Different types of contractions in rat colon and their
modulation by oxidative stress
Asensio
Gonzalez and
Sushil K.
Sarna
Departments of Surgery and Physiology, Medical College of
Wisconsin and Zablocki Veterans Affairs Medical Center, Milwaukee,
Wisconsin 53226
 |
ABSTRACT |
The aim of this study was to investigate the
modulation of in vitro rat colonic circular muscle contractions by
dextran sodium sulfate (DSS)-induced inflammation and in spontaneous
inflammation in HLA-B27 rats. We also examined the potential role of
hydrogen peroxide (H2O2) in modulating
excitation-contraction coupling. The muscle strips from the middle
colon generated spontaneous phasic contractions and giant contractions
(GCs), the proximal colon strips generated primarily phasic
contractions, and the distal colon strips were mostly quiescent. The
spontaneous phasic contractions and GCs were not affected by
inflammation, but the response to ACh was suppressed in DSS-treated
rats and in HLA-B27 rats. H2O2 production was
increased in the muscularis of the inflamed colon. Incubation of
colonic muscle strips with H2O2 suppressed the
spontaneous phasic contractions and concentration and time dependently
reduced the response to ACh; in the middle colon, it also increased the
frequency of GCs. We conclude that H2O2 mimics
the suppression of the contractile response to ACh in inflammation. H2O2 also selectively suppresses phasic
contractions and increases the frequency of GCs, as found previously in
inflamed dog and human colons.
colonic inflammation; dextran sodium sulfate; giant contraction; hydrogen peroxide; giant migrating contractions
 |
INTRODUCTION |
IN SEVERAL SPECIES,
in vivo studies (38-40) have shown that the small
intestinal and colonic circular muscle cells generate three distinct
types of contractions: rhythmic phasic contractions, giant migrating
contractions (GMCs), and tone. The cellular mechanisms of initiation of
these contractions and their motility functions are markedly different
(41-43). The rhythmic phasic contractions are
regulated by slow waves superimposed with spikes. Their maximum frequency is the same as that of slow waves. These contractions partially or completely occlude the lumen, propagate over short distances, and cause the mixing and propulsive movements in the fasting
and the postprandial states (9). The GMCs are severalfold longer in duration and larger in amplitude than the phasic
contractions, and in dogs and humans they propagate uninterruptedly
over very long distances, sometimes over the entire length of the small intestine or the colon (15, 17, 20, 30, 37). Strong occlusion of the lumen coupled with long distances of uninterrupted propagation by these contractions causes mass movements (17, 23). GMCs are not regulated by slow waves, as their duration is
much longer than that of a single slow wave cycle (37).
The tone regulates the lumen size, and the tone is also not regulated by rhythmically occurring slow waves. The precise role of tone in
propulsion is not known, but a decrease in the lumen size may enhance
the efficacy of phasic contractions in mixing and propulsion.
Rodents are now increasingly used in gastrointestinal motility studies
because of the availability of transgenic and knockout models. However,
it is not known whether the colonic circular muscle of these species
generates the same types of contractions as the higher species, such as
dogs and humans, in vivo and in vitro. The stooling habits and stool
shape and texture in rats and mice differ from those in dogs and humans.
Inflammation suppresses rhythmic phasic contractions and tone and, at
the same time, increases the frequency of GMCs (19, 31, 33,
44). The suppression of phasic contractions prevents the normal
mixing and propulsive movements of the gut, whereas frequent mass
movements due to the increased frequency of GMCs cause diarrhea. The
differences in cellular mechanisms that differentially suppress phasic
contractions and tone, while at the same time stimulating GMCs, are not
completely understood. However, it is thought that these alterations in
cellular function are due to the production of reactive oxygen species
(ROS), cytokines, and lipid mediators in the muscularis during
inflammation. Main et al. (29) and Hurst and Collins
(18) reported that some cytokines, such as tumor necrosis
factor-
and interleukin-1
(IL-1
) can damage the enteric
neurons to produce dysfunction similar to that seen in inflammation. In
contrast, the effects of ROS on smooth muscle contractility are not
completely understood. It is known that the levels of ROS are elevated
in the mucosa of patients with inflammatory bowel disease as well as in
experimental models of inflammation (13, 22, 46). If ROS
contribute to smooth muscle and enteric neural dysfunction in
inflammation, their production also must be increased in the
muscularis. It is unlikely that the elevation of ROS levels in the
mucosa can directly affect smooth muscle or enteric neural function.
The objectives of this study were to 1) define the types of
contractions the rat colon generates in a muscle bath, 2)
determine whether the production of hydrogen peroxide
(H2O2) is increased in the muscularis of
dextran sodium sulfate (DSS)-treated rats and transgenic HLA-B27 rats
that develop spontaneous inflammation, and 3) examine the
effect of incubation of muscle strips with H2O2
on the occurrence of different types of colonic contractions.
 |
MATERIALS AND METHODS |
Animal model of inflammation.
Male Sprague-Dawley rats (200-250 g, Harlan, Indianapolis, IN)
were used. Colonic inflammation was induced by giving 5% (wt/vol) DSS
(mol wt >40,000, ICN Biochemicals, Aurora, OH) in drinking water for 7 days. The mortality rate was 8.3%. Transgenic HLA-B27 rats from a
Fisher 344 genetic background, which develop spontaneous enterocolitis
(1), were purchased from Taconic (Germantown, NY). By
28-32 wk of age, rats had developed loose stools and were killed.
Age-matched Fisher 344 rats, also purchased from Taconic, were used as
controls for transgenic HLA-B27 rats.
Muscle bath studies.
