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HORMONES AND SIGNALING
1Department of Internal Medicine, Section of Digestive Diseases, and the 2Department of Cell Biology, Veterans Administration Connecticut Healthcare, West Haven, and Yale University School of Medicine, New Haven, Connecticut
Submitted 18 November 2005 ; accepted in final form 28 November 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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trypsin(ogen); chymotrypsin(ogen); procarboxypeptidase B; cathepsin B; zymogen granule-enriched fraction; microsomal fraction
Studies have described pancreatic acinar cell zymogen activation taking place in zymogen granules and other vesicular compartments. Two studies have suggested that initial zymogen activation might occur in zymogen granules (18, 22). Recent work from our laboratory and others concluded that initial zymogen activation takes place in a vesicular, nonzymogen granule compartment (13, 20). The latter studies used antibodies that selectively label the trypsinogen activation peptide, a surrogate for trypsinogen activation, in confocal and immunoelectron microscopy to detect the site of trypsinogen activation in the caerulein hyperstimulation model of acute pancreatitis. Trypsinogen activation peptide was initially localized to small vesicles in a supranuclear region of the acinar cell that contained markers of lysosomes and recycling endosomes. Whether the dissimilar findings relating to the site of zymogen activation are due to differences in detection methods or experimental systems remains unclear.
Similar to the in vivo caerulein hyperstimulation model of acute pancreatitis, zymogen activation can be initiated by supraphysiological stimulation of G protein-coupled receptors on the pancreatic acinar cell. Thus supraphysiological concentrations of agonists such as cholecystokinin and acetylcholine analogs cause pathological zymogen activation within acinar cells (13, 15, 20). In this study, we report that, in the in vivo caerulein hyperstimulation model, this activation occurs in zymogen granule-enriched and a microsomal fraction.
Although reconstituted cell systems have been used to define many molecular mechanisms, technical limitations have precluded their use to study zymogen activation in the pancreatic acinar cell. To further explore the mechanisms responsible for the activation and define relevant cellular compartments, we have reconstituted zymogen activation in vitro. Using this system, we showed that zymogen activation 1) requires both cytosol and cellular organelles, 2) can take place in both zymogen-granule enriched and microsomal fractions, and 3) exhibits ATP-dependent and independent mechanisms.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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All experiments and procedures using animals were approved by the Veterans Affairs International Animal Care and Use Committee (West Haven, CT; approved protocol FG0006). Experimental pancreatitis was initiated in 150- to 200-g Sprague-Dawley rats (anesthetized intraperitoneally with ketamine-xylazine) via a tail vein injection of 40 µg/kg caerulein (hyperstimulatory dose), a cholecystokinin analog used to cause pancreatitis, and pathologic pancreatic zymogen activation (4, 26). Controls received saline. After 15 min, rats were euthanized in a CO2 chamber, and the pancreas was harvested. Particulate fractions and cytosolic fractions were assayed for enzyme content as described below.
Pancreatic Fractionation: Isolation of Zymogen Granule-Enriched, Microsomal and Cytosolic Fractions
Pancreatic fractionation followed the protocol of Tartakoff and Jamieson (28) with several modifications. The pancreas was divided in two; one-half was homogenized on ice in 10 vol (
5 ml) of 0.3 M sucrose and 1 mM DTT in the absence of protease inhibitors and used for isolation of the zymogen granule-enriched and microsomes (preparation A). The other one-half was homogenized in 10 vol (5 ml) of 0.3 M sucrose, 1 mM DTT, 25 mM Tris (pH 7.2), 5 mM aprotonin, soybean trypsin inhibitor (0.1 mg/ml), 5 mM benzamidine, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and complete EDTA-free protease inhibitor cocktail (20 µl/ml of 25x stock solution). All reagents were purchased from Sigma, except for the Roche protease inhibitor cocktail, used for isolation of cytosol (cytosolic buffer; denoted preparation B). The high-molecular-weight protease inhibitors were added to inhibit enzyme activity in the cytosol and to ensure that enzyme activity observed in the assay was within the organelles. Homogenates were centrifuged at 500 g for 5 min. All preparations and storage were at 4°C unless otherwise stated.
