Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis

Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. The mechanism responsible for the intrapancreatic activation of digestive zymogens is unknown, but a recent hypothesis predicts that a redistribution of lysosomal cathepsin B (CTSB) into a zymogen-containing subcellular compartment triggers this event. To test this hypothesis, we used CTSB-deficient mice in which the ctsb gene had been deleted by targeted disruption. After induction of experimental secretagogue–induced pancreatitis, the trypsin activity in the pancreas of ctsb^–/– animals was more than 80% lower than in ctsb^+/+ animals. Pancreatic damage as indicated by serum activities of amylase and lipase, or by the extent of acinar tissue necrosis, was 50% lower in ctsb^–/– animals. These experiments provide the first conclusive evidence to our knowledge that cathepsin B plays a role in intrapancreatic trypsinogen activation and the onset of acute pancreatitis.

Acute pancreatitis has long been considered to be an autodigestive disorder, in which the parenchymal tissue of the organ is destroyed by its own digestive proteases (1). Under physiological conditions, pancreatic proteases are synthesized as inactive precursor zymogens and stored by the acinar cells in zymogen granules. Autodigestion of the gland therefore requires premature activation of these zymogens. How and where such a premature and intrapancreatic activation of digestive proenzymes is initiated in the course of pancreatitis has been the subject of several investigations (2). Recent studies strongly suggest that the early pathophysiological events that eventually lead to necrosis of pancreatic tissue originate in the acinar cell (3, 4) and involve the intracellular presence of active trypsin (4, 5), a serine proteinase capable of activating other pancreatic zymogens. In pancreatic acinar cells, cytoplasmic vacuoles have been identified as the subcellular compartment in which premature trypsinogen activation begins within minutes after induction of experimental pancreatitis (6, 7). The molecular mechanisms responsible for the intracellular activation of trypsinogen, however, have remained elusive. One hypothesis predicts that the lysosomal cysteine proteinase cathepsin B (CTSB) plays an essential role in this process (8). The largely circumstantial evidence for this “cathepsin B hypothesis” is based on the following observations: (a) CTSB was shown to activate trypsinogen in vitro (9); (b) during the initial phase of acute pancreatitis in several animal models, a redistribution of CTSB into a zymogen granule-containing subcellular compartment was detected by density-gradient centrifugation (10); (c) in the same pancreatitis models, lysosomal enzymes were detected by immunogold electron microscopy in secretory organelles that also contained digestive enzymes, e.g., trypsinogen (11). Experimental approaches to show an essential role of CTSB in premature zymogen activation by inhibition of this lysosomal enzyme with synthetic inhibitors rendered contradictory results either increasing (12) or decreasing (13) premature zymogen activation, or failing to improve the course of experimental pancreatitis (14). To test the cathepsin B hypothesis more directly and to overcome the shortcomings of lysosomal enzyme inhibitors, which have only limited specificity for CTSB, we used a CTSB-deficient mouse strain that was generated by targeted disruption of the ctsb gene (15). CTSB-deficient mice were found to be viable and fertile, and they did not show any spontaneous pathological phenotype. Experimental pancreatitis was induced in these animals and their wild-type controls by injection of supramaximal concentrations of the secretagogue caerulein. When the course of pancreatitis in both groups of animals was compared, we found that the premature and intracellular activation of trypsinogen that is followed by acinar cell necrosis largely depends on the presence of CTSB.

Targeted disruption of the ctsb gene and generation of CTSB-deficient mice.

The strategy for generating CTSB-deficient mice is summarized in Figure 1, a–c, and its respective legend. To test for inactivation of the ctsb gene in homozygous mutant mice, its expression was studied by Northern and Western blot analyses. Neither ctsb transcript was detectable in RNA from homozygous mutant mice (Figure 1d) nor was CTSB protein present in lysosomal protein extracts (Figure 1e). Using Z-Arg-Arg-4-methyl-coumarin-7-amide as substrate, no CTSB-activity was detected in liver of ctsb^–/ctsb^– mice (Figure 1f). These data indicate that the ctsb gene was successfully inactivated.

