The long-term goal of this project is a thorough understanding of the molecular and cellular pathophysiology of sickle disease. The current molecular focus pertains to the hemoglobin polymerization process and the cellular focus is on the membrane transport abnormalities of sickle RBC.
Specific Aim 1 will examine reticulocyte ion transport heterogeneity and generation of dense sickle cells, studies that fall into three categories. Studies of ion transport in reticulocytes explore the hypothesis that marked heterogeneity of Hb concentration, volume, and ion content of circulating RBC is largely determined by early cation permeabilization of reticulocytes or earlier precursors with a diversity of expression of ion transporters. Using a combination of tracer flux methods, a novel high-precision osmotic lysis method, and a new flow cytometric technology, the applicant will identify and separate sickle reticulocyte subpopulations with different transport properties, including stress reticulocytes, and characterize their major ion transporters at different stages of maturity, testing the hypothesis that these differences affect susceptibility to rapid dehydration. As part of this work, he will further define the pH sensitivity of the K:Cl co- transporter and the effect of inhibitors on its pH-activated component. Secondly, he will follow up his novel observation that magnesium therapy may confer benefit by examining in vitro correlates of the in vivo studies ongoing. These studies will include in vivo (rat mesenteric system) examination for vasodilatory effects of magnesium on sickle RBC microvascular flow behavior. Thirdly, the flow cytometry system will be used to test the hypothesis that a determinant of the early fall in dense cells during vaso-occlusive crisis comprises decreased release of new stress reticulocytes into the circulation. The second Specific Aim will examine the nature of sickling-induced permeability in red cells and reticulocytes, with emphasis on distinguishing the roles of calcium- induced inhibition of sodium permeability and calcium-induced K channel activation in the dehydration process. In these experiments, he will use heparin as a marker of this pathway, and he will attempt to define the mechanism of heparin stimulation of sickling-induced leak in reticulocytes. He will test the hypothesis that cation leak is not localized to spicules and he will attempt to use the ex vivo rat mesocecum to test the effect of microcirculatory shear on calcium permeability of normal and sickle RBC. The third Specific Aim will continue studies of the structure of the HbS polymer, with emphasis on the intracellular effects of its formation and breakdown.
This aim will employ a new method developed by the investigator more accurately measuring polymer concentration and the size of the polymer water compartment. He will test the variability of these parameters as influenced by non-S hemoglobins, and examine the distribution of cytoplasmic lower MW substances in the PWC, expecting polymer formation to influence cell metabolism. The applicant's new method also will be used to further define the intermolecular interactions of the polymer by testing recombinant mutants. The second area of emphasis will be to use EPR assays of hemichrome generation in sickle cells during incubation, testing the hypothesis that this results from oxyhemoglobin S instability rather than polymerization. Additionally, the magnitude of sickling-induced endocytosis in reticulocytes and discocytes will be tested to examine mechanisms of development of calcium-accumulating vesicles, with an emphasis on determining whether at least some of these are retic-derived organelles.
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