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1 The rat CCK A and CCK B receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and e ects of protein kinase C (PKC) thereupon. 3 Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK A cells. By contrast, none of these agonists increased cyclicAMP in CCK B cells. 4 Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca 2+ mobilizing e ect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK A cells. In both cases, the inhibitory e ect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-a-phorbol 12-myristate 13-acetate. 5 During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca 2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKCa, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6 This study demonstrates that following expression in CHO cells (i) both CCK A and CCK B receptors are coupled to Ca 2+ mobilization, (ii) only CCK A receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.

Recently a molecular model was proposed lor the binding site of the antagonist 3M -)-/V-(2,3-dihydro-l-mediyl-2-oxo-5~phenyl-l H-1,4-benzodia/epine-3 s I) I // mdole 2-c^'hoxamide (deva/epide) on the cholecystokinin-A (CCK A) receptor (Van der Bent et al., 1994. Drug Design Discov. 12, 129 14X). Fifteen amino acids were identified, including hydrophilic ones such as Seru '\ Asivw and S e r'71\ that might interact with the caihoxamide moiety in deva/epide. To provide mutational evidence for this model, wild-type and mutant receptors (S139A, N349A and S379A) were transiently expressed and compared with respect to the ability of devazepide to inhibit binding of radiolabelled cholecystokinin <2(> 33)^peptkle amide (CCK^S) mid CCK-H-evoked (V 1 mobilization. The data presented suggest the involvement of the three residues in antagonist binding, although to a different extent. However, it does not seem likely that hydrogen bonds are the driving force in view of the relatively minor changes in receptor affinity and activity. £9 1997 Elsevier Science B.V.

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