Protein kinase C-mediated inhibition of transmembrane signalling through CCK_A and CCK_B receptors

Abstract

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 Ca²⁺ concentration ([Ca²⁺]_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) (EC₅₀ values of 0.19 nM and 0.18 μM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK_B cells (EC₅₀ values of 0.86 nM and 1.18 nM, respectively). The CCK_A receptor agonist JMV-180 increased [Ca²⁺]_i only in CCK_A cells. Likewise, pentagastrin increased [Ca²⁺]_i only in CCK_B cells. Finally, CCK-8-S-induced Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ mobilization, (ii) only CCK_A receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.

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