Inflammation modulates in vitro colonic myoelectric and
contractile activity and interstitial cells of Cajal
G.
Lu1,
X.
Qian1,
I.
Berezin2,
G. L.
Telford1,
J. D.
Huizinga2, and
S. K.
Sarna1
1 Departments of Surgery and
Physiology, Medical College of Wisconsin and Zablocki Veterans Affairs
Medical Center, Milwaukee, Wisconsin 53226; and
2 Intestinal Disease Research
Unit, McMaster University, Hamilton, Ontario, Canada L8N
3ZS
 |
ABSTRACT |
Inflammation suppresses phasic contractile
activity in vivo. We investigated whether inflammation also suppresses
in vitro phasic contractile activity and, if so, whether this could in part be due to the alteration of specific slow wave characteristics and
morphology of the interstitial cells of Cajal (ICC). Circular muscle
strips were obtained from normal and inflamed distal canine colon.
Inflammation was induced by mucosal exposure to ethanol and acetic
acid. The amplitudes of spontaneous, methacholine-induced, substance
P-induced, and electrical field stimulation-induced contractions were
smaller in inflamed muscle strips than in normal muscle strips.
Inflammation reduced the resting membrane potential and the amplitude
and duration of slow waves in circular muscle cells. Inflammation did
not affect the amplitude of inhibitory junction potentials but did
decrease their duration. Ultrastructural studies showed expansion of
the extracellular space between circular muscle cells, reduction in the
density of ICC and associated neural structures, damage to ICC
processes, vacuolization of their cytoplasm, and blebbings of the
plasma membrane. We conclude that inflammation-induced alterations of
slow wave characteristics contribute to the suppression of phasic
contractions. These alterations may, in part, be due to the damage to
ICC. Inflammation impairs both the myogenic and neural regulation of
phasic contractions.
slow waves; electrical control activity; intracellular recording; inhibitory junction potential; colitis; substance P; acetylcholine
 |
INTRODUCTION |
COLONIC SLOW WAVES REGULATE excitation-contraction
coupling of phasic contractions in vivo and in vitro (2, 26, 27a, 32, 34). The phasic contractions occur only when the membrane
depolarizes beyond an excitation threshold during slow waves. However,
the precise electrical events associated with phasic contractions vary,
depending on the amplitude of these contractions under different experimental conditions. The amplitude of contractions recorded concurrently with intracellular electrophysiological tracings depends
primarily on the amplitude and duration of the plateau potential of the
slow wave (25). The amplitude of these contractions is typically in the
range of 0.25 to 1 g (
1.5 to 3 × 10 mm muscle tissue). These
contractions are not associated with any spike bursts during the
plateau potential and are called spike-independent contractions (25).
When the in vitro contractions are stimulated by the addition of an
agonist such as acetylcholine or substance P to the muscle bath and the
electrical recordings are made concurrently with extracellular
electrodes or sucrose gap, the phasic contractions are associated with
spike bursts superimposed on the plateau potential of the slow waves
(7, 9, 11). These contractions are typically 3-5 g in amplitude
(
1.5 to 3 × 10 mm muscle tissue). The intracellular electrodes cannot be maintained inside the cells with these stronger contractions. Similarly, the phasic contractions recorded in vivo in
the intact animals or in the ex vivo colon are always accompanied by
spike bursts recorded by extracellular electrodes (17, 26, 28, 29).
These are spike-dependent contractions. These contractions are
typically 75-150 g in amplitude (
5 × 15 mm muscle
tissue). The amplitude of spike-dependent contractions is correlated
with the number, frequency, and duration of the spike bursts.
Previous reports have indicated that spike-dependent in vivo phasic
contractions in patients with ulcerative colitis (15, 35) as well
as in animal models of inflammation (31) are suppressed during
inflammation. The generation of isometric tone in response to stretch
and cholinergic stimulation is also decreased in muscle strips taken
from patients with ulcerative colitis and from animal models of
inflammation (8, 10, 33). However, the increase in isotonic tone is a
sustained contraction and it does not depend on rhythmic slow wave
depolarizations. The excitation-contraction coupling and signal
transduction for the tonic and phasic contractions are likely to be
different. It is not known whether colonic inflammation also suppresses
the in vitro spike-independent phasic contractions. If so, does
inflammation alter the slow wave characteristics that may account, at
least partially, for the suppression of phasic contractions?
Our objectives in this study were to determine the effects of colonic
inflammation on spontaneous and agonist-induced in vitro phasic
contractions as well as the intracellular electrical activity. The
pacemaker cells for slow waves in the colon are thought to be the
interstitial cells of Cajal (ICC) associated with a specialized layer
of circular muscle cells near the submucosal border (1, 20, 30, 40,
44). These cells are interconnected by a network of gap junctions. We
therefore also investigated whether inflammation damages the ICC and
their processes, which could affect the characteristics of colonic slow
waves and the excitation-contraction coupling in the circular muscle
layer.
 |
EXPERIMENTAL METHODS |
Intracellular recordings and contractile activity.
The dogs were anesthetized with 30 mg/kg pentobarbital sodium. We
removed 5-cm-long segments of the distal colon, 5-10 cm orad to
the peritoneal reflection, from 25 normal dogs and 17 dogs in which
colonic inflammation was induced by mucosal exposure to ethanol and
acetic acid. In three additional dogs, the tissues were removed 30 min
after the mucosal exposure to ethanol and acetic acid to determine if
mucosal injury itself had any effect on slow wave and contractile
parameters. The mucosa was removed, and a full-thickness (1.5 × 10 mm) muscle strip was dissected parallel to the long axis of the
circular muscle cells. The strip was pinned down in a muscle bath with
the cross-sectional surface facing up. One-half of the length of the
muscle strip was immobilized by pinning along the edges to record
intracellular electrical activity, as described previously (23). The
loose end of the other half was connected to an isometric force
transducer (Grass Instruments, Quincy, MA) to record contractile
activity concurrently.
The muscle bath volume was 2 ml. The bath was perfused continuously
with prewarmed and oxygenated Krebs solution at 5 ml/min (137.4 mM
Na+, 5.9 mM
K+, 2.5 mM
Ca2+, 1.2 mM
Mg2+, 134 mM
Cl
, 15.5 mM
1.2 mM
H2
and 11.5 mM glucose). The solution was bubbled with 95%
O2-5% CO2 to maintain a pH of 7.4. The
bath temperature was maintained at 38 ± 0.5°C. The muscle
strips were stretched to 1 g and left to equilibrate for at least 2 h.
