Brain specific non-AT1, non-AT2 angiotensin binding site

描述(申请人提供)：关于脑血管紧张素系统有许多不确定性，其功能存在争议。然而，人们普遍认为脑血管紧张素会导致高血压和其他心血管疾病。随着对血管紧张素II的多肽片段功能的认识，一些片段有自己的受体，现在人们知道脑血管紧张素具有多种功能，远远超出了对心血管系统的调节。我们最近在大脑中发现了一个非AT1、非AT2结合部位，这可能会给大脑血管紧张素系统增加一层额外的复杂性。它的存在数量高于任何一种主要的血管紧张素受体亚型，可能是一种新的血管紧张素受体，可能仅限于大脑，也可能是一种高度特异的血管紧张素酶，在血管紧张素的代谢中发挥关键作用。为了更好地了解这个结合位点，本项目旨在进一步表征这个新的脑特异性非AT1、非AT2血管紧张素肽结合位点。第一个具体目的是通过聚丙烯酰胺凝胶纯化和测序技术确定其生化特性，以确定其结合部位的氨基酸序列和结构。第二个具体目的是确定其药理学特异性，以确定该结合位点是否为血管紧张素肽所特有，或者它是否具有更广泛的底物/配体特异性。第三个具体目标是研究结合位点在大脑中的分布，并确定与脑血管紧张素系统调节心血管系统的改变相关的条件或病理生理条件，如中风、遗传性或实验性高血压以及缺乏血管紧张素受体的转基因小鼠，是否可以改变该结合位点在特定脑区的表达。这些变化应该使我们能够对其参与生理和病理生理环境的可能性进行功能神经解剖学评估。预计这种蛋白将被证明在脑血管紧张素系统的功能中具有相当重要的作用。由于脑血管紧张素系统因导致心血管疾病而臭名昭著，这项研究可能会导致开发治疗高血压和中风的新治疗策略。然而，这个结合位点在大脑区域，如Meynert基底核、黑质、杏仁核、伏隔核和脊髓胶状质中的高浓度出现，可以解释脑血管紧张素系统参与阿尔茨海默氏症和帕金森病、应激和焦虑症、药物滥用和慢性疼痛。公共卫生相关性：心血管疾病，特别是高血压，是美国的主要死亡原因(2006年为62.9万人)。除了导致心力衰竭和肾脏损伤外，高血压还是中风的主要原因。2006年，13.7万美国人死于中风，其他许多人则患有严重的运动和认知障碍。与心血管疾病发病率最相关的激素是血管紧张素II(Ang II)。它可以说是最有效的血压升高荷尔蒙。但除此之外，它的病理生理作用与其引起高血压的能力是分开的。现在已经知道，血管紧张素II是在大脑中制造的，对调节血压的大脑系统产生强大的影响。也许血管紧张素II升高血压的能力的一半可能是通过它在大脑中的作用来实现的。至少有一家制药公司正在研究专门针对脑血管紧张素系统治疗高血压的药物。然而，我们仍然不知道脑血管紧张素系统是如何工作的。我们最近发现了一个新的Ang II结合位点，它在包括人类在内的几个物种的大脑中高度表达和广泛分布。本研究旨在确定脑内这一新的血管紧张素结合部位的结构特征和功能，以评估其在调节或拮抗脑内血管紧张素转换酶II的病理生理作用中的作用。

DESCRIPTION (provided by applicant): There are many uncertainties about the brain angiotensin system and its functionality is controversial. However, it is generally accepted that brain angiotensin causes hypertension and other cardiovascular disorders. With the recognition of functionality of peptide fragments of angiotensin II some having their own receptors, it is now known that brain angiotensins have a multitude of functions extending well beyond regulation of the cardiovascular system. We recently discovered a non-AT1, non-AT2 binding site in the brain that may add an additional layer of complexity to the brain angiotensin system. It is present in higher quantities than either of the major angiotensin receptor subtypes and could function either as a novel receptor for angiotensins that may be limited to the brain, or it could be a highly specific angiotensinase that plays a critical role in the metabolism of angiotensin peptides. To better understand this binding site, this project is directed to further characterizing this novel, brain-specific non-AT1, non-AT2 binding site for angiotensin peptides. The first specific aim is to define its biochemical characteristics through polyacrylamide gel purification and sequencing techniques to determine the amino acid sequence and structure of the binding site. The second specific aim is to determine its pharmacological specificity to determine if this binding site is specific for angiotensin peptides or if it has a broader substrate/ligand specificity. The third specific aim will study the distribution of the binding site in the brain and ascertain whether conditions associated with altered brain angiotensin system regulation of the cardiovascular system or pathophysiological conditions, e.g., brain ischemia as in stroke, genetic or experimental hypertension and in transgenic mice lacking angiotensin receptors, can alter the expression of this binding site in specific brain regions. The changes should enable us to do a functional neuroanatomical assessment of its possible participation in physiological and pathophysiological circumstances. It is anticipated that this protein will prove to be of considerable importance in the functionality of the brain angiotensin system. Since the brain angiotensin system is notorious for causing cardiovascular disease, this research could lead to the development of novel therapeutic strategies for treatment of hypertension and stroke. However the occurrence of this binding site in high concentrations in brain regions such as the nucleus basalis of Meynert, substantia nigra, amygdala, nucleus accumbens and substantia gelatinosa of the spinal cord could explain the proposed involvement of the brain angiotensins system in Alzheimer's and Parkinson's Disease, stress and anxiety disorders, drug abuse and chronic pain. PUBLIC HEALTH RELEVANCE: Cardiovascular disease, especially hypertension, is the leading cause of death (629,000 in 2006) in the US. In addition to causing heart failure and kidney damage, hypertension is the primary cause of strokes, which killed 137,000 Americans in 2006, and leaves many others with severe motor and cognitive impairments. The hormone most commonly associated with cardiovascular morbidity is angiotensin II (Ang II). It is arguably the most potent blood pressure-raising hormone. But in addition, it has pathophysiological effects separate from its ability to cause high blood pressure. It is now known that Ang II is made in the brain and exerts powerful influences on the brain systems that regulate blood pressure. Perhaps half of Ang II's ability to raise blood pressure may occur via its actions in the brain. At least one drug company is investigating drugs that specifically target the brain angiotensin system as a treatment for high blood pressure. However, we still do not know how the brain angiotensin system works. We recently discovered a novel binding site for Ang II that is highly expressed and widely distributed throughout the brain in several species including humans. This proposal aims to determine the structural identity and functionality of this novel angiotensin binding site in the brain to assess its role in mediating or antagonizing the pathophysiological actions of Ang II in the brain.