BRAIN ANGIOTENSIN II RECEPTOR IN HYPERTENSION

该提案的长期目标是研究血管紧张素II（AII） 受体调节在原代神经元富集（PNC）和血管平滑肌 来自SHR、WKY和Sprague-Dawley（SD）大鼠的肌细胞（VSMC）培养物， 检查卡托普利和其他转化酶抑制剂的作用 (CEI)对AII受体调节的影响，并确定 CEI在SHR中的抗高血压作用与下调 脑AII受体。 我们将利用新生儿的PNC和VSMC培养物 比较SHR与正常血压大鼠的AII受体调节， 确定CEI对中枢和外周AII受体的影响。 这 该项目分为两个具体目标。 具体目标1：测试 假设脑AII受体在生物化学和功能上 SHR与正常血压大鼠品系的差异， SHR与WKY和SD大鼠的海马神经元的调节方式不同。 我们将 利用放射性配体结合技术表征AII受体数量 和亲和力在PNC（下丘脑/脑干共培养物）从SHR，WKY和 SD幼鼠，并将其与从相同培养物中培养的VSMC中的AII受体进行比较。 小狗 将在完整细胞和膜中进行结合研究 从这些细胞中提取。 我们将用已知的试剂孵育细胞 刺激或减少AII结合，并确定是否存在 SHR与WKY和PNC与WKY中AII受体反应的差异 VSMC培养。 我们将在功能上评估PNC和VSMC中的AII受体 SHR与WKY和SD相比，通过监测45 Ca 2+流出，细胞内 [Ca2+]、磷酸肌醇周转和AII刺激血管加压素释放 （副总统）。 我们认为，介导AII作用的一条主要信号途径是 神经元组织中的磷酸肌醇/Ca 2+途径。 具体目标二： 为了验证对SHR给予CEI与SHR的高血压相关的假设， 下调脑AII受体。 我们将a）比较AII受体 SHR、WKY和SD幼鼠PNC和VSMC的数量和亲和力 在子宫内用CEI治疗，B）我们将确定短期的效果， 用不同化学品CEI孵育PNC和VSMC培养物 AII结合动力学的结构。 我们将研究这些影响 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受体与其第二受体的偶联 使者 为了确定CEI对细胞增殖的影响的特异性， 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 增加我们对大脑AII受体及其调节的理解， 这些受体在高血压的遗传模型中是如何改变的。

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.