Thereafter, the muscle strips were stretched incrementally until the
amplitude of spontaneous phasic contractions was maximal.
Circular smooth muscle cells were impaled with glass microelectrodes
(20-35 M
) filled with 3 M KCl. Recordings were accepted when a
sharp drop in voltage greater than
55 to
35 mV was
observed, depending on the distance of the electrode from the border of the circular muscle layer and the submucosa. Membrane potentials were
amplified (model Duo 773, World Precision Instruments, New Haven, CT)
and displayed on an oscilloscope (Tektronix 5111A, Tektronix,
Beaverton, OR). The myoelectric and contractile activities were
recorded on an FM tape recorder (Hewlett-Packard 3968A, Santa Clara,
CA) for later off-line analysis. A square wave stimulator (model S48,
Grass Instruments) and a stimulus isolation unit (model SIU5B, Grass
Instruments) were used to apply electrical field stimulation (EFS) to
the muscle strips. The EFS was applied through two platinum electrode
wires oriented parallel to the muscle strip.
Induction of colonic inflammation.
An intraluminal catheter (ID, 2.6 mm; OD, 4.9 mm) was implanted
surgically in the proximal colon under general pentobarbital sodium
anesthesia. The dogs were allowed to recover for at least 7 days. The
colon was cleansed by infusing 1 liter Colyte through the intraluminal
catheter. The dogs were fasted overnight. The next day, the dogs were
anesthetized with 10 mg/kg Telazol (Fort Dodge Laboratories, Ford
Dodge, IA), and 75 ml of 95% ethanol were infused intraluminally at 5 ml/min. Ten minutes later, 10 ml of 75% acetic acid were infused over
a 1-min period. Simultaneously, the same volumes of ethanol and acetic
acid were infused through a Silastic tube inserted anally. The end of
the anal tube was 15 cm from the external anal sphincter. Five minutes
after acetic acid infusions concluded, 100 ml of 0.9% saline were
infused through the proximal intraluminal catheter. This method induces
diffuse pan colitis that lasts for ~10 days (31). Two days after the induction of inflammation, the dogs were anesthetized with 30 mg/kg
pentobarbital sodium and the tissue was harvested from the distal
colon, as described above. All dogs had diarrhea on the day of tissue
harvesting. The dogs were given 500 ml of 5% dextrose in Ringer by
daily intravenous infusion to compensate for fluid loss by diarrhea.
Electron microscopy studies.
Three additional dogs in which inflammation was induced as described
above were anesthetized with 30 mg/kg pentobarbital sodium. The abdomen
was opened along the midline, and the mesenteric artery supplying blood
to a 5-cm-long segment in the proximal colon was identified. An
Angiocath was inserted into the artery, and the perfused segment was
identified by infusing 0.9% saline. The mesenteric vein draining blood
from this segment was identified and cut. The segment was then fixed by
perfusing a solution of 2% glutaraldehyde in 0.075 M cacodylate buffer
containing 4.5% sucrose and 1 mM CaCl2, pH 7.4, as rapidly as
possible and until the segment became rigid. The segment was then
removed, opened lengthwise, cleansed, and submerged in the same
glutaraldehyde fixative in a petri dish (19). The tissue was cut into
strips along the circular muscle axis (1.5 × 2 to 3 cm long) and
fixed further in the fixative for 4-5 h at room temperature. The
tissue was then washed two to three times in 0.1 M cacodylate buffer
containing 6% sucrose and 1.25 mM
CaCl2, pH 7.4. Tissues from three
normal colons fixed as described above were used as control.
Measurement of myeloperoxidase activity.
Myeloperoxidase (MPO) was measured as described by Castro et al. (5).
Tissue samples from six normal dogs and six dogs with colonic
inflammation were chosen randomly from the whole group. In addition,
the five tissue samples that showed no spontaneous slow wave activity
in muscle strips were also processed for MPO activity. The MPO was
determined in the lamina propria scraped with a glass slide and in the
muscularis externa separately. The tissues were weighed and homogenized
with hexadecyltrimethylammonium bromide (HTAB) buffer [0.5% HTAB in
50 mM phosphate buffer, pH 6.0, 4°C; 1:20 (wt/vol)]. The
homogenate was freeze thawed three times and then centrifuged at 35,000 g for 30 min. The supernatant was
processed for MPO as described previously (5). One unit of MPO activity
was defined as that degrading 1 µM hydrogen peroxide per minute at
25°C. The MPO activity was expressed per gram of wet tissue.
Test substances.
The following agents were used: acetyl-
-methacholine chloride,
atropine sulfate, phentolamine hydrochloride,
propranolol-DL-hydrochloride, and N
-nitro-L-arginine methyl
ester (L-NAME). All these
substances were purchased from Sigma Chemical (St. Louis, MO),
dissolved in a stock solution of 0.9% saline, and diluted in Krebs
solution. [Sar9,Met(O2)11]substance
P was purchased from Research Biochemicals International (Natick, MA)
and also dissolved as described above.
Data analysis.
The contractile response was quantified as the mean amplitude of phasic
contractions. Each dose of the agonist was infused for 5 min, and the
mean amplitude of contractions was determined during the 5th min of the
infusion and expressed per gram wet weight of the tissue. The
amplitudes were measured from the baseline before the start of infusion
of the test substance. The amplitudes of the spontaneous phasic
contractions were also averaged over 1-min periods. All data are
expressed as means ± SE. One-way analysis of variance with repeated
measures was used for normally distributed data. Kruskal-Wallis one-way
analysis of variance on ranks was used if the data failed the normality
test. Multiple comparisons were done by Student-Newman-Keuls method.
P
0.05 was considered statistically
significant. This study was approved by the Animal Care Committee at
the Zablocki Veterans Affairs Medical Center.
 |
RESULTS |
Effect of mucosal injury on slow wave and contractile parameters.
All muscle strips taken from the colon 30 min after mucosal exposure to
ethanol and acetic acid exhibited spontaneous slow waves and
contractions. The slow waves and contractile parameters of these muscle
strips were not significantly different from those of strips taken from
the normal dogs (Table 1). For all further work, the strips from normal colon were used as control.
Inflammatory modulation of in vitro phasic contractile activity.
All muscle strips from the normal colon exhibited spontaneous phasic
contractions after a 2-h equilibration period (Fig.
1A). Twelve of seventeen muscle strips from the inflamed colon also exhibited spontaneous phasic contractions (Fig.
