1 Department of Surgery, Beth
Israel Deaconess Medical Center and Harvard Medical School, and
Harvard Digestive Diseases Center, Boston, Massachusetts 02215;
2 Department of Medicine B, Supramaximal
stimulation of the pancreas with the CCK analog caerulein causes acute
edematous pancreatitis. In this model, active trypsin can
be detected in the pancreas shortly after the start of supramaximal
stimulation. Incubation of pancreatic acini in vitro with a
supramaximally stimulating caerulein concentration also results in
rapid activation of trypsinogen. In the current study, we have used the
techniques of subcellular fractionation and both light and electron
microscopy immunolocalization to identify the site of trypsinogen
activation and the subsequent fate of trypsin during caerulein-induced
pancreatitis. We report that trypsin activity and
trypsinogen-activation peptide (TAP), which is released on activation
of trypsinogen, are first detectable in a heavy subcellular fraction.
This fraction is enriched in digestive enzyme zymogens and lysosomal
hydrolases. Subsequent to trypsinogen activation, both trypsin activity
and TAP move to a soluble compartment. Immunolocalization studies
indicate that trypsinogen activation occurs in cytoplasmic vacuoles
that contain the lysosomal hydrolase cathepsin B. These observations suggest that, during the early stages of pancreatitis, trypsinogen is
activated in subcellular organelles containing colocalized digestive
enzyme zymogens and lysosomal hydrolases and that, subsequent to its
activation, trypsin is released into the cytosol.
pancreas; immunolocalization; trypsinogen activation peptide; trypsin; subcellular fractionation; cathepsin B
ACUTE PANCREATITIS is generally believed to be an
autodigestive disease in which the pancreas is injured by enzymes that
are normally secreted by acinar cells. Under physiological conditions, most of these potentially harmful digestive enzymes are synthesized and
secreted by acinar cells as inactive proenzymes or zymogens with
activation occurring in the duodenum. There, the brush-border enzyme
enteropeptidase cleaves the
NH2-terminal
trypsinogen-activation peptide (TAP) from trypsinogen, releasing active
trypsin that can then catalyze the activation of the other zymogens. In
contrast, during the early stages of pancreatitis, digestive enzyme
zymogens, including trypsinogen, are believed to be prematurely
activated in the pancreas (1, 7, 17, 24, 25, 30, 35).
The location in the pancreas of premature trypsinogen activation and
the fate of activated trypsin during the early stages of pancreatitis
are issues of considerable importance and interest, but a clear
understanding of these phenomena has not yet been reached. Studies
exploring these issues in clinical pancreatitis have not been possible,
because pancreatic tissue is generally not available for examination
during the early stages of pancreatitis in humans. To overcome this
limitation, investigators have employed a number of experimental models
of pancreatitis, which permit examination of pancreas samples during
the very early stages of pancreatitis.
Secretagogue (caerulein)-induced pancreatitis is, perhaps, the most
frequently employed model of experimental pancreatitis (6). Caerulein
is an analog of the pancreatic secretagogue CCK. Rats infused with a
dose of caerulein exceeding that required for maximal digestive enzyme
secretion develop acute edematous pancreatitis (16). In time-dependence
studies performed by our laboratory (7), we have recently noted that
activated trypsinogen and elevated TAP levels can be detected in the
pancreas within 15-30 min of supramaximal secretagogue
stimulation. We have suggested (32) that the intrapancreatic activation
of trypsinogen is catalyzed by the lysosomal hydrolase cathepsin B,
which, during the early stages of secretagogue-induced pancreatitis, as
well as in other models of pancreatitis, is colocalized with digestive
enzyme zymogens.
In the present study, we report our findings using the model of
secretagogue-induced pancreatitis to identify the intrapancreatic site
of trypsinogen activation and the fate of activated trypsin. To address
these questions, we have used the techniques of subcellular fractionation and both light and electron microscopy
immunolocalization. Our studies were performed under both in vivo and
in vitro conditions. We report that, in response to supramaximal
secretagogue stimulation, trypsinogen is activated in a particulate
intracellular compartment that is, most likely, composed of cytoplasmic
vacuoles. Subsequent to its activation, trypsin moves from this
compartment to a soluble intracellular compartment that, we suggest, is
the acinar cell cytosol.
