Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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Intrapancreatic activation of trypsinogen is believed to play a critical role in the initiation of acute pancreatitis, but mechanisms responsible for intrapancreatic trypsinogen activation during pancreatitis have not been clearly defined. In previous in vitro studies, we have shown that intra-acinar cell activation of trypsinogen and acinar cell injury in response to supramaximal secretagogue stimulation could be prevented by the cell permeant cathepsin B inhibitor E64d (Saluja A, Donovan EA, Yamanaka K, Yamaguchi Y, Hofbauer B, and Steer ML. Gastroenterology 113: 304-310, 1997). The present studies evaluated the role of intrapancreatic trypsinogen activation, this time under in vivo conditions, in two models of pancreatitis by using another highly soluble cell permeant cathepsin B inhibitor, L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl-L-isoleucyl-L-proline methyl ester (CA-074me). Intravenous administration of CA-074me (10 mg/kg) before induction of either secretagogue-elicited pancreatitis in mice or duct infusion-elicited pancreatitis in rats markedly reduced the extent of intrapancreatic trypsinogen activation and substantially reduced the severity of both pancreatitis models. These observations support the hypothesis that, during the early stages of pancreatitis, trypsinogen activation in the pancreas is mediated by the lysosomal enzyme cathepsin B. Our findings also suggest that pharmacological interventions that inhibit cathepsin B may prove useful in preventing acute pancreatitis or reducing its severity.
zymogens
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INTRODUCTION |
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OVER 100 YEARS AGO, Chiari suggested that acute pancreatitis is an autodigestive disease in which the pancreas is injured by enzymes that it normally synthesizes and secretes. Under physiological conditions, most of the potentially harmful digestive enzymes produced by pancreatic acinar cells are synthesized and secreted as inactive zymogens that become activated only after they reach the duodenum. Considerable interest, therefore, has been focused on the mechanisms by which those enzymes might become activated, prematurely, within the pancreas and cause pancreatic injury. Our own studies have suggested that intrapancreatic activation of trypsinogen, and presumably other zymogens, occurs within acinar cells and that it is mediated by the lysosomal hydrolase cathepsin B (19). Although controversial, several recent publications have supported this proposal. In one, genetic deletion of cathepsin B was shown to partially protect mice from pancreatitis and to reduce intrapancreatic activation of trypsinogen, which occurs during pancreatitis (8). In another study, one of the mutated forms of cationic trypsinogen shown to be associated with hereditary pancreatitis was noted to display increased sensitivity to cathepsin B-mediated activation (22).
We (19) have previously shown that, under in vitro conditions, the cell permeant cathepsin B inhibitor E64d can prevent the intra-acinar cell activation of trypsinogen, which follows exposure of freshly isolated pancreatic acini to a supramaximally stimulating concentration of the cholecystokinin analog cerulein. The present studies were designed to examine the role of cathepsin B in the in vivo rather than in vitro intrapancreatic activation of trypsinogen and the importance of this phenomenon in two dissimilar models of pancreatitis. We did not use E64d for these studies because of its relatively short half-life in vivo (23). Instead, we used another cathepsin B inhibitor, [L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester (CA-074me), which is known to be highly soluble, cell permeant, and effective for prolonged periods. CA-074me was tested in two dissimilar models of pancreatitis, the secretagogue and the duct infusion model, and in both, it was found to prevent intrapancreatic activation of trypsinogen as well as to markedly reduce the severity of pancreatitis. These studies suggest that pharmacological methods of inhibiting cathepsin B activity may offer promise in the prevention and/or treatment of clinical pancreatitis.
