ENDOTHELIN RECEPTOR--PURIFICATION AND CHARACTERIZATION

内皮素受体——纯化和表征

• 批准号: 3087702
• 负责人: MICHAEL D GUNN
• 金额: $ 6.46万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3087702
• 关键词: Xenopus; affinity chromatography; biological signal transduction; blood vessel disorder; calcium flux; chemical structure function; cow; gel electrophoresis; heart cell; high performance liquid chromatography; hypertension; ion exchange chromatography; molecular cloning; muscle tone; nuclear magnetic resonance spectroscopy; nucleic acid probes; peptide chemical synthesis; protein purification; protein structure function; pulmonary hypertension; radionuclides; receptor binding; receptor expression; site directed mutagenesis; tissue /cell culture; vascular resistance; vascular smooth muscle; vasoconstrictors; vasospasm

Numerous cardiovascular diseases involve alterations in vascular tone and resistance to blood flow. These include systemic and pulmonary hypertension and acute coronary and cerebral artery vasospasm. It is now known that the vascular endothelium produces important vaso-regulatory substances which mediate vascular smooth muscle tone. One such substance is endothelin, the most potent vasoconstrictor polypeptide known to date. The long term goal of this proposal is to understand the role of endothelin in vascular disease and to develop methods of blocking its pathological effects. Endothelin acts by binding to high affinity receptors on the surface of vascular smooth muscle cells. The aim of this project is to purify the endothelin receptor and use it to study the mechanism of endothelin action. In order to do this, our specific aims are to determine the important structural features of the endothelin molecule, to determine the endothelin binding characteristics of various tissues, to purify, clone, and sequence the endothelin receptor, and to use mutated forms of the receptor to study endothelin's signaling pathways. This work will utilize newer biochemical techniques including the use of nuclear magnetic resonance spectroscopy to determine the three dimensional structure of endothelin and expression cloning as a means to isolate the endothelin receptor. We have the ability to synthesize functional endothelin derivatives, which may allow the production of specific endothelin antagonists. Preliminary studies have shown that endothelin receptor stimulation causes a marked increase in intracellular calcium and may be coupled to opening of the voltage activated calcium channel. Site directed mutagenesis will allow us to study the mechanism of this and other signaling pathways important to cell biology. The results of these investigations should provide a better understanding of the basic aspects of endothelin action and should aid in the development of new approaches to the study and treatment of vascular disease.

许多心血管疾病涉及血管张力的改变， 血液流动的阻力。 这些包括全身和肺部 高血压和急性冠状动脉和脑动脉血管痉挛。 现在 已知血管内皮细胞产生重要的血管调节作用， 调节血管平滑肌张力的物质。 一种这样的物质 是内皮素，迄今为止已知的最有效的血管收缩多肽。 本提案的长期目标是了解 内皮素在血管疾病中的作用，并开发阻断其作用的方法。 病理影响。 内皮素通过与血管内皮细胞表面上的高亲和力受体结合而起作用。 血管平滑肌细胞 该项目的目的是净化 内皮素受体，并利用其研究内皮素的作用机制 行动上 为了做到这一点，我们的具体目标是确定 内皮素分子的重要结构特征，以确定 各种组织的内皮素结合特性，纯化， 克隆内皮素受体，并对其进行测序， 研究内皮素信号通路的受体。 这项工作将 利用更新的生化技术，包括使用核 磁共振波谱法来确定 内皮素的结构和表达克隆作为分离内皮素的手段， 内皮素受体 我们有能力合成 内皮素衍生物，这可能允许特定的生产 内皮素拮抗剂。 初步研究表明，内皮素受体刺激 引起细胞内钙的显著增加，并可能与 打开电压激活的钙通道。定点 诱变将使我们能够研究这一机制和其他 对细胞生物学很重要的信号通路。 的结果予以 调查应使人们更好地了解 内皮素的作用，并应有助于开发新的方法 血管疾病的研究和治疗。