Male Wistar rats (125-200 g), obtained from Charles River
Laboratories (Wilmington, MA), were housed in temperature-controlled (23 ± 2°C) rooms with a 12:12-h light-dark cycle. They were fed standard laboratory chow and given water ad libitum. Caerulein was
purchased from Research Plus (Bayonne, NJ), pentobarbital from
Veterinary Laboratories (Lenexa, KS), V/3 tubing from Biolab Products
(Lake Havasu, AZ), collagenase from Worthington Biochemical (Freehold,
NJ), trypsin substrate
(t-butyloxycarbonyl-L-glutaminyl-L-alanyl-L-arginine 4-methyl-coumaryl-7-amide) from Peptides International (Louisville, KY), Superfrost/Plus slides from Fisher Scientific (Pittsburgh, PA),
Vectashield mounting medium, fluorescein anti-rabbit IgG, and Texas red
anti-sheep IgG from Vector Laboratories (Burlingame, CA), and sheep
anti-human cathepsin B from ICN Pharmaceuticals (Costa Mesa, CA). All
other chemicals and reagents were purchased from Sigma Chemical (St.
Louis, MO). All experimental protocols were approved by the
Institutional Animal Use Committee of the Beth Israel Deaconess Medical
Center.
Animal and tissue preparation.
For in vivo studies, animals were prepared and pancreatic tissue was
processed as described previously (7). Similarly, for in vitro studies
pancreatic acini were freshly prepared, using collagenase digestion
according to the method of Powers et al. (23). The acini were incubated
in 10 mM HEPES buffer (pH 7.4) containing 0.1 µM caerulein for
15-120 min. They were separated from the extracellular fluid by
centrifugation (60 g for 30 s), and
the pellet, containing the acini, was used for subcellular fractionation (see below).
Subcellular fractionation.
For subcellular fractionation of in vivo-derived samples taken from
whole pancreas, portions of the pancreas were minced in ice-cold 5 mM
MOPS buffer (pH 6.5) containing 250 mM sucrose and 1 mM
MgSO4. The minced pancreas was
homogenized with a motorized glass-Teflon grinder (0.10- to 0.15-mm
clearance) using three up and down strokes at a motor speed of 3,000 rpm. For subcellular fractionation of freshly prepared
acini studied in vitro, the acini were washed twice by repeated
centrifugation and resuspension in the same MOPS buffer and homogenized
using the same tissue grinder but with seven up and down strokes. In
both cases, a sample of the homogenate was used to measure DNA content.
The remaining portion was subjected to low-speed centrifugation (150 g for 10 min) to remove nuclei and
unbroken cells. The resulting supernatant (postnuclear supernatant) was
used for subcellular fractionation, according to the method of
Tartakoff and Jamieson (33) as slightly modified by Saluja et al. (28).
Three subcellular fractions were obtained as follows: a zymogen
granule-enriched fraction (1,300 g for
15 min pellet), a lysosome/mitochondria-enriched fraction (12,000 g for 13 min pellet), and a
microsome/cytosol-soluble fraction (12,000 g for 13 min supernatant). The
identity of these fractions was confirmed by assay of marker enzymes as
previously described (28). In preliminary experiments, the possibility that either trypsin or TAP was located within microsomes as opposed to
being in the nonsedimentable soluble portion of the 12,000 g supernatant fraction was evaluated
by subjecting the 12,000 g supernatant
to high-speed centrifugation (105,000 g for 60 min). All of the active
trypsin and TAP was subsequently recovered in the resulting
supernatant, indicating that they are soluble and not contained in
microsomes.
Electron microscopy immunolocalization of TAP.
