Neural regulation of in vitro giant contractions in the rat
colon
Asensio
Gonzalez and
Sushil K.
Sarna
Departments of Surgery and Physiology, Medical College of
Wisconsin, Milwaukee 53266; and Zablocki Veterans Affairs Medical
Center, Milwaukee, Wisconsin 53295
 |
ABSTRACT |
The rat middle colon spontaneously generates regularly
occurring giant contractions (GCs) in vitro. We investigated the
neurohumoral and intracellular regulation of these contractions in a
standard muscle bath. cGMP content was measured in strips and single
smooth muscle cells. The circular muscle strips generated spontaneous GCs. Their amplitude and frequency were significantly increased by
tetrodotoxin (TTX),
-conotoxin,
N
-nitro-L-arginine
(L-NNA), and the dopamine D1 receptor
antagonist Sch-23390. The GCs were unaffected by hexamethonium,
atropine, and antagonists of serotonergic (5-HT1-4),
histaminergic (H1-2), and tachykininergic
(NK1-2) receptors but enhanced by NK3
receptor antagonism. The guanylate cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ) also enhanced GCs
to the same extent as TTX and L-NNA, and each of the three agents prevented the effects of the others. GCs were abolished by
electrical field stimulation,
S-nitroso-N-acetyl-penicillamine, and
8-bromo-cGMP. BAY-K-8644 and apamin enhanced the GCs, but they were
abolished by D-600. Basal cGMP content in strips was decreased by TTX,
L-NNA, or ODQ, but these treatments had no effect on cGMP
content of enzymatically dissociated single smooth muscle cells. We
conclude that spontaneous contractions in the rat colonic muscle strips
are not generated by cholinergic, serotonergic, or histaminergic input.
Constitutive release of nitric oxide from enteric neurons sustains cGMP
synthesis in the colonic smooth muscle to suppress spontaneous in vitro GCs.
nonadrenergic, noncholinergic; enteric neurons; nitric oxide; guanosine 3',5'-cyclic monophosphate; dopamine; cholinergic; tachykinins; apamin; calcium channels
 |
INTRODUCTION |
THE IN VIVO COLONIC
MOTOR activity in most species, including dogs, humans, and rats,
is characterized by three distinct types of contractions: 1)
rhythmic phasic contractions, 2) giant migrating contractions (GMCs), and 3) tone (18, 22, 38,
39). The GMCs are large-amplitude and long-duration contractions
that migrate uninterruptedly over long distances and are associated
with mass movements and defecation (23, 24, 41). The
spontaneous GMCs occur once or twice a day in the human and the dog
colon. In the in vivo rat colon, however, they occur regularly at a
frequency of ~40/h (22). In this species, the GMCs
migrate infrequently and over much shorter distances than in humans or
dogs. Previous studies have shown that the GMCs are not regulated by
slow waves and their smooth muscle signaling pathways are different
from those of phasic contractions (37, 40).
The role of enteric neurons in the regulation of colonic GMCs is not
well understood. One of the limitations has been that the GMCs are not
known to occur in canine and human in vitro muscle strips. A recent
report by Gonzalez and Sarna (12) indicates, however, that
circular muscle strips prepared from the middle rat colon generate
regular giant contractions (GCs) as well as phasic contractions. This
in vitro finding provides an opportunity to investigate the enteric
neural regulation of GCs in a muscle bath environment. The specific
aims of this study were to determine whether 1) there is a
constitutive release of neurotransmitters to stimulate or inhibit GCs
and 2) there is a differential enteric neural regulation of
concurrently occurring GCs and phasic contractions. Our results
indicated that there is constitutive release of nitric oxide (NO) and
dopamine that suppresses the GCs. Therefore, our third aim was to
determine the cellular pathways for the inhibition of spontaneous GCs
by NO.
 |
MATERIALS AND METHODS |
Recording of spontaneous contractile activity.
Male Sprague-Dawley rats (300-350 g in weight) fed with a standard
chow diet were used for the study. The animals were given 50 mg/kg
pentobarbital sodium intraperitoneally. The middle colon (5 cm from the
cecum and 7 cm from the pelvic brim) was removed and immediately placed
in Krebs solution at 37°C preequilibrated with carbogen. Mucosa-free
strips, 3 mm wide × 10 mm long, were cut along the circular
muscle axis and mounted in a 3-ml muscle bath filled with Krebs at
37°C and bubbled with carbogen. The tissues were left to equilibrate
for at least 60 min, and the bath solution was changed every 15-20
min until GC frequency and amplitude stabilized. Both phasic
contractions and GCs were observed at low levels of stretch. However,
preliminary experiments were performed by stepwise stretching and
measuring the contraction to 2 µM ACh. Optimal stretching was found
at ~8-10 mN passive tension, and for subsequent experiments the
strips were routinely stretched to 10 mN tension.
Pharmacological antagonists and neurotoxins were added to the muscle
bath in volumes of <1% of the bath volume, and the effects were
recorded for 10 min unless stated otherwise. The frequency was measured
over the 10-min period before addition of drugs and during the 10 min
after the addition, unless noted otherwise. In experiments with
combined addition of antagonists and agonists, the antagonist was
allowed to act for 10 min before adding the agonist. The contractions
were recorded on a Grass 7D polygraph and analyzed by a digital data
acquisition system (DATAQ Instruments, Akron, OH).
