1 Division of Experimental Surgery, Department of Surgery, Otto-von-Guericke-Universität, 39120 Magdeburg; 2 Division of Medical Biology, Department of Pathology, Universität Rostock, 18057 Rostock; 3 Department of Medicine B, Westfälische Wilhelms-Universität, 48129 Münster; and 4 Center for Vascular Biology and Medicine Erfurt, Friedrich-Schiller-Universität Jena, 99089 Erfurt, Germany
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ABSTRACT |
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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
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INTRODUCTION |
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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 itself from an inactive (trypsinogen) to an active (trypsin) protease (19). On the basis of these fundamental observations, others have postulated an activation cascade in which trypsinogen is initially activated by the brush border protease enterokinase (enteropeptidase) in the gut and then proceeds to activate all other digestive proteases including itself (20). Whereas this protease cascade with trypsin as the crucial trigger enzyme has been shown to be operative in the small intestine, the conditions within the pancreas are different in several respects. Because enterokinase as an initial activator of trypsinogen is absent in the pancreas, other mechanisms must account for the conversion from trypsinogen to trypsin before a proteolytic cascade, which would be analogous to the one in the gut, could be expected to occur. Therefore, several hypotheses have been proposed to explain trypsinogen activation within the pancreas in the absence of enterokinase. The two most prominent theories that have addressed this issue appear to be mutually exclusive. The first predicts that cathepsin B, a lysosomal enzyme that has been found to colocalize with digestive zymogens in the pancreas during early pancreatitis, accounts for the initial activation of trypsinogen (25, 26, 29). We and others have already confirmed that a significant proportion of intrapancreatic trypsinogen activation depends on the presence of cathepsin B in the pancreas (4, 24). The second hypothesis predicts that trypsinogen autoactivates (31) and, therefore, a trypsin-induced trypsinogen activation represents the triggering event for acute pancreatitis. In the study presented here, we have tested the latter hypothesis.
With the use of a cell-permeant, highly specific, and reversible trypsin inhibitor, we were able to completely inhibit trypsin activity in isolated rat pancreatic acini or lobules. Because of the reversibility of the inhibitor, however, we were able to determine not only to what extent trypsin had been activated, by measuring trypsinogen activation peptide (TAP), but also how much free intracellular trypsin activity had been generated by supramaximal secretagogue stimulation at the end of the experiment. Our data indicate that the conversion of trypsinogen to trypsin in response to supramaximal caerulein remains completely unaffected by the presence of a specific trypsin inhibitor and thus by the presence or absence of free trypsin activity within the acinar cells. Autoactivation of trypsinogen is therefore not an initiating factor for the intrapancreatic proteolytic cascade. Intracellular trypsin, on the other hand, was responsible for the rapid inactivation of ~80% of the prematurely generated free trypsin activity in the pancreas.
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MATERIALS AND METHODS |
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Chemicals and reagents.
The reversible cell-permeant and highly specific trypsin inhibitor
N-(2-naphthylsulfonyl)-3-amidinophenylalanine-carboxymethylpiperazide (S124), which belongs to the class of benzamidine-type serine protease
inhibitors but has a much higher affinity to trypsin than benzamidine,
was kindly provided by Dr. P. Wikström (Basel, Switzerland). The
protease substrates (CBZ-Ile-Pro-Arg)2-rhodamine 110, BOC-Gln-Ala-Arg-AMC, and Z-Arg-Arg-AMC were purchased from Molecular
Probes (Eugene, OR) and Bachem (Heidelberg, Germany), respectively.
Caerulein was obtained from Pharmacia & Upjohn (Erlangen, Germany).
