Somatostatin sst2 receptors inhibit peristalsis in
the rat and mouse jejunum
Faiza
Abdu1,
Gareth A.
Hicks2,
Grant
Hennig3,
Jeremy P.
Allen4, and
David
Grundy1
1 Department of Biomedical Science, Alfred Denny
Building, University of Sheffield, Western Bank, Sheffield S10 2TN;
2 GlaxoSmithKline, Gastrointestinal Department,
Neurology CEDD, New Frontiers Science Park, Harlow CM19 5AW;
4 Laboratory of Cognitive and Developmental
Neuroscience, The Babraham Institute, Babraham, Cambridge, CB2 4AT,
United Kingdom; and 3 Department of
Physiology and Cell Biology, College of Medicine, University of
Nevada, Reno, Nevada 89557
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ABSTRACT |
Somatostatin [somatotropin
release-inhibitory factor (SRIF)] has widespread actions
throughout the gastrointestinal tract, but the receptor mechanisms
involved are not fully characterized. We have examined the effect of
selective SRIF-receptor ligands on intestinal peristalsis by studying
migrating motor complexes (MMCs) in isolated segments of jejunum from
rats, mice, and sst2-receptor knockout mice. MMCs were
recorded in 4- to 5-cm segments of jejunum mounted horizontally in
vitro. MMCs occurred in rat and mouse jejunum with intervals of
104.4 ± 10 and 131.2 ± 8 s, respectively. SRIF,
octreotide, and BIM-23027 increased the interval between MMCs, an
effect fully or partially antagonized by the sst2-receptor antagonist Cyanamid154806. A non-sst2 receptor-mediated
component was evident in mouse as confirmed by the observation of an
inhibitory action of SRIF in sst2 knockout tissue. Blocking
nitric oxide generation abolished the response to SRIF in rat but not
mouse jejunum. sst2 Receptors mediate inhibition of
peristalsis in both rat and mouse jejunum, but a non-sst2
component also exists in the mouse. Nitrergic mechanisms are
differentially involved in rat and mouse jejunum.
knockout; migrating motor complex; enteric nervous system; somatotropin release-inhibitory factor
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INTRODUCTION |
ISOLATED SEGMENTS OF
BOWEL can perform complex and coordinated contractile activity,
which is dependent on interactions between myogenic and local neural
mechanisms. The polarized reflex responses to intestinal stimuli, first
described by Bayliss and Starling (3), are now recognized
to involve activation of ascending and descending enteric pathways
leading to contraction and relaxation of smooth muscle
(6). A variety of different transmitters and neuromodulators in these enteric pathways contributes to the
coordination of this peristaltic activity. Somatostatin [somatotropin
release-inhibitory factor (SRIF)] is present in a subpopulation of
descending interneurones that project caudally within the myenteric
plexus but not into either the longitudinal or circular muscle layers
of the intestine (10, 31, 35). SRIF is also found in
submucous plexus neurones, around submucosal blood vessels, and is
present in mucosal endocrine cells in human (31), guinea
pig (10), and rodent intestine (12, 13). SRIF
is released by intestinal distension (11) and participates
in the coordination of descending relaxation (21). The
effects of exogenous SRIF on intestinal motility are complex. In the
guinea pig ileum, both excitatory and inhibitory effects, operating
through prejunctional mechanisms (14, 18, 24, 36, 45),
have been described. The contractile response of the rat colon to SRIF
also appears to involve activation of noncholinergic nerves
(33), possibly by a process of disinhibition of vasoactive
intestinal peptide (VIP) ergic/nitrergic interneurones supplying
longitudinal muscle motoneurones (22). In contrast, the
activity of VIPergic/nitregic neurons supplying the circular muscle
layer is augmented by SRIF leading to relaxation (21). Thus SRIF plays a neuromodulatory role by regulating transmitter release (45), although postjunctional effects on enteric
neuronal excitability have also been described (30, 34).
The diverse effects of SRIF are mediated by specific, high-affinity,
membrane-bound receptors termed sst1-5
(28). Expression of all five of these receptors has been
described in the wall of the gastrointestinal tract (32).