Preparation of Membrane Fractions
The pellet from the 500-g centrifugation was discarded, and the resulting postnuclear supernatant was centrifuged at 3,000 g for 10 min over a 2 M sucrose cushion. The sucrose cushion was necessary to prevent organelle rupture during centrifugation. After the 3,000-g centrifugation, the layer at the interface of preparation A was retained, diluted with 2 vol of 0.3 M sucrose, and washed at 3,000 g over a 2 M sucrose cushion (further described below in KCl and Sucrose Wash). The layer at the interface was retained; this was the zymogen granule-enriched fraction, which was subsequently diluted with distilled water to give a concentration of 0.3 M sucrose (using a refractometer; Leica, Buffalo, NY). The supernatant from the initial 3,000-g centrifugation (preparation A) was then centrifuged at 15,000 g for 15 min over a 2 M sucrose cushion. The layer at the interface (mitochondrial fraction) was retained. The remaining supernatant was centrifuged at 180,000 g for 60 min over a 2 M sucrose cushion. The layer at the interface was removed, diluted with 2 vol of 0.3 M sucrose, and washed at 288,000 g over a 2 M sucrose cushion. The layer at the interface was retained; this was the microsomal fraction, which was subsequently diluted with water to give a concentration of 0.3 M sucrose.
KCl and Sucrose Wash
To eliminate associated proteins from the organelle surface, zymogen granule-enriched and microsomal fractions were diluted fivefold in 0.3 M sucrose (control) or 0.5 M KCl (21). They were centrifuged at 3,000 g and 180,000 g, respectively, over a 2 M sucrose cushion. The layer at each interface was collected and diluted with water to 0.3 M sucrose. These were denoted as "washed" fractions.
Preparation of the Cytosolic Fraction
After the 500-g centrifugation, the supernatant (preparation B) was retained and centrifuged at 180,000 g for 60 min on a 2 M sucrose cushion. The supernatant was removed, and the layer at the interface was discarded. Endogenous ATP and low-molecular-weight protease inhibitors were removed from the cytosol by dialysis (molecular weight cut-off 3,500) in 0.3 M sucrose, 25 mM Tris (pH 7.2), and 100 mM KCl for 60 min with a dialysis cassette (Slide-A-Lyzer dialysis cassette, 3,500 molecular weight cut off, 0.5- to 3-ml capacity; Pierce Biotech). After dialysis, the fraction was centrifuged at 288,000 g for 15 min in tubes precoated with cytosolic buffer to remove small vesicles.
The zymogen granule-enriched fraction and the microsomal fraction from preparation A (lacking protease inhibitors) and the cytosolic fraction (containing high-molecular-weight protease inhibitors) from preparation B were used in activation assays described elsewhere.
Zymogen Activation Assays
In this study, the conversion of zymogens to mature enzymes is taken as either an increase in enzyme activity, as determined with fluorogenic assays (16, 17), or the appearance of mature enzyme forms, as determined with immunoblot analysis (15).
Enzymatic Assays
Assay buffer (350 µl) [50 mM Tris (pH 7.6) and 150 mM KCl] was added to each well of a 24-well plate (Greiner Bio-one Cellstar TC-plate; Greiner Bio-One, Monroe, NC) at room temperature (25°C). Zymogen granule-enriched or microsomal fractions (50 µl), previously stored on ice, were then added to the assay buffer in each well at room temperature. Next, 50 µl of 400 µM enzyme substrate (trypsin or chymotrypsin substrate; trypsin was MAA3135-v and chymotrypsin was MAA3114-v, from Peptides International, Louisville, KY) diluted in assay buffer at room temperature (40 µM final) were added to each well. The plate was lightly agitated from side to side to mix the contents of each well and then left at room temperature under normal lighting conditions. In preliminary studies, incubation times of 0–60 min were used to allow uptake of fluorogenic substrate by the organelles; 15 min was found to be the optimal value. For in vivo experiments, stimulated zymogen granule-enriched and microsomal fractions were assayed alone, in the absence of any cytosol or ATP, after immediate addition of substrate.
For in vitro experiments, after the 15-min incubation of substrates with organelles, 50 µl of buffer (control) or cytosol (50 µl) were added to each well and incubated for a further 15 min. In preliminary studies, these conditions resulted in a linear increase in fluorometric emission. Preliminary studies also demonstrated that the cytosol-dependent generation of protease activity was linear when 50 µl was added to the assay; this amount was used unless otherwise stated. After a total incubation of 30 min, ATP (5 mM) or AMP-PNP (5 mM) was added, and fluorescence emissions were recorded at an excitation wavelength of 380 nm and an emission of 440 nm for 11 min (20 reads) with an HTS 7000 fluorimeter (Perkin-Elmer Analytical Instruments, Shelton, CT). The fluorometric emission slope (relative fluorescence units/s) represented enzymatic activity. Organelles were lysed with Triton X-100, and amylase content was determined (Phaebadas kit; Pharmacia, Rochester, NY). The enzyme activity was normalized to amylase content (expressed as fold activation vs. the zymogen granule/microsomal fraction plus cytosol plus ATP condition). Because preliminary studies demonstrated that normalization to amylase or protein content were equivalent, data are normalized to amylase content.
