Departments of Surgery and Physiology, Medical College of
Wisconsin, and Zablocki Veterans Affairs Medical Center, Milwaukee,
Wisconsin 53295
We investigated the in vivo signal-transduction
pathways to stimulate phasic contractions in normal and inflamed ileum
by close intra-arterial infusions of test substances. Methacholine stimulated phasic contractions dose dependently. This response was
suppressed during inflammation. Verapamil inhibited the response to
methacholine dose dependently in both normal and inflamed ileum. Neomycin inhibited the response partially in normal ileum and almost
completely in inflamed ileum. H-7 and chelerythrine partially inhibited
the methacholine response in normal ileum but had no significant effect
in inflamed ileum. Ryanodine stimulated phasic contractions that were
blocked by TTX, hexamethonium, atropine, or ruthenium red. Ruthenium
red, however, had no significant effect on the contractile response to
methacholine. Conclusions: 1)
Ca2+ influx through the L-type
channels may be the primary source of
Ca2+ to stimulate in vivo phasic
contractions. 2)
Phosphatidylinositol hydrolysis enhances the stimulation
of in vivo phasic contractions in the normal ileum. In the inflamed
ileum, phosphatidylinositol hydrolysis may be essential to stimulate
phasic contractions. 3) Inflammation
may downregulate the protein kinase C pathway. 4) Ryanodine stimulates phasic
contractions by the release of ACh.
gastrointestinal motility; smooth muscle; calcium; protein kinase
C; ryanodine; ruthenium red; neomycin; H-7; W-7; chelerythrine; inflammation
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INTRODUCTION |
AN INCREASE in intracellular
Ca2+ concentration
([Ca2+]i)
is an essential step for smooth muscle cells to contract. The increase in
[Ca2+]i
is part of a series of signal-transduction steps that result in the
phosphorylation of contractile proteins. The two sources of
Ca2+ to increase cytosolic
concentration are the extracellular medium and the rapidly exchanging
intracellular stores.
The gut smooth muscle generates at least three different types of
contractions to perform the complex motility functions of mixing and
propulsion: 1) rhythmic phasic
contractions, 2) giant migrating
contractions (GMCs), and 3) tone.
The rhythmic phasic contractions produce mixing and net slow distal
propulsion of chyme in the postprandial state and of residual food and
secretions in the fasting state (31). The GMCs produce mass movements
(29). The precise role of the generation of tone in circular muscle cells is not known, but the resulting decrease in the diameter of the
gut may enhance the efficiency of the phasic contractions in mixing and
propulsion. The time course, frequency, and force generated by each of
the above three types of contractions are significantly different from
each other. The phasic contractions occur rhythmically at a few cycles
per minute in different species, last for about 3-5 s,
and generate a moderate force (~75-100 g), and their occurrence
in time and space is controlled by slow waves (30, 37). The GMCs occur
infrequently (about once or twice a day), last for about 20 s, and
generate a very strong force (>150 g), and these contractions are
independent of slow waves (29). The tone is normally of small to
moderate force; it is independent of slow waves and it can last for
prolonged periods of time (several minutes to hours). It is remarkable
that, using a limited number of second messengers, the same smooth
muscle cells can generate so many different types of contractions. Our hypothesis is that the signal transduction in smooth muscle cells to
stimulate different types of contractions is different. A significant amount of work has been done to identify the signal-transduction pathways to generate tone in dispersed small intestinal smooth muscle
cells (4, 5, 13, 16, 20, 23, 24). However, very little is known about
the signal-transduction pathways for the stimulation of in vivo phasic
contractions and GMCs.
The two major effects of inflammation on small intestinal motility are
the suppression of phasic contractions and the stimulation of GMCs (1,
2, 17). The suppression of phasic contractions compromises the mixing
and slow orderly distal propulsion of the ingested meal, whereas the
stimulation of GMCs produces frequent mass movements that are
associated with diarrhea and abdominal cramping (2, 17, 34). The
inflammatory response has been shown previously to alter the enteric
neuronal function (6, 10) as well as the characteristics of slow waves
in smooth muscle cells (21). The effects of inflammation on signal
transduction in small intestinal circular smooth muscle cells are not
known.
We investigated the hypothesis that the influx of
Ca2+ through L-type channels is
the primary source for the stimulation of in vivo ileal phasic circular
muscle contractions by muscarinic receptor activation. The hydrolysis
of phosphatidylinositol (PI) by muscarinic receptor activation enhances
these contractions. Our second hypothesis is that inflammation
modulates signal-transduction pathways to suppress the phasic
contractions in ileal circular smooth muscle cells. These hypotheses
were tested in conscious dogs by close intra-arterial infusions of test
substances in short segments of the ileum. We chose methacholine, a
stable analog of ACh, as the agonist to stimulate phasic contractions.
