1 Gut Hormone Laboratory, To test the hypothesis that the
changes in intestinal contractility, which accompany inflammation of
the gut, are agonist specific, we compared the response of inflamed
strips to substance P (SP), motilin, ACh, and
K+ as a function of time. In
parallel experiments, changes in the general mechanical properties
(passive tension, optimal stretch) of the colitic tissue were
evaluated. Colitis was induced by trinitrobenzenesulfonic acid, and
rabbits were killed after 1, 2, 3, 5, or 8 days. Passive tension was
increased starting from day 2 until
day 8, and maximal active tension
(Tmax) was
generated at less stretch from day 5. A 50% decrease in
Tmax was observed
for ACh and K+ between
days 2 and
3 and for motilin and SP between
days 3 and 5. For all compounds,
Tmax returned to
normal after 8 days. The pEC50
value (negative logarithm of the concentration that induces 50% of the
maximal contractile activity) for ACh was increased from
day 3 until day
8 and for SP at day 3,
whereas for motilin it was decreased at day
1. The changes in passive tension and optimal stretch
indicate generalized structural alterations of smooth muscle tissue.
However, the different time profiles of the changes in active tension
and contractile potency for different contractile agents suggest that
inflammation specifically affects receptor-mediated mechanisms.
inflammation; 2,4,6-trinitrobenzenesulfonic acid; in vitro smooth
muscle contraction; length-tension
IT IS NOW WELL ESTABLISHED that intestinal inflammation
is associated with disturbed motility. However, it remains unclear whether inflammation causes nonspecific generalized damage to the
neuromuscular apparatus or whether specific contractile mechanisms are affected.
Altered contractility has been demonstrated in muscle tissue resected
from patients with inflammatory bowel disease but was attributed to
various mechanisms. Snape et al. (17, 18) reported a significant
reduction in the development of maximal tension by circular muscle from
ulcerative colitis patients, which appeared to be agonist nonspecific,
whereas Vermillion et al. (20) demonstrated agonist-dependent changes
of the contractility of muscle from the small intestine of Crohn's
disease patients, which were also different between circular and
longitudinal muscle. It may also be noted that another study found no
difference between patients and controls (11).
Conflicting data were also obtained in animal models of intestinal
inflammation. One study, in rabbits, failed to find changes in the
muscle layer (15), but all the others noted either increased or
decreased contractility. A receptor-independent decrease in tension
development was reported in longitudinal strips from rats with colitis
induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS), acetic acid,
Trichinella spiralis larvae, or
intraperitoneal injection of mitomycin c (10). The increased
responsiveness of jejunal longitudinal muscle in
Trichinella-infected rats (19) and the decreased contractility in jejunal circular muscle from
nematode-infected rats (6) were also found to be pharmacologically
nonselective. However, another study using TNBS-induced
ileitis in guinea pigs found that nonreceptor-mediated contraction is
not modified by inflammation, whereas receptor-mediated contractions
are differentially altered in the longitudinal and circular layers
(13).
The above-mentioned studies mainly compared the effects of cholinergic
agents, adrenergic agents, or histamine with KCl-mediated responses at
one particular time point, eventually in both circular and longitudinal
muscle, and used the KCl response to classify observed changes as due
to nonreceptor or receptor-mediated mechanisms. Such an approach
overlooks the possibility that both mechanisms may play a role. We
hypothesized that if receptor-mediated mechanisms would be involved the
time course of inflammation-induced changes would differ for different
agonists. We therefore decided to compare the responses to different
agonists as a function of time in a model of rabbit TNBS colitis.
Because few studies have determined the effect of inflammation on the
general mechanical properties of smooth muscle tissue, this effect was
studied in parallel. We selected four stimuli: substance P (SP),
motilin, ACh, and KCl. ACh is the classical excitatory neurotransmitter
in the gut, whereas with KCl receptor-independent mechanisms can be
monitored. SP was chosen because it may act as a mediator of neurogenic
inflammation during inflammatory bowel disease, and motilin was chosen
because it is an important endocrine regulator of gastrointestinal
motility. Furthermore, motilin's levels have been shown to be
increased in patients with Crohn's disease and ulcerative colitis (1). The model of rabbit colitis was selected because the pharmacological responses of rabbit colonic smooth muscle to inflammatory mediators closely resemble those of the human colon (14). The rabbit is also the
best model to study the effect of motilin in vitro because only in this
species have motilin receptors been demonstrated in the colon (7).
