NICOTINIC RECEPTORS IN NONNEURONAL CELLS AS TARGETS FOR NICTOINE TOXICITY

非神经元细胞中的烟碱受体作为尼古丁毒性的目标

• 批准号: 6104195
• 负责人: BIANCA M CONTI-FINE
• 金额: --
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6104195
• 关键词: RNase protection assay; acetylcholine; acetylcholinesterase; cell adhesion; cell motility; cell proliferation; choline acetyltransferase; confocal scanning microscopy; immunocytochemistry; in situ hybridization; keratinocyte; membrane channels; nicotinic receptors; peripheral blood vessel disorder; protein structure function; rabies virus; receptor sensitivity; respiratory epithelium; respiratory pharmacology; tissue /cell culture; vascular endothelium; voltage /patch clamp

Bronco epithelial cells, endothelial cells and esophagus keratinocytes express nicotinic acetylcholine receptors of neuronal type (nAChR) sensitive to nicotine (Nic): the overall goal of the proposed studies is to identify new mechanisms of Nic toxicity resulting from a direct effect on those nAChRs. Block of the nAChR in bronchial epithelial and endothelial cells causes cell paralysis and cell-cell detachment. Long term exposure to Nic causes nAChR desensitization, and will likely result in cell-cell detachment, leading to bronchitis and esophagitis, atherosclerotic lesions, and facilitated entrance of carcinogenic compounds. Acute exposure of bronchial epithelial cells to Nic causes apoptosis, that might be an additional mechanism of Nic toxicity. The proposed studies will have three major (1 to 3), and three minor (4-6) specific aims: 1) to investigate the structural and functional properties of nAChRs expressed by bronchial epithelial cells, endothelial cells and esophagus keratinocytes; to extend these studies to lung alveolar epithelial cells; by patch clamp and binding studies using ligands specific for different nAChR subtypes; by immunohistochemistry, using antibodies and protein probes specific for different nAChR subtypes; by PCR using subunit specific primers, followed by cloning and sequencing of the products; by in situ hybridization using subunit specific probes. 2) to demonstrate in these cells the presence of the enzymes that metabolize ACh; by immunohistochemical and to demonstrate in these cells the presence of the enzymes that metabolize ACh; by immunohistochemical and biochemical assays of the enzymes choline acetyltransferase and acetylcholinesterase. 3) to investigate the cellular functions modulated by Nic and ACh binding , and the effects of acute and chronic Nic exposure in vitro, using functional assays of cell adhesion, motility, and proliferation, and by measuring the rate of cell death and apoptosis after exposure to Nic. 4) to investigate the possibility that the nAChRs expressed by bronchial epithelial cells are a port of entry for rabies virus, explained rabies cases that result from airborne exposure. 5) to investigate the characteristics of the endothelia nAChRs expressed in patients with Buerger's disease, a vasculitis of the limb blood vessels caused or triggered by tobacco usage. 6) to do pilot investigations to test whether nAChRs are expressed at other non-neuronal locations involved in tobacco toxicity.

野马上皮细胞，内皮细胞和食道角质形成细胞 神经元类型（NACHR）的表达烟碱乙酰胆碱受体 对尼古丁敏感（NIC）：拟议研究的总体目标是 确定直接效应引起的NIC毒性的新机制 在那些nachrs上。支气管上皮和 内皮细胞会导致细胞瘫痪和细胞细胞脱落。长的 接触NIC的期限会导致NACHR脱敏，并可能导致 在细胞细胞脱离中，导致支气管炎和食道炎， 动脉粥样硬化病变，并促进致癌的入口 化合物。支气管上皮细胞急性暴露于NIC原因 凋亡，这可能是NIC毒性的另一种机制。这 拟议的研究将有三个主要（1至3），三个次要（4-6） 具体目的： 1）研究NACHR的结构和功能特性 由支气管上皮细胞，内皮细胞和食道表达 角质形成细胞；将这些研究扩展到肺肺泡上皮细胞； 通过贴片夹和结合研究，使用特定于不同的配体 NACHR亚型；通过免疫组织化学，使用抗体和蛋白质 针对不同NACHR亚型的探针；通过PCR使用亚基 特定的引物，然后进行克隆和测序。经过 使用亚基特定探针原位杂交。 2）在 这些细胞的存在代谢ACH的酶的存在；经过 免疫组织化学并在这些细胞中证明 代谢ACH的酶；通过免疫组织化学和生化测定 酶胆碱乙酰转移酶和乙酰胆碱酯酶的酶。 3）到 研究由NIC和ACH结合调节的细胞功能，以及 急性和慢性NIC在体外的影响，使用功能 细胞粘附，运动性和增殖的测定，并通过测量 暴露于NIC后的细胞死亡和凋亡率。 4）调查 支气管上皮细胞表达的NACHR的可能性 狂犬病病例解释 来自空中暴露。 5）研究 内皮nachrs在Buerger病患者中表达 肢体血管的血管炎是由烟草使用引起或触发的。 6）进行试点调查以测试是否在 涉及烟草毒性的其他非神经元位置。