Arredondo J, Hall L L, Ndoye A, Chernyavsky A I, Jolkovsky D L, Grando S A
Department of Dermatology, School of Medicine, University of California, Davis, California, USA.
J Periodontal Res. 2003 Feb;38(1):79-89. doi: 10.1034/j.1600-0765.2003.01006.x.
We have previously reported the presence in human gingival keratinocytes (GKC) of choline acetyltransferase, the acetylcholine (ACh) synthesizing enzyme, acetylcholinesterase, the ACh degrading enzyme, and alpha 3, alpha 5, alpha 7, beta 2 as well as alpha 9 nicotinic ACh receptor subunits. To expand the knowledge about the role of ACh in oral biology, we investigated the presence of the muscarinic ACh receptor (mAChR) subtypes in GKC. RT-PCR demonstrated the presence of m2, m3, m4, and m5 mRNA transcripts. Synthesis of the respective proteins was verified by immunoblotting with the subtype-specific antibodies that revealed receptor bands at the expected molecular weights. The antibodies mapped mAChR subtypes in the epithelium of human attached gingiva and also visualized them on the cell membrane of cultured GKC. The whole cell radioligand binding assay revealed that GKC have specific binding sites for the muscarinic ligand [3H]quinuclidinyl benzilate, Bmax = 222.9 fmol/106 cells with a Kd of 62.95 pM. The downstream coupling of the mAChRs to regulation of cell cycle progression in GKC was studied using quantitative RT-PCR and immunoblotting assays. Incubation of GKC for 24 h with 10 micro m muscarine increased relative amounts of Ki-67, PCNA and p53 mRNAs and PCNA, cyclin D1, p21 and p53 proteins. These effects were abolished in the presence of 50 micro m atropine. The finding in GKC of mAChRs coupled to regulation of the cell cycle progression demonstrate further the structure/function of the non-neuronal cholinergic system operating in human oral epithelium. The results obtained in this study help clarify the role for keratinocyte ACh axis in the physiologic control of oral gingival homeostasis.
我们之前报道过，在人牙龈角质形成细胞（GKC）中存在胆碱乙酰转移酶（一种合成乙酰胆碱的酶）、乙酰胆碱酯酶（一种降解乙酰胆碱的酶）以及α3、α5、α7、β2和α9烟碱型乙酰胆碱受体亚基。为了进一步了解乙酰胆碱在口腔生物学中的作用，我们研究了GKC中M型乙酰胆碱受体（mAChR）亚型的存在情况。逆转录聚合酶链反应（RT-PCR）证明存在m2、m3、m4和m5 mRNA转录本。用亚型特异性抗体进行免疫印迹验证了相应蛋白质的合成，该抗体在预期分子量处显示出受体条带。这些抗体在人附着龈上皮中定位了mAChR亚型，并在培养的GKC细胞膜上也使其可视化。全细胞放射性配体结合试验表明，GKC具有M型配体[3H]喹核醇基苯甲酸酯的特异性结合位点，Bmax = 222.9 fmol/106个细胞，解离常数（Kd）为62.95 pM。使用定量RT-PCR和免疫印迹试验研究了mAChRs与GKC细胞周期进程调节的下游偶联。用10 μM毒蕈碱孵育GKC 24小时可增加Ki-67、增殖细胞核抗原（PCNA）和p53 mRNA以及PCNA、细胞周期蛋白D1、p21和p53蛋白的相对含量。在存在50 μM阿托品的情况下，这些作用被消除。在GKC中发现mAChRs与细胞周期进程调节偶联，进一步证明了在人口腔上皮中起作用的非神经元胆碱能系统的结构/功能。本研究获得的结果有助于阐明角质形成细胞乙酰胆碱轴在口腔牙龈稳态生理控制中的作用。
