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 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.

Generation of Site-Specific Retargeting Platform Cell Lines for Drug Discovery Using phiC31 and R4 Integrases

“…3A, B). 16,17,20 Similar to the retargeted HEK293 cells, these pools displayed stable responses over 2 dozen passages (data not shown).…”

“…Calcium was released in a concentration-dependent manner that was consistent with expected results with an EC 50 of 4.52E-10 M (hill slope 1.6). 20 All samples were performed in quadruplicates.…”

Characterization of human 5‐HT4(d) receptor desensitization in CHO cells

“…One inhibitor of PKA, H‐89 (10 μ M ) and one of inhibitor of PKC, GF 109203X (10 μ M ) were used to test the role of PKA and PKC in 5‐HT‐induced receptor desensitization. At the concentration used in our study, these protein kinase inhibitors are known to be effective in CHO cells (Smeets et al ., 1998; May et al ., 1999; Robert et al ., 2001). We investigated the effects of two inhibitors of internalization, hypertonic sucrose (0.45 M ) and Con A (0.25 mg ml −1 ) on the agonist‐induced desensitization of the 5‐HT 4(d) receptor.…”

Cholecystokinin Exerts an Effect via the Endocannabinoid System to Inhibit GABAergic Transmission in Midbrain Periaqueductal Gray

“…Sulfated cholecystokinin-(26-33) (CCK-S), but not nonsulfated cholecystokinin-(26-33) (CCK-NS) produced an inward current in PAG neurons at concentrations between 100-300 nM. This suggests that the effects of cholecystokinin were mediated by CCK1 receptors because CCK-S has low nanomolar affinity for both CCK1 and CCK2 receptors, whereas CCK-NS only has low nanomolar affinity for CCK1 receptors (eg, Smeets et al, 1998). To confirm this, we also examined the effect on CCK-S-induced currents of the selective antagonists devazepide and CI-988, which have low nanomolar affinity for CCK1 and CCK2 receptors, respectively (Noble et al, 1999).…”