Phenotypic analysis of ctsb^–/ctsb^– mice.

Homozygous CTSB-deficient mice exhibited normal development at birth and showed no apparent neurological or behavioral deficits. Postnatal development was indistinguishable from that of heterozygous and wild-type littermates. Inspection of external and internal organs at different ages did not reveal any macroscopic abnormalities in ctsb^–/ctsb^– mice. On histopathological investigation of various organ systems including brain, heart, lung, liver, spleen, thymus, pancreas, stomach, intestine, ovary, kidney, skeletal muscle, and lymph nodes, no pathological alterations could be detected in CTSB-deficient mice (data not shown). Homozygous mutant mice were fertile and reproduced normally. FACS analysis of lymphoid tissues (thymus, spleen and lymph nodes) did not reveal any abnormalities of T or B lymphocytes (data not shown).

Isolated pancreatic acini that were prepared from the pancreas of CTSB-deficient and wild-type mice responded to stimulation with caerulein in parallel manner, i.e., with increasing rates of amylase secretion up to a caerulein concentrations of 10^–11 M, and with decreasing rates of amylase secretion at supramaximal caerulein concentrations of up to 10^{– 9} M (Figure 2a). These results indicate that the CCK-receptor signaling pathway was not affected by the deletion of the ctsb gene and that the biphasic stimulus response to caerulein had remained intact in CTSB-deficient mice.

Figure 2

Trypsinogen activation during experimental pancreatitis. In the experiment shown in a, dispersed acini were generated from the pancreas of CTSB-deficient and wild-type animals as described in Methods and incubated in different concentrations of caerulein. Amylase secretion over the course of 30 minutes was expressed in percent of total amylase content. In b–d, the pancreatitis time course is shown over a 24-hour period for (b) pancreatic trypsinogen content, (c) free trypsin activity in the pancreas, and (d) trypsinogen activation peptide (TAP) in the pancreas. Data points represent the means of four or more pancreatitis animals (n ≥ 3 for controls) at each interval ± SEM. ^ASignificant differences (P < 0.05) between the CTSB^+/+ and the CTSB^–/– mice.

Intrapancreatic trypsinogen activation during experimental pancreatitis.

Because the premature activation of trypsinogen as an initiating event of pancreatitis depends on the amount of trypsinogen that is available in the pancreas, the pancreatic trypsinogen content was determined in CTSB-deficient and control mice. The resting levels of pancreatic trypsinogen in CTSB^–/– mice amounted to about twice those found in control mice (Figure 2b). During the course of experimental pancreatitis, the trypsinogen content rose in both groups of animals, which is due to a blockage of enzyme secretion (34) in the presence of ongoing protein synthesis. When trypsin activity rather than trypsinogen content was determined, only very little substrate cleavage was found in the pancreas of untreated or saline-injected CTSB^+/+ or CTSB^–/– mice (Figure 2c). The intraperitoneal injection of caerulein was followed by a significant activation of trypsinogen in the pancreas of CTSB^+/+ and in that of CTSB^–/– mice as well. At all time intervals during the course of experimental pancreatitis, however, the pancreatic trypsin activity was significantly lower in CTSB^–/– mice and reached only 18–24% of the activity levels found in wild-type controls. When, instead of measuring trypsin activity, the presence of TAP, the NH₂-terminal pentapeptide that is cleaved from trypsinogen during the conversion to active trypsin, was measured as an indicator of premature trypsinogen activation, a similar reduction in premature trypsinogen activation during the pancreatitis of CTSB^–/– mice was found (Figure 2d). The TAP-immunoreactivity levels of CTSB^–/– mice reached only 38–50% of those found in the pancreas of CTSB^+/+ animals during the course of pancreatitis. Less than 1% of the total trypsinogen content in the pancreas participated in this premature and intrapancreatic conversion to active trypsin.

