Departments of 1 Surgery, 2 Medicine, 3 Pathology, and 4 Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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We examined whether the capsaicin vanilloid receptor-1 (VR1) mediates substance P (SP) release from primary sensory neurons in experimental pancreatitis. Pancreatitis was achieved by 12 hourly injections of caerulein (50 µg/kg ip) in mice. One group received capsazepine (100 µmol/kg sc), a competitive VR1 antagonist, at 4-h intervals. Neurokinin-1 receptor (NK1R) internalization in acinar cells, used as an index of endogenous SP release, was assessed by immunocytochemical quantification of NK1R endocytosis. The severity of pancreatitis was assessed by measurements of serum amylase, pancreatic myeloperoxidase (MPO) activity, and histological grading. Caerulein administration caused significant elevations in serum amylase and pancreatic MPO activity, produced histological evidence of pancreatitis, and caused a dramatic increase in NK1R endocytosis. Capsazepine treatment significantly reduced the level of NK1R endocytosis, and this was associated with similar reductions in pancreatic MPO activity and histological severity of pancreatitis. These results demonstrate that repeated caerulein stimulation causes experimental pancreatitis that is mediated in part by stimulation of VR1 on primary sensory neurons, resulting in endogenous SP release.
capsazepine; neurogenic inflammation; neurokinin-1 receptor; pancreatic acinar cells; primary sensory neurons
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INTRODUCTION |
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THE PATHOPHYSIOLOGICAL EVENTS causing acute pancreatitis are incompletely understood. Experimentally, secretagogue-induced pancreatitis and other models have been extremely useful for defining many of the organ-specific and cellular processes that are involved in the induction of the disease. Substantial in vivo and in vitro data (13, 25) suggest that the early insult in experimental pancreatitis may involve the intra-acinar activation of digestive enzymes. After the initial insult, the severity of pancreatitis is thought to depend on a number of factors, including ischemia, pancreatic glutathione, oxygen-derived free radicals, mode of cell death (ischemia vs. necrosis), and proinflammatory mediators (25). A major proinflammatory factor believed to play a central role in exacerbating the inflammatory process after the initial insult is substance P (SP), a neuropeptide released from primary sensory nerve endings (19).
Neurogenic inflammation mediated by SP has been shown to play a significant role in the severity of experimental pancreatitis. It has previously been demonstrated (8) that SP stimulates plasma extravasation from postcapillary venules in the mouse gastrointestinal tract and pancreas. This effect is blocked by administration of an antagonist to the SP neurokinin-1 receptor (NK1R) (8). Furthermore, it has been shown (3) that genetic deletion of the NK1R in mice results in a marked reduction in the severity of secretagogue-induced pancreatitis compared with wild-type mice. These findings demonstrate that SP is an important mediator of pancreatic inflammation.
Although it is clear that SP release and subsequent activation of the NK1R play a central role in the neurogenic inflammatory process, the mechanism underlying SP release requires elucidation. Specifically, the stimuli triggering primary sensory neurons to release SP in experimental pancreatitis have not been investigated. Administration of capsaicin, an excitotoxin that is highly specific for SP- and calcitonin gene-related peptide-containing primary sensory neurons, causes plasma extravasation in the gastrointestinal tract and pancreas of rodents (8). Thus activation of the capsaicin receptor in pancreatitis may be responsible for primary sensory neuron depolarization and the subsequent release of SP, resulting in NK1R activation and parenchymal injury.
A recently cloned capsaicin receptor, the vanilloid receptor subtype-1
(VR1), has been demonstrated (6) to be a nonselective cation channel expressed by primary sensory neurons. Noxious heat and
capsaicin directly activate the VR1 (29). Furthermore,
moderate acidity (pH 6.4) decreases the temperature threshold
and has been shown to cause VR1 activation at 37°C (29).
Capsazepine, a synthetic competitive antagonist of capsaicin
(2), is a VR1 antagonist capable of blocking activation
induced by capsaicin, heat, and protons (29). Therefore,
if SP release and subsequent NK1R activation in experimentally induced
pancreatitis are mediated by VR1 activation, capsazepine should inhibit
these events.
Endogenous SP release and stimulation of the NK1R can be assessed in vivo by evaluating cell-specific SP-induced NK1R endocytosis (14), because both ligand and receptor are rapidly internalized after binding of SP to the NK1R (4). With the use of an antiserum specific for the COOH-terminal 15 amino acids of the rat NK1R (30), functional NK1R activation by endogenous SP release can be assessed by immunocytochemical methods. Thus we quantified NK1R-immunoreactive endosomes in pancreatic acinar cells as an index of endogenous SP release in our model of secretagogue-induced experimental pancreatitis. This technique offers the advantage of measuring endogenous SP release at the cellular level, which is not reflected by measurements of total tissue concentration of peptide.