E-64d, bovine trypsin (T-8003; Sigma), cathepsin B, and soybean trypsin
inhibitor were obtained from Sigma (Deisenhofen, Germany). The TAP
enzyme immunoassay kit was purchased from Biotrin International
(Sinsheim-Reihen, Germany). The protein assay was obtained from BioRad
(Munich, Germany)
Preparation and incubation of pancreatic acini and lobules. For all experiments involving animals, the permissions of the respective animal care and use committees were obtained. Male Wistar rats (HsdCpb:WU; 150-180 g) were purchased from Harlan-Winckelmann (Borchen, Germany), kept in nalgene shoebox cages at a 12:12-h light-dark cycle, and left for a minimum of 5 days to adjust to laboratory conditions. Acini or lobules from rat pancreas were prepared after an overnight fast of the animals with access to water ad libitum. Acini were prepared by collagenase (Serva, Heidelberg, Germany), as previously reported (16, 32), and incubated in a medium containing (in mM) 24.5 HEPES, 96 NaCl, 6 KCl, 1 MgCl2, 2.5 Na2HPO4, 0.5 CaCl2, 11.5 glucose, 5 Na pyruvate, 5 Na glutamate, and 5 Na fumarate, with minimum essential medium (1% vol/vol) and bovine serum albumin, fraction V (1% wt/vol), at pH 7.4.
Lobules were prepared by needle dissection according to Lerch et al. (15) in an ice-cold medium consisting of (in mM) 140 NaCl, 2.8 KCl, 1.3 CaCl2, 0.6 MgCl2, 0.8 Na2HPO4, 10 glucose, 2 Na glutamine, and 10 mM HEPES with essential amino acids (pH 7.4) gassed with oxygen. Incubations of lobule preparations (10-12 mg protein/ml) were performed at 37°C. After the incubation, aliquots of the lobule incubations were transferred into four volumes of iced medium containing 5 mM MOPS, 1 mM MgSO4, and 250 mM sucrose at pH 6.5, homogenized using an Ultra-Turrax, sonicated, and centrifuged for 5 min at 16,000 g. Sample preparation for TAP assay was performed by supplementing the lobule homogenate with 1 mM EDTA and Triton X-100 (0.1% vol/vol) and boiling the solution for 10 min. Supernatants were stored atTrypsin activity and TAP immunoreactivity measurements. Trypsin activity was determined in supernatants of lobule homogenates with the fluorogenic substrate BOC-Gln-Ala-Arg-AMC (64 µM) at 37°C. AMC fluorescence was recorded at 360 nm (excitation)/450 nm (emission) wavelength using a Perkin-Elmer LS50 B fluorometer. Trypsin activity was calculated as soybean trypsin inhibitor (SBTI)-sensitive fluorescence increase in relation to bovine trypsin as standard and expressed as picomoles of trypsin.
Trypsin activity in intact living acini was quantitated as previously reported in detail (9). In brief, after an equilibration of 30 min, the cholecystokinin analog caerulein was added at either supramaximal (10 nM) or maximal (0.1 nM) concentrations for up to 60 min and at 37°C. Acini were then washed and resuspended in medium without secretagogue but in the presence of the cell-permeant synthetic trypsin substrate (CBZ-Ile-Pro-Arg)2-rhodamine 110 (10 µM). To quantitate substrate cleavage, acini, together with the substrate, were transferred to 96-well microtiter plates and theStatistical analysis of data. Data are given as mean values (±SD) for at least four separate experiments in each group. Treatment and control values at the different time intervals were tested for statistically significant differences by means of Student's t-test with Bonferroni correction for multiple comparisons wherever appropriate. A P value of <0.05 was considered to be statistically significant.
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RESULTS |
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One of the difficulties in investigating the role of trypsin in its own activation or degradation has been the absence of appropriate research tools. Although trypsin autoactivation as a triggering event for acute pancreatitis appeared an attractive hypothesis, it could not be tested. The available trypsin inhibitors used in the in vitro studies of the past were either nonspecific, would not readily cross the acinar cell membrane, or inhibited the enzyme in a nonreversible manner. Moreover, trypsin activity was the only available parameter to determine trypsinogen activation, and inhibition of trypsin would therefore prevent any assessment of autoactivation. A number of recent developments has greatly aided the design of studies addressing trypsinogen activation. The first is the generation of specific antibodies directed against the NH2-terminal activation (penta)-peptide of trypsinogen (TAP) and an according assay based on immunoreactivity (7). The antibody detects neither trypsinogen nor trypsin but only TAP after cleavage from trypsinogen. The advantage of this approach is that trypsinogen activation can be determined without the need to measure catalytic trypsin activity and even in the presence of trypsin inhibitors. The second is the development of cell-permeant and highly specific trypsin inhibitors that block enzymatic activity completely but reversibly in the nanomolar range.
