Biochemical regulation of the three different states of the cholecystokinin (CCK) receptor in pancreatic acini

Abstract

We used rat pancreatic acini and measured binding of [ 125I]CCK-8 and [ 3H]L-364,718 to the three different states of the CCK receptor to examine potential biochemical regulation of ligand binding for each receptor state. Binding of [ 125I]CCK-8 to the high affinity state of the receptor was measured as carbachol-inhibitable binding of [ 125I]CCK-8, whereas binding of [ 125I]CCK-8, whereas binding of [ 125I]CCK-8 to the low affinity state was measured as carbachol-resistant binding of [ 125I]CCK-8. Interaction of CCK-8 with the very low affinity state of the CCK receptor was measured as CCK-8-inhibitable binding of [ 3H]L-364,718. [ 125I]CCK-8 that was bound to the high affinity state dissociated slowly at a rate of 0.20%/min and this dissociation was not altered by 30 mM NaF. Dissociation of [ 125I]CCK-8 bound to the low affinity state was biphasic — 22% of the bound radioactivity dissociated completely within 3 min and the remaining 78% dissociated slowly at a rate of 0.19%/min. Dissociation of [ 125I]CCK-8 from the low affinity state was not altered by 30 mM NaF. The pattern of dissociation of bound [ 125I]CCK-8 from the pancreatic CCK receptor expressed in COS cells was also biphasic and closely resembled that observed in pancreatic acini. CCK-8 that was bound to the very low affinity state dissociated completely during a 20-min period of washing and resuspension of acini that had been first incubated with CCK-8. We found extensive biochemical regulation of the different states of the CCK receptor in pancreatic acini. Bombesin, TPA, NaF, CCCP and trifluoperazine each altered binding of [ 125I]CCK-8 to the high affinity state and to the low affinity state, and except for bombesin each agent was more potent in affecting the high affinity state than the low affinity state. No agent tested affected the low affinity state but not the high affinity state. In contrast, a number of agents affected the high affinity state but not the low affinity state. These included receptor-mediated agonists (carbachol, secretin, VIP), 8Br-cAMP, NEM, agents that affect microtubules or microfilaments (cytochalasin B, vinblastine), calmodulin inhibitors (W-7, chlorpromazine) and genistein. Experiments with EGTA, A23187 and thapsigargin indicated that none of the three receptor states was influenced by intracellular or extracellular calcium. No agent tested altered the interaction of CCK-8 with the very low affinity state of the CCK receptor. The effects on binding of [ 125I]CCK-8 by NaF, TPA, secretin, bombesin and carbachol were enhanced by okadaic acid, indicating that the abilities of these agents to influence CCK receptor binding involves phosphorylation. The effects of TPA, but not those of NaF, secretin, bombesin or carbachol, were prevented by staurosporine and H-7 (inhibitors of protein kinase C and other kinases). Genistein (inhibitor of protein tyrosine kinase), W-7 and trifluoperazine (calmodulin antagonists) did not alter the effects of TPA, secretin, bombesin or carbachol on binding of [ 125I]CCK-8.