Pancreatic damage during experimental pancreatitis

Serum pancreatic enzyme activities and interstitial edema. Elevated amylase and lipase activities appeared as early as 1 hour after the first caerulein injection in the serum of CTSB^+/+ as well as of CTSB^–/– mice (Figure 3, a and b). One hour after the last intraperitoneal injection of caerulein, which in this model of pancreatitis represents the peak of hyperamylasemia and lipasemia, respectively, the serum amylase and lipase activities were 46% lower in CTSB^–/– mice compared with their wild-type controls. The pancreatic content of either amylase or lipase was not reduced in the CTSB^–/– animals (data not shown).

Figure 3

Pancreatic damage during experimental pancreatitis. The time course over 24 hours is shown for (a) serum amylase activity, (b) serum lipase activity, and (c) pancreatic edema. Enzyme activities were measured as indicated in Methods, and the increase in pancreatic water content was determined indirectly as increase in pancreatic weight. Data points represent the means of six or more pancreatitis animals (n ≥ 4 for controls) at each interval ± SEM. ^ASignificant differences (P < 0.05) between the CTSB^+/+ and the CTSB^–/– mice.

A further parameter of pancreatic damage in clinical as well as in experimental pancreatitis is the formation of intercellular fluid collections termed pancreatic edema. Although the development of pancreatic edema in mice is much less prominent than in the equivalent rat model of pancreatitis, and the differences between the increases in pancreatic water content of CTSB^–/– and CTSB^+/+ mice were not large, they reached significant levels at 1 hour and 8 hours of the evolution of acute pancreatitis (Figure 3c).

Pancreatic tissue necrosis and apoptosis. The course of clinical acute pancreatitis and that of various animal models of the disease can be complicated by significant necrosis of pancreatic tissue, and the extent of necrosis correlates with disease severity (35). In our study, the first ultrastructural change in acinar cells was a progressive dilatation of the endoplasmic reticulum and the formation of cytoplasmic vacuoles that either contained a homogeneous or a partially condensed protein content or, alternatively, fragments of organelles. Some of these vacuoles could thus clearly be identified as autophagosomes, whereas others may have derived from secretory vesicles. Although the number and size of these vacuoles increased over time, their occurrence was not nearly as frequent as in the equivalent rat model of secretagogue-induced pancreatitis (36). Neither the quality of these changes nor their frequency in affected cells differed in the course of pancreatitis of CTSB^–/– and CTSB^+/+ animals (data not shown). The most severe morphological alteration observed during experimental pancreatitis was the necrosis of either individual acinar cells or the appearance of small tissue areas composed of necrotic acinar cells (Figure 4).

Figure 4

Pancreatic necrosis and apoptosis during experimental pancreatitis. The time course over 24 hours is shown for (a) the percentage of acinar cells that had undergone necrosis and (b) the percentage of cells that had undergone apoptosis after the induction of pancreatitis. The appearance of necrotic acinar cells was quantitated in electron microscopy sections and the number of apoptotic cells was determined using fluorescence labeling of DNA-strand brakes and was expressed as percentage of all DAPI-positive nuclei. Data points represent the means of three or more animals at each interval ± SEM. ^ASignificant differences (P < 0.05) between the CTSB^+/+ and the CTSB^–/– mice. In c, a representative micrograph is shown from the pancreas of a wild-type animal after 24 hours of pancreatitis. Note the prominent areas of acinar cell necrosis with resolution of cellular membranes and organelles as well as dark condensed nuclei (asterisks). In d, a corresponding section from a CTSB^–/– animal is shown. Here more intact acinar cells with densely packed zymogen granules around the acinar lumen (arrow) are seen, and the area of necrosis is confined to the right-hand margin of the micrograph. Calibration bars = 10 μm.

