PROTEIN-KINASE C-MEDIATED INHIBITION OF TRANSMEMBRANE SIGNALING THROUGH CCKA AND CCKB RECEPTORS

1 The rat CCKA and CCKB receptors were stably expressed in Chinese ham ster ovary (CHO-09) cells in order to compare modes of signal transduc tion and effects of protein kinase C (PKC) thereupon. 2 Spectrofluorop hotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cel ls. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increase d the cytosolic Ca2+ concentration ([Ca2+](i)) in CCKA 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 n M and 0.18 mu M, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCKB cells (EC50 values of 0.86 nM and 1.18 nM, res pectively). The CCKA receptor agonist JMV-180 increased [Ca2+](i) only in CCKA cells. Likewise, pentagastrin increased [Ca2+](i) only in CCK B cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCKA rec eptor was most potently inhibited by the CCKA receptor antagonist L364 ,718, whereas the CCKB receptor antagonist L365,260 was more potent in CCKB cells. 3 Receptor-mediated activation of adenylyl cyclase was me asured in the presence of the inhibitor of cyclic nucleo tide phosphod iesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a les ser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCKA cells. By contrast, none of these ag onists increased cyclicAMP in CCKB cells. 4 Short-term (3 min) pretrea tment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA ) evoked a rightward shift of the dose-response curve for the Ca2+ mob ilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulati on in CCKA cells. In both cases, the inhibitory effect of TPA was abol ished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-alpha-phorb ol 12-myristate 13-acetate. 5 During prolonged TPA treatment, the cell s gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca2+ mobilization complete recovery was achieved afte r 24 h of TPA treatment. Western blot analysis revealed that this reco very was paralleled by down-regulation of PKC-alpha, suggesting the in volvement 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 CCKB receptors are coupled to Ca2+ mobilization, (ii) only CCKA receptors are coupled to cyclicAMP formation and (iii) with both recep tors signalling is inhibited by PKC.