Induction of Colitis
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Histological Evaluation of Colitis
Selected segments of distal colon taken at 5 cm from the rectum (part I) and 25 cm from the proximal colon (part II) were snap-frozen in 2-methylbutane atMyeloperoxidase Activity
Myeloperoxidase (MPO), a marker for tissue neutrophil content, was measured in mucosal sections taken 5 cm from the rectum using the procedure described by Bradley et al. (2).Contraction Studies
Approximately 8 cm from the rectum, a piece of colon of 5 cm was removed. Circular strips, freed from mucosa, of 0.2 × 2.5 cm were cut and suspended along their circular axis in a tissue bath filled with HEPES buffer (pH 7.4), and the response of the strips was measured either isotonically or isometrically.Isotonic measurements.
Strips were mounted with a preload of 1 g. After a stabilization
period, cumulative dose-response curves
(108 to
10
4 M) toward ACh were
established. After a washout period, the response of strips to
increasing concentrations of motilin
(10
9 to
10
6M) and SP
(10
10 to
10
6M) was measured. Results
were expressed relative to a supramaximal dose of ACh
(10
4 M) added at the end of
the dose-response curves. The contractions were measured using HP
7DCDT-1000 transducers from Hewlett-Packard (Palo Alto, CA) with a
displacement transducer control unit obtained from Janssen Scientific
Instrument Division (Beerse, Belgium) and recorded on
multirecorder MC6601 (Watanabe Instruments, Tokyo, Japan)
and on a Pentium computer with a DI-200 PGL ADC card and using the
WINDAQ/200 acquisition software (DATAQ Instruments, Akron, OH).
Calculations were performed with the WINDAQ/EX playback software.
Values of negative logarithm of the concentration that induces 50% of
the maximal contractile activity
(pEC50) were derived from the
concentration-response curves by linear interpolation.
Isometric measurements.
After an equilibration period, length-tension relationships of the
strips were established. Transducers (Harvard Apparatus, Edenbridge,
UK) were raised via a micrometer system to a point such that the
tissues were held rigidly but no tension was recorded by the system.
The length of the strip under these conditions was designated as the
initial length. Muscle strips were then stretched in 5% increments of
initial length to a maximum stretch of 100% initial length. The
passive tension that developed in response to stretch was measured for
each increment in length. A contraction was then elicited by adding
maximally active doses, as determined in a previous study (7), of ACh
(104 M), motilin
(10
7 M), SP
(10
7 M), or
K+ (140 mM) to the bathing medium.
This contraction above the passive tension was considered the active
tension. Optimal stretch
(Lo) was
defined as that degree of stretch that gave the maximum response to a
contractile agent. The signals were recorded on a BD112 recorder (Kipp
and Zonen) and on a Pentium computer using the same data acquisition
system as described above. The magnitude of the tension was expressed
in grams and normalized for cross-sectional area of the strip using the
following equation: cross section
(mm2) = tissue wet weight
(mg)/[tissue length (mm) × density
(mg/mm3)]. The density of
smooth muscle was assumed to be 1.05 mg/mm3.
Statistical Analysis
Data are presented as means ± SE. Time-dependent changes in contractility were compared using one-way ANOVA. Specific comparisons were made by calculating appropriate t-test values. Significance was accepted at the 5% level. ![]() |
RESULTS |
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Evaluation of Colitis
Appearance of colitis.
TNBS-treated rabbits lost weight after the induction of inflammation.
At day 8, they had lost 375 ± 50 g, ~13% of their initial weight, whereas control rabbits showed a
gain of 70 ± 20 g during the same period. The cross-sectional area
of the distal colon gradually increased from 30.7 ± 3.4 mm2 (control) to 65.3 ± 3.7 mm2 (day
3) and persisted until day
8 (62.4 ± 7.2 mm2). The data are illustrated
in Fig. 1. A similar increase in
cross-sectional area was observed in the second, more proximal, part of
the distal colon (data not shown).
|
Histology.
Tissue sections, stained with hematoxylin-eosin, from the colon of
control rabbits and TNBS-treated rabbits obtained 5 days after the
induction of inflammation are shown in Figs.
2 and
3. The wall of the colon
of the TNBS-treated rabbit is diffusely thickened with loss of mucosal
haustration and folding. There is evidence of submucosal edema and
distortion of the mucosal architecture. Crypts are branched and no
longer follow a parallel course. The distance between the crypt base
and muscularis mucosae is increased, and the intercryptal distance is
highly variable. The lamina propria cellular infiltrate is increased in
intensity, mainly basal in location, and mixed in composition (with
eosinophils, neutrophils, and mononuclear cells). Vasodilatation,
thickening of the muscularis mucosae, and segmentation of the circular
muscle layer are also clearly seen. Changes in histological features after the induction of the inflammation, as reflected in the scores of
the parameters that were observed, are summarized for several time
points in Tables 1 and
2. Desquamation of epithelial cells started at day 1 and lasted until
day 5, leaving a general defect in the
structure of the mucosa. Reepithelialization was observed from
day 3 on. Complete healing was
observed after 8 days. There was a marked increase in edema in the
submucosa and within the muscularis propria from day
1 until day 5 after
the induction of inflammation. This was accompanied by an unraveling or
segmentation of the circular muscle layer. A thickening of the
muscularis mucosae and the muscularis propria became apparent at
day 5 postinjury and was most
pronounced for the circular and longitudinal muscle layers after 8 days. The intensity of the inflammatory infiltrate increased toward a
maximum 5 days after the induction of the injury and was still elevated
at day 8. The composition of the
infiltrate was mononuclear during the first 3 days and then consisted
of a mixed population of polymorphonuclear granulocytes at
days 5 and
8. The distribution of the
inflammatory cells was limited to the mucosa during the first 3 days,
with an increased intensity in the basal part compared with the top
part. In addition, a few inflammatory cells were observed in the
muscularis propria at days 5 and
8 after the induction of inflammation.
