Surgical Laboratories, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
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ABSTRACT |
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To investigate the debated role of intracellular
trypsinogen activation and its relation to lysosomal enzyme
redistribution in the pathogenesis of acute pancreatitis, rats were
infused with the cholecystokinin analog caerulein at 5 µg · kg1 · h
1
for intervals up to 3 h, and the changes were contrasted with those in
animals receiving saline or 0.25 µg · kg
1 · h
1
caerulein. Saline or 0.25 µg · kg
1 · h
1
caerulein did not induce significant changes. In contrast, 5 µg · kg
1 · h
1
caerulein caused significant hyperamylasemia and pancreatic edema within 30 min. Pancreatic content of trypsinogen activation peptide (TAP) increased continuously (significant within 15 min). TAP generation was predominantly located in the zymogen fraction during the
first hour but expanded to other intracellular compartments thereafter.
Cathepsin B activity in the zymogen compartment increased continuously
throughout the experiments and correlated significantly with TAP
generation in the same compartment. Total trypsinogen content increased
to 143% with marked interstitial trypsinogen accumulation after 3 h.
Supramaximal caerulein stimulation causes trypsinogen activation by 15 min that originates in the zymogen compartment and is associated with
increasing cathepsin B activity in this subcellular compartment.
However, a much larger pool of trypsinogen survives and accumulates in
the extracellular space and may become critical in the evolution of
necrotizing pancreatitis.
pancreas; protease activation; acinar cell; experimental; cathepsin B
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INTRODUCTION |
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ONE HUNDRED YEARS AGO Chiari (5) suggested that autodigestion by premature extraintestinal activation of the digestive enzyme precursors is responsible for the histopathological changes in acute pancreatitis. Only recently has proof of intrapancreatic protease activation been found in both human (12) and experimental pancreatitis of different etiologies (3, 28, 33, 34, 58). However, the events leading to this extraintestinal activation are still obscure. Multiple biochemical and morphological abnormalities have been detected within the acinar cell in models of acute pancreatitis, including alterations of enzyme sorting and compartmentation (1, 23, 42-45, 54, 55), and current investigations are focusing on possible intracellular trigger mechanisms for premature zymogen activation (1, 14, 23, 26, 28, 42-44, 54-56). Immunocytochemical studies have described subcellular colocalization of digestive enzyme precursors and lysosomal enzymes in crinophagic-autophagic vacuoles in several experimental models of acute pancreatitis (1, 45, 54, 55). Subcellular fractionation techniques demonstrated a coincident increase of lysosomal enzyme activity in the zymogen compartment, which includes crinophagic vacuoles (29, 43, 56). Because previous studies have demonstrated the capability of the lysosomal enzyme cathepsin B to activate trypsinogen in vitro (11, 15), it has been suggested that such an intracellular admixture of cathepsin B and trypsinogen may cause intracellular activation of trypsinogen (43, 49, 50, 54). Inasmuch as trypsin triggers the activation of the pancreatic enzyme cascade (40), it is speculated that cathepsin B-induced intracellular trypsinogen activation may be an early event in the pathophysiological processes leading to autodigestion of the gland (43, 50). However, evidence of trypsinogen activation in association with the subcellular changes of cathepsin B activity has never been demonstrated, partly because of the technical difficulty of reliably quantitating subcellular trypsin activity (17). Moreover, because colocalization of zymogens and lysosomal enzymes has recently been demonstrated in normal pancreatic cells (29, 45) and because the extent of this phenomenon does not seem to correlate with the severity of the disease (29), its relevance in the development of acute pancreatitis and its progression to a panglandular autodigestive disease is currently a matter of debate (13, 17, 51).
The conversion of trypsinogen to active trypsin occurs by cleavage of a small trypsinogen activation peptide (TAP), which is easily assayed and provides a quantitative index of active trypsin generation (10, 19). Using TAP as a stable, equimolar measure of trypsinogen activation in nanomolar quantities, we studied the kinetics and subcellular localization of early pancreatic trypsinogen activation, its possible association with the intracellular changes of lysosomal enzyme activity, and its relationship to intrapancreatic trypsinogen distribution in experimental pancreatitis induced by supramaximal secretagogue stimulation.