The whole colon from the cecum to the pelvic brim was removed after
asphyxiation of the animal with CO2 and immediately
immersed in warm, carbogenated Krebs solution (in mM: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1 NaH2PO4, 1.2 MgCl2, 11 D-glucose, and 25 NaHCO3). Segments (3 cm long) of the proximal (~1 cm from
the cecum), middle (~7 cm from the cecum), and distal colon (~15 cm
from the cecum) were removed, opened along the mesenteric border,
cleaned, and pinned flat on a petri dish with Sylgard base. The mucosal
layer, facing up, was removed under a magnifying glass. Strips (2 mm × 10 mm) were cut along the circular muscle axis and mounted
in 3-ml muscle baths filled with carbogenated Krebs solution at 37°C. Contractions were measured with Grass isometric force transducers connected to a Grass polygraph (Quincy, MA) that was interfaced to a
digital data acquisition system (DATAQ Instruments, Akron, OH). The
strips were stretched in steps until the ACh-induced contractions were
of maximal amplitude. They were left to equilibrate for at least 50 min
while the bath solution was replaced every 10-15 min.
The effect of H2O2 on ACh-induced contractions
was tested by first obtaining a control response to 2 µM ACh that was
taken as 100%. Then H2O2 was added to the bath
to give final concentrations of 0.3, 1, and 3 mM. The bathing solution
was replaced every 15 min to compensate for
H2O2 degradation. Control strips without H2O2 were incubated in parallel to monitor
changes in contractility over time. The contraction to 2 µM ACh was
measured again at 30, 60, 90, 180, and 360 min after incubation with
H2O2 and compared with the initial response.
Five minutes before ACh, the bathing solution was replaced with warm
Krebs solution so that ACh responses were recorded in the absence of
H2O2. Because H2O2 may
be degraded by muscle strips to an unknown and variable degree, we also
tested an alternative protocol. The strips were continuously superfused with H2O2 at rates of 60, 200, and 600 nmol/min, and the contractility to 2 µM ACh was checked at the same
intervals as given above. ACh was added 5 min after stopping
H2O2 superfusion and replacing it with normal
Krebs solution
Measurement of H2O2 in muscularis
homogenates.
A modification of the method described by Ravindranath
(35) was used. The method is based on the oxidation of
2',7'-dichlorofluorescin (DCFH) to dichlorofluorescein (DCF) by
H2O2. Before oxidation, DCFH diacetate
(DCFH-DA) is deacetylated to DCFH by intracellular esterases.
Rats anesthetized with 50 mg/kg pentobarbital were perfused through the
left ventricle with carbogenated Krebs solution to remove vascular
blood. A 3-cm-long segment of the distal colon was removed, and the
mucosa was dissected as described above. The tissue was frozen
immediately in liquid nitrogen and kept at
80°C for up to a week
until processing. Before analysis, the tissue was quickly
weighed and homogenized at 4°C in buffer (in mM: 120 KCl, 33 Na2HPO4, and 1 EDTA, pH 7.4) in a smooth glass homogenizer for 3 min. An aliquot was obtained to determine protein concentration (in triplicate) with the bicinchoninic acid method (Pierce, Rockford, IL). The homogenates were incubated at 37°C in the
presence of 0.5 µM DCFH-DA (Sigma Chemical, St. Louis, MO). The
incubation was stopped after 30 min by the addition of 4 vol of
ice-cold buffer and subsequent centrifugation. The supernatant was
separated for photon counting in an Aminco-Bowman spectrofluorometer (Spectronics, Rochester, NY). Peak wavelengths for excitation and
emission were 500 and 520 nm, respectively. The contribution of
nonoxidized DCFH-DA to the total signal was negligible. The autofluorescence of every sample was determined in parallel and subtracted. The excitation and emission spectra from DCFH-DA oxidized by the homogenates were identical to those from oxidized DCF obtained commercially. Commercial DCF was used to calibrate the measurements (expressed as nM oxidized DCF/mg protein).
Myeloperoxidase assay and visual assessment of mucosal injury.
Myeloperoxidase (MPO) activity was measured by the protocol employed by
Castro and Arntzen (6). The tissue was thawed and homogenized at 0.5 g/10 ml homogenization buffer (50 mM
KH2PO4 and 0.5% hexadecyltrimethylammonium
bromide, titrated with 0.1 M Na2HPO4 to pH 6).
The homogenate was then immersed in liquid N2, freeze
thawed three times, and centrifuged at 2,000 g. The supernatant was used to assay for MPO activity.
The assay was performed in cuvettes containing 1 ml guaiacol (0.22 ml/100 ml H2O), 2 ml phosphate buffer (0.01 M
KH2PO4 titrated with 0.1 M
Na2HPO4 to get pH 6.0), and 0.1 ml sample. The
spectrophotometer was set to 0, and 5 µl H2O2
(0.44 µmol) were added to start the reaction. The optical density
values were recorded and converted to MPO activity using the standard
curve of horseradish peroxidase type II (Sigma Aldrich, St. Louis, MO).
The protein in the supernatant was determined with Bio-Rad reagent
(4). The calculated specific activity is expressed as
myeloperoxide activity per milligram of protein. One unit of MPO
activity was defined as that degrading 1 µmol of
H2O2 per minute at room temperature.
Data analysis.
The contractile response of circular muscle strips to ACh was measured
as the area under contractions for 2 min after the addition of ACh to
the bath (expressed in N · s). The contractile area was
normalized by the cross-sectional area, which was calculated as
follows: weight in mg/(1.05 × length in mm). Multiple comparisons were performed by one-way ANOVA followed by the Student-Newmann-Keuls test. The comparisons between two means were performed by parametric Student's t-test. All data populations were normally
distributed. The curve fittings for all calibrations had the squared
correlation coefficient as >0.99.
 |
RESULTS |
Gross morphology, clinical symptoms, and MPO activity in
inflammation.
All rats treated with DSS developed diarrhea by day 7, and
their weight was 19 ± 6% less than that of their age-matched
siblings. The entire colon and cecum contained liquid, bloody feces.
Macroscopically, the mucosa was thicker and extensively ulcerated in
the distal colon; the middle and proximal colon had fewer lesions. An
enlargement of the submucosal layer throughout the colon was evident.