When the percentage of necrotic acinar cells was quantified in CTSB^+/+ and CTSB^–/– mice, the amount of tissue necrosis was up to 48% lower in the latter (Figure 4a). The greatest extent of acinar tissue necrosis and the greatest difference between the CTSB^+/+ and the CTSB^–/– groups of mice were found after 24 hours of secretagogue-induced pancreatitis. Because cell death during the course of acute pancreatitis is known to occur not only as a consequence of autodigestion and necrosis but also owing to a greatly increased rate of acinar cell apoptosis (37, 38), and given that CTSB has been implicated in the development of apoptosis (39), we compared the rate of apoptosis in the pancreas of CTSB^+/+ and CTSB^–/– mice. Based on the number of fluorescein-labeled DNA-strand brakes, approximately 9% of all cells had undergone cell death by apoptosis at the end of the 24-hour observation period. However, no difference in the overall rate of apoptosis between CTSB^+/+ and CTSB^–/– mice was found (Figure 4b).

Extrapancreatic damage during experimental pancreatitis. To study whether CTSB plays a role in determining the extrapancreatic severity of the disease process, we studied serum levels of IL-6, a proinflammatory cytokine that is known to increase during pancreatitis (40), the degree of leukocyte infiltration of the pancreas and lungs in terms of tissue levels of MPO, and the morphological changes in the lungs during the course of the disease (29). In both CTSB^+/+ and CTSB^–/– mice, the induction of pancreatitis was associated with increased serum IL-6 concentrations (Figure 5a) and with increased tissue levels of MPO in the pancreas (Figure 5b) and lungs (Figure 5c). The measurement of neither parameter resulted in statistically significant or biologically meaningful differences between the two groups, with the sole exception of serum IL-6 measurements at 24 hours. Here, the declining IL-6 levels toward the end of the experiment were somewhat lower (P = 0.035) in the CTSB-deficient animals. The morphological changes in the pulmonary tissue consisted of alveolar fluid accumulation and a progressive thickening, hyperemia, and neutrophil infiltration of the interalveolar tissue. Neither of these alterations was found to differ between the lungs of CTSB^+/+ and CTSB^–/– animals in the course of pancreatitis (Figure 5d).

Figure 5

Extrapancreatic manifestations of experimental pancreatitis. The time course over 24 hours is shown for (a) serum concentrations of IL-6, (b) MPO levels in the pancreas, (c) MPO levels in the lungs, and (d–g) morphological changes in the lungs. Data points represent the means of five or more animals at each interval ± SEM. ^ASignificant differences (P < 0.05) between CTSB^+/+ and CTSB^–/– animals. (d–g) Representative examples for the lung histology of saline-injected control (CTSB^+/+, d; CTSB^–/–, e) mice, and pancreatitis animals at 24 hours of the disease (CTSB^+/+, f; CTSB^–/–, g) are shown. Note that the thickening of the interalveolar tissue, its hyperemia, and the infiltration by leukocytes are indistinguishable between the CTSB^+/+ and CTSB^–/– group of animals. Calibration bar = 100 μm.

