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.
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