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 lumenal 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 feet-AMP 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 compare the kinetic cinstants of these enzymes, in vivo, to those in endothelial cells in culture; we will study conditions in the internal environment (pH, pCO2, pO2, flow, viscosity) that may affect ACE and NCT kinetics, in vivo; we will compare the known action of inhibitors of these enzymes in vitro, with their properties in vivo. Furthermore, we will investigate the effects of endothelial injury (ionizing radiation) on enzyme kinetics in vivo and in culture, factors contributing to these effects, and the effects of lung toxicity on the mechanism of action of enzyme inhibitors, in vivo. Finally, we will study the temporal development of radiation-induced changes in enzyme function as means of developing a technique for the early assessment of lung toxicity. Enzyme activities in vivo, will be determined using multiple indicator-dilution techniques. The indicators will be the enzyme substrates (3H-Benzoyl-Phe-Ala-Pro for ACE and 14C-5 feet-AMP for NCT) and metabolism will be studied during a single transpulmonary passage under first order and mixed order conditions. Enzyme kinetics in culture will be determined in small vessel rabbit pulmonary endothelial cell monolayers using standard techniques. Lung injury will be produced by exposing the thorax of rabbits to 3,000 rads ionizing radiation. Results from these studies should be of considerable usefulness to the development of pharmacologic inhibitors of enzyme activity. Furthermore, these studies may provide novel means of diagnosing lung microvascular injury and could be of particular importance in lung diseases of known or suspected endothelial involvement.
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