A century ago, when the digestive properties of pancreatic secretions became known, the morphological appearance of pancreatitis on autopsy sections suggested that the disease represents an autodigestive process (1). This view has not changed, and according to our current understanding, the onset of the disease involves a premature and intrapancreatic activation of proteolytic enzymes (2). Digestive proteases are, however, synthesized and secreted as inactive precursor zymogens, and the pancreatic acinar cell possesses a whole variety of protective mechanisms that either prevent premature zymogen activation or inhibit protease activity (41). The question, therefore, remains as to which molecular factors either trigger the initial protease activation or permit the cellular defenses to be overcome. One of the hypotheses that have attempted to address this issue predicts that CTSB, a lysosomal cysteine proteinase, is critically involved in the initial trypsinogen activation (8). This cathepsin B hypothesis is based on the observations that CTSB can activate trypsinogen in vitro (9), that CTSB is redistributed to a zymogen-granule–enriched subcellular compartment (10), and that lysosomal enzymes colocalize with digestive zymogens during the early course of experimental pancreatitis (11). Although this cathepsin-hypothesis appears attractive from a cell biologic point of view, a number of experimental observations appear to be incompatible with its assumptions: (a) a colocalization of cathepsins with digestive zymogens has not only been observed in the initial phase of acute pancreatitis, but also under physiological control conditions and in secretory vesicles that are destined for regulated secretion from healthy pancreatic acinar cells (42); (b) a redistribution of CTSB into a zymogen-enriched subcellular compartment can be induced in vivo by experimental conditions that interfere with lysosomal sorting and are neither associated with nor followed by the development of acute pancreatitis (43); (c) the administration of potent lysosomal enzyme inhibitors in vivo does not prevent the onset of acute experimental pancreatitis (14); (d) in experiments that used lysosomal enzyme inhibitors in vitro, an increase in the rate of intracellular trypsinogen activation, as well as a decrease in the rate of intracellular trypsinogen activation, has been reported (12, 13), and even a protective role against premature zymogen activation has been considered for CTSB (44, 45). In view of the limited specificity and bioavailability of the existing inhibitors for lysosomal hydrolases, the only remaining option to address the cathepsin-hypothesis conclusively was therefore to use CTSB-deficient animals and to study them in an experimental model of acute pancreatitis.

We have chosen this approach and have used a strain of mice in which the ctsb gene was deleted by targeted disruption (15). In an extensive anatomical and functional characterization, we could show that the resulting CTSB^–/– animals are phenotypically indistinguishable from their wild-type controls, that they reproduce normally, and that they carry no abnormalities in either their organ development or immune system. We used these CTSB-deficient animals to induce an experimental variety of acute pancreatitis. This secretagogue-induced model of the disease appears ideally suited to address the cathepsin-hypothesis for a number of reasons: (a) it is noninvasive and can be induced in mice and other small rodents (46); (b) it is associated with significant intrapancreatic trypsinogen activation that precedes acinar cell injury (6, 13); and (c) many of the local and systemic alterations known to be relevant for the human disease are found in this animal model including hyperamylasemia, significant pancreatic tissue necrosis, systemic inflammatory response, and lung injury (26, 28).

The targeted disruption of the ctsb gene and the complete absence of functional CTSB in the pancreas of these mice altered the course of acute pancreatitis in a number of ways. The most dramatic change in comparison to wild-type control animals, and also the most relevant in regard to the cathepsin-hypothesis of acute pancreatitis, was a significant reduction in the premature and intrapancreatic trypsinogen activation. In terms of substrate-defined trypsin activity, this reduction amounted to more than 90% over the course of 24 hours, when the greater pancreatic trypsinogen content of CTSB^–/– animals is taken into account.

This prevention of premature trypsinogen activation was, however, incomplete. In terms of the presence of TAP in the pancreas, which indicates the total amount of trypsinogen that has been activated over time rather than the actual activity at a given point in time, the reduction of trypsinogen conversion that can be attributed to the absence of CTSB was approximately 50% and thus corresponded to the reduction of acinar cell necrosis. CTSB is therefore clearly not the only factor involved in premature intrapancreatic trypsinogen activation or cell injury and additional mechanisms such as trypsinogen autoactivation or a trypsinogen activation by other lysosomal cysteine proteinases, e.g., cathepsin L, H, and S, must be considered as potential alternatives.

Although the reduction of trypsinogen activation corresponded to the decrease in acinar cell injury, the amount of cells undergoing apoptosis and, more surprisingly, the systemic inflammatory response did not. Neither the degree of leukocyte infiltration in the pancreas or lungs nor the histological degree of lung injury during pancreatitis was affected by the absence of CTSB. This may indicate that the presence or absence of CTSB is irrelevant for the development of systemic and inflammatory complications associated with acute pancreatitis. Alternatively, this observation might indicate that as long as pancreatic damage above a certain threshold level has occurred, the systemic complications of the disease evolve in a manner that is completely independent of the initial degree of trypsinogen activation or the extent of acinar cell injury.

