Epithelial Acetylcholine Oral Biology and Pathology

描述(由申请人提供)：目的：识别医疗和 新的非神经性胆碱能系统的生物学意义 上消化道，并阐明纯尼古丁(NIC)与 烟草制品对烟碱型和毒酪碱型乙酰胆碱受体(nAChR和 口腔和食道角质形成细胞(KC)表达mAChR)。背景：ACH 调节KC的重要功能，包括增殖、黏附、迁移、 分化和细胞凋亡。字母3、字母5、字母7、字母9、字母2、 和β4nAChR亚基，m2、m3、m4和m5mAChR亚型，以及 胆碱乙酰转移酶及其降解酶的合成 乙酰胆碱酯酶在人类口腔KC中表达和功能，并可能 将KC作为NIC的直接影响的目标。初步观察：伴随而来 KC分化过程中不同ACh受体的出现 ACh对发育中的粘膜表现出多样化和时间性的调节 上皮组织。ACh对nAChR介导的钙内流的刺激作用 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)口服KC表达的每个ACh受体唯一地调节 细胞周期进程使分化决定的变化在 单个角质形成细胞中的ACh受体谱系可以使 ACh的生物学效应。2)长期接触网卡会扰乱动态 角质形成细胞烟碱途径和毒碱途径之间的平衡 由ACh控制，导致细胞周期异常。具体目标：1)确定 每种ACh受体在细胞周期进程中的作用 亚型选择性激动剂和拮抗剂、反义寡核苷酸和 基因敲除老鼠。2)确定NIC与水提物的长期效果 无烟烟草与环境烟草烟雾对其表达/功能的影响 角质形成细胞ACh受体及参与ACh信号转导的钙通道 KC。方法学：结合分子生物学(RT-PCR，实时荧光定量 TaqMan PCR，Northern blots)，免疫学(免疫组织化学，Western 将采用印迹、酶联免疫吸附试验、药理和生物检测等方法 定量测定ACh受体介导的细胞周期进程的变化(目标1) 以及NIC和烟草制品对神经细胞结构和功能的影响 角质形成细胞ACh受体(目标2)。意义：非神经性ACh的作用 在口腔细胞生物学和病理学方面将得到阐明，这将有助于解释 中上层烟草产品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.