DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

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Cellular cations play fundamental roles in the mediation of hormonal signaling, and are also involved in the mediation of cell injury. The development of intracellular indicators for cytosolic cations and other parameters of interest has had a major impact on the field of cell biology. Our goals in this area have included the development of more sensitive intracellular calcium indicators, and more selective intracellular magnesium indicators. During the last year, several improved fluorinated BAPTA derivatives were developed for the measurement of intracellular calcium. These have improved relaxation characteristics, leading to greater sensitivity/unit time, and also additional fluorine nuclei, resulting in improved sensitivity. One of the most useful niches which physiological NMR studies can fill involves the analysis of sub-cellular ion distributions. The realization of this objective requires the development of fast exchange NMR indicators which will effectively load into both the cytosol and other subcellular compartments. During the past year, we demonstrated that the tetrafluoroBAPTA (TFBAPTA) NMR calcium indicator previously developed by our group can be effectively loaded into both the cytosol and the sarcoplasmic reticulum of perfused rabbit hearts. Subsequent physiological studies involving chemical agents which selectively reduce SR calcium uptake (cyclopiezonic acid) or promote calcium release (caffeine) have allowed assignment of these resonances to the two pools. Gating experiments have allowed measurement of the SR calcium pool as a function of the cardiac cycle. Work has continued on the preparation of a new series of Mg2+ selective indicators, and although progress has been made in the preparation of these molecules, useful indicators have not yet been prepared. We anticipate evaluation of further derivatives in the next year. Recent experiments on the transport of fluorinated compounds which are based on the intra/extracellular shift difference phenomenon which we initially observed, have focused on the drug 2'-fluoro-5-methyl-beta-L- arabinofuranosyl uracil (L-FMAU), a fluorinated L-nucleoside which has been shown to inhibit the growth of Epstein-Barr virus under in vitro conditions. These studies show that the uptake of this analog occurs by both the nucleoside transporter mediated as well as by a non-facilitated diffusion pathway. Equilibrium exchange studies of glucosyl fluoride in human erythrocytes show that the beta-anomer is transported more rapidly than the ` anomer, in contrast with 2-, 3-, 4-,. and 6-fluorodeoxy glucose analogs. It was proposed that this reversal reflects a reduced interaction of the glucosyl fluoride with the externally oriented carrier.