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MATERIALS AND METHODS |
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All experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center. Male Wistar rats (100-125 g), obtained from Charles River Laboratories (Wilmington, MA) and male CD-1 mice (18-20 g) obtained from Taconic (Germantown, NY) were housed in temperature-controlled (23 ± 2°C) rooms with a 12:12-h light-dark cycle. They were fed standard laboratory chow, given water ad libitum, fasted overnight before each experiment, and randomly assigned to control or experimental groups. Cerulein, the decapeptide analog of the potent pancreatic secretagogue cholecystokinin, and the cathepsin B substrate were purchased from Research Plus (Bayonne, NJ). Boc-Glu-Ala-Arg-4-methylcoumaryl-7-amide (MCA; the trypsin substrate) and CA-074me were from Peptides International (Louisville, KY). Anti-rabbit trypsinogen activation peptide (TAP) antibody was a kind gift from D. McNally (Abbott Laboratories, North Chicago, IL), and biotinylated anti-rabbit IgG and streptavidin alkaline phosphatase were purchased from Vector Laboratories (Burlingame, CA). Ketamine and xylazine were obtained from Abbott Laboratories and Bayer, respectively. Na+-taurocholate, as well as other chemicals and reagents, were purchased from Sigma (St. Louis, MO).
Pretreatment of animals with CA-074me. Stock solutions of the cathepsin B inhibitor CA-074me were made at a concentration of 10 mg/ml in dimethyl sulfoxide. This was diluted 1:10 in saline and administered at a dose of 10 mg/kg body wt by tail vein injection 1 h before the induction of pancreatitis. Control animals received vehicle alone.
Induction of pancreatitis. CD-1 mice were given hourly (×6) intraperitoneal injections of cerulein (50 µg/kg) to elicit secretagogue-induced pancreatitis, whereas control animals received intraperitoneal injections of a comparable amount of saline. At selected times, animals were killed by CO2 asphyxia and tissue samples were obtained for study. To elicit duct infusion-induced pancreatitis, male Wistar rats were anesthetized with ketamine and xylazine and subjected to midline laparotomy. A blunt needle was introduced transduodenally into the common biliopancreatic duct by the method of Heinkel (11) and secured in place with a ligature around the duct. The hepatic duct was closed at the hilum of the liver with a bulldog clamp. Sodium taurocholate (5% in saline) was infused using a compact infusion pump (model 975; Harvard Apparatus) at a rate of 0.052 ml/min, and each rat received a total volume of 1 ml/kg body wt. The needle, ligature, and clamp were then removed, and the laparotomy incision was closed. Control animals underwent sham operation, which, under an identical anesthesia, consisted of laparotomy and puncture of the duodenum. Six hours after duct infusion or sham operation, animals were killed by CO2 asphyxia and samples were taken for study.
Trypsin activity in pancreas homogenates. Preliminary studies were done to decide the time at which trypsin activity peaked in the pancreas during each form of experimental pancreatitis. In the secretagogue-induced model, this was found to be 30 min after the start of cerulein administration, after which time the levels declined. In the duct infusion model, trypsin activity peaked 45 min after taurocholate infusion and remained elevated to the same extent until 6 h after duct infusion. After tissue harvest, the pancreas samples were homogenized in cold (4°C) MOPS buffer, pH 7.0 (250 mM sucrose, 5 mM MOPS, 1 mM MgSO4) using a motorized glass-Teflon homogenizer. The resulting homogenate was centrifuged (50 g, 5 min), and the supernatant was taken for assay. Trypsin activity was measured fluorometrically using Boc-Glu-Ala-Arg-MCA as the substrate according to the method of Kawabata et al. (14). DNA was measured using Hoechst dye 33258, according to the method of Labarca and Paigen (17). The trypsin results were expressed as trypsin activity per microgram of DNA in the homogenate, as described previously (12).
TAP levels. Immediately after death, the pancreas was removed and a portion was resuspended in TAP assay buffer, pH 7.4 (20 mM Tris · HCl, 5 mM EDTA, 150 mM NaCl). The samples were boiled for 10 min, homogenized in the same buffer, and centrifuged; the supernatant was frozen and stored for later TAP measurement. TAP levels were determined by direct competitive ELISA using affinity-purified rabbit polyclonal anti-TAP antibodies, according to the method of Gudgeon et al. (7). TAP levels were expressed as picograms of TAP per microgram of DNA.
