The long term objective of the proposed research is to analyze the mechanisms by which endothelial and epithelial cells regulate the permeability properties of the pulmonary air-blood barrier. To accomplish this two model systems are employed. 1. The plasma protein-free fluorocarbon exchange transfused rat and 2. epithelial cells in culture. Biochemical and electrophysiological data are correlated with observations made by ultrastructural immunocytochemistry, morphometry and freeze fracture.
The specific aims i nclude studies utilizing the fluorocarbon exchange transfused rat, to determine the ability of immunoglobulin G (IgG), alpha1- acid glycoprotein (alpha1-AGP), fibrinogen (FAN), plasma fibronectin (FN) and dibutyryl cAMP, alone and in combination, to augment the action of albumin in preventing the increased macromolecular transport and edema that occurs in a protein-free preparation. The localization of administered test proteins on the pulmonary endothelium will be determined immunocytochemically, and correlated with morphometric measurements of ferritin movement, a measure of macromolecular transport. The distribution of ZO-1, a TJ associated protein, is mapped immunocytochemically in microvascular endothelial cells under conditions of normal and augmented fluid filtration. The plasmid pZipSVtsA58, a construct that confers temperature sensitive growth potential to transfected epithelial cells at 33oC, but allows expression of differentiated functions at 39oC, is used to immortalize alveolar type II cells. Clones that develop a transepithelial resistance >100 omega cm2 are selected for study of the regulation and composition of TJs. Novel strategies using biotinylating agents and a heterobifunctional crosslinking agent are employed to identify and characterize TJ protein(s) to which monoclonal antibodies will be raised. The role of membrane lipids in the regulation and function of TJs will be examined in cultured epithelial cells which, when made cholesterol deficient by administration of Lovastatin, develop steady state transepithelial resistances two to three-fold greater than controls. Results of the proposed research will: i. Demonstrate the key role played by specific plasma proteins, in addition to albumin, in regulating endothelial permeability. ii. Identify TJ proteins and provide reagents with which to study their structure using tools of molecular biology and iii. aid in resolving controversy concerning the role of membrane lipids in TJs. A better understanding of the nature of this important structure will provide a rational basis for the management of pulmonary edema.
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