A number of synthetic peptides have been identified that displays
selectivity for recombinant SRIF receptors. Octreotide and BIM-23027
are agonists with some selectivity for the sst2 receptor,
but each has additional agonist activity at sst5 receptors,
whereas Cyanamid154806 (Cyn) and BIM-23056 are antagonists for
sst2 and sst5 receptors, respectively (2, 42). In this respect, BIM-23027 has been shown to inhibit
neurogenically mediated contraction in the guinea pig ileum
(15), whereas octreotide modulates gastrointestinal
motility in both animals and human tissue (29, 40). The
aims of this study were therefore to characterize the spontaneous
peristaltic activity observed in isolated preparations of rat and mouse
small intestine and to examine its modulation by SRIF and selective
sst-receptor ligands. The availability of an sst2-receptor
knockout mouse provided an opportunity to further characterise the role
of sst2 receptors and also to compare the effects of SRIF
in rat and mouse tissues.
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METHODS |
Experiments were performed on in vitro segments of jejunum from
Sheffield-strain female hooded Lister rats (250-350 g) and 10- to
12-wk-old male C57BL/6 mice (25-37 g). The
sst2-receptor knockout mice
(Sstr2
/
) were generated at the
Babraham Institute (Cambridge, UK) by gene targeting (1).
Briefly, the Sstr2 coding sequence was replaced
by homologous recombination in HM-1 embryonic stem cells (129/OlaHsd)
with a cassette comprising a neomycin selectable marker and a
lacZ reporter gene. Chimeras were produced by blastocyst injection and then mated to C57BL/6 to achieve germline transmission. No sst2-receptor expression was detectable in
Sstr2
/
by RT-PCR. The mutation
was back-crossed onto C57BL/6 for three generations. Heterozygous
intercrossing was then employed to generate the wild-type and
sst2-receptor knockout animals examined in the current study.
Animals were stunned by a blow to the head and killed by cervical
dislocation. A midline laparotomy was performed, and a segment of
proximal jejunum was rapidly excised beginning 2-3 cm from the
ligament of Treitz. The excised segment was placed in gassed (95%
O2 and 5% CO2) Krebs bicarbonate buffer
solution (composition in mM: 117 NaC1, 4.7 KC1, 25 NaHCO3,
2.5 CaC12, 1.2 MgCl2, 2 NaH2PO4, 1.2 H2O, and 11 D-glucose), cleared of any mesenteric connective tissue,
and the lumen was flushed with Krebs solution. Two jejunal segments
~5 cm in length were prepared from each animal, and four in total
were mounted horizontally in separate 20-ml perfusion chambers. The
oral and aboral ends of each segment were secured to two metal
catheters fixed at either end of the chamber and adjusted to maintain
the segments at their resting length. For each segment, the oral end
was connected to a perfusion pump for intrajejunal infusion of Krebs
solution at a rate of 0.16 ml/min, and the aboral end was attached to a
pressure transducer (Elcomatic EM 760, Elcomatic Ltd, Glasgow, UK) to
record contractile activity as changes in intraluminal pressure under
isovolumetric conditions. Tissues were maintained at 37°C, perfused
with Krebs solution at a rate of 5 ml/min, and allowed to equilibrate
for at least 30 min before experiments started. In some intestinal
segments, spontaneous contractile activity developed during this
equilibration period, but in others it did not. However, in preliminary
experiments, it was found that activity developed more readily when the
segment was distended. We, therefore, standardized the experimental
setup by routinely infusing Krebs buffer into the closed segment to an
initial intraluminal pressure of 10-11 cmH2O in the
rat and 2.5-3.5 cmH2O in the mouse. Regular aborally
propagating waves of contraction [migrating motor complexes (MMCs)]
developed under these conditions and could be maintained for several
hours. The output from the pressure transducers was relayed to a
data-acquisition system (CED 1401+, Cambridge Electronic Design,
Cambridge, UK) and from there to a computer running Spike 2 software
(CED), which displayed the four-channel pressure recordings online and
also stored the data for subsequent offline analysis.
Experimental protocol.
Only preparations in which regular migrating motor complexes were
maintained were used for subsequent experiments. Drugs or the
appropriate vehicle was added to the chambers 20 min after stopping
perfusion and recording continued for a further 10 min before washing
out the drugs and reinstating perfusion.
SRIF or the sst2-receptor agonists octreotide and BIM-23027
were added to the baths to produce final concentrations between 0.l and
1,000 nM. In experiments to study concentration-response relationships,
only one concentration of a single agonist was used in each tissue
segment to avoid complications produced by response desensitization.
