Pathologic processes occurring in the human lung commonly involve a component of injury to pulmonary endothelial cells or the response of these cells to locally released mediators. Because of its substantial content of mast cells and the scope of the pulmonary vascular bed, the lung is both is rich source of histamine and a locus of potential importance for the clearance of histamine via interaction with cells of the vasculature. Histamine exerts its biologic activity in the lung by causing H1 and/or H2 receptor-mediated vasoconstriction or vasodilation and by triggering the production of prostaglandins, leukotrienes, and hydroxy fatty acids. Both the changes in vascular tone and the release of inflammatory mediators may play a role in the progression of lung disease or injury. This project will explore the nature of the enzymatic mechanisms for histamine metabolism present in cultured human cells of the pulmonary vasculature compared with cells from the systemic circulation. The objectives are to determine how human pulmonary endothelial cells metabolize histamine, to define which enzymes in the established ring methylation/amine oxidation or direct side-chain oxidation pathways are present, and to characterize potential interaction of metabolizing enzymes at the endothelial cell surface and involvement of an endocytic process in cellular uptake of histamine. Metabolic products of 3H histamine in endothelial cells will be identified by acetic acid: ethanol extraction and thin layer chromatography in solvent systems which distinguish metabolites arising from methylation versus oxidation. The cellular locations of histamine conversion will be delineated by measuring total 3H histamine radioactivity in subcellular compartments after cell lysis and sucrose density gradient centrifugation, and determining the chemical nature of the labeled products by thin-layer chromatography. Potential binding and cell surface activity of enzymes catalyzing the oxidation step will be examined in human pulmonary endothelial cells by measuring cell-associated radioactivity of a heterologous fetal bovine 125I-diamine oxidase and by identifying 3H histamine metabolites generated in its presence. The participation of human pulmonary vascular smooth muscle cells in histamine metabolism will also be characterized by identification pf enzymes and 3H histamine metabolic products. Metabolizing enzymes which may be released in association with cellular injury may ultimately be useful as markers for denoting human pulmonary endothelial cell injury in vivo.