The MPO activity was significantly increased in the distal and the proximal colon compared with age-matched normal rats (Table
1). The percent increase in MPO activity
was greatest in the distal colon (~550% increase). The MPO activity
in the middle colon of DSS-treated rats increased by about ~50%, but
the difference did not reach statistical significance (Table 1). The
MPO activity in the distal colon of HLA-B27 rats was also greater than
that in Fisher 344 controls (0.32 ± 0.07 vs. 0.02 ± 0.006 U/mg protein, respectively; n = 6; P < 0.05).
Types of spontaneous colonic circular muscle contractions in
normal, DSS-treated, and HLA-B27 rats.
The muscle strips exhibited spontaneous rhythmic phasic contractions at
a frequency similar to that of short spike bursts that are regulated by
the slow waves (12). The frequency of phasic contractions
did not differ in the proximal, middle, and distal colon strips (Table
2), but under identical experimental conditions, the mean amplitude in the middle colon was about
one-seventh the mean amplitude of that in the proximal colon and in the
distal colon it was about one-thirtyfifth of that in the proximal colon (Fig. 1). The distal colon muscle strips
were mostly quiescent. In addition, the middle colon strips generated
regular giant contractions (GCs) at a frequency of 0.3 ± 0.1 contractions/min. GCs were defined as contractions with duration
>150% and amplitude >300% of the corresponding mean values of
phasic contractions. The GCs were sometimes superimposed with phasic
contractions, particularly in their falling phase (Fig. 1). The
proximal and the distal colon strips rarely generated GCs. In
muscle strips, both the phasic contractions and the GCs were
insensitive to 1 µM atropine. This same dose of atropine completely
blocked the response to 30 µM ACh.
View this table:
[in this window]
[in a new window]
|
Table 2.
Frequency and amplitude of spontaneous contractions of colonic circular
muscle in vitro in normal and DSS-treated rats
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 1.
A: spontaneous contractions of the circular
muscle in vitro in the different regions of the rat colon. Phasic
contractions of similar frequency were observed throughout the colon,
although with different amplitudes (see Table 2). In addition, the
middle colon exhibited giant contractions (GCs). B:
spontaneous contractions in mucosa and submucosa-free strips. Note that
the spontaneous phasic contractions are absent.
|
|
The phasic contractions are regulated by slow waves whose pacemaker
cells have been reported to reside at the submucosal border in the
feline and canine colons (10, 11, 47). We sought to
determine whether the pacemaker cells at the submucosal border also
regulate GCs. The mucosa and submucosa were removed in 20 muscle strips
from the middle colon. This completely blocked the spontaneous phasic
contractions as has been reported in other species (10, 11,
47), but it had no effect on the frequency of GCs (Fig.
1B).
No differences were noted in the characteristics of spontaneous phasic
contractions between strips from the normal and DSS-treated proximal,
middle, and distal colons (Table 2). The frequency of spontaneous GCs
in the middle colon was also not affected by inflammation.
We also compared the spontaneous contractions of the chronically
inflamed distal colon of transgenic HLA-B27 rats with those of
age-matched control Fisher 344 rats. Circular muscle strips from the
distal colon of Fisher 344 rats exhibited spontaneous phasic
contractions (amplitude: 0.4 ± 0.2 mN; frequency: 9 ± 1.2 contractions/min; n = 5). They were not different from
those seen in the distal colon of HLA-B27 rats (amplitude: 0.5 ± 0.2 mN; frequency: 10 ± 1.8 contractions/min; n = 5; P > 0.05). As with Sprague-Dawley rats, the
circular muscle strips from the distal colons of Fisher 344 rats and
HLA-B27 rats were usually quiescent and rarely exhibited spontaneous GCs.
ACh-induced contractions in normal, DSS-treated, and HLA-B27 rats.
Low concentrations of ACh (up to 2 µM) increased the amplitude of
ongoing phasic contractions in the distal rat colon (Fig. 2). Higher concentrations induced a tonic
contraction superimposed with increased amplitude of phasic
contractions. The phasic contractions were usually ablated during the
peak of tonic contraction, but reappeared when the tone began to
decrease from the maximum (Fig. 2). The contractile response, measured
as the combined area under tonic and phasic contractions, was increased
by ACh concentration dependently in strips from normal and DSS-treated
rats, but the increase was significantly suppressed in the inflamed
strips (~50% reduction in maximal response; n = 5;
P < 0.01; Fig. 3).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of low and high concentrations of ACh on rat
distal colonic circular muscle strips. Low concentrations increase the
amplitude of the phasic contractions. High concentrations also induce a
superimposed tonic contraction. DSS, dextran sodium sulfate.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Suppression of ACh-induced contractions in the circular
muscle from the distal colon. A: normal rats,
; DSS-treated rats, . B:
Fisher 344 rats, ; HLA-B27 rats, .
|
|
To eliminate the potential mechanical constraint due to the thickened
submucosa in inflamed colon strips, ACh responsiveness was tested in
distal colon muscle strips in which the mucosa and submucosa were
carefully removed. In addition, TTX (0.3 µM) and hexamethonium (0.1 mM) were included in these experiments to block any neural effects of
ACh. The contractile response of circular muscle strips devoid of
submucosa was also suppressed in DSS-treated rats compared with normal
rats (maximal effect: 17.9 ± 4.2 vs. 42.1 ± 6.9 N · s · mm
2; n = 4;
P < 0.05), indicating a defect in
excitation-contraction coupling rather than a mechanical artifact. The
mean cross-sectional areas of the strips devoid of submucosa from the
two groups were not statistically different (0.21 ± 0.03 vs.
0.17 ± 0.02 mm2 for inflamed vs. normal,
respectively; P > 0.05), ruling out the presence of
significant muscle hyperplasia of the muscularis in this model.
