NSF Award
8818863
The goals of this research are: (1) to investigate the transport kinetics of anions and cations in single erythroblastic cells at various stages of maturation by means of cryosectioning and x-ray microanalysis, and (2) to demonstrate the applicability of these techniques in studying transport of materials labeled with identifiable elements such as bromine or iodine in a multicompartment system. Investigation of membrane transport of erythroid cells is complicated by the heterogeneity of the cell population. Without physical separation of the cells into distinct homogeneous populations conventional methods for measuring transport cannot be applied. The techniques of cryomicrotomy and x-ray microanalysis provide a solution to this problem. They will be used to study rubidium and bromide uptake as well as changes in potassium and sodium concentrations in dog, sheep, and rabbit erythroid cells at various stages of maturation. The different species of animals selected are distinct in their red cell membrane transport properties. The stage of maturation of the cells will be identified either morphologically or from the amount of iron (i.e. hemoglobin) present. Pump/leak as well as co-transport and counter-transport systems will be examined by multielemental analysis and with the aid of inhibitors such as ouabain, furosemide, bumetanide, DIDS, and SITS. Changes in surface to volume ratios of the cells will be determined by stereological techniques and the contribution of these changes to uptake will be examined. In addition, x-ray microanalysis will be applied to examine the intracellular distribution of model iodine or bromine labeled compounds in order to test the feasibility of using this technique for studying transport, distribution and targeting of other materials in general. The techniques of rapidly freezing cells, cutting frozen thin sections, and studying their elemental content using x-ray microanalysis provides a unique approach to the study of the transport and compartmentation of inorganic ions and small molecules in cells. This procedure permits simultaneous quantitative analysis of their elemental content and study of their morphology so that the transport properties of individual cell types in a mixed population can be deduced from very small samples. This research has already provided new insights into the development of transport processes in maturing red blood cells. Extension of this technique to study of the distribution in cells of model compounds labeled with identifiable tracer elements will have many potential applications in basic and applied research.
