EPITHELIAL ACETYLCHOLINE IN ORAL BIOLOGY AND PATHOLOGY

描述(由申请人提供)：申请资助，以支持我们正在进行的研究，以确定乙酰胆碱(ACh)、其药理同系物和烟草产品对口腔角质形成细胞(OKC)的调节作用的分子机制。角质形成细胞出生和死亡的连续周期是一个自我维持的过程，部分受局部激素ACh控制，通过信号通路将每种类型的ACh受体连接到特定细胞功能的调节。游离细胞递质ACh以生理上相关的浓度存在于上消化道衬里的上皮中。OKC既表达ACh合成酶和降解酶，也表达烟碱型和毒鼠型ACh受体。在对胆碱能蛋白SLULP(分泌型哺乳动物Ly-6/尿激酶型纤溶酶原激活剂受体相关蛋白)-1和-2的研究中，发现了一种通过烟碱型ACh受体(NAChRs)调节细胞的新范式。初步结果表明，slurp-1和slurp-2对角质形成细胞的增殖、凋亡和分化具有调节作用。最重要的是，口香糖和专业的尼古丁拮抗剂可以部分消除尼古丁衍生的亚硝胺4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)和N‘-亚硝基烟碱(NNN)引起永生化OKC转化的能力。我们将检验下列工作假设：1)NNK的病理生物学效应主要通过A7和/或A9nAChR(S)介导，而NNN-主要通过A3合成的nAChR(S)介导；2)SLUP蛋白在体内和体外都可以阻止亚硝胺依赖的口腔细胞转化，并取消在角质形成细胞周期、生长和分化中依赖烟草/尼古丁的改变；以及3)SLUP-1主要与NNK竞争结合同质突触nAChR(S)和slurp-2-与NNN在异质突触nAChR(S)的结合部位，并两者都干扰亚硝胺诱导的nAChR信号转导。其具体目的是确定：1)角质形成细胞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免受烟草毒性的生理保护中的作用；以及3)受体介导的信号机制介导slurp-1和-2对OKC的作用。该应用的主要意义在于其目的在于阐明烟碱型乙酰胆碱受体如何介导烟草亚硝胺的病理生物学效应，以及口水如何预防亚硝胺的毒性效应。整合唾液和上皮乙酰胆碱轴的结构和功能信息将有助于更好地了解上消化道衬里上皮的正常发育和功能。了解唾液与亚硝胺对口腔角质形成细胞作用的药理学，将有助于制定有效的预防计划，在该计划中，烟草产品的有害影响可以通过作为解毒剂的特定烟碱型乙酰胆碱受体的药理配体来预测，甚至消除。

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.