Hypoxia is a consequence of many pathological conditions and one of the most important factors leading to organ failure and death. The deleterious effects of hypoxia can be ameliorated by compensatory mechanisms that occur along the oxygen transport system and those that are manifested at cell level. The mechanisms that occur along the oxygen transport system are fairly well understood. Much less is known of the mechanisms that work at the cell level. The purpose of this proposal is to examine the effects of short and long term in vivo hypoxia on the distribution f the energy producing pathways and on the supply of energy to support membrane functions. These studies are relevant because the evidence indicates that among the most critical functions that are lost early when cells are exposed to hypoxia is their capacity to maintain normal ionic gradients. Most likely this is due to an insufficient energy supply to the membrane. Experiments will be performed in hepatic and renal cells isolated from hypoxic and normoxic animals. Studies will focus on the effects of hypoxia on the distribution and morphometry of mitochondria, on the oxygen dependence of mitochondrial function, on the relative contribution of oxidative and glycolytic energy to support membrane transport functions and on Na/K active transport. The assumptions to be tested are: 1) that hypoxia changes the distribution and/or anatomy of the mitochondria, 2) that as a consequence of the above, diffusion distances for oxidative ATP to the plasma membrane are increased and that the membrane dependence on glycolytic ATP is increased and 3) that these changes are coupled with a reduction in the energy requirements for ion transport of the membrane. Cells will be studied using dual wavelength spectrophotometry to determine oxygen dependence of mitochondrial function. Mitochondrial distribution and anatomy will be estimated by morphometric techniques in electron micrographs. Oxidative and glycolytic capacities will be evaluated by measuring enzyme activities and by the capacity of isolated cells to utilize different substrates. Na permeability and Na/K active transport and their oxygen dependence will be measured by using radioisotopes. The contribution of oxidative and glycolytic energy for ion transport functions will be estimated through the inhibition of oxygen consumption and lactate production induced by ouabain. Results from these studies are crucial to the understanding of adaptive responses to hypoxia at tissue level and will provide important information on the mechanism and management of hypoxia- associated diseases.
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