Matozaki T, Göke B, Tsunoda Y, Rodriguez M, Martinez J, Williams J A
Department of Physiology, University of Michigan, Ann Arbor 48109.
J Biol Chem. 1990 Apr 15;265(11):6247-54.
A new hepatapeptide cholecystokinin (CCK) analog, JMV-180 (Boc-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester), acts as an agonist at high affinity CCK receptors on rat pancreatic acini to stimulate amylase release but unlike cholecystokinin octapeptide (CCK8) does not act on low affinity CCK receptors to inhibit amylase release (Galas, M. D., Lignon, M. F., Rodriguez, M., Mendre, C., Fulcrand, P., Laur, J., and Martinez, J. (1988) Am. J. Physiol. 254, G176-G188). To investigate the biochemical mechanisms initiated by CCK acting on each class of CCK receptor, the effects of JMV-180 and CCK8 on amylase release, Ca2+ mobilization, and phospholipid hydrolysis were studied in isolated rat pancreatic acini. When acini were loaded with the intracellular Ca2+ chelator BAPTA, amylase release stimulated by both JMV-180 and CCK8 was reduced. Measurement of 45Ca2+ efflux and cytosolic free calcium concentration ([Ca2+]i) by the fluorescence of fura-2-loaded acini in a stirred cuvette showed that JMV-180 induced a concentration-dependent increase but with a maximal response only two-thirds that induced by CCK8. When [Ca2+]i of individual fura-2-loaded acinar cells was measured by microspectrofluorometry, all concentrations of JMV-180 (1 nM-10 microM) induced repetitive transient [Ca2+]i spikes (Ca2+ oscillations). By contrast, stimulation with a high concentration of CCK8 (1 nM) caused a large increase in [CA2+]i followed by a small sustained elevation of [Ca2+]i. The measurement of inositol trisphosphate (IP3) production by both [3H]inositol labeling and 1,4,5-IP3 radioreceptor assay showed that JMV-180 had only minimal effects at 10 microM in contrast to the large increase induced by high concentrations of CCK8 (more than 1 nM). JMV-180 blocked the effect of a high concentration of CCK8 on both [Ca2+]i and 1,4,5-IP3 productions but did not affect the response to carbamylcholine. JMV-180 caused a delayed monophasic stimulation of 1,2-diacylglycerol (DAG) sustained to 60 min without the early increase in DAG observed in response to CCK8. Furthermore, JMV-180 stimulated the release of [3H]choline metabolites, primarily phosphorylated choline, from [3H]choline-labeled acini at low concentrations and to the same extent as CCK8. Since JMV-180 interacts not only with high affinity CCK receptors as an agonist but also with low affinity CCK receptors as a functional antagonist, the present results indicate that the occupancy of high affinity state receptors by CCK induces Ca2+ oscillations, DAG formation from phosphatidylcholine hydrolysis, and amylase release with minimal phosphatidylinositol 4,5-bisphosphate hydrolysis.(ABSTRACT TRUNCATED AT 400 WORDS)
一种新的肝肽胆囊收缩素（CCK）类似物JMV-180（Boc-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-2-苯乙酯），作为大鼠胰腺腺泡高亲和力CCK受体的激动剂，刺激淀粉酶释放，但与八肽胆囊收缩素（CCK8）不同，它不作用于低亲和力CCK受体来抑制淀粉酶释放（加拉斯，M.D.，利尼翁，M.F.，罗德里格斯，M.，门德雷，C.，富尔克朗，P.，劳尔，J.，和马丁内斯，J.（1988年）《美国生理学杂志》254卷，G176 - G188页）。为了研究CCK作用于每一类CCK受体引发的生化机制，在分离的大鼠胰腺腺泡中研究了JMV-180和CCK8对淀粉酶释放、Ca2+动员和磷脂水解的影响。当腺泡加载细胞内Ca2+螯合剂BAPTA时，JMV-180和CCK8刺激的淀粉酶释放均减少。通过在搅拌小室中用fura-2加载的腺泡的荧光测量45Ca2+外流和胞质游离钙浓度（[Ca2+]i）表明，JMV-180诱导浓度依赖性增加，但最大反应仅为CCK8诱导反应的三分之二。当通过显微分光荧光测定法测量单个fura-2加载的腺泡细胞的[Ca2+]i时，所有浓度的JMV-180（1 nM - 10 microM）均诱导重复性瞬时[Ca2+]i尖峰（Ca2+振荡）。相比之下，用高浓度CCK8（1 nM）刺激导致[Ca2+]i大幅增加，随后[Ca2+]i有小幅持续升高。通过[3H]肌醇标记和1,4,5-IP3放射受体测定法测量肌醇三磷酸（IP3）生成表明，与高浓度CCK8（超过1 nM）诱导的大幅增加相比，JMV-180在10 microM时只有最小的影响。JMV-180阻断高浓度CCK8对[Ca2+]i和1,4,5-IP3生成的影响，但不影响对氨甲酰胆碱的反应。JMV-180引起1,2-二酰基甘油（DAG）延迟的单相刺激，持续到第60分钟，没有观察到对CCK8反应时DAG的早期增加。此外，JMV-180在低浓度下刺激[3H]胆碱标记的腺泡释放[3H]胆碱代谢物，主要是磷酸化胆碱，且程度与CCK8相同。由于JMV-180不仅作为激动剂与高亲和力CCK受体相互作用，还作为功能性拮抗剂与低亲和力CCK受体相互作用，目前的结果表明，CCK占据高亲和力状态受体诱导Ca2+振荡、磷脂酰胆碱水解形成DAG以及淀粉酶释放，同时磷脂酰肌醇4,5-二磷酸水解最少。（摘要截于400字）
