Felder C C, Kanterman R Y, Ma A L, Axelrod J
Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
J Biol Chem. 1989 Dec 5;264(34):20356-62.
The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.
将毒蕈碱型乙酰胆碱受体m1基因转染至A9 L细胞并使其稳定表达。毒蕈碱受体激动剂卡巴胆碱可刺激这些细胞中肌醇磷酸生成、花生四烯酸释放及环磷酸腺苷（cAMP）积聚。卡巴胆碱刺激花生四烯酸和肌醇磷酸释放的效力相似，而cAMP生成则需要更高浓度。开展研究以确定卡巴胆碱刺激的cAMP积聚是由于m1毒蕈碱受体通过鸟苷三磷酸（GTP）结合蛋白直接与腺苷酸环化酶偶联，还是由其他第二信使介导。卡巴胆碱未能刺激A9 L细胞膜中的腺苷酸环化酶活性，而前列腺素E2可刺激，提示为间接刺激。佛波酯，即佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA），可刺激花生四烯酸释放，但抑制卡巴胆碱诱导的cAMP积聚。PMA还抑制卡巴胆碱诱导的肌醇磷酸释放，提示磷脂酶C的激活可能参与cAMP积聚。PMA不抑制前列腺素E2、霍乱毒素或福斯高林刺激的cAMP积聚。磷脂酶A2抑制剂二十碳四烯酸以及环氧化酶抑制剂吲哚美辛和萘普生对卡巴胆碱刺激的cAMP积聚无影响。卡巴胆碱刺激的cAMP积聚可被细胞内钙释放抑制剂TMB - 8及钙调蛋白拮抗剂W7抑制。这些观察结果表明，卡巴胆碱刺激的cAMP积聚并非通过m1毒蕈碱受体直接偶联或通过花生四烯酸及其代谢产物的释放发生，而是由磷脂酶C的激活介导。通过肌醇1,4,5 - 三磷酸生成胞质钙，随后m1毒蕈碱受体刺激磷脂酶C激活钙调蛋白，似乎可导致cAMP积聚。