1B). The remaining five muscle
strips did not show any identifiable contractile activity (Fig.
1C).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Intracellular recordings indicate reduction in resting membrane
potential and amplitude and duration of slow waves in inflamed colon
(B) compared with normal colon
(A). The amplitude of spontaneous
phasic contractions in inflamed colon was also decreased. Tracings in
C show absence of slow wave activity
and spontaneous contractions observed in 5 of 17 inflamed muscle
strips.
|
|
The mean weight of the normal muscle strips (6.6 ± 0.2 mg;
n = 25) was not different from that of
the inflamed muscle strips (7.0 ± 0.3 mg;
n = 17;
P > 0.05). The mean amplitude of the
spontaneous phasic contractions in the inflamed muscle strips (17.7 ± 2.6 mg/mg wet tissue wt; n = 12)
was significantly less than that in the normal muscle strips (77.2 ± 3.6 mg/mg wet tissue wt; n = 25;
P < 0.05). However, the frequency of
the spontaneous phasic contractions was not different between the
inflamed and the normal muscle strips (4.3 ± 0.3 vs. 4.2 ± 0.3 cycles/min, respectively).
Methacholine at 10
9 to
10
5 M and substance P at
10
11 to 5 × 10
6 M dose dependently
increased the amplitude of phasic contractions in both normal and
inflamed muscle strips (Fig. 2). The
response in the inflamed muscle strips was significantly less than that in the normal strips (Fig. 2, P < 0.05 for methacholine as well as substance P).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 2.
Methacholine (A) and substance P
(B) dose dependently increased the
amplitude of phasic contractions in normal and inflamed muscle strips.
However, the response in inflamed muscle strips was less than that in
normal strips at all doses.
|
|
Inflammatory modulation of slow wave activity.
The thickness of the circular muscle layer in normal muscle strips was
1.5 ± 0.04 mm (n = 16). The
thickness increased to 1.9 ± 0.06 mm
(n = 11, P < 0.05) in the inflamed muscle
strips. The location of the recording site in the circular muscle layer was normalized as the percent distance from the submucosal border. Slow
waves were recorded at 5%, 50%, and 95% of the circular muscle thickness from the submucosal border. The resting membrane potential in
the inflamed muscle strips was significantly less than that in the
normal muscle strips at 5% and 50% locations, but not at the 95%
location in the circular muscle layer (Fig.
3A). The
percent decrease in the resting membrane potential from the 5% to 95% distance from the submucosal border was significantly greater in the
normal (38 ± 3%; n = 4) than in
the inflamed muscle strips (25 ± 2%;
n = 4;
P < 0.05).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
A: resting membrane potentials
decreased from the 5% to the 95% distance in circular muscle layer
from submucosal border in both normal and inflamed muscle strips.
Inflammation also decreased resting membrane potentials at the 5% and
50% distances but not at the 95% distance.
B: tracings of slow wave activity from
circular muscle at the 5% (a), 50%
(b), and 95%
(c) distances from myenteric
border.
|
|
Whereas all normal muscle strips exhibited spontaneous slow wave
activity (Fig. 1A), the slow waves
were absent in 5 of 17 inflamed muscle strips (Fig.
1C). The resting membrane potentials in these strips at 5%, 50%, and 95% distances were, however, not significantly different from those in the 12 muscle strips that exhibited spontaneous slow waves (Table 2,
P > 0.05). The muscle strips that
had no slow waves also showed no spontaneous contractions (Fig.
1C).
The mean amplitude of the slow waves in the inflamed muscle
strips was significantly less than that in the normal muscle strips at
the 5%, 50%, and 95% distance in the circular muscle layer (Table
3). The mean maximum attained level of
depolarization decreased significantly at the 5% distance in the
circular muscle layer during inflammation, but there was no significant
change at the 50% and 95% distances (Table 3).
The mean duration of slow waves at 50% amplitude was
significantly shorter in the inflamed strips than in the normal strips at the 5% and 50% distances in the circular muscle layer (Table 3).
The slow wave duration at the 95% distance could not be measured reliably due to the instability and small amplitude of slow waves at
this location. However, the frequency of slow waves in the inflamed
muscle strips (4.3 ± 0.3 cycles/min) was not different from that in
the normal strips (4.2 ± 0.3 cycles/min). The slow waves associated
with spontaneous contractions were not superimposed with spikes in
normal or inflamed muscle strips.
Effect of inflammation on EFS-induced contractions.
EFS at 5, 10, and 15 Hz, with pulse duration of 0.35 ms,
amplitude of 150 V, and train duration of 1 min, stimulated phasic contractions that increased in amplitude with frequency (Fig. 4A). The
amplitude of EFS-induced contractions in inflamed muscle strips was
significantly less than that in normal muscle strips at all frequencies
of stimulation (Fig. 4A). Atropine
(1 µM) significantly reduced the amplitude of contractions in both
normal and inflamed muscle strips (Fig. 4,
B and
C). In contrast,
L-NAME (1 µM) had no
significant effect on the contractile response to EFS in either case
(Fig. 4, B and
C).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
A: EFS at 5, 10, and 15 Hz frequency
dependently increased the amplitude of phasic contractions in normal
and inflamed muscle strips. Response in inflamed strips was
significantly less than in normal muscle strips.
B and
C: atropine significantly inhibited
amplitude of phasic contractions in both normal (B)
and inflamed (C) strips, but
N -nitro-L-arginine
methyl ester (L-NAME) had no
significant effect.
|
|
Effect of inflammation on inhibitory junction potentials.
Under nonadrenergic, noncholinergic (NANC) conditions (1 µM atropine,
1 µM phentolamine, and 1 µM propranolol), EFS at frequencies of 1, 5, 10, 15, 20, 25, and 30 Hz, with pulse duration of 0.35 ms, amplitude
of 150 V, and train duration of 1 s, induced inhibitory junction
potentials (IJPs) in all normal muscle strips and in 12 of 17 inflamed
muscle strips (Fig. 5,
A and
B). The IJPs were blocked almost
completely by 1 µM L-NAME, as
has been reported previously in the canine colon (Ref. 39; data not
shown). The amplitude (Fig.
6A) and
duration (Fig. 6B) of the IJPs were
frequency dependent in both the normal and the inflamed muscle strips.