Preparation of single smooth muscle cells.
The muscularis externa from the middle colon was dissected free of
mucosa and submucosa with fine tweezers under a dissecting microscope.
The tissue was incubated at 37°C in digestion solution containing 25 mM HEPES and 0.4 mg/ml papain (Sigma Chemical, St. Louis, MO) for
20-30 min and then in collagenase Worthington type II (0.2 mg/ml)
until cells were released by pipetting of the digested tissue as
assessed microscopically (30-60 min). The tissue was further
pipetted with a silicone-coated Pasteur pipette, and the cells were
washed twice with sterile DMEM (GIBCO, Rockville, MD) and incubated for
4 h in DMEM at 37°C in an atmosphere of 10% CO2 to
allow the cells to recover from the digestion procedure. For cGMP
measurement, the cells were lysed following the protocol described in
the enzyme immunoassay kit (Amersham Life Science, Chicago, IL). The
cell dispersions were filtered through a 200-µm nylon mesh, and the
suspensions were checked with a phase contrast microscope to confirm
the presence of smooth muscle cells in the absence of enteric ganglia,
which were much larger in size.
Measurement of cGMP.
The mechanical effect of pharmacological agents on muscle strips was
checked before measuring their cGMP content. Then the strips were dried
by blotting immediately (<10 s), frozen in liquid nitrogen, and stored
at
80°C until assay. For analysis, the frozen tissue pieces were
quickly weighed and homogenized in 6% trichloroacetic acid, the debris
was pelleted by centrifugation, and the supernatants were washed three
times with water-saturated ethyl ether to extract residual
trichloroacetic acid. The extracts were lyophilized and reconstituted
in 1 M potassium phosphate buffer, pH 7.4 (37). cGMP was
determined by enzyme immunoassay (Amersham Life Sciences) following the
acetylation protocol, with a lower limit of detection in the femtomolar range.
Analysis of data.
Comparisons between two groups were performed by parametric Student's
test; P < 0.05 was considered statistically
significant. The n values represent the number of strips
tested, and they were taken from at least three different animals. For
biochemical measurements, standard curves were generated by nonlinear
regression analysis of the experimental data points, and goodness of
fit (r2) was always >0.99. All data are
expressed as means ± SE.
Drugs.
Tetrodotoxin (TTX),
-conotoxin GVIA (
-CTX),
N
-nitro-L-arginine
(L-NNA), MDL-12330A, apamin, 8-bromo-cGMP (8-BrcGMP), D-600 phentolamine, domperidone, mepyramine,
1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ), vasoactive
intestinal peptide (VIP), VIP(10---28), and ranitidine were purchased
from Sigma Chemical. L-703606,
L-659977,
Trp7,
-Ala8-NKA4-10,
suramin, pyridoxal phosphate-6-azophenyl-2',4'-disulfonoic acid
(PPADS), reactive blue,
S-nitroso-N-acetyl-penicillamine (SNAP),
BAY-K-8644, NAN-190 HBr, LY-53857, SDZ-205557, and tropisetron were
obtained from RBI-Sigma. KT-5823 was purchased from Calbiochem (San
Diego, CA), atropine from Elkins-Sinn (Cherry Hill, NJ), and Sch-23390
from Schering-Plough (Atlanta, GA).
 |
RESULTS |
The circular muscle strips from the rat middle colon generated
regular phasic contractions and GCs (Fig.
1). The amplitude of phasic contractions
occurring in between GCs was nearly uniform. In addition, there was
typically a burst of two to six phasic contractions that occurred at
the tail end of GCs and had a larger amplitude than of those that
occurred in between GCs (Fig. 1).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1.
Enhancement of spontaneous giant contractions (GCs) in
rat colonic circular muscle strips by -conotoxin GVIA ( -CTX, 0.5 µM), tetrodotoxin (TTX, 1 µM), and
N -nitro-L-arginine
(L-NNA, 0.1 mM).
|
|
Neural regulation of spontaneous contractions.
The spontaneous phasic contractions occurred at a frequency of 7.1 ± 0.5/min with a mean amplitude of 6 ± 1 mN and GCs at a
frequency of 0.21 ± 0.02/min with a mean amplitude of 30 ± 6 mN (n = 47). The blockade of fast sodium channel
enteric neural conduction by 1 µM TTX or N-type Ca2+
channels by 0.5 nM
-CTX immediately increased the frequency of GCs
to 0.78 ± 0.05/min and 0.65 ± 0.05/min, respectively (Figs. 1 and 2), and amplitude to 61 ± 1 mN and 57 ± 1 mN, respectively (n = 5, P < 0.01 both). The amplitude and frequency of phasic contractions were
unaltered except for those that occurred at the tail end of the GCs.
Their amplitude was increased in 65% of the preparations by 47 ± 12% (n = 5, Fig. 1).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of different neural blockers, receptor antagonists, second
messengers, and ion channel blockers on the frequency (A)
and amplitude (B) of GCs in the circular muscle of the rat
middle colon (P < 0.05 vs. control). ODQ,
1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one.
|
|
L-NNA (0.1 mM) also increased the GC frequency and
amplitude (Fig. 1) to 0.79 ± 0.04/min and 60 ± 1 mN,
respectively (P < 0.01, n = 5, Fig.
