The hemolytic anemia, which occurs in patients with sickle cell disease, is related to the formation of irreversibly sicked cells (ISCs). In contrast to reversibly sickled cells, ISCs exhibit stabilized sickle shape, dehydration and poor deformability following reoxygenation. These changes probably result from the higher intracellular calcium content of ISCs. Results from normal red blood cells suggest that polyphosphoinesitide (polyPI) metabolism is important in regulating intracellular calcium levels. Higher levels of polyPIs in erythrocyte membranes are associated with increased Ca++ Mg++ ATPase activity and increased pumping of calcium out of the cell. Phosphatidic acid (PA), the product of polyPI breakdown, has calcium ionophoric properties which would enhance the leak of calcium into the cell. Thus, I hypothesize that during sickling, membrane polyPIs are hydrolyzed to PA in a progressive and irreversible manner. This conversion would reduce active calcium efflux by reduction of Ca++ Mg++ ATPase activity and enhance passive calcium influx through increased PA formation causing the high intracellular calcium concentrations which mediate the altered properties of ISCs. The hypothesis will be tested using four specific aims. 1. The metabolism of polyPIs, ATP content, 45Ca level and calcium pump activity will be correlated with the formation of ISCs in vitro at 6 hr. intervals over 24 hrs. 2. The extent of polyPI breakdown in ISCs formed in vivo will be assessed by measuring the ratio of polyPIs to PA in light and dense cell fractions obtained from stractan gradients. 3. The possible involvement of polyPIs in the defective Ca++ Mg++ ATPase of sickle cells will be studied. The enzymes will be purified by affinity chromatography from normal and sickle cell membranes and reconstituted into liposomes of defined composition. The Ca++ Mg++ ATPase activity will be assayed for polyPI and calmodulin stimulation. 4. The effects of exogenously added PA on 45Ca flux across normal and sickle cells will be compared. The long-term objective of this research is to elucidate the molecular processes that result in the formation of ISCs. An understanding of these processes will provide a rationale for the development of future therapies aimed at reducing hemolysis and vaso-occlusive crises observed in sickle cell anemia.
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