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 variations can significantly impact the interpretation of the magnesium ion levels. We have recently developed a new series of highly selective fluorescent magnesium ion indicators which is based on the carboxyquinolizone structure. Some of these exhibit a significant shift in the emission maximum, making them of potential use as ratioable magnesium ion indicators. Further, since intra and extracellular magnesium levels are qualitatively similar, these can be applied to both intra- and extracllular measurements. A preliminary patent application on these structures has been filed. A second project involves the determination of intracellular and particularly intra-organelle ionized calcium levels. We had previously found that the indicator TFBAPTA or tetrafluoroBAPTA that has a KDCa = 65 uM could be loaded into the sarcoplasmic reticulum (SR) of intact rabbit hearts and provide an estimate of the SR calciujm concentration. However, the SR calcium level was found to be well above 65 uM, so that the indicator was not functioning in its most sensitive range. We have recently developed higher KDCa indicators for this purpose. The new indicator was successfully loaded into both perfused rat and mouse hearts, although in these species, very little indicator is present in the SR. The indicators which have been developed have been applied by our collaborator, Dr. Elizabeth Murphy, in a project designed to investigate the role of altered ion homeostasis and ion signaling in cell injury and protective adaptation, using the perfused heart as a model system. The development of an artificial liver represents a long standing goal of toxicology as well as medicine. Attempts to utilize various bioreactor models have had only moderate success, with systems being relatively short lived and showing poor expression of drug metabolizing enzymes such as cytochrome P450. However, recent advances in the development of stem cell technology have brought this goal considerably closer to reality. Efforts are in progress to study the metabolism of hepatic cells in these bioreactors using NMR spectroscopy.
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