2). These values were not significantly different from those after
neural or N-type Ca2+ channel blockade (P > 0.05). The blockade of NO synthesis also increased the amplitude of
phasic contractions in between GCs by 68 ± 17% in 81% of the
strips (n = 5).
Table 1 lists the pharmacological
antagonists and their concentrations used to investigate the role of
spontaneous neurotransmitter release in the genesis or modulation of
GCs, phasic contractions, and tone. Atropine (1 µM), a nonspecific
muscarinic receptor antagonist, reduced the GC frequency transiently,
but it recovered in 10 min (Fig. 3). Atropine had no
transient or long-term effect on spontaneous phasic contractions. The
concentration of 1 µM atropine totally blocked the excitatory effect
of 30 µM ACh, confirming that this dose was effective in blocking
muscarinic receptors (Fig. 3).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
A: lack of antagonism of spontaneous rat
colonic GCs by 1 µM atropine. ACh (30 µM) was added in the presence
of atropine to illustrate that muscarinic blockade was complete.
B: typical response of a different colonic strip to 30 µM
ACh without atropine.
|
|
Hexamethonium, a nicotinic-receptor antagonist, and antagonists for
adrenergic, serotonergic, histaminergic, VIP, pituitary adenylate
cyclase-activating polypeptide (PACAP), and tachykininergic receptors did not affect the frequency or amplitude of GCs and phasic
contractions with the exception of selective NK3-receptor antagonist
Trp7,
-Ala8-NKA4-10, which
elevated the tone of muscle strips and increased the frequency
of GCs to 0.51 ± 0.05/min (Fig. 4).
This effect was resistant to 1 µM TTX, indicating that
NK3 tachykininergic regulation of tone and GCs is nonneural
in origin.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
A: effect of NK3-receptor
antagonist Trp7,
-Ala8-NKA4-10 (10 µM) on spontaneous
contractions of the rat colonic circular muscle strips. The amplitude
of the phasic contractions was suppressed, whereas GC frequency and
tone were increased. B: dopamine D1-receptor
antagonist Sch-23390 (10 µM) increased the frequency and amplitude of
spontaneous GCs.
|
|
The nonspecific purinergic antagonist suramin did not alter the GC
frequency or amplitude in the 10-min period after its addition to the
bath. However, it increased the amplitude of the GCs to 41 ± 6 mN
(n = 8, P < 0.05) 30 min after being
added to the bath (Fig. 2). The frequency of GCs was not altered at
this time. The role of purinergic receptors was examined further with
selective antagonists. PPADS (P2X receptor antagonist) did
not affect the spontaneous phasic contractions or GCs. Reactive blue
(P2Y receptor antagonist) actually decreased the amplitude
of GCs to 13 ± 1.3 mN (n = 4, P < 0.05). By contrast, dopamine D1 receptor antagonist Sch-233990 (10 µM) consistently increased the frequency to 0.71 ± 0.05/min and amplitude to 51 ± 12 mN of GCs (n = 7, P < 0.05, Fig. 2B and Fig.
4B). The selective D2 receptor antagonist
domperidone (10 µM) caused an increase in GC amplitude to 39 ± 5 mN (n = 6, P < 0.05) without
affecting the frequency. These antagonists had no effect on phasic contractions.
Intracellular regulation of GCs and phasic
contractions.
When the activation of soluble guanylate cyclase was blocked by 1 µM
ODQ, the GC frequency and amplitude exhibited a similar immediate
increase to 0.73 ± 0.06/min and 63 ± 2 mN
(n = 9, P < 0.01) as was seen after
L-NNA, TTX, and
-CTX. The amplitude of phasic
contractions in between the GCs was not affected, but that of
contractions superimposed on the trailing edge of GCs was increased by
32 ± 5% in 45% of the strips (Fig.
5A). In contrast, the
inhibition of adenylate cyclase by 10 µM MDL-12330 had no effect on
the frequency or amplitude of GCs or phasic contractions 10 min after
its addition to the muscle bath (Fig. 5B).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
A: enhancement of rat colonic spontaneous GCs
by guanylate cyclase inhibitor ODQ (1 µM). ODQ increased both the
frequency and amplitude of spontaneous GCs. B: by contrast,
adenylate cyclase inhibitor MDL-12330A was without effect.
|
|
To elucidate whether TTX, L-NNA, and ODQ act through common
pathways, the effect of each of these agents was examined in the presence of the others. Neither L-NNA nor ODQ further
altered the frequency and amplitude of GCs after neural blockade with TTX (Fig. 6). Similarly, TTX had no
effect on GCs in the presence of L-NNA or ODQ (Fig. 6).
Furthermore, electrical field stimulation (EFS) at 10 Hz, 0.4 ms
duration, 150 V for 5 min or the NO donor SNAP (100 µM) in the
presence of atropine, phentolamine, and propranolol (1 µM each)
completely abolished the GCs (Fig. 7,
A and B). The phasic contractions persisted in
the presence of EFS, but they were abolished by SNAP. The GCs were
abolished by the membrane-permeable cGMP analog 8-BrcGMP (10 µM, Fig.
