The primary objective is to study liver plasma membrane zinc transport/binding and relate it to early events in hepatocyte zinc metabolism, i.e. changes in labile, intracellular zinc pools. Zinc-membrane transport/binding and the labile intracellular pools may serve as critical intermediates in the processes by which various physiological stimuli (e.g. stress, bacterial infection, diabetes, carcinogenesis) perturb body zinc pools. In addition, this research will provide new information on trace metal interactions at the plasma membrane. A spectroscopic technique using plasma membrane vesicles which entrap selected chromophoric/fluophoric chelators provides (1) excellent time resolution for transport studies and (2) a means to distinguish external membrane-bound Zn(II) from the transported cation. Such a technique has been developed in this laboratory with model egg phosphatidyl choline vesicles and will be applied to study the unidirectional Zn(II) flux into rat liver plasma membrane vesicles. Kinetic analysis will employ both conventional and stopped-flow spectrophotometers. The results of these in vitro transport studies will be compared with in vivo Zn(II) uptake/efflux kinetics in rat hepatocytes using rapid pulse/chase techniques. Such techniques allow identification of a labile, intracellular zinc pool. Labile pool characteristics will be correlated with the transport studies as a function of the presence of external physiological ligands and other trace metal ions. The ability to define an active/labile component of the intracellular zinc pool allows critical analysis of transient zinc metabolism and its relationship to overall metabolism. The results of this work will contribute to our understanding of trace metal metabolism and, specifically, to an understanding of the relationship between transient zinc fluxes and the physiological stimuli which initiate them.