For these studies, pancreatitis was induced by infusion of a
supramaximally stimulating caerulein dose as described above for 30 min
or 3.5 h. Control animals received an equal amount of saline. Animals
were killed at the respective time intervals, the pancreas was quickly
removed, and 2-mm tissue blocks from the head, body, and tail of the
organ were fixed by incubation in PBS (pH 7.4, at 4°C) containing
formaldehyde (2%) and glutaraldehyde (0.01%) for 60 min. For
preparation of ultrathin cryosections, the blocks were immersed in PBS,
infiltrated with increasing concentrations of sucrose (up to 2.3 M) at
pH 7.4, and frozen in liquid nitrogen. Sections were prepared as
previously described (11). In brief, after blocking with horse serum
and BSA, we incubated thin sections for 60 min with primary antibody
(affinity-purified rabbit anti-TAP-antibody, a kind gift from D. McNally, Abbott Laboratories). These antibodies were raised against
TAP. They do not bind to either the zymogen (trypsinogen) or the active
enzyme (trypsin) (7, 10). They were diluted (1:100) in PBS (containing
0.5% BSA) at 4°C. After repeated rinsing in PBS, incubation with
goat-anti-rabbit IgG conjugated with 15-nm gold particles (Amersham,
Buckinghamshire, UK) followed. Sections were finally stained with
0.05% uranylacetate and embedded in 2% methylcellulose before viewing
on a Zeiss 109 electron microscope. For controls, pancreatic tissue
from animals infused with saline instead of caerulein was used. A
second control consisted of cryosections incubated without primary TAP
antibody but under otherwise identical conditions. A third control
consisted of cryosections incubated with TAP antibody that had been
kept overnight at 4°C with a 50-fold excess of antigen (TAP
peptide). Micrographs were taken at calibrated magnifications of
×20,000, and prints were coded for morphometry. To determine the
relative concentration of TAP label in the different subcellular
compartments, 100 coded prints from each experiment were
morphometrically evaluated in a blinded fashion (20) by placing a
graticule with random fields over the areas occupied by cytoplasmic
vacuoles, zymogen granules, cytosol (including endoplasmic reticulum),
and the nucleus (as an internal control). The relative concentration of
TAP label was expressed as the number of 15-nm gold grains/0.04
µm2.
Light microscopy immunolocalization of TAP and cathepsin B.
For these studies, pancreatitis was induced by infusion of a
supramaximally stimulating dose of caerulein, as described above, for
30 min. Pancreata were fixed in neutral-buffered Formalin for 12 h and
after processing were embedded in paraffin wax. Immunocytochemical localization of cathepsin B and TAP, at the light microscopic level,
was carried out on consecutive sections. Sections 4 µm thick were cut
and mounted on Superfrost/Plus slides. They were deparaffinized by
passing them through two changes of xylene and a graded series of
alcohol followed by rinses in tap water and PBS (0.01 M phosphate
buffer, pH 7.4, 0.154 M NaCl), respectively. Nonspecific binding was
blocked by incubating sections in 1% BSA in PBS for 30 min. Sections
were then incubated for 1 h in the two primary antibodies applied
together (anti-TAP, 1:120 or anti-cathepsin B, 1:100), diluted in
BSA-PBS. Control sections were incubated in BSA-PBS. After rinsing in
PBS, the sections were incubated for 30 min with the corresponding
fluorescent secondary antibodies, i.e., FITC-conjugated anti-rabbit IgG
and Texas red anti-sheep IgG. After three rinses in PBS, the sections
were mounted in Vectashield mounting medium.
Assays.
Amylase activity was determined according to the method of Pierre et
al. (22), cathepsin B by the method of McDonald and Ellis (21) with
modifications (27), and trypsin by the method of Kawabata et al. (12).
In the trypsin assay, the slope of rising fluorescence emission was
calculated as arbitrary units and expressed per milligram of DNA in the
homogenate of that particular tissue or acini sample. To allow for
pooling of data from different experiments, we expressed the trypsin
activity in each fraction as a percentage of the total trypsin activity
determined by combining the activity found in all of the fractions. We
measured DNA, using Hoechst dye 33258, according to the method of
Labarca and Paigen (15), with calf thymus DNA as the standard. TAP
levels were determined using a direct competitive ELISA according to
the method of Gudgeon et al. (9).
Analysis of data.
Results represent means ± SE obtained from multiple determinations
in three or more separate experiments. In all figures, vertical bars
denote SE and the absence of such bars indicates that the SE is too
small to depict. The significance of changes was evaluated using
Student's t-test when the data
consisted of only two groups or by ANOVA when comparing three or more
groups. If ANOVA indicated a significant difference, the data were
analyzed using Tukey's method as a post hoc test for the difference
between groups. P Subcellular distribution of trypsin activity.
We have previously noted that supramaximal stimulation with caerulein
for 15-30 min causes trypsinogen activation in the pancreas, but
that in the absence of supramaximal stimulation trypsin activity cannot
be detected in the gland (7). A small portion (20-30% of total)
of the trypsin activity detected after 30 min of supramaximal stimulation is recovered in the lysosome/mitochondria-enriched fraction, and this component does not change with continued
supramaximal stimulation for 210 min (data not shown). On the other
hand, the major portion (60-65%) of the trypsin activity detected
after 30 min of supramaximal stimulation is recovered from the heavier zymogen granule-enriched (1,300 g
pellet) fraction (Fig. 1). With further
supramaximal stimulation for up to 210 min, trypsin activity gradually
falls in the 1,300 g pellet and,
during the same period, trypsin activity in the soluble (12,000 g supernatant) fraction rises (Fig.
