EPITHELIAL ACETYLCHOLINE IN ORAL BIOLOGY AND PATHOLOGY

描述（由申请人提供）：申请资金用于支持我们正在进行的研究，以确定介导乙酰胆碱（ACh），其药理学同系物和烟草制品对口腔角化细胞（OKC）影响的分子机制。角化细胞出生和死亡的连续循环是一个自我维持的过程，部分由局部激素乙酰胆碱通过信号通路控制，这些信号通路将每种类型的乙酰胆碱受体偶联以调节特定的细胞功能。游离细胞递质乙酰胆碱以生理相关浓度存在于上消化道上皮内。OKC既表达乙酰胆碱合成和降解酶，也表达烟碱类和毒蕈碱类乙酰胆碱受体。在对胆碱能蛋白SLURP（哺乳动物分泌的Ly-6/尿激酶纤溶酶原激活物受体相关蛋白）-1和-2的研究中，发现了一种通过尼古丁乙酰胆碱受体（nAChRs）进行细胞调节的新模式。初步结果表明，SLURP-1和-2调控角质细胞增殖、凋亡和分化。最重要的是，slurp和专业的尼古丁拮抗剂可以部分地破坏尼古丁衍生的亚硝胺4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK）和N'-亚硝基烟碱（NNN）引起永生化OKC转化的能力。我们将检验以下工作假设：1)NNK的病理作用主要通过a7和/或a9 nAChR介导，而nnn的病理作用主要通过a3-制造的nAChR介导；2) SLURP蛋白可以在体内和体外阻止口腔细胞亚硝胺依赖性转化，并消除角化细胞周期、生长和分化中的烟草/尼古丁依赖性改变；3) SLURP-1主要与NNK竞争同源性nAChR(s)的结合，slurp -2主要与NNN竞争异源性nAChR(s)的结合位点，两者都干扰亚硝胺诱导的nAChR信号。具体目的将是确定：1)角化细胞nAChRs在介导烟草亚硝胺的病理生物学效应中的作用；2) SLURP-1和-2在OKC抗烟草毒性生理保护中的作用；3)受体介导的SLURP-1和-2作用于OKC的信号传导机制。该应用的主要意义在于阐明烟碱乙酰胆碱受体如何介导烟草源性亚硝胺的病理作用，以及SLURP如何预防亚硝胺的毒性作用。整合slurp和上皮乙酰胆碱轴的结构和功能信息将有助于更好地理解上消化道上皮的正常发育和功能。了解SLURP与亚硝胺对口腔角化细胞作用的药理学将有助于制定有效的预防方案，其中烟草制品的有害影响是通过特定的尼古丁乙酰胆碱受体的药理学配体作为解毒剂来预测甚至消除的。

DESCRIPTION (provided by applicant): Funding is requested to support our ongoing studies toward identification of molecular mechanisms mediating effects of acetylcholine (ACh), its pharmacologic congeners and tobacco products on oral keratinocytes (OKC). The continuous cycle of keratinocyte birth and death is a self-sustained process controlled, in part, by the local hormone ACh through the signaling pathways that couple each type of ACh receptors to regulation of a particular cell function. Free cytotransmitter ACh is present in physiologically-relevant concentrations in the epithelium lining the upper digestive tract. OKC express both the ACh synthesizing and degrading enzymes, and both nicotinic and muscarinic classes of ACh receptors. A novel paradigm of cell regulation via nicotinic ACh receptors (nAChRs) has been discovered in studies of the cholinergic proteins termed SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein)-1 and -2. Preliminary results indicate that SLURP-1 and -2 regulate keratinocyte proliferation, apoptosis and differentiation. Most importantly, SLURPs and professional nicotinic antagonists can abolish, in part, the abilities of the nicotine- derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) to cause transformation of immortalized OKC. We will test the following working hypotheses: 1) the pathobiologic effect of NNK is mediated predominantly via a7 and/or a9 nAChR(s), and that of NNN-via a3- made nAChR(s); 2) SLURP proteins can prevent nitrosamine-dependent transformation of oral cells both in vivo and in vitro, and abolish tobacco/nicotine-dependent alterations in the keratinocyte cell cycle, growth and differentiation; and 3) SLURP-1 competes mainly with NNK for binding to the homopentameric nAChR(s) and SLURP-2-with NNN at the binding site of heteropentameric nAChR(s), and both SLURPs interfere with the nitrosamine-induced nAChR signaling. The Specific Aims will be to determine: 1) the role of keratinocyte nAChRs in mediating the pathobiologic effects of tobacco nitrosamines; 2) the roles for SLURP-1 and -2 in the physiologic protection of OKC from tobacco toxicity; and 3) the receptor-mediated signaling mechanisms mediating SLURP-1 and -2 actions on OKC. The primary significance of the application lies in its goal to elucidate how nicotinic acetylcholine receptors mediate pathobiologic effects of tobacco-derived nitrosamines and how SLURP can prevent the toxic effects of nitrosamines. Integrating structural and functional information about SLURPs and the epithelial acetylcholine axis will facilitate a better understanding of normal development and function of the epithelium lining the upper digestive tract. Learning the pharmacology of the SLURP vs. nitrosamine action on oral keratinocytes will help develop an effective prevention programs wherein hazardous effects of tobacco products are anticipated, or even abolished, by a pharmacologic ligand of a specific nicotinic acetylcholine receptors acting as an antidote.