Characterization of trypsin inhibition by S124.
One of these novel high-affinity inhibitors is S124, a benzamidine type
inhibitor that does not cross-react with other pancreatic serine
proteases (5, 27). When we tested the sensitivity and
specificity of S124 in purified trypsin preparations in vitro, we found
a concentration-dependent inhibition of the catalytic activity
beginning at 10 nM and complete inhibition at 1 µM (Fig. 1A). The in vitro activity of
purified cathepsin B, on the other hand, remained completely unaffected
by the presence or absence of S124 over the entire range of
concentrations.
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Reversibility of trypsin inhibition by S124.
To this end, we stimulated isolated pancreatic acini for 30 min with
supramaximal concentrations of caerulein and subsequently studied
intracellular trypsin activity over 60 min by fluorometry of the living
cell using the cell-permeant substrate
(CBZ-Ile-Pro-Arg)2-rhodamine 110. When S124 (20 µM) was
present during caerulein stimulation and during subsequent activity
measurements, no substrate cleavage could be detected, and fluorescence
levels corresponded to control acini (Fig.
2). Supramaximal caerulein alone led to a
rapid increase of fluoresence followed by a subsequent decline, the
latter indicating a loss of trypsin activity rather than substrate
depletion as previously shown (9). When S124 was present
during caerulein stimulation only and not during the subsequent
incubation for cytofluorometry, an even greater fluorescence increase
than with caerulein alone was found, and no subsequent decline in
fluorescence occurred over 60 min (Fig. 2). This already indicates that
S124 can rapidly dissociate from trypsin and that a single wash of acini before the transfer to cytofluorometry is sufficient to recover
trypsin activity. When we studied these events by direct digital
imaging of living acini, no activity was detected at the start of the
experiment in acini after exposure to either caerulein alone (10 nM) or
to caerulein + S124 (10 µM, Fig.
3, A and D, respectively). Thirty minutes after caerulein stimulation alone, a
maximum of rhodamine 110 fluorescence had developed in the apical compartment of the acinar cells (Fig. 3B), whereas no
fluorescence could be detected in acini that had been exposed to
caerulein + S124 simultaneously and in whose buffer S124 was still
present (Fig. 3E). When caerulein-stimulated acini were
followed for up to 120 min, fluorescence progressively and completely
disappeared (Fig. 3C). When the S124-containing buffer was
continuously replaced by S124-free buffer (washout of S124), bright
apical fluorescence rapidly developed, was still detectable after 120 min of incubation, and was more prominent than the fluorescence
generated in response to caerulein alone (Fig. 3F). This
indicates that trypsin inhibition by S124 is completely reversible and
that the trypsin activity that is generated in response to supramaximal
caerulein stimulation can be recovered by washout of S124 from living
acinar cells. It also suggests that the actual trypsin activity that is
detected as cleavage of a fluorescent trypsin substrate at any given
point in time is the result of two independent processes, the
activation of trypsinogen and the degradation of active trypsin, and
that S124 can affect both of these events.
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Role of trypsin in trypsinogen activation.
To study the role of trypsin and cathepsin B in converting trypsinogen
to active trypsin in pancreatic acinar cells, we used an isolated
lobule assay because of the easier comparison between trypsin activity
measurements and TAP generation data and because the preparation of
lobules does not involve proteolytic digestion of pancreatic tissue. In
analogy to the experiments with acini, pancreatic lobules were
stimulated with supramaximal concentrations of caerulein for 60 min and
S124 (10 µM) was either present or absent during the entire duration
of the assay. TAP generation, as determined by immunoreactivity assay,
was minimal in control lobules and in lobules incubated with S124 alone
(Fig. 4A). Surprisingly, the
generation of TAP that followed supramaximal caerulein stimulation was
completely unaffected by trypsin inhibition with S124. This indicates
that the conversion from trypsinogen to trypsin and thus the
intracellular activation of trypsinogen is totally independent of the
presence or absence of available free trypsin activity in acinar cells.