The sst2-receptor antagonist Cyn was added 3 min before
exposure to the test agonist. Nitro-L-arginine methyl ester
(L-NAME) and its enantiomer nitro-D-arginine
methyl ester (D-NAME) were added 15 min before the test agonist.
Spatiotemporal maps.
To determine the nature of the contractile activity generated by these
isolated jejunal segments, we used an imaging analysis system as
described previously (25). Briefly, a digital video camera
(Sony, DCR-PC100E) was mounted above the preparation, and brief
sequences of contractile activity were recorded for subsequent analysis
and construction of spatiotemporal maps as described in detail
elsewhere (25). Movement of the intestinal wall are mapped
as changes in diameter along the entire length of the segment and
plotted over time. The widest diameter is coded black, and the
narrowest is coded white, enabling contractions to appear as dynamic
changes in shading in the spatiotemporal maps.
Data analysis.
MMCs were quantified in terms of their peak amplitude above baseline
(cmH2O), duration (s), and interval between them (s; Fig.
1). Baseline values were taken during the
10 min before drug application and the response effect in the 10 min
following application. In preliminary studies, it was established that
SRIF-receptor ligands influenced the interval between contraction
complexes without significantly affecting the contraction amplitude.
This effect was quantified by calculating the maximum interval between MMCs in the 10-min period before and after agonist administration. If
contractions were abolished, then an interval of 600 s was recorded and used in subsequent statistical analysis. Responses are
expressed as absolute values ± SE, with n being the
number of animals. Paired data were compared using Student's
t-test or Wilcoxon's rank sum test as appropriate. Grouped
data from wild-type and sst2-receptor knockout animals were
compared using repeated-measures ANOVA. In all cases, a probability of
P < 0.05 was considered as significant.

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Fig. 1.
Spatiotemporal maps. Spatiotemporal maps
illustrating motor activity in an isolated segment of mouse jejunum. On
the right running top to bottom is a
representative trace of intraluminal pressure showing periodic
increases in intraluminal pressure separated by periods of relative
quiescence. The parameters used to quantify this activity are
illustrated. An expanded recording of 1 contraction complex is shown
together with the associated spatiotemporal map obtained from the video
sequence, a single frame of which is shown above. The maps are
generated by measuring the diameter of the segment at each point along
its length and converting this value into a grayscale (calibration bar)
ranging from white (the minimum diameter) to black (the maximum). The
grayscale coded pixels from each frame of the video sequence were used
to construct a single row. Sequential rows were stacked 1 below the
previous image to produce the spatiotemporal map of intestinal
diameters. An expanded map showing activity during the contractile
period is shown on the far left. Note that light bands
represent waves of contractions that propagate from the oral end of the
segment toward the aboral end at an interval of ~2 s.
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Drugs.
SRIF, L-NAME, D-NAME, atropine sulphate,
nifedipine,
-conotoxin GVIA, and TTX were purchased from Sigma
Chemical. Octreotide acetate "Sandostatin" [D-Phe-c
(Cys-Phe-D-Trp-Lys-Thr-Cys) Thr-ol] was obtained from a
pharmaceutical supplier. BIM-23027
[c(N-Me-Ala-Tyr-D-Trp-Lys-Abu-Phel)], Cyn
[AcNH-4-NO2-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)-Tyr-NH2],
and BIM-23056
[(D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH2)]
were custom synthesized by Neosystem Laboratoire (Strasbourg).
All the peptides were dissolved in distilled water with the exception
of SRIF, which was dissolved in 1% bovine serum albumen in distilled
water, and BIM-23056, which was initially dissolve in 10%
dimethylsulfoxide. Atropine sulfate and
-conotoxin were dissolved in
saline (0.9% NaCl). All drugs were stored at
20°C. Freshly diluted
aliquots were maintained on ice during the course of the experiments
and added to the bath in microlitre volumes.
 |
RESULTS |
Migrating motor complexes.
Luminal distension of isolated segments of rat and mouse jejunum
evoked a regular pattern of contractile activity. The activity consisted of periodic increases in intraluminal pressure separated by
periods of relative quiescent (Fig. 1). Spatiotemporal maps of both
mouse and rat jejunal contractile activity revealed a similar pattern
of activity. The increase in intraluminal pressure coincided with waves
of contraction, seen as parallel lines on the maps that originated at
the oral end of the segment and propagated aborally (Fig. 1). These
contractions occurred at ~2-s intervals and traveled at about 5 mm/s.