ACh is the physiological neurotransmitter of spontaneous small
intestinal phasic contractions at the neuroeffector junction. Atropine,
a nonspecific muscarinic receptor antagonist, completely blocks spontaneous phasic contractions in the conscious state (25, 32).
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MATERIALS AND METHODS |
Surgical procedure.
The experiments were performed on 11 healthy conscious dogs of either
sex. Each dog weighed 20-29 kg (mean wt 24 ± 1.7 kg). The dogs
were anesthetized with 30 mg/kg pentobarbital sodium (Abbott
Laboratories, Chicago, IL). Access to the abdominal cavity was obtained
by a midline ventral laparotomy. An intraluminal catheter (2.6 mm ID,
4.9 mm OD) was implanted 158 ± 4 cm proximal to the ileocolonic
junction to infuse ethanol and acetic acid for the induction of
inflammation, as described below. A stainless steel fistula was
implanted 20 cm proximal to the ileocolonic junction to drain ethanol
and acetic acid and prevent them from reaching the colon.
Two mesenteric arteries were identified in the segment between the
intraluminal catheter and the fistula. The arteries were freed
completely from the mesentery, preserving the nerves. A Silastic
catheter (0.75 mm ID, 1.63 mm OD) was inserted in the centripetal direction in a branch artery so that its tip rested 1-2 mm from the junction of the branch artery and the main artery. The boundaries of the perfused segment were identified by infusing saline at 15-20 ml/min for 10-15 s. The segment
refilled with blood within 2-3 s after the end of the infusion.
Infusion of saline at 1 ml/min for up to 10 min produced no apparent
change in color of the segment and did not stimulate any contractions. The length of the infused segment was limited to 5-6 cm by
ligating the secondary branch arteries, if necessary. The catheters
were secured by ties to the branch artery and the mesentery.
One electrode-strain-gauge pair and two strain-gauge transducers were
attached to the seromuscular layer in the infused segment by 3-O
Surgilon sutures (Davis & Geck, Danbury, CT). An additional strain-gauge transducer was attached to the seromuscular layer 10 cm
distal to each infused segment. All transducers were oriented to record
circular muscle contractions.
The intraluminal and intra-arterial catheters were exteriorized
subcutaneously in the subscapular region. The catheters were housed in
jackets that the dogs wore at all times. Each intra-arterial catheter
was flushed twice daily with 2,000 IU of heparin. The dogs were allowed
5 days to recover from surgery.
Experimental protocol.
All experiments were performed in the conscious state after an
overnight fast. At least one phase III activity was recorded to
establish the fasting state. The contractile and electrical signals
were recorded on a 12-channel Grass recorder (model 7D; Grass
Instruments, Quincy, MA), with lower and upper cut-off frequencies set
at direct current and 15, 0.1, and 15 Hz, respectively.
All test substances were infused at 1 ml/min during phase I or a
quiescent period during phase II activity of the migrating myoelectric/motor complex cycle. Preliminary experiments were done to
establish the duration at which infusion of each test substance was
maximally effective. The agonist, methacholine, was infused for 1 min.
The antagonists were infused for 1-, 5-, or 10-min periods. When an
antagonist was infused for 1 min, methacholine was infused about 2 min
after the end of the antagonist infusion. When an antagonist was
infused for 5- or 10-min periods, the 1-min infusion of methacholine
started 2 or 5 min after the beginning of the antagonist infusion,
respectively. A waiting period of at least 30 min was allowed between
successive infusions. Preliminary experiments indicated that the
responses to repeated infusions after this interval were the same. All
experiments were done first in the normal state and then during
inflammation.
Induction of inflammation.
Ileal inflammation was induced by mucosal exposure to ethanol and
acetic acid, as described previously (17). Briefly, 75 ml of 95%
ethanol were infused intraluminally on day
1. The same amount of ethanol was infused on
days 3 and
5, followed 1 h later by infusions of
50 ml of 20% acetic acid. Mucosal exposure to ethanol and acetic acid
induces an inflammatory response that lasts for about 10 days (17).
Myeloperoxidase activity and neutrophil infiltration are increased in
both the mucosa and the muscle layers (17). The experiments in the
inflamed state were done on days 3 and
4 and
days
6-9 after the first exposure to
ethanol. Shi and Sarna (34) reported recently that there is no
difference in response to methacholine on
days
3 and
6 of inflammation. The performance of
experiments with different antagonists was distributed randomly during
the period of inflammation on days
3 and
4 and days
6-9.
Test substances.