It further proves that the mechanism responsible for caerulein-induced
trypsinogen activation is independent of S124-inhibitable serine
proteases and therefore rules out an autoactivation of trypsin. When
lobules exposed to supramaximal caerulein concentrations for up to 60 min were homogenized at different time points during the experiment and
the homogenates were diluted 103-fold to wash out S124, the
substrate-specific trypsin activity could readily be recovered and
quantitated. Lobules that had been supramaximally stimulated in the
presence of S124 were found to contain 400% of the trypsin activity
that had been generated after caerulein exposure alone (Fig.
4B). When the extent of TAP generation and free trypsin
activity were compared and calculated in molar terms, it was found that
TAP immunoreactivity corresponded to trypsin activity in the lobules
that were incubated in the presence of S124. The trypsin activity
detected in lobules that were treated with caerulein alone amounted to
only ~20% of TAP. This indicates not only that trypsin inhibition
with S124 has no effect on trypsinogen activation but that it prevents
the degradation of 80% of the trypsin activity that is generated in
response to supramaximal caerulein stimulation. Trypsin activity within
acinar cells is therefore not involved in trypsinogen activation or
autoactivation but represents an extremely potent factor for the
elimination of free intracellular trypsin activity (Figs. 2-4).
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DISCUSSION |
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In a series of elegant experiments that go back to the beginning of the last century, it was firmly established that trypsin, once activated by the brush-border enzyme enterokinase, can activate all other digestive proteases of the exocrine pancreas, including its own precursor zymogen trypsinogen (11, 19). Trypsin was therefore regarded as the "key activator" of the proteolytic cascade of pancreatic zymogens, and its premature and intracellular activation was thought to represent a critical factor for the development of acinar cell injury (3, 23) and the onset of pancreatitis. This conclusion, however, was entirely based on experiments using either pancreatic juice or pancreatic homogenates or purified pancreatic enzyme preparations (21). Our current concepts with which we try to explain the premature and intrapancreatic activation of digestive proteases, and hence pancreatic autodigestion during pancreatitis, are therefore derived entirely from in vitro observations and may not reflect the complex and compartimentalized conditions within the pancreatic acinar cell. Only recent developments in biotechnical methods have allowed the intracellular events involved in premature zymogen activation in living pancreatic acinar cells to be addressed directly (14).
Support for a crucial role of trypsin in the onset of pancreatitis came from genetic studies that have linked the phenotype of hereditary pancreatitis to point mutations in the cationic trypsinogen gene (31). In view of the role of trypsin in activating all other digestive proteases in the gut, it was proposed that hereditary pancreatitis could be caused by either a higher rate of trypsin autoactivation, an extended period of intracellular trypsin activity, or a greater resistance to degradation of the mutated trypsin (30). All of these possibilities would require that trypsin is, indeed, the trigger of an activation cascade in the pancreas. Studies that have addressed the functional role of disease-relevant trypsin mutations had to rely, as of today, on in vitro experiments in which the activation kinetics of recombinant mutant trypsin were compared with the wild-type enzyme (22, 28).
By using a technique that allows for studying trypsinogen activation in living pancreatic acini, and thus under physiological intracellular in vivo conditions, and by employing a novel, highly specific, and cell-permeant inhibitor for trypsin, we were able to completely block the intracellular trypsin activity that is generated within pancreatic acinar cells in response to supramaximal caerulein concentrations. In vivo administration of supramaximal concentrations of the cholecystokinin analog caerulein is known to result in an experimental variety of acute pancreatitis (12, 17), and in vitro exposure to supramaximal caerulein has been shown to be associated with premature and intracellular activation of digestive proteases (6, 9, 10, 13) and subsequent proteolytic damage (3, 23). When we inhibited trypsin activity in acinar cells, the rate at which trypsinogen was converted to trypsin (as indicated by the release of TAP) was found to be completely unaffected. Moreover, when we recovered the trypsin activity at the end of the experiment, which was possible because trypsin inhibition by S124 is a reversible process, it was up to 400% higher than the activity recovered after supramaximal caerulein stimulation alone. When, on the other hand, we measured trypsinogen activation in response to caerulein in the presence of the cysteine protease inhibitor E-64d, both trypsin activity and TAP release were found to be significantly reduced.
