Muscarinic activation of mitogen-activated protein kinase in PC12 cells
Muscarinic activation of mitogen-activated protein kinase in PC12 cells
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DOI：
10.1046/j.1471-4159.2000.0750487.x
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发表时间：
2000-08-01
影响因子：
4.7
通讯作者：
Levey, AI
中科院分区：
文献类型：
--
作者：
Berkeley, JL;Levey, AI
毒蕈碱乙酰胆碱受体（mAChRs）激活许多下游信号通路，其中一些可导致丝裂原活化蛋白激酶（MAPK）磷酸化和活化。MAPKs在调节细胞生长、分化和突触可塑性中发挥作用。本研究在内源性表达machr的PC12细胞中检测了MAPK的激活。使用磷酸化特异性MAPK抗体的Western blot分析显示，在甲氨基酚（CCh）刺激的细胞中，MAPK磷酸化呈剂量依赖性和阿托品敏感性增加。最大反应发生在5分钟后，并迅速降低到基线水平。为了研究PC12细胞中负责CCh激活MAPK的受体，我们使用RT-PCR和免疫沉淀技术确定了PC12细胞中存在的mAChR亚型。采用RT-PCR扩增m1、m4和m5相应大小的片段，用限制性酶切法确认条带的身份。亚型特异性抗体免疫沉淀显示，95%的表达受体与m4相似，其余5%的表达受体与mi和m5相似。一种高度特异性的mi毒素完全阻断了MAPK在CCh刺激下的磷酸化。machr诱导的MAPK活化被蛋白激酶C下调所消除，部分被百日咳毒素所抑制。虽然mi只占总mAChR群体的一小部分，但药理学证据表明mi负责PC12细胞中MAPK的激活。
Muscarinic acetylcholine receptors (mAChRs) activate many downstream signaling pathways, some of which can lead to mitogen-activated protein kinase (MAPK) phosphorylation and activation. MAPKs play roles in regulating cell growth, differentiation, and synaptic plasticity. Here, the activation of MAPK was examined in PC12 cells endogenously expressing mAChRs. Western blot analysis using a phosphospecific MAPK antibody revealed a dose-dependent and atropine-sensitive increase in MAPK phosphorylation in cells stimulated with carbachol (CCh). The maximal response occurred after 5 min and was rapidly reduced to baseline. To investigate the receptors responsible for CCh activation of MAPK in PC12 cells, the mAChR subtypes present were determined using RT-PCR and immunoprecipitation. RT-PCR was used to amplify fragments of the appropriate sizes for m1, m4, and m5, and the identities of the bands were confirmed with restriction digests. Immunoprecipitation using subtype-specific antibodies showed that similar to 95% of the expressed receptors were m4, whereas the remaining similar to 5% were mi and m5. A highly specific mi toxin completely blocked MAPK phosphorylation in response to CCh stimulation. The mAChR-induced MAPK activation was abolished by protein kinase C down-regulation and partially inhibited by pertussis toxin. Although mi represents a small proportion of the total mAChR population, pharmacological evidence suggests that mi is responsible for MAPK activation in PC12 cells.