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. Cellular cations play fundamental roles in 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 primary recent goal has been the development of a more selective intracellular indicator for magnesium ions. The need for such an indicator arises because changes in intracellular Mg2+ concentration are generally quite small, so that even weak interactions with Ca2+ or other ions which are subject to large changes can significantly impact the interpretation of the magnesium ion levels. During the past year, we have synthesized new fluorescent Mg2+ indicators, several of which exhibit an emission shift upon Mg2+ complexation. We are currently working to develop improved loading and fluorescent properties of these molecules. We have also continued our studies of sarcoplasmic calcium in perfused rabbit heart using our previously developed fluorinated, high KD calcium indicator tetrafluoroBAPTA, which can be loaded preferentially into the SR. Studies were performed to investigate the regulation of the SR Ca2+ gradient, particularly the relationship to the energy state of the cell under a variety of physiological and pathological conditions. Among other issues, the effect of ischemia on SR calcium is also of potential interest due to the possible contribution of SR Ca2+ cycling. It was found that preconditioning attenuated the fall in SR Ca2+ during ischemia. Pathways involved in glucose uptake during ischemia have also been evaluated. The metabolism of propionate is of interest since it can involve a range of potential pathways, and since it has been of interest recently in terms of the evaluation of metabolite channeling involving the enzymes of the TCA cycle. We have recently evaluated propionate metabolism in E. coli MG 1655 cells using [1,2-13C2] propionate. This labeling pattern allows separation of the flux through the methylmalonate pathway and pathways which preserve the propionate carbon skeleton. Consistent with a recent report, we found that metabolism proceeds via the methylcitrate pathway, allowing conversion of propionate to pyruvate with retention of the carbon skeleton. During this study, we were also able to observe in cell extracts resonances arising from peptides involved in cell wall biosynthesis, suggesting that this approach may be useful for studying the effects of antibiotics which interfere with this process. - magnesium; fluorescent indicator; palladium; chelators; NMR; cell injury; calcium; sarcoplasmic reticulum; propionate metabolism; methylcitrate cycle