7C). In the presence of 10 µM 8-BrcGMP, neither TTX, nor
L-NNA, nor ODQ were able to revive the GCs. As noted
earlier, all three agents increased the frequency and amplitude of GCs
without 8-BrcGMP.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 6.
A and B: presence of TTX (1 µM)
prevented L-NNA (0.1 mM) or ODQ (1 µM) from further
increasing the frequency of GCs. C and D:
likewise, ODQ (1 µM) or L-NNA (0.1 mM) prevented further
enhancement of GCs by TTX (1 µM).
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
Blockade of GCs by electrical field stimulation
(A, EFS, 10 Hz, 0.4-ms duration, 150 V for 5 min), nitric
oxide donor S-nitroso-N-acetyl-penicillamine
(B, SNAP, 0.1 mM), and the permeable cGMP analog
8-bromo-cGMP (8-BrcGMP, C).
|
|
The inhibition of cGMP-dependent kinase (protein kinase G, PKG) with
KT-5823 (1 µM) had no effect on GCs, indicating that cGMP inhibits
GCs independent of the activation of PKG.
An alternate pathway for the inhibition of cell contractions by cGMP
involves the K+ channels (21, 27). The
blockade of Ca2+-activated K+ channels with 1 µM apamin increased the frequency of GCs to 0.63 ± 0.04/min and
amplitude to 69 ± 9 mN (n = 4, P < 0.01, Fig. 8C). In the
presence of TTX or L-NNA, apamin did not further affect the
frequency, but it further increased the GC amplitude to 87 ± 10 mN (n = 3, P < 0.05). In the presence
of apamin, ODQ further increased the GC frequency to 0.98 ± 0.04/min (n = 3, P < 0.05).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 8.
A: enhancement of GCs in the rat colon by
BAY-K-8644 (1 µM). B: blockade by L-type calcium channel
blocker D-600 (1 µM) of all mechanical activity. C:
increase by Ca2+-activated small-conductance K+
channel blocker apamin (1 µM) of the frequency and amplitude of GCs.
D: lack of effect of the nonspecific blocker of cation
channels gadolinium on spontaneous GCs.
|
|
The Ca2+ channel opener BAY-K-8644 (1 µM) increased the
GC frequency to 0.76 ± 0.07/min and amplitude to 67 ± 3 mN
(P < 0.05, n = 5). All GCs and phasic
contractions were totally blocked by 1 µM D-600 (Fig. 8B).
Thus Ca2+ influx through L-type channels is
necessary for both phasic contractions and GCs. In contrast, gadolinium
chloride (10 µM), a nonspecific blocker of cation channels (20,
29) had no effect on the GCs or phasic contractions.
The origin of NO inducing cGMP
synthesis.
The cGMP content in the muscularis externa containing the myenteric
plexus was 39 ± 3 pmol/g tissue weight. When the muscle strips
were incubated with 1 µM TTX, 0.1 mM L-NNA, or 1 µM
ODQ, the cGMP content significantly decreased to 25 ± 3, 19 ± 2, and 22 ± 3 pmol/g tissue weight, respectively
(n = 5, P < 0.05 for each, Fig.
9A).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 9.
cGMP synthesis in rat colonic muscle strips
(A) and dispersed smooth muscle cells (B) in
response to 1 µM TTX, 0.1 mM L-NNA, and 1 µM ODQ. In
B, the cells treated with 10 µM SNAP increased their cGMP
content by 86%.
|
|
We then measured cGMP in dispersed smooth muscle cells devoid of
enteric ganglia. The cGMP content in dispersed cells was 0.24 ± 0.05/106 cells. When the cells were incubated with 1 µM
TTX, 0.1 mM L-NNA, or 1 µM ODQ, the cGMP content was
0.22 ± 0.07, 0.23 ± 0.02, and 0.19 ± 0.04/106 cells, respectively (n = 5, P > 0.05 for each, Fig. 9B). We checked whether the dispersed cells retained their ability to synthesize cGMP
in response to NO. An aliquot of dispersed cells was incubated for 10 min with the NO donor 0.1 mM SNAP; the cGMP level increased to
0.41 ± 0.06/106 cells (n = 4, P < 0.05, Fig. 9B), thus confirming that
guanylate cyclase activation by NO is maintained in the dispersed cells.
 |
DISCUSSION |
Inhibitory neural regulation of GCs.
We (12) and Middleton et al. (25) have noted
that circular muscle strips prepared from the rat middle colon generate
spontaneous GCs as well as rhythmic phasic contractions. The generation
of spontaneous phasic contractions by muscle strips is well
established, but the generation of spontaneous and regularly occurring
GCs was novel. We therefore used these strips to investigate the
enteric neural regulation of GCs. Our findings show that in the rat
colon, the GCs are tonically inhibited by nonadrenergic,
noncholinergic (NANC) inhibitory neurons. The inhibition of fast
Na+ channel enteric neural conduction by TTX or the
blockade of N-type neural Ca2+ channels by
-CTX
increased the frequency and amplitude of GCs.
