Trypsin activity is not involved in premature, intrapancreatic trypsinogen activation

Abstract

Halangk, Walter, Burkhard Krü ger, Manuel Ruthenbü rger, Jö rg Stü rzebecher, Elke Albrecht, Hans Lippert, and Markus M. Lerch. Trypsin activity is not involved in premature, intrapancreatic trypsinogen activation. Am J Physiol Gastrointest Liver Physiol 282: G367-G374, 2002. First published September 21, 2001 10.1152/ajpgi.00315.2001.-A premature and intracellular activation of digestive zymogens is thought to be responsible for the onset of pancreatitis. Because trypsin has a critical role in initiating the activation cascade of digestive enzymes in the gut, it has been assumed that trypsin also initiates intracellular zymogen activation in the pancreas. We have tested this hypothesis in isolated acini and lobules from rat pancreas. Intracellular trypsinogen activation was induced by supramaximal secretagogue stimulation and measured using either specific trypsin substrates or immunoreactivity of the trypsinogen activation peptide (TAP). To prevent a trypsin-induced trypsinogen activation, we used the cell-permeant, highly specific, and reversible inhibitor N␣-(2-naphthylsulfonyl)-3-amidinophenylalanine-carboxymethylpiperazide (S124), and to prevent cathepsin-induced trypsinogen activation, we used the cysteine protease inhibitor E-64d. Incubation of acini or lobules in the presence of S124 completely prevented the generation of trypsin activity in response to supramaximal caerulein but had no effect whatsoever on the generation of TAP. Conversely, when trypsin activity was recovered at the end of the experiment by either washout of S124 from acini or extensive dilution of lobule homogenates, it was up to 400% higher than after caerulein alone and corresponded, in molar terms, to the generation of TAP. Both trypsin activity and TAP release were inhibited in parallel by E-64d. We conclude that caerulein-induced trypsinogen activation in the pancreas is caused by an E-64d-inhibitable mechanism such as cathepsin-induced trypsinogen activation, and neither involves nor requires intracellular trypsin activity. Specific trypsin inhibition, on the other hand, prevents 80% of trypsin inactivation or autodegradation in the pancreas. autoactivation; cathepsin B; acute pancreatitis ACUTE PANCREATITIS HAS LONG been thought to represent an autodigestion of the pancreas by its own digestive proteases (1). Recent evidence suggests that a premature activation of digestive proteases does indeed occur within pancreatic acinar cells (6, 10, 13) and has been closely associated with the proteolytic damage to the organ (3, 23). What have remained controversial are the triggering events that initiate the premature and intrapancreatic activation of these proteolytic enzymes that are physiologically synthetized, stored, and secreted from the pancreas as inactive precursor zymogens.In a series of elegant studies from the first half of the last century, Kunitz and Northrop (11) have firmly established that highly purified trypsin cannot only activate other serine protease precursors, such as chymotrypsinogen, but can also convert itsel...

Biochemical Models of Hereditary Pancreatitis

“…Destruction of this "failsafe mechanism" by the R122H mutation would increase intrapancreatic trypsin activity and eventually precipitate pancreatitis. Halangk et al demonstrated that autodegradation of trypsin mitigates cathepsin B-mediated trypsinogen activation during cerulein-induced zymogen activation in isolated rat acini, suggesting that an autolytic failsafe mechanism might be operational in the mammalian pancreas [21]. Biochemical evidence supports the notion that Arg122 is important for autolysis of trypsin [15,16,[22][23][24][25][26] and mutations of this amino-acid result in increased trypsin stability [16,23,26].…”

“…The complex properties of the R122C mutant were first interpreted as an example of a loss-offunction PRSS1 mutation associated with hereditary pancreatitis, suggesting that a trypsinogen mutation could cause pancreatitis by eliminating a trypsin-dependent protective mechanism [36]. Additional support for this hypothesis came from earlier studies indicating that trypsin autodegradation might play a protective role in cerulein-induced zymogen activation [21]. Although this model is provocative, the bulk of results from in vitro experiments using recombinantly expressed trypsinogen mutants remains inconsistent with it, as a loss of function could not be demonstrated for the large majority of mutations (Table 2).…”

Chymotrypsin C (caldecrin) promotes degradation of human cationic trypsin: Identity with Rinderknecht's enzyme Y

“…6 and references therein). Inactivation of intrapancreatic trypsin through trypsin-mediated trypsin degradation (autolysis) or by an unidentified serine protease (enzyme Y) was therefore proposed to be protective against pancreatitis (7)(8)(9)(10)(11). This notion received support from the discovery that the R122H mutation, which eliminates the Arg 122 autolytic site in cationic trypsinogen, causes autosomal dominant hereditary pancreatitis in humans (9).…”

Early events in acute pancreatitis

“…In inhibitor wash-out experiments, it was determined that, following hormone-induced trypsinogen activation in pancreatic acinar cells, 80% of the active trypsin is inactivated immediately and directly by trypsin itself. Taken together, these experiments suggest that trypsin activity is neither required nor involved in trypsinogen activation, that trypsinogen does not autoactivate in living pancreatic acinar cells, and that its most prominent role is in autodegradation [55]. This, in turn, suggests that intracellular trypsin activity might have a role in the defense against other, potentially more harmful digestive proteases.…”

“…Therefore, isolated pancreatic acini and lobules offer an alternative to study the role of trypsin in pancreatitis. In a recent study that used a specific, cell permeant and reversible trypsin inhibitor, it was found that complete inhibition of trypsin activity does not prevent, or even reduce the conversion of trypsinogen to trypsin [55]. A cell permeant cathepsin B inhibitor, on the other hand, prevented trypsinogen activation completely.…”