alpha-chymotrypsin and Shock

Recent evidence indicates that shock is accompanied by a failure of the mucosal barrier in the intestine and entry of pancreatic digestive enzymes into the wall of the intestine. To investigate the formation of cytotoxic mediators produced by enzymatic digestion of the intestine, we applied homogenates of rat small intestinal wall to human neutrophils and used flow cytometry measurements of propidium iodide uptake to determine cytotoxicity. We show that homogenates of the small intestine after ischemia by occlusion of the superior mesenteric and celiac arteries for 3 h, but not without ischemia, are cytotoxic. Digestion of homogenates of nonischemic intestinal wall with purified trypsin, chymotrypsin, or elastase, proteases normally present in the intestinal lumen, yielded cytotoxic mediators. Before cell death, we saw cell damage in the form of bleb formation and flow cytometry measurements of cell size changes due to blebbing. Cytotoxicity was prevented by serine protease inhibition with phenylmethylsulfonyl fluoride (PMSF) before, but not after proteolytic digestion of the wall homogenates, indicating that enzymatic action of proteases on the homogenate is necessary for cytotoxicity. Cytotoxicity of wall homogenates digested by enzymes in the fluid collected from the lumen of the intestine was greater than digests by the individual purified proteases. Cytotoxicity is undetectable if digestive enzymes in the luminal fluid are inhibited with a combination of enzyme inhibitors PMSF and 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate before addition of wall homogenates. Passage of digested intestinal wall homogenates across a hydrophobic glass-fiber filter reduced cytotoxicity. Furthermore, we found that luminal fluid itself may be cytotoxic, possibly because of digestion of ingested food. To test whether digested food can be cytotoxic, we homogenized rat food and digested it in vitro with chymotrypsin or endogenous enzymes in luminal fluid. Cytotoxicity was significantly increased after digestion of food by luminal fluid compared with luminal fluid or undigested food. These results indicate the presence of a previously unknown mechanism for hemorrhagic necrosis in shock.

Topics: Animals; Apoptosis; Chymotrypsin; Cytotoxins; Flow Cytometry; Humans; Inflammation; Intestines; Ischemia; Male; Neutrophils; Pancreas; Rats; Rats, Wistar; Shock

Shock is associated with a dramatic rise in the level of inflammatory mediators found in plasma. The exact source of these mediators has remained uncertain. We recently examined a previously undescribed mechanism for production of inflammatory mediators in shock involving pancreatic digestive enzymes. The current in vitro study was designed to identify particular pancreatic enzymes and organs that may potentially produce inflammatory mediators. A selection of different organs from the rat (heart, liver, brain, spleen, pancreas, intestine, diaphragm, kidney, and lung) were homogenized and incubated with purified trypsin, chymotrypsin, elastase, lipase, nuclease, or amylase and the supernatant was incubated with fresh naïve leukocytes for 15 min. The level of leukocyte activation in the form of pseudopod formation and the fraction of cell death were measured. Without the addition of purified enzymes, only the homogenate of the pancreas yielded enhanced cell activation. Organs incubated with physiological concentrations of trypsin also stimulated significantly higher levels of pseudopod formation as compared with the undigested organs or enzymatic controls. Lipase and chymotrypsin were able to elicit cellular activation from selected organs such as the heart, intestine, liver and diaphragm. Undigested pancreatic homogenates were capable of producing substantial cell death, as compared with all other undigested organs. Intestinal digests with elastase, lipase, trypsin and chymotrypsin also stimulated significant cell mortality. Lipase-treated heart, liver, intestine, diaphragm, kidney, and lung stimulated cell death as well. We conclude that the intestine, as well as several other organs, may serve as a major source of inflammatory mediators during shock if exposed to digestive enzymes.

Topics: Amylases; Animals; Cell Death; Chymotrypsin; Flow Cytometry; Humans; Inflammation; Leukocytes; Male; Neutrophils; Pancreas; Pancreatitis; Rats; Rats, Wistar; Shock; Time Factors; Tissue Distribution

Topics: Amylases; Animals; Ascitic Fluid; Chymotrypsin; Dogs; Female; Immunoassay; Macroglobulins; Male; Pancreatitis; Protein Binding; Shock; Trypsin; Trypsin Inhibitors; Trypsinogen

Topics: Acid Phosphatase; Animals; Anti-Arrhythmia Agents; Aprotinin; Blood Pressure; Cats; Celiac Artery; Chymotrypsin; Enzyme Activation; Female; Hydrolases; Ligation; Lysosomes; Male; Mesenteric Arteries; Methylprednisolone; Microscopy, Electron; Pancreas; Phospholipases; Shock; Trypsin

Topics: Acute Disease; Amylases; Animals; Arteries; Blood Pressure; Chymotrypsin; Dogs; Female; Male; Pancreas; Pancreatitis; Peptide Hydrolases; Protease Inhibitors; Shock; Time Factors; Tissue Extracts; Trypsin

Topics: Chymotrypsin; Dogs; Electrocardiography; Enteritis; Models, Theoretical; Pancreatic Ducts; Pathology; Research; Shock; Shock, Hemorrhagic; Suture Techniques; Trypsin