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 暴露对多种尼古丁的表达和功能的调节 乙酰胆碱受体(NAChR)亚型。 正常肌肉和转化肌肉表达不同的nAChR亚型 细胞或由不同的神经元及其克隆系类似物。对于每个 正常或克隆性细胞类型和nAChR亚型的组合，慢性 尼古丁处理对nAChR表达有明显影响。这个 这一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都是通过 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由 翻译后。我们假设，对于所有类型的细胞，尼古丁 与nAChR上开放通道阻滞点的相互作用诱导初始， 细胞表面nAChR和nAChR功能活性迅速丧失。在……里面 此外，对于大脑神经元和选定的细胞系，我们假设 NAChR基因转录随后被激活，而 至少在功能上沉默的细胞表面nAChR的再现 最初是这样。我们还假设，这些功能沉默的nAChR具有 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一个成熟的过程，并且 NAChR的内化(和/或nAChR磷酸化的变化 状态)是尼古丁早期作用的机制。 这项拟议的研究将通过克隆细胞系来验证这些假设。 稳定而自然地表达不同的nAChR亚型 神经或肌肉特征。的时间和剂量依赖效应 尼古丁和其他尼古丁药物的治疗将被量化 关于(A)nAChR功能活性水平(由离子评估 外排分析和单通道记录)、(B)nAChR水平和位置 表达(通过配体或抗体结合试验和亚细胞鉴定 分布分析)，(C)nAChR亚基组成(通过 免疫印迹和亲和分离分析)，(D)nAChR水平 亚单位mRNA表达和基因转录活性(使用 Northern和核连续分析)，以及(E)磷酸化 NAChR多肽的状态(用~(32)P原位标记和抗- NAChR多肽的磷酸化状态(32P原位标记) 和反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).