This grant proposal concerns mechanisms by which red blood cells achieve and maintain their content of water and small ions. The experiments are divided into two parts. The first involves dog red blood cells, in which movements of sodium, calcium, protons, and potassium across the membrane are strongly dependent on cell volume. The proposed work seeks to examine the nature of the stimulus-response coupling, so as to clarify how certain membrane transporters are turned on and off by minor degrees of cell shrinkage or swelling. Methods will involve the use of cross-linking agents, membrane-expanding treatments, and selective proteolyic digestion to influence the relationship between cell volume and ion transport. Membrane protein separations will be performed by gel electrophoresis, using cells that have had specific transporters """"""""turned on"""""""" or """"""""turned off"""""""" by volume manipulation. Net and isotopic flux measurements will be used to monitor transport. The second project is a one-year, preliminary study designed to evaluate, by the techniques of electron probe microanalysis, a unifying hypothesis that would explain the generation of dense, viscous red blood cells in sickle cell disease, hereditary spherocytosis, and other hemolytic states. The hypothesis states that red blood cells in all these conditions are subject to fragmentation or budding. The vesicles that bud off the cells are believed to be richer in water and salts and poorer in hemoglobin content than the parent cells, thus leaving the latter dense, viscous, and poorly deformable. The work proposed here involves a one year feasibility study during which red blood cell fragments produced in vitro would be assayed for ion, water, and hemoglobin content by standard techniques of atomic absorption and/or flame photometry. The same fragments would be subjected to electron probe microanalysis, to determine whether the method has the sensitivity and specificity to approach the problem with blood samples from patients. The hypothesis cannot be evaluated in the blood of patients in any other way than by electron probe microanalysis: this is because cell fragments in the circulation may originate from platelets, leukocytes, and possibly other non-red cell sources. Only by combining the techniques of electron microscopy and microanalysis can one be sure of the origin of the fragments in which quantitative elemental determinations are to be made.
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