---start--- bbd 9/28 biochemical basis of acute pancreatitis - Dr. Washabau Q: while interning at the AMC, you dicover a new antibiotic you are using produces acute pancreatitis in 50% of the treated animals. While no one at AMC cares, your curiousity motivates you to pursue this. What experiements would you devise to show this form of spontaneous pancreatitis is similar to experimental pancreatitis. what are the mechanisms by which the pancreatic acinar cell protects itself from autodigestion. --- This is really a disease of dog/cat/man, not large animals. Take home points: history: inflammatory, multisystemic, life threatening pathogenesis: intra-acinar activation of digestive enzymes models: dietary, hormonal, obstructive presentation can vary from mild clinical signs to extremely inflammatory multisystemic life threatening process. It's an inflammation of the pancreas and can extend out of the pancreas to involve other organ systems. People and animals can have multisystemic complications - there is a lot of morbidity and mortality with respect to some formss of this disease. spontaneous pancreatitis pathogenesis has been hard to understand. from studies we find it is probably intra-acinar activation of digestive enzymes. the cells that make these digestive enzymes, we think, are destroyed when the enzymes are accidentally activated IN the cell. we think that is what is going on. we have three models of experimental pancretitis - one induced by diet, one induced hormonally, and then one due to obstruction of pancreatic ductal flow - probably more clinically relevant. these three dissimilar models produce remarkably similar changes on the cellular level. review points: remember the pancreas is a bilobed structure located with the left and right limbs in intimate apposition iwth pylorus and duodenum as well as ascending and transverse colon. the exocrine portion of the pancreas vs endocrine portion - two major exocrine cell types - acinar cells, and duct cells. acinar cells make and secrete enzymes, which go through ducts to duodenum where they are activated. the duct is lined by duct epithelium which secretes bicarbonate rich fluid, which neutralizes gastric acid so that lipase and such can carry out digestion of lipids in a higher pH. we think what happens is there is premature activation of the zymogens (inactive enzymes) in the acinar cells. this leads to cell death and pancreatitis. the endocrine part of the pancreas has islet cells which secrete a number of hormones. these probably autoregulate acinar and duct cell development take the acinar cell and look at it more closely - it has the ability to secrete several zymogens - it has all the protein synthesis mahcinery - rough ER to synthesize de novo proteins - and it makes zymogens like trypsinogen (which later becomes trypsin and is a proteolytic enzyme). the cell protect itself from autodigestion by several mechanismss. if the cell made active trypsin on rough ER, it would proteolyse the proteins of the cell and kill it. so making zymogens is protective. tthe zymogens go to golgi and get modified- glycosylations in golgi apparatus occur, so that lysosomal hydrolases are phosphorylated at 6 mannose residue, and transported into a maturing lyosome. this is another mechanism - selective phosphorylation at 6 mannose residue, to target the enzyme into a maturing lysosome. the digestive zymogens do not have selective mannose phosphorylation, and are pjackaged into zymogen granules. synthesize of inactive enzyme transport of structures segregation into vesicles exocytosis something stimulates secretion - like feeding. neuronal and hormonal stimulation causing fusion of zymogen granule wwith cell membrane, contents released into duct. these zymogens go through ducts into small intestine. there, enterokinase activates trypsin (cleaves away trypsinogen activating peptide), trypsin activates other enzyme. so there ar efour ways the cell protects itself, and any perturbation can result in intraacinar activation. there is also trypsin inhibitor in the zymogen granules, by the way. so that's another protective mechanism. so cell really tries to protect itself. what happens in the cell that permits premature activation and cell necrosis? one quick example: signalment: 8 yr old female mini schnauzer hx: acute onset vomiting, complete anorexia, lethargy, bloody diarrhea PE: fever, depression, dehydration, icterus, abdominal tenderness, hematochezia. so this presentation is one of the very inflammatory, life threatening ones. fever is due to the inflammation. so is the abdominal tenderness. remember from CLM that there can be obstruction of pancreatic and common bile ducts leading to jaundice or icterus. why hematochezia? remember, the pancreas is in apposition