The overall aim of this proposal is to develop our understanding of the behavior of spin labels in living cells so that this potentially powerful tool can be utilized fully in studies of membrane dynamics and redox mechanisms in cells, including the use of spin labels in conjunction with in vivo NMR studies. In particular, we need to gain sufficient knowledge of the distribution and reactivity of spin labels in cells so the existing experimental data can be interpreted more satisfactorily and future experiments can take into account the location and reactions of spin labels in cells. The current state of knowledge on the distribution of specific spin labels and/or their reduction in living systems has sufficient uncertainties and gaps to preclude drawing firm conclusions from many experiments employing this approach. In spite of these problems (and often in ignorance of their existence) increasing numbers of such studies are being carried out and reported.
Our specific aims are: 1) to determine unequivocally the location of various spin labels, including fatty acid analogs, within cells; 2) to determine the types and sites of spin label reactions within cells; 3) determine the effects of physiological variables on the distribution and reactions of spin labels in cells; 4) to design and perform experiments that will utilize spin labels to detect metabolic changes during physiological and pathological alterations; 5) to examine the potential efficacy of spin labels as NMR contrast agents, including their use to enhance contrast selectively on the basis of metabolic states (e.g., to provide contrast that indicates hypoxic areas). Our experimental approach is to use several different methods (e.g., double labeled spin labels, spin labels with different physical properties, use of paramagnetic metal ions, alterations of cellular metabolism) to examine the validity of the prevalent assumptions about the behavior of spin labels in cells and modify these as indicated. We shall then apply these findings to determine the effects of physiological variables on spin labels in cells (e.g., oxygen, redox metabolism, and pH). We shall then use spin labels in our studies including: measurement of intracellular oxygen, including measurements in particular compartments of cells; use of nitroxides as selective contrast agents for in vivo NMR; and measurement of some of the physical-chemical changes associated with cellular transformation.
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