The results support our working hypothesis that plasma purines present at micromolar concentrations play a significant metabolic role in red cell energetics. In evaluating this hypothesis, we will use non-glycolytic pig and glycolytic human red cells as models. Pig red cells cannot use glucose due to the loss of membrane permeability to glucose during transition from reticulocytes to erythrocytes. Inosine, produced by the liver, has been identified as an in vivo substrate. To understand the development of purine-dependent red cells, effects of the insulin and Beta-adrenergic system will be ascertained in terms of membrane transport and protein synthesis in immature bone marrow cells and reticulocytes undergoing in vitro maturation. The most remarkable metabolic feature of the pig red cell is that inosine plasma level of 2 MuM is sufficient to meet its energy requirements. However, inosine by itself cannot replenish the purine moiety of adenine nucleotides. To turnover adenine nucleotides the red cells must encounter adenosine or adenine. Since no adenosine or adenine has been found in pig plasma, we hypothesize that red cells must receive adenine nucleotide precursors during the transit through the liver or other body organs. This will be ascertained using an in situ organ perfusion system. Moreover, the possible in vivo metabolic role of trioses and pentoses, each of which is a known in vitro substrate, will be delineated. Although human red cells are glycolytic, their ability to survive solely by trace amounts of purines also has been found. To understand the physiological significance of purine metabolism we plan to delineate the mechanism by which human red cells utilize multiple substrates for their energy needs. In addition to a direct metabolic substrate role, certain purine compounds can act in a sense like hormones in that they can increase ongoing glycolytic flux in human cells. The underlying mechanism for this purine mediated glycolytic activation will be ascertained in terms of purinergic receptors, membrane permeability and glycolytic enzyme stimulation. A better understanding of purine metabolism will shed more light on the pathogenesis of such human diseases as Lesch-Nyhan syndrome, gout and certain immune deficiencies. Finally, the investigation of the liver perfusion may lead to a better means by which to preserve the liver in relation to transplantation.
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