Departments of 1 Medicine and 2 Pharmacology, University of Illinois at Chicago and Chicago Veterans Affairs Medical Center, West Side Division, Chicago, Illinois 60612
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ABSTRACT |
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Galanin is widely
distributed in enteric nerve terminals and acts to modulate intestinal
motility by altering smooth muscle contraction. This ligand causes
Cl secretion when colonic epithelial cells express
the galanin-1 receptor (Gal1-R) subtype. Because Gal1-R expression by
colonic epithelia is upregulated by the transcription factor nuclear
factor-
B (NF-
B), increasingly appreciated as critical in the
pathophysiology of inflammatory bowel disease, we wondered whether the
diarrhea associated with this condition could be due to
NF-
B-mediated increases in Gal1-R expression. To test this
hypothesis, we provided oral dextran sulfate sodium (DSS) to C57BL/6J
mice. Although Gal1-R are not normally expressed by epithelial cells
lining the mouse colon, DSS treatment resulted in increased NF-
B
activation and Gal1-R expression. Whereas galanin had no effect on
murine colonic tissues studied ex vivo, it progressively increased
short-circuit current and colonic fluid secretion in DSS-treated mice.
Concomitant parenteral administration of the NF-
B inhibitor
dexamethasone attenuated the activation of this transcription factor by
DSS, inhibiting the increase in Gal1-R expression. Although
Gal1-R-specific antagonists do not exist, intracolonic administration
of commercially available galanin antibody diminished the DSS-induced
increase in colonic fluid accumulation. Overall, these data demonstrate that a significant component of the excessive fluid secretion observed
in DSS-treated mice is due to increased Gal1-R expression.
galanin; secretion; inflammatory bowel disease
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INTRODUCTION |
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GALANIN IS A NEUROPEPTIDE originally isolated from porcine small intestine (37) that is 30 amino acids long in humans (7) and 29 amino acids long in all other species (for review, see Ref. 21). Galanin is widely distributed along the length of the gastrointestinal (GI) tract, with immunoreactivity to this peptide found in the enteric nerve terminals of all species studied (4, 19, 42). Recent molecular studies indicate that galanin acts by binding to one of three different receptor subtypes now identified as the galanin-1 (Gal1-R), galanin-2, and galanin-3 receptors. All three subtypes have been cloned and sequenced, including the human (16) and murine Gal1-R (41).
Although pharmacological studies indicate that all three galanin
receptor subtypes are expressed by smooth muscle cells lining the GI
tract (14, 15), we have previously shown (5, 6, 23) that only Gal1-R
mRNA is found in epithelial cells lining the human colon. When galanin
binds to receptors on smooth muscle cells, it causes both contraction
and relaxation, thereby acting to modulate intestinal motility (for
review, see Ref. 30). In contrast, we have recently demonstrated (5)
that galanin activation of Gal1-R expressed by the human colon
epithelial cell line T84 results in Cl secretion.
This finding therefore suggested the possibility that galanin
activation of Gal1-R expressed by epithelial cells lining the GI tract
could be important in regulating intestinal fluid secretion.
Our cloning of the human GAL1R gene (6, 24) revealed the
presence of multiple recognition sites for the inflammation-associated transcription factor nuclear factor-B (NF-
B) in the
5'-flanking region, which we have shown to be functional using
reporter gene studies (24). This transcription factor is activated in
many inflammatory disorders of the GI tract, including idiopathic
inflammatory bowel disease (IBD) (26, 27, 36), and subsequent to
infection with many different enteric pathogens (13, 34, 46). We have therefore hypothesized that the excessive colonic fluid secretion observed in diarrheal diseases associated with inflammation could at
least partly be due to NF-
B-induced upregulation of Gal1-R. In
support of this hypothesis, we recently have shown (17) that infection
of human T84 cells in vitro, or murine colonocytes in vivo, with
pathogenic Escherichia coli results in NF-
B activation and
Gal1-R upregulation. Increased Cl
secretion
consequently occurs (17), thereby supporting a role for galanin and the
Gal1-R in mediating the excessive fluid secretion observed in
infectious diarrhea. In contrast, nothing is known about whether Gal1-R
expression is increased in noninfectious inflammatory disorders
affecting the colon. The aim of this study, therefore, was to determine
whether enhanced Cl
secretion induced by
noninfectious inflammatory conditions could also be attributable to
increased expression and subsequent activation of Gal1-R.
