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Differential regulation of cholecystokinin- and muscarinic-receptor-mediated phosphoinositide turnover in Flow 9000 cells.

ABSTRACT: We have explored the hypothesis that the apparent greater efficiency of cholecystokinin (CCK-8) receptor-second messenger coupling compared with that of muscarinic receptor in Flow 9000 cells is due to differential feedback inhibitory control mechanisms. Pretreatment of Flow 9000 cells with the tumour-promoting protein kinase C (PKC)-activating agent 12-O-tetradecanoylphorbol 13-acetate (TPA) produced a time- and dose-dependent inhibition of CCK-8 and acetylcholine (ACh) stimulation of inositol phosphate production. The inhibition by TPA of ACh-induced PI (phosphoinositide) response involved reduction of the maximal response, but no change in the concentration of ACh required to evoke a half-maximal response. In contrast, TPA inhibition of CCK-8 responses could be overcome by increasing the CCK-8 concentrations. Flow 9000 cells pretreated with TPA exhibited a 52-68% reduction in [3H]quinuclidinyl benzilate ([3H]QNB) binding capacity, whereas [125I]CCK-8 binding was unchanged. In saponin-permeabilized Flow 9000 cells, TPA pretreatment had no effect on guanosine 5'-[gamma-thio]triphosphate (GTP[S])-induced inositol phosphate formation, indicating that G-protein linkage to phosphoinositidase C (PIC) was not affected. However, TPA significantly inhibited the potentiating effect of GTP[S] on CCK-8 and ACh activation of PI response, suggesting that the coupling between the receptors and the G-protein was impaired. The PKC-activator 1-oleoyl-2-acetylglycerol (OAG), a diacylglycerol analogue, also significantly reduced CCK-8 and ACh stimulation of inositol phosphate accumulation in these cells. Our results are consistent with the hypothesis that muscarinic activation of PI hydrolysis is subjected to rapid feedback inhibition via the 1,2-diacylglycerol-PKC pathway. CCK-receptor activation of PI turnover is modulated to a lesser extent, and this may partially explain apparent differences in the efficiency of receptor-second messenger coupling. It is proposed that TPA acting through PKC exerts its inhibitory action on muscarinic-agonist-mediated PI response mainly at the receptor level, whereas the inhibitory effect on CCK-8 response is at a site close to the receptor-G-protein coupling step.

SUBMITTER: Lo WW

PROVIDER: S-EPMC1149052 | biostudies-other | 1988 May

REPOSITORIES: biostudies-other

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Project description:Cholecystokinin (CCK) is a neuropeptide that affects growth rate in chickens by regulating appetite. CCK peptides exert their function by binding to two identified receptors, CCKAR and CCKBR in the GI tract and the brain, respectively, as well as in other organs. In mammals, CCK/CCKAR interactions affect a number of immunological parameters, including regulation of lymphocytes and functioning of monocytes. Thus, food intake and growth can potentially be altered by infection and the resulting inflammatory immune response. It is uncertain, however, whether chicken express CCKAR in immune organs and cells, and, if so, whether CCKAR expression is regulated by pathogen derived inflammatory stimuli. Herein, we identify expression of CCKAR protein in chicken peripheral blood mononuclear cells (PBMC) including monocytes, and expression of the CCKAR gene in PBMC, thymus, bursa, and spleen, in selected commercial and pure chicken breeds. Further, stimulation with various types of E. coli heat-labile enterotoxins or lipopolysaccharide significantly regulated expression of CCKAR on monocytes in the different breeds. Ligation of CCKAR with antibodies in PBMC induced mobilization of Ca2+, indicating that CCKAR is signal competent. Injection with polyinosinic: polycytidylic acid (poly I:C), a synthetic analogue of double stranded viral RNA that binds Toll-Like Receptor-3 (TLR3), also regulated gene expressions of CCKAR and proinflammatory cytokines, in the different breeds. Interestingly, variations in the expression levels of proinflammatory cytokines in the different breeds were highly correlated with CCKAR expression levels. Taken together, these findings indicate that the physiological function of CCKAR in the chicken is tightly regulated in immune organs and cells by external inflammatory stimuli, which in turn regulate growth. This is the first report CCKAR expression in immune organs and cells, in any species, and the initial observation that CCKAR is regulated by inflammatory stimuli associated with bacterial and viral infection.

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Differential regulation of cholecystokinin- and muscarinic-receptor-mediated phosphoinositide turnover in Flow 9000 cells.

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