ANTAGONIST ACTIONS OF NICOTINE

本建议中所述研究的目的是比较 神经元烟碱受体几种亚型的特性 并确定尼古丁如何影响这些细胞的数量和功能， 受体。 神经元烟碱受体由α和β 亚基，哺乳动物脑表达编码至少6种不同的 α和3种不同的β亚基尽管存在这种潜在的多样性， 只有两种亚型的神经元烟碱受体的性质， 哺乳动物的大脑已经有了比较好的特征。两个重要 缺乏关于其他神经元烟碱的信息的原因 受体亚型是：l）直到最近，放射性标记的烟碱 可用的配体只能可靠地测量 含有α 4和β 2亚基或α 7亚基，以及2） 没有哺乳动物细胞模型表达单一定义的 神经元烟碱受体亚型。本文中描述的研究 这些建议旨在克服这些限制。我们将使用 [~ 3 H] E_3，一种新的高亲和力配体 （约5 pM- 1 nM），用于几种不同的神经元亚型 烟碱受体和烟碱受体介导的86 Rb + 比较烟碱外排的特点和调控 受体在现有的克隆细胞系和新的， 表达确定的神经元烟碱亚型的哺乳动物细胞系 受体。 本研究的具体目的是：1）比较 大鼠原代培养物中α 4/β 2烟碱受体的调节 在脑和哺乳动物细胞系中稳定表达这些受体， 在体大鼠脑内受体的调节。2)表征 药理学性质和两种或更多种烟碱的调节 脑内[~ 3 H]地棘蛙素标记的受体结合位点。3)比较 非α 4的药理学性质和亚基组成 大鼠PC 12细胞、IMR-32细胞和SHSY-5 Y细胞中的烟碱受体。 四、 探讨非α 4功能和数量的调节 烟碱受体 5)构建哺乳动物细胞系文库 用至少两种不同神经元的组合稳定转染 烟碱受体亚单位cDNA克隆。6)使用稳定转染的 细胞，以研究每种定义的受体亚型的急性脱敏， 为了确定每个α和β亚基如何影响急性 脱敏。7)为了确定长期接触尼古丁和 其他尼古丁药物会影响神经元数量和功能的调节 每一种都有明确的受体亚型。

The objectives of the studies described in this proposal are to compare the characteristics of several subtypes of neuronal nicotinic receptors and to determine how nicotine affects the number and function of these receptors. Neuronal nicotinic receptors are assembled from alpha and beta subunits, and mammalian brain expresses mRNA encoding at least 6 different alpha and 3 different beta subunits. Despite this potential diversity, the properties of only two subtypes of neuronal nicotinic receptors in mammalian brain have been relatively well-characterized. Two important reasons for this lack of information about other neuronal nicotinic receptor subtypes are that l) until recently the radiolabeled nicotinic ligands that were available could reliably measure only receptors that contained either alpha4 and beta2 subunits or alpha7 subunits, and 2) there were no mammalian cell models that expressed a single defined subtype of neuronal nicotinic receptor. The studies described in this proposal are designed to overcome these limitations. We will use [3H)epibatidine ([3H]E3), a new ligand with very high affinity (approximately 5pM - 1 nM) for several different subtypes of neuronal nicotinic receptors, and measurements of nicotinic receptor-mediated 86Rb+ efflux to compare the characteristics and the regulation of nicotinic receptors in existing clonal cell lines and new, stably transfected mammalian cell lines that express defined subtypes of neuronal nicotinic receptors. The specific aims of this research are: 1) To compare the regulation of alpha4/beta2 nicotinic receptors in primary cultures of rat brain and in mammalian cell lines that stably express these receptors to the regulation of receptors in rat brain in vivo. 2) To characterize the pharmacological properties and the regulation of the two or more nicotinic receptor binding sites labeled by [3H]epibatidine in brain. 3) To compare the pharmacological properties and subunit composition of the non-alpha4 nicotinic receptors in rat PC12 cells, IMR-32 cells and SHSY-5Y cells. 4) To examine the regulation of the function and number of non-alpha4 nicotinic receptors. 5) To construct a library of mammalian cell lines stably transfected with combinations of at least two different neuronal nicotinic receptor subunit cDNA clones. 6) To use the stably transfected cells to study acute desensitization of each defined receptor subtype and to determine how each alpha and beta subunit influences acute desensitization. 7) To determine how chronic exposure to nicotine and other nicotinic drugs affects the regulation of the number and function of each defined receptor subtype.