The overall objective of this renewal application remains elucidation of the pathogenesis of acute lung injury resulting in increased permeability pulmonary edema. The focus is on study of the interaction of neutrophils (PMN's) with vascular endothelial cells, with emphasis on changes in potential mechanisms protective against PMN-induced endothelial cell injury. The central hypothesis of the proposal is that endothelial cell injury is due to loss of balance between protective mechanisms, which normally prevent PMN-induced injury, and factors which exacerbate PMN- induced injury. A potential protective mechanism is production by endothelial cells of adenosine. The potentially injurious effect to be studied is the effect on PMN adherence to endothelium of extracellular adenine nucleotides, released with vascular injury. We will compare the effects of adenosine and adenine nucleotides on isolated PMN adherence to cultured endothelium and the mechanisms of these effects, including mediation by receptors, changes in cell surface adherence proteins, effects on cation fluxes, and effects of uptake or metabolism of these ligands. In order to assess whether agents or conditions which alter PMN adherence also change endothelial cell adenosine metabolism, we will study the effects of the inflammatory cytokine, tumor necrosis factor-alpha (TNFalpha) on endothelial cell production and metabolism of adenosine and adenine nucleotides. The studies outlined in this proposal will determine how TNFalpha alters adenosine and adenine nucleotide production by endothelium and whether changes in these correlate with PMN adherence. We will determine the mechanism responsible for changes in adenosine and adenine nucleotide production and metabolism by investigating effects of TNFalpha on adenosine uptake, rapid transport, release, and ectonucleotidase activity. We will compare intracellular metabolism of adenosine into purines and nucleotides after stimulation of endothelial cells with TNFalpha. Because of the potential therapeutic importance of extracellular adenosine as protection against PMN-induced endothelial cell injury, we will ascertain whether pharmacologic agents which alter adenosine uptake or metabolism, also change PMN adherence to endothelium in response to TNFalpha. These studies will determine the effects of TNFalpha on endothelial cell adenosine and adenine nucleotide production and metabolism and will assess the biologic effects of adenosine and adenine nucleotides on PMN adherence to endothelium, a crucial early step in PMN-induced vascular injury.
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