The role of complement and cytokines as mediators of intracellular sodium accumulation and increased energy demand during critical illness will be studied in collaboration with the other members of the center grant application. The experiments described in this section are based on the hypothesis that complement activation and abnormal cytokine production facilitate and maintain potentially lethal alterations in transmembrane sodium gradients that necessarily affect energy metabolism within the cell. Thus, the role of complement activation, cytokine production, and free radicals in the genesis changes in: 1) sodium and water distribution across the cell membrane; 2) cellular bioenergetics; and 3)intracellular and extracellular pH will be examined. In established rodent models of sepsis, and ischemia and reperfusion injury, the specific aims are to: 1) determine how complement activation and cytokine elaboration affect sodium and water distribution, Na+-K+ ATPase activity, and pH in the gastrocnemius muscle and liver; 2) examine the effect of complement and cytokine inhibition on the transmembrane sodium gradient and secondary changes in high energy phosphate metabolism; and 3) correlate changes in complement activity and cytokine elaboration, as markers of the severity of injury, with changes in phosphocreatine utilization and other in vivo indices of cellular energy state as markers of changes in energy supply and demand within the cell. These relationships will be explored in the gastrocnemius muscle and level of normal rats and in the thigh (vastus lateralis) muscle of normal, complement deficient mice. Changes in sodium and water distribution will be measured using in vivo sodium magnetic resonance spectroscopy (MRS) and the shift reagent triethylenetetraminehexaacetate dysprosium (III) and phosphorus MRS and the MRS-visible water space markers dimethylmethylphosphonate and phenylphosphonate respectively. Phosphocreatine breakdown rates will be measured using in vivo 31P-MRS magnetization transfer techniques. Experimental interventions that will be used to understand the mechanisms of the observed changes will include the administration of endotoxin and TNF and complement blockade using sCR1. These in vivo experiments should clarify the mechanisms by which intracellular sodium content increases during critical illness, improve our understanding of the regulation of cellular bioenergetics and facilitate the development of strategies to ameliorate the effects of sepsis and ischemia-reperfusion injury.
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