MOLECULAR MECHANISMS OF NICOTINE DEPENDENCE

作为药物成瘾和滥用的分子基础的模型， 拟议的研究将建立慢性尼古丁 暴露调节多种烟碱的表达和功能 乙酰胆碱受体（nAChR）亚型。 正常和转化肌肉表达不同的nAChR亚型 细胞或不同的神经元及其克隆系类似物。 为每个 正常或克隆细胞类型和nAChR亚型的组合，慢性 尼古丁处理对nAChR的表达具有独特的影响。 的 该建议的中心假设是尼古丁对nAChR的影响 在脑中和在选定的细胞系中的表达是在两个细胞系中介导的。 转录和翻译后水平，而对nAChR的影响， 肌肉、自主神经节和类似的细胞系主要介导 事后的 我们假设，对于所有类型的细胞，尼古丁 与nAChR上的开放通道阻断位点的相互作用诱导初始的， 细胞表面nAChR和nAChR功能活性的快速丧失。 在 此外，对于脑神经元和选定的细胞系，我们假设 有一个随后的激活nAChR基因转录和 细胞表面nAChR的重新出现，至少在功能上是沉默的， 一开始 我们还假设，这些功能沉默的nAChR 独特的亚基组成或磷酸化状态和/或需要 在它们表现出功能活性之前经历成熟过程， nAChR的内化（和/或nAChR磷酸化的变化） 状态）是尼古丁早期效应的机制。 这项研究将通过克隆细胞系来验证这些假设 稳定和天然表达不同的nAChR亚型， 神经元或肌肉特征。 时间和剂量依赖性效应 将量化尼古丁和其他烟碱类药物的治疗 关于（a）nAChR功能活性的水平（通过离子分析评估）， 外排试验和单通道记录），（B）nAChR的水平和位点 表达（通过配体或抗体结合测定和亚细胞免疫测定评估） 分布分析），（c）nAChR的亚基组成（通过 蛋白质免疫印迹和亲和分离分析）。（d）nAChR水平 亚基mRNA表达和基因转录活性（使用 北方和核连续试验），以及，如果需要，（e）磷酸化 nAChR肽的状态（通过原位32 P标记和抗- nAChR肽的磷酸化状态（通过原位32 P标记评估 和抗磷酸酪氨酸免疫印迹分析）。

As a model for the molecular basis of drug addiction and abuse, the proposed studies will establish mechanisms by which chronic nicotine exposure regulates expression and function of diverse nicotinic acetylcholine receptor (nAChR) subtypes. Different nAChR subtypes are expressed by normal and transformed muscle cells or by different neurons and their clonal line analogs. For each combination of normal or clonal cell type and nAChR subtype, chronic nicotine treatment has distinctive effects of nAChR expression. The central hypothesis of this proposal is that effects of nicotine on nAChR expression in brain and in selected cell lines are mediated at both transcriptional and post-translational levels, whereas effects on nAChR in muscle, autonomic ganglia, and analogous cell lines are mediated primarily post-translationally. We hypothesize that, for all cell types, nicotine interaction with open channel blocking sites on nAChR induces an initial, rapid loss of cell surface nAChR and nAChR functional activity. In addition, for brain neurons and for selected cell lines, we hypothesize that there is a subsequent activation of nAChR gene transcription and the reappearance of cell surface nAChR that are functionally silent, at least initially. We also postulate that these functionally silent nAChR have either unique subunit compositions or phosphorylation states and/or need to undergo a maturation process before they exhibit functional activity, and that internalization of nAChR (and/or a change in nAChR phosphorylation state) is the mechanism for early nicotine effects. The proposed study will test these hypotheses by using clonal cell lines that stably and naturally express different nAChR subtypes and either neuronal or muscle characteristics. Time- and dose-dependent effects of treatment with nicotine, and with other nicotinic drugs, will be quantified with respect to (a) levels of nAChR functional activity (assessed by ion efflux assays and single channel recording), (b) levels and sites of nAChR expression (assessed by ligand or antibody binding assays and subcellular distribution analysis), (c) subunit composition of nAChR (assessed by Western immunoblot and affinity isolation analyses), (d) levels of nAChR subunit mRNA expression and gene transcriptional activity (measured using Northern and nuclear run-on assays), and, if warranted, (e) phosphorylation state of nAChR peptides (assessed by 32P labeling in situ and anti- phosphorylation state of nAChR peptides (assessed by 32P labeling in situ and anti-phosphotyrosine immunoblot analyses).