Serum amylase.
Harvested blood was allowed to clot and was centrifuged, and the serum
obtained was used for measurement of amylase activity. Serum amylase
activity was measured using 4,6-ethylidene
(G7)-p-nitrophenyl (G1)-1D-maltoheptoside as the
substrate according to the method of Kruse-Jarres et al.
(16).
Cathepsin B activity.
Cathepsin B activity was determined according to the method of McDonald
and Ellis (18), with minor modifications, using CBZ-arginyl-arginine--naphthylamide as the substrate. Cathepsin B results were expressed as cathepsin B activity (slope of fluorescence emission) per microgram DNA in the homogenate.
Neutrophil sequestration. The sequestration of neutrophils within the pancreas was evaluated by quantitating tissue myeloperoxidase (MPO) activity using a modification of the bromide-dependent chemiluminescence technique as described by Haqqani et al. (10).
Morphology. The extent of pancreatic acinar cell necrosis and interstitial edema were morphometrically quantitated by an observer who was not aware of the sample identity. For these studies, paraffin-embedded samples were sectioned (5 µm) and stained with hematoxylin and eosin. Ten randomly chosen microscopic fields (×125) were examined for each tissue sample, and the extent of acinar cell injury/necrosis was expressed as a percentage of total acinar tissue as described previously (13). Using the same slides, tissue edema was scored using a 0-3 scale in which 0 = none and 3 = severe.
In vitro studies. For these studies, dispersed rat pancreatic acini were prepared by collagenase digestion and gentle shearing as described previously (4). Acini were resuspended in HEPES-Ringer buffer (pH 7.4), which contained (in mM) 115 NaCl, 5 KCl, 1 MgSO4, 1 CaCl2, 10 HEPES, and 15 glucose and 0.1% bovine serum albumin. Viability of acini was >95% as assessed by trypan blue exclusion. After incubation of acini with various agents, the cells were washed twice with HEPES-Ringer buffer and processed for either trypsin activity or TAP measurements as described above. Amylase secretion from pancreatic acini was determined as described previously (19).
Data presentation. Results reported represent means ± SE for multiple determinations from four or more animals in each group. In the figures, vertical bars denote SE. Student's t-test was used to evaluate the significance of changes. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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We and others (1, 6) have previously reported that
both supramaximal secretagogue stimulation and retrograde duct infusion result in acute pancreatitis. Both models are characterized by a rise
in serum amylase activity, pancreatic edema, and acinar cell necrosis
(Figs. 1 and
2). Intrapancreatic TAP
levels and trypsin activity increased in both models, indicating that
intrapancreatic trypsinogen activation occurred (Figs.
3 and 4).
In addition, pancreatic MPO activity in the pancreas increases in both
models, indicating that sequestration of neutrophils within the
pancreas occurred (Figs. 1 and 2).
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Effects of CA-074me on pancreatic cathepsin B activity.
As shown in Fig. 5, A and
B, cathepsin B activity in the pancreas was not
significantly changed during either model of pancreatitis. Pretreatment
of animals with CA-074me resulted in a profound decrease in pancreatic
cathepsin B activity in both the secretagogue- and the duct
infusion-induced model. Under these conditions cathepsin B activity was
barely detectable. A similar reduction in pancreatic cathepsin B
activity was observed when control animals were given CA-074me (data
not shown).
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Effects of CA-074me on cerulein-induced in vitro trypsinogen
activation.
As shown in Fig. 6, incubation of freshly
prepared acini with 1 mM CA-074me completely prevented cerulein-induced
in vitro activation of trypsinogen activation as determined by
measurement of trypsin activity and TAP levels in cellular
homogenates.
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Effects of CA-074me on secretagogue-induced pancreatitis.