The contractile responsiveness to ACh was also significantly suppressed
in HLA-B27 rats (Fig. 3). The maximal effective response was 32.1 ± 3.2 and 22.4 ± 2.9 N · s · mm
2
in Fisher 344 control rats and HLA-B27 rats, respectively
(n = 5, P < 0.05).
H2O2 production in DSS-inflamed and HLA-B27
rat colonic muscle.
Muscle strip homogenates from the distal colons of normal, DSS-treated,
and HLA-B27 rats generated fluorescence when incubated with the
peroxide-sensitive compound DCFH-DA. The fluorescence increased
linearly with time from 0 to 30 min. Autofluorescence from both normal
and inflamed samples was always <5% of the signal. The fluorescent
signal was almost abolished if 5,000 U/ml catalase was included in the
incubation medium, in both normal (86 ± 5% inhibition) and
DSS-treated rats (91 ± 7% inhibition).
Muscle homogenates from DSS-treated rat distal colons oxidized
significantly more DCFH (72 ± 3.0 nM DCF/mg protein) than those from normal distal colons (32 ± 11 nM DCF/mg protein;
P < 0.05; n = 5; Fig.
4). Because catalase decomposes
H2O2 very rapidly, it is possible that part of
the H2O2 formed during the incubation was
degraded by endogenous catalase present in the homogenates before it
could oxidize DCFH, in which case H2O2
generation would have been underestimated in the above experiments.
Therefore, we included the catalase inhibitor 3-amino-1,2,4-triazole
(3-AT) during the incubation to avoid H2O2
decomposition by endogenous catalase. In the presence of 5 mM 3-AT, the
oxidation of DCFH by inflamed muscle homogenates (241 ± 58 nM
DCF/mg protein) was much more marked than that by the control muscle
homogenates (27 ± 8 nM DCF/mg protein; P < 0.01;
Fig. 4).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 4.
Oxidation of 2',7'-dichlorofluorescin diacetate (DCFH-DA)
by distal colonic muscle homogenates from normal and DSS-treated rats.
Inflamed samples (I) oxidized DCFH-DA more than normal samples (N).
Catalase (Cat; 5,000 U/ml) inhibited fluorescence generation by >90%.
When the catalase inhibitor 3-amino-1,2,4-triazole (3-AT) was included
in the incubation medium, an 8-fold increase was observed in the
inflamed vs. normal muscle. DCF, dichlorofluorescein. Values are
means ± SE of 5 experiments. * P < 0.05;
** P < 0.01.
|
|
Generation of H2O2 in muscle homogenates from
the distal colons of HLA-B27 rats produced a 1.7-fold increase in
H2O2 production (118 ± 28 in HLA-B27
colon vs. 67.5 ± 15 in control colon). However, this difference
was not statistically significant (n = 5, P = 0.149).
Effects of exogenous H2O2 on normal colonic
muscle contractility.
Incubation of muscle strips with 1 mM H2O2
suppressed the spontaneous contractions in the proximal, middle, and
distal colon within 90 min in 69% of the strips (Fig.
5). The response of the strips to 2 µM
ACh decreased in a time-dependent pattern when incubated with
the above concentration of H2O2 (Fig.
6A). The suppression
of the response to ACh by incubation with H2O2
was more marked in the distal than in the proximal or the middle colon (Fig. 6B). In muscle strips from the middle colon, the
response to 2 µM ACh declined smoothly over time when incubated with
0.3 mM H2O2, and it decreased abruptly with 1 mM or higher concentrations of H2O2 (Fig.
6A). In contrast, the responsiveness of normal strips (used
as time controls) to ACh was maintained or even slightly increased
during the 360-min period (Fig. 6A). Additionally, in an
attempt to simulate continuous H2O2 production
as it may happen during an inflammatory episode, we superfused the
strips with H2O2 at constant rates, rather than
replacing H2O2 in the muscle bath at fixed
intervals. At a superfusion rate of 60 nmol/min for up to 3 h,
H2O2 did not alter the response to ACh (data
not shown). However, at superfusion rates of 200 nmol/min and higher, H2O2 induced a time-dependent suppression of
response to ACh, similar to that in the previous experiments.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of 90 min incubation with 1 mM
H2O2 on spontaneous phasic contractions in the
proximal (A), middle (B), and distal normal colon
(C).
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
A: time course of the suppression of
ACh-induced contraction in normal rat middle colon circular muscle by
H2O2. Successive response to 2 µM ACh,
measured after 30, 60, 90, 180, and 360 min of incubation with 0.3, 1, and 3 mM H2O2. The responsiveness of strips
incubated in parallel with Krebs solution did not decrease throughout
the experiment. Values are means ± SE of 5 independent
experiments. B: suppression of ACh-induced contraction by 1 mM H2O2 in strips from proximal, middle, and
distal colon.
|
|
After incubation and washout of H2O2 and a
recovery period of 3 h, the spontaneous activity and the
responsiveness to ACh reappeared in the middle colon (Fig.
7). The spontaneous contractions were
primarily GCs without any superimposed or independent phasic contractions. The frequency of GCs in the postincubation period (0.8 ± 0.2 contractions/min) was significantly greater than that before incubation (0.3 ± 0.1 contractions/min; P < 0.05). The response to ACh showed partial recovery at this time
(Fig. 7B).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of H2O2 on phasic
contractions, GCs, and ACh-induced contractions. The spontaneous
contractions before incubation with H2O2 are
shown at left. After incubation with 1 mM
H2O2, very little or no activity is observed
(middle). However, 3 h after washout of
H2O2 the GCs reappear at a higher frequency
(A, right) and responsiveness to ACh is partially
recovered (B, right).
|
|
To rule out the influence of inhibitory neurons and other submucosal
elements in the inhibitory effect of H2O2, we
repeated these experiments in strips without the mucosa and the
submucosa and in the presence of 0.3 µM TTX or 0.1 mM
NG-nitro-L-arginine
(L-NNA). Incubation with
H2O2 in these strips suppressed the contractile
response to 2 µM ACh with a time course similar to that found in
strips containing the mucosa and the submucosa (n = 3, data not shown).
