Nicotine Addiction: ACh Receptors and Secretion

描述(申请人提供)：尼古丁成瘾会导致 大大缩短了数百万人和数十亿人的寿命 每年花在烟草相关疾病上的医疗保健费用。尽管 成瘾的机制还不清楚，有越来越多的证据 指出高亲和力神经元尼古丁受体的参与 (NAChR)成瘾。我们的项目将探索可能的机制， 尼古丁成瘾是其根本原因。我们的研究将在肾上腺进行 嗜铬细胞，这可能是尼古丁的主要外周靶点。 嗜铬细胞表达神经元nAChR，与神经元相似； 随后由尼古丁引发的儿茶酚胺释放可能参与了 上瘾的过程。儿茶酚胺的释放可能会影响血压和心脏 速度，并可能参与了一些不利的生理变化 吸烟者。此外，我们还将研究生理释放机制。 通过ACh(或尼古丁)。我们已经帮助开发了一种新的模型系统，将允许 美国将直接测试神经元尼古丁受体在启动 神经递质释放。类似的研究将在嗜铬细胞中进行 细胞。这笔赠款的具体目的是： 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的释放。人体内的尼古丁水平 吸烟者足以使受体显著脱敏， 这就改变了释放。因为nAChRs对钙离子有很高的渗透性，我们的目标是 就是确定这条钙离子内流途径在触发 使用生理上相关的尼古丁释放。这些 实验将在短期和长期(20 mm。至6天) 暴露在低水平尼古丁(100-500 NM)中的情况与在 吸烟者。因为细胞内Ca~(2+)的升高动员小泡 释放后，我们将尝试确定低水平的尼古丁(100-500 Nm)影响囊泡动员和脱敏在其中的作用 进程。 AIM 2(小鼠嗜铬细胞瘤细胞)-假设：多种细胞因子的激活 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)最近发现一种小鼠嗜铬细胞瘤(MPC)细胞 有分泌能力但不表达尼古丁受体和 内源性钙通道很少或没有。我们将用基因转染这些细胞 不同类型的尼古丁受体来确定它们是否可以触发 分泌或动员囊泡及其相关机制。亚基将会 检测对象包括A7(单独)、A3 b2、A3/b4和A4/b2。在这些实验中 我们将使用不表达内源性钙电流的细胞(由 短暂地使每个细胞去极化)。生理上，nAChRs的激活 使细胞去极化，激活钙通道。在其他实验中，我们将 NAChRs与A1b/a2d/b3a(N型)钙通道亚基顺序共转染 研究这些钙离子-INF1ux信号通路之间的协同作用。这一新目标是 对我们来说是非常令人兴奋的，只有因为发现了这个 非凡的细胞系。

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.