Pancreatic Zymogens
For determination of total pancreatic trypsinogen and chymotrypsinogen in the zymogen granule-enriched and microsomal fractions, samples were incubated with excess enterokinase (5 µg/100 µl) in the presence of 10 mM CaCl2 at 37°C for 60 min, allowing for complete activation of trypsinogen and chymotrypsinogen in the sample (18). The samples were assayed with fluorogenic substrate for either trypsin or chymotrypsin (values for total activity determined as relative fluorescence units per second as a function of total protein content, with protein determined by the Bradford assay). To determine the percentage activation induced by cytosol and ATP in the zymogen granule-enriched and microsomal fractions, the rfu/sec values obtained in these samples were expressed as a function of total protein content and the values divided by the total activity in that fraction.
Organelle Lysis
To determine whether active enzymes from lysed organelles contributed to measured enzyme activities, the cytosolic preparation was modified. High-molecular-weight protease inhibitors (0.1 mg/ml soybean trypsin inhibitor, 5 mM aprotinin) were replaced with lower-molecular-weight inhibitors (5 mM benzamidine, 5 mM leupeptin). Before dialysis, the soybean trypsin inhibitor (0.1 mg/ml) and 5 mM aprotonin were added to one-half of the preparation. Both cytosolic preparations were dialyzed to remove low-molecular-weight inhibitors, centrifuged at 288,000 g, and used in trypsin and chymotrypsin activity assays. Hence, the cytosolic preparation with soybean trypsin inhibitor has the capacity to inhibit free protease and the other does not. Furthermore, cytosol treated with membrane-permeable complete EDTA-free protease inhibitor cocktail (20 µl/ml of 25x stock solution; Roche) was used in additional assays to demonstrate that zymogen activation was occurring within membrane-bound compartments.
Proteasomal Inhibitors
Proteasomal inhibitors lactacystin (20 µM) and MG-132 (0.75 µM) (Calbiochem) were added at the same time as substrate in enzyme assays to examine the possible involvement of the proteasome in chymotrypsin activation.
Immunoblot Assays for Zymogen Activation
Zymogen granule-enriched fraction or microsomal fraction was incubated alone, in the presence of cytosol, or in the presence of cytosol and ATP. After 1 min, 10 µl of complete EDTA-free protease inhibitor cocktail and 100 µl of 6x Laemmli sample buffer were added to each well. Because carboxypeptidase degraded rapidly in this system, procarboxypeptidase processing was assayed after 1-min incubations. Samples were heated to 95°C for 5 min. Equal protein was loaded as determined by the Bradford assay, and proteins were separated by SDS-PAGE (12.5% polyacrylamide) and processed for immunoblot analysis on polyvinylidene difluoride membranes as described (15). To detect proteolytic zymogen activation, membranes were probed with an antibody that detected the zymogen and active forms of procarboxypeptidase B (gift from G. Scheele).
Protein Kinase Inhibitors
The general protein kinase inhibitors K252a (100 nM) and H-89 (100, 30, and 1 µM) (Calbiochem) and the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM) and LY-294002 (25 µM) (Sigma) were added at the same time as substrate in enzyme assays to determine the effects of protein kinases in trypsinogen and chymotrypsinogen activation. Controls were incubated with the same concentration of drug vehicle (DMSO).
Role of Cathepsin B in Zymogen Activation
To examine the effects of cathepsin B inhibition on zymogen activation, trypsin and chymotrypsin were assayed in the presence of the cathepsin B inhibitor E64d (Sigma). E64d (1 mM) was added at time 0 (24). Control experiments used an equal volume of the vehicle (1:1 methanol-water). Effects of E64d on 100 ng of pure trypsin and chymotrypsin (Sigma) were evaluated using the same assay technique as described above.
Statistical Analysis
Statistical significance was determined using the Wilcoxon rank sum test. Significance was assumed for P < 0.05. Deviation bars in all figures represent 25th and 75th quartiles.