Finally, we determined whether the suppression of contractility to ACh
in the inflamed strips was due to the presence of
H2O2 in the musculature at the time of the
experiments or to a fundamental change in excitation-contraction
coupling due to the long-term exposure of the muscle to
H2O2. We found that the addition of 5,000 U/ml
catalase to inflamed muscle strips did not immediately reverse the
suppression of response to 100 µM ACh. The contractile response after
catalase was 98 ± 4.3% of that in the untreated strips
(n = 8, P > 0.05).
 |
DISCUSSION |
Types of spontaneous in vitro circular muscle contractions in rat
colon.
The circular muscle of the colon of higher species, such as dogs and
humans, generates rhythmic phasic contractions, GMCs, and tone in the
intact conscious state (39, 40). However, in muscle bath,
the colons of these species generate only the phasic contractions and
tone. Our present findings show that, unlike the higher species, muscle
strips from the rat middle colon generate spontaneous GCs. In muscle
bath, the propagation of GCs cannot be examined, but recent studies by
Li et al. (25) show that the GCs do propagate in the
intact rat colon and are therefore similar to the GMCs found in the dog
and the human colon (2, 20, 30, 34, 44). We did not
concurrently record myoelectric activity, but the duration and
frequency of phasic contractions and GCs correspond to those of short
and long spike bursts, respectively, as previously reported by Castex
et al. (5).
Compared with the intact conscious state, a basic change in the
generation of different types of spontaneous colon contractions seems
to occur when the circular muscle strips are prepared for muscle bath.
The intact rat colon in the conscious state generates GCs in the
proximal, middle, and distal colon at a frequency of ~0.5-0.7
GCs/min (25). In addition, the rhythmic phasic
contractions are present at about the same amplitude (but much smaller
than that of GCs) and frequency (~12 contractions/min) in all three segments (25). In contrast, in muscle bath, the
spontaneous GCs were noted predominantly in the middle colon and their
frequency was about one-half of that seen in the intact conscious state (25). The frequency of rhythmic phasic contractions was
also about one-half of that seen in the intact state. In addition, the
amplitude of phasic contractions decreased dramatically from the
proximal to the middle to the distal colon (~35-fold decrease in
amplitude). The precise reasons for these changes from the intact
conscious state to muscle strips are not known, but they are likely to
be due to the effects of dissection and the nutritional environment of
the muscle bath compared with the nutrition derived from regular blood
flow. The muscle strips from all three regions of the colon were
prepared in an identical manner.
Several studies (10, 11, 24, 47, 50) have reported that
the pacemaker cells for the generation of slow waves in the colonic
muscularis reside at the submucosal border. These cells have been
identified as the interstitial cells of Cajal (ICC). Accordingly, the
phasic contractions were absent in our muscle strips when the mucosa
and the submucosa, along with the ICC layer, were removed. However, the
GCs were not affected by the removal of the submucosal border
containing the ICC, confirming that they are not regulated by slow
waves or ICC (37). This finding also confirms that the GCs
are distinct from the phasic contractions and, particularly, they are
not due to tetany produced by phasic contractions.
The rat colon strips did not exhibit spontaneous variations in tone.
Smaller doses of ACh primarily accentuated the amplitude of phasic
contractions. Larger doses, however, increased the muscle tone, and the
phasic contractions were superimposed on it. In the intact conscious
state, the rat colon also does not show spontaneous variations in tone
(25). The lower doses of ACh may therefore be more
physiological in reference to the generation of spontaneous colonic
contractions. It has not been feasible to measure the concentration of
ACh at the neuroeffector junction to determine the physiological range
of neurotransmitter release. However, because the smaller doses of ACh
in our experiments mimicked the generation of phasic contractions seen
in intact rats, we used these concentrations (2 µM) to evaluate the
effect of inflammation in muscle strips. Because the cellular
mechanisms of initiation of tone, phasic contractions, and GCs differ
(42, 43) and their motility functions are also markedly
different, our findings suggest that the effects of pharmacological
agents should be examined for each type of contraction separately.
Effects of inflammation on spontaneous and ACh-induced contractions
in DSS-treated and HLA-B27 rats.
The frequency of spontaneous in vitro phasic contractions was not
affected by inflammation in the rat colon, which is similar to that
reported for the canine colon (26). However, the response to ACh was significantly suppressed in the rat colon inflamed by DSS
treatment. This is similar to the suppression of muscarinic response in
muscle strips from the human ulcerative colitis colon and
experimentally inflamed rabbit and dog colons (8, 26, 28,
48). The response to ACh was also suppressed in circular muscle
strips from the colon of HLA-B27 rats, as previously reported by
Venkova and Greenwood-Van Meerveld (52). Grossi et al.
(14) found that the suppression of in vitro contractility
of the circular muscle in the colon does not depend on the method of
inducing inflammation. In the intact conscious state (27)
and also in enzymatically single dispersed cells (45), the
muscarinic response of circular muscle cells is suppressed in the
inflamed colon.
The response to ACh was suppressed in muscle strips even when they were
devoid of submucosa and ICC and the inhibitory neural input was blocked
by TTX or L-NNA. Therefore, it seems that the suppression
of contractility during inflammation is due to a defect in
excitation-contraction coupling in smooth muscle cells. However, this
does not preclude additional abnormalities due to neural dysfunction
and damage to ICC. Other studies (26, 36) found that ICC
processes are damaged but not totally absent in ulcerative colitis and
in the inflamed canine colon.
