The cholecystokinin analogues JMV-180 and CCK-8 stimulate phospholipase C through the same binding site of CCK(A) receptor in rat pancreatic acini

Abstract

1. This study was designed to address the controversy related to the involvement of phospholipase C in the signalling pathway linked to CCK(A) receptor stimulation by the cholecystokinin analogue JMV-180, a full agonist for amylase release, in rat pancreatic acini. 2. JMV-180 was shown to stimulate phospholipase C by measuring the incorporation of [(32)P]-orthophosphoric acid ([(32)P]-Pi) into phosphatidic acid (PtdOH) and phosphatidylinositol (PtdIns). Both responses elicited by JMV-180 were time and concentration dependent. Maximal effects elicited by JMV-180 were 39.08+/-0.72 and 8.02+/-0.40% for the labelling of [(32)P]-PtdIns and [(32)P]-PtdOH, respectively, as compared to the maximal effects of CCK-8, a full agonist of the CCK(A) receptor. 3. [(32)P]-Pi incorporation into PtdOH and PtdIns was sensitive to lithium, demonstrating that both responses are a consequence of phospholipase C activation. However, since lithium blocks the phosphoinositide cycle by an uncompetitive mechanism, its effect was only apparent at high concentrations of CCK-8 (>10 pM), which elicited stimuli above 20 and 60% of the maximal [(32)P]-PtdOH and [(32)P]-PtdIns labelling, respectively. 4. JMV-180 inhibited the incorporation of [(32)P]-Pi into PtdOH and PtdIns as stimulated by CCK-8, down to its own maximal effect. The estimated IC(50) values for the inhibition curves were not significantly different from those calculated assuming the same single binding site for both agonists. These results indicated that the well established role of JMV-180 as a partial agonist for CCK(A) receptor-linked signalling responses, also applies for the stimulation of phospholipase C. 5. The comparison of CCK-8 and JMV-180 dose-response curves of amylase release to those of PtdIns and PtdOH labelling with [(32)P]-Pi showed the existence of an amplification mechanism between phospholipase C and amylase release for both agonists. 6. In conclusion, we show that JMV-180, as well as CCK-8, stimulate phospholipase C upon interaction with the same binding site at the CCK(A) receptor in rat pancreatic acini.

Figure 1

Time course of [³²P]-PtdIns (a) and [³²P]-PtdOH (b) increase in rat pancreatic acini. [³²P]-Pi-prelabelled rat pancreatic acini were incubated for the times indicated with 1 nM CCK-8, 1 μM JMV-180, or under basal conditions. Results are mean±s.e.mean of three experiments in triplicate.

Figure 2

Dose-response curves for [³²P]-Pi incorporation into PtdIns (a) and PtdOH (b) in the absence and in the presence of lithium. [³²P]-Pi-prelabelled rat pancreatic acini were incubated for 60 min with the indicated concentrations of CCK-8 or JMV-180 in the absence or presence of 10 mM LiCl. Values are mean±s.e.mean of at least three experiments performed in triplicate and are represented as percentages of maximal effect of CCK-8 in the absence of LiCl.

Figure 3

Effect of lithium on the dose-response curves of [³²P]-PtdIns (a) and [³²P]-PtdOH (b) increase stimulated by Cch. [³²P]-Pi-prelabelled rat pancreatic acini were incubated for 60 min with the indicated concentrations of Cch in the absence or presence of 10 mM LiCl. Values are mean±s.e.mean of three experiments performed in triplicate, and are represented as a percentage of the maximal effect of CCK-8 in the absence of LiCl (Figure 2).

Figure 4

Competition curves for [³²P]-Pi incorporation into PtdIns (a) and PtdOH (b) [³²P]-Pi-prelabelled rat pancreatic acini were incubated for 60 min with increasing concentrations of JMV-180 in the presence of an indicated concentration of CCK-8. Values are mean±s.e.mean of at least three experiments performed in triplicate and are represented as percentages of maximal effect of CCK-8.

Figure 5

Dose-response curves for [³²P]-Pi incorporation into PtdOH and PtdIns, and amylase release stimulated by CCK-8 (a) and JMV-180 (b) [³²P]-Pi-prelabelled, except for the amylase release experiments, rat pancreatic acini were incubated for 60 min with the indicated concentrations of CCK-8 or JMV-180. Values are mean±s.e.mean of at least three experiments performed in triplicate, and are represented as a percentage of the maximal effect of CCK-8 for each response.