Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells--ionic dependence and effects of local anesthetics.
Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells--ionic dependence and effects of local anesthetics.
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培养神经细胞毒蕈碱受体介导的环 GMP 形成——离子依赖性和局部麻醉剂的作用。
DOI：
10.1016/0006-2952(78)90064-3
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发表时间：
1978
影响因子：
5.8
通讯作者：
S. Divinetz
中科院分区：
文献类型：
--
作者：
E. Richelson;F. Prendergast;S. Divinetz
一种新的测定技术，用于测量受体介导的环鸟苷酸形成培养的小鼠成神经细胞瘤细胞的作用，离子和局部麻醉剂的影响，毒蕈碱受体的功能进行了研究。该技术涉及通过用[3 H]鸟嘌呤孵育细胞并用阳离子交换树脂（Dowex-50+）柱分离[3 H]环GMP来放射性标记GTP的细胞内储存。细胞提取物和Dowex柱洗脱液的高压液相色谱显示，45分钟后，细胞提取物中的大部分放射性是[3 H]GTP，对于氨甲酰胆碱刺激的细胞，洗脱液中90%以上的放射性是[3 H]环GMP。在没有外部Na+（[Na+]e，与氯化铯作为渗透填料）或外部（[Ca 2 +]e），氨甲酰胆碱刺激形成的[3 H]环GMP分别约为60%和10%的控制，而其他离子的去除没有显着的影响。10 mM与110 mM−[Na+]e的反应几乎没有差异，而最佳[Ca 2 +]约为5 mM。Ca 2+增加了[3 H]环GMP形成对氨甲酰胆碱的反应，而不影响这种激动剂对受体的表观亲和力。局麻药对氨甲酰胆碱有明显的竞争性抑制作用，其平衡解离常数（KB）在6-250 μM范围内。局部麻醉药对毒蕈碱受体的表观亲和力的排序为丁卡因=布他卡因=普鲁卡因>地布卡因=利多卡因>氨基苯甲酸乙酯。
A new assay technique for measuring receptor-mediated cyclic GMP formation by cultured mouse neutroblastoma cells was used to study the role of ions in, and the effects of local anesthetics on, the function of the muscarinic receptor. The technique involved radioactively labeling intracellular stores of GTP by incubating cells with [3H]guanine and isolating [3H]cyclic GMP with a cation exchange resin (Dowex-50+) column. High-pressure liquid chromatography of cell extracts and of eluates from the Dowex column showed that after 45 min the majority of the radioactivity in the cell extracts was [3H]GTP and that, for carbamylcholine-stimulated cells, greater than 90 per cent of the radioactivity in the eluates was [3H]cyclic GMP. In the absence of external Na+([Na+]e, with cesium chloride as osmotic filler) or external ([Ca2+]e), the carbamylcholine-stimulated formation of [3H]cyclic GMP was about 60 and 10 per cent of control, respectively, while removal of other ions had no significant effect. There was little difference in the responses at 10 mM vs 110 mM−[Na+]e, whereas the optimal [Ca2+], was around 5 mM. Ca2+increased [3H]cyclic GMP formation in response to carbamylcholine without affecting the apparent affinity of this agonist for the receptor. Local anesthetics were apparently competitive inhibitors of carbamylcholine with equilibrium dissociation constants (KB) in the range of 6–250 μM. The rank order for the apparent affinity of local anesthetics for the muscarinic receptor was tetracaine = butacaine = procaine > dibucaine = lidocaine > ethyl aminobenzoate.