Li et al. (25) reported recently that the spontaneous
colonic phasic contractions and GMCs in intact conscious rats are blocked by atropine, whereas in vitro these contractions are
insensitive to muscarinic receptor blockade. It therefore seems that
the stimulus for the spontaneous contractions differs in vivo and in
vitro. The stimulus for in vitro spontaneous contractions has not been identified, but it may be stretch. Li et al. (25) also
found that the overall contractile activity is reduced in the inflamed rat colon. This is consistent with our findings that the response to
ACh is suppressed in muscle strips. The lack of suppression of
spontaneous phasic contractions in vitro may be because these contractions are independent of release of ACh and the signaling pathways for ACh and stretch-induced contractions may differ. However,
in the intact state, a decrease in neurotransmitter release may also
contribute to the suppression of overall contractile activity. Myers et
al. (33) also found a decrease in rat circular muscle
contractility in response to ACh in colonic inflammation induced by
mucosal exposure to acetic acid.
H2O2 production in muscularis and its role
in alteration of colon contractions in inflammation.
Previous studies (13, 22, 46) have reported that
H2O2 production is increased in the mucosa of
patients with ulcerative colitis as well as in animal models of
inflammation. Our findings show that H2O2
production is also increased in the muscularis of DSS-treated rats,
which makes this ROS a potential candidate to contribute to some of the
alterations in motility seen during inflammation. This role was
supported by the finding that incubation of the normal muscle strips
with H2O2 suppressed ACh-induced response similar to that seen in strips from the inflamed colon. Interestingly, after incubation with H2O2 and washout, the
frequency of GCs was significantly increased at the same time that the
spontaneous phasic contractions and the response to ACh were
suppressed. Both of these alterations in motility patterns are seen in
colonic inflammation in dogs and in patients with ulcerative colitis
(7, 21, 44). It is unlikely, however, that the total
effect of inflammation on motility patterns is due to a single
inflammatory response mediator. Several cytokines and lipids, such as
IL-1
and platelet-activating factor, are also increased in the
muscularis during inflammation, and they may contribute to the motility
abnormalities as well (18, 29, 31).
H2O2 production also increased about twofold in
the HLA-B27 rats, but it did not reach statistical significance. The
production of H2O2 in spontaneous inflammation
may be less, but it may occur over a longer period of time. Our data
with normal strips showed that the effect of
H2O2 on circular muscle contractions depends on
both the concentration of H2O2 as well as the
duration of incubation.
Previous studies (16, 32, 51) investigating the effect of
oxidative stress on gut smooth muscle examined the direct and immediate
effect of addition of oxidants to the muscle bath. Our aim was to
determine whether H2O2 alters the
excitation-contraction coupling on its exposure to the smooth muscle
cells. Therefore, in our experiments, H2O2 was
washed out before the addition of ACh. We found that incubation with
H2O2 time and concentration dependently
suppressed the response to ACh. The suppression was, however,
reversible over a period of a few hours. Our hypothesis that
H2O2 causes a fundamental longer lasting change
in excitation-contraction coupling was confirmed by the finding that
the addition of catalase to inflamed muscle strips did not immediately
reverse the suppression of contractility to ACh. Also, the addition of
H2O2 to the muscle bath altered the
excitation-contraction coupling for several hours after
H2O2 was washed out. A similar situation may
exist for other inflammatory response mediators.
The exposure to H2O2 had a greater effect on
the distal colon than on the proximal or the middle colon. This is in
agreement with the reports (51) that the catalase activity
decreases from the proximal to the middle colon. Therefore, the distal
colon has less defense against the deleterious effects of
H2O2. Nitric oxide, which has been proposed as
both a protective and a deleterious molecule in inflammation, is also
more abundant in the myenteric plexus of the proximal colon than that
of the distal colon (49).
It should be noted, however, that H2O2 is only
an intermediate, and not especially reactive, compound in the cascade
of ROS formation that begins with the generation of superoxide anion. It is therefore likely that the cytotoxic effects of
H2O2 seen in our study were actually produced
by the breakdown products of H2O2, such as
hydroxyl radical and hypochlorous acid, which are considerably more reactive.
Our findings show that the rat colon may be a useful model to study the
in vitro cellular mechanisms of GCs. Particularly, the effects of
genetic manipulation on the stimulation or suppression of GCs can be
investigated in transgenic and gene knockout rodent models. So far,
this is the only species reported that generates GCs in muscle bath. A
decrease in the frequency of GCs has been associated with constipation
(3) and an increased frequency with diarrhea (7, 21,
44).
Our findings also show that H2O2 production is
increased in the muscularis during inflammation, which may be a
contributing factor to the suppression of ACh-induced contractions seen
in inflammation. In addition, incubation of normal muscle strips with
H2O2 suppresses phasic contractions and
generation of tone but increases the frequency of GCs. These effects
are similar to those seen in patients with ulcerative colitis and in
experimental models of inflammation (7, 21, 44).
H2O2 may therefore be one of the inflammatory
response mediators that contributes to motility defects and digestive
dysfunction in acute and chronic inflammation of the colon.
 |
ACKNOWLEDGEMENTS |
This study was suppported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-32346 and by the Department of
Veterans Affairs Medical Research Service.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: S. K. Sarna, General Surgery, Medical College of Wisconsin-FWC, 9200 West Wisconsin Ave., Milwaukee, WI 53226 (E-mail:
ssarna{at}mcw.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 5 April 2000; accepted in final form 8 November 2000.
 |
REFERENCES |
1.
Aiko, S,
and
Grisham MB.
Spontaneous intestinal inflammation and nitric oxide metabolism in HLA-B27 transgenic rats.
Gastroenterology
109:
142-150,
1995[ISI][Medline].
2.
Bassotti, G,
and
Gaburri M.
Manometric investigation of high-amplitude propagated contractile activity of the human colon.
Am J Physiol Gastrointest Liver Physiol
255:
G660-G664,
1988[Abstract/Free Full Text].
3.
Bassotti, G,
Gaburri M,
Imbimbo BP,
Rossi L,
Farroni F,
Pelli MA,
and
Morelli A.
Colonic mass movements in idiopathic chronic constipation.