Anuras and Christensen (2) reported tonic inhibition of in
vitro rhythmic phasic contractions by NANC neurons in cat colonic circular muscle strips. They found that TTX enhanced the amplitude of
spontaneous contractions of colonic circular but not longitudinal muscle strips in this species (2). Wood (49)
found that TTX enhanced the spontaneous phasic contractions in isolated
segments of the cat jejunum. By contrast, Rae et al. (34)
found that TTX had no effect on the spontaneous contractions of the
human colonic circular muscle strips, whereas it enhanced the amplitude of phasic contractions in the canine colon (19). In rat
middle colon, TTX had no significant effect on phasic contractions, but at the same time it enhanced the GCs. TTX did increase the amplitude of
phasic contractions that occurred on the falling phase of the GCs.
However, the basal conditions for these phasic contractions may be
different from those that occur in between GCs. For example, the
membrane is likely to be depolarized during a GC (37).
Interestingly, the phasic contractions persisted in the presence of EFS
at 10 Hz, 0.4 ms, 150 V for 5 min, even though it blocked the GCs. On the other hand, SNAP, an NO donor, and 8-BrcGMP both blocked the phasic
contractions as well as GCs. The differences between the effects of TTX
and EFS on spontaneous phasic contractions and GCs is likely to be due
to a difference in the thresholds of inhibitory effects on the two
types of contractions. The ongoing spontaneous inhibitory input or that
on EFS is enough to suppress the GCs but not the phasic contractions.
The GCs are large-amplitude and long-duration contractions and
therefore they may maximally utilize the cellular resources such as
cytosolic Ca2+ to contract, hence their reduced
susceptibility to inhibitory input.
In muscle strips, it seems that the spontaneous generation of GCs is
not dependent on the release of a neurotransmitter from the known
excitatory cholinergic, serotoninergic, histaminergic, or
tachykininergic neurons. The antagonists of their receptors (or
receptor subtypes) had no effect on the frequency or amplitude of GCs.
By contrast, Li et al. (22) found that in the in vivo intact conscious state, the spontaneous GCs in the rat colon are blocked by atropine. In muscle bath, atropine transiently reduced the
frequency of GCs, but it recovered within 10 min. One possibility is
that stretch, which is an essential feature of muscle bath and induces
Ca2+ influx, is able to trigger the same cellular
mechanisms in vitro that ACh does in vivo to stimulate GCs. No
spontaneous rhythmic contractions were observed visually in strips when
they were not stretched. Other reports have indicated that stretch
opens nonselective cation channels in smooth muscle cells
(16). We found, however, that gadolinium chloride, a
blocker of nonspecific cation channels, did not block the GCs or phasic
contractions. On the other hand, the blockade of L-type
Ca2+ channels totally inhibited all spontaneous
contractions. Farrugia et al. (10) have reported
stretch-activated L-type Ca2+ channels in human intestinal
smooth muscle cells. The stretch on muscle strips therefore may open
the L-type channels to activate the cellular processes that produce
these contractions. This hypothesis is supported by the observation
that the opening of L-type channels by BAY-K-8640 increased the
frequency and amplitude of GCs.
The inhibition of NK3 receptor increased the basal tone of
muscle strips as well as the frequency of GCs. This was a direct effect
of NK3 antagonist on smooth muscle cells because it was not
blocked by TTX. Of all the antagonists used in this study, NK3 antagonist was the only one that increased muscle tone.
The others selectively affected the frequency and amplitude of GCs or
had no effect. This confirms other reports that the cellular processes
for the generation of different types of contractions may differ
(40).
Inhibitory neurotransmitters.
Further examination of the tonic inhibitory input to suppress GCs
indicated that both NO and dopamine may be involved in this phenomenon.
The inhibition of NO synthesis immediately increased the amplitude and
frequency of GCs to the same extent as that by TTX or
-CTX.
Middleton et al. (25) also found that a constitutive release of NO enhances the frequency of what they called summation contractions in the rat distal colon. We (12) showed,
however, that the GCs are distinct contractions and that they are not
due to the summation of phasic contractions. The inhibition of
D1 receptors by Sch-233990 had a similar effect. On the
other hand, the blockade of D2 receptors by domperidone
increased only the amplitude of GCs without affecting their frequency.
The reasons for this difference in the response between the blockade of
D1 and D2 receptors are not known, but it is
likely that each receptor is coupled to a different signal transduction
pathway to produce its inhibitory effect. By contrast, in the dog and
the human colon dopamine has been found to be excitatory rather than
inhibitory (5, 48).
The addition of ODQ, Sch-233990, or domperidone to the muscle bath
enhanced the GCs, but it had no significant effect on the phasic
contractions. Franck et al. (11) also found that ODQ has
no effect on spontaneous phasic contractions in the canine proximal
colon. These observations highlight the differences in the regulation
of spontaneous GCs and phasic contractions by tonic inhibitory input.
The exception to the above observations is that nitro-L-arginine methyl ester (L-NAME)
increased the frequency of phasic contractions in ~65% of the muscle
strips. The precise reasons for this anomaly are not known.
In contrast to NO and dopamine antagonists, the antagonism of VIP
receptors by VIP(10---28) and purinergic receptors by suramin, PPADS
(P2x), and reactive blue (P2y) did not increase
the frequency of GCs. The inhibition of adenylate cyclase by MDL-12330
also had no effect on spontaneous GCs. It therefore seems that ATP and
VIP-PACAP are not released spontaneously by the NANC inhibitory neurons
to inhibit GCs. Plujà et al. (32) found, however,
that suramin increases the amplitude and frequency of phasic
contractions in the rat colon. They did not specify the part of the
colon used in their study. It is possible that the expression of ATP
may be different in different parts of the colon, as is the case with NO (43).
