Beta-adrenergic agonists have been shown to attenuate the edema and increased vascular permeability that various agents can produce in systemic and pulmonary vascular beds. The objectives of this renewal are to: (1) determine if beta-agonists reduce water and protein fluxes by a direct effect on endothelial permeability of water and albumin and/or by changes in hemodynamics; (2) determine the validity of beta-agonists as potential therapeutic agents for pulmonary edema and increased vascular permeability, and (3) determine the mechanism, from stimulation of adenylate cyclase to cytoskeletal and junctional organization, for enhancement of the barrier function of the endothelium. We hypothesize that beta-agonists, especially when administered as an aerosol, can prevent or reduce increased fluid and protein fluxes with minimal alterations in hemodynamics, gas exchange, and lung compliance. We also hypothesize that these agents modify permeability by their action on junctional associated microfilaments to prevent alterations in junctional architecture, cell shape, and permeability. The overall design of this renewal utilizes a structure-function approach and models of cell culture, isolated organ, and whole animal. These models focus on the pulmonary endothelium and utilize albumin uptake as the common indicator of protein permeability. Measurements of capillary pressure, hydraulic conductivity, diffusive albumin flux, and the albumin reflection coefficient in the isolated, perfused guinea pig lung will be used to differentiate between hemodynamic and direct permeability effects of beta- agonists administered via the circulation or airway. Measurements of inositol phosphate metabolism and cytosolic calcium will be made to determine if beta-agonists affect these processes in opposition to the permeability - increasing action of alpha-thrombin. Sheep will be used to determine if beta-agonists are potential therapeutic agents for pulmonary edema and increased vascular permeability, the latter assessed by the calculation of the reflection coefficient. Studies will compare intravenous versus airway administration of the agents on fluid and protein fluxes, hemodynamics, gas exchange, and lung compliance. Endothelial cell monolayers cultured from bovine pulmonary artery endothelial cells will be used to continue to determine the mechanism for the permeability - decreasing action of beta-agonists. Drugs designed to activate or inhibit membrane cyclases and kinases will also determine if the action of cAMP is shared by cGMP-enhancing agents. Correlative light, immunofluorescence, and electron microscopy will determine if beta-agonists increase the number and interaction of the peripheral band of actin microfilaments with junctional elements of endothelial cells and, thereby, prevent the alterations in the endothelial junction, in cell shape, and in permeability induced by alpha-thrombin. In parallel with these objectives is the long- term goal of determining the transcellular signalling pathways that may alter the shape of endothelial and epithelial cells to influence their function as semipermeable membranes.
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