We seek to expand a new nuclear magnetic resonance (NMR) approach which we have developed. It employs new shift reagents, paramagnetic anions, for the study of the NMR of the physiological alkali (Na+,K+) and alkaline earth (Mg++, Ca++) metal aquo cations as well as NH4+. It allows discrimination of intra- and extracellular ions and thus quantitative measurement of their cytoplasmic concentrations and physical state in vivo and of their fluxes across biological membranes in a continuous manner. We want to expand this approach along a number of parallel lines. These include testing new, more effective, shift reagents and quantifying the NMR measurements of intracellular concentrations, but our main emphasis is to explore the kinds of transport measurements which can be made and to demonstrate their quantitative accuracy. To this end, we propose; to use vesicles to study effluxes and influxes induced by ionophores, detergents, and membrane proteins (such as (Na+, K+)ATPase, and epithelial Na+/H+ antiporter); to study natural transport into and out of isolated organelles (such as mitochondria and yeast vacuoles); to study physiological and non-physiological effluxes and influxes of cellular systems (such as in vitro suspensions of human erythrocytes, cultured heart cells, and yeasts); and to study transport in in vitro tissue preparations (such as toad urinary bladder, perfused shark rectal gland and perfused rat hearts). Emphasis is given in the proposal to the elucidation of the mechanisms of NH4+ transport across simple lipid bilayers and cation transport in yeast and in the epithelial systems of toad urinary bladder and shark rectal gland. These experiments mostly employ the standard methodology of high-resolution NMR, at high fields (7T), of the stable magnetic isotopes of the physiological cations (23Na, 39K, 25Mg, 43Ca, and 14NH4) present in natural abundance. Our long-term objective is to demonstrate that this approach will become a routine technique for quantitating cytoplasmic concentrations and transmembrane fluxes and that its application to living systems will find its place in the growing repertoire of in vivo and topical NMR. This work involves aspects of physical and inorganic chemistry, biochemistry, biophysics, and physiology and has ramifications in the study of a number of pathological conditions including hypertension, affective disorders, and cancer.
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