Epithelial Acetylcholine Oral Biology and Pathology

描述（由申请人提供）：目的：确定医疗和 新的非神经元胆碱能系统的生物学意义 上消化道，并阐明纯尼古丁（Nic）与 烟草制品对烟碱和毒蕈碱乙酰胆碱受体（nAChR和 mAChR）。背景：乙酰胆碱 调节KC的重要功能，包括增殖、粘附、迁移， 分化和凋亡。α 3，α 5，α 7，α 9，β 2， 和β 4 nAChR亚基，m2、m3、m4和m5 mAChR亚型，以及 合成酶胆碱乙酰转移酶和降解酶 乙酰胆碱酯酶在人类口腔KC中表达和发挥功能， 以KC为目标，让Nic直接生效。初步观察结果：伴随 在KC分化过程中出现不同的ACh受体， ACh对发育中的粘膜细胞表现出不同的时间调节作用， 上皮ACh对nAChR介导的Ca 2+内流的刺激作用， 平衡mAChR介导的抑制作用和同时激活 这两种受体的结合在KC中产生了一种阴阳调节平衡。 假设：1）由口腔KC表达的每个ACh受体独特地调节 细胞周期的进展，使分化决定的变化， 单个角质形成细胞中的ACh受体库可以使 ACh的生物效应2)长期暴露于Nic干扰了动态 角质形成细胞烟碱和毒蕈碱途径之间的平衡 由乙酰胆碱控制，导致细胞周期异常。具体目标：1）识别 每个ACh受体对细胞周期进程的贡献， 亚型选择性激动剂和拮抗剂，反义寡核苷酸，和 敲除小鼠2)确定Nic与水提取物的长期影响 无烟烟草与环境烟草烟雾对 角质形成细胞ACh受体和ACh信号转导的Ca 2+通路 KC.方法学：采用分子生物学（RT-PCR，实时荧光定量PCR） TaqMan PCR，北方印迹），免疫学（免疫组织化学，Western印迹 印迹，ELISA）、药理学和生物学测定将用于 定量ACh受体介导的细胞周期进展变化（目标#1） 以及尼克和烟草制品对结构和功能的影响， 角质形成细胞ACh受体（目标#2）。意义：非神经元ACh的作用 在口腔细胞生物学和病理学将阐明，这将有助于解释 烟草制品在上层大气中有害作用的一些机制 消化道

DESCRIPTION (provided by applicant): PURPOSE: To identify the medical and biological significance of the novel non-neuronal cholinergic system of the upper digestive tract, and elucidate the effects of pure nicotine (Nic) vs. tobacco products on nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) expressed by oral and esophageal keratinocytes (KC). BACKGROUND: ACh regulates vital functions of KC including proliferation, adhesion, migration, differentiation and apoptosis. The alpha3, alpha5, alpha7, alpha9, beta2, and beta4 nAChR subunits, the m2, m3, m4 and m5 mAChR subtypes, as well as the synthesizing enzyme choline acetyltransferase and the degrading enzyme acetylcholinesterase are expressed and function in human oral KC, and may target KC for direct effects of Nic. PRELIMINARY OBSERVATIONS: Concomitant emergence of different ACh receptors in the course of KC differentiation allows ACh to exhibit diverse and temporally regulation of the developing mucosal epithelium. A stimulatory effect of ACh on Ca2+ influx, mediated by nAChR, balances an inhibitory effect, mediated by mAChR, and simultaneous activation of both receptors produces a kind of a yin yang regulatory balance in KC. HYPOTHESES: 1) Each ACh receptor expressed by oral KC regulates uniquely the cell cycle progression so that the differentiation-determined changes in the repertoire of ACh receptors in a single keratinocyte can diversify the biological effects of ACh. 2) Chronic exposure to Nic disturbs the dynamic equilibrium between the nicotinic and the muscarinic pathways of keratinocyte control by ACh, leading to an aberrant cell cycle. SPECIFIC AIMS: 1) Identify contribution of each ACh receptor to the cell cycle progression using subtype-selective agonists and antagonists, antisence oligonucleotides, and knockout mice. 2) Identify long-term effects of Nic vs. aqueous extract of smokeless tobacco vs. environmental tobacco smoke on expression/function of keratinocyte ACh receptors and the Ca2+ pathways subserving ACh signaling in KC. METHODOLOGY: A combination of molecular biological (RT-PCR, real-time TaqMan PCR, Northern blots), immunological (immunohistochemistry, Western blots, ELISA), pharmacological and biological assays will be employed to quantitate ACh receptor-mediated changes of the cell cycle progression (Aim #1) as well as effects of Nic and tobacco products on the structure and function of keratinocyte ACh receptors (Aim #2). SIGNIFICANCE: The role of non-neuronal ACh in oral cell biology and pathology will be elucidated, which will help explain some of the mechanisms of deleterious effects of tobacco products in the upper digestive tract.