sincalide and 2-(4-amylcinnamoyl)amino-4-chlorobenzoic-acid

In an attempt to examine the structure-activity relationship of the cholecystokinin (CCK) peptide, we examined the structural motif of CCK truncated peptides responsible for rat pancreatic acinar amylase secretion and signal transduction. CCK-6 (Met28-Gly29-Trp30-Met31-Asp32-Phe33 -NH2), CCK-5 [CCK-(29-33)], and CCK-4 [CCK-(30-33)] caused monophasic amylase secretion with EC50 values of 3, 20, and 30 nM, respectively. CCK-7 [Tyr(SO3H)27 plus CCK-(28-33)] evoked biphasic secretion with an EC50 of 0.7 pM, whereas CCK-3 [CCK-(31-33)] had no effect, suggesting the importance of Tyr(SO3H)27 and Trp30. Whereas CCK-7 [Tyr(SO3H)27/ Met28] evoked biphasic amylase secretion, CCK-OPE [Tyr(SO3H)27/Nle28] and CCK-6 (Met28) caused monophasic secretion. Thus Tyr(SO3H)27/Met28 appears to be required for biphasic secretion. CCK-8-OH and CCK-5-OH did not cause amylase secretion and Ca2+ spiking, suggesting the importance of Phe33 amidation. Similar to CCK-8, CCK-6 and CCK-4 caused Ca2+ oscillations at low doses and large Ca2+ transients at high doses. In contrast, similar to JMV-180 and CCK-OPE, CCK-5 elicited Ca2+ oscillations at all concentrations. Phospholipase C (PLC) inhibitor inhibited amylase secretion induced by high doses of CCK-6 and CCK-4 but not by CCK-5. Protein tyrosine kinase (PTK) inhibitor only inhibited the action of high doses of CCK-4. Neither PLC inhibitor nor PTK inhibitor affected amylase secretion evoked by low doses of CCK-6, CCK-5, and CCK-4. In contrast to its actions on JMV-180 and CCK-OPE, phospholipase A2 (PLA2) inhibitor had no effect on the action evoked by all CCK short peptides. CCK-6, CCK-5, and CCK-4 caused a 2.1- to 3.2-fold increase in intracellular inositol 1,4,5-trisphosphate levels over basal. None of these CCK short peptides increased intracellular arachidonic acid levels. CCK-6 and CCK-5 did not stimulate PTK activity, whereas CCK-4 evoked a 3.2- to 5.3-fold increase over basal. We conclude that Tyr(SO3H)27, Trp30, and Phe33-CONH2 of the CCK peptide are key amino acids in evoking amylase secretion. At low doses, CCK-6, CCK-5, and CCK-4 utilize some yet to be identified pathways to evoke Ca2+ oscillations and amylase secretion. At high doses, CCK-6 and CCK-4 utilize PLC but not PLA2 pathways. CCK-4 possesses the minimal essential amino acids to fully activate the PTK pathway.

Topics: Aminobenzoates; Amylases; Animals; Calcium; Cells, Cultured; Chlorobenzoates; Cholecystokinin; Cinnamates; Enzyme Inhibitors; Estrenes; Kinetics; Male; ortho-Aminobenzoates; Pancreas; Peptide Fragments; Phosphodiesterase Inhibitors; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Signal Transduction; Sincalide; Tetragastrin; Type C Phospholipases

In pancreatic acini, administration of the phospholipase C inhibitor, U-73122, abolished Ca2+ oscillations and amylase secretion induced by CCK but had much less effect on the action of CCK analog JMV-180. In contrast, the phospholipase A2 inhibitor, ONO-RS-082, inhibited both Ca2+ spikes and amylase secretion induced by JMV-180, but it had little effect on the action of CCK-8. Both arachidonic acid (AA) and a cytochrome P-450 inhibitor, SKF-96365, generated Ca2+ spikes from the agonist-sensitive pool. AA was capable of releasing Ca2+ from the endoplasmic reticulum (ER), suggesting the direct Ca2+ releasing pathway. There is no evidence of Ca(2+)-induced Ca2+ release (CICR) since neither caffeine, a CICR potentiator, nor ryanodine, a CICR inhibitor, modulated agonist-induced Ca2+ oscillations and Ca2+ release from the ER. On the contrary, increasing concentrations of caffeine abolished agonist-induced Ca2+ spikes. Therefore we have demonstrated that depending on the agonists used, CCK receptor activation may result in the differential involvement of the phosphoinositol and arachidonic acid pathways to mediate calcium oscillation and amylase secretion.

Topics: Aminobenzoates; Amylases; Animals; Arachidonic Acid; Calcium; Chlorobenzoates; Cinnamates; Estrenes; Imidazoles; Inositol 1,4,5-Trisphosphate; Male; ortho-Aminobenzoates; Pancreas; Periodicity; Phospholipases A; Phospholipases A2; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Cholecystokinin; Ryanodine; Signal Transduction; Sincalide; Type C Phospholipases