In conclusion, we used CTSB-deficient mice to study whether CTSB is critically involved in premature zymogen activation and pancreatic damage in an experimental model of pancreatitis. Our results show that the presence or absence of CTSB is, indeed, an important factor that determines the degree of premature trypsinogen activation and the extent of acinar cell necrosis, but not the systemic inflammatory response associated with acute pancreatitis. To our knowledge, these data provide the first direct and conclusive experimental evidence for a role of cathepsin B in the onset of acute pancreatitis.

The authors thank P. Schmidt and A. Hafner (München, Germany) for evaluating histological sections; S. Dollwet (Freiburg, Germany), V. Krause (Magdeburg, Germany), N. Hartelt (Göttingen, Germany), and U. Breite (Münster, Germany) for excellent technical assistance; P. Saftig (Göttingen, Germany) for providing wild-type control mice; K. Rajewski (Köln, Germany) for the E-14-1 cell line; and E. Weber for a CTSB-specific antiserum. J. Deussing was supported by a fellowship grant of the Fonds der Chemischen Industrie. This study was supported by the DFG, the BMBF of Germany, the ZFKI Freiburg, and the IZKF Münster.

Walter Halangk and Markus M. Lerch contributed equally to this work.

1. Chiari, H. Über die Selbstverdauung des menschlichen Pankreas. Zeitschrift für Heilkunde 1896. 17:69-96.
2. Whitcomb, DC. Early trypsinogen activation in acute pancreatitis. Gastroenterology 1999. 116:770-772.
   View this article via: PubMed Google Scholar
3. Lerch, MM, et al. Acute necrotising pancreatitis in the opossum: earliest morphologic changes involve acinar cells. Gastroenterology 1992. 103:205-213.
   View this article via: PubMed Google Scholar
4. Whitcomb, DC, et al. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet 1996. 14:141-145.
   View this article via: PubMed CrossRef Google Scholar
5. Gudgeon, AM, et al. Trypsinogen activation peptides assay in the early prediction of severity of acute pancreatitis. Lancet 1990. 335:4-8.
   View this article via: PubMed CrossRef Google Scholar
6. Hofbauer, B, et al. Intra-acinar cell activation of trypsinogen during caerulein-induced pancreatitis in rats. Am J Physiol 1998. 275:G352-G362.
   View this article via: PubMed Google Scholar
7. Otani, T, Chepilko, SM, Grendell, JH, Gorelick, FS. Codistribution of TAP and the granule membrane protein GRAMP-92 in rat caerulein-induced pancreatitis. Am J Physiol 1998. 275:G999-G1009.
   View this article via: PubMed Google Scholar
8. Steer, ML, Meldolesi, J. The cell biology of experimental pancreatitis. N Engl J Med 1987. 316:144-150.
   View this article via: PubMed Google Scholar
9. Figarella, C, Miszczuk-Jamska, B, Barrett, A. Possible lysosomal activation of pancreatic zymogens. Activation of both human trypsinogens by cathepsin B and spontaneous acid. Activation of human trypsinogen-1. Biol Chem Hoppe Seyler 1988. 369:293-298.
   View this article via: PubMed Google Scholar
10. Saluja, A, et al. Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis. Am J Physiol 1987. 253:G508-G516.
    View this article via: PubMed Google Scholar
11. Watanabe, O, Baccino, FM, Steer, ML, Meldolesi, J. Supramaximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell: early morphological changes during development of experimental pancreatitis. Am J Physiol 1984. 246:G457-G467.
    View this article via: PubMed Google Scholar
12. Leach, SD, Modlin, IM, Scheele, GA, Gorelick, FS. Intracellular activation of digestive zymogens in rat pancreatic acini. Stimulation by high does of cholecystokinin. J Clin Invest 1991. 87:362-366.