Gut
29:
1172-1179,
1988.
4.
Bradford, MM.
A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein dye binding.
Anal Biochem
72:
248-252,
1976[ISI][Medline].
5.
Castex, N,
Fioramonti J,
Fargeas MJ,
More J,
and
Bueno L.
Role of 5-HT3 receptors and afferent fibers in the effects of mast cell degranulation on colonic motility in rats.
Gastroenterology
107:
976-984,
1994[ISI][Medline].
6.
Castro, GA,
and
Arntzen CJ.
Immunophysiology of the gut: a research frontier for integrative studies of the common mucosal immune system.
Am J Physiol Gastrointest Liver Physiol
265:
G599-G610,
1993[Abstract/Free Full Text].
7.
Chaudhary, NA,
and
Truelove SC.
Human colonic motility: a comparative study of normal subjects, patients with ulcerative colitis, and patients with the irritable colon syndrome. I. Resting patterns of motility.
Gastroenterology
40:
1-36,
1961[ISI].
8.
Cohen, JD,
Kao HW,
Tan ST,
Lechago J,
and
Snape WJ, Jr.
Effect of acute experimental colitis on rabbit colonic smooth muscle.
Am J Physiol Gastrointest Liver Physiol
251:
G538-G545,
1986[ISI][Medline].
9.
Cowles, VE,
and
Sarna SK.
Relation between small intestinal motor activity and transit in secretory diarrhea.
Am J Physiol Gastrointest Liver Physiol
259:
G420-G429,
1990[Abstract/Free Full Text].
10.
Du, C,
and
Conklin JL.
Origin and slow waves in the isolated proximal colon on the cat.
J Auton Nerv Syst
28:
167-178,
1989[ISI][Medline].
11.
Durdle, NG,
Kingma YJ,
Bowes KL,
and
Chambers MM.
Origin of slow waves in the canine colon.
Gastroenterology
84:
375-382,
1983[ISI][Medline].
12.
Ferre, JP,
and
Ruckebusch Y.
Myoelectrical activity and propulsion in the large intestine of fed and fasted rats.
J Physiol (Lond)
362:
93-106,
1985[Abstract].
13.
Grisham, MB.
Oxidants and free radicals in inflammatory bowel disease.
Lancet
344:
859-861,
1994[ISI][Medline].
14.
Grossi, L,
McHugh K,
and
Collins SM.
On the specificity of altered muscle function in experimental colitis in rats.
Gastroenterology
104:
1049-1056,
1993[ISI][Medline].
15.
Hardcastle, JD,
and
Mann CV.
Study of large bowel peristalsis.
Gut
9:
512-520,
1968[ISI][Medline].
16.
Hermann, BW,
Cullen JJ,
Ledlow A,
Murray JA,
and
Conklin JL.
The effect of peroxynitrite on sphincter of Oddi motility.
J Surg Res
81:
55-58,
1999[ISI][Medline].
17.
Holdstock, DJ,
Misiewicz JJ,
Smith T,
and
Rowlands EN.
Propulsion (mass movements) in the human colon and its relationship to meals and somatic activity.
Gut
11:
91-99,
1970[ISI][Medline].
18.
Hurst, SM,
and
Collins SM.
Mechanism underlying tumor necrosis factor-alpha suppression of morenpinephrine release from rat myenteric plexus.
Am J Physiol Gastrointest Liver Physiol
266:
G1123-G1129,
1994[Abstract/Free Full Text].
19.
Jouët, P,
Sarna SK,
Singaram C,
Ryan RP,
Hillard CJ,
Telford GL,
Fink J,
and
Henderson JD.
Immunocytes and abdominal gastrointestinal motor activity during ileitis in dogs.
Am J Physiol Gastrointest Liver Physiol
269:
G913-G924,
1995[Abstract/Free Full Text].
20.
Karaus, M,
and
Sarna SK.
Giant migrating contractions during defecation in the dog colon.
Gastroenterology
92:
925-933,
1987[ISI][Medline].
21.
Kern, FJ,
Almy TP,
Abbot FK,
and
Bogdonoff MD.
The motility of the distal colon in nonspecific ulcerative colitis.
Gastroenterology
19:
492-503,
1951[ISI].
22.
Keshavarzian, A,
Sedghi S,
Kanofsky J,
List T,
Robinson C,
Ibrahim C,
and
Winship D.
Excessive production of reactive oxygen metabolites by inflamed colon: analysis by chemiluminescence probe.
Gastroenterology
103:
177-185,
1992[ISI][Medline].
23.
Kruis, W,
Azpiroz F,
and
Phillips SF.
Contractile patterns and transit of fluid in canine terminal ileum.
Am J Physiol Gastrointest Liver Physiol
249:
G264-G270,
1985[ISI][Medline].
24.
Lee, JCF,
Thuneberg L,
Berezin I,
and
Huizinga JD.
The generation of slow waves in membrane potential is an intrinsic property of interstitial cells of Cajal.
Am J Physiol Gastrointest Liver Physiol
277:
G409-G423,
1999[Abstract/Free Full Text].
25.
Li, M,
Johnson CP,
Adams MB,
and
Sarna SK.
Ileal and colonic motor activity in dextran sodium sulfate-induced colitis in conscious rats (Abstract).
Gastroenterology
116:
G4470,
1999.
26.
Lu, G,
Qian X,
Berezin I,
Telford GL,
Huizinga JD,
and
Sarna SK.
Inflammation modulates in vitro colonic myoelectric and contractile activity and interstitial cells of Cajal.
Am J Physiol Gastrointest Liver Physiol
273:
G1233-G1245,
1997[Abstract/Free Full Text].
27.
Lu, G,
and
Sarna SK.
In vivo modulation of Ca2+ mobilization by colonic inflammation in regulating phasic contractions (Abstract).
Gastroenterology
110:
A710,
1996.
28.