In this study, we tested the hypothesis that VR1-mediated SP release plays a role in experimental pancreatitis by evaluating the effects of capsazepine on SP release and the severity of pancreatic inflammation. We demonstrate that capsazepine treatment significantly inhibits SP release and diminishes pancreatic inflammation, suggesting that secretagogue-induced experimental pancreatitis in mice is mediated, in part, by stimulation of the VR1 in primary sensory neurons, resulting in endogenous SP release.
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MATERIALS AND METHODS |
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Animal protocol and experimental design. Male C57/BL6 mice, 4 wk of age and weighing 12-15 g, were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were housed in climate-controlled rooms with a 12:12-h light-dark cycle. All animals were fed standard laboratory chow until a 14- to 15-h overnight fast before the experiment. Mice were permitted water ad libitum throughout the experiment. Mice were assigned to the following groups (n = 8): control (vehicle), caerulein only, and caerulein plus capsazepine. All procedures were approved by the institutional animal care and use committee.
Caerulein-induced pancreatitis. The CCK analog caerulein was purchased from Bachem California (Torrance, CA), and the VR1 antagonist capsazepine was purchased from Sigma RBI (Natick, MA). Caerulein was dissolved in 0.1 M NaHCO3 and then diluted in isotonic saline. Capsazepine was dissolved in 100% DMSO and then diluted in absolute ethanol, Tween 80 (Sigma, St. Louis, MO), and isotonic saline (10:10:80, vol/vol/vol). Both solutions were prepared the morning of the experiment and stored on ice.
Caerulein was administered as 12 hourly intraperitoneal injections at a supramaximal stimulating dose of 50 µg/kg per injection (3). Control mice received 12 hourly intraperitoneal injections of isotonic saline. Capsazepine (100 µmol/kg) was administered to the caerulein plus capsazepine group via subcutaneous injections at 4-h intervals commencing 1 h before the first caerulein injection. As an additional control, one group of caerulein-treated mice (n = 8) received subcutaneous capsazepine vehicle injections with a dosing schedule identical to capsazepine administration. One hour after the last caerulein or vehicle injection, animals were euthanized in a CO2 precharged chamber. Mixed arteriovenous blood was collected by decapitation for measurement of serum amylase concentration. The pancreas was then quickly removed and divided for histological grading, measurement of tissue myeloperoxidase (MPO) activity, and immunocytochemical analysis of SP release.Serum amylase concentration.
Mixed arteriovenous blood was centrifuged for 10 min at 1,500 g. The serum amylase concentration was measured using the
procion yellow starch assay as previously described (10).
Briefly, serum samples diluted with isotonic saline were added to glass
culture tubes containing 800 µl of the 3% procion yellow starch
solution in a buffer composed of 0.2 M NaH2PO4
and 0.2 M Na2HPO4 (pH 6.9). Blank tubes
did not contain serum, and standard tubes contained a known amount of
-amylase. The blank, standard, and serum sample tubes were placed in
a shaker bath and incubated at 37°C for 20 min. The reaction was
stopped by the addition of 1.6 ml of 0.1 M HCl. The tubes were then
centrifuged for 10 min, and the absorbance of the supernatant was read
at 420 nm without disturbing the pellet. The standard curve was
prepared using crude type VI-B
-amylase (Sigma).
MPO activity.
Portions of the harvested pancreata were immediately frozen at
80°C. Tissue MPO activity was measured as previously described (5). Briefly, the tissue was homogenized in 0.5%
hexadecyltrimethylammonium bromide (Sigma) in 50 mM
KH2PO4 (pH 6). The homogenate was subjected to
three freeze-thaw cycles and centrifuged at 4°C for 2 min. Absorbance
was read at 460 nm at 0, 30, and 60 s after the addition of 2.9 ml
of o-dianisidine dihydrochloride (Sigma) to 100 µl of the
supernatant. The maximal change in absorbance per minute was used to
calculate the units of MPO activity based on the oxidized o-dianisidine molar absorbency index of 1.13 × 104 M
1 · cm
1.
Histological grading.
