The major goals of this research are: (1) to study the kinetics and regulation of anion and cation transport in single erythroblastic cells during differentiation and maturation using the technique of x-ray microanalysis, and to investigate the effect of erythropoietic stimulating factors and cofactors in modulating these changes. (2) to demonstrate the applicability of this technique in investigating transport of materials labelled with identifiable elements such as platinum, iodine or bromine in a multi-compartment system. (3) to better understand and improve the technique of cryosectioning for preparation of samples for x-ray microanalysis. Investigation of membrane transport of the erythroid cells in the bone marrow is complicated by the heterogeneity of the cell population. Without physical separation of the cells into distinct homogeneous populations conventional radioisotopic tracer methods for measuring transport cannot be applied. The techniques of cryomicrotomy and x-ray microanalysis provide a solution to this problem. Changes in sodium potassium chloride as well as rubidium and bromide uptake in individual erythroid cells from sheep, dog and rabbit at various stages of maturation will be studied. The different species of animals selected have different gene expressions in their red cell membrane transport properties. Relationships among morphological changes, membrane transport alteration and hemoglobin production during maturation will be studied. Pump/leak, cotransport and counter transport systems will be examined by multielemental analysis. In addition, the maturational transport characteristics of an erythroid progenitor cell type from the spleen of mice infected with Friend's virus will be studied to examine the effect of erythropoietin on the changes in membrane transport function and hemoglobin production. These cells can be obtained inadequately large quantity and can survive in culture to allow for transport measurement using radioisotopic tracers as well as x-ray microanalysis. Finally this project includes a section where we will try to better understand the cryosectioning technique and extend the application of x-ray microanalysis to the study of uptake and intracellular distribution of larger labeled molecules in bone marrow cells. %%% One of the fundamental properties of living cells is their ability to control their internal salt content and composition by controlling the passage of inorganic ions across their membranes. This is important because different salts play different physiological roles in different cells at different developmental stages. This research uses a sophisticated technique known as electron probe x-ray microanalysis to study the inorganic salt content of individual, identified cells as visualized by the electron microscope. The cell type and its stage of maturity can be determined and correlated with its salt content. This information can be used to deduce the ion transport mechanisms at work. This research will contribute to the basic understanding of biological ion transport processes.***//