Lu, G,
Vanderwinden JM,
Rumessen JJ,
Telford GL,
and
Sarna SK.
Electrophysiological, morphological and ultrastructural changes in ulcerative colitis (UC) and idiopathic constipation (IC) (Abstract).
Gastroenterology
114:
A796,
1998.
29.
Main, C,
Blennerhasset P,
and
Collins SM.
Human recombinant interleukin 1 beta suppresses acetylcholine release from rat myenteric plexus.
Gastroenterology
104:
1648-1654,
1993[ISI][Medline].
30.
Malcolm, A,
and
Camilleri M.
Coloanal motor coordination in association with high-amplitude colonic contractions after pharmacological stimulation.
Am J Gastroenterol
95:
715-719,
2000[ISI][Medline].
31.
Morteau, O,
More J,
Pons L,
and
Bueno L.
Platelet-activating factor and interleukin 1 are involved in colonic dysmotility in experimental colitis in rats.
Gastroenterology
104:
47-56,
1993[ISI][Medline].
32.
Moummi, C,
Gullikson GW,
Grisham MB,
and
Gaginella TS.
Differential effects of reactive oxygen metabolites on neurally stimulated and nonstimulated guinea pig ileum.
J Pharmacol Exp Ther
256:
266-271,
1991[Abstract].
33.
Myers, BS,
Martin JS,
Dempsey DT,
Parkman HP,
Thomas RM,
and
Ryan JP.
Acute experimental colitis decreases colonic circular smooth muscle contractility in rats.
Am J Physiol Gastrointest Liver Physiol
273:
G928-G936,
1997[Abstract/Free Full Text].
34.
Narducci, F,
Bassotti G,
Gaburri M,
and
Morelli A.
Twenty-four hour manometric recording of colonic motor activity in healthy man.
Gut
28:
17-25,
1987[Abstract].
35.
Ravindranath, V.
Animal models and molecular markers for cerebral ischemia reperfusion injury in brain.
Methods Enzymol
233:
610-618,
1994[ISI][Medline].
36.
Rumessen, JJ.
Ultrastructure of interstitial cells of Cajal at the colonic submuscular border in patients with ulcerative colitis.
Gastroenterology
111:
1447-1455,
1996[ISI][Medline].
37.
Sarna, SK.
Giant migrating contractions and their myoelectric correlates in the small intestine.
Am J Physiol Gastrointest Liver Physiol
253:
G697-G705,
1987[Abstract/Free Full Text].
38.
Sarna, SK.
In vivo myoelectric activity: methods, analysis and interpretation.
In: Handbook of Physiology. The Gastrointestinal System. Motility and Circulation. Bethesda, MD: Am. Physiol. Soc, 1989, sect. 6, vol. I, pt. 2, chapt. 1, p. 817-863.
39.
Sarna, SK.
Physiology and pathophysiology of colonic motor activity (Part one of two).
Dig Dis Sci
36:
827-862,
1991[ISI][Medline].
40.
Sarna, SK.
Physiology and pathophysiology of colonic motor activity (Part two of two).
Dig Dis Sci
36:
998-1018,
1991[ISI][Medline].
41.
Sarna, SK.
In vivo signal-transduction pathways to stimulate phasic contractions in normal and inflamed ileum.
Am J Physiol Gastrointest Liver Physiol
274:
G618-G625,
1998[Abstract/Free Full Text].
42.
Sarna, SK.
Molecular mechanisms of giant migrating contractions (GMCs) in normal and inflamed ileum (Abstract).
Gastroenterology
116:
G4665,
1999.
43.
Sarna, SK.
Differential signal transduction pathways to stimulate colonic giant migrating and phasic contractions (Abstract).
Gastroenterology
118:
4404,
2000.
44.
Sethi, AK,
and
Sarna SK.
Colonic motor activity in acute colitis in conscious dogs.
Gastroenterology
100:
954-963,
1991[ISI][Medline].
45.
Shi, X-Z,
and
Sarna SK.
Impairment of Ca2+ mobilization in circular muscle cells of the inflamed colon.
Am J Physiol Gastrointest Liver Physiol
278:
G234-G242,
2000[Abstract/Free Full Text].
46.
Simmonds, NJ,
Allen RE,
Stevens TRJ,
Van Someren RNM,
Blake DR,
and
Rampton DS.
Chemiluminescence assay of mucosal reactive oxygen metabolites in inflammatory bowel disease.
Gastroenterology
103:
186-196,
1992[ISI][Medline].
47.
Smith, TK,
Reed JB,
and
Sanders KM.
Origin and propagation of electrical slow waves in circular muscle of canine proximal colon.
Am J Physiol Cell Physiol
252:
C215-C224,
1987[Abstract/Free Full Text].
48.
Snape, WJ, Jr,
and
Kao HW.
Role of inflammatory mediators in colonic smooth muscle function in ulcerative colitis.
Dig Dis Sci
33 Suppl:
65S-70S,
1988[Medline].
49.
Takahashi, T,
and
Owyang C.
Regional differences in the nitrergic innervation between the proximal and the distal colon in rats.
Gastroenterology
115:
1504-1512,
1998[ISI][Medline].
50.
Torihashi, S,
Gerthoffer WT,
Kobayashi S,
and
Sanders KM.
Identification and classification of interstitial cells in the canine proximal colon by ultrastructure and immunocytochemistry.
Histochemistry
101:
169-183,
1994[ISI][Medline].
51.
Van der Vliet, A,
Tuinstra TJ,
and
Bast A.
Modulation of oxidative stress in the gastrointestinal tract and effect on rat intestinal motility.
Biochem Pharmacol
38:
2807-2818,
1989[ISI][Medline].
52.
Venkova, K,
and
Greenwood-Van Meerveld B
Dysfunction of colonic circular muscle in HLA-B27 rats (Abstract).
Dig Dis Sci
43:
1590,
1998.
Am J Physiol Gastrointest Liver Physiol 280(4):G546-G554