Structural requirements of CCK analogues to differentiate second messengers and pancreatic secretion

We previously demonstrated that, in rat pancreatic acinar cells, the high-affinity cholecystokinin (CCK) receptor agonist JMV-180 utilizes the phospholipase A2 (PLA2) cascade to mediate Ca2+ oscillations and amylase secretion. In contrast, the low-affinity CCK receptor utilizes the phospholipase C beta 1 (PLC beta 1) pathway. We have investigated structural requirements of CCK analogues to activate different intracellular pathways. CCK analogues such as CCK-8 [Met28,31; half-maximal effective concentration (EC50) = 0.4 pM], CCK-7 (Met28,31; EC50 = 0.7 pM), and NONA (Thr28/Nle31; EC50 = 5 pM) caused a biphasic amylase secretion. Reduction of secretion occurred with high doses of these peptides (> 100 pM). In contrast, CCK-5 (Met31; EC50 = 20,000pM), JMV-180 (Nle28,31; 1,500 pM), and OPE (Nle28,31; 200 pM) caused a monophasic secretion. CCK-8, but not JMV-180, increased protein kinase C (PKC) activities. The PKC activator phorbol ester inhibited an increase in myo-inositol 1,4,5-trisphosphate levels induced by CCK-8 and abolished monophasic amylase secretion induced by OPE. CCK-8, CCK-7, and NONA caused Ca2+ oscillations (< 100 pM) or large Ca2+ transients (> 100 pM). In contrast, JMV-180 and OPE evoked Ca2+ oscillations, even in high doses. Ca(2+)-signaling modes induced by CCK-5 were intermediate types between CCK-8 and JMV-180. CCK-8- and CCK-7-stimulated Ca2+ spikes were inhibited by the PLC inhibitor U-73122, but not by the PLA2 inhibitor ONO-RS-082. The action of CCK-5 was only partially sensitive to the PLC inhibitor. In contrast, JMV-180- and OPE-stimulated Ca2+ oscillations were inhibited by the PLA2, but not by the PLC, inhibitor. NONA was sensitive to PLC and PLA2 inhibitors. Although JMV-180 differs from CCK-8 by having an Asp-2 phenylethylester, rather than an Asp-phenylalanine amide, it is unlikely that these differences in the carboxyl terminus are important in determining which second-messenger systems will be activated. This is because CCK-5 (Phe33-CONH2) causes monophasic amylase secretion and Ca2+ oscillation in a manner similar to those induced by JMV-180 (2-phenylethylester). Meanwhile NONA (Phe33-CONH2) appeared to activate PLC and PLA2 pathways. The actions of all CCK analogues were abolished by L-364,718, indicating mediation by CCK-A receptors. Therefore, depending on the agonists used, CCK-A receptor activation in pancreatic acini may result in differential involvement of second-messenger systems, Ca2+ signal transduction, and amylase secretion. On the basis of the amino acid sequence of the carboxy terminus of CCK analogues, it appears that key amino acids for this differentiation are Met28 (or Thr28) for PLC pathways and Nle28 for PLA2 pathways.