Hyperoxic injury complicates therapy of many acute respiratory distress syndromes. Loss of mitochondrial aconitase activity and respiration are early events in O2 toxicity. To maintain energy and survive in hyperoxia, lung cells must increase their glucose consumption. Previous exposures which decrease respiration, including hypoxia, sublethal hyperoxia or TNF, confer pulmonary tolerance to lethal hyperoxia in rats. Some of these stimuli can upregulate lung hexokinase (HK), especially HK-II. HK phosphorylates glucose, facilitating its entry into the cell and rate-limiting glycolysis in the lung. HK also can bind to mitochondria and, thereby, could modulate their function. We hypothesize that adaptation to oxidant stress in lung requires elevated hexokinase expression. In the proposed studies, we will: (1) Determine the differential expression of hexokinase I, II, and III mRNA's and proteins in models of tolerance to hyperoxia in rat lungs and cultured cells, (2) Define the mitochondrial association or lack thereof of these hexokinase isoforms and the expression of porin proteins to which they bind, and (3) Clarify the potential importance of HK-II expression in adaptation and tolerance to hyperoxia. In rat and cell models, we will measure HK mRNA's, total and isoform-specific activities. Mitochondrial association of these isoforms will be defined by isolating lung mitochondrial and cytosolic fractions by differential centrifugation and measuring the relevant HK's by Western blot and activity gels. For confirmation, mitochondrial association of HK-I and -II isoforms will be assessed in cultured cells and lungs of animals exposed to hyperoxia or air using immunolocalization with electron microscopy. These studies will be complemented by in situ immunolocalization using fluorescence microscopy while concomitantly employing fluorescent mitochondrial marker dyes. Expression of isoforms of the mitochondrial HK-binding protein porin, the outer membrane voltage-dependent anion channel (VDAC), will be measured by Northern and Western blots. To determine the role of HK-II expression, lung epithelial cells will be transfected with an HK- II antisense vector, or with an HK-II expression vector, before acute exposure to hyperoxia, and cell viability, glycolytic rate, ATP and energy charge will be measured. Studies in which mitochondrial membrane potential is estimated by flow cytometry, using intact and permeabilized cells and various inhibitors and/or substrates, will be used to determine the impact of HK-II up- and downregulation on mitochondrial electron flow and energy homeostasis. Together, these approaches will define the role of HK in adaptation to hyperoxia.

R01HL052732

1995-09-15

2003-08-31