These observations have a number of important implications. 1) Whether or not trypsin activity is inhibited within the pancreatic acinar cell is completely immaterial for the rate at which the conversion from the zymogen trypsinogen to the active protease trypsin occurs. This effectively rules out that a trypsin-induced trypsinogen activation or an autoactivation of trypsinogen plays any significant role as the triggering event for premature protease activation within the pancreas. Although the zymogen trypsinogen itself has some autocatalytic property that is independent of a prior cleavage of the activation peptide (8), this autocatalytic potential is much too small to account for the trypsin activity and TAP release that are generated in response to supramaximal caerulein. 2) The premature intracellular trypsin activity as well as the generation of TAP are both highly dependent on the activity of lysosomal cysteine proteases such as cathepsin B. This observation is in line with earlier reports using either lysosomal enzyme inhibitors (24) or knockout strains of mice in which the cathepsin B gene was deleted (4). Although our present experiments cannot exclude that nonlysosomal cysteine proteases (such as calpain) or lysosomal hydrolases other than cathepsin B (e.g., cathepsin L, K, and H) account for some of the E-64d effect reported here and elsewhere, our studies using cathepsin B-deleted animals have firmly established the role of this particular cysteine protease in triggering trypsinogen activation (4). 3) The predominant role of prematurely activated trypsin appears to be the elimination of prematurely activated trypsin. Whereas trypsin activity appears to play no role in triggering intracellular trypsinogen activation, the trypsin-induced inactivation of trypsin, most probably by means of autodegradation, appears to be highly effective. Because TAP and recovered free trypsin activity were found to be equimolar only in the presence of S124, >80% of the trypsin activity that was generated within 1 h of supramaximal caerulein stimulation must have been inactivated by an S124-sensitive protease. Although we cannot completely rule out the possibility that another, as yet unidentified, serine protease of the exocrine pancreas is responsible for this rapid degradation of activated trypsin, the high specificity and affinity of S124 for trypsin makes this assumption unlikely.
As far as human pancreatitis is concerned, the most critical conclusion of our study, that trypsin is not involved in trypsinogen activation but accounts for 80% of trypsin degradation, must be interpreted with caution. Whereas this interpretation appears unequivocal as far as secretagogue-induced trypsinogen activation in the pancreas of rodents is concerned, it remains unclear whether frequent human causes of pancreatitis such as alcohol or gallstones are associated with an equivalent intracellular proteolytic cascade. The human pancreas, although also susceptible to secretagogue-induced pancreatitis, may contain a different composition of cysteine as well as serine proteases, and human cationic trypsin is known to have a higher capacity for autoactivation at a pH <6 than trypsin from, e.g., the bovine pancreas (2). What remains unequivocal is the fact that trypsinogen activation, as determined by the release of TAP into the serum and urine of affected patients, is a frequent characteristic of clinical pancreatitis and occurs independent of the etiology of pancreatitis, and its extent reflects disease severity (18). Whether it represents a triggering event or a protective defense mechanism in pancreatitis remains to be shown.
For the pathophysiology of hereditary pancreatitis, the results from our study suggest the possibility of a previously unrecognized mechanism: that disease-causing trypsinogen mutations change the enzyme's conformation in a manner that, although completely immaterial to the events that lead to activation, could interfere with the autodegradation of free intracellular trypsin. If that were the case, any physiological or pathological stimulus associated with premature intracellular protease activation could result, analogous to our experiments, in a 5- to 10-fold increase in uninhibited free trypsin activity within acinar cells.
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ACKNOWLEDGEMENTS |
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We thank V. Krause, U. Naumann, and S. Rackow for expert technical assistance.
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FOOTNOTES |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Interdisciplinary Center of Clinical Research, Münster.