In this study of acute edematous pancreatitis induced by supramaximal caerulein stimulation, we report that there is very early ectopic activation of trypsinogen that originates in the zymogen compartment and that may be caused by the increasing cathepsin B activity in the same subcellular compartment. However, a large pool of not yet activated trypsinogen survives and accumulates in the extracellular compartment. Our observations suggest that interstitial rather than intracellular protease activation may be critical in the evolution from the mild edematous pancreatitis of this model to severe necrotizing pancreatitis.
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MATERIALS AND METHODS |
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Materials.
H-arginyl-arginine--naphthylamide was purchased from Bachem
Biosciences (Philadelphia, PA). Biotin-goat anti-rabbit
immunoglobulin G (B-9642), phosphatase-labeled extravidin (E-2636), and
p-nitrophenyl phosphate (N-9389) were
obtained from Sigma (St. Louis, MO). Rabbit serum
albumin-(Asp)4-Lys conjugate and
Ca-independent rabbit
anti-(Asp)4-Lys antiserum were
kindly provided by J. Hermon-Taylor. Caerulein was obtained from
Farmitalia.
Experimental protocol.
The study was approved by the Subcommittee on Research Animal Care of
the Massachusetts General Hospital and performed according to the
Guide for the Care and Use of Laboratory Animals,
[Department of Health and Human Services Publication No. (NIH)
85-23]. Experiments were performed on 110 male Sprague-Dawley
rats (280-400 g) obtained from Charles River Laboratories
(Wilmington, MA). Anesthesia was initiated with vaporized ether and
maintained by injections of pentobarbital (20 mg/kg iv; Anpro
Pharmaceuticals, Arcadia, CA) and ketamine (40 mg/kg im, Ketalar;
Parke-Davis, Morris Plains, NJ). On the day before the experiment, a
polyethylene catheter (Intramedic, ID 58 mm, Clay Adams, Parsippany,
NJ) was introduced into the left internal carotid artery and advanced
into the aorta for blood sampling and infusions. The catheter was
tunneled subcutaneously to the suprascapular region and brought out
through a steel tether, permitting unrestrained activity of the
conscious animal. Animals were then allowed to stabilize and fasted
overnight with water ad libitum. On the next day animals were randomly
allocated to receive either continuous infusion of 0.9% NaCl
(n = 33), the cholecystokinin (CCK)
analog caerulein at 0.25 µg · kg1 · h
1
(n = 29), which causes maximal
physiological stimulation (25, 47), or caerulein at 5 µg · kg
1 · h
1
(n = 48) for 3 h, known as
supramaximal stimulation, which causes acute edematous pancreatitis.
Serum amylase activity. Serum amylase activity was determined according to the method of Ceska et al. (4) using the Phadebas amylase test (Pharmacia Diagnostics, Uppsala, Sweden).
Wet-to-dry weight ratio. At the designated time points animals were killed by pentobarbital overdose (200 mg/kg iv), and the entire gland was removed in a standardized fashion by the same investigator, trimmed of fat and mesentery, blotted dry, and weighed. Pancreatic water content was determined by calculating the ratio of the initial weight of the pancreatic specimen (wet weight) to its weight after incubation at 210°C for 6 h (dry weight).
Subcellular fractionation.
Isolation of subcellular acinar fractions was achieved by differential
centrifugation as described by Tartakoff and Jamieson (52) and DeLisle
et al. (6) with slight modifications. Briefly, samples of resected
pancreas (0.2-1.8 g) were quickly placed in ice-cold (4°C)
homogenization buffer containing 0.3 M sucrose and 20 mM EDTA at pH 7.0 and supplemented with soybean trypsin inhibitor and aprotinin (both at
0.01%) to prevent artificial protease activation and proteolytic
consumption of native trypsinogen during the fractionation process.
