ENDOTHELIN RECEPTOR--PURIFICATION AND CHARACTERIZATION

许多心血管疾病涉及血管张力和血管张力的改变 对血液流动的阻力。这些包括全身和肺脏 高血压和急性冠状动脉和脑动脉血管痉挛。现在是时候了 已知血管内皮细胞产生重要的血管调节 调节血管平滑肌张力的物质。一种这样的物质 内皮素是迄今已知的最有效的血管收缩多肽。 这项提议的长期目标是理解 内皮素与血管疾病及其阻断方法的研究进展 病理效应。 内皮素通过与血管表面高亲和力受体结合发挥作用 血管平滑肌细胞。这个项目的目的是净化 内皮素受体及其在内皮素机制研究中的应用 行动。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 不同组织的内皮素结合特性，为了提纯， 克隆内皮素受体并对其进行测序，并使用突变形式的 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 磁共振波谱学确定三维 内皮素的结构和表达克隆作为分离血管内皮细胞的手段 内皮素受体。我们有能力合成功能性的 内皮素衍生物，这可能允许产生特定的 内皮素拮抗剂。 初步研究表明，内皮素受体刺激 导致细胞内钙显著增加，并可能与 开放电压激活的钙通道。站点定向 突变将使我们能够研究这种和其他的机制 对细胞生物学很重要的信号通路。这些研究的结果 调查应提供对基本方面的更好了解 内皮素的作用，应该有助于开发新的方法 对血管疾病的研究和治疗。

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.