The pertinent parameter for most processes in cells which involve the concentration of oxygen ([O2]) is the intracellular [O2]. While this has been difficult to measure accurately in the past, recently the investigators have developed methods based on the use of electron paramagnetic resonance (EPR or ESR) which have permitted such measurements in functional cells. They found that under certain circumstances that there can be significant gradients between intracellular and extracellular [O2]. These results imply both the need to make such measurements (because the conventional methods to measure [O2] dependent processes in cells usually assume that the measurement of extracellular [O2] will accurately reflect intracellular [O2]), and the need to understand how cells can establish and maintain such gradients (simple calculations, based on the usual assumptions about the diffusion of oxygen in biological systems do not predict the occurrence of the observed gradients). The overall goals of the research in the next period are: to add further proof on the existence of substantial gradients in [O2] between the intracellular and extracellular compartments; to achieve a fundamental understanding as to the factors that affect the observed gradients; to extend these measurements to specific intracellular compartments; and to determine the mechanisms that enable cells to have such gradients. In order to achieve these goals they will perform the following specific aims: 1. To improve the methodology for measuring intracellular [O2]. 2. To measure [O2] in the intracellular and extracellular compartments of macrophages under various physiological and pathophysiological conditions (the rationale for this specific aim includes seeking information on the mechanisms, whereby, cells are able to maintain the gradient in [O2], as well as determining conditions which can change the gradient). 3. To develop and apply methodology for the measurement of [O2] in specific regions of the cell (the rationale for this specific aim is the potential value of obtaining site-specific information on [O2] in order to relate events occurring in those locations to the actual [O2] rather than and average [O2] and also the need to know whether large variations in [O2] can occur in cells).
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