1).
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
0.05 was
considered to indicate a significant difference.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
In vivo and in vitro subcellular distribution of trypsin activity. For
in vivo studies, rats were infused with caerulein (10 µg · kg 1 · h
1)
for up to 210 min. Subcellular fractions were prepared by differential
centrifugation, and trypsin activity was measured in each fraction
(A and
B) as described in the text.
A: 1,300 g pellet.
B: 12,000 g supernatant. For in vitro studies,
pancreatic acini were prepared by collagenase digestion and stimulated
with 0.1 µM caerulein for up to 60 min. Acini were homogenized, and
subcellular fractions were prepared as described in the text. Trypsin
activity was measured in each fraction
(C and
D).
C: 1,300 g pellet.
D: 12,000 g supernatant. Trypsin activity in
each fraction was calculated as % of that noted in the postnuclear
supernatant. Results are means ± SE obtained from 4 different
experiments. * P
0.05 compared with 30-min values.
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Subcellular distribution of cathepsin B. We have previously noted that under in vivo conditions supramaximal secretagogue stimulation changes the sedimentation characteristics of lysosomal hydrolases such as cathepsin B and, as a result, a greater portion is recovered in the zymogen granule-enriched fraction and a lesser portion is recovered in the lysosome/mitochondria-enriched fraction (27). This shift in cathepsin B distribution can be detected within 15-30 min of the start of supramaximal stimulation and is believed to be accounted for by the formation of cytoplasmic vacuoles that contain colocalized digestive zymogens and lysosomal hydrolases (27, 29). We now report that a similar change in the subcellular distribution of cathepsin B is noted when freshly prepared acini are incubated in vitro with a supramaximally stimulating concentration of caerulein. The cathepsin B activity in the 12,000 g pellet falls and the activity in the 1,300 g pellet rises. As a result, the ratio of cathepsin B in the 1,300 g pellet to that in the 12,000 g pellet more than doubles (Fig. 3). This subcellular redistribution of cathepsin B is detected within 15 min of the start of in vitro supramaximal stimulation (Fig. 3).
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Subcellular distribution of TAP. TAP is the NH2-terminal peptide of trypsinogen that is cleaved from the zymogen during activation (9). Using the techniques of subcellular fractionation and light and electron microscopy immunolocalization, we have evaluated the distribution of TAP in the pancreas after in vivo supramaximal stimulation. In control samples, a small amount of TAP can be detected in the absence of supramaximal secretagogue stimulation, but this level of TAP increases markedly within 30 min of supramaximal stimulation (7). As shown in Fig. 4, roughly 50% of the TAP can be recovered and measured, by ELISA, in the zymogen granule-enriched 1,300 g pellet within 15 min of supramaximal stimulation. With more prolonged supramaximal stimulation, TAP levels in this heavy subcellular fraction fall. Roughly 35% of the TAP can also be detected in the soluble 12,000 g supernatant within 15 min of supramaximal stimulation. However, with more prolonged stimulation, this value gradually increases and by 210 min nearly all of the TAP measured by ELISA is in the soluble compartment (Fig. 4).
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Immunolocalization of TAP at electron microscopic level. Immunogold labeling of pancreatic cryosections from control animals with anti-TAP antibody results in only a weak background gold decoration (Fig. 5A). This background label does not appear to indicate that TAP is present in acinar cells under physiological conditions, because it is also found on sections that are incubated without primary antibody (Fig. 5B). When the primary antibody is preincubated with a 50-fold excess of antigen (TAP peptide), no specific gold decoration beyond background levels results, regardless of whether or not the cryosections are from caerulein-infused or control animals. Even the cytoplasmic vacuoles that are characteristic of experimental pancreatitis remain negative under these conditions (Fig. 5C), but they are strongly TAP positive (Fig. 5D, inset) when the primary antibody is not preincubated with antigen. After 30 min of supramaximal secretagogue stimulation, these vacuoles represent the principal TAP-containing compartment and neither zymogen granules nor the acinar lumen (Fig. 5D) is found to be significantly labeled with anti-TAP antibodies. Even the cytosol in the immediate vicinity of TAP-positive cytoplasmic vacuoles (Fig. 6A) and the adjacent endoplasmic reticulum (Fig. 6B) contain only background gold decoration at 30 min of supramaximal stimulation. After 3.5 h of caerulein infusion, the TAP-specific gold label becomes much less distinct. The surrounding cytoplasm near the cytoplasmic vacuoles becomes strongly TAP positive (Fig. 6C) and only the nucleus, mitochondria, and zymogen granules (Fig. 6C, inset) remain without gold decoration beyond background levels. At this time, the endoplasmic reticulum appears progressively dilated (Fig. 6D) and its TAP label is comparable to that of other cytoplasmic structures.
