Protein kinase C‐mediated inhibition of transmembrane signalling through CCKA and CCKB receptors

• Published in: British journal of pharmacology Vol. 123; no. 6; pp. 1189 - 1197
• Main Authors: Smeets, R L L., Fouraux, M A., Emst‐de Vries, S E., De Pont, J J H H M., Willems, P H G M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.03.1998; Nature Publishing
• Subjects: Biological and medical sciences; calcium mobilization; CCK‐8; Cell physiology; Chinese hamster ovary cells; CholecystokininA receptor; cholecystokininB receptor; cyclicAMP formation; Fundamental and applied biological sciences. Psychology; JMV‐180; Medical sciences; Molecular and cellular biology; Neuropharmacology; Neurotransmitters. Neurotransmission. Receptors; Peptidergic system (neuropeptide, opioid peptide, opiates...). Adenosinergic and purinergic systems; Pharmacology. Drug treatments; protein kinase C down‐regulation; Signal transduction; TPA; Protein kinase C; Enzyme; Transferases; Rodentia; Phosphorus-oxygen lyases; Cyclic AMP; Lyases; Activation; Gene expression; Adenylate cyclase; Signal transduction; Vertebrata; Mammalia; Animal; Established cell line; Peptidergic receptor; Cholecystokinin; Mechanism of action; Tumor cell; Hamster; Calcium Cations
• DOI: 10.1038/sj.bjp.0701713
• ISSN: 0007-1188

1 The rat CCKA and CCKB 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 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 nM and 0.18 μM, 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, respectively). The CCKA receptor agonist JMV‐180 increased [Ca2+]i only in CCKA cells. Likewise, pentagastrin increased [Ca2+]i only in CCKB cells. Finally, CCK‐8‐S‐induced Ca2+ signalling through the CCKA receptor 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 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 CCKA cells. By contrast, none of these agonists increased cyclicAMP in CCKB 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 CCKA 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 CCKA and CCKB receptors are coupled to Ca2+ mobilization, (ii) only CCKA 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