Address for reprint requests and other correspondence: M. M. Lerch, Dept. of Medicine B, Westfälische Wilhelms-Universität, Albert-Schweitzer-Str. 33, 48129 Münster, Germany (E-mail: markus.lerch{at}uni-muenster.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00315.2001
Received 18 July 2001; accepted in final form 22 October 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Chiari, H.
über die Selbstverdauung des menschlichen pankreas.
Zeitschrift für Heilkunde
17:
69-96,
1896.
2.
Figarella, C,
Amouric M,
and
Guy-Crotte O.
Proteolysis of human trypsinogens. I. Pathogenetic implications in chronic pancreatitis.
Biochem Biophys Res Commun
118:
154-161,
1984[ISI][Medline].
3.
Grady, T,
Otani T,
Mah'moud M,
Rhee S,
Lerch MM,
and
Gorelick FS.
Zymogen proteolysis within the pancreatic acinar cell is associated with cellular injury.
Am J Physiol Gastrointest Liver Physiol
275:
G1010-G1017,
1998
4.
Halangk, W,
Lerch MM,
Brandt-Nedelev B,
Roth W,
Ruthenbuerger M,
Reinheckel T,
Domschke W,
Lippert H,
Peters C,
and
Deussing J.
Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis.
J Clin Invest
106:
773-781,
2000
5.
Halangk, W,
Stürzebecher J,
Matthias R,
Schulz HU,
and
Lippert H.
Trypsinogen activation in rat pancreatic acinar cells hyperstimulated by caerulein.
Biochim Biophys Acta
1362:
243-251,
1997[ISI][Medline].
6.
Hofbauer, B,
Saluja AK,
Lerch MM,
Bhagat L,
Bhatia M,
Lee HS,
Frossard JL,
Adler G,
and
Steer ML.
Intra-acinar cell activation of trypsinogen during caerulein-induced pancreatitis in rats.
Am J Physiol Gastrointest Liver Physiol
275:
G352-G362,
1998
7.
Hurley, PR,
Cook A,
Jehanli A,
Austen BM,
and
Hermon-Taylor J.
Development of radioimmunoassays for free tetra-aspartyl-L-lysine trypsinogen activation peptides (TAP).
J Immunol Methods
111:
195-203,
1988[ISI][Medline].
8.
Kassel, B,
and
Kay J.
Zymogens of proteolytic enzymes.
Science
180:
1022-1027,
1973[ISI][Medline].
9.
Krüger, B,
Albrecht E,
and
Lerch MM.
The role of intracellular calcium signaling in premature protease activation and the onset of pancreatitis.
Am J Pathol
157:
43-50,
2000
10.
Krüger, B,
Lerch MM,
and
Tessenow W.
Direct detection of premature protease activation in living pancreatic acinar cells.
Lab Invest
78:
763-764,
1998[ISI][Medline].
11.
Kunitz, M,
and
Northrop JH.
Crystalline chymotrypsin and chymotrypsinogen. I. Isolation, crystallization, and general properties of a new proteolytic enzyme and its precursor.
J Gen Physiol
18:
433-453,
1935.
12.
Lampel, M,
and
Kern HF.
Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue.
Virchows Arch
373:
97-117,
1977.
13.
Leach, SD,
Modlin IM,
Scheele GA,
and
Gorelick FS.
Intracellular activation of digestive zymogens in rat pancreatic acini. Stimulation by high doses of cholecystokinin.
J Clin Invest
87:
362-366,
1991[ISI][Medline].
14.
Lerch, MM,
and
Gorelick FS.
Trypsinogen activation in acute pancreatitis.
Med Clin North Am
84:
549-563,
2000[ISI][Medline].
15.
Lerch, MM,
Hoppe-Seyler P,
and
Matern S.
In vitro perfusion and incubation of rat pancreatic lobules in a kinetic system.
Biomed Biochim Acta
50:
1115-1125,
1991[ISI][Medline].
16.
Lerch, MM,
Saluja AK,
Dawra R,
Saluja M,
and
Steer ML.
The effect of chloroquine administration on two experimental models of acute pancreatitis.
Gastroenterology
104:
1768-1779,
1993[ISI][Medline].
17.
Lerch, MM,
Weidenbach H,
Gress TM,
and
Adler G.