There was no significant difference in the amplitude of IJPs between the normal and the inflamed strips at any frequency. However, the
duration of IJPs was shorter in the inflamed muscle strips compared
with the normal muscle strips. The 5 of 17 muscle strips that did not
show slow waves (Fig. 1C) had no
IJPs at any frequency of stimulation (Fig.
5C).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibitory junction potentials (IJPs) in normal
(A) and inflamed
(B) muscle strips in frequency range
of 1-30 Hz are shown. C: 5 of 17 inflamed muscle strips with no slow wave activity also did not show
IJPs.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
A: amplitude of IJPs increased
dependently with frequency in normal and inflamed muscle strips. There
was no difference in IJP amplitude between normal and inflamed strips
at any frequency. B: duration of IJPs
also increased dependently with frequency in normal and inflamed muscle
strips. However, duration of IJPs in inflamed muscle strips was
significantly less than that in normal strips at all frequencies.
|
|
Effect of inflammation on MPO activity.
The MPO activity in the muscularis externa and the lamina propria of
the inflamed muscle strips that exhibited spontaneous slow waves with
altered characteristics was significantly greater than that in normal
muscle strips (Table 4). The MPO activity in the muscularis externa of the inflamed muscle strips without spontaneous slow waves was significantly greater than that in the
muscularis externa of normal muscle strips, as well as in the inflamed
muscle strips with slow waves (Table 2). The MPO activity in the lamina
propria of muscle strips without slow waves was greater than in normal
strips but not different from those that had slow waves (Table 2).
View this table:
[in this window]
[in a new window]
|
Table 4.
MPO activity in muscularis externa and lamina propria of normal and
inflamed colon and colon with acute injury
|
|
Effect of inflammation on ICC and immunocyte infiltration.
At the submucosal surface of the circular muscle layer of the proximal
dog colon, a dense network of overlapping ICC and nerve fibers adjoined
the circular muscle cells (Fig. 7), as has
been reported previously (3). ICC were interconnected to one another and to adjacent innermost circular muscle cells by gap junctions and
close apposition contacts (Figs. 7B,
8B, and
9C).
Elsewhere in the circular muscle layer, the gap junctions were seen
extremely rarely, but these were present between innermost circular
muscle cells (within the 1-7 innermost layers of cells) (Fig.
9B). ICC differed from
smooth muscle cells by a branched pattern of cellular processes and the
presence of numerous mitochondria, free ribosomes, bundles of
intermediate filaments, and caveolae (Fig.
7B). Submucosal fibroblasts were
distinguished from ICC by a lack of caveolae and the presence of a
well-developed cisternae of rough endoplasmic reticulum (Fig.
8A). ICC were situated in close
proximity to nerve fibers (Fig. 7) and frequently formed close contacts
with them.

View larger version (194K):
[in this window]
[in a new window]
|
Fig. 7.
Cross section through circular muscle-submucosa interface of control
dog colon. A: a low-magnification
micrograph shows a typical dense network of interstitial cells of Cajal
(ICC) processes (arrows) and nerves (N) closely associated with
innermost circular muscle layer (CM) present in dog colon. At low
magnification, ICC are distinguished by a high concentration of
mitochondria (m) and the branched character of their processes. Note
absence of immune cells in submucosa. F, fibroblast. Original
magnification, ×5,280. Bar, 2 µm.
B: high-magnification micrograph
showing arrangement between small and large overlapping ICC processes
and nerves in inner border of circular muscle layer. ICC processes
interconnected by gap junction (large arrow) and close apposition
contacts (small arrows) were positioned at close proximity to nerve and
muscle cells. ICC is recognized by its branched profile, presence of
numerous mitochondria, and plasma membrane caveolae (arrowheads).
Original magnification, ×21,150. Bar, 1 µm.
|
|

View larger version (185K):
[in this window]
[in a new window]
|
Fig. 8.
Cross section through the circular muscle-submucosa interface of
inflamed dog colon. A: a
low-magnification micrograph shows an infiltration of neutrophils (Nu)
into circular muscle layer (CM) and the effect of inflammation on ICC
network and ultrastructure of smooth muscle cells. There is injury to
some smooth muscle cells. A damaged muscle cell (dCM) is characterized
by a dark electron-dense nucleus (Nu), light cytoplasm containing
secondary lysosomes (L), and confluent cytoplasmic vacuoles (-x-).
Extracellular space between cells is enlarged and filled with collagen
(Co). Note accumulation of single or confluent lipid droplets (arrows)
in some smooth muscle cells (arrows). Most of the ICC network at the
inner border of circular muscle layer is absent in this section. Only a
small group of ICC processes is seen (boxed area). F, fibroblast.
Original magnification, ×5,870. Bar, 2 µm.
B: high-magnification micrograph of
ICC seen in boxed area in A. There are
no obvious changes to these ICC with respect to mitochondria (m),
filaments, and caveolae (arrowheads). ICC processes still form close
apposition contacts (arrows) with neighboring circular muscle cells,
similar to control tissue. The most conspicuous structural change in
these ICC processes is the presence of membrane-bounded structures
between two ICC process (-x-). Note cytological abnormalities in two
neighboring smooth muscle cells: presence of a large lipid droplet (L)
in cell at top and proliferation of
ribosomes (R) in cell at left.
Original magnification, ×27,600. Bar, 1 µm.
|
|

View larger version (191K):
[in this window]
[in a new window]
|
Fig. 9.
A: low-magnification micrograph of the
inner border of circular muscle layer (CM). No apparent injury to
smooth muscle cells is seen in this region. However, most of the ICC
network at the inner border of the CM has disappeared. Note a group of
injured ICC processes (boxed area) adjacent to innermost circular
muscle cells. N, nerve. Nu, a neutrophil in submucosa. Original
magnification, ×6,700. Bar, 2 µm.
B: high-magnification micrograph of
boxed area in A shows structural abnormalities in injured
ICC processes: single and multiple merging membrane-bounded vacuoles
(v) and plasma membrane blebbings (*). Mitochondria (m) and filament
organization appear normal. ICC processes are connected by close
apposition contacts (arrows). Co, collagen, E, elastin. Original
magnification, ×17,100. Bar, 1 µm.
C: gap junction (arrow) between two
ICC processes from lesion area. Original magnification, ×63,150.
