Physiological And Metabolic Magnetic Resonance Studies

Environmental Health Sciences

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Abstract

NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. This project investigates how chemical toxins or physical factors alter metabolic processes, with primary emphasis on perfused mouse heart preparations. Studies have focused on intracellular ions, but other cellular metabolites are also evaluated. Intracellular measurements of Ca2+ and Mg2+ in perfused organs have generally been made by NMR studies utilizing flourinated BAPTA and APTRA derivatives which provide NMR-sensitive indicators for these ions. The recent emphasis on studies of hearts derived from transgenic mice rather than from rats and other larger species has resulted in a significant loss of signal intensity due to the smaller amounts of tissue, leading to a significant loss of S/N in the NMR measurements. In order to deal with this problem, recent efforts have been directed toward the synthesis of more sensitive indicators, e.g. carbon-13 labeled 5,5'-dimethylBAPTA, which would in principle provide substantially greater sensitivity than 5FBAPTA. An additional motivation for this work is that an indicator with greater sensitivity can be loaded to lower concentrations, reducing the ionic perturbation resulting from indicator buffering of the ion concentration. In addition to the indicator work, we have continued to investigate the molecular basis for the reduced sensitivity of female hearts to ischemia/reperfusion injury. Recent studies utilizing carbon-13 labeled metabolites indicate that protection in females may involve an increase in phosphorylated glycogen synthase kinase-3, which results in enhanced oxidation of glucose. A second area of research has focused on determining the mechanisms by which borate exerts physiological and toxicological effects. Previous NMR studies of borate in the presence of trypsin indicated that in addition to the interaction with the active enzyme site, an additional interaction with a second with site was present. More extensive NMR and X-ray crystallography studies have now demonstrated a binding interaction with Ser164 and Ser167 residues that are located ~ 24 ? from the active site. This binding interaction probably is representative of similar binding interactions in other proteins. The identification of such interactions is important for understanding the physiological behavior of boron, as well as for the development of boronate ligands.