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.

支气管上皮细胞、内皮细胞和食道角质形成细胞 表达神经元型烟碱乙酰胆碱受体 (nAChR) 对尼古丁（Nic）敏感：拟议研究的总体目标是 确定由直接作用引起的尼克毒性的新机制 关于那些 nAChR。支气管上皮细胞中 nAChR 的阻断 内皮细胞导致细胞麻痹和细胞-细胞脱离。长的 长期接触 Nic 会导致 nAChR 脱敏，并可能导致 细胞与细胞分离，导致支气管炎和食管炎， 动脉粥样硬化病变，并促进致癌物质的进入 化合物。支气管上皮细胞急性暴露于 Nic 的原因 细胞凋亡，这可能是 Nic 毒性的另一个机制。这 拟议的研究将包括三个专业（1至3）和三个辅修（4-6） 具体目标： 1) 研究nAChRs的结构和功能特性 由支气管上皮细胞、内皮细胞和食管表达 角质形成细胞；将这些研究扩展到肺泡上皮细胞； 通过膜片钳和使用不同配体特异性的结合研究 nAChR 亚型；通过免疫组织化学，使用抗体和蛋白质 针对不同 nAChR 亚型的特异性探针；使用亚基通过 PCR 特异性引物，然后对产物进行克隆和测序；经过 使用亚基特异性探针进行原位杂交。 2) 证明在 这些细胞存在代谢乙酰胆碱的酶；经过 免疫组织化学并证明这些细胞中存在 代谢乙酰胆碱的酶；通过免疫组织化学和生化检测 胆碱乙酰转移酶和乙酰胆碱酯酶的酶。 3）到 研究 Nic 和 ACh 结合调节的细胞功能，以及 使用功能性方法研究体外急性和慢性 Nic 暴露的影响 细胞粘附、运动和增殖的测定，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 nAChR 的可能性 是狂犬病病毒的入境口岸，解释了导致狂犬病病例的原因 来自空气中的暴露。 5）研究特征 布尔格病患者中表达的内皮细胞 nAChR 由吸烟引起或引发的四肢血管炎。 6) 进行试点调查以测试 nAChR 是否在 与烟草毒性有关的其他非神经元位置。