Bar, 200 nm.
|
|
Infiltration of neutrophils and macrophages occurred in the circular
muscle layer and the submucosal region of all sections of the inflamed
tissues (Figs. 8A and
9A). Structural changes were
observed in some circular muscle cells, indicating cell injury. Damaged
smooth muscle cells were mostly restricted to regions with an extensive
infiltration of neutrophils and macrophages (Fig. 8). The structural
abnormalities in injured muscle cells consisted of the appearance of
many secondary lysosomes, large lipid droplets, and empty
membrane-bounded vacuoles (Fig. 8). Some smooth muscle cytoplasm
displayed the proliferation of ribosomes, indicating that a moderate
myofibroblast transformation occurred in some cells (Fig.
8B). In most sections, there were a
few electron lucent smooth muscle cells, indicating irreversible
injury. They were characterized by dark, necrotic nuclei, prominent
vacuolization of cytoplasm, and the partial depletion of myofilaments,
resulting in reduction of electron density of the cytoplasm (Fig.
8A). In some samples, there was an
expansion of the extracellular space between circular muscle cells
(Fig. 8A), which might be due to edema and may account for the increase in the total thickness of the
circular muscle layer.
In all sections of the inflamed tissues, there was an apparent
reduction in the density of ICC at the inner border of the circular
muscle layer (Figs. 8A,
9A, and
10A)
compared with control tissue (Fig.
7A). The network of ICC with
associated neural structures was disrupted. The density of nerve fibers
in the submuscular plexus appeared qualitatively to be decreased. In
most sections, only small groups of ICC processes were detected (Figs.
8 and 9, A and
B). In other sections, several ICC
were undamaged, with normal ultrastructure of their cell bodies (Fig.
10A) and cellular processes (Figs.
8B and
9C). They formed gap junctions (Fig.
9C) and close apposition contacts
with each other and with adjacent smooth muscle cells (Fig.
8B). Most of the structural
abnormalities in ICC were seen in the cellular processes, consisting of
the vacuolization of the cytoplasm (Figs. 9,
A and
B, and
10A), blebbings of the plasma
membrane, and the appearance of multiple membranes in the zones of
contacts between ICC processes (Figs.
8B and
10B). A few ICC processes showed
irreversible damage, which was characterized by a large number of
ruptured mitochondria, the depletion of many thin and intermediate
filaments, the presence of large lipid droplets, and empty
membrane-bounded vacuoles (Fig.
10C). Direct contacts or close appositions between ICC and nerves were rare in the inflamed colon.

View larger version (176K):
[in this window]
[in a new window]
|
Fig. 10.
A: longitudinal section through the
circular muscle-submucosa interface from another inflamed colonic
segment showing normal ICC cell body and injured processes (dICC). No
structural abnormalities are seen in ICC body (ICC). The cytoplasm of a
damaged ICC process is dominated by multiple membrane-bounded empty
vacuoles (*). N, nerve; Co, collagen; CM, circular muscle cell.
Original magnification, ×11,790. Bar, 1 µm.
B: a group of overlapping ICC
processes at close proximity to muscle cells (CM). Note gap junction
contact (large arrow) between two innermost circular muscle cells, and
small close apposition contacts (small arrows) between partially
injured ICC processes. Original magnification, ×33,210.
Structural abnormalities in ICC processes include multiple
membrane-bounded vacuoles (*) merging with plasma membrane present in
zones of contacts between ICC processes. Bar, 500 nm.
C: irreversibly injured ICC process is
characterized by presence of many ruptured mitochondria (dm), partial
depletion of filaments (*), empty vacuoles (v), and a large lipid
droplet (L). An ICC process is recognized by the presence of numerous
caveolae. Original magnification, ×19,225. Bar, 1 µm.
|
|
MPO activity.
The MPO activity in the tissue removed 48 h after the mucosal exposure
to ethanol and acetic acid was significantly greater than that in
normal tissue (Table 4). The MPO activity was increased in both the
lamina propria and the muscularis externa. There was no significant
difference in MPO activity between the tissue that exhibited
spontaneous slow waves and the tissue that had no slow waves. The MPO
activity in the tissue that was removed 30 min after the mucosal
exposure to ethanol and acetic acid was also significantly greater than
that in the normal tissue (Table 4), but it was not significantly
different from that in the tissue removed after 48 h in both the
muscularis externa and the lamina propria (Table 4).
 |
DISCUSSION |
Previous studies have reported that spontaneous in vivo spike-dependent
phasic contractions of colonic circular muscle cells are suppressed in
patients with ulcerative colitis (15, 35) as well as in animal models
of colonic inflammation (31). Our present findings show that
spontaneous in vitro spike-independent phasic contractions of colonic
circular muscle strips are also suppressed significantly during
inflammation. The suppression of these contractions is associated with
a significant reduction in the resting membrane potential and in the
amplitude and duration of slow waves.
The resting membrane potential in circular muscle cells from normal
muscle strips exhibited a gradient of ~29 mV from the submucosal to
the myenteric border, as has been reported previously (20). This
gradient decreased significantly during inflammation to ~10 mV. The
decrease in the gradient was due to depolarization of the resting
membrane potential at 5% and 50% distances in the circular muscle
layer. There was no significant change in membrane potential at the
95% distance from the submucosal border. The precise mechanisms
underlying the generation of the resting membrane potential or its
gradient are not understood completely. However, two hypotheses have
been proposed. According to the first hypothesis (19), the circular
muscle cells have a uniform intrinsic resting membrane potential of
about
60 mV. The coupling of circular muscle cells near the
submucosal border with the ICC pulls up the resting membrane potential
of circular muscle cells in the inner one-third of the layer.
Similarly, the coupling of the circular muscle cells with the
longitudinal muscle cells near the myenteric border pulls down the
resting membrane potential of the cells in the outer one-third, giving
rise to the observed gradient in the full-thickness intact muscle
strips. The intrinsic resting membrane potential of the longitudinal
muscle cells is about
44 mV. In support of this hypothesis,
removal of the ICC and longitudinal muscle layers produces a uniform
membrane potential in the remaining circular muscle layer (19). The
removal of the ICC layer alone flattens the gradient only in the inner
one-third of the circular muscle layer (19).
Ultrastructural findings in our study showed that the ICC and their
processes were partially damaged by the inflammatory response. Whereas
the physical removal of the entire ICC layer may completely eliminate
the gradient of resting membrane potential in the submucosal one-third
of the circular muscle layer, partial damage reduced the gradient
partially but significantly. The gradient in the outer one-half of the
circular muscle layer was not affected, because this is maintained by
the interaction between the longitudinal and circular muscle layers
(19) and this did not seem to be affected by inflammation. There was
little or no damage to the longitudinal muscle cells during
inflammation. Recently, Rumessen (24) also reported damage to ICC in
the colon of ulcerative colitis patients similar to that observed in
the acetic acid model of colonic inflammation. The density of ICC is
also decreased in the aganglionic segment of patients with
Hirschprung's disease (42), resulting in a loss of slow waves (18).