Cellular mechanisms of NO-mediated inhibition of
GCs.
Previous studies have reported that NO activates guanylate cyclase to
inhibit cell contractility (7, 13, 45, 47). Our findings
show that this cellular pathway is utilized by the spontaneous release
of NO to inhibit GCs. ODQ that inhibits the activation of guanylate
cyclase increased the frequency and amplitude of GCs similar to that
seen after TTX,
-CTX, and L-NNA. In addition, 8-BrcGMP
inhibited the GCs.
cGMP has been reported to inhibit smooth muscle contractility by the
activation of PKG and by the opening of Ca2+-dependent
small conductance K+ channels (35). In our
study, the inhibition of PKG by KT-5823 did not affect the spontaneous
GCs. Smaller concentrations of KT-5823 have been shown to inhibit the
activation of PKG in other cell types (44). On the other
hand, the inhibition of Ca2+-dependent small- conductance
K+ channels by apamin increased the frequency and amplitude
of GCs to the same extent as that seen after TTX, L-NNA, or
ODQ. There may, however, be an additional pathway, such as the
regulation of the sensitivity of the contractile proteins to
Ca2+ (29) for the inhibition of GCs by cGMP
because in the presence of apamin, ODQ further increased the frequency
of GCs. This pathway is undefined.
The spontaneous release of NO and subsequent formation of cGMP is a
constitutive phenomenon independent of stretch. In unstretched muscle
strips TTX, L-NNA, or ODQ reduced the basal levels of cGMP in tissues containing longitudinal muscle, circular muscle, and the
myenteric plexus. By contrast, these agents had no effect on the basal
cGMP content of dispersed circular smooth muscle, even though these
cells maintained their ability to synthesize cGMP in response to NO.
These findings confirmed that NO was being released from the enteric neurons.
Physiological and clinical relevance.
Most previous studies (3, 4, 6, 9, 28, 31, 42) have
investigated the inhibitory role of NO in gut motility by monitoring
the relaxation of stretch or agonist-induced tone in muscle strips or
by monitoring inhibitory junctional potentials. Whereas a physiological
role of tone is well defined in sphincteric muscle (1, 36,
46), its physiological role in the main organs of the
gastrointestinal tract is not fully understood. For this reason, we
focused our studies on the potential role of NO in the generation of
spontaneous phasic contractions and GCs whose motility roles in mixing
and propulsion of digesta are well defined (8, 17, 33,
41). The phasic contractions cause the orderly slow distal
propulsion of luminal contents in the fasting and postprandial states,
whereas GMCs cause mass movements. Our findings show that a
constitutive release of NO and dopamine may suppress the amplitude and
frequency of spontaneous in vitro GCs in the rat middle colon.
Mizuta et al. (26) found that the inhibition of NO
synthesis by L-NAME in intact rats retards colonic transit.
Studies in our laboratory have shown that L-NAME increases
the frequency of GCs in intact rats (22), which is similar
to that seen in muscle strips. Mizuta et al. (26) proposed
that the slower transit after L-NAME may be due to the
impairment of descending relaxation. Our findings suggest that the
delayed transit after the inhibition of NO synthase may also be due to
the uncontrolled stimulation of spontaneous GCs in this species. The
suppression of GCs in the untreated colon may produce spatial
coordination so that they are more effective in propulsion. If so, the
inhibitory neurons may have an important role in producing not only
descending inhibition (14, 15) but also partially
inhibiting the spontaneous contractions to facilitate propulsion.
Orihata and Sarna (30) also found that the inhibition of
NO synthase increases the frequency of pyloric and duodenal
contractions without a concurrent coordination of contractions across
the pylorus and therefore delays gastric emptying. The degree of this
nitrergic inhibition may, however, vary among different species because
available evidence indicates that the neural inhibition of spontaneous
contractions in dog and human colon may be less pronounced than that in
the rat colon.
We conclude that a constitutive release of NO and dopamine inhibit the
GCs but not the phasic contractions in the rat mid-colon. The
NO-induced inhibition is mediated by cGMP and
Ca2+-dependent small conductance K+ channels.
PKG does not seem to be involved. These findings, together with those
observed in vivo (26), suggest that this inhibition may be
important in normal transit in the rat colon.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by the National Institute of
Diabetes and Digestive and Kidney Diseases Grant DK-32346 and 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 28 August 2000; accepted in final form 7 February 2001.
 |
REFERENCES |
1.
Allescher, HD,
Tougas G,
Vergara P,
Lu S,
and
Daniel EE.
Nitric oxide as a putative nonadrenergic noncholinergic inhibitory transmitter in the canine pylorus in vivo.
Am J Physiol Gastrointest Liver Physiol
262:
G695-G702,
1992[Abstract/Free Full Text].
2.
Anuras, S,
and
Christensen J.
Effects of autonomic drugs on cat colonic muscle.
Am J Physiol Gastrointest Liver Physiol
240:
G361-G364,
1981[Abstract/Free Full Text].
3.
