RENAL ENDOTHELIAL AND SMOOTH MUSCLE CELLS INTERACTIONS

本研究的长期目标是阐明肾脏的调节。 内皮(ECs)和平滑肌细胞(SMC)的血流动力学 作为一个功能单位。拟议研究的重点是 肾脏之间可能的前馈和反馈相互作用 微血管内皮细胞和平滑肌细胞与血管内皮生长因子的产生和作用 一氧化氮(NO)和内皮素(ET)。需要检验的主要假设 是基于最初的观察结果，这表明 集成ECS-SMCS单元固有的振荡驱动。具体的 建议研究的目的包括调查营运模式。 通过探索不同级别的系统，对ECS-SMCS单位进行 组织，从分子遗传学到整个器官功能，如下所示： 1.阐明ET/B受体在内皮细胞中的作用。 2.阐明细胞内信使和物理作用力的作用 作用于内皮细胞，调节结构性一氧化氮合酶的活性。 3.确定SMC生理性收缩是否激活内皮细胞 NO和ET-1的产生，以及这一作用的可能介体。 4.阐明与变形相关的因素(拉伸、 压力)和与流动相关的因素(剪切力)在时间过程中 连续同时监测ET-1和NO的合成。 为了实现研究的目标，几项新的技术 开发了以下方法：1)应用非选择性 微电极连续监测内皮细胞和灌流的NO释放 抗性血管；2)定点生物素化ET-2的合成 1和生物素标记的IRL-1620，它们允许执行生命荧光 ET/A和ET/B受体的定位；3)永生化的建立 大鼠肾阻力动脉内皮细胞和平滑肌细胞的建立 基因工程中国仓鼠卵巢细胞稳定表达 ET/B受体和/或内皮型一氧化氮合酶；5) “三明治”共培养系统，以模拟内皮细胞和 SMCS。拟议研究中将使用的其他方法包括 肾微血管的摄像显微镜，细胞形状的图像分析 细胞内钙离子浓度的变化及定量荧光 ET受体的定位，ET-1的放射免疫测定和生物测定。 这些研究将阐明身体因素和感官的作用。 根据拟议的建议，调节内皮细胞产生NO和ET-1的投入 “乒乓球”模式--收缩反馈调节内皮细胞功能 SMCS，以及综合ECS-SMCS单元的能力，用于自动- 震荡。

The long-term goal of this study is to elucidate the regulation of renal hemodynamics by the endothelial (ECs) and smooth muscle cells (SMCs) acting as a functional unit. The focus of the proposed study is on the possible feed-forward and feedback interactions between renal microvascular ECs and SMCs with regard to the production and action of nitric oxide (NO) and endothelin (ET). The major hypothesis to be tested is based on original observations suggesting the possibility of the oscillatory drive intrinsic to the integrated ECs-SMCs unit. The specific aims of the proposed study comprise investigations of the operation mode of the ECs-SMCs unit by exploring the system at various levels of organization, from molecular genetics to whole organ function, as follows: 1. To elucidate the functional role of the ET/B receptor in ECs. 2. To elucidate the role of intracellular messengers and physical forces acting on ECs in regulating the activity of constitutive NO synthase. 3. To determine whether physiological contraction of SMCs activates ECs production of NO and ET-1, and the possible mediators of this action. 4. To elucidate the contribution of deformation-related factors (stretch, pressure) and flow-related factors (shear) on the time-course of continuously and simultaneously monitored ET-1 and NO synthesis. Toward accomplishing the goals of the study, several novel technical approaches were developed: 1) The application of an NO-selective microelectrode to continuously monitor NO release from ECs and perfused resistance vessels; 2) The synthesis of the site-specific biotinylated ET- 1 and biotinylated IRL-1620 which allow to perform vital fluorescence mapping of ET/A and ET/B receptors; 3) The establishment of immortalized ECs and SMCs from rat renal resistance arteries; 4) The establishment of genetically-engineered Chinese hamster ovary cells stably expressing the ET/B receptor and/or endothelial NO synthase; 5) The development of a "sandwich" coculture system to model the interaction between the ECs and SMCs. Other approaches to be used in the proposed study include videomicroscopy of renal microvasculature, image analysis of cell shape changes and cytosolic calcium concentration, quantitative fluorescence mapping of ET receptors, and radioimmunoassay and bioassay for ET-1. These studies will elucidate the role of physical factors and sensory inputs regulating ECs production of NO and ET-1, according to the proposed "ping-pong" model, feedback regulation of ECs function by contracting SMCs, and the capacity of the integrated ECs-SMCs unit for auto- oscillations.