The following substances were used: methacholine, verapamil, neomycin,
ruthenium red, TTX, and hexamethonium, all dissolved in 0.9% saline;
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)
and chelerythrine, dissolved in sterile water and diluted in 0.9%
saline;
N-(6-aminohexyl)-5-chloro-1-naphthalene
sulfonamide (W-7), dissolved in DMSO and diluted in sterile water; and
ryanodine, dissolved in ethanol and diluted in 0.9% saline. All of
these substances were purchased from Sigma Chemical (St. Louis, MO). Atropine sulfate was purchased from Lymphomed (Deerfield, IL) and
dissolved in 0.9% saline. The infusion of vehicle alone had no effect
on the contractile activity.
Data analysis.
The contractile response was quantified as the area under contractions
(WINDAQ/EX program; DATAQ Instruments, Akron, OH). The area under
contractions was measured from the beginning of the first contraction
after the start of infusion to the point at which the tracing returned
to the baseline and contractions ceased to occur.
All data are means ± SE. The n
value represents the number of dogs. Statistical analysis was done by
analysis of variance with repeated measures. Multiple comparisons were
done by Student-Newman-Keul's test. P
0.05 was considered statistically significant. This study was
approved by the Animal Studies Subcommittee at the Zablocki Veterans
Affairs Medical Center.
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RESULTS |
Inflammatory modulation of the ileal phasic contractile response to
methacholine.
Close intra-arterial infusions of methacholine (0.05-8
nmol · ml
1 · min
1
for 1 min) stimulated a series of phasic contractions in a
dose-dependent fashion. Inflammation significantly reduced the response
to methacholine (Fig.
1A;
F = 17.2, degrees of freedom = 69, P < 0.001). The
half-maximal effective dose during inflammation, 1.7 ± 0.33 nmol,
was significantly greater than that in the normal state, 0.55 ± 0.26 nmol. The dose of methacholine (4 nmol · ml
1 · min
1
for 1 min) that produced nearly maximum response was used in the
following experiments.

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Fig. 1.
A: methacholine dose dependently
increased the phasic contractile response in both normal and inflamed
ileum. Response in inflamed ileum was significantly less than that in
normal ileum. B: contractile response
to methacholine was not affected significantly by prior close
intra-arterial infusions of TTX and hexamethonium but was blocked
completely by atropine in both normal and inflamed ileum.
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The contractile response to methacholine was not affected significantly
by prior close intra-arterial infusions of TTX (75 nmol · ml
1 · min
1
for 1 min) or hexamethonium (70 µmol · ml
1 · min
1
for 1 min), but it was blocked completely by prior infusion of atropine
(150 nmol · ml
1 · min
1
for 1 min) (Fig. 1B;
n = 5). These doses of TTX,
hexamethonium, and atropine have been reported previously to
effectively block Na+ channel
enteric neural conduction, nicotinic receptors, and muscarinic receptors, respectively (11, 32). Thus methacholine, given close
intra-arterially, acted primarily on muscarinic receptors on circular
smooth muscle cells in the normal and the inflamed ileum to stimulate
phasic contractions.
Role of Ca2+ influx through
L-type channels and PI hydrolysis in stimulating the phasic contractile
response to methacholine in normal and inflamed ileum.
Close intra-arterial infusions of verapamil, an L-type
Ca2+ channel blocker (0.1-800
nmol · ml
1 · min
1
for 5 min, n = 6; Fig.
2), and of neomycin, an antagonist of phospholipase C (PLC; 1-12
µmol · ml
1 · min
1
for 10 min, n = 5; Fig.
3), dose dependently inhibited the phasic contractile response to methacholine in both the normal and the inflamed ileum. In each case, the response was expressed as a percentage of the control response to methacholine in the normal or the
inflamed ileum. The absolute value of the control response in the
inflamed ileum, however, was less than that in the normal ileum (Fig.
1A). At the maximum dose,
verapamil blocked the response almost completely in both the normal and
the inflamed ileum. There was no significant difference between the
half-maximal inhibitory dose values of verapamil between the normal and
the inflamed states (42.4 ± 20.2 and 23.0 ± 16 nmol · ml
1 · min
1
for 5 min, respectively). In normal ileum, the highest dose of neomycin
inhibited the response by ~50%. In the inflamed ileum, the same dose
of neomycin completely inhibited the contractile response to
methacholine (Fig. 3).

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Fig. 2.
Close intra-arterial infusions of verapamil dose dependently inhibited
the phasic contractile response to methacholine in normal and inflamed
ileum. In both states, response to methacholine with saline infusion
was taken as 100%. There was no significant difference between the two
inhibitory dose-response curves.
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Fig. 3.
Close intra-arterial infusion of neomycin dose dependently inhibited
the contractile response to methacholine in normal and inflamed ileum.
