Pandya P K, Huang S C, Talkad V D, Wank S A, Gardner J D
Department of Internal Medicine, Saint Louis University Health Sciences Center, MO 63104.
Biochim Biophys Acta. 1994 Oct 20;1224(1):117-26. doi: 10.1016/0167-4889(94)90119-8.
We used rat pancreatic acini and measured binding of [125I]CCK-8 and [3H]L-364,718 to the three different states of the CCK receptor to examine potential biochemical regulation of ligand binding for each receptor state. Binding of [125I]CCK-8 to the high affinity state of the receptor was measured as carbachol-inhibitable binding of [125I]CCK-8, whereas binding of [125I]CCK-8 to the low affinity state was measured as carbachol-resistant binding of [125I]CCK-8. Interaction of CCK-8 with the very low affinity state of the CCK receptor was measured as CCK-8-inhibitable binding of [3H]L-364,718. [125I]CCK-8 that was bound to the high affinity state dissociated slowly at a rate of 0.20%/min and this dissociation was not altered by 30 mM NaF. Dissociation of [125I]CCK-8 bound to the low affinity state was biphasic--22% of the bound radioactivity dissociated completely within 3 min and the remaining 78% dissociated slowly at a rate of 0.19%/min. Dissociation of [125I]CCK-8 from the low affinity state was not altered by 30 mM NaF. The pattern of dissociation of bound [125I]CCK-8 from the pancreatic CCK receptor expressed in COS cells was also biphasic and closely resembled that observed in pancreatic acini. CCK-8 that was bound to the very low affinity state dissociated completely during a 20-min period of washing and resuspension of acini that had been first incubated with CCK-8. We found extensive biochemical regulation of the different states of the CCK receptor in pancreatic acini. Bombesin, TPA, NaF, CCCP and trifluoperazine each altered binding of [125I]CCK-8 to the high affinity state and to the low affinity state, and except for bombesin each agent was more potent in affecting the high affinity state than the low affinity state. No agent tested affected the low affinity state but not the high affinity state. In contrast, a number of agents affected the high affinity state but not the low affinity state. These included receptor-mediated agonists (carbachol, secretin, VIP), 8Br-cAMP, NEM, agents that affect microtubules or microfilaments (cytochalasin B, vinblastine), calmodulin inhibitors (W-7, chlorpromazine) and genistein. Experiments with EGTA, A23187 and thapsigargin indicated that none of the three receptor states was influenced by intracellular or extracellular calcium. No agent tested altered the interaction of CCK-8 with the very low affinity state of the CCK receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
我们使用大鼠胰腺腺泡，测量了[125I]CCK - 8和[3H]L - 364,718与CCK受体三种不同状态的结合情况，以研究每种受体状态下配体结合的潜在生化调节。[125I]CCK - 8与受体高亲和力状态的结合通过卡巴胆碱抑制的[125I]CCK - 8结合来测量，而[125I]CCK - 8与低亲和力状态的结合则通过卡巴胆碱抗性的[125I]CCK - 8结合来测量。CCK - 8与CCK受体极低亲和力状态的相互作用通过[3H]L - 364,718的CCK - 8抑制性结合来测量。与高亲和力状态结合的[125I]CCK - 8以0.20%/分钟的速率缓慢解离，且30 mM NaF不会改变这种解离。与低亲和力状态结合的[125I]CCK - 8的解离是双相的——22%的结合放射性在3分钟内完全解离，其余78%以0.19%/分钟的速率缓慢解离。30 mM NaF不会改变[125I]CCK - 8从低亲和力状态的解离。在COS细胞中表达的胰腺CCK受体上结合的[125I]CCK - 8的解离模式也是双相的，与在胰腺腺泡中观察到的模式非常相似。与极低亲和力状态结合的CCK - 8在最初用CCK - 8孵育的腺泡洗涤和重悬20分钟期间完全解离。我们发现胰腺腺泡中CCK受体的不同状态存在广泛的生化调节。蛙皮素、佛波酯、NaF、CCCP和三氟拉嗪均改变了[125I]CCK - 8与高亲和力状态和低亲和力状态的结合，除蛙皮素外，每种药物对高亲和力状态的影响比对低亲和力状态的影响更强。没有测试的药物只影响低亲和力状态而不影响高亲和力状态。相反，许多药物影响高亲和力状态但不影响低亲和力状态。这些药物包括受体介导的激动剂（卡巴胆碱、促胰液素、血管活性肠肽）、8 - 溴 - cAMP、N - 乙基马来酰亚胺、影响微管或微丝的药物（细胞松弛素B、长春碱）、钙调蛋白抑制剂（W - 7、氯丙嗪）和染料木黄酮。用乙二醇双（2 - 氨基乙醚）四乙酸、A23187和毒胡萝卜素进行的实验表明，三种受体状态均不受细胞内或细胞外钙的影响。没有测试的药物改变CCK - 8与CCK受体极低亲和力状态的相互作用。（摘要截于400字）
