ELECTRON MICROPROBE ANALYSIS OF EPITHELIAL NA TRANSPORT

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Intracellular electrolyte concentrations are determined by electron microprobe analysis to further elucidate the mechanisms underlying active transepithelial Na transport. This work, performed on isolated, well defined model epithelia like the frog skin and toad urinary bladder, may contribute to a better understanding of more complex epithelial structures, such as the mammalian kidney. The epithelia are incubated in Ussing-type chambers to monitor the functional state of transepithelial Na transport. Electron microprobe analysis is performed on thin freeze-dried cryosections in a scanning electron microscope using an energy dispersive x-ray detection system. Quantification of the intracellular electrolyte concentrations is achieved by the internal standard method. In parallel microelectrode impalements the intracellular electrical potential will be determined. The investigation will address different aspects of transepithelial Na transport in high resistance epithelia, regulation of transport, interaction between epithelial cells, and localization of transport pathways. Individual projects are designed to answer the following questions: (1) is the natriferic effect of aldosterone limited to an effect on the apical membrane and, if not, is the stimulation of the Na pump secondary to an intracellular Na increase, (2) by which mechanisms are the passive and active transport steps synchronized to maintain cellular homeostasis of ions in the face of widely varying transport rates, and (3) how do epithelial cells in a histologically complex epithelium cooperate in transepithelial transport? For this purpose the intracellular electrolyte concentrations will be determined after defined perturbations of transepithelial transport. These include the addition of stimulators and inhibitors of Na transport like aldosterone, amiloride and ouabain, procedures that affect the intracellular pH, pCa, or metabolic state, and conditions that lead to uncoupling of the cell-to-cell pathway.