sincalide and arachidonyltrifluoromethane

High-affinity cholecystokinin (CCK) receptors were reported to be coupled with phospholipase A2 (PLA2)-arachidonic acid (AA) pathways to mediate Ca2+ oscillations and amylase secretion in rat pancreatic acinar cells. To investigate which types of PLA2 were involved in PLA2-AA pathways, the effects of specific inhibitors for type II and type IV PLA2 on Ca2+ oscillations and amylase secretion were studied in isolated rat pancreatic acini. An inhibitor of type IV (cytosolic) PLA2, AACOCF3 inhibited Ca2+ oscillations elicited by CCK-8 (30 pM) and JMV-180 (100 nM). AACOCF3 inhibited amylase secretion stimulated by JMV-180 and low concentrations of CCK-8 (< or =30 pM). On the other hand, an inhibitor of type II (secretory, nonpancreatic) PLA2 had no effects on Ca2+ oscillations and amylase secretion stimulated by CCK-8 and JMV-180. These results suggest that high-affinity CCK receptors are coupled to cytosolic PLA2 to mediate Ca2+ oscillations and amylase secretion in rat pancreatic acinar cells.

Topics: Amylases; Animals; Arachidonic Acid; Arachidonic Acids; Calcium Signaling; Enzyme Inhibitors; Isoenzymes; Male; Pancreas; Phospholipases A; Phospholipases A2; Rats; Rats, Wistar; Receptors, Cholecystokinin; Sincalide

In rat pancreatic acini, we previously demonstrated that depending on the agonist used, activation of cholecystokinin type A (CCKA) receptor (CCK-AR) results in the differential involvement of the cytosolic phospholipase A2 (cPLA2), phospholipase Cbeta1 (PLCbeta1) and Src/protein tyrosine kinase (PTK) pathways. The high-affinity CCK-AR appears to be coupled to the Gbeta/cPLA2/arachidonic acid (AA) cascade in mediating Ca2+ oscillations. The low-affinity CCK-AR is coupled to both the Galphaq/11/PLCbeta1/inositol 1,4,5-trisphosphate (IP3) to evoke intracellular Ca2+ release and the Src/PTK pathway to mediate extracellular Ca2+ influx. The objectives of this study were to provide evidence that cPLA2 is present in pancreatic acini and to evaluate the possibility that its activation results in Ca2+ oscillations and amylase secretion. Furthermore, we investigated the mechanism of Ca2+ oscillations mediated by the high-affinity CCK-AR. In rat pancreatic acini, immunoprecipitation studies using an anti-cPLA2 monoclonal antibody, demonstrated a cPLA2 band at the location of 110 kDa. A selective inhibitor of cPLA2, AACOCF3 (100 microM), inhibited production of AA metabolites, Ca2+ oscillations and amylase secretion elicited by the high-affinity CCK-AR agonist, CCK-OPE (10-1000 nM). In addition, through the repetitive release of intracellular Ca2+, CCK-OPE enhanced phosphotransferase activities of Ca2+/calmodulin-dependent protein kinase type IV (CaMK IV), which were inhibited by AACOCF3. The CaMK inhibitor, K252-a (1-3 microM), also abolished basal and CCK-OPE-stimulated CaMK IV activities. The CaM inhibitor, W-7 (100 microM), and K252-a inhibited Ca2+ oscillations and amylase secretion evoked by CCK-OPE without affecting the AA formation. Therefore, it appears that Ca2+ oscillations elicited by the high-affinity CCK-AR/Gbeta/cPLA2/AA pathway activate CaMK IV. Activated CaMK, in turn, regulates Ca2+ oscillations through a positive feedback mechanism to mediate pancreatic exocytosis.

Topics: Affinity Labels; Amylases; Animals; Antibodies, Monoclonal; Arachidonic Acid; Arachidonic Acids; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cytosol; Enzyme Inhibitors; Feedback; Indole Alkaloids; Kinetics; Leukotriene C4; Leukotriene D4; Leukotriene E4; Male; Models, Biological; Pancreas; Peptide Fragments; Phospholipases A; Phospholipases A2; Precipitin Tests; Rats; Rats, Sprague-Dawley; Receptor, Cholecystokinin A; Receptors, Cholecystokinin; Sincalide; Sulfonamides

A specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), was found to inhibit the regulated exocytosis of pancreatic acinar AR42J cells. When AR42J cells were stimulated with cholecystokinin octapeptide (CCK-8), the regulated exocytosis monitored by amylase release was rapidly activated and increased by 2.5-fold during one hour. After AR42J cells were treated with AACOCF3, amylase release by CCK-8 remained at the basal level. Thus, changes in the composition of membrane phospholipids before and after stimulation were investigated. Within 1 min after CCK-8 stimulation, lysophosphatidylethanolamine (lysoPE) in the cellular membranes of AR42J was increased while lysophosphatidylcholine stayed unchanged. In the presence of AACOCF3, lysoPE was not increased by CCK-8. Those results indicate that the increment of lysoPE is linked to the regulated exocytosis.

Topics: Amylases; Animals; Arachidonic Acids; Cell Membrane; Cells, Cultured; Dexamethasone; Dose-Response Relationship, Drug; Enzyme Inhibitors; Exocytosis; Lysophospholipids; Membrane Lipids; Pancreas; Phospholipases A; Phospholipases A2; Phospholipids; Rats; Sincalide