Nicotine Addiction: ACh Receptors and Secretion

尼古丁成瘾：乙酰胆碱受体和分泌

• 批准号: 6481785
• 负责人: AARON P. FOX
• 金额: $ 11.34万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-20 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6481785
• 关键词: calcium channel; calcium flux; cell line; chromaffin cells; drug addiction; electrophysiology; neurotransmitter transport; nicotinic receptors; pharmacokinetics; receptor coupling; receptor expression; receptor sensitivity; secretion; transfection

DESCRIPTION (provided by applicant): The addiction to nicotine results in a significantly shortened life span in millions of people and in billions of health care dollars spent every year in tobacco related illnesses. Although the mechanism of addiction is not understood there is a growing body of evidence pointing to the involvement of high-affinity neuronal nicotinic receptors (nAChR) in addiction. Our project will explore possible mechanisms that underlie nicotine addiction. Our studies will be carried out in adrenal chromaffin cells, which may be a primary peripheral target for nicotine. Chromaffin cells express neuronal nAChR similar to those in neurons; the subsequent catecholamine release triggered by nicotine may be involved in the addiction process. Release of catecholamine may affect blood pressure and heart rate and may be involved in some of the adverse physiological changes seen in smokers. In addition, we will study physiological release mechanisms mediated by ACh (or nicotine). We have helped develop a new model system that will allow us to directly test the efficacy of neuronal nicotinic receptors in initiating neurotransmitter release. Similar studies will be carried out in chromaffin cells. The specific aims of this grant are: Aim 1 (Chromaffin Cells) - Hypothesis: Activation of nicotinic receptors directly triggers or modulates neurotransmitter release. Nicotine levels in smokers are sufficient to produce significant desensitization of receptors, which alters release. Because nAChRs have a high permeability to Ca2+ our goal is to determine the importance of this Ca2+-influx pathway in triggering release using physiologically relevant applications of nicotine. These experiments will be repeated after both short- and long-term (20 mm. to 6 days) exposure to low levels of nicotine (100 -- 500 nM) similar to those observed in smokers. Because elevations of intracellular Ca2+ mobilize vesicles for release, we will try to determine whether low levels of nicotine (100 -- 500 nM) affect vesicular mobilization and the role of desensitization in this process. Aim 2 (mouse pheochromcytoma cells) - Hypothesis: Activation of a variety of different nAChR can directly trigger secretion. My lab (in collaboration with Dr. Art Tischler's lab) recently identified a mouse pheochromocytoma (MPC) cell line that is secretion competent but which expresses no nicotinic receptors and few or no endogenous Ca2+ channels. We will transfect these cells with different types of nicotinic receptors to determine whether they can trigger secretion or mobilize vesicles and the mechanisms involved. Subunits that will be tested include a7 (alone), a3 b2, a3 / b4 and a4/b2. For these experiments we will employ cells that express no endogenous Ca2+ currents (determined by briefly depolarizing every cell). Physiologically, activation of nAChRs depolarizes cells and activates Ca2+ channels. In other experiments we will co-transfect nAChRs and the a1b/a2d/b3a(N-type) Ca2+ channel subunits in order to study the synergy between these Ca2+-inf1ux pathways. This new aim is extremely exciting to us and is only possible because of the discovery of this remarkable cell line.

描述（由申请人提供）：尼古丁成瘾导致 数百万人和数十亿人的寿命显着缩短 每年用于治疗烟草相关疾病的医疗保健费用。虽然 成瘾的机制尚不清楚，有越来越多的证据 指出高亲和力神经元烟碱受体的参与 (nAChR) 成瘾。我们的项目将探索可能的机制 尼古丁成瘾的根源。我们的研究将在肾上腺进行 嗜铬细胞，可能是尼古丁的主要外周靶标。 嗜铬细胞表达与神经元相似的神经元 nAChR；这 随后由尼古丁引发的儿茶酚胺释放可能与 成瘾过程。儿茶酚胺的释放可能会影响血压和心脏 率并可能涉及一些不良的生理变化 吸烟者。此外，我们将研究介导的生理释放机制 通过乙酰胆碱（或尼古丁）。我们帮助开发了一个新的模型系统，该系统将允许 我们直接测试神经元烟碱受体在启动中的功效 神经递质释放。类似的研究将在嗜铬细胞中进行 细胞。这笔赠款的具体目标是： 目标 1（嗜铬细胞）- 假设：烟碱受体的激活 直接触发或调节神经递质释放。尼古丁含量 吸烟者足以产生显着的受体脱敏作用， 这会改变释放。因为 nAChR 对 Ca2+ 具有高渗透性，我们的目标 是为了确定该 Ca2+ 流入途径在触发中的重要性 使用尼古丁的生理相关应用来释放。这些 短期和长期（20毫米至6天）后将重复实验 接触低水平的尼古丁（100 - 500 nM），类似于在 吸烟者。因为细胞内 Ca2+ 的升高会动员囊泡 释放后，我们将尝试确定尼古丁含量是否低（100 - 500 nM）影响囊泡动员以及脱敏在此过程中的作用 过程。 目标 2（小鼠嗜铬细胞瘤细胞）- 假设：激活多种 不同的nAChR可以直接触发分泌。我的实验室（与 Art Tischler 博士的实验室）最近鉴定出一种小鼠嗜铬细胞瘤 (MPC) 细胞 具有分泌能力但不表达烟碱受体的细胞系 很少或没有内源性 Ca2+ 通道。我们将转染这些细胞 不同类型的烟碱受体以确定它们是否可以触发 分泌或动员囊泡以及所涉及的机制。亚基将 被测试的包括a7（单独）、a3 b2、a3/b4 和a4/b2。对于这些实验 我们将使用不表达内源 Ca2+ 电流的细胞（由 短暂地使每个细胞去极化）。生理学上，nAChR 的激活 使细胞去极化并激活 Ca2+ 通道。在其他实验中我们将 按顺序共转染 nAChR 和 a1b/a2d/b3a(N 型) Ca2+ 通道亚基 研究这些 Ca2+-inf1ux 途径之间的协同作用。这个新目标是 对我们来说非常令人兴奋，并且只有因为这一发现才成为可能 非凡的细胞系。