Portions of the pancreata were fixed overnight at room temperature in a
pH-neutral, phosphate-buffered, 10% formalin solution. The tissue was
then embedded in paraffin, sectioned at 5 µm, stained with
hematoxylin and eosin, and coded for examination by a pathologist blinded to the experimental design. The pathologist graded the severity
of pancreatitis using modified scoring criteria as previously described
(23). The results were expressed as a score of 0 to 3 for
the histological parameters of edema and neutrophil infiltration and as
a score of 0 to 7 for the parameter of tissue necrosis (Table
1).
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Immunocytochemical analysis of SP release. SP release was assessed by analysis of NK1R endocytosis as described previously (14) with modifications. Briefly, portions of pancreata from control, caerulein-treated, and capsazepine-pretreated caerulein-treated mice were fixed overnight in ice-cold freshly depolymerized paraformaldehyde (4% in PBS) at 4°C and then placed in ice-cold PBS-30% sucrose for 24 h. The tissue was then embedded in Tissue Tek OCT (Sakura, Torrance, CA), frozen, sectioned at 20 µm, mounted on Superfrost Plus glass slides (Fisher, Pittsburgh, PA), and dried with desiccant at room temperature for 4 h. After being washed, the sections were stained overnight at room temperature using a rabbit antiserum (no. 11886-5) specific for the COOH-terminal 15 amino acids of the rat NK1R (SPR393-407) at a dilution of 1:3,000 (30). The sections were then washed and incubated with cyanine 3-conjugated donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:600 for 3 h at room temperature. The sections were washed and coverslipped using one drop of Aquamount (Lerner Laboratories, Pittsburgh, PA). Control sections were incubated with primary antiserum preabsorbed with 10 µmol/l SPR393-407 overnight at 4°C; specific NK1R immunostaining was abolished in these controls (data not shown).
Quantification of NK1R endocytosis. Immunostained sections were analyzed using a Zeiss LSM-410 inverted krypton-argon confocal laser scanning system coupled to a Zeiss Axiovert 100 microscope. Optical sections (0.5 µm) of 512 × 512 pixels were obtained and processed using Adobe PhotoDeluxe. Quantification of NK1R endocytosis was performed by analyzing 10 NK1R-immunoreactive acinar cells per mouse (n = 5) and determining the number of these cells containing >50 NK1R-immunoreactive endosomes. Cytoplasmic endosomes were distinguished from plasma membrane-associated NK1R immunoreactivity by ensuring that the nucleus of the acinar cells was in the same optical section as the NK1R-immunoreactive endosomes.
Statistical analysis. Results are expressed as means ± SE. Statistical comparisons among groups were examined by one-way ANOVA with the Tukey post test, using GraphPad Prism version 2.00 (GraphPad Software, San Diego, CA). Statistical significance was set at P < 0.05.
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RESULTS |
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Administration of supramaximal stimulating doses of caerulein (12 hourly injections of 50 µg · kg1 · dose
1)
resulted in endogenous SP release as indicated by SP-induced endocytosis of the NK1R in pancreatic acinar cells (Fig.
1, top). Administration of the
specific VR1 antagonist capsazepine to caerulein-treated mice
significantly inhibited endogenous SP release. Endogenous SP release
was quantified by immunocytochemical assessment of SP-induced NK1R
endocytosis in pancreatic acinar cells. In control mice, only rare
acinar cells (6%) contained more than 50 NK1R-immunoreactive endosomes
per cell (Fig. 1, bottom). After caerulein administration, nearly all (94%) of the acinar cells displayed >50
NK1R-immunoreactive endosomes per cell. Capsazepine treatment
significantly reduced SP release as determined by the level of NK1R
endocytosis (P < 0.001); only 32% of acinar cells
contained >50 NK1R-immunoreactive endosomes per cell.
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Repeated caerulein administration produced evidence of pancreatitis as
determined by serum amylase levels, tissue MPO activity, and
histological grading. Mice receiving caerulein exhibited a 10-fold
increase in serum amylase concentration compared with control animals
(Fig. 2). In addition, caerulein
treatment caused a significant increase in the pancreatic activity of
MPO (Fig. 3), an enzyme produced by
neutrophils and used as a marker of inflammation associated with
neutrophil infiltration. Histologically, caerulein treatment produced
moderately severe pancreatitis characterized by pancreatic edema,
neutrophil infiltration, and necrosis (Fig. 4). The histological parameters of edema,
neutrophil infiltration, and necrosis were significantly elevated in
mice receiving caerulein alone (Table 2).