After sharp fragmentation into 2- to 3-mm specimens, samples were
homogenized in ice-cold homogenization buffer (15 ml/g tissue) by four
up-and-down strokes in a Teflon glass pestle. Unbroken cells, nuclei,
and other heavy debris were removed from the homogenate by low-speed
centrifugation (150 g for 10 min,
4°C). The resulting supernatant was centrifuged at 1,300 g for 15 min (4°C), yielding a
zymogen granule-rich pellet (ZRF). Centrifugation of the decanted
supernatant at 12,000 g for 12 min
(4°C) resulted in sedimentation of a lysosome-mitochondria-rich pellet (LRF) and respective supernatant that was harvested and centrifuged at 105,000 g for 60 min
(4°C) to obtain a microsome-rich pellet (MRF) and final
supernatant. This final soluble fraction, defined as the postmicrosomal
fraction (PMF), therefore, represents a combination of cytosol,
interstitium, and intraluminal compartment. The individual pellets were
separately resuspended in 5 ml of homogenization buffer containing 2%
Triton X-100 for 30 min at 4°C, immediately frozen, and stored at
70°C for later measurements. The fractional parameters of
each respective experiment were assayed simultaneously and total values
were calculated from individual fractions.
TAP.
For assay of this equimolar measure of trypsinogen activation animals
were killed at the designated time points, the pancreas was resected,
and two tissue portions (10-240 mg) were rapidly excised.
Specimens were immersed in 0.2 M tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.3) containing 20 mM EDTA and immediately boiled
at 100°C for 15 min to denature remaining proteases and halt
further TAP generation. Each sample was then homogenized in a Brinkman
Polytron (Brinkman Instruments, Westbury, NY) for 30 s. After
subsequent centrifugation (1,500 rpm, 10 min, 4°C) the resulting
supernatants were coded and stored at 70°C until assayed.
For measurements of TAP in the isolated pancreatic fractions, samples
were obtained from each individual fraction diluted in Tris buffer
(100°C; 1:5, vol/vol) and boiled, coded, and stored as above.
Aliquots were simultaneously assayed for free TAP by competitive
enzyme-linked immunosorbent assay. Briefly, samples were
added into microtiter plates, coated for 12 h (4°C) with albumin-(Asp)4-Lys conjugate, and
incubated with rabbit
anti-(Asp)4-Lys antiserum (1:400)
at 25°C for 60 min. Plates were washed three times and then
incubated with biotin-labeled goat anti-rabbit immunoglobulin G
(1:1,000) at 25°C for 30 min. After additional washing, plates were
incubated with alkaline phosphatase-extravidin conjugate (1:750) for 30 min. With p-nitrophenyl phosphate used as a substrate, color development was followed for 30 min; absorption at 405 nm was measured in a Dynatech MR 5000 microplate reader (Dynatech Laboratories, Chantilly, VA) and compared with a standard curve. As previously described, this assay has a lower detection limit
of TAP of 5 × 10
12 M
(10, 16, 19, 35).
Pancreatic trypsinogen. For determination of subcellular changes of pancreatic trypsinogen, samples from isolated subcellular fractionations were incubated with excess enterokinase (10 U/l) in Tris-HCl containing 7.5 mM CaCl2 at pH 8.0 (25°C) for 60 min, allowing for complete activation of trypsinogen contained within the sample (35). The concentration of the released TAP was then assayed as described, and trypsinogen content was calculated by subtracting spontaneous TAP levels from the value measured after enterokinase activation.
Cathepsin B.
Fractional activity of cathepsin B was measured according to the
fluorometric technique described by McDonald and Ellis (32), using
H-arginyl-arginine--naphthylamide as substrate and defined concentrations of unconjugated fluorescent
-naphthylamine for fluorometric calibration. The enzyme was allowed to activate in 50 mM
phosphate buffer containing 10 mM dithiothreitol and 10 mM EDTA
(37°C, pH 6.5) for 5 min. After addition of the substrate, changes
of fluorescence intensity were continuously monitored in a
SPEX-Fluorolog 2 spectrofluorometer system (SPEX Industries, Edison,
NJ) using emission and excitation maxima of 410 and 335 nm,
respectively (2). Rates of hydrolysis were established by comparing the
increase of fluorescence to the standard curve relating fluorescence
intensity to
-naphthylamine concentration.