    View this article via: JCI PubMed CrossRef Google Scholar
13. Saluja, AK, et al. Cerulein-induced in vitro activation of trypsinogen in rat pancreatic acini is mediated by cathepsin B. Gastroenterology 1997. 113:304-310.
    View this article via: PubMed CrossRef Google Scholar
14. Steer, M.L., and Saluja, A.K. 1993. Experimental acute pancreatitis: studies of the early events that lead to cell injury. In The pancreas: biology, pathobiology, and disease. V.L.W. Go, et al., editors. Raven Press. New York, New York, USA. 489–500.
    View this article via: PubMed Google Scholar
15. Deussing, J, et al. Cathepsin B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation. Proc Natl Acad Sci USA 1998. 95:4516-4521.
    View this article via: PubMed CrossRef Google Scholar
16. Qian, F, Frankfater, A, Chan, SJ, Steiner, DF. The structure of the mouse cathepsin B gene and its putative promoter. DNA Cell Biol 1991. 10:159-168.
    View this article via: PubMed Google Scholar
17. Thomas, KR, Capecchi, MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 1987. 51:503-512.
    View this article via: PubMed CrossRef Google Scholar
18. Hooper, M, Hardy, K, Handyside, A, Hunter, S, Monk, M. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature 1987. 326:292-295.
    View this article via: PubMed CrossRef Google Scholar
19. Kühn, R, Rajewski, K, Müller, W. Generation and analysis of interleukin-4 deficient mice. Science 1991. 254:707-710.
    View this article via: PubMed CrossRef Google Scholar
20. Köster, A, et al. Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins. EMBO J 1993. 12:5219-5223.
    View this article via: PubMed Google Scholar
21. Chirgwin, JM, Przybla, AE, MacDonald, RJ, Rutter, WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Biochemistry 1979. 18:241-245.
22. Isbrandt, D, et al. Muccopolysaccharidosis VI (Maroteaux-Lamy syndrome): six unique arylsulfatase gene alleles causing variable disease phenotypes. Am J Hum Genet 1994. 54:454-463.
    View this article via: PubMed Google Scholar
23. Lyons, K, et al. Vgr-1, a mammalian gene related to Xenopus Vg-1, is a member of the transforming growth factor beta superfamily. Proc Natl Acad Sci USA 1989. 86:4554-4558.
    View this article via: PubMed CrossRef Google Scholar
24. Owens, JW, Gammon, KL, Stahl, PD. Multiple forms of beta-glucuronidase in rat liver lysosomes and microsomes. Arch Biochem Biophys 1975. 166:258-272.
    View this article via: PubMed CrossRef Google Scholar
25. Saftig, P, et al. Mice deficient for the lysosomal proteinase cathepsin D exhibit progressive atrophy for the intestinal mucosa and profound destruction of lymphoid cells. EMBO J 1995. 14:3599-3608.
    View this article via: PubMed Google Scholar
26. Barrett, A.J. and Kirschke, H. 1981. Cathepsin B, cathepsin H and cathepsin L. In Methods in enzymology. Vol. 80. L. Lorand, editor. Academic Press. New York, New York, USA. 535–561.
    View this article via: PubMed Google Scholar
27. Niederau, C, Ferrell, LD, Grendell, JH. Caerulein-induced acute necrotizing pancreatitis in mice: protective effects of proglumide, benzotript, and secretin. Gastroenterology 1985. 88:1192-1204.
    View this article via: PubMed Google Scholar
28. Grady, T, Saluja, AK, Kaiser, A, Steer, ML. Edema and intrapancreatic trypsinogen activation precedes glutathione depletion during caerulein pancreatitis. Am J Physiol 1996. 271:G20-G26.
    View this article via: PubMed Google Scholar
29. Bhatia, M, et al. Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury. Proc Natl Acad Sci USA 1998. 95:4760-4765.
    View this article via: PubMed CrossRef Google Scholar