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RESULTS |
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Zymogen activation in particulate fractions.
Zymogen granule-enriched and microsomal fractions were harvested from rat pancreas stimulated in vivo with supraphysiological concentrations of caerulein for 15 min. This treatment caused approximately threefold and fivefold increases in trypsin activity in both the zymogen granule-enriched and microsomal fractions, respectively, when compared with unstimulated pancreas. (Fig. 1A). Similarly, chymotrypsin activity was 10- and 6-fold higher in the zymogen granule-enriched and microsomal fractions, respectively, when compared with controls from unstimulated pancreas (normalized to amylase content) (Fig. 1B). On the basis of evidence that zymogen activation might occur in distinct cellular organelles, we developed a cell-free assay system to determine whether activation could be reconstituted using isolated pancreatic fractions.
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Total zymogen and amylase content of particulate fractions. The relative trypsinogen, chymotrypsinogen, and amylase contents were similar between the zymogen granule-enriched and microsomal fractions (Fig. 2). Therefore, results were normalized to amylase content unless noted otherwise.
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Neither wortmannin nor LY-294002 inhibited cytosol-dependent or cytosol/ATP-dependent activation of chymotrypsin. Trypsinogen activation, although ATP insensitive, was studied, and no inhibition of activity was seen. Studies with K252a at 100 nM, a concentration that inhibits many kinases, showed no inhibition of activity. However, the general protein kinase inhibitor H-89 caused a concentration-dependent inhibition of cytosol/ATP-dependent chymotrypsin activity in both the zymogen granule-enriched and microsomal fractions (Fig. 6). These data indicate that ATP-dependent mechanisms in zymogen granule-enriched and microsomal fractions may be mediated by protein kinases.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To further investigate the mechanism of this activation, we developed a reconstitution system using pancreatic cytosol and isolated organelles. In this system, we used fractions from unstimulated tissue and introduced factors to determine whether they could induce activation within organelles. Although it could be argued that activating zymogens in subcellular fractions from unstimulated tissue may not fully reflect pathological enzyme activation seen in pancreatitis, the approach of using inactive proteins and adding back stimulatory factors has been used in many reconstitution systems (29–31).
With the use of this system, we have made several key observations. First, zymogen activation occurs within intact organelles and is cytosol dependent. This is supported by the finding that little activation is observed in either isolated organelles or cytosol. When isolated organelles are incubated with cytosol, a significant increase in protease activity is seen (Figs. 3 and 4). The presence of high-molecular-weight membrane-impermeable serine protease inhibitors in the cytosol ensures that the protease activities detected by the fluorogenic assays are not being generated in the cytosol but within organelles (Table 2). The low level of activity detected in cytosol is possibly due to contamination from light vesicles, as this activity was markedly diminished by very high-speed centrifugation. Furthermore, there is a lack of an effect on activity by removal of adherent proteins from membrane fractions with potassium chloride or treatment with proteasomal inhibitors. Because cytosol-dependent zymogen activation is observed with isolated organelles from unstimulated cells, it might represent basal cellular activity (as seen in whole cell studies; Refs. 16, 17), or it could result from activation of the machinery, such as protein kinases, required for zymogen activation during preparation of cytosol.
Second, cytosol-stimulated zymogen activation occurs in both zymogen granule-enriched and microsomal compartments, suggesting that more than one compartment might support zymogen activation. Although the levels of activation seen in these compartments represent a small percentage of the total zymogen content (Table 1), the values are consistent with those from studies that have used intact cells (9, 15, 24). Our data suggest a more efficient processing of zymogens in microsomes than with zymogen granule-enriched fraction and a more efficient processing of chymotrypsinogen than of trypsinogen (Table 1). The proportions of total zymogen in zymogen granule-enriched fraction are greater than those seen in the microsomal fraction (Fig. 2) and comparable to levels described in another study (18). Thus, when normalized to the distribution of active enzymes between the two compartments, the greater contribution to enzyme activity comes from the zymogen granule-enriched fraction. Whether a similar distribution is observed in intact cells remains unclear.
Studies from our laboratory and another group have both reported that initial zymogen processing is localized to a vesicular compartment containing markers of lysosomes and recycling endosomes (13, 20). In our present study, immunoblot analysis demonstrates that the lysosomal and endosomal marker GRAMP-92 is present predominantly in the zymogen granule-enriched fraction and to a lesser extent in the microsomal fraction (data not shown). Because these two have substantially different densities, it is unlikely that GRAMP-92 is associated with the same organelle in the zymogen granules and microsomes. Additional fractionation studies will be required to identify the compartments ultimately responsible for zymogen processing.
