Lin Alan L, Zhu Bing, Zhang WanKe, Dang Howard, Zhang Bin-Xian, Katz Michael S, Yeh Chih-Ko
Department of Dental Diagnostic Science, University of Texas Health Science Center, San Antonio, TX, USA.
Am J Physiol Cell Physiol. 2008 Jun;294(6):C1454-64. doi: 10.1152/ajpcell.00151.2007. Epub 2008 Apr 2.
Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca2+ ([Ca2+]i by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca2+]i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M1) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M3, but not M1, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms-pilocarpine acting via M3 receptors and Src-dependent transactivation of EGF receptors, and carbachol via M1/M3 receptors and PKC-converging on the ERK pathway.
胆碱能 - 毒蕈碱受体激动剂用于缓解口干，尽管介导这些药物对唾液腺作用的细胞信号尚未明确。我们研究了两种毒蕈碱激动剂毛果芸香碱和卡巴胆碱对人唾液腺细胞系（HSY）中ERK1/2的激活作用。免疫印迹分析显示，两种激动剂均能诱导ERK1/2的瞬时激活。毛果芸香碱可诱导表皮生长因子（EGF）受体磷酸化，而卡巴胆碱则不能。此外，EGF受体抑制剂AG - 1478可消除毛果芸香碱诱导的ERK激活，但对卡巴胆碱无此作用。用佛波酯PMA长时间处理细胞使PKC下调，可减少卡巴胆碱诱导的ERK磷酸化，但对毛果芸香碱的反应性无影响。用EGTA耗尽细胞内Ca2+（[Ca2+]i）对两种药物诱导的ERK激活均无影响。与卡巴胆碱不同，毛果芸香碱不会引起HSY细胞内[Ca2+]i动员。用毒蕈碱受体亚型3（M3）拮抗剂N -（3 - 氯丙基）- 4 - 哌啶基二苯乙酸处理细胞可降低ERK对两种药物的反应性，而亚型1（M1）拮抗剂哌仑西平仅降低卡巴胆碱的反应。M3受体小干扰RNA也可降低毛果芸香碱对ERK的刺激作用，但M1受体小干扰RNA无此作用。Src抑制剂PP2可阻断毛果芸香碱诱导的ERK激活和EGF受体磷酸化，而不影响卡巴胆碱诱导的ERK激活。我们的结果表明，毛果芸香碱和卡巴胆碱在唾液腺细胞中的作用是通过两种不同的信号传导机制介导的——毛果芸香碱通过M3受体和EGF受体的Src依赖性转活化起作用，而卡巴胆碱则通过M1/M3受体和PKC起作用，最终汇聚于ERK途径。