Boeckxstaens, GE,
Pelckmans PA,
Bult H,
de Man JG,
Herman AG,
and
van Maercke YM.
Evidence for nitric oxide as mediator of non-adrenergic, non-cholinergic relaxations induced by ATP and GABA in the canine gut.
Br J Pharmacol
102:
434-438,
1991[Abstract].
4.
Boeckxstaens, GE,
Pelckmans PA,
Herman AG,
and
van Maercke YM.
Involvement of nitric oxide in the inhibitory innervation of the human isolated colon.
Gastroenterology
104:
690-697,
1993[ISI][Medline].
5.
Bueno, L,
Fargeas MJ,
Fioramonti J,
and
Honde C.
Effects of dopamine and bromocriptine on colonic motility in dog.
Br J Pharmacol
82:
35-42,
1984[Abstract].
6.
Burleigh, DE.
N G-nitro-L-arginine reduces nonadrenergic, noncholinergic relaxations of human gut.
Gastroenterology
102:
679-683,
1992[ISI][Medline].
7.
Conklin, JL,
and
Du C.
Guanylate cyclase inhibitors: effect on inhibitory junction potentials in esophageal smooth muscle.
Am J Physiol Gastrointest Liver Physiol
263:
G87-G90,
1992[Abstract/Free Full Text].
8.
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].
9.
Dalziel, HH,
Thornbury KD,
Ward SM,
and
Sanders KM.
Involvement of nitric oxide synthetic pathway in inhibitory junction potentials in canine proximal colon.
Am J Physiol Gastrointest Liver Physiol
260:
G789-G792,
1991[Abstract/Free Full Text].
10.
Farrugia, G,
Holm AN,
Rich A,
Sarr MG,
Szurszewski JH,
and
Rae JL.
A mechanosensitive calcium channel in human intestinal smooth muscle cells.
Gastroenterology
117:
900-905,
1999[ISI][Medline].
11.
Franck, H,
Sweeney KM,
Sanders KM,
and
Shuttleworth CW.
Effects of a novel guanylate cyclase inhibitor on nitric oxide-dependent inhibitory neurotransmission in canine proximal colon.
Br J Pharmacol
122:
1223-1229,
1997[Abstract].
12.
Gonzalez, A,
and
Sarna SK.
Different types of contractions in rat colon and their modulation by oxidative stress.
Am J Physiol Gastrointest Liver Physiol
280:
G546-G554,
2001[Abstract/Free Full Text].
13.
Goyal, RK,
and
Xue DH.
Evidence for NO · redox form of nitric oxide as nitrergic inhibitory neurotransmitter in gut.
Am J Physiol Gastrointest Liver Physiol
275:
G1185-G1192,
1998[Abstract/Free Full Text].
14.
Grider, JR.
Interplay of VIP and nitric acid in regulation of the descending relaxation phase of peristalsis.
Am J Physiol Gastrointest Liver Physiol
264:
G334-G340,
1993[Abstract/Free Full Text].
15.
Hata, F,
Ishii T,
Kanada A,
Yamano N,
Kataoka T,
Takeuchi T,
and
Yagasaki O.
Essential role of nitric oxide in descending inhibition in the rat proximal colon.
Biochem Biophys Res Commun
172:
1400-1406,
1990[ISI][Medline].
16.
Hisada, T,
Walsh JV, Jr,
and
Singer JJ.
Stretch-inactivated cationic channels in single smooth muscle cells.
Pflügers Arch
422:
393-396,
1993[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.
Karaus, M,
and
Sarna SK.
Giant migrating contractions during defecation in the dog colon.
Gastroenterology
92:
925-933,
1987[ISI][Medline].
19.
Keef, KD,
Murray DC,
Sanders KM,
and
Smith TK.
Basal release of nitric oxide induces an oscillatory motor pattern in canine colon.
J Physiol (Lond)
499:
773-786,
1997[Abstract].
20.
Kirber, M,
Guerrero-Hernandez A,
Bowman DS,
Fogarty KE,
Tuft RA,
Singer JJ,
and
Fay FS.
Multiple pathways responsible for the stretch-induced increase in Ca2+ concentration in toad stomach smooth muscle cells.
J Physiol (Lond)
524:
3-17,
2000[Abstract/Free Full Text].
21.
Koh, SD,
Campbell JD,
Carl A,
and
Sanders KM.
Nitric oxide activates multiple channels in canine colonic smooth muscle.
J Physiol (Lond)
489:
735-743,
1995[Abstract].
22.
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.
23.
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].
24.
Matsufuji, H,
Yokoyama J,
Hirabayashi T,
Wantanabe S,
and
Sakurai K.
Cooperative roles of colon and anorectum during spontaneous defecation in conscious dogs.
Dig Dis Sci
43:
2042-2047,
1998[ISI][Medline].
25.
Middleton, SJ,
Cuthbert AW,
Shorthouse M,
and
Hunter JO.
Nitric oxide affects mammalian distal colonic smooth muscle by tonic neural inhibition.
Br J Pharmacol
108:
974-979,
1993[Abstract].
26.
Mizuta, Y,
Takahashi T,
and
Owyang C.
Nitrergic regulation of colonic transit in rats.
Am J Physiol Gastrointest Liver Physiol
277:
G275-G279,
1999[Abstract/Free Full Text].