Inhibition at maximum dose was partial in normal ileum but complete in
inflamed ileum. Inhibition was significantly greater in inflamed than
in normal ileum (F = 4.37, degrees of
freedom = 29, P = 0.0075). Control
response to methacholine in normal and inflamed ileum was taken as
100%.
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The infusion of H-7 (5-200
nmol · ml
1 · min
1
for 5 min) or chelerythrine (5-400
nmol · ml
1 · min
1
for 5 min), inhibitors of protein kinase C (PKC), dose dependently inhibited the phasic contractile response to methacholine in the normal
ileum (Fig. 4). The inhibition
at the maximum dose was about 40% of the control response. In the
inflamed state, H-7 and chelerythrine in the same dose range had no
significant effect on the contractile response to methacholine (Fig.
4).

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Fig. 4.
H-7 ( A) and chelerythrine ( B) dose
dependently inhibited the contractile response to methacholine in
normal ileum. In inflamed ileum, they had no significant effect.
Control response to methacholine in normal and inflamed ileum was taken
as 100%.
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The role of ryanodine-sensitive intracellular
Ca2+ stores and calmodulin in
the contractile response to methacholine in normal and inflamed ileum.
Close intra-arterial infusion of ryanodine at 20 nmol · ml
1 · min
1
for 5 min stimulated a series of phasic contractions in the normal ileum (n = 4). The mean maximum
amplitude of these contractions was 23 ± 4% of the maximum
amplitude of phasic contractions stimulated by methacholine. However,
the total duration of occurrence of ryanodine-induced contractions (405 ± 120 s) was about threefold longer than that of
methacholine-induced contractions (128 ± 13 s). As a result, the
total area under ryanodine-induced contractions was not significantly
different from that under methacholine-induced contractions (Fig.
5A). The
response to ryanodine began 265 ± 60 s after the start of infusion,
as opposed to 24 ± 4 s for methacholine (P < 0.05). The contractile response
to ryanodine was blocked completely by a 5-min concurrent infusion of
1.0 nmol · ml
1 · min
1
ruthenium red, an antagonist of ryanodine receptors. However, the 5-min
infusion of ruthenium red in the dose range of 0.5-1.5 nmol · ml
1 · min
1
had no significant effect on the contractile response to methacholine (Fig. 5B). The contractile response
to ryanodine was also blocked by TTX, atropine, and hexamethonium
infusions (data not shown). Ryanodine did not stimulate phasic
contractions in the inflamed ileum (Fig.
5A).

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Fig. 5.
A: close intra-arterial infusion of
ryanodine (hatched bars) stimulated a phasic contractile response in
normal ileum that was not significantly different from response to
methacholine (open bars; P > 0.05).
Response to methacholine was suppressed significantly in inflamed
ileum. However, ryanodine hardly stimulated any contractions in
inflamed ileum. Response to combined infusions of ryanodine and
methacholine (stippled bars) was slightly greater than that to
methacholine alone in both normal and inflamed ileum, but they were not
significantly different (P > 0.05).
However, response to combined infusions in inflamed ileum was
significantly less than that in normal ileum
(* P < 0.05).
B: ruthenium red had no significant
effect on contractile response to methacholine. Control response to
methacholine in normal and inflamed ileum was taken as 100%.
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The contractile response to close intra-arterial infusion of
methacholine during a concurrent infusion of ryanodine in normal and
inflamed ileum exhibited a slight increase, but it was not statistically significant (Fig. 5A).
The infusion of W-7, a calmodulin antagonist, at 0.4 or 1.6 µmol · ml
1 · min
1
for 5 min had no significant effect on the phasic contractile response
to methacholine in the normal or the inflamed ileum (data not shown).
Infusion of W-7 at 1.6 µmol · ml
1 · min
1
for 5 min has been reported previously to inhibit the contractile response to 5-hydroxytryptamine (11).
Relationship between myoelectrical activity and phasic contractions
in normal and inflamed ileum.
Each spontaneous phasic contraction in the ileum was associated with a
slow wave and a spike burst (Fig.
6B).
Only one spike burst occurred in each slow wave cycle. No significant
contractile activity was recorded when spike bursts were absent (Fig.
6A; n = 6). The methacholine-induced phasic contractions were related similarly to the spike bursts and slow wave cycles (Fig.
6C). This 1:1:1 relationship among
phasic contractions, spike bursts, and slow wave cycles did not change
during inflammation. Inflammation had no significant effect on the
frequency of slow waves (15.7 ± 0.2 vs. 15.9 ± 0.2 cycles/min
in normal and inflamed ileum, respectively;
n = 6, P > 0.05).