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Capsazepine administration was very effective in reducing MPO activity
in caerulein-treated mice. As depicted in Fig. 3, animals receiving
both caerulein and capsazepine exhibited an 84% reduction in MPO
activity compared with caerulein treatment alone (P < 0.001). In contrast to the caerulein-treated group, the mice receiving both caerulein and capsazepine developed significantly less edema, neutrophil infiltration, and necrosis by histological criteria (Fig.
4). As demonstrated in Table 2, capsazepine treatment significantly reduced the scores of edema by 42% (P < 0.001),
neutrophil infiltration by 40% (P < 0.001), and
necrosis by 67% (P < 0.05). The total histological
severity score was diminished by 47% in mice that received capsazepine
(P < 0.001) (Fig. 5).
Although the serum amylase concentration tended to be lower in the
animals receiving capsazepine treatment, there was no statistically
significant difference between the caerulein only and the caerulein
plus capsazepine groups. Capsazepine vehicle had no effect on
caerulein-induced pancreatitis (data not shown).
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There was no mortality during the study period. The persistent elevation in serum amylase seen with significant reduction in tissue MPO activity and histological severity scores suggests that the initiating insult to the pancreas induced by caerulein was not affected by capsazepine.
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DISCUSSION |
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Recent studies (3, 8) have suggested that SP plays a critical role in experimental pancreatitis. SP has been demonstrated to stimulate plasma extravasation in the pancreas, and this effect is blocked by administration of NK1R antagonists (8). Furthermore, genetic deletion of the NK1R in mice ameliorates secretagogue-induced pancreatitis, suggesting a significant role for SP stimulation of the NK1R in the pathogenesis of experimental pancreatitis (3). However, the mechanism of SP release in experimental pancreatitis has not yet been reported.
It has previously been shown (27) that stimulation of the capsaicin VR1 results in depolarization of the primary sensory neuron and subsequent activation of both afferent and efferent functions. SP is an undecapeptide neurotransmitter found in primary sensory neurons throughout the gastrointestinal tract and is released from nerve endings in both the spinal cord and gut after nerve stimulation and depolarization. We hypothesized that antagonism of the capsaicin VR1 would inhibit primary sensory neuronal activity (i.e., SP release) and thus reduce the severity of tissue inflammation.
In this study, we used an antiserum specific for the COOH-terminal 15 amino acids of the rat NK1R (30) to study the effects of repeated caerulein administration on endogenous SP release in the pancreas. Studies in vitro and in vivo (4, 15, 16) have demonstrated that after binding of SP, the NK1R is rapidly internalized by endocytosis and recycled to the plasma membrane after degradation of bound SP. By quantifying NK1R-immunoreactive endosomes in pancreatic acinar cells, we were able to assess the amount of endogenous SP stimulation after repeated caerulein administration and in caerulein-treated mice receiving the VR1 antagonist capsazepine.
The current study demonstrates that repeated caerulein administration in mice causes SP release and biochemical and histological evidence of acute pancreatitis. Furthermore, pharmacological antagonism of the capsaicin VR1 significantly inhibits SP release and reduces the severity of secretagogue-induced pancreatitis in mice. These findings suggest that primary sensory neurons play a critical role in the tissue inflammatory response to injury in pancreatitis. Thus we propose that repeated caerulein stimulation of the pancreas generates a signal that activates the capsaicin VR1 on primary sensory neurons, resulting in SP release and subsequent propagation of the inflammatory cascade, and that inhibition of primary sensory neurons through VR1 antagonism diminishes tissue inflammation in the pancreas via reduction in SP release.
Although supramaximal secretagogue stimulation causes mild pancreatitis in rats (12), it has been shown in mice to cause moderately severe pancreatitis characterized by tissue damage and elevated serum enzyme concentrations (9, 11). Capsazepine reduced the severity of tissue damage observed with caerulein administration by significantly diminishing pancreatic edema, neutrophil infiltration, and parenchymal injury and necrosis. Although serum amylase concentrations tended to be lower in the animals receiving capsazepine, these data were not significantly different. This finding suggests that caerulein-stimulated amylase release is an early event in the induction of pancreatitis and is not mediated by primary sensory neurons or correlated with the severity of experimental pancreatitis. Thus the initial insult to the pancreas (i.e., supramaximal caerulein stimulation) and consequent increased serum enzyme concentration are not blocked by capsazepine administration. However, capsazepine significantly diminished the severity of tissue damage and the extent of neutrophil infiltration, indicating that neural signaling participates in the entire complement of inflammatory changes, which is consistent with an important role for neurogenic inflammation.