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Light microscopy immunolocalization of TAP and cathepsin B. To determine whether the vacuoles that contain TAP shortly after the onset of supramaximal stimulation also contain the lysosomal hydrolase cathepsin B, we performed immunolocalization studies at the light microscopic level of resolution using antibody directed against either TAP or cathepsin B. As shown in Fig. 8, both TAP (Fig. 8A) and cathepsin B (Fig. 8B) are localized to cytoplasmic vacuoles. Figure 8C, which is the merged image obtained when both anti-TAP and anti-cathepsin B antibodies are used, indicates that both enzymes are localized in the same vacuoles.
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DISCUSSION |
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The pathogenesis of acute pancreatitis has been the recent subject of intensive investigation and considerable controversy. Although most investigators agree that the disease is initiated by digestive enzyme activation, the location at which activation occurs and the fate of the enzymes, once activated, have not been unequivocably established. Some have suggested that enzyme activation occurs at an extracellular site in the gland parenchyma (2) and that pancreatic injury begins in the periductal or perilobular areas (13, 25). We (32), on the other hand, have argued that digestive enzyme activation occurs in acinar cells as a result of the colocalization of digestive enzyme zymogens with lysosomal hydrolases. Evidence supporting this so-called "colocalization hypothesis" includes the following observations: 1) the earliest morphological changes in acute biliary pancreatitis are seen in acinar cells (18); 2) digestive enzyme zymogens and lysosomal hydrolases are colocalized in cytoplasmic vacuoles that appear, in several experimental models of pancreatitis, before other evidence of cell injury can be detected (14, 26, 27, 29); 3) under appropriate in vitro conditions, the lysosomal hydrolase cathepsin B can activate trypsinogen (3, 8, 19); 4) activated digestive zymogens, including trypsin, can be detected in the pancreas in clinical pancreatitis (5) and, in several models of experimental pancreatitis, activated trypsin can be detected before other evidence of cell injury (7, 24); 5) hereditary pancreatitis results from mutation of the trypsinogen gene with expression of trypsinogen that, once activated, is resistant to inactivation (34); and 6) supramaximal secretagogue stimulation, which is known to induce acute pancreatitis in animals, can cause activation of trypsinogen in isolated pancreatic acini studied in vitro and this activation can be prevented by the cell-permeant, specific cathepsin B inhibitor E64D (30).
The present study was undertaken to elucidate 1) the location of trypsinogen activation in acinar cells during the early stages of pancreatitis and 2) the fate of trypsin after its activation. We have chosen to explore these issues using the secretagogue model of pancreatitis, because it is a model that evolves in a rapid as well as highly reproducible fashion and because we have recently noted (7) that activation of trypsinogen along with release of TAP can be detected in the pancreas as early as 15-30 min after the start of supramaximal stimulation. Bialek et al. (1) have already shown, using enzyme-blotting techniques, that activated zymogens can be found in an acinar cell particulate fraction 2 h after the onset of supramaximal secretagogue stimulation. We reasoned that measurement of trypsin activity in subcellular fractions prepared at varying but earlier times after the start of supramaximal stimulation would allow us to define the subcellular compartment in which activation occurs and to track the fate of trypsin after it becomes activated. Furthermore, we reasoned that immunolocalization of the activation peptide released during trypsinogen activation (i.e., TAP) would enhance our ability to define the location of trypsin activation. In designing these studies, we recognized the importance of making determinations at the earliest times possible after enzyme activation, because intracellular transport or organelle disruption could result in altered localization of both trypsin and TAP at later times.