As shown in Fig. 3, the extent of trypsinogen activation within the
pancreas during secretagogue-induced pancreatitis was markedly reduced
when mice were pretreated with CA-074me before the start of cerulein
administration. Under these conditions, intrapancreatic TAP levels were
the same in control and cerulein-treated animals, whereas
intrapancreatic trypsin activity was only slightly greater in the
cerulein-treated group than in the controls. Pancreatic MPO activity,
pancreatic edema, and the extent of acinar cell necrosis were also
markedly reduced by CA-074me pretreatment (Fig. 1). These effects of
CA-074me pretreatment noted in our biochemical studies (Fig. 1) were
also observed in morphological studies (Fig. 7) that showed a marked reduction in the
changes that characterize this model. In contrast to these rather
profound changes, CA-074me pretreatment caused only modest, but
significant, reduction in the magnitude of secretagogue-induced
hyperamylasemia (Fig. 1).
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Effects of CA-074me on duct infusion-induced pancreatitis. As shown in Figs. 2 and 4, the administration of CA-074me before retrograde pancreatic duct infusion with Na+-taurocholate reduces the intrapancreatic rise in trypsin activity and MPO activity as well as the extent of acinar cell necrosis in this model of pancreatitis. Morphological changes that characterize this model, including inflammation and necrosis, are also reduced by CA-074me pretreatment (Fig. 7). In contrast to findings with the secretagogue model, a more profound reduction in hyperamylasemia was noted after pretreatment with CA-074me in this model (Fig. 2). On the other hand, little or no change in the magnitude of pancreatic edema was observed (Fig. 7).
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DISCUSSION |
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In the present studies, we used a pharmacological approach to inhibit acinar cell cathepsin B, a lysosomal cysteine protease belonging to the papain family of endopeptidases (15). Studies by others (2) using the agent we chose for our experiments, CA-074, have shown that it is relatively specific for cathepsin B and highly potent. It is believed to act by irreversibly binding to Cys29, causing permanent inactivation of the enzyme. In addition to inhibiting cathepsin B, CA-074 can inhibit other lysosomal cysteine proteases, including cathepsins H and L, but only with a potency that is two to three orders of magnitude less than that noted for cathepsin B (2). CA-074 is highly negatively charged and, as a result, relatively impermeant to cells. To increase the likelihood that we would achieve effective intracellular levels of CA-074 in our studies, we have administered the cathepsin B inhibitor in its methyl ester form. CA-074me is readily taken up by cells, and it remains intracellularly active after deesterification by intracellular esterases (2). For in vivo studies, others used doses ranging from 4 to 80 mg/kg body wt (24, 25). In our studies, we chose to use a dose of 10 mg/kg body wt.
We have found that the administration of CA-074me markedly reduces
pancreatic cathepsin B activity under resting conditions and in two
dissimilar models of pancreatitis. This reduction in cathepsin B
activity is associated, in both models, with 1) a reduction
in intrapancreatic trypsinogen activation and 2) a reduction in the severity of pancreatitis. The observation that CA-074me pretreatment results in these changes in both models would strongly indicate that the protective effects we noted are not model specific. Rather, they suggest that our findings are applicable to the general issue of pancreatitis. Thus our observation that cathepsin B inhibition with CA-074me prevents trypsinogen activation and reduces the severity
of pancreatitis indicates that cathepsin B inhibition, with this or
other agents, might reduce the severity of clinical pancreatitis as
well. Interestingly, we also found that CA-074me pretreatment prevents
nuclear factor (NF)-B activation during cerulein-induced
pancreatitis (data not shown). This effect on NF-
B activation is not
surprising, because cysteine proteases are known to play an important
role in proteasome function and proteasome function is required for
NF-
B activation (9). This reduction in NF-
B
activation, observed after CA-074me administration, may contribute to
the reduction in pancreatitis severity we observed, because, as
recently noted by Chen et al. (3), direct NF-
B activation in the pancreas can induce pancreatic inflammation. This
mechanism, however, cannot explain the prevention of trypsinogen activation noted in our studies to follow CA-074me pretreatment, because direct activation of NF-
B leads to little, if any,
trypsinogen activation (3). In contrast to its effects on
NF-
B activation, CA-074me was not observed in our studies to alter
the upregulated expression of several cytokines/chemokines (e.g.,
monocyte chemoattractant protein-1 and macrophage inflammatory
protein-1
) in the pancreas during pancreatitis (data not
shown). Presumably, this indicates that expression of those
inflammatory mediators is not regulated by NF-
B activation and that
at least these mediators are not, by themselves, sufficient to induce
pancreatitis in the absence of trypsinogen activation.
