Schmidt M, Bienek C, van Koppen C J, Michel M C, Jakobs K H
Institut für Pharmakologie, Universitätsklinikum Essen, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 1995 Nov;352(5):469-76. doi: 10.1007/BF00169379.
We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca2+]i with EC50 values of about 2 microM and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP3) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca2+]i were 300-350 nM and mainly, almost 90%, due to influx of extracellular Ca2+. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca2+]i with EC50 values of about 20 microM and 7 microM, respectively. Maximal InsP formation was only 10-15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca2+]i were similar in both cell types. Formation of InsP3 was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca2+]i increases induced by m2 mAChR activation were exclusively due to Ca2+ mobilization from intracellular stores. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca2+]i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca2+ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.
我们比较了毒蕈碱型乙酰胆碱受体（mAChR）与磷脂酶C（PLC）的偶联以及人胚胎肾（HEK）细胞中细胞质Ca2+浓度[Ca2+]i的增加情况，这些细胞稳定表达人m3或m2受体亚型。在表达m3 mAChR的细胞中，卡巴胆碱刺激肌醇磷酸（InsP）形成并增加[Ca2+]i，其EC50值分别约为2 microM和30 nM。最大肌醇1,4,5-三磷酸（InsP3）产量（约四倍）迅速（15秒）且在2分钟内保持稳定。[Ca2+]i的最大增加量为300 - 350 nM，主要（几乎90%）是由于细胞外Ca2+的内流。毛果芸香碱刺激InsP和Ca2+反应的效力与卡巴胆碱无显著差异。所有m3 mAChR介导的反应均对百日咳毒素（PTX）不敏感。在表达m2 mAChR的细胞中，卡巴胆碱刺激InsP形成并增加[Ca2+]i，其EC50值分别约为20 microM和7 microM。最大InsP形成量仅为表达m3 mAChR细胞中观察到的10 - 15%，而两种细胞类型中[Ca2+]i的最大升高相似。InsP3的形成迅速（15秒至2分钟），比基础水平高出约两倍。与m3 mAChR激活相反，m2 mAChR激活诱导的[Ca2+]i增加完全是由于细胞内储存的Ca2+释放。毛果芸香碱刺激InsP和Ca2+反应的效力分别为卡巴胆碱效力的50%和20%。PTX处理不影响m2 mAChR诱导的PLC刺激，但将m2 mAChR介导的[Ca2+]i增加降低至50%。总之，在HEK细胞中稳定表达的m3和m2 mAChRs可诱导相似的细胞反应；然而，它们通过激活明显不同的信号通路来实现。虽然m2 mAChR与PLC的偶联以PTX不敏感的方式发生，但与细胞内储存的Ca2+释放的偶联部分对PTX敏感，这可能至少部分独立于PLC激活发生。
