Research proposed in this application aims to unravel the complex mechanisms which underlie the molecular pathogenesis of lethal membrane damage during cell injury due to depletion of adenosine triphosphate (ATP). The investigations will use a comprehensive approach covering the metabolic, lipid biochemical and structural aspects of cellular pathology caused by energy depletion. The major thrust of the effort will be to study how catabolism of membrane lipids relates to membrane damage. The experimental design hinges on the use of inhibitory interventions which retard specific aspects of the injury process. A restricted set of small amino acids (glycine, alanine and their analogs) powerfully inhibit the cellular degeneration which occurs during energy deprivation in a manner unrelated to their metabolism (FASEB J, in press Dec 1990). Use of these protective amino acids in conjunction with techniques to """"""""clamp"""""""" cytosolic Ca++ at fixed levels has led to the development of experimental models appropriate for critical examination of the role played by Ca++ dependent and Ca++ independent mechanisms of cell damage. One mechanism, triggered by Ca++, damages the mitochondria and the plasma membrane, and is accompanied by polyphosphoinositide hydrolysis. The other is independent of calcium, primarily damages nonmitochondrial intracellular membranes, and appears to be lethal in an unknown fashion. Prevention of intracellular Ca++ increase inhibits the first process, and protective amino acids inhibit the second. The amino acids also have an overriding protective role and retard cell death by either mechanism. Selective or simultaneous manipulations of these two factors were found to specifically and uniquely alter the structural patterns of injury. Using these well-established protocols, we will address the roles played by phospholipid hydrolysis, unesterified fatty acid release, and fatty acid cytotoxicity in the injury process. Firstly, we will ask if the protective amino acids cause decreased hydrolysis of phospholipids and decreased release of unesterified fatty acids and thereby ameliorate membrane damage. Secondly, we will also investigate whether the protective amino acids promote the action of fatty acid binding proteins to bind and thereby segregate fatty acids away from membranes and other important proteins. Thirdly, since our experiments indicate that Ca++ dependent and amino acid responsive mechanisms of injury are associated with clearly identified cellular compartments, we will determine the structural and biochemical basis for such effects. These investigations will target the catabolism of phospholipids which are enriched in organelles such as mitochondria and the brush border membrane. In related experiments, we will also use fluorescent probes to label specific membrane compartments in living cells, and trace the sequence of membrane disintegration during injury, with emphasis on how inhibitory interventions modulate these events.
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