The second hypothesis is that the resting membrane gradient is
maintained by differences in the activities of the electrogenic Na+-K+
pump. Inhibition of the
Na+-K+
pump by ouabain or removal of K+
from the extracellular medium decreases the resting membrane potential
of circular muscle cells near the submucosal border and in the middle
but not near the myenteric border (4). However, the expression of the
Na+ pump in the circular muscle
layer is not affected during inflammation (14). It seems, therefore,
that the changes in the resting membrane potential in colonic circular
muscle cells during inflammation may primarily be due to the damage to
the ICC, rather than due to the decreased expression of the
Na+ pump.
Five of the seventeen inflamed muscle strips exhibited no slow waves at
any location in the circular muscle layer and no spontaneous phasic
contractions in vitro. The MPO activity in the muscle strips without
slow waves was, however, not significantly different from that in
inflamed muscle strips that showed slow waves. Physical removal of the
ICC layer in muscle strips (19), the destruction of ICC by methylene
blue and exposure to light (20), the reduction of these cells in BALB/c
mice (22), or lack of development of ICC (12, 43) also lead to the
absence of slow waves and spontaneous in vitro contractions. However,
the resting membrane potential in the inflamed muscle strips without
spontaneous slow waves was not different from that in muscle strips
with slow waves. Thus additional factors, such as the increased
distance between circular muscle cells and direct injury to circular
muscle cells noted during inflammation, may also be involved.
Colonic inflammation reduced the amplitude of slow waves at 5% and
50%, but not 95%, thickness of the circular muscle layer from the
submucosal border. Previous studies (6, 19, 32) have noted a direct
relationship between the resting membrane potential and the slow wave
amplitude in colonic circular smooth muscle cells. The amplitude of
slow wave depolarizations decreases as the resting membrane potential
decreases. Therefore, it is likely that the reduction of slow wave
amplitude during inflammation is secondary to the depolarization of the
membrane potential. The lack of a change in slow wave amplitude at the
95% distance correlates with no change in the resting membrane
potential at this location. Due to the concurrent decrease in resting
membrane potential and slow wave amplitude during inflammation, the
maximum depolarization at the slow wave plateau in the inflamed colon at the 50% location was not different from that in the normal colon.
However, the maximum amplitude of depolarization at the 5% location
was significantly greater than that in the normal colon. Although it is
known that the slow waves regulate contractile activity by depolarizing
the membrane above an excitatory threshold potential (38), it is not
known whether this excitatory threshold is a fixed potential or if it
is a function of the resting membrane potential. Therefore, the precise
effects of the concurrent decrease of the resting membrane potential
and slow wave amplitude on excitation-contraction coupling cannot be
predicted at this time.
Inflammation had no significant effect on the frequency of colonic slow
waves or spontaneous in vitro phasic contractions. Koch et al. (16)
also reported no change in the frequency of colonic contractions in
muscle strips taken from patients with ulcerative colitis. Our findings
are also in agreement with those of Cohen et al. (8), who reported
depolarization of the resting membrane potential in the circular muscle
cells of the rabbit colon during inflammation.
Inflammation reduced the duration of slow waves at 50% amplitude,
which represents a reduction in the duration of the plateau potential.
This reduction may be a major factor in the reduction of phasic
contractile activity during inflammation. The amplitude of
spike-independent in vitro phasic contractions is correlated directly
with the amplitude and duration of the plateau potential. The precise
mechanisms of reduction of the plateau potential during inflammation
are not known. However, removal of the ICC layer from muscle strips has
been reported to reduce the duration of the plateau potential (19).
Damage to the ICC and their processes noted in inflamed muscle strips
may therefore contribute to the reduction in the duration of the
plateau potential.
Inflammation decreased the amplitude of the phasic contractile response
induced by methacholine and substance P in muscle strips. We have
reported a similar decrease in the contractile response to close
intra-arterial infusions of methacholine and substance P during
inflammation in conscious dogs (21, 41) (S. R. Jadcherla and S. K. Sarna, unpublished results). The phasic contractions in vivo are spike
dependent (25, 26, 27). These data indicate that inflammation may have
similar effects on in vivo spike-dependent and in vitro
spike-independent contractions.
Inflammation had no significant effect on the amplitude of the IJPs
stimulated by EFS in the frequency range of 1-30 Hz in the strips
that displayed spontaneous slow waves. However, the duration of the
IJPs was reduced significantly at all frequencies in the above range.
The colonic IJPs in our study as well as in those reported previously
(39) were largely nitronergic: they were blocked by
L-NAME. The reduction in the
duration of IJP suggests a reduction in the synthesis of neuronal NO.
Therefore, the inhibition of phasic contractions during inflammation is
unlikely to be due to an excessive nitronergic inhibitory input from
the NANC neurons. The reduction in the synthesis or the release of
neuronal nitric oxide is supported by our findings that the "on
response" to EFS that depends on the release of acetylcholine was
also reduced. Ultrastructural observations also indicated damage to the
enteric neurons during inflammation.
The precise mechanisms by which mucosal exposure to ethanol and acetic
acid induces inflammation have not yet been established (36, 45).
However, a protonated form of acid is required to induce inflammation,
because mucosal exposure to HCl of pH similar to that of acetic acid
does not induce inflammation (37, 46). The factors involved in inducing
inflammation by acetic acid include breakdown of the mucosal barrier,
subsequent infiltration of luminal antigens, and, perhaps, the
dissociation of acetic acid within the epithelium to liberate protons
(45). Our data indicate, however, that the changes in slow wave and
contractile parameters are not due to initial mucosal injury induced by
acetic acid but due to the subsequent inflammatory response. Our data
also suggest that the damage to the neuromuscular apparatus for
motility may require an increase in immunocyte infiltration beyond a
certain threshold and a prolonged exposure of the inflammatory response mediators to the enteric neurons and smooth muscle cells. The MPO
activity was increased significantly 30 min after the mucosal exposure
to ethanol and acetic acid, but no significant change in slow wave or
contractile parameters was observed until 48 h after the beginning of
the inflammatory response.