27.
Mule, F,
D'Angelo S,
and
Serio R.
Tonic inhibitory action by nitric oxide on spontaneous mechanical activity in rat proximal colon: involvement of cyclic GMP and apamin-sensitive K+ channels.
Br J Pharmacol
127:
514-520,
1999[Abstract/Free Full Text].
28.
Murray, J,
Du C,
Ledlow A,
Bates JN,
and
Conklin JL.
Nitric oxide: mediator of nonadrenergic noncholinergic responses of opossum esophageal muscle.
Am J Physiol Gastrointest Liver Physiol
261:
G401-G406,
1991[Abstract/Free Full Text].
29.
Nishimura, J,
and
van Breemen C.
Direct regulation of smooth muscle contractile elements by second messengers.
Biochem Biophys Res Commun
163:
929-935,
1989[ISI][Medline].
30.
Orihata, M,
and
Sarna SK.
Inhibition of NO synthase delays gastric emptying of solid meals.
J Pharmacol Exp Ther
271:
660-670,
1994[Abstract].
31.
Osthaus, LE,
and
Galligan JJ.
Antagonists of nitric oxide synthesis inhibit nerve-mediated relaxations of longitudinal muscle in guinea pig ileum.
J Pharmacol Exp Ther
260:
140-145,
1992[Abstract].
32.
Plujà, L,
Fernández E,
and
Jiménez M.
Neural modulation of the cyclic electrical and mechanical activity in the rat colonic circular muscle: putative role of ATP and NO.
Br J Pharmacol
126:
883-892,
1999[Abstract/Free Full Text].
33.
Quigley, EMM,
Phillips SF,
and
Dent J.
Distinctive patterns of interdigestive motility of the canine ileocolonic junction.
Gastroenterology
87:
836-844,
1984[ISI][Medline].
34.
Rae, MG,
Fleming N,
McGregor DB,
Sanders KM,
and
Keef KD.
Control of motility patterns in the human colonic circular muscle layer by pacemaker activity.
J Physiol (Lond)
510:
309-320,
1998[Abstract/Free Full Text].
35.
Rapoport, RM,
and
Murad F.
Agonist induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cyclic GMP.
Circ Res
52:
352-357,
1983[Abstract].
36.
Rattan, S,
and
Chakder S.
Role of nitric oxide as a mediator of internal anal sphincter relaxation.
Am J Physiol Gastrointest Liver Physiol
262:
G107-G112,
1992[Abstract/Free Full Text].
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.
Physiology and pathophysiology of colonic motor activity (1).
Dig Dis Sci
36:
827-862,
1991[ISI][Medline].
39.
Sarna, SK.
Physiology and pathophysiology of colonic motor activity (2).
Dig Dis Sci
36:
998-1018,
1991[ISI][Medline].
40.
Sarna, SK.
Differential signal transduction pathways to stimulate colonic giant migrating and phasic contractions (Abstract).
Gastroenterology
118:
4404,
2000.
41.
Sethi, AK,
and
Sarna SK.
Contractile mechanisms of colonic propulsion.
Am J Physiol Gastrointest Liver Physiol
268:
G530-G538,
1995[Abstract/Free Full Text].
42.
Shuttleworth, CWR,
Murphy R,
and
Furness JB.
Evidence that nitric oxide participates in non-adrenergic inhibitory transmission to intestinal muscle in the guinea-pig.
Neurosci Lett
130:
77-80,
1991[ISI][Medline].
43.
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].
44.
Tejedo, J,
Bernabe JC,
Ramirez R,
Sobrino F,
and
Bedoya FJ.
NO induces a cGMP-independent release of cytochrome C from mitochondria which precedes caspase 3 activation in insulin producing RINm5F cells.
FEBS Lett
459:
238-243,
1999[ISI][Medline].
45.
Thornbury, KD,
Ward SM,
Dalziel HH,
Carl A,
Westfall DP,
and
Sanders KM.
Nitric oxide mimics non-adrenergic, non-cholinergic hyperpolarization in gastrointestinal muscles.
Am J Physiol Gastrointest Liver Physiol
261:
G553-G557,
1991[Abstract/Free Full Text].
46.
Tottrup, A,
Svane D,
and
Forman A.
Nitric oxide mediating NANC inhibition in opossum lower esophageal sphincter.
Am J Physiol Gastrointest Liver Physiol
260:
G385-G389,
1991[Abstract/Free Full Text].
47.
Ward, SM,
Dalziel HH,
Bradley ME,
Buxton ILO,
Keef K,
Westfall DP,
and
Sanders KM.
Involvement of cyclic GMP in non-adrenergic, non-cholinergic inhibitory neurotransmission in dog proximal colon.
Br J Pharmacol
107:
1075-1082,
1992[Abstract].
48.
Wiley, J,
and
Owyang C.
Dopaminergic modulation of rectosigmoid motility: action of domperidone.
J Pharmacol Exp Ther
242:
548-551,
1987[Abstract].
49.
Wood, JD.
Excitation of intestinal muscle by atropine, tetrodotoxin, and xylocaine.
Am J Physiol
222:
118-125,
1972[ISI][Medline].
Am J Physiol Gastrointest Liver Physiol 281(1):G275-G282