Interstitial trypsinogen/TAP ratio.
The edematous interstitial fluid that had accumulated in five animals
receiving caerulein stimulation at 5 µg · kg1 · h
1
for 3 h was collected by fine-needle aspiration (ID 0.7 mm) after microdissection of the pancreatic interstitial septae with careful sparing of the pancreatic acinar tissue. The aspirated fluid was immersed in Tris-HCl buffer as above (1:1, vol/vol), and the
interstitial trypsinogen and TAP concentration was measured as
described earlier.
Data analysis. The data are presented as means ± SE of at least four separate experiments in each group at each time point. Statistical significance was determined using Student's t-test or Bonferroni two-tailed analysis of variance. Association between variables was tested using linear regression and correlation analysis. Significance was assumed for P < 0.05. Deviation bars in the figures indicate SE values, whereas their absence indicates a SE value too small for illustration.
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RESULTS |
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Serum amylase activity. No significant changes of serum amylase activity were observed in animals receiving saline infusion (31.4 ± 4.7 U/l). A transient increase of serum amylase was observed until 30 min of lowdose caerulein infusion, but levels returned to baseline values after 3 h (35.0 ± 3.0 U/l) (Fig. 1). In comparison, significant hyperamylasemia developed within 30 min of supramaximal caerulein stimulation (122.7 ± 19.8 U/l, P < 0.05), increasing even further to 157.0 ± 21.3 U/l (P < 0.01) at the end of the observation period (Fig. 1).
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Pancreatic edema formation.
Neither saline infusion nor 0.25 µg · kg1 · h
1
caerulein caused significant pancreatic edema (wet-to-dry weight ratio;
2.58 ± 0.2 and 2.75 ± 0.2, respectively). In
contrast, pancreatic water content increased continuously in animals
receiving caerulein at 5 µg · kg
1 · h
1.
The difference became significant by 30 min (3.67 ± 0.24, P
0.001) and increased
significantly further up to 3 h (6.14 ± 0.40, P < 0.001, 30 min vs. 3 h). At this time the accumulation of interstitial fluid was clearly
visible, with up to an 8.5-fold increase of pancreatic wet-to-dry
weight ratio (Fig. 2).
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Pancreatic trypsinogen content and distribution.
At baseline 51.7 ± 1.8% of total pancreatic trypsinogen was
located in the zymogen-rich compartment. The remaining distribution was
12.1 ± 3.5% in the LRF, 3.9 ± 1.1% in the
microsomal fraction, and 28.4 ± 1.4% in the soluble fraction. This
distribution pattern confirmed the quality of the subcellular
fractionation procedure, as high levels of trypsinogen are expected to
be physiologically present in the zymogen compartment (ZRF) and lumina
of pancreatic ducts (PMF). No significant changes of pancreatic
trypsinogen status were observed in animals infused with saline or 0.25 µg · kg1 · h
1
caerulein. At 3 h of physiological caerulein stimulation, the trypsinogen distribution was 49.4 ± 7% (ZRF), 9.0 ± 3.2%
(LRF), 4.9 ± 2.4% (MRF), and 36.5 ± 5.2% (PMF), perhaps
reflecting a slight increase in intraluminal trypsinogen secondary to
stimulated exocrine secretion. Supramaximal caerulein stimulation
increased overall pancreatic trypsinogen content to 143 ± 53%
[not significant (NS)] of baseline values at 3 h. The main
increase was observed in the PMF, which contained 44.6 ± 9.5% (NS)
of the total fractional trypsinogen at 3 h (Fig.
3), whereas zymogen-rich (49.8 ± 7.9%), lysosome-rich (4.3 ± 1.4%), and microsomal
fractions (1.8 ± 0.9%) did not show a relative increase.
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Pancreatic TAP content and distribution.
Pancreatic TAP content in control animals was 279 ± 31 nmol · l1 · g
1.
Values were not significantly different after low-dose caerulein infusion (308 ± 35 nmol · l
1 · g
1).
There were no significant changes in the distribution of TAP among the
individual fractions in the control or low-dose caerulein group.