In this study, we used the dextran sulfate sodium (DSS) model of murine
colitis (9, 28) to determine whether Gal1-R expression was upregulated
in association with colonic inflammation and to evaluate the ability of
galanin to increase colonic fluid secretion. We show here that
DSS-induced colonic inflammation is associated with increased NF-B
activation and Gal1-R expression. The increase in Gal1-R expression
allows galanin to increase the amount of colonic fluid generated. These
data therefore support a role for Gal1-R expression in mediating the
diarrhea associated with colonic inflammation.
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METHODS |
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Materials.
Specific pathogen-free C57BL/6J male mice, at least 8 wk old, were
obtained from Jackson Laboratory (Bar Harbor, ME) and used after
reaching weights of 25-30 g. DSS (mol wt = 40,000) was obtained from ICN Biomedicals (Aurora, OH), and galanin was from Bachem (Torrance, CA). The antibody specific for the p65 subunit (active conformation) of NF-B was from Boehringer Mannheim (Mannheim, Germany), and the monoclonal antibody to human galanin, which has 100%
cross-reactivity with murine galanin, was from Peninsula Labs (San
Francisco, CA). Pentobarbital was from Abbott (North Chicago, IL), and
2-0 silk was from Johnson and Johnson, (Somerville, NJ). All other
supplies were from Sigma Chemical (St. Louis, MO).
Animal care and induction of colitis. All animals were studied at 8 wk of age. Mice were kept in microisolator cages and provided free access to food and water. Colitis was induced by allowing animals access to 2% (wt/vol) DSS in their drinking water ad libitum as previously described (9, 28). Animals were fasted for 24 h before euthanasia, while retaining free access to water with DSS. This study was approved by the University of Illinois at Chicago Animal Care and Use Committee.
Histological analysis. Freshly resected specimens were placed in 10% formalin and fixed in paraffin-embedded blocks. Blocks were sectioned at a thickness of 5 µm and stained with hematoxylin and eosin using standard techniques (1). The severity of colitis was graded on a scale from 0 to 3, as previously described (28), by three separate investigators blinded as to the conditions of the animal from which the colon was obtained. Briefly, 0 indicated normal mucosa; 1, focal inflammatory cell infiltration including neutrophils; 2, inflammatory cell infiltration, gland dropout, and cryptitis; and 3, frank mucosal ulceration.
Immunohistochemistry.
A standard three-stage indirect immunoperoxidase technique was used for
all immunohistochemistry (20). We utilized an anti-peptide antibody
specific for the Gal1-R at a dilution of 1:500 as previously described
for 60 min at 22°C (17). The commercially available antibody for
the p65 subunit of NF-B, which only detects this molecule when
biologically active, was used as previously described at a dilution of
1:200 (27).
Chromogen quantification.
We have developed a novel algorithm for quantitative
immunohistochemistry that is based on calculating the cumulative signal strength, or energy, of the digital file representing any portion of an
image (25). This algorithm allows us to determine the absolute amount
of antibody-specific chromogen per pixel for any cellular region or
structure. In brief, digital images are acquired at ×1,000 using
a Microlumina ultraresolution scanning digital camera (3,380 × 2,700 pixels; Leaf Systems, Fort Washington, PA) attached to a Nikon
E600 microscope system. Chromogen abundance is quantified by
calculating the cumulative signal strength within three randomly
selected areas. For any image file of dimensions N1
by N2 pixels, and where n1 and
n2 identify the specific position of the data
within the digital image file, the cumulative signal strength (or
mathematical energy, E) is defined as
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Electrophysiology.
Mice were euthanized by CO2 asphyxiation. The cecum and
rectum were identified, and a full-thickness piece of proximal colon was resected and immediately mounted in an Ussing chamber. The colon
was mounted as a sheet on a 4-mm-internal diameter ring and incubated
at 37°C in Ringer solution that was continually gassed with 95%
O2-5%CO2. Transepithelial resistance was
determined as described previously (17). Briefly, electrical current
(100 µA) was applied as a 2-s pulse under current-clamped conditions across the tissue using Ag-AgCl electrodes connected to the reservoirs via salt bridges, and the resultant potential difference was recorded every 5 min for 30-40 min. The short-circuit current
(Isc) was then measured and recorded every minute
after the addition of secretagogue under voltage-clamped conditions.
Tissues were equilibrated for 15-30 min to allow for the
development of a stable baseline. Mucosal and serosal sides were then
exposed to 1 µM galanin, a dose previously shown to maximally yet
specifically activate Gal1-R-induced Cl secretion
(5). After return to stable baseline, the tissues were then stimulated
by adding 100 µM carbachol to the basal reservoir.