Effect of kinin inhibition in experimental acute pancreatitis.
Am J Physiol Gastrointest Liver Physiol
269:
G490-G499,
1995
18.
Neoptolemos, JP,
Kemppainen EA,
Mayer JM,
Fitzpatrick JM,
Raraty MG,
Slavin J,
Beger HG,
Hietaranta AJ,
and
Puolakkainen PA.
Early prediction of severity in acute pancreatitis by urinary trypsinogen activation peptide: a multicentre study.
Lancet
355:
1955-1960,
2000[ISI][Medline].
19.
Northrop, JH,
and
Kunitz M.
Isolation of protein crystals possessing tryptic activity.
Science
73:
262-263,
1931.
20.
Rinderknecht, H.
Activation of pancreatic zymogens, normal activation, premature intrapancreatic activation, protective mechanisms against inappropriate activation.
Dig Dis Sci
31:
324-321,
1986.
21.
Rinderknecht, H.
Pancreatic secretory enzymes.
In: The Pancreas: Biology, Pathobiology, and Disease, edited by Go VLW,
DiMagno EP,
Gardner JD,
Lebenthal E,
Reber HA,
and Scheele GA.. New York: Raven, 1993, p. 219-251.
22.
Sahin-Toth, M,
and
Toth M.
Gain-of-function mutations associated with hereditary pancreatitis enhance autoactivation of human cationic trypsinogen.
Biochem Biophys Res Commun
278:
286-289,
2000[ISI][Medline].
23.
Saluja, AK,
Bhagat L,
Lee HS,
Bhatia M,
Frossard JL,
and
Steer ML.
Secretagogue-induced digestive enzyme activation and cell injury in rat pancreatic acini.
Am J Physiol Gastrointest Liver Physiol
276:
G835-G842,
1999
24.
Saluja, AK,
Donovan EA,
Yamanaka K,
Yamaguchi Y,
Hofbauer B,
and
Steer ML.
Cerulein-induced in vitro activation of trypsinogen in rat pancreatic acini is mediated by cathepsin B.
Gastroenterology
113:
304-310,
1997[ISI][Medline].
25.
Saluja, A,
Sadamitsu H,
Saluja M,
Powers M,
Meldolesi J,
and
Steer M.
Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis.
Am J Physiol Gastrointest Liver Physiol
253:
G508-G516,
1987
26.
Steer, ML,
and
Meldolesi J.
The cell biology of experimental pancreatitis.
N Engl J Med
316:
144-150,
1987[ISI][Medline].
27.
Stürzebecher, J,
Prasa D,
Hauptmann J,
Vieweg H,
and
Wikstroem P.
Synthesis and structure-activity relationships of potent thrombin inhibitors: piperazides of 3-amidinophenylalanine.
J Med Chem
40:
3091-3099,
1997[ISI][Medline].
28.
Teich, N,
Ockenga J,
Hoffmeister A,
Manns M,
Moessner J,
and
Keim V.
Chronic pancreatitis associated with an activation peptide mutation that facilitates trypsin activation.
Gastroenterology
119:
461-465,
2000[ISI][Medline].
29.
Watanabe, O,
Baccino FM,
Steer ML,
and
Meldolesi J.
Supramaximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell: early morphological changes during development of experimental pancreatitis.
Am J Physiol Gastrointest Liver Physiol
246:
G457-G467,
1984
30.
Whitcomb, DC.
New insights into hereditary pancreatitis.
Curr Gastroenterol Rep
1:
154-160,
1999[Medline].
31.
Whitcomb, DC,
Gorry MC,
Preston RA,
Furey W,
Sossenheimer MJ,
Ulrich CD,
Martin SP,
Gates LK,
Amann ST,
Toskes PP,
Liddle R,
McGrath K,
Uomo G,
Post JC,
and
Ehrlich GD.
Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene.
Nat Genet
14:
141-145,
1996[ISI][Medline].
32.
Williams, JA,
Korc M,
and
Dormer RL.
Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.
Am J Physiol Endocrinol Metab Gastrointest Physiol
235:
E517-E524,
1978.