As shown in Fig. 1, the trypsin activity detected in the pancreas after in vivo supramaximal stimulation and in acinar cells after in vitro supramaximal stimulation is first seen in a heavy subcellular fraction that sediments at 1,300 g. This fraction is normally enriched in zymogen granules and, after either in vivo or in vitro supramaximal stimulation, it is the fraction that is also enriched in the lysosomal hydrolase cathepsin B (32) (Fig. 3). This 1,300 g pellet is also the fraction that contains most of the measurable TAP after 30 min of supramaximal stimulation (Fig. 4). With prolonged supramaximal stimulation, both trypsin activity and measurable TAP decrease in this heavy subcellular fraction and a corresponding increase in both trypsin and TAP is noted in the soluble compartment (Figs. 1 and 4). With in vivo supramaximal stimulation, this shift in trypsin activity from the particulate to the soluble fraction occurs after 60 min, whereas it is first seen after 30 min of supramaximal stimulation in vitro (Fig. 1). This difference may reflect the influence of studying the same phenomenon under different experimental conditions. The value of the soluble compartment may also differ between the in vivo and in vitro studies. In samples prepared from portions of whole pancreas (i.e., in vivo experiments), this soluble compartment would be expected to include both cytosol and extracellular interstitial edema fluid, but in samples prepared from suspended acini, the soluble compartment should include only the cytosol.
Taken together, these results suggest that trypsinogen activation during supramaximal secretagogue stimulation occurs in a particulate compartment that includes zymogen granules, and, during pancreatitis, also contains lysosomal hydrolases. Furthermore, these results suggest that, once activated, trypsin moves from this particulate location to the acinar cell cytosol.
In general, results of studies performed using the technique of subcellular fractionation must be interpreted with caution because of the possibility that enzyme redistribution during or after the time of tissue processing may alter the sedimentation characteristics of many components (31). Furthermore, subcellular fractionation experiments do not permit discrimination between different organelles that have similar sedimentation characteristics. Thus, using only subcellular fractionation, we would have been unable to establish whether trypsin is activated within zymogen granules, lysosomes, or cytoplasmic vacuoles, which cosediment along with the zymogen granules after supramaximal secretagogue stimulation.
We have used the techniques of light and electron microscopy immunolocalization to resolve these areas of uncertainty. At present, probes for pancreatic proteolytic activity at the ultrastructural level are not available and antibodies to digestive enzymes would not discriminate between the zymogens and their activated enzymes. For this reason, we have chosen to use purified antibodies raised against TAP. Those antibodies are specific for TAP and have little or no cross reactivity with either trypsin or trypsinogen. It could be anticipated, therefore, that the site of highest TAP content at early times would correspond to the initial site of trypsinogen activation.1
Our initial studies were performed using the technique of electron microscopy immunogold localization and anti-TAP antibodies. When we labeled the pancreas of saline-infused control animals for TAP, we found a sparse but even distribution of gold grains over the entire acinar cell. Although this could indicate that a small amount of trypsinogen activation takes place under physiological conditions, we reject this interpretation for two reasons. First, we would have expected to see a greater concentration of the TAP-specific label in compartments that are known to contain large amounts of trypsinogen (e.g., endoplasmic reticulum and zymogen granules), but these compartments were not labeled at a greater density than, for example, the nucleus. The second reason for rejecting this interpretation is the fact that the same background gold decoration was also found when no primary TAP antibody was used. Thus we conclude that trypsinogen activation either does not occur under physiological conditions or, alternatively, that it occurs at a level impossible to detect by immunogold labeling.
