MOLECULAR APPROACHES TO NICOTINE ADDICTION

尼古丁是一种滥用药物，可能通过以下方式发挥其作用： 它与大脑中特定尼古丁受体的相互作用。 慢性 暴露于尼古丁导致尼古丁丰度增加 在老鼠和人类大脑中的结合位点。 我们发现， 克隆并测序了10个编码 神经元烟碱受体的亚基。在本申请中，我们 提出一种利用这些克隆来研究新陈代谢的方法， 调节大脑中的尼古丁结合位点。 这种方法 使用我们的cDNA克隆作为合成胞外 每个受体亚基的结构域，然后使用这些 细菌表达的蛋白质在兔子体内产生抗体。 在含有亚基特异性肽的柱上的亲和层析 导致抗体能够区分细胞外 每个受体亚基的结构域。 这些抗体和放射性 肽用于产生对以下足够敏感的定量测定： 以毫克为单位测定特定受体亚单位的量 的大脑。 我们将使用这些测定来探索 烟碱受体和产生上调的机制 慢性尼古丁使用后的尼古丁结合位点。 我们的目标是 区分不同的机制，可能产生更多的 尼古丁结合位点。 我们还将使用抗体在一个更 定性的方式来确定在神经元上的烟碱 特定亚基组成的受体。 长期目标是 研究的目的是了解结合位点的位置， 在慢性药物治疗过程中上调与以下过程有关， 导致上瘾。 这些实验将解决一些基本问题， 尼古丁成瘾和导致依赖的细胞机制 在药物上。

Nicotine is a drug of abuse that presumably exerts its effects through its interactions with specific nicotine receptors in the brain. Chronic exposure to nicotine results in an increase in the abundance of nicotine binding sites in the brains of both rats and humans. We have discovered, cloned, and sequenced ten members of the gene family that encodes subunits of the neuronal nicotinic receptors. In this application we propose a means of using these clones to study the metabolism and regulation of the nicotine binding sites in the brain. This approach uses our cDNA clones as templates for the synthesis of the extracellular domains of each receptor subunit in bacteria and then uses these bacterially expressed proteins to generate antibodies in rabbits. Affinity chromatography on columns containing subunit specific peptides results in antibodies able to distinguish between the extracellular domains of each receptor subunit. These antibodies and radioactive peptides are used to create quantitative assays sufficiently sensitive to determine amounts of specific receptor subunits in milligram quantities of brain. We will use these assays to explore the metabolism of the nicotinic receptor and the mechanisms that produce the up-regulation of nicotine binding sites following chronic nicotine use. Our goal is to distinguish between the different mechanisms that might generate more nicotine binding sites. We will also use the antibodies in a more qualitative fashion to determine the location on neurons of nicotinic receptors of specific subunit composition. The long-term goal of this research is to understand how the location of the binding sites that are up-regulated during chronic drug treatment relates to the processes that lead to addiction. These experiments will address issues fundamental to nicotine addiction and to the cellular mechanisms that lead to dependence on the drug.