However, pancreatic TAP content in animals infused with 5 µg · kg
1 · h
1
caerulein increased to 822 ± 128 nmol · l
1 · g
1
within 15 min (P < 0.01) and to
5,081 ± 960 nmol · l
1 · g
1
at 3 h (P < 0.001) (Fig.
4). Starting from an almost equal
distribution of TAP between the isolated fractions at baseline, an
increase of TAP generation, reflecting trypsinogen activation, was
observed in all isolated fractions of animals treated with supramaximal caerulein (Fig.
5A). An
immediate increase was observed in the ZRF, which became significant
after 30 min (294 ± 61%, P < 0.05) and continued throughout the experimental period (539 ± 102%
at 3 h, P < 0.01). Although changes
of TAP levels were minor in the PMF within the first hour, a
significant increase to 395 ± 63% (P < 0.01) was observed during the
subsequent experimental period. The biphasic character of the changes
of fractional TAP distribution within the 3 h of supramaximal caerulein
stimulation became more obvious when the data were plotted for relative
changes. As Fig. 5B demonstrates,
early TAP generation predominantly occurred in the ZRF, reaching
significance after 30 min (P < 0.05)
and continuing until 1 h, when 50.6 ± 4.1% of all TAP were
localized in the ZRF (P < 0.01).
However, whereas TAP generation in the zymogen compartment continued after this early phase, TAP concentrations increased most
rapidly in the PMF between 1 (16.0 ± 3.2%) and 3 h (31.5 ± 4.0%) (P < 0.05, 1 vs. 3 h).
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Subcellular cathepsin B activity.
The pattern of subcellular distribution of cathepsin B activity at
baseline was 25 ± 3.8% (ZRF), 55 ± 2.5% (LRF), 8.5 ± 2.2% (MRF), and 12 ± 2.9% (PMF) and did not significantly change
in animals receiving infusion of 0.9% NaCl or caerulein at 0.25 µg · kg1 · h
1.
In contrast, supramaximal caerulein stimulation caused a relative increase of cathepsin B activity in the ZRF to 50 ± 5.2%
(P < 0.01) and in the PMF to 21 ± 2.3% (P < 0.05) of total
pancreatic activity, changes paralleled by a significant relative
decrease of enzyme activity to 26 ± 2.7%
(P < 0.01) in the LRF after 3 h
(Fig. 6). When the relative increase of
cathepsin B activity in the ZRF was plotted against the relative
increase of TAP concentration in the same compartment, a positive
correlation between both values was obtained
(r = 0.87, P < 0.001; Fig.
7).
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Interstitial trypsinogen-to-TAP ratio. Only minute amounts of free TAP were detected in the edema fluid, which accumulated in the pancreatic interstitium after 3 h of supramaximal caerulein stimulation (7.5 ± 2.8 nmol/l). Subsequent addition of enterokinase to the aspirated fluid resulted in a massive increase of TAP to 1,807 ± 740 nmol/l, indicating the presence of a large quantity of inert trypsinogen in the extracellular space in acute edematous pancreatitis (Fig. 8). Inasmuch as the edema fluid in these circumstances constitutes most of the tissue weight, the total interstitial trypsinogen content is very substantial.
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DISCUSSION |
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Pancreatitis induced by supramaximal exocrine stimulation with the synthetic CCK analog caerulein, first described by Lampel and Kern (25), is characterized by marked interstitial edema, leukocyte infiltration, hyperamylasemia, and severe disturbances of acinar cell morphology and the secretory process. Similar changes are seen in human pancreatitis, which in the great majority of cases presents as an edematous inflammatory process that regresses without development of fulminant necrotizing disease (8). Because of its self-limited character, caerulein-induced pancreatitis offers the attractive possibility to observe the sequence of events that may lead to and cause premature protease activation before widespread autodigestion prevents any systematic investigation.
