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 大大缩短了数百万人和数十亿人的寿命， 每年花费在烟草相关疾病上的医疗费用。虽然 成瘾的机制尚不清楚，有越来越多的证据表明， 这表明高亲和力神经元烟碱受体参与了 （nAChR）成瘾。我们的项目将探索可能的机制， 是尼古丁成瘾的基础我们的研究将在肾上腺 嗜铬细胞，这可能是尼古丁的主要外周靶点。 嗜铬细胞表达的神经元nAChR类似于神经元中的nAChR; 尼古丁引发的随后的儿茶酚胺释放可能参与了 成瘾过程释放儿茶酚胺可能影响血压和心脏 率，并可能参与一些不利的生理变化，看到在 吸烟者。此外，我们将研究生理释放机制介导的 乙酰胆碱（或尼古丁）我们帮助开发了一个新的模型系统， 我们直接测试神经元烟碱受体在启动 神经递质释放类似的研究将在嗜铬细胞中进行 细胞这项赠款的具体目标是： 目的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的释放。中的尼古丁含量 吸烟者足以使受体显着脱敏， 从而改变释放。由于nAChR对Ca2+具有高渗透性，我们的目标是 是为了确定这种Ca2+内流途径在触发 使用生理学相关的尼古丁应用释放。这些 在短期和长期（20 mm至6天）后重复实验 暴露于低水平尼古丁（100 - 500 nM），与在 吸烟者。因为细胞内Ca2+的升高动员了囊泡， 释放，我们将试图确定是否低水平的尼古丁（100 - 500 nM）影响囊泡动员以及脱敏在其中的作用 过程 目的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 博士Art Tischler的实验室）最近发现了一种小鼠嗜铬细胞瘤（MPC）细胞 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。我们将用 不同类型的烟碱受体，以确定它们是否可以触发 分泌或动员囊泡和相关机制。下属单位将 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性Ca2+电流的细胞（通过测定细胞内Ca2+浓度来确定）。 短暂地使每个细胞去极化）。生理上，nAChRs的激活 使细胞去极化并激活Ca2+通道。在其他实验中，我们将 共转染nAChR和a1b/a2d/b3a（N型）Ca2+通道亚单位， 研究这些Ca2 +-influx途径之间的协同作用。这个新目标是 这对我们来说是非常令人兴奋的， 非凡的细胞系