Evaluation of colonic fluid secretion. Animals were fasted for 24 h before surgery while continuing to have free access to water containing 2% DSS. Anesthesia was achieved using pentobarbital sodium (50 µg/mg ip). The adequacy of anesthesia was assessed by interdigital toe pinch, as previously described (43). Under sterile conditions using a dissecting microscope, a small laparotomy incision was made, the cecum identified, and the most distal segment of colon exposed. Using 2-0 silk, a ligature was first placed on the most distal aspect of this segment of colon, followed by placement of a second ligature at the most proximal portion of the colon to generate a loop that was ~5 cm in length. Before the proximal suture was completely tied off, a 26-gauge TB needle was inserted through the area defined by the proximally placed yet loosely tied suture. The knot was closed around the needle now inside the colonic lumen. Loops were injected with 200 µl of 1 µM galanin or saline (control), the needle was withdrawn, and the suture was tightened. The bowel was placed back into the peritoneal cavity, and the laparotomy incision was closed using a running (mattress) suture. The mice were placed back in their cage with free access to water without DSS but not food. After 4 h, the mice were killed by CO2 asphyxiation and the colonic loops were removed and their lengths and weights recorded. Fluid secretion was calculated by determining the ratio of loop weight (mg) to length (cm), as previously described (8).
Statistical evaluations. All comparisons between multiple groups of animals (i.e., Table 1) were performed by ANOVA, whereas comparisons between paired groups of animals (i.e., Table 2) were performed using the Student's paired t-test. In all cases, data were analyzed using StatView (Abacus Concepts, Berkeley, CA), with P < 0.05 being considered significant.
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RESULTS |
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DSS causes a progressive and severe colitis.
Mice given 2% DSS ad libitum in their drinking water developed a
progressively worsening colitis (Table
1). Overall, animals lost
nearly one-third of their body weight after 6 days, with losses seen as
early as 24 h after 2% DSS was started. In contrast, histological
evidence of inflammation was not observed until 3 days after DSS
administration, with significant shortening in overall colonic length
not observed for another 2 days (Table 1). The development
of gross bleeding and loose stools paralleled the development of the
histological colitis, which was not detected until mice had been
exposed to DSS for 4 or more days (Table 1). We found that DSS-induced
inflammation was so rapidly progressive that animals were unable to
reliably survive beyond 7 days of DSS exposure. For ethical reasons,
therefore, all mice were killed after 6 days of exposure to DSS.
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Galanin increases Isc only in inflamed colonic tissues.
Consistent with the absence of Gal1-R on colonic epithelial cells by
immunohistochemistry in control mice, normal colons mounted in Ussing
chambers failed to show any increase in Isc when
exposed to ligand. Specifically, basal Isc in
control animals was 3.4 ± 0.2 µA/cm2 and remained
unchanged at 3.6 ± 0.2 µA/cm2 after exposure to 1 µM
galanin. We used carbachol as a control secretagogue because, similar
to galanin, this drug causes Cl secretion via a
Ca2+-dependent mechanism after binding to a specific
heptaspanning receptor. In contrast to what was observed with galanin,
these tissues rapidly responded to 100 µM carbachol so that
Isc rapidly rose from 3.5 ± 0.7 to 11.2 ± 0.5 µA/cm2 (n = 4). For the first 2 days of DSS
exposure, galanin continued to have no effect on altering
Isc in mouse colon (Fig.
2A). However, with the onset of
detectable inflammation after 3 days of exposure to DSS and associated
with the immunohistochemical evidence of extranuclear Gal1-R
expression, galanin increased Isc. Indeed, the
galanin-induced increase in Isc (Fig. 2A)
paralleled the degree of colonic inflammation and the amount of Gal1-R
detectable by immunohistochemistry (Fig. 1). After 6 days of DSS, basal
Isc was unchanged (3.3 ± 0.5 µA/cm2) from that observed in mice not exposed to DSS
(3.4 ± 0.2 µA/cm2). However, 1 µM galanin markedly
increased Isc (10.7 ± 0.8 µA/cm2)
to an extent similar to that observed with stimulation with carbachol
(basal, 3.4 ± 0.4 µA/cm2; 100 µM carbachol, 9.1 ± 0.7 µA/cm2).
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DSS-induced inflammation decreases transepithelial resistance.
Although basal Isc was not changed in DSS-treated
mice compared with control mice, resistance was markedly diminished in
a manner consistent with that recently reported for human colonic epithelium obtained from patients with IBD (35). Although tissue resistance was 530 ± 40 · cm2 for
untreated control animals, that of mice treated with DSS for 6 days was
310 ± 30
· cm2. Although galanin
acted to increase Isc, it had no effect on resistance (330 ± 30
· cm2 in mice
treated with 2% DSS for 6 days and exposed to 1 µM galanin). Thus
these data indicate that galanin acts primarily by increasing Isc.