to stomach, intestine, colon. when inflamed, direct extension of inflammation can involve colon and produce a colitis associated with the pancreatitis. slide: sad, painful hunched schnauzer imaging is usually done this contrast study shows an area of increased opacity in right upper cranial abdominal quadrant. abdominal ultrasound shows a hypoechoic change in the area of the pancreas. hypoechogenicity of pancreas is consistent with infiltration of inflammatory cells. this animal went on to have multisystemic complications - v-tach due to release of substances into circulation. this animal died and the pancreas at necropsy was hemorrhagic and necrotic. there was also fat necrosis and caseation. the pancreas releases vasoactive substances and enzymes. histopath: fat necrosis, PMN infiltrate, coagulation necrosis. the challenge is, how to keep animal alive. what happened here? pathophysiology: trypsin - broad range of proteolytic activity kallikrein - converts kininogen to kinin elastase- blood vessel elastin digestion, vascular damage and hemorrhage lipase, phospholipase A- fat necrosis, membrane dissolution, pulmonary edema amylase, carboxypeptidase - no significant pathology all these things get activated in the pancreatic cell theories of pathogenesis of this syndrome: 1. common channel theory 2. sphincter of oddi incompetence 3. hypertension of pancreatic duct 4. intraacinar enzyme activation. #4 seems to be the one. models: 1. diet induced pancreatitis: if you feed a choline deficient, (m)ethionine supplemented diet to mice/rats, they quickly develop hemorrhagic, necrotizing pancreatitis and die. feeding less, or for shorter time - they get sick but do not die. is this relevant to us? no. but what we see here is similar to other types. 2. secretagogue induced pancreatitis - when a mouse, rat or dog is treated with or infused with a hormone like CCK (a hormone that stimulates acinar cells to secrete enzymes) or a CCK analogue, in a dose in excess of that which induces maximal enzyme secretion, they will get a less severe form of edematous, non necrotizing pancreatitis. 3. pancreatic ductal obstruction - more relevant to what we see. if you take mouse, rat, or rabbit and temporarily occlude pancreatic duct flow - like, place a cannula in the duct and obstruct the flow for some time, the animal will get an edematous form of acute pancreatitis. sometimes we see obstruction of the duct clinically due to tumor, or stone, or foreign body in proximal small intestine. so this is pretty relevant. point is, all these models are different but the cell biology changes are very similar. review points in health,w e have synthesis of zymogen on RER, intracellular transport to golgi, glycosilation, mannose 6 phosphorylation for lysosomal enzymes, targeting and maturation of zymogens into condensing vacuoles and mature zymogen granules, then exocytosis. in diagram B and C - dietary form and secretagogue form both show that there is co-localization of digestive zymogens and lysosomal hydrolases in large vacuoles - in both experimental models. so, the lysosomal hydrolases very effectively cleave the activation peptide from trypsinogen. trypsin can then activate other enzymes and there is proteolysis and lipolysis of the cell. Dietary model: amylase production charted for several diets. more amylase is produced when animal is fed the special experimental diet. they have predictable increases in activity (it says amount in handout...) of amylase, trypsinogen, and chymotrypsinogen within the cells and in the circulation. why? is it increase in synthesis? transport defect? secretory defect? experiments show us that after or during feeding, when radiolabelled phenylalanine was given, and the pancreases were analyzed, control aniamls (on normal diet) have increased synthesis of enzymes right after feeding, and then the radioactivity decreases with secretion postprandially. choline depleted animals mimic controls. ethionine supplemented animals have same synthesis, but prolonged secretory phase, suggesting inhibition of secretion. so synthesis rate is like control, but secretion rate is markedly prolonged - and this is prolonged still more in the ethionine supplemented, choline deficient animal. in fact, there was total ihibition of secretion in these animals. so this suggests that the increase has nothing to do with synthesis, but everything to do with decreased secretion. EM slides: zymogen granules fusing with plasma membrane - healthy cell. em supplemented, choline deficient diet - no exocytosis occuring. no fusion of zymogen granules. many large zymogen granules accumulating in cell. seems to be inhibition of exocytosis, and accumulation of large granules. there are other studies which show us tht some cells respond