At baseline ~80% of pancreatic trypsinogen was contained within the zymogen-rich compartment (52%) and the PMF (28%), which includes zymogens already secreted into the lumen. This physiological distribution pattern confirms the validity and quality of our subcellular fractionation procedure. The increase of overall pancreatic trypsinogen content we observed in the pancreatitis model has been previously described and likely reflects in part the caerulein-induced blockage of apical exocytosis in the face of continuing protein synthesis within the acinar cell (42, 44). Although trypsinogen levels increased also in the ZRF, the most prominent increase was observed in the soluble PMF after 3 h. Using the same fractionation technique, Saluja et al. (44) also observed a progressive accumulation of [3H]phenylalanine pulse-labeled proteins and amylase in the soluble final supernatant after 3.5 h of supramaximal caerulein infusion. Because the final supernatant fraction reflects the combined changes in the cytosol, acinar lumina, and interstitium, they posited that the observed accumulation could be located 1) in the extracellular space (interstitium or intraluminal), 2) the cytosol as a result of intracellular discharge from intact organelles, or 3) inside intact but fragile intracellular organelles that rupture during the homogenization procedure. Because the authors did not observe basolateral exocytosis by radioautography after 2.5 h of supramaximal caerulein infusion and detected pulse-labeled proteins only in zymogen granules and large cytoplasmic vacuoles, they concluded that the recovery of zymogens in the PMF would result from accumulation within fragile intraacinar organelles and seemingly excluded interstitial or cytosolic zymogen accumulation. However, a recent study from our laboratory suggested that a significant interstitial trypsinogen pool exists in caerulein pancreatitis and that this repository of trypsinogen can be activated by enterokinase to produce lethal necrohemorrhagic pancreatitis (10). Because the interstitial pool of trypsinogen may thus represent a critical component in the progression from acute edematous pancreatitis to fulminant necrotizing pancreatitis, as seen in about 15% of human cases (8), we wished to distinguish between the trypsin and trypsinogen contents of the components of the PMF, that is, the cytosolic and interstitial components. This analysis was only possible at 3 h when edema was marked enough for safe collection of interstitial fluid without acinar cell damage (as confirmed by the minute amounts of TAP in the interstitial aspirates), but this is probably the time point of greatest importance. Our findings clearly demonstrate that large amounts of native (uncleaved) trypsinogen are present in the intercellular space in acute pancreatitis at 3 h. This finding suggests that much of the observed increase of the zymogen in the PMF was caused by its interstitial accumulation.
Although this finding might seem to conflict with the cited work of Saluja et al. (44), a later study from the same group (42) demonstrated that maturation of condensing vacuoles into zymogen granules and intracellular protein transport is impaired during caerulein hyperstimulation, thereby delaying the appearance of newly synthesized, labeled zymogens in the mature zymogen granules. Their observation that newly synthesized proteins were still contained within maturing or crinophagic organelles at 3 h may explain their failure to detect labeled proteins in the interstitium after 2.5 h in the earlier study (44). In addition, previously stored unlabeled zymogens could have already accumulated in the interstitium at 2.5 h and would not be detected without detection by radioautography. By 3 h Saito et al. (42) had begun to detect labeled proteins in the interstitium.
Supramaximal stimulation of the pancreatic acinar cell with caerulein induces a block of the normal secretion of zymogens from the cell into the duct lumen but does not, as noted, prevent the ongoing synthesis of zymogens. Because blocked normotopic discharge with persisting zymogen synthesis cannot indefinitely continue without overwhelming the storage capacity of the acinar cell, abnormal export or leakage of the accumulated zymogens is predictable. Basolateral exocytosis of zymogen granules or vacuoles derived from intracellular zymogen fusion has in fact been described as early as 10-15 min after the start of caerulein hyperstimulation (1, 47) and offers a plausible mechanism for the high interstitial trypsinogen concentration found in the present study. Such interstitial discharge might also provide a morphological basis for the increase of serum amylase levels consistently observed by us and others during excessive secretagogue infusion (1, 10, 30, 42-44, 54). Demonstration of increasing serum levels of the zymogen granule protein GP2 in caerulein-induced pancreatitis further supports this interpretation (27). Alternatively, increased ductal permeability secondary to structural changes in the pancreatic duct tight junction seal (7) with consequent leakage of intraluminal content back into the interstitium could also contribute to the observed trypsinogen accumulation and may thus explain the early absence of secretory products in the luminal space (25, 47).