30. Williams, JA, Korc, M, Dormer, RL. Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini. Am J Physiol 1978. 235:E517-E524.
31. Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976. 72:248-254.
    View this article via: PubMed CrossRef Google Scholar
32. Gavrieli, Y, Shearman, Y, Ben-Sasson, SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992. 119:493-501.
    View this article via: PubMed CrossRef Google Scholar
33. Lerch, MM, Saluja, AK, Rünzi, M, Dawra, R, Steer, ML. Luminal endocytosis and intracellular targeting by acinar cells during early biliary pancreatitis in the opossum. J Clin Invest 1995. 95:2222-2231.
    View this article via: JCI PubMed CrossRef Google Scholar
34. Jensen, RT, Lemp, GF, Gardner, JD. Interaction of cholecystokinin with specific membrane receptors on pancreatic acinar cells. Proc Natl Acad Sci USA 1980. 77:2079-2083.
    View this article via: PubMed CrossRef Google Scholar
35. Balthazar, EJ, Robinson, DL, Megibow, AJ, Ranson, JH. Acute pancreatitis: value of CT in establishing prognosis. Radiology 1990. 174:331-336.
    View this article via: PubMed Google Scholar
36. Lerch, MM, et al. Dissociation and reassembly of adherens junctions during experimental acute pancreatitis. Gastroenterology 1997. 113:1355-1366.
    View this article via: PubMed CrossRef Google Scholar
37. Gukovskaya, AS, et al. Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat. Gastroenterology 1996. 110:875-884.
    View this article via: PubMed CrossRef Google Scholar
38. Kaiser, AM, Saluja, AK, Sengupta, A, Saluja, M, Steer, ML. Relationship between severity, necrosis, and apoptosis in five models of experimental acute pancreatitis. Am J Physiol 1995. 269:C1295-C1304.
    View this article via: PubMed Google Scholar
39. Roberts, LR, et al. Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes. Gastroenterology 1997. 113:1714-1726.
    View this article via: PubMed CrossRef Google Scholar
40. Leser, HG, et al. Elevation of serum interleukin-6 concentration precedes acute-phase response and reflects severity in acute pancreatitis. Gastroenterology 1991. 101:782-785.
    View this article via: PubMed Google Scholar
41. Lerch, MM, Adler, G. Pathophysiology of acute pancreatitis. Dig Surg 1994. 11:186-192.
    View this article via: CrossRef Google Scholar
42. Tooze, J, Hollinshead, M, Hensel, G, Kern, HF, Hoflack, B. Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells. Eur J Cell Biol 1991. 56:187-200.
    View this article via: PubMed Google Scholar
43. Lerch, MM, Saluja, AK, Dawra, R, Saluja, M, Steer, ML. The effect of chloroquine administration on two experimental models of acute pancreatitis. Gastroenterology 1993. 104:1768-1779.
    View this article via: PubMed Google Scholar
44. Gorelick, FS, Modlin, IM, Leach, SD, Carangelo, R, Katz, M. Intracellular proteolysis of pancreatic zymogens. Yale J Biol Med 1992. 65:407-420.
    View this article via: PubMed Google Scholar
45. Klonowski-Stumpe, H, et al. Inhibition of cathepsin B does not affect the intracellular activation of trypsinogen by cerulein hyperstimulation in isolated rat pancreatic acinar cells. Pancreas 1998. 16:96-101.
    View this article via: PubMed Google Scholar
46. Lerch, MM, Adler, G. Experimental animal models of acute pancreatitis. Int J Pancreatol 1994. 15:159-170.
    View this article via: PubMed Google Scholar
47. Ferrara, M, et al. Gene structure of mouse cathepsin B. FEBS Lett 1990. 273:195-199.
    View this article via: PubMed CrossRef Google Scholar
48. Rhaissi, H, Béchet, D, Ferrara, M. Multiple leader sequences for mouse cathepsin B mRNA. Biochemistry 1993. 75:899-904.
    View this article via: CrossRef Google Scholar