The long term objective of the proposed research is to analyze the mechanisms by which the endothelial glycocalyx, pinocytotic vesicles and tight junctions determine the normal and experimentally altered permeabilty properties of the pulmonary endothelium and the epithelium which lines airways and alveoli. To accomplish this, lungs from fetal and adult rats and cultured epithelial cells (Type II pneumocytes, MDCK cells) will be examined at the ultrastructural level using immunocytochemistry, rapid freezing, freeze substitution, freeze fracture and morphometry. The results of the morphological studies will be correlated with biochemical and immunochemical analyses as well as electrophysiological measurements.
The specific aims i nclude a study, in the plasma protein-free fluorocarbon exchange transfused rat, of the mechanisms involved in the interaction of albumin with the glycocalyx by the use of chemically modified albumins. Rapid freezing combined with a newly developed freeze substitution method will be used to examine directly the ultrastructure of the endothelial glycocalyx and the effect albumin (modified or unmodified) has on its organization. The distribution of anionic sites and glycoconjugates on the luminal surface of pulmonary capillary endothelium will be probed by means of cationized ferritin and lectin-gold complexes. The role of membrane lipids in tight junction formation, structure and function will be examined in epithelial cells cultured in vitro either by increasing the unsaturation of membrane phospholipid fatty acids or by selectively removing cholesterol from the membrane. Data from electrophysiological measurements will be correlated with results of biochemical analysis of membrane lipids and the ultrastructure of the tight junctions. The role of circulating proteins in endothelial pinocytosis will be examined in control and fluorocarbon exchange transfused rats by rapid freezing methods, to avoid possible fixation artefacts. In fetal rats the numerical density of intramembranous particles and filipin-cholesterol complexes will be correlated with the rise in numerical density of pinocytotic vesicles which occurs during gestation. Results from these studies should lead to a better understanding of the barrier function of anatomical structures comprising the air-blood barrier and provide a base-line for future studies of these structures in a variety of pathological conditions.
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