G-PROTEIN DIFFUSION DURING MUSCARINIC ACTIVATION

我们研究了大鼠泪腺细胞对 乙酰胆碱与紧密密封的全细胞技术。要测量 我们监测的那个细胞内部储存的钙的释放 钙依赖钾和氯电流的应用 乙酰胆碱。细胞内包涵体IP2释放钙离子。展示 聚磷脂酰肌醇(PIP2)的水解是毒扁豆碱的一个步骤 激活我们添加了新霉素，它与PIP2结合并通过以下方式阻断激活 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，人们的反应是 稳定了几分钟，然后呈指数下降。延迟和时间 冲刷的常量与冲刷的大小成正比 这是扩散的途径，与细胞体积成反比。它 出现了一些可溶性的细胞内因子，需要后受体 激活，是释放细胞内钙所必需的。尝试 通过加入CTP、ATP和肌醇来不断补充PIP2 停止冲刷。细胞内包含ATP、GTP、cAMP、cGMP，一起或 独自一人，没有停止冲刷。我们在一间牢房里，轻轻地观察了洗涤 在对乙酰胆碱没有进一步反应后取出移液管， 并将含有IP3的新移液管密封到同一个电池中。一个快速而又 启动全细胞模式后可见持续的反应。 细胞内包合的GTP-S增强了M反应和 冲刷速度变慢了。我们的结论是，这次冲刷是由于一个 M受体激活后作用的扩散性因子和 在多磷肌醇释放之前。我们怀疑这一因素对 涉及一种G蛋白。

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.