Cong X, Min S N, Wu L L, Cai Z G, Yu G Y
Center for Salivary Gland Diseases, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China.
Department of Physiology and Pathophysiology, Peking University School of Basic Medical Sciences, Beijing 100191, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2019 Jun 18;51(3):390-396. doi: 10.19723/j.issn.1671-167X.2019.03.003.
Muscarinic acetylcholine receptors (mAChRs), including M1-M5 subtypes, are classic receptors in regulating water, ion, and solute transport in salivary gland. Our work focuses on the studies on the expression pattern and function of mAChR in the submandibular gland (SMG), and the underlying mechanism involved in the mAChR-regulated secretion, together with the effect of parasympathectomy on the salivary secretion. Microvascular autotransplantation of SMG into the temporal fossa provides a continuous and endogenous source of fluids, and is currently an effective method for treating severe keratoconjunctivitis sicca. By using RT-PCR, Western blotting, and immunofluorescence, our data demonstrated that the expression of M1 and M3 subtypes were decreased in latent period in rabbit SMG autotransplantation model, whereas carbachol stimulation promoted the salivary secretion, as well as M1 and M3 expressions. By contrast, mAChRs were hypersensitive in epiphora SMGs, whereas atropine gel and botulinum toxin A application significantly inhibited the hypersecretion in both animal models and patients. Furthermore, the possible intracellular signal molecules involved in the mAChR-modulated salivary secretion were explored. Activation of mAChR upregulated the expression of aquaporin 5 (AQP5), the main transporter that mediated water secretion through transcellular pathway, and led to AQP5 trafficking from lipid rafts to non-lipid microdomain. Extracellular signal-regulated kinase 1/2 (ERK1/2) was involved in the mAChR-regulated AQP5 content. mAChR activation also modulated the expression, distribution, and function of tight junction proteins, and increased paracellular permeability. ERK1/2/β-arrestin2/clathrin/ubiquitin signaling pathway was responsible for the mAChR-regulated downregulation of tight junction molecule claudin-4. Cytoskeleton filamentous actin (F-actin) was also involved in the distribution and barrier function of epithelial tight junctions. Besides, endothelial tight junctions were opened by mAChR agonist-evoked salivation in the mice. Furthermore, parasympathetic denervation increased resting salivary secretion in the long terminrats and minipigs. Taken together, our work demonstrated that mAChR regulated saliva secretion via transcellular and paracellular pathways in SMG epithelium as well as tight junction opening in SMG endothelium. Modulation of mAChR might be a promising strategy to ameliorate SMG dysfunction.
毒蕈碱型乙酰胆碱受体（mAChRs），包括M1 - M5亚型，是调节唾液腺中水、离子和溶质转运的经典受体。我们的工作重点是研究下颌下腺（SMG）中mAChR的表达模式和功能，以及mAChR调节分泌的潜在机制，以及交感神经切除术对唾液分泌的影响。将SMG微血管自体移植到颞窝可提供持续的内源性液体来源，目前是治疗严重干燥性角结膜炎的有效方法。通过逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹法和免疫荧光法，我们的数据表明，在兔SMG自体移植模型的潜伏期，M1和M3亚型的表达降低，而卡巴胆碱刺激促进了唾液分泌以及M1和M3的表达。相比之下，在溢泪的SMG中mAChRs过敏，而应用阿托品凝胶和肉毒杆菌毒素A在动物模型和患者中均显著抑制了分泌过多。此外，还探索了参与mAChR调节唾液分泌的可能的细胞内信号分子。mAChR的激活上调了水通道蛋白5（AQP5）的表达，AQP5是通过跨细胞途径介导水分泌的主要转运蛋白，并导致AQP5从脂筏转运至非脂微区。细胞外信号调节激酶1/2（ERK1/2）参与了mAChR调节的AQP5含量。mAChR激活还调节了紧密连接蛋白的表达、分布和功能，并增加了细胞旁通透性。ERK1/2/β-抑制蛋白2/网格蛋白/泛素信号通路负责mAChR调节的紧密连接分子claudin-4的下调。细胞骨架丝状肌动蛋白（F-肌动蛋白）也参与上皮紧密连接的分布和屏障功能。此外，在小鼠中，mAChR激动剂诱发的唾液分泌可打开内皮紧密连接。此外，去交感神经支配增加了长期大鼠和小型猪的静息唾液分泌。综上所述，我们的工作表明，mAChR通过SMG上皮细胞的跨细胞和细胞旁途径以及SMG内皮细胞紧密连接的开放来调节唾液分泌。调节mAChR可能是改善SMG功能障碍的一种有前景的策略。
