Physiological shock and multi-organ failure is one of the most important medical problems with high mortality. A powerful inflammatory cascade accompanies shock, but there is no consensus for the trigger mechanisms of the inflammation. It is our long-term objective to identify the origin of the inflammation in shock and develop new interventions to minimize the inflammation and multi-organ failure. We recently developed a new line of research that has served to identify pancreatic digestive enzymes in the intestine as key players in shock. This family of enzymes is usually restricted to the lumen of the intestine as part of normal digestion. The digestive enzymes are present in comparatively high concentrations as part of normal digestion, capable to degrade most biological molecules and entire tissues within hours. Under normal circumstances, the digestive enzymes are retained within the lumen of the intestine by the mucosal barrier. But under conditions of shock, the same digestive enzymes are transported from the lumen into the wall of the intestine. Once inside the wall, they initiate an auto-digestion process with release of pancreatic enzymes as well as inflammatory digestive products into the central circulation where they cause cell injury and multi-organ failure. We obtained preliminary evidence to indicate that blockade of the digestive enzymes in the lumen of the intestine dramatically reduces the production of inflammatory mediators and significantly improves survival after severe forms of shock. It is our hypothesis that in hemorrhagic shock the protective barrier normally provided by the brush border epithelium is compromised and allows access of preexisting digestive enzymes into interstitial tissue in the wall of the intestine. The digestive enzymes are carried through multiple pathways into the central circulation where they cause microvascular inflammation and major cell dysfunctions by enzymatic cleavage of membrane receptors, e.g. cleavage of the extracellular domain of tight junction proteins and irreversible elevation of epithelial and endothelial permeability or cleavage of the extracellular binding site of the insulin receptor and insulin resistance. Blockade of the pancreatic enzymes and temporary prevention of digestion in the lumen of the intestine serves to prevent inflammation and reduces mortality due to multi-organ failure. Thus we propose to investigate the following three important Specific Aims: 1. Determine in hemorrhagic shock the activity and transport of the pancreatic digestive enzymes from the lumen of the intestine along multiple pathways into the peripheral microcirculation and the level of the associated microvascular inflammatory reaction. 2. Determine by enzyme blockade in the lumen of the intestine the role of pancreatic digestive enzymes in generation of inflammatory and cytotoxic mediators and in long-term survival after hemorrhagic shock. 3. Measure the level of extracellular receptor cleavage by proteases associated with failure of tight junctions and loss of key cell functions during the early stage of shock. These studies will determine the mechanisms for the origin of the powerful cell and organ injury mechanisms in shock. We will test a new form of intervention against the high mortality in shock that may have clinical utility. PUBLIC HEALTH RELEVANCE: The overall objective of this research is to develop a new intervention against inflammation and cell injury in shock and multi-organ failure, one of the most important problems in terms of mortality. The work is based on the new hypothesis, that in shock the pancreatic digestive enzymes play a central role in the development of inflammation, cell dysfunction and organ failure. These extraordinary powerful digestive enzymes are usually retained in the lumen of the intestine as part of normal digestion and they are prevented from entry into the wall of the intestine by the mucosal barrier. But under conditions of ischemia in the intestine and shock, the mucosal barrier becomes permeable and enzymes pass across the mucosal epithelium into the wall of the intestine and from there into other tissue compartments where they rapidly auto-digest healthy tissue. We provide preliminary evidence that blockade of digestive enzymes in the lumen of the intestine is highly effective to reduce inflammation in diverse forms of shock. But little is known about the details of the transport and the action of pancreatic digestive enzymes under shock conditions in order to optimize blockade of the digestive enzymes. In this project we will determine quantitatively with a new microzymographic technique in hemorrhagic shock the transport of digestive enzymes across the intestinal epithelium into the wall of the intestine, into lymphatics, the peritoneal fluid and into the peripheral microcirculation. We will determine to what degree blockade of the digestive enzymes in the lumen of the intestine reduces mortality in shock. Furthermore we will investigate a new hypothesis for the acute cell dysfunctions in shock in form of receptor cleavage by digestive proteases, e.g. cleavage of tight junction proteins as a mechanisms to cause irreversible increase in epithelial or endothelial permeability or insulin resistance due to cleavage of the extracellular domain of insulin receptors. This research will provide essential information for development of a new intervention against multi-organ failure by transient blockade of the pancreatic digestive enzymes.