G-PROTEIN DIFFUSION DURING MUSCARINIC ACTIVATION

毒蕈碱激活过程中 G 蛋白的扩散

• 批准号: 3964318
• 负责人: J ZIMMERBERG
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3964318
• 关键词: acetylcholine; adenosine triphosphate; anticholinergic agent; calcium; cell morphology; cyclic nucleoside monophosphate; drug interactions; inositol; lacrimal apparatus; lipid metabolism; neomycin; neuropharmacology; phosphatidylinositols; potassium

We have studied the muscarinic response of rat lacrymal gland cells to acetylcholine with the tight-seal whole-cell technique. To measure the liberation of calcium from internal stores in that cell we monitored the calcium dependent K and Cl currents resulting from application of acetylcholine. Intracellular inclusion of IP2 liberated calcium. To show that polyphosphoinositide (PIP2) hydrolysis is a step in muscarinic activation we added neomycin, which binds to PIP2 and blocked activation by acetylcholine. The response to agonist diminished over several minutes after initiation of whole-cell dialysis. We found that the response was stable for some minutes, then decreased exponentially. The delay and time constant of the washout was directly proportional to the size of the pathway for diffusion and inversely proportional to the cell volume. It appeared that some soluble intracellular factor, needed after receptor activation, was needed for liberation of intracellular calcium. Attempts to constantly replenish PIP2 by inclusion of CTP, ATP, and inositol did not halt washout. Intracellular inclusion of ATP, GTP, cAMP, cGMP, together or alone, did not stop washout. We observed washout in one cell, gently removed the pipette after no further response to acetylcholine was seen, and sealed to the same cell a new pipette containing IP3. A rapid and sustained response was seen upon initiation of the whole cell mode. Intracellular inclusion of GTP-S potentiated the muscarinic response and slowed washout. We conclude that the washout is due to the loss of a diffusible factor which acts after muscarinic receptor activation and before polyphosphoinositol release. We suspect the action of the factor to involve a G protein.

我们研究了大鼠泪腺细胞的毒蕈碱反应， 乙酰胆碱与紧密密封的全细胞技术。 测量 我们监测了细胞内钙从内部储存中的释放， 钙依赖性钾和氯电流的应用， 乙酰胆碱 IP 2的细胞内包涵体释放钙。 展示 聚磷酸肌醇（PIP 2）水解是毒蕈碱 我们加入了新霉素，它与PIP 2结合，并通过 乙酰胆碱 对激动剂的反应在几分钟内减弱 开始全细胞透析后。 我们发现， 稳定几分钟，然后呈指数下降。 延迟和时间 洗出常数直接正比于 扩散途径，并与细胞体积成反比。 它 似乎一些可溶性细胞内因子，需要后受体 激活，是细胞内钙释放所必需的。 尝试 通过包括CTP、ATP和肌醇来不断补充PIP 2， 停止冲洗。 ATP、GTP、cAMP、cGMP一起或 独自一人，并没有停止冲刷。 我们在一个细胞中观察到洗脱， 在没有观察到对乙酰胆碱的进一步反应后取出移液管， 并将含有IP 3的新移液管密封到相同的池中。 一种快速 在开始全细胞模式时观察到持续的反应。 细胞内包含GTP-S增强了毒蕈碱反应， 慢洗。 我们的结论是，洗脱是由于失去了一个 在毒蕈碱受体活化后起作用的扩散因子， 在聚磷酸肌醇释放之前。 我们怀疑因子的作用是 涉及G蛋白。