In preliminary experiments, we have observed a dose dependence for effects of capsazepine in reducing the severity of pancreatitis. Low doses of capsazepine (30 µmol/kg sc once, 1 h before caerulein administration) had no effect on pancreatic MPO activity or histological score. However, more frequently administered doses of capsazepine (30 µmol/kg sc every 4 h) significantly diminished pancreatic MPO activity (data not shown). The highest doses reported here (100 µmol/kg sc every 4 h) significantly reduced MPO activity and all histological parameters (Fig. 3 and Table 2). Similar doses of capsazepine have been shown (20, 28) to block the capsaicin VR1 in vitro, and selective blockade of the capsaicin receptor by capsazepine (100 µmol/kg sc) has been reported previously (7, 18, 21) in rats and mice. These findings of dose-dependent inhibitory effects of capsazepine provide further support for the hypothesis that capsazepine acts via a specific receptor (e.g., VR1) and that stimulation of the VR1 mediates neurogenic inflammation.
The mechanism of VR1 activation in caerulein-induced experimental
pancreatitis is currently unknown. The VR1, a nonselective cation
channel expressed by primary sensory neurons, is directly activated by
noxious heat (29). However, it is known that protons, even
at moderately acidic conditions (pH 6.4), are capable of lowering the
temperature threshold for activation of the VR1 at 37°C
(29). Because pancreatic lysosomal and vacuole
compartments have a high proton concentration and are increased in
experimental pancreatitis (31), it is conceivable that the
intra-acinar activation of trypsinogen (13, 25) damages
these acidic compartments, subsequently lowering the tissue pH enough
to activate the VR1 at 37°C. In addition to protons originating from
pancreatic vacuoles and lysosomes, it is known that high proton
concentrations are physiologically attainable in several injurious
processes, including ischemia, infection, and inflammation
(1, 24, 26). Therefore, the inflammatory process
associated with experimental pancreatitis may also serve as a source of
protons, thereby lowering the temperature threshold enough to activate
the VR1 and efferent function of the primary sensory neuron. It is also
possible that in addition to increasing proton concentration, a yet to
be identified endogenous capsaicin-like molecule may bind and activate
the VR1 in pancreatitis (32), as depicted in Fig.
6. Activation of the neuron leads to the
release of various neuropeptides, particularly SP, thus mediating
neurogenic inflammation through activation of target cell receptors,
such as the NK1R.
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The measurement of NK1R endocytosis in pancreatic acinar cells as an index of endogenous SP release is an extremely useful tool, as it allows the evaluation of neuropeptide secretion at a cellular level, which is not reflected in the quantification of total SP levels in tissue homogenates. The physiological significance of SP stimulation and subsequent NK1R endocytosis in acinar cells in experimental pancreatitis is unclear: is it involved in the inflammatory cascade of acute pancreatitis, or is it simply a secretagogue effect and thus irrelevant to pancreatic parenchymal damage? In vitro studies utilizing rat (17) and guinea pig (22) pancreatic acini have demonstrated that exposure to SP results in amylase release from acinar cells. Whether the interaction of SP with pancreatic acinar cells serves an additional proinflammatory role in experimental pancreatitis requires elucidation. However, a proinflammatory role for other nonacinar SP target cells (e.g., vascular endothelial cells) in pancreatitis has been suggested. Frossard et al. (9) demonstrated that endothelial cell surface expression of intracellular adhesion molecule 1 (ICAM-1) is increased in the pancreas in caerulein-induced pancreatitis and that ICAM-1 deficiency and neutrophil depletion reduce the severity of pancreatitis and pancreatitis-associated lung injury (9). These results, together with our biochemical and histological findings of a capsazepine-induced reduction in pancreatic neutrophil infiltration in caerulein-treated mice, suggest that the ability of capsazepine to reduce the severity of pancreatitis may occur by virtue of limiting the SP-induced extravasation of neutrophils.
In conclusion, the results of this study demonstrate that repeated caerulein stimulation in mice causes experimental acute pancreatitis that is mediated in part by stimulation of the capsaicin VR1 on primary sensory neurons, resulting in endogenous SP release. Endogenous SP release results in NK1R activation on target cells and subsequent propagation of the inflammatory cascade. Further study is required to determine the mechanism of VR1 activation in caerulein-induced pancreatitis.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. A. Liddle, Dept. of Medicine, Box 3913, Duke Univ. Medical Center Durham, NC 27710 (E-mail: liddl001{at}mc.duke.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 May 2001; accepted in final form 3 July 2001.
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