Pulmonary endothelium is involved in numerous transport and metabolic functions which are susceptible to toxic and pharmacologic modulations. The enzymes responsible for many of these functions are located on the luminal surface of the microvasculature and catalyze the synthesis and/or disposition of vasoactive substances. The long term objective of this application is the elucidation of the properties and functions of pulmonary endothelial enzymes, in vivo, under normal and toxicant - altered homeostasis. Two enzymes will be studied: angiotensin converting enzyme (ACE) and 5""""""""-nucleotidase (NCT); these enzymes uniformly line the luminal surface of the pulmonary endothelium, are responsible for the synthesis and/or metabolism of potent vasoactive compounds (angiotensin, bradykinin, adenosine) and can be assayed easily. We will estimate kinetic constants of interaction of these enzymes with substrates and inhibitors, in vivo, under various physiologic conditions. Utilizing an animal model which allows for the control of perfused vascular surface area, in vivo, we will investigate endothelial enzyme dysfunction induced by ionizing radiation, bleomycin, paraquat, endotoxin, complement activation and phorbol ester. We will correlate levels of exposure to toxin with changes in enzyme kinetics and investigate the ability of known antagonists of lung injury in attenuating toxin- induced ACE and NCT dysfunction. We will contrast the effects of each toxin on endothelial ectoenzyme kinetics in vivo, to those in cultured arterial and venous endothelial cells. Enzyme activities in vivo will be determined using multiple indicator dilution techniques. The indicators will include the enzyme substrates (3H-benzoyl-Phe-Ala-Pro for ACE and 14C-5'-AMP for NCT) and metabolism will be studied during a single transpulmonary passage under first order and mixed order reaction conditions. Transpulmonary binding of radiolabelled enzyme inhibitors in vivo will provide additional information on enzyme dysfunction or loss. Enzyme kinetics in culture will be determined in rabbit pulmonary arterial and venous endothelial cell monolayers using standard techniques. Additional studies will aim towards developing novel means of diagnosing lung microvascular injury and could be of particular importance in lung diseases of known or or suspected endothelial involvement.
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