The goal is to obtain comprehensive information about the volume changes which occur in sickle cells during the sickling process, about the ionic basis of these volume changes, and about the changes in calcium homeostasis of the sickle cell which result from sickling. Since it is known that sickle cells shrink with aging, and since it is known that rate of sickling is a very strong function of the concentrate of SS hemoglobin, it is expected that understanding of the underlying mechanisms involved in sickling may allow intervention in the process and consequently modification of the clinical course of sickle cell disease. We expose experiments of three general types. 1) We will directly measure the volume changes which occur during the sickling process and during recovery from sickling using resistive cell sizing equipment. The effect on volume changes of alterations in initial cell volume, alteration of ionic composition of the suspending medium, and of transport inhibitors will provide information which will allow us to make some preliminary judgement about the nature of the transport mechanisms involved. 2) Guided by the results of these experiments, we will perform experiments to directly measure ion transport rates through various pathways during sickling and recovery from sickling. We will be particularly interested in measuring the activity of the C1 dependent, volume sensitive K fluxes shown to occur in human red cells under circumstances likely to alter the association of the cytoskeleton with the lipid bilayer, an event thought to occur during sickling. 3) The potential for altered Ca2+ homeostasis in the sickling process will be investigated in inside out vesicles and spicules generated during sickling. Specifically, Ca2+ permeability, Ca2+ active transport, cytoskeletal composition and lipid organization will be studied through biochemical and biophysical analysis of the above subcellular fragments. It is hoped that these experiments will lead to a clarification of the role of Ca2+ in the sickling process. Taken together, the data from the above studies will be applied towards the determination of the mechanisms for ion flux and ion homeostasis alterations and volume regulation associated with sickling.
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