RENAL ENDOTHELIAL AND SMOOTH MUSCLE CELLS INTERACTIONS

本研究的长期目标是阐明肾脏的调节 内皮细胞（EC）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 肾间质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的生产和行动， 一氧化氮（NO）和内皮素（ET）。 待检验的主要假设 是基于最初的观察， 集成ECs-SMC单元固有的振荡驱动。 具体 建议研究的目的包括调查 通过在各个层面探索该系统， 组织，从分子遗传学到整个器官功能，如下所示： 1.阐明内皮细胞ET/B受体的功能作用。 2.阐明细胞内信使和物理力的作用 作用于内皮细胞调节组成型NO合酶的活性。 3.为了确定SMC的生理收缩是否激活EC NO和ET-1的产生，以及这种作用的可能介质。 4.为了阐明变形相关因素（拉伸， 压力）和流动相关因素（剪切）的时间过程， 连续并同时监测ET-1和NO的合成。 为了实现研究的目标，一些新的技术 方法开发：1）应用NO选择性 微电极连续监测从EC和灌注的NO释放 2）合成位点特异性生物素化的ET- 1和生物素化的IRL-1620，其允许进行活体荧光 ET/A和ET/B受体的定位; 3）建立永生化的 大鼠肾阻力动脉内皮细胞和平滑肌细胞的培养; 稳定表达的基因工程中国仓鼠卵巢细胞 ET/B受体和/或内皮型NO合酶; 5）内皮型NO合酶的发展。 “三明治”共培养系统，以模拟EC和 SMC。 拟议研究中采用的其他方法包括 肾微血管的视频显微镜，细胞形状的图像分析 变化和胞浆钙浓度，定量荧光 ET受体定位，ET-1的放射免疫测定和生物测定。 这些研究将阐明物理因素和感觉因素在 输入调节内皮细胞产生NO和ET-1，根据建议 “乒乓”模型，通过收缩反馈调节内皮细胞的功能 SMC，以及集成的ECs-SMC单元的自动 振荡

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.