Although our finding of large quantities of uncleaved trypsinogen in the interstitium is potentially of great importance in the pathogenesis of necrotizing pancreatitis, this latter development occurs only in a minority of human cases and is likely to be a second-tier phenomenon. Much evidence has accumulated that ectopic intracellular trypsinogen activation plays a role in the early evolution of pancreatic cell injury. Activation of serine proteases in caerulein pancreatitis has been previously described (3, 28, 56, 58). Protease activity in these earlier studies was measured indirectly by radioimmunoassay or spectrophotometry of colorimetric or fluorometric products liberated by proteolysis. However, under assay conditions, which included delays of up to 4 h between sampling and measurements, trypsin autodegradation occurs readily (31, 41) and has in fact been observed (3). That problem may well explain the failure to detect trypsin activity after CCK stimulation in vitro by Leach et al. (26). Also, the presence of other active proteases or intrinsic protease inhibitors in pancreatic tissue samples affects the sensitivity of these assay techniques and impairs their ability to provide accurate quantitative data (20). We therefore sought an alternative trypsin-specific method for investigating the subcellular events of pancreatic trypsinogen activation in caerulein pancreatitis.
The activation of trypsinogen occurs by highly specific cleavage after the P1 lysine residue of the amino terminal (Asp)4-Lys recognition sequence of trypsinogen, with subsequent release of an activation peptide (TAP), and active conformation of trypsin (19, 20). Measurement of the small and stable activation peptide, which does not influence the activation process (22), by a well-established enzyme-linked immunosorbent assay (10) provides a reliable, equimolar index of subcellular changes of trypsinogen activation at nanomolar quantities. This method is free of the shortcomings inherent to the previously used techniques (9, 19, 33, 34).
A small amount of TAP was found in both pancreatic tissue homogenates and fractions at baseline. As in previous studies (10, 33, 34, 36), these levels remained unchanged in control and low-dose caerulein animals throughout the experimental period and may reflect the small amount of limited physiological activation that occurs in the normal pancreas (3, 14, 28, 58). A significant increase of trypsinogen activation was seen in pancreatic tissue homogenates as early as 15 min after the start of supramaximal caerulein infusion, and TAP levels increased progressively until the end of the experiments. The early and rapid increase of pancreatic TAP content supports the hypothesis that premature intrapancreatic activation of digestive enzymes may be an early or even an initiating event in the evolution of secretagogue-induced experimental pancreatitis (49, 56).
Within the first hour TAP generation was largely confined to the ZRF, reaching significance at 30 min. These results are supported by the study of Bialek et al. (3), who also detected an increase of serine protease activity in their subcellular fraction of secretory granules and vacuoles in caerulein pancreatitis. Although TAP generation in the ZRF continued until the end of the experiments, a proportionally greater increase of TAP was observed in the soluble PMF between 1 and 3 h. As the analysis of the interstitial aspirates at 3 h shows, this fractional TAP increase reflects an intracellular process because TAP levels in the interstitial component of the PMF were negligible. Thus TAP could be either located in the cytosol after discharge or spillage from degrading organelles or still contained inside intracellular organelles that become increasingly fragile and rupture during the fractionation process. Diffuse staining of the cytoplasm with antizymogen immunofluorescence during exocrine hyperstimulation (54) favors the first option, whereas the latter possibility is supported by the finding of increasing membrane fragility of crinophagic organelles with their rapid disruption during homogenization in caerulein pancreatitis (44). The present study cannot answer this question. However, the protection afforded by supplemental protease inhibitors, which reduce hyperamylasemia, edema, and the changes of intracellular organelle integrity, lysosomal enzyme distribution, and the secretory pathway (18, 37, 38, 57), supports the likelihood of free cytosolic proteases. These are known to promote cytoskeletal disassembly and cellular dysfunction by proteolytic depolymerization of intracellular microtubules and microfilaments (21, 53).