Galanin potentiates fluid collection in colonic closed loops.
The ability of galanin to increase Isc, which we
have previously shown is due to increased Cl
secretion in T84 cells (5), does not necessarily mean that this peptide
causes increased fluid secretion into the colonic lumen. To determine
the ability of galanin to induce fluid secretion, we evaluated the
effect of this peptide using closed colonic loops. After 4 h, closed
loops in untreated control animals weighed 29 ± 4 mg/cm (Fig.
3). As expected given the absence of
detectable Gal1-R in colonic epithelial cells of control animals,
instilling 1 µM galanin into the colonic loop did not
significantly alter the amount of fluid secretion (Fig. 3). After 6 days of exposure to DSS, however, closed colonic loop weights increased
by 2.3 ± 0.2-fold compared with that observed in untreated control
animals. This increase in closed loop weight was markedly potentiated
by galanin so that after 6 days of DSS, this peptide increased colonic loop weights by an additional 2.2 ± 0.4-fold compared with loops injected only with saline (Fig. 3, inset). Therefore, overall galanin increased fluid secretion in animals exposed to DSS for 6 days
by 5.1 ± 0.4-fold compared with control animals not given DSS.
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Effect of dexamethasone-induced inhibition of NF-B
activation and Gal1-R expression on colonic fluid secretion.
Gal1-R-specific antagonists do not currently exist, as all previously
described galanin antagonists have been shown to act as partial
agonists at this receptor subtype (5, 18, 29, 40, 45). To explore the
contribution of the Gal1-R to the excessive fluid secretion observed in
DSS murine colitis, we attempted to inhibit the expression of this
receptor by treating mice with a NF-
B inhibitor. Steroids are known
to have multiple different effects, including inhibiting the activation
of NF-
B present in the colonic epithelia of patients with IBD (3).
We also have demonstrated (17) that dexamethasone inhibits NF-
B
activation, and associated Gal1-R expression, in the colons of C57BL/6J
mice subsequent to infection with enteric pathogens. Because this agent can be readily given to animals without reservation, we investigated whether parenterally administered dexamethasone had any effect on the
colitis caused by 2% DSS in mice. Mice were given 0.15 µg/mg
dexamethasone in 0.9% saline intraperitoneally every 12 h, a
concentration similar to that used in the treatment of acute idiopathic
colitis in humans, with the first dose administered 2 h before their
initial exposure to DSS. In the absence of dexamethasone, and as
described above, 6 days of DSS caused a marked colitis (Table 1) that
resulted in significant increases in NF-
B activation and Gal1-R
expression (Fig. 1). In contrast, dexamethasone markedly attenuated the
amount of inflammation, NF-
B activation, and Gal1-R expression
observed (Fig. 4, Table
2). Finally, although increased fluid
secretion was seen in DSS-treated animals not given dexamethasone (Fig.
3), concomitant administration of this anti-inflammatory agent
decreased fluid secretion to levels similar to those observed for
untreated control animals (Table 2). Not unexpectedly given the absence
of Gal1-R in DSS-treated animals also given dexamethasone, galanin had
no effect on colonic fluid secretion in mice given DSS as well as this
steroid (Table 2).
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Intraluminal administration of antibody to galanin inhibits fluid secretion. To confirm the contribution of Gal1-R activation to the excessive fluid secretion observed, we intraluminally instilled galanin antibody in DSS-treated mice. Although colonic fluid secretion in mice exposed to DSS for 5 days was approximately twofold greater than observed in control mice, intraluminal injection of galanin antibody completely eliminated the DSS-induced increase in colonic fluid secretion (Table 2). We instilled sufficient antibody in 200 µl of saline to achieve a 1:500 final dilution based on the amount of colonic fluid secretion normally observed over a 4-h period. The amount of colonic fluid generated over 4 h from the colonic loops of DSS-treated mice in the presence of galanin antibody (27 ± 3 mg/cm) was not significantly different from that observed in untreated control mice processed in parallel (29 ± 4 mg/cm).
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DISCUSSION |
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In this study we show that in the DSS model of murine colitis, a
significant proportion of the increase in colonic fluid secretion is
due to Gal1-R upregulation. We previously showed (5) that Gal1-R are
expressed by the human colonic epithelial cell line T84 and that when
activated cause Cl secretion by a
Ca2+-dependent mechanism. We also have shown (17) that
although murine colonic epithelial cells do not normally express
Gal1-R, infection with pathogenic E. coli results in
upregulation of this receptor via an NF-
B-mediated process. This
physiological observation therefore is in accordance with our prior
biochemical finding that GAL1R gene expression is regulated by
this inflammation-associated transcription factor (24).