The histological profile in the proximal part of the distal colon
(part II) was similar to the distal
part of the distal colon (part I),
confirming the uniformity of the inflammation.
|
|
|
|
MPO activity.
Colonic mucosal MPO activity in healthy controls was 0.233 ± 0.096 U · mg wet
wt1 · min
1.
Intracolonic administration of TNBS resulted in a time-dependent increase in MPO activity (Fig. 4). At
day 8, MPO activity was still 3.2-fold
higher than in controls.
|
Contractile Response of Colitic Strips
Passive tension. In control rabbits, passive tension at 70% stretch amounted to 1.86 ± 0.82 g/mm2 and was not significantly (P = 0.49) different from the tension at day 1 (1.89 ± 1.39 g/mm2). Passive tension at 70% stretch started to increase at day 2 to 13.09 ± 2.39 g/mm2 (P < 0.0005) and remained unchanged for the rest of the observation period (day 3: 11.29 ± 6.87 g/mm2, day 5: 19.54 ± 4.32 g/mm2, day 8: 14.98 ± 8.20 g/mm2).
Length-tension relationships to ACh and motilin.
Inflammation markedly affected the length-tension relationship for the
response to ACh (104 M) and
motilin (10
7 M), and
changes gradually developed during the observation period. As an
example, Fig. 5 shows the results obtained
with ACh in a control rabbit and in a rabbit on day
5. In both cases, the response to ACh increased to a
maximum with increasing stretch (maximal active tension reached at
Lo) and
declined thereafter. However, in inflamed strips, maximum active
tension was lower (more than 60%), and less stretch was needed to
reach it. Lo,
expressed as the percent increase of the initial muscle length, was
decreased from 63% (control) to 40% (inflamed).
|
|
|
Comparison of maximum active tension induced by ACh, motilin, SP,
and K+.
Figure 7 compares the maximum active
tension generated by inflamed strips in response to ACh, SP, motilin,
and KCl at different days after the induction of inflammation. For all
agents, inflammation resulted in a decrease of the maximum active
tension, but the magnitude of the decrease and its time course were not
identical. Maximum active tension induced by ACh and KCl decreased in
parallel and was already decreased by 50% between day
2 and day 3. The response to motilin and SP decreased more slowly and was only decreased
by 50% between day 3 and
day 5. Eight days after the induction
of inflammation, maximum active tension returned to its normal value
with all contractile agents used.
|
Contractile potency of inflamed strips to ACh, motilin, and SP.
The contractile potency of ACh, motilin, and SP was determined from
dose-response curves under isotonic conditions. Figure 8 shows those toward ACh obtained 1, 2, 3, 5, and 8 days after TNBS treatment. The
pEC50 values deduced from these
curves indicate an increase from 5.87 ± 0.15 (control) to 6.45 ± 0.09 (day 3), 6.60 ± 0.08 (day 5), and 6.74 ± 0.23 (day 8). All data are summarized in Table
4. There is a significant increase of the
pEC50 for SP at
day 3 (from 8.23 ± 0.18 to 8.72 ± 0.09) and a decrease for motilin at day
1 (from 8.13 ± 0.13 to 7.78 ± 0.01).
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DISCUSSION |
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This study demonstrates that TNBS-induced colitis in the rabbit changes the contractile response of circular colonic smooth muscle tissue, but the extent of the change and its time course depends on the stimulus. Moreover, the time courses of mucosal damage and of changes in the passive tension and in the Lo to induce maximal tension differ as well. Our results demonstrate that, although TNBS-induced colitis in the rabbit changes the general mechanical properties of the smooth muscle, TNBS-induced colitis also has specific effects on receptor-mediated pathways.
The mechanical properties of smooth muscle depend on the amount of elastic components and connective tissue of the muscle relative to the contractile components of the preparation. It has previously been shown that muscles with a high passive tension at lengths below Lo have a greater connective tissue content than muscles with a low passive tension (9). Therefore, our data indicate that inflammation increases connective tissue content. However, the increase in edema, as reflected in the histological analysis and in the increased cross-sectional area, could also contribute to the increased passive tension. Edema, eventually accompanied by a change in connective tissue content, may reduce contact between neighboring smooth muscle cells and in this way change the contractile properties of the tissue.
