BRAIN ANGIOTENSIN II RECEPTOR IN HYPERTENSION

该提案的长期目标是研究血管紧张素II(AII)。 初级神经元丰富(PNC)和血管平滑中的受体调节 SHR、WKY和SD大鼠的肌细胞培养 检查卡托普利和其他转换酶抑制剂的效果 (CEI)对这些大鼠AII受体的调节，并确定 CEI对自发性高血压大鼠的降压作用与下调血管紧张素转换酶 脑部AII受体。我们将利用新生儿的PNC和VSMC培养 比较SHR与正常血压大鼠AII受体的调节 测定CEI对中枢和外周AII受体的影响。这 项目分为两个具体目标。具体目标1：测试 大脑AII受体在生化和功能上的假说 自发性高血压大鼠与正常血压大鼠品系的差异及其脑内AII受体 在自发性高血压大鼠中的调节与WKY和SD大鼠不同。我们会 利用放射性配基结合技术鉴定AII受体数目 SHR、WKY和PNC(下丘脑/脑干共培养)的亲和力 并将这些受体与从相同来源培养的VSMC中的AII受体进行比较 小狗。结合研究将在完整的细胞和膜上进行。 从这些细胞中制备的。我们将用已知的药物来培养细胞 刺激或减少所有结合，并确定是否有 SHR与WKY、PNC与SHR对AII受体反应的差异 VSMC培养。我们将从功能上评估PNC和VSMC中的AII受体 SHR与WKY和SD的比较，通过监测胞浆中45个钙的外流 [Ca~(2+)]、肌醇磷脂周转和ALI刺激血管加压素释放 (副总裁)。我们认为，一个主要的信号通路介导所有的作用在 神经元组织是肌醇磷酸/钙离子途径。具体目标2： 为了验证给SHR注射CEI与 下调脑部AII受体。我们将a)比较AII受体 应激后SHR、WKY和SD仔鼠PNC和VSMC的数量和亲和力 在宫内使用CEI治疗，b)我们将确定短期效果 不同化学成分的CEI对PNC和VSMC培养的影响 关于所有结合动力学的结构。我们将研究这些措施的影响 试剂对Kd和Bmax以及缔合和解离常数的影响。 我们还将研究这些药物对AII受体的影响。 AII受体的内化及其与其第二受体的偶联 信使。为了确定CEI影响的特异性 我们将同时监测CEI对AII受体的影响 去甲肾上腺素(NE)和VP结合。拟议研究的结果应该是 加深对脑内AII受体及其调控的了解 在高血压的遗传模型中，这些受体是如何改变的。

The long-term goals of this proposal are to study angiotensin II (AII) receptor regulation in primary neuronal enriched (PNC) and vascular smooth muscle cell (VSMC) cultures from SHR, WKY and Sprague-Dawley (SD) rats, to examine the effects of captopril and other converting enzyme inhibitors (CEI) on AII receptor regulation in these rats and to determine whether the anti-hypertensive effect of CEI in SHR is related to a down-regulation of brain AII receptors. We will utilize PNC and VSMC cultures from neonates to compare AII receptor regulation in SHR vs normotensive rats and to determine the effects of CEI on central and peripheral AII receptors. This project is divided into two specific aims. Specific Aim 1: To test the hypothesis that brain AII receptors are biochemically and functionally different in SHR vs normotensive rat strains and that brain AII receptors in SHR are regulated differently from those of WKY and SD rats. We will utilize radioligand binding techniques to characterize AII receptor number and affinity in PNC (hypothalamus/brain stem co-cultures) from SHR, WKY and SD pups and compare these to AII receptors in VSMC cultured from the same pups. Binding studies will be performed in intact cells and in membranes prepared from these cells. We will incubate cells with agents known to stimulate or decrease AII binding and determine whether there are differences in the responses of AII receptors in SHR vs WKY and in PNC vs VSMC cultures. We will functionally assess AII receptors in PNC and VSMC from SHR as compared to WKY and SD by monitoring 45 Ca2+ efflux, cytosolic [Ca2+], phosphoinositide turnover and AII stimulated release of vasopressin (VP). We propose that a major signal pathway mediating AII action in neuronal tissue is the inositol phosphate/Ca2+ pathway. Specific Aim 2: To test the hypothesis that administration of CEI to SHR is associated with down regulation of brain AII receptors. We will a) compare AII receptor number and affinity in PNC and VSMC from SHR, WKY and SD pups subjected to in utero treatment with CEI, b) we will determine the effects of short-term incubation of PNC and VSMC cultures with CEI of different chemical structures on AII binding kinetics. We will study the effects of these agents on the Kd and Bmax and the association and dissociation constants. We will also study the effect of these agents on AII receptor internalization and the coupling of the AII receptor to its second messenger. In order to determine the specificity of the effect of CEI on the AII receptor we will simultaneously monitor the effect of CEI on norepinephrine (NE) and VP binding. Results from the proposed study should increase our understanding of brain AII receptors and the regulation and how these receptors are altered in a genetic model of hypertension.