Matozaki T, Sakamoto C, Nishisaki H, Suzuki T, Wada K, Matsuda K, Nakano O, Konda Y, Nagao M, Kasuga M
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
J Biol Chem. 1991 Nov 25;266(33):22246-53.
The effects of cholecystokinin (CCK) and other pancreatic secretagogues on phosphatidylcholine (PC) synthesis were studied in isolated rat pancreatic acini. When acini were incubated with [3H]choline in the presence of 1 nM CCK-octapeptide (CCK8) for 60 min, the incorporations of [3H]choline into both water-soluble choline metabolites and PC in acini were reduced by CCK8 to 74 and 41% of control, respectively. Pulse-chase study revealed that CCK8 reduced both the disappearance of phosphocholine and the synthesis of PC. Other Ca(2+)-mobilizing secretagogues such as carbamylcholine, bombesin, and Ca2+ ionophore A23187 also reduced PC synthesis to the same extent as did CCK8. When combined with 1 nM CCK8, A23187 or carbamylcholine did not further inhibit PC synthesis. Furthermore, W-7 or W-5, a calmodulin antagonist, reversed the inhibition by CCK8 of PC synthesis, suggesting that a Ca(2+)-calmodulin-dependent pathway may be involved in CCK-induced inhibition of PC synthesis in acini. By contrast, neither cAMP-dependent secretagogues such as secretin and dibutyryl cAMP nor a phorbol ester had any effect on PC synthesis in acini. Staurosporine or H-7, a protein kinase C inhibitor, did not affect the inhibition by CCK of PC synthesis. The analysis of enzyme activity involved in PC synthesis via CDP-choline pathway showed that CCK treatment of acini reduced CTP:phosphocholine cytidylyltransferase activity in both cytosolic and particulate fraction, a finding consistent with the delayed disappearance of phosphocholine induced by CCK in pulse-chase study. By contrast, CCK treatment of acini did not alter the activities of choline kinase and phosphocholine transferase in acini. The extent of inhibition by CCK of cytidylyltransferase activity became much larger when subcellular fractions of acini were prepared in the presence of phosphatase inhibitors. In addition, W-7 reversed the inhibitory effect of CCK treatment on cytidylyltransferase activity in acini. When acini were labeled with [3H]myristic acid and chased, CCK8 (1 nM) reduced the synthesis of [3H]myristic acid-labeled PC to 27% of control after a 60-min chase period. This inhibition of PC synthesis induced by CCK was accompanied by a delayed disappearance of [3H]diacylglycerol, the radioactivity of which was 225% of control at 60 min. These results indicate that CCK inhibits PC synthesis by inducing both the reduction of choline uptake into acini and the inhibition of CTP:phosphocholine cytidylyltransferase activity. Furthermore, the results suggest the possibility that the activation of Ca(2+)-calmodulin-dependent kinase in response to CCK may phosphorylate cytidylyltransferase thereby decreasing this enzyme activity in pancreatic acinar cells.(ABSTRACT TRUNCATED AT 400 WORDS)
在分离的大鼠胰腺腺泡中研究了胆囊收缩素（CCK）和其他胰腺促分泌素对磷脂酰胆碱（PC）合成的影响。当腺泡在1 nM八肽胆囊收缩素（CCK8）存在下与[3H]胆碱孵育60分钟时，CCK8使腺泡中[3H]胆碱掺入水溶性胆碱代谢产物和PC的量分别降至对照的74%和41%。脉冲追踪研究表明，CCK8减少了磷酸胆碱的消失以及PC的合成。其他能动员钙离子的促分泌素，如氨甲酰胆碱、蛙皮素和钙离子载体A23187，也使PC合成减少到与CCK8相同的程度。当与1 nM CCK8联合使用时，A23187或氨甲酰胆碱不会进一步抑制PC合成。此外，钙调蛋白拮抗剂W - 7或W - 5可逆转CCK8对PC合成的抑制作用，提示钙（2+）-钙调蛋白依赖性途径可能参与CCK诱导的腺泡中PC合成的抑制。相比之下，促胰液素和二丁酰环磷腺苷等依赖环磷腺苷的促分泌素以及佛波酯对腺泡中PC合成均无影响。蛋白激酶C抑制剂星形孢菌素或H - 7不影响CCK对PC合成的抑制作用。通过CDP - 胆碱途径参与PC合成的酶活性分析表明，CCK处理腺泡会降低胞质和微粒体部分的CTP：磷酸胆碱胞苷转移酶活性这一发现与脉冲追踪研究中CCK诱导的磷酸胆碱延迟消失一致。相比之下，CCK处理腺泡不会改变腺泡中胆碱激酶和磷酸胆碱转移酶的活性。当在磷酸酶抑制剂存在下制备腺泡的亚细胞部分时，CCK对胞苷转移酶活性的抑制程度变得更大。此外，W - 7逆转了CCK处理对腺泡中胞苷转移酶活性的抑制作用。当腺泡用[3H]肉豆蔻酸标记并进行追踪时，在60分钟的追踪期后，1 nM CCK8使[3H]肉豆蔻酸标记的PC合成降至对照的27%。CCK诱导的这种PC合成抑制伴随着[3H]二酰甘油的延迟消失，其放射性在60分钟时为对照的225%。这些结果表明，CCK通过诱导腺泡中胆碱摄取减少和抑制CTP：磷酸胆碱胞苷转移酶活性来抑制PC合成。此外，结果提示了一种可能性，即响应CCK激活的钙（2+）-钙调蛋白依赖性激酶可能使胞苷转移酶磷酸化，从而降低胰腺腺泡细胞中该酶的活性。（摘要截短至400字）
