (cPLA(2)), phospholipase C-beta 1 (PLCbeta 1) and Src/protein tyrosine kin ase (PTK) pathways, The high-affinity CCK-AR appears to be coupled to the G beta/cPLA(2)/arachidonic acid (AA) cascade in mediating Ca2+ oscillations. The low-affinity CCK-AR is coupled to both the G alpha(q/11)/PLCbeta 1/ino sitol 1,4,5-trisphosphate (IP3 to evoke intracellular Calf release and the Src/PTK pathway to mediate extracellular Ca2+ influx. The objectives of thi s study were to provide evidence that cPLA(2) is present in pancreatic acin i and to evaluate the possibility that its activation results in Ca2+ oscil lations and amylase secretion. Furthermore, we investigated the mechanism o f Ca2+ oscillations mediated by the high-affinity CCK-AR. In rat pancreatic acini, immunoprecipitation studies using an anti-cPLA(2) monoclonal antibo dy, demonstrated a cPLA(2) band at the location of 110 kDa. A selective inh ibitor of cPLA(2), AACOCF(3) (100 CIM), inhibited production of AA metaboli tes, Ca2+ oscillations and amylase secretion elicited by the high affinity CCK AR agonist, CCK OPE (10-1000 nM). In addition, through the repetitive r elease of intracellular Ca2+ CCK-OPE enhanced phospho-transferase activitie s of Ca2+/calmodulin-dependent protein kinase type TV (CaMK IV), which were inhibited by AACOCF(3). The CaMK inhibitor, K252-a (1-3 mu M), also abolis hed basal and CCK-OPE-stimulated CaMK IV activities. The CaM inhibitor, W-7 (100 mu M), and K252-a inhibited Ca2+ oscillations and amylase secretion e voked by CCK-OPE without affecting the AA formation. Therefore, it appears that Ca2+ oscillations elicited by the high-affinity CCK-AR/G beta/cPLA(2)/ AA pathway activate CaMK IV. Activated CaMK, in turn, regulates Ca2+ oscill ations through a positive feedback mechanism to mediate pancreatic exocytos is.