As early as 30 min after the start of caerulein infusion, the distribution of TAP-specific gold label changes remarkably. At that time cytoplasmic vacuoles have formed and these pancreatitis-associated structures represent the principal and exclusive compartment in which significant concentrations of gold label are consistently found. Although occasional gold label is observed in other areas of the cell (zymogen granules, cytosol, nucleus), the density of that label is not significantly above the background level. This observation strongly suggests that the initial conversion of trypsinogen to active trypsin occurs in the cytoplasmic vacuoles. Furthermore, our findings all but exclude some other potential or additional sites of premature protease activation, such as zymogen granules, the acinar or ductal lumena, or the interstitial space between acinar cells. After 3.5 h of caerulein infusion, the density of gold label associated with cytoplasmic vacuoles has still further increased, but the vacuoles no longer represent the exclusive site of TAP localization. Large areas of the acinar cell cytosol, including the endoplasmic reticulum, are now found to be gold labeled, but this redistribution of TAP still excludes zymogen granules and nuclei. From these data we conclude that, very early after supramaximal stimulation, a premature and intrapancreatic activation of proteases occurs and this event is initially confined to the cytoplasmic vacuoles in acinar cells. Later, TAP can be found over large areas of the cytosol in addition to the vacuoles. This subsequent redistribution of TAP could, theoretically, be explained in a number of ways. The explanation we consider most likely is that the cytoplasmic vacuoles, which are known to be unstable cellular organelles in vitro (27, 29), disintegrate in vivo and their content, which includes TAP and by inference active trypsin, is released into the cellular cytosol. An alternative explanation for our observed redistribution of TAP would be the passage of this small peptide, as opposed to the much larger trypsin, through the membrane of intact cytoplasmic vacuoles and, thus, into the cytosol. Unfortunately, probes that discriminate between activated digestive enzymes and their inactive zymogens are not currently available and, for this reason, we cannot unequivocably conclude that trypsin is released into the cytoplasm along with TAP.
The final issue we have addressed is the mechanism by which trypsinogen becomes activated during the early stages of pancreatitis. In previous reports (27), we have suggested that trypsinogen may be activated by the lysosomal hydrolase cathepsin B during pancreatitis. Indeed, cosedimentation of TAP, active trypsin, and cathepsin B in the same subcellular fraction during the early stages of caerulein-induced pancreatitis and after in vitro supramaximal stimulation with caerulein (Figs. 1, 3, and 4; Ref. 27) is compatible with this conclusion. To further evaluate this issue, we have performed light microscopy studies to immunolocalize cathepsin B and TAP 30 min after the start of in vivo supramaximal stimulation with caerulein. As shown in Fig. 8, cytoplasmic vacuoles located primarily on the luminal side of the nucleus are apparent under these conditions and these vacuoles contain both cathepsin B and TAP. Based on their location, one might conclude that these vacuoles are derived from the Golgi apparatus, but further studies will be needed to more clearly define their genesis. The colocalization of cathepsin B and TAP within these vacuoles is consistent with the conclusion that cathepsin B catalyzes trypsinogen activation in these organelles.
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In summary, our findings lead us to conclude that intrapancreatic activation of trypsinogen occurs at a very early stage in the evolution of secretagogue-induced pancreatitis and that that activation occurs in cytosolic vacuoles containing both digestive enzymes and the lysosomal hydrolase cathepsin B. On the basis of these and previously reported observations (30), we conclude that intra-acinar cell activation of trypsinogen in the cytoplasmic vacuoles is catalyzed by cathepsin B. At later times, trypsin appears to be released from the cytoplasmic vacuoles into the cytoplasm. We suggest that the presence of activated trypsin in the cytoplasmic space is a critical event that eventually leads to cell injury, but further studies evaluating the relationship between cytoplasmic trypsin and cell injury will be needed to unequivocally establish this relationship. In the present study, we found that the increase in soluble (i.e., cytoplasmic) trypsin activity and TAP levels was not apparent before 60-120 min of caerulein infusion, yet, in earlier studies (6), we found that hyperamylasemia and pancreatic edema were present within 30-60 min of supramaximal stimulation. Although these time-dependent differences may merely reflect the inherent technical difficulty encountered in monitoring small but potentially critical changes in trypsin or TAP distribution, these differences may also indicate that other, as yet unidentified, phenomena contribute to cell injury in this model.
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ACKNOWLEDGEMENTS |
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We thank Dan Brown, E. Wolff-Hieber for expert technical assistance with the immunolocalization experiments, and L. Edelmann and E. Morgenstern for electron microscopy support.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-31396, Deutsche Forschungsgemeinschaft Grant Le 325/4-1, and by Boehringer Ingelheim.
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. §1734 solely to indicate this fact.
1 It should be noted that morphometric studies quantitating immunogold anti-TAP labeling allow one to define the concentration of TAP in specified compartments. The actual content of TAP in a compartment would depend on the volume of that compartment. However, it is highly unlikely that a mechanism exists in acinar cells that results in the taking up of TAP against a concentration gradient. Thus the site of greatest TAP concentration is most likely to correspond with the site of initial TAP release (i.e., trypsinogen activation).
Address for reprint requests: M. Steer, Dept. of Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215.
Received 13 March 1998; accepted in final form 7 April 1998.
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