A decrease in maximal tension has also been observed in a rabbit model of Formalin-immune complex colitis (4) and in patients with ulcerative colitis (18). In the rabbit model, a decrease in maximal tension was accompanied by a decreased membrane potential, which could not be ascribed to a change in the Na+-K+-ATPase and was therefore suggested to be due to altered intracellular Ca2+ fluxes. However, another study found that the decreased force development in this model was due to an abnormal rate of actin myosin cross-bridge cycling (21). It remains to be investigated whether similar changes are involved in rabbit TNBS colitis.
The most interesting finding of our study is that the decrease in maximum active tension development is time and agonist dependent, which leads to the conclusion that the effects of inflammation are not limited to generalized damage of the contractile apparatus or generalized disturbance of postreceptor events. Indeed, although the decrease in response to ACh parallels the decrease in response to KCl, suggesting that events at the receptor level are not important, the decrease of the response to SP and motilin develops more slowly. This decrease may reflect a decreased number of receptors. In fact, for motilin, we have shown that colitis in rabbits is accompanied by a downregulation of motilin receptors (8).
In addition, temporal changes were also observed in the pEC50 values for the respective contractile agents studied, suggesting that inflammation also affects either the affinity of the agonist for its receptor or the conformational changes involved in the interaction between the receptor and its effector system. The fact that the time course for the changes in pEC50 values differs for the contractile agents studied again supports the hypothesis that specific receptor-dependent mechanisms are affected. A practical consequence is that when different studies are compared care should be taken to take into account the stimulus and the time point at which effects were evaluated.
Together with the observation that plasma motilin levels are increased during inflammation (1), our study for the first time also emphasizes a role for motilin during inflammation. Also, SP is actively involved in the disturbed motility effects. Although previous studies have shown that SP receptor binding sites are upregulated in the small arterioles and venules of the intestine in patients with inflammatory bowel disease (12), our study indicates that the contractile response to SP is decreased. A downregulation of SP receptors at the smooth muscle level remains therefore to be investigated.
The present study shows that the different parameters that determine the contractile activity follow different time courses. The decrease in active tension induced by all contractile agents studied is maximal at day 5 and normalized at day 8, whereas the increase in passive tension is already maximal at day 2 and remains increased for the rest of the observation period. In contrast, changes in Lo become only significant from day 5 on. The above-mentioned alterations cannot be simply correlated to histological changes. For instance, at day 8, the architecture of the mucosa is normal, although the cellular infiltrate and MPO levels are still increased. Inflammatory mediators still present at that time may mediate the changes in passive tension and Lo but not the changes in active tension. From a therapeutic point of view, these observations suggest that anti-inflammatory substances should be evaluated on all parameters that determine the contractile activity induced by several agonists.
Cominelli et al. (5) have shown the ability of the interleukin-1 receptor antagonist to suppress inflammation, tissue damage, and eicosanoid production in the rabbit model of Formalin-immune complex colitis in a dose-dependent manner. In the same model, it was shown that administration of a novel inhibitor of proinflammatory cytokine synthesis, CGP-47969A, reduces inflammation and tissue damage (3). Furthermore, it has been demonstrated that in vivo production of leukotrienes B4 and C4 correlates with indications of inflammation in rabbit colitis (22). Together, these studies suggest that proinflammatory cytokines and eicosanoids are involved in the inflammatory process in rabbit colitis. It remains to be investigated whether they also trigger the changes in smooth muscle contractility and mechanical properties that were observed in the present study.
In conclusion, our results suggest that the changes in contractility observed in TNBS-induced colitis in the rabbit are not only due to nonspecific smooth muscle damage but also to disturbances of specific neuropeptide-mediated receptor mechanisms. Motilin and SP are actively involved in the changed motility effects induced by inflammation.
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ACKNOWLEDGEMENTS |
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We thank L. Nijs for her skillful technical assistance.
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FOOTNOTES |
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This work was supported by grants from the Fund for Scientific Research-Flanders (Belgium) (Nationaal Fonds voor Wetenschappelijk Onderzoek Grant 3.0187.96) and the Belgian Ministry of Science (GOA 92/96-04 and IUAP P4/16).
I. Depoortere is a postdoctoral research fellow of the Fund for Scientific Research-Flanders (Belgium).
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: T. L. Peeters, Gut Hormone Lab, Gasthuisberg O & N, B-3000 Leuven, Belgium (E-mail: theo.peeters{at}med.kuleuven.ac.be).
Received 9 June 1998; accepted in final form 23 March 1999.
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