Third, on the basis of the effects of ATP and E64d in our system, the requirements for trypsinogen activation may be distinct from those necessary for activation of chymotrypsinogen and procarboxypeptidase. The ATP requirement for conversion of chymotrypsinogen and procarboxypeptidase and kinase inhibitor studies indicates a potential role for one or more protein kinases. Our data with protein kinase inhibitors indicates a lack of involvement for phosphatidylinositol 3-kinase in the cell-free system. The results with the inhibitor H-89 suggest that a protein kinase inhibited in the range from 30 to 100 µM may regulate processing. Protein kinase A and protein kinase G are inhibited at submicromolar concentrations of H-89 and are therefore unlikely candidates (11). However, other protein kinases, such as casein kinases I and II, calmodulin-dependent kinases, and protein kinase C, require inhibitory H-89 concentrations in the ranges observed in this study (11). Notably, preliminary studies suggest that H-89 also selectively inhibits chymotrypsin but not trypsin activity in acini simulated by supraphysiological concentrations of caerulein (unpublished data). Studies with selective protein kinase inhibitors and specific agonists may identify the protein kinases that mediate this activation.
The inhibitory effects of E64d on trypsinogen activation are consistent with the published effects of cathepsin B on trypsinogen activation, but its lack of effectiveness in inhibiting chymotrypsinogen or procarboxypeptidase B processing is surprising. Together with the selective effects of ATP on chymotrypsinogen or procarboxypeptidase B activation, these findings strongly suggest that the activation of some zymogens is trypsin independent. Indeed, one study even suggests that trypsin has a more relevant role in autodegradation rather than in activation of itself and other zymogens (10). However, we cannot exclude the possibility that undetectable increases in trypsin activity might be responsible for the activation of the other zymogens. Most other studies with pancreatic cathepsin B and trypsinogen activation have not examined activation of other zymogens (10, 12, 24). In this context, in cathepsin B knockout mice, despite a significant reduction in trypsinogen activation, cell injury and other measures of pancreatitis are still prominent (9). Furthermore, in transgenic mice with increased levels of pancreatic secretory trypsin inhibitor 1, active trypsin is inhibited and the severity of caerulein-induced pancreatitis is reduced but not eliminated (19). One possible explanation for the persistent injury seen in both the cathepsin B knockout and the transgenic pancreatic secretory trypsin inhibitor 1 animals is that activation of proteases other than trypsinogen may not be reduced.
In summary, this report used an in vivo model of acute pancreatitis and a reconstituted system to examine the mechanisms of zymogen activation in the pancreatic acinar cell. From these studies, we conclude that pathological zymogen activation can occur in more than one cellular organelle and requires cytosolic factors. Furthermore, these studies demonstrate that zymogen activation is complex and that distinct pathways may lead to activation of specific subsets of proteases. In this context, activation within the acinar cell may not follow the pattern that has been established for intestinal activation, with trypsinogen activation leading to proteolytic activation of other zymogens. Elucidation of the cytosolic factors and pathways that mediate zymogen activation may lead to the rational design of preventive therapies.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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regulates key pathologic responses to cholecystokinin in pancreatic acinar cells. Gastroenterology 126: 554–566, 2004.[CrossRef][Web of Science]
B and trypsinogen activation in rat pancreas after supramaximal caerulein stimulation. Biochem Biophys Res Commun 280: 388–395, 2001.[CrossRef][Web of Science][Medline]
liwiec P, Gryko M, Ostrowska H, and Baltaziak M. Chymotrypsin-like activity in rat tissues in experimental acute pancreatitis. Rocz Akad Med Bialymst 48: 61–65, 2003.[Medline]This article has been cited by other articles:
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  E. C. Thrower, S. Osgood, C. A. Shugrue, T. R. Kolodecik, A. M. Chaudhuri, J. R. Reeve Jr, S. J. Pandol, and F. S. Gorelick The novel protein kinase C isoforms -{delta} and -{varepsilon} modulate caerulein-induced zymogen activation in pancreatic acinar cells Am J Physiol Gastrointest Liver Physiol, June 1, 2008; 294(6): G1344 - G1353. [Abstract] [Full Text] [PDF]
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