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首页> 外文学位 >Studies of neuronal nicotinic acetylcholine receptors in the rat pineal gland: A model for native receptors composed of alpha-3 and beta-4 subunits.
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Studies of neuronal nicotinic acetylcholine receptors in the rat pineal gland: A model for native receptors composed of alpha-3 and beta-4 subunits.

机译：大鼠松果体神经元烟碱乙酰胆碱受体的研究：由α-3和β-4亚基组成的天然受体的模型。

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摘要

Neuronal nicotinic acetylcholine receptors (nAChRs) are located throughout the central and peripheral nervous systems. They are ligand-gated ion channels composed of alpha and beta subunits. To date, nine alpha (2--10) and three beta (2--4) subunit genes have been identified. Due to the presence of multiple subunits, there are many possible combinations that can form functional receptor subtypes, each displaying distinct pharmacological properties.; Studies of nAChRs in heterologous expression systems have confirmed receptor diversity and provided important information on the pharmacology, function, and regulation of defined receptor subtypes. However, the precise subtypes of nAChRs in native tissues are less understood. Identifying the subunit composition of native nAChRs is important to establishing the physiological roles played by different subtypes in vivo.; The presence of nAChRs in the rat pineal gland was previously suggested by immunocytochemistry and autoradiographic studies. Furthermore, the expression of mRNA encoding for the alpha3, beta2, and beta4 subunits has been demonstrated. The objectives of this research were to assess the pharmacology, subunit composition, and regulation of nAChRs in the rat pineal.; Radioligand binding experiments indicated the pineal gland expresses a high density of [3H]EB labeled binding sites with a K d of 103 pM. Nicotinic drugs competed for [3H]EB binding sites with a rank order of A85380 > cytisine > nicotine > DHBE. In parallel experiments using alpha3beta4 HEK cells, the rank order of potencies, and absolute Ki values were similar to those measured in the rat pineal.; We utilized whole-cell patch clamp to study the functional pharmacology of nAChRs in dissociated rat pinealocytes. The rank order of nicotinic agonists' affinities for current activation was A85380 > DNWP > cytisine ∼ nicotine > acetylcholine. In terms of drug efficacy, cytisine was equally efficacious to nicotine and acetylcholine in activating current. The antagonists, mecamylamine, curare, and DHbetaE inhibited nicotine-activated currents, but MLA and alpha-Bgtx, were ineffective.; Immunoprecipitation with subunit selective antibodies raised against the alpha3, beta2, and beta4 subunits was performed to identify subunit associations of nAChRs from rat pineal. Immunoprecipitation of virtually all [3H]EB labeled receptors was seen for the anti-alpha3 and anti-beta4 antibodies but not the anti-beta2 antibody. Furthermore, initial incubation with anti-alpha3 or anti-beta4 antibodies eliminated virtually all specific immunoprecipitation by a second incubation with either the anti-alpha3 or anti-beta4, indicating the alpha3 and beta4 subunits are associated in the majority of nAChRs in the rat pineal. Additionally, the alpha3beta4 receptor subtype appeared to decrease following chronic treatment with the cholinesterase inhibitor, DFP.; Our findings indicate the rat pineal gland serves as an excellent model in which to study the pharmacology and functional roles of native nAChR containing the alpha3 and beta4 subunit combination.
机译：神经元烟碱型乙酰胆碱受体（nAChRs）位于整个中枢和周围神经系统。它们是由α和β亚基组成的配体门控离子通道。迄今为止，已经鉴定出九个α（2--10）和三个β（2--4）亚基基因。由于存在多个亚基，有许多可能的组合可以形成功能性受体亚型，每个亚型表现出不同的药理特性。在异源表达系统中对nAChRs的研究已经证实了受体的多样性，并提供了有关定义的受体亚型的药理学，功能和调节的重要信息。但是，对天然组织中nAChRs的确切亚型了解较少。鉴定天然nAChRs的亚基组成对于确定体内不同亚型发挥的生理作用很重要。先前通过免疫细胞化学和放射自显影研究提示大鼠松果体中存在nAChRs。此外，已经证明了编码α3，β2和β4亚基的mRNA的表达。这项研究的目的是评估大鼠松果体中nAChRs的药理作用，亚基组成和调节。放射性配体结合实验表明，松果体表达高密度的[3H] EB标记的结合位点，K d为103 pM。烟碱类药物竞争[3H] EB结合位点，其排列顺序为A85380>胱氨酸>烟碱> DHBE。在使用alpha3beta4 HEK细胞的平行实验中，效价的等级顺序和绝对Ki值与在大鼠松果体中测得的相似。我们利用全细胞膜片钳研究解离的大鼠松果细胞中nAChRs的功能药理。烟碱激动剂对当前激活的亲和力的等级顺序为：A85380> DNWP>胱氨酸〜尼古丁>乙酰胆碱。就药物功效而言，胱氨酸在激活电流方面对尼古丁和乙酰胆碱同样有效。拮抗剂美卡明胺，咖喱和DHbetaE抑制了尼古丁激活的电流，但MLA和α-Bgtx无效。用针对α3，β2和β4亚基的亚基选择性抗体进行免疫沉淀，以鉴定来自大鼠松果体的nAChRs的亚基缔合。几乎所有[3H] EB标记的受体的免疫沉淀都针对抗α3和抗β4抗体，但未抗β2抗体。此外，与抗α3或抗β4抗体的初始孵育通过与抗α3或抗β4的第二次孵育消除了几乎所有的特异性免疫沉淀，表明α3和β4亚基与大鼠松果体中的大多数nAChR相关。 。此外，用胆碱酯酶抑制剂DFP长期治疗后，α3beta4受体亚型似乎减少。我们的发现表明，大鼠松果体是研究包含α3和β4亚基的天然nAChR的药理作用和功能作用的出色模型。

著录项

  • 作者

    Hernandez, Susan Carolyn.;

  • 作者单位

    Georgetown University Medical Center.;

  • 授予单位 Georgetown University Medical Center.;
  • 学科 Biology Neuroscience.; Health Sciences Pharmacology.
  • 学位 Ph.D.
  • 年度 2001
  • 页码 126 p.
  • 总页数 126
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 神经科学;药理学;
  • 关键词

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