to extracellular stimuli through activation of receptors - say pancreatic acinar cell responding to stimulation by CCK - receptor coupled to G protein and further to phospholipase C, DAG, and IP3. IP3 liberates calcium from ER. Ca++ activates secretory machinery. in this dietary model, there is clearly diminished activation of phospholipase C. no problem with receptor activation. there is reduced IP3 and DAG production. so exocytotic events do not occur. see diagram in handout. COntrast this with the secretogogue induced pancreatitis which is usually milder, and edematous. what happens when you take a mouse and give too much CCK? look at amylase activity in blood or pancreas - you see the same thing. after the treatment there is amarked elevation of amylase activity in blood or pancreas. what is the mechanism? does this cause increased synthesis, transport defect, or secretory inhibition? similar study was done - radiopulse labellign with radioactive AA. synthesis rates and secretion rates - in health, there is protein synthesis and then secretion. in treated animals (with excess CCK), we see synthesis rates similar to normal, but inhibition of secretion. this is why the enzymes accumulate and cause necrosis. if you go on to do differential centrifugation and look at enzyme activities within different fractions of homogenate zymogen, lysosomes, misochromes, soluble elements (left to right on chart) loking for amylase, in health - most amylase activity is in zymogen fraction cytochrome oxidase is mostly in mitochondrial/lysosome fraction RNA is mostly in the microsomal fraction cathepsin B is mostly in lysosomal/mitochrondrial fraction this is normal but, in the CCK treated animals, you see a shifting of these things. you see cathepsin B a lysosomal enzyme moving into the zymogen fraction! more evidence for colocation of lysosomal hydrolases like cathepsin into the zymogen granules. this is important b/c cathepsin b is a hydrolase that will carry out activation of zymogens. biochemical mechanism not fully characterized but it seem sthat - and remember this is after excessive CCK infusion - in health, CCK is released after feeding, it binds receptor which is coupled to things leading to secretion. excess CCK, though, binds an inhibitory CCK receptor on the cell! the inhibitory receptor inhibits the whole secretory cascade. the diet model has uncoupling of phospholipase c from receptor this model has an inhibitory receptor inhibiting the whole thing so, diet induced pancreatitis - normal synthesis and transport, then inhibition of secretion with zymogen accumulation and colocalization of zymogens and lysosomal hydrolases leading to activation of trypsin and cell death. similar findings in hormonal form. normal synthesis and transport and glycosylation, with inhibition of secretion and colocalization. these are two totally different models but very similar biology ductal obstruction model: now what happens? we've done it in rabbits, g.pigs, and mice. duct is occluded temporarily. same experiments are run as before. when outflow is obstructed, pancreatic and blood amylase contents rise dramatically. same things are seen - synthesis rates are similar for controls and experimental animals, but secretion rates are different - obstructed duct animals have inhibition of secretion but normal synthetic rates. differential centrifugation - in health, the cathepsin is in the lysosomal fraction. in the obstructed animal, the cathepsin is shifted into the zymogen fraction. the zymogen marker used is glucosaminidase. again, we see ihibition of secretion and colocalization of zymogens and lysosomal enzymes. again - it's an inflammatory condition- all these enzymes within the cell are activated, cause autodigestion, elicits tremendous inflammatory response. dissemination of enzymes and vasoactive substances causes damage at distant sites. life threatening. intraacinar activation of these enzymes is what's happening, and we'll go over it more in medsurg3. think about therapy. what could you do to promote secretion or diminish secretory inhibition? case: dog ate a jar of peanut butter and almost died. severe, acute fulminating pancreatitis w/in 6 hrs with complications. animal had 4 major multisystemic complications and almost died. clinician asked dr washabau what to do. often not much. but he was asked "if too much enzyme is being made, maybe we need to inhibit it? and there is a somatostatin analogue which inhibits gastric and pancreatic secretions and motility - can we use this?" it has been suggested it might inhibit enzyme activation. would it? well, no. the problem isn't increased synthesis. the problem is lack of secretion. the somatostatin analogue will reduce synthesis but won't promote secretion. ---end----