Protein kinase C‐mediated inhibition of transmembrane signalling through CCK A and CCK B receptors

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 effects of protein kinase C (PKC) thereupon. 2 Spectrofluorophotometry of Fura‐2‐loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin‐(26‐33)‐peptide amide (CCK‐8‐S) increased the cytosolic Ca 2+ concentration ([Ca 2+ ] i ) in CCK A cells, measured as an increase in Fura‐2 fluorescence emission ratio, 1000 fold more potently than its non‐sulphated form (CCK‐8‐NS) (EC 50 values of 0.19 n M and 0.18 μ M , respectively). By contrast, CCK‐8‐S and CCK‐8‐NS were equally potent in CCK B cells (EC 50 values of 0.86 n M and 1.18 n M , respectively). The CCK A receptor agonist JMV‐180 increased [Ca 2+ ] i only in CCK A cells. Likewise, pentagastrin increased [Ca 2+ ] i only in CCK B cells. Finally, CCK‐8‐S‐induced Ca 2+ signalling through the CCK A receptor was most potently inhibited by the CCK A receptor antagonist L364,718, whereas the CCK B receptor antagonist L365,260 was more potent in CCK B cells. 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 effect 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 effect of TPA was abolished by the PKC inhibitors, GF‐109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4‐α‐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 PKC‐α, 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.British Journal of Pharmacology (1998) 123 , 1189–1197; doi: 10.1038/sj.bjp.0701713