The question remains as to how the activation of trypsinogen is
initiated. Previous investigators have speculated that intracellular colocalization of lysosomal enzymes and trypsinogen may be the initiating event because previous studies have demonstrated the capability of cathepsin B to activate trypsinogen in vitro (11, 15). In
accordance with other reports, no significant changes of enzyme
distribution were induced by infusion of saline or 0.25 µg · kg1 · h
1
caerulein (43), but during caerulein hyperstimulation there was a
continuous increase of cathepsin B activity in the ZRF and PMF in
parallel with a relative decrease of its activity in the LRF.
Comparable distribution changes have been reported by others (24, 43).
Although the hypothesis that exposure of trypsinogen to cathepsin B is responsible for activation of the former logically should require that increased cathepsin B activity be demonstrable before or at least coincident with TAP generation, that temporal relationship is not delineated precisely in the present experiments. Both cathepsin B and TAP were increased in the ZRF at 30 min, although the change in cathepsin B did not achieve statistical significance until the following determination. There was a high baseline activity of cathepsin B (25%) in the ZRF, which is the product both of the well-recognized limitation of the ultracentrifugation technique for separating "heavy" lysosomes from zymogen granules (18, 24, 43, 45, 52) and of the physiological colocalization of zymogens and lysosomal enzymes (56). Whatever the explanation of the high baseline cathepsin B, it did not cause trypsinogen activation or TAP generation in either the saline or physiological caerulein controls. The data, supported by the correlation depicted in Fig. 7, show that only the further increase above baseline is associated with trypsinogen cleavage in the zymogen granules, although the high baseline limits the sensitivity of the method for detecting initial small increments in cathepsin B activity that might be meaningful.
The finding that preincubation of isolated acinar cells with the cysteine protease inhibitor E-64 did not prevent CCK-induced activation of procarboxypeptidase has previously raised doubts that cathepsin B is the agent of protease activation (26). However, a recent in vitro study has demonstrated that E-64 achieves only incomplete inhibition of cathepsin B. Complete inhibition by the more potent agent E-64d prevented protease activation induced by caerulein hyperstimulation of pancreatic acinar cells (46).
Despite the intracellular trypsinogen activation documented in this study, pancreatic autodigestion did not occur in caerulein pancreatitis by 3 h. Because the observation period is relatively short, one could conclude that at this early point the activation process is still contained within the acinar cell (49). However, in caerulein pancreatitis no progression to necrotizing disease occurs even later. In fact, edema is completely reabsorbed, and only minimal single-cell necrosis develops (25). It therefore appears that intracellular trypsinogen activation in this model does not reach the level that will cause panglandular autodigestion. Thus, even if there is a true causal relationship between cathepsin B redistribution and trypsinogen activation, the phenomenon is not powerful enough to cause fulminant necrotizing pancreatitis, as previously proposed (51). Our interpretation is also consistent with the lack of correlation between the extent of cathepsin B redistribution and disease severity (29). The observation that ischemia superimposed on hyperstimulation causes progression to severe autodigestion (39), whereas ischemia alone does not cause such extensive damage (33), may suggest that intracellular derangements such as those induced by caerulein hyperstimulation sensitize and predispose the gland to necrosis. In this setting the accumulation of trypsinogen in the interstitium may assume critical pathophysiological significance because its activation has been shown to result in the development of massive pancreatic necrosis (10), but the trigger for the activation of the interstitial pool of trypsinogen remains unknown. If the second injury fails to occur within a limited time frame, the interstitial zymogens are presumably washed out by the increased pancreatic capillary perfusion in edematous pancreatitis (48).
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical assistance of Uma Mandavilli. The kind provision of rabbit serum albumin-(Asp)4-Lys conjugate and anti-(Asp)4-Lys antiserum by John Hermon-Taylor (London, UK) is greatly appreciated. Caerulein was a generous gift from Farmitalia, Carlo Erba (Freiburg, Germany).
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FOOTNOTES |
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K. Mithöfer was supported by Deutsche Forschungsgemeinschaft Grant Mi 450/1-2.
Address for reprint requests: A. L. Warshaw, Dept. of Surgery, Massachusetts General Hospital, 15 Parkman St., WAC 336, Boston, MA 02114.
Received 2 June 1997; accepted in final form 1 October 1997.
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