ROLES OF THE PROXIMAL TUBULE RENIN ANGIOTENSIN SYSTEM

描述：(改编自申请者的摘要)还有很多问题 近端小管(PT)肾素血管紧张素系统(RAS) 细胞内构型及其作用机制。此应用程序 使用两株永生化的大鼠PT细胞(IRPTC) 应用程序定义PT RAS旁分泌、自分泌和 内分泌效应。据推测，Ang II和其他血管紧张素 PT中的本地产品通过以下方式影响盐和水的重吸收 通过特定的细胞内信号机制调节转运蛋白和 核内机制尚未完全推论。Ang II似乎 降低cAMP，刺激钙交换活性；增加细胞内 钙通过蛋白激酶C和刺激钠/钙转运，并增加 磷脂酶A2，刺激磷脂酰胆碱水解为 溶血磷脂酰胆碱和脂肪酸，影响钠转运。是这样的 血管紧张素Ⅱ效应可能通过表面和细胞内两种途径实现。 PT中的受体，如最近来自申请人的证据所提示的 IRPTC有核Ang II受体的实验室。具体目标是 定义：I：细胞内RAS的构型和空间排列 IRPTC。虽然所有RAS组件在PT(和两者)中都清楚地展示 IRPTC的品系)，实际的细胞内定位尚不完全清楚。 RAS组分的胞内生产、定位和运输 将由免疫电子显微镜、共聚焦显微镜、 亚细胞分离加上分子和细胞技术。II： 血管紧张素转换酶Ⅱ对溶质运移的影响机制Ang II展示了一个 水盐运移的双相影响。血管紧张素转换酶II对人的影响 将定义转运体调节，使用Na/H交换器作为 包括分子生物学和细胞的平行研究中的原型 生理学。将寻求血管紧张素II反应元件；血管紧张素转换酶对 NHEs的信使核糖核酸和蛋白质调控将被描绘出来[例如，信使核糖核酸稳定 状态水平、稳定性和局部性；免疫染色强度和 模式[例如，贩运和免疫分析]；以及细胞信号和 生理学将被定义。III：血管紧张素II及其自分泌和内分泌 影响，因为这些影响PT的功能，如生长和修复，以及蛋白质 加工，假设Ang II调节细胞生长和修复。这个 血管紧张素Ⅱ在IRPTCs中的功能作用将通过阻断或 刺激RAS成分解剖自分泌效应。IRPTC有 细胞内Ang II受体；因此，可能的细胞内处理 血管紧张素原(肾素底物)将被研究。

DESCRIPTION: (Adapted from the Applicant's Abstract) Many questions still remain concerning both proximal tubule (PT) renin angiotensin system (RAS) intracellular configuration and mechanisms of its effects. This application uses two lines of immortalized rat PT cells (IRPTC) developed in this application's first cycle to define PT RAS paracrine, autocrine and intracrine effects. It is hypothesized that Ang II and other angiotensins produced locally within PT influence salt and water reabsorption by modulating transporters via specific intracellular signaling mechanisms and intranuclear mechanisms as yet incompletely deduced. Ang II appears to reduce cAMP and stimulate Ca2+ exchanger activity; to increase intracellular calcium via PKC and stimulate Na+/Ca++ transport; and to increase phospholipase A2, stimulating hydrolysis of phosphatidylcholine to lysophosphatidylcholine and fatty acid, influencing sodium transport. Such Ang II effects may be carried out both via surface and intracellular receptors in PT, as suggested by recent evidence from the applicant's laboratory that IRPTC have nuclear Ang II receptors. Specific Aims are to define: I: Intracellular configuration and spatial arrangement of RAS in IRPTC. While all RAS components are clearly demonstrated in PT (and in both lines of IRPTC), actual intracellular localization is incompletely known. Intracellular production, localization and trafficking of RAS components will be defined by immunoelectron microscopy, confocal microscopy, subcellular fractionation plus molecular and cellular techniques. II: Mechanisms of Ang II effect on solute transport in PT. Ang II exhibits a biphasic influence on water and salt transport. The influence of Ang II on transporter regulation will be defined, using Na+/H+ exchanger as a prototype in parallel studies encompassing both molecular biology and cell physiology. Ang II-responsive elements will be sought; effects of Ang II on mRNA and protein regulation of NHEs will be delineated [e.g., mRNA steady state level, stability and localization; immunostaining intensity and pattern [e.g., with trafficking and immunoassay]; and cell signaling and physiology will be defined. III: Ang II and its autocrine and intracrine effects, as these affect PT functions such as growth and repair, and protein processing, hypothesizing that Ang II modulates cell growth and repair. The functional role of Ang II in IRPTCs will be defined by blocking or stimulating RAS components to dissect autocrine effects. IRPTCs have intracellular Ang II receptors; thus, possible intracellular processing of angiotensinogen (renin substrate) will be studied.