In conclusion, our findings show that inflammation alters specific
parameters of slow waves in the colon and reduces the amplitude of
spontaneous spike-independent in vitro phasic contractions. The changes
in the electrical activity of the cell membrane, including the
depolarization of the resting membrane potential and reduction in the
amplitude and duration of slow waves, may be related to the damage to
the ICC and their processes. Inflammation also suppresses the
contractions induced by muscarinic and neurokinin receptor stimulation.
The duration of IJPs in response to EFS in the colon was decreased
significantly by the inflammatory response, indicating a reduction in
the neuronal release of NO.
 |
ACKNOWLEDGEMENTS |
This study was supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Grant DK-32346 (S. K. Sarna),
Veterans Affairs Medical Research Service and Medical College of
Wisconsin Research Affairs Committee Grant (G. Lu), and the
Advisory Board of the Digestive Disease Research Center (S. K. Sarna).
 |
FOOTNOTES |
Portions of this work have been presented previously in abstract form
(Gastroenterology 110 (6): A710, 1996.
Address for reprint requests: S. K. Sarna, General Surgery, Medical
College of Wisconsin, 9200 West Wisconsin Ave., Milwaukee, WI 53226.
Received 19 November 1996; accepted in final form 7 August 1997.
 |
REFERENCES |
1.
Barajas-Lopez, C.,
I. Berezin,
E. E. Daniel,
and
J. D. Huizinga.
Role of the interstitial cells of Cajal in generation of pacemaker activity.
Am. J. Physiol.
257 (Cell Physiol. 26):
C830-C835,
1989[Abstract/Free Full Text].
2.
Barajas-Lopez, C.,
and
J. D. Huizinga.
Different mechanisms of contraction generation in circular muscle of canine colon.
Am. J. Physiol.
256 (Gastrointest. Liver Physiol. 19):
G570-G580,
1989[Abstract/Free Full Text].
3.
Berezin, I.,
J. D. Huizinga,
and
E. E. Daniel.
Interstitial cells of Cajal in the canine colon: a special communication network at the inner border of the circular muscle.
J. Comp. Neurol.
273:
42-51,
1988[Medline].
4.
Burke, E. P.,
J. B. Reed,
and
K. M. Sanders.
Role of Na+ pump in membrane potential gradient of canine proximal colon.
Am. J. Physiol.
254 (Cell Physiol. 23):
C475-C483,
1988[Abstract/Free Full Text].
5.
Castro, G. A.,
S. A. Roy,
and
R. D. Stockstill.
Trichinella spiralis: peroxidase activity in isolated cells from the rat intestine.
Exp. Parasitol.
36:
307-315,
1974[Medline].
6.
Chambers, M. M.,
Y. J. Kingma,
and
K. L. Bowes.
Intracellular electrical activity in circular muscle of canine colon.
Gut
25:
1268-1270,
1984[Abstract].
7.
Christensen, J.,
S. Anuras,
and
C. Arthur.
Influence of intrinsic nerves on electromyogram of cat colon in vitro.
Am. J. Physiol.
234 (Endocrinol. Metab. Gastrointest. Physiol. 3):
E641-E647,
1978[Medline].
8.
Cohen, J. D.,
H. W. Kao,
S. T. Tan,
J. Lechago,
and
W. J. Snape, Jr.
Effect of acute experimental colitis on rabbit colonic smooth muscle.
Am. J. Physiol.
251 (Gastrointest. Liver Physiol. 14):
G538-G545,
1986[Medline].
9.
Conklin, J. L.,
and
C. Du.
Pathways of slow-wave propagation in proximal colon of cats.
Am. J. Physiol.
258 (Gastrointest. Liver Physiol. 21):
G894-G903,
1990[Abstract/Free Full Text].
10.
Grossi, L.,
K. McHugh,
and
S. M. Collins.
On the specificity of altered muscle function in experimental colitis in rats.
Gastroenterology
104:
1049-1056,
1993[Medline].
11.
Huizinga, J. D.,
G. Chang,
N. E. Diamant,
and
T. Y. El-Sharkawy.
Electrophysiological basis of excitation of canine colonic circular muscle by cholinergic agents and substance P.
J. Pharmacol. Exp. Ther.
231:
692-699,
1984[Abstract].
12.
Huizinga, J. D.,
L. Thuneberg,
M. Llüppel,
J. Malysz,
H. B. Mikkelsen,
and
A. Bernstein.
W/kit gene required for interstitial cells of Cajal and pacemaker activity.
Nature
373:
347-349,
1995[Medline].
13.
Jadcherla, S.R.,
and
S.K. Sarna.
Inflammation modulates the colonic contractile response to muscarinic receptor activation (Abstract).
Dig. Dis. Sci.
41:
A71,
1996.
14.
Kahn, I.,
and
S. M. Collins.
Altered expression of Na+ pump isoforms in the inflamed intestine of Trichinella spiralis-infected rats.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G1160-G1168,
1993[Abstract/Free Full Text].
15.
Kern, F. J.,
T. P. Almy,
F. K. Abbot,
and
M. D. Bogdonoff.
The motility of the distal colon in nonspecific ulcerative colitis.
Gastroenterology
19:
492-503,
1951.
16.
Koch, T. R.,
J. A. Carney,
V. L. W. Go,
and
J. H. Szurszewski.
Spontaneous contractions and some electrophysiologic properties of circular muscle from normal sigmoid colon and ulcerative colitis.
Gastroenterology
95:
77-84,
1988[Medline].
17.
Kocylowski, M.,
K. L. Bowes,
and
J. Y. Kingma.
Electrical and mechanical activity in the ex vivo perfused total canine colon.
Gastroenterology
77:
1021-1026,
1979[Medline].
18.
Kubota, M.,
Y. Ito,
and
K. Ikeda.
Membrane properties and innervation of smooth muscle cells in Hirschsprung's disease.
Am. J. Physiol.
244 (Gastrointest. Liver Physiol. 7):
G406-G415,
1983[Abstract/Free Full Text].
19.
Liu, L. W. C.,
and
J. D. Huizinga.
Electrical coupling of circular muscle to longitudinal muscle and interstitial cells of Cajal in canine colon.
J. Physiol. (Lond.)
470:
445-461,
1993[Abstract].
20.
Liu, L. W. C.,
L. Thuneberg,
and
J. D. Huizinga.
Selective lesioning of interstitial cells of Cajal by methylene blue and light leads to loss of slow waves.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G485-G496,
1994[Abstract/Free Full Text].
21.