Recent studies have indicated that NF-B is activated in many
inflammatory disorders of the human colon in addition to infection (13,
34, 46), including Crohn's disease and ulcerative colitis (26, 36).
This has led to an awareness that many therapeutic agents used to treat
patients with IBD, including sulfasalazine (39) and steroids (3, 31,
32, 44), mediate their effects by acting as NF-
B inhibitors. In this
study, we show for the first time that DSS activates NF-
B and that
the increased activation of this transcription factor is associated
with increased Gal1-R expression. Although we do not provide definitive
proof in this study that NF-
B is directly responsible for the
DSS-induced increase in Gal1-R expression, we do show that the increase
in the expression of this receptor permits galanin to increase
Isc (Fig. 2) and net colonic fluid secretion (Fig.
3). At the very least, these data support the possibility that
increased Gal1-R expression may be an important component of the
excessive fluid secretion observed in IBD.
Our electrophysiological findings are in agreement with the few such studies performed on inflamed human tissues. In patients with IBD it has come to be appreciated that significant colonic fluid accumulation occurs via increases in paracellular permeability (35). We likewise show that the colonic resistance decreased by >40% in mice given 2% DSS for 6 days, similar to the ~50% decrease reported in patients with active colitis (35). It also has been reported that basal Isc is not increased in humans with active IBD, supporting the argument that the associated diarrhea is largely a problem of increased paracellular permeability. Similarly, we did not find any significant change in basal Isc in DSS-treated vs. control mice. However, we did find that whereas galanin had no effect on control murine colonic epithelium, this peptide hormone markedly increased Isc in DSS-treated mice (Fig. 2). The failure to see elevations in basal Isc, along with our showing that galanin increases Isc in DSS-treated mice, is possibly due to the fact that this ligand is present in enteric nerve terminals lining the GI tract of all species studied, including humans (4, 19). Removal of colonic epithelial tissues for study ex vivo either displaces them from the source of stimulatory ligand or dilutes the ligand available when they are placed in an Ussing chamber. That galanin is important in mediating fluid secretion is nonetheless demonstrated by its ability to potentiate colonic fluid secretion in vivo (Fig. 3), with galanin antibody markedly diminishing the amount of fluid secretion observed (Table 2), in DSS-treated mice.
It should be emphasized that the basic mechanism of action of DSS in causing colitis has yet to be established. Studies suggest that DSS mediates its effects by altering mucosal prostanoid concentrations (38) and host immunologic responsiveness (2, 11, 12) and by being directly toxic to the gut epithelial cells (9, 10). However, and particularly in light of our recent finding that enteric pathogens increase Gal1-R expression in gut epithelia (17), it is the ability of DSS to alter the composition of the colonic microflora that may be of greatest significance (28). DSS has been shown to alter the composition of the intestinal flora by two different mechanisms. First, DSS can be phagocytosed by gut macrophages, causing them to have reduced phagocytic ability of normal commensal organisms, allowing expansion of the Gram-negative anaerobic population (22). Second, DSS also has been shown, by virtue of it directly injuring intestinal epithelia, to permit increased uptake of bacterial endotoxin and peptidoglycan-polysaccharide polymers (33). Thus DSS may cause colonic injury, as well as increase Gal1-R expression, by altering the composition of the host bacterial flora.
This study does not attempt to validate the use of DSS-treated mice as
a model for the study of IBD. Rather, this study is the first to show
that DSS activates NF-B and increases Gal1-R expression in the
colonic epithelium and that the increase in Gal1-R expression is
directly responsible for a significant proportion of the colonic fluid
secretion observed. Overall, these findings support a possible role for
galanin and the Gal1-R in the pathophysiology of IBD-associated diarrhea.
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ACKNOWLEDGEMENTS |
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This study was supported by an American Digestive Health Foundation/Astra Merck Advanced Research Training Award (J. A. Marrero), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50694 (G. Hecht) and DK-51168 (R. V. Benya), Veterans Administration Merit Reviews (G. Hecht and R. V. Benya), and the Veterans Administration Research Enhancement Awards Program (G. Hecht and R. V. Benya).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. V. Benya, Dept. of Medicine, Univ. of Illinois at Chicago, 840 South Wood St. (M/C 787), Chicago, IL 60612 (E-mail: rvbenya{at}uic.edu).
Received 11 August 1999; accepted in final form 14 December 1999.
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