Lo W W, Hughes J
Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge, U.K.
Biochem J. 1988 May 1;251(3):625-30. doi: 10.1042/bj2510625.
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.
在Flow 9000细胞中，胆囊收缩素（CCK-8）受体与第二信使的偶联效率明显高于毒蕈碱受体，这是由于不同的反馈抑制控制机制所致。用促肿瘤蛋白激酶C（PKC）激活剂12-O-十四烷酰佛波醇13-乙酸酯（TPA）预处理Flow 9000细胞，可产生时间和剂量依赖性地抑制CCK-8和乙酰胆碱（ACh）刺激的肌醇磷酸生成。TPA对ACh诱导的PI（磷脂酰肌醇）反应的抑制作用涉及最大反应的降低，但引起半最大反应所需的ACh浓度没有变化。相反，通过增加CCK-8浓度可克服TPA对CCK-8反应的抑制作用。用TPA预处理的Flow 9000细胞，其[3H]喹核醇基苯甲酸酯（[3H]QNB）结合能力降低了52%-68%，而[125I]CCK-8结合未发生变化。在皂素通透的Flow 9000细胞中，TPA预处理对鸟苷5'-[γ-硫代]三磷酸（GTP[S]）诱导的肌醇磷酸形成没有影响，表明G蛋白与磷脂酶C（PIC）的连接未受影响。然而，TPA显著抑制了GTP[S]对CCK-8和ACh激活PI反应的增强作用，提示受体与G蛋白之间的偶联受损。PKC激活剂1-油酰-2-乙酰甘油（OAG），一种二酰基甘油类似物，也显著降低了这些细胞中CCK-8和ACh刺激的肌醇磷酸积累。我们的结果与以下假说一致：毒蕈碱激活PI水解通过1,2-二酰基甘油-PKC途径受到快速反馈抑制。CCK受体激活PI周转受到的调节程度较小，这可能部分解释了受体与第二信使偶联效率的明显差异。有人提出，TPA通过PKC起作用，主要在受体水平对毒蕈碱激动剂介导的PI反应发挥抑制作用，而对CCK-8反应的抑制作用则位于靠近受体-G蛋白偶联步骤的位点。