Lu, G.,
and
S. K. Sarna.
In vivo modulation of Ca2+ mobilization by colonic inflammation in regulating phasic contractions (Abstract).
Gastroenterology
110:
A710,
1996.
22.
Maeda, H.,
A. Yamagata,
S. Nishikawa,
K. Yoshinaga,
S. Kobayashy,
K. Nishi,
and
S. Nishikawa.
Requirement of c-kit for development of interstitial pacemaker system.
Development
116:
369-375,
1992[Abstract/Free Full Text].
23.
Morgan, K. G.,
and
J. H. Szurszewski.
Mechanisms of phasic and tonic actions of pentagastrin on canine gastric smooth muscle.
J. Physiol. (Lond.)
301:
229-242,
1980[Abstract].
24.
Rumessen, J. J.
Ultrastructure of interstitial cells of Cajal at the colonic submuscular border in patients with ulcerative colitis.
Gastroenterology
111:
1447-1455,
1996[Medline].
25.
Sanders, K. M.,
and
T. K. Smith.
Enteric neural regulation of slow waves in circular muscle of the canine proximal colon.
J. Physiol. (Lond.)
377:
297-313,
1986[Abstract].
26.
Sarna, S. K.
Myoelectric correlates of colonic motor complexes and contractile activity.
Am. J. Physiol.
250 (Gastrointest. Liver Physiol. 13):
G213-G220,
1986[Abstract/Free Full Text].
27.
Sarna, S. K.
Physiology and pathophysiology of colonic motor activity. Part I.
Dig. Dis. Sci.
36:
827-862,
1991[Medline].
27a.
Sarna, S. K.
Physiology and pathophysiology of colonic motor activity. Part II.
Dig. Dis. Sci.
36:
998-1018,
1991[Medline].
28.
Sarna, S. K.,
P. R. Latimer,
D. Campbell,
and
W. E. Waterfall.
Effect of stress, meal and neostigmine on rectosigmoidal electrical control activity (ECA) in normals and irritable bowel syndrome patients.
Dig. Dis. Sci.
27:
582-591,
1982[Medline].
29.
Sarna, S. K.,
W. E. Waterfall,
B. L. Bardakjian,
and
J. F. Lind.
Types of human colonic electrical activities recorded post-operatively.
Gastroenterology
81:
61-70,
1981[Medline].
30.
Serio, R.,
C. Barajas-Lopez,
E. E. Daniel,
I. Berezin,
and
J. D. Huizinga.
Slow-wave activity in colon: role of network of submucosal interstitial cells of Cajal.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G636-G645,
1991[Abstract/Free Full Text].
31.
Sethi, A. K.,
and
S. K. Sarna.
Colonic motor activity in acute colitis in conscious dogs.
Gastroenterology
100:
954-963,
1991[Medline].
32.
Smith, T. K.,
J. B. Reed,
and
K. M. Sanders.
Interaction of two electrical pacemakers in muscularis of canine proximal colon.
Am. J. Physiol.
252 (Cell Physiol. 21):
C290-C299,
1987[Abstract].
33.
Snape, Jr., W. J., and H. W. Kao. Role
of inflammatory mediators in colonic smooth muscle function in
ulcerative colitis. Dig. Dis. Sci. 33, Suppl.: 65S-70S, 1998.
34.
Snape, W. J., Jr.,
and
S. T. Tan.
Effect of tetraethylammonium on an evoked spike potential in feline colonic muscle.
Am. J. Physiol.
252 (Gastrointest. Liver Physiol. 15):
G791-G796,
1987[Abstract/Free Full Text].
35.
Spriggs, E. A.,
C. F. Code,
J. A. Bargen,
R. K. Curtiss,
and
N. C. Hightower, Jr.
Motility of the pelvic colon and rectum of normal persons and patients with ulcerative colitis.
Gastroenterology
19:
480-491,
1951.
36.
Stenson, W. F.
Animal models of inflammatory bowel disease.
In: Inflammatory Bowel Disease from Bench to Bedside, edited by S. R. Targan,
and F. Shanahan. Baltimore, MD: Williams and Wilkins, 1994, p. 180-192.
37.
Strober, W. Animal models of inflammatory bowel disease: an
overview. Dig. Dis. Sci. 30, Suppl.: 3S-10S, 1985.
38.
Szurszewski, J. H.
Electrical basis for gastrointestinal motility.
In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1981, p. 1435-1466.
39.
Thornbury, K. D.,
S. M. Ward,
H. H. Dalziel,
A. Carl,
D. P. Westfall,
and
K. M. Sanders.
Nitric oxide and nitrosocysteine mimic nonadrenergic, noncholinergic hyperpolarization in canine proximal colon.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G553-G557,
1991[Abstract/Free Full Text].
40.
Thuneberg, L.
Interstitial cells of Cajal: intestinal pacemaker cells?
Adv. Anat. Embryol. Cell Biol.
71:
1-130,
1982[Medline].
41.
Tsukamoto, M.,
S. K. Sarna,
and
R. E. Condon.
A novel motility effect of tachykinins in normal and inflamed colon.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G1607-G1614,
1997[Abstract/Free Full Text].
42.
Vanderwinden, J.-M.,
J. J. Rumessen,
H. Liu,
D. Descamps,
M.-H. De Laet,
and
J.-J. Vanderhaeghen.
Interstitial cells of Cajal in human colon and in Hirschsprung's disease.
Gastroenterology
111:
901-910,
1996[Medline].
43.
Ward, S. M.,
A. J. Burns,
S. Torihashi,
and
K. M. Sanders.
Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.
J. Physiol. (Lond.)
480:
91-97,
1994[Abstract].
44.
Ward, S. M.,
and
K. M. Sanders.
Pacemaker activity in septal structures of canine colonic circular muscle.
Am. J. Physiol.
259 (Gastrointest. Liver Physiol. 22):
G264-G273,
1990[Abstract/Free Full Text].
45.
Yamada, T.,
S. Marshall,
R. D. Specian,
and
M. B. Grisham.
A comparative analysis of two models of colitis in rats.
Gastroenterology
102:
1524-1534,
1992[Medline].
46.
Zeitlin, I. J.,
and
A. A. Norris.
Animal models of colitis.
In: British Society of Gastroenterology: Smith Klein and French Laboratories Workshop. Stanstead Abbotts, UK: Smith Klein, 1983, p. 70-73.
AJP Gastroint Liver Physiol 273(6):G1233-G1245
0193-1857/97 $5.00
Copyright © 1997 the American Physiological Society