A series of in vitro studies were performed to be certain that the cysteine protease inhibitor CA-074me does not interfere with intrapancreatic trypsinogen activation by nonspecific mechanisms. In those studies, we found that CA-074me prevents in vitro cerulein-induced trypsinogen activation (i.e., it inhibits the cerulein-induced rise in intra-acinar cell trypsin activity and TAP levels) (Fig. 6) as it does under in vivo conditions. On the other hand, CA-074me does not appear to alter acinar cell function, i.e., cerulein-stimulated acinar cell secretion of amylase is not altered in the presence of CA-074me (Fig. 8). Furthermore, CA-074me does not directly alter either trypsin activity (Fig. 8) or measurable TAP levels when evaluated in vitro in a cell-free system. From these studies, we conclude that the in vivo changes noted with CA-074me are not the result of nonspecific effects of CA-074me on either acinar cell function or the measurement of trypsinogen activation.
Mechanisms by which cathepsin B might mediate intracellular trypsinogen activation and regulate the severity of pancreatitis are poorly understood. The theory we favor suggests that, during the early stages of pancreatitis before intracellular zymogen activation, digestive enzyme zymogens and lysosomal hydrolases, including cathepsin B, become colocalized, and, within that colocalization compartment, cathepsin B catalytically activates trypsinogen (20). Although this theory remains unproven, there is a gradually enlarging body of evidence that has supported it. Work by several groups has clearly shown that, under appropriate conditions, cathepsin B can cleave the activation peptide from trypsinogen and, as a result, activate the zymogen (5, 19). Our own studies have indicated that colocalization of lysosomal hydrolases with digestive enzyme zymogens occurs before zymogen activation in experimental pancreatitis and that the colocalization of these two types of enzyme occurs within the compartment in which zymogen activation occurs (12, 21). Furthermore, in an in vitro system, we found that the cathepsin B inhibitor E64d could prevent secretagogue-induced intra-acinar cell activation of trypsinogen (19). The results of the present studies are consistent with these prior observations, and they extend our earlier work by demonstrating that, in addition to preventing trypsinogen activation, cathepsin B inhibition reduces the severity of pancreatitis when evaluated under in vivo conditions.
Halangk et al. (8) recently reported the results of studies evaluating the role of cathepsin B in pancreatitis using mice with a genetic deletion of cathepsin B. They found that deletion of cathepsin B was associated with ~50% reduction in intrapancreatic trypsinogen activation and in the severity of pancreatitis. In contrast, in the present studies, we noted a more profound reduction in both trypsinogen activation and in pancreatitis severity. These somewhat discrepant observations may indicate that other cathepsins, sensitive to CA-074me inhibition but not lost by a genetic deletion of cathepsin B, may contribute to trypsinogen activation and disease severity in pancreatitis. Further studies using agents targeting other lysosomal enzymes will be needed to address this issue. Regardless of the results of those studies, however, our present observations suggest that pharmacological interventions resulting in cathepsin B inhibition might prove useful in either preventing acute pancreatitis or reducing its severity.
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ACKNOWLEDGEMENTS |
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A. K. Saluja and M. L. Steer contributed equally to this work.
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FOOTNOTES |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-31396 (to M. L. Steer) and DK-58694 (A. K. Saluja).
Address for reprint requests and other correspondence: M. L. Steer, Dept. of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02215 (E-mail: Msteer{at}bidmc.harvard.edu).
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.
April 10, 2002;10.1152/ajpgi.00363.2001
Received 15 August 2001; accepted in final form 1 April 2002.
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