Histamine exerts its vascular biologic activity by causing direct receptor-mediated vasoconstriction or vasodilation and by triggering the production of lipid mediators. Subsequent clearance mechanisms for this agonist appear to differ in the pulmonary vs. systemic circulation. This project seeks to define the nature of the enzymatic mechanisms for histamine metabolism, the role of receptor-mediated endocytosis in the histamine degradation process, and the impact of these elements on histamine receptor regulation, in human endothelial cells derived from the pulmonary vasculature compared with cells from the systemic circulation. It will characterize in a tissue culture model system the means by which these cells and histamine mutually interact with one another, and explore the participation in histamine metabolism by other cell types in the subendothelial structure of blood vessels and interstitial connective tissue - smooth muscle cells, fibroblasts, and cells of the host defense system such as granulocytes, representing both the pulmonary and systemic vascular beds. The objectives are to determine how human pulmonary endothelial cells metabolize histamine, to define which types of enzymes known in the established ring methylation/amine oxidation or direct side-chain oxidation pathways are utilized under various environmental situations, 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 will be identified by thin layer chromatography and ion exchange chromatography. The cellular locations of histamine conversion and histamine-metabolizing enzymes will be delineated in subcellular compartments after cell lysis and sucrose or Percoll density gradient centrifugation. Potential binding and cell surface activity of enzymes catalyzing the metabolic steps will be examined in a homologous human system. We have provided evidence that histamine metabolism in cultured human systemic vascular endothelial cells takes place in a cell-surface-associated manner as a two-stage sequence, involving an initial degradative step by an endogenous enzyme and a subsequent one by an exogenous enzyme. A cellular uptake process is tightly coupled to the enzymatic degradation pathway. This proposal's cell biologic approach to biogenic amine metabolism will seek to define at the cellular level the mechanistic basis for the exclusion of the pulmonary circulation in histamine clearance.
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