The contraction region itself migrated more slowly (~1 cm/min) and
coincided with the maintained rise in intraluminal pressure. The
pressure returned to baseline as the burst of peristaltic activity came
to an end.
MMCs in the rat jejunum.
Baseline activity in a sample of 16 control tissues consisted of
periodic increases in intraluminal pressure of 31 ± 2.1-s duration and 10.5 ± 0.7-cmH2O amplitude separated by
periods of relative inactivity. The mean interval between such MMCs was
104.4 ± 9.6 s. MMCs were completely abolished (Fig.
2) by TTX (0.6 µM, n = 3),
conotoxin (0.1 µM, n = 5), atropine (1 µM,
n = 3), and nifedipine (1 µM, n = 7).
In contrast, the nitric oxide synthase (NOS) inhibitor
L-NAME (100 µM) produced an increase in both MMC frequency and amplitude (Fig. 5A), such that maximum
intervals decreased from 194.4 ± 27 to 75.1 ± 7 s and
amplitude increased from 12.2 ± 1.5 to 17.8 ± 1.7 cmH2O (n = 9, P < 0. 01).
The inactive isomer D-NAME was without effect (100 µM,
n = 4, data not shown).

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Fig. 2.
Contractile activity in the rat jejunum. Migrating motor complexes
(MMCs) in distended segments of rat jejunum consisted of periodic
increases in intraluminal pressure separated by periods of relative
quiescence. Representative traces showing the abolition of MMCs by TTX
(0.6 µM), -conotoxin (0.1 µM), atropine (1 µM), and nifedipine
(1 µM).
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SRIF inhibits contractions via activation of sst2
receptors in rat jejunum.
SRIF (1-1,000 nM, n = 8-12) produced a
concentration-dependent reduction in the frequency of MMCs by
increasing the interval between them (Fig.
3, A and B). This
inhibition appeared as a period of contractile quiescence followed by a
return of activity at the rate observed before the addition of drug
rather than a long-term reduction in contraction frequency. Octreotide
and BIM-23027 mimicked the effect of SRIF, producing an increase in the
interval between MMCs. When tested at a concentration of 10 nM, the
increase in interval produced by both BIM-23027 (n = 8, P < 0.01) and octreotide (n = 4, P < 0.05) was greater than that produced by SRIF at
the same concentration (Fig.
4A), suggestive of an action
at sst2 receptors. In the presence of the selective
sst2-receptor antagonist Cyn (1 µM, n = 7), which had no effect on MMCs itself, the inhibitory action of SRIF
(300 nM) was abolished (Fig. 4B). Indeed, after Cyn, there
was a trend (P = 0.1) toward a decreased interval
between MMCs following SRIF.

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Fig. 3.
Effect of somatotropin release-inhibitory factor (SRIF)
on MMC intervals in the rat jejunum. A: representative trace
showing the transient increase in the interval between MMCs produced by
SRIF (300 nM). B: SRIF (1-1,000 nM) evoked a
concentration-dependent increase in MMC interval. Means ± SE for
8-12 preparations before ( ) and after
( ) administration of SRIF. ** P < 0.01, *** P < 0.001 compared with predrug
control.
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Fig. 4.
Effect of sst2-receptor agonists and
antagonists on MMC intervals in the rat jejunum. A:
histograms showing the maximum interval between MMCs before
( ) and after ( ) addition of SRIF (10 nM, n = 8), octreotide (10 nM, n = 4),
and BIM-23027 (10 nM, n = 8); * P < 0.05, ** P < 0.01 compared with predrug control.
The effects of BIM-23027 and octreotide were significantly greater than
that of SRIF. #P < 0.05, ##P < 0.01 compared with SRIF (10 nM).
B: histograms showing the effect of Cyanamid154806 (Cyn) on
MMCs and on the inhibitory effect of SRIF. The left
histogram shows the MMC interval before ( ) and after
( ) SRIF (300 nM, n = 9). On the
right are data showing the prevention of the inhibitory
effect of SRIF (300 nM, n = 7) by Cyn (1 µM,
n = 7). Note that the antagonist alone had no effect on
MMC intervals. ** P < 0.01 compared with predrug
control.
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Inhibition of contractions in rat jejunum by
sst2-receptor activation involves an NOS pathway.
As described above, 100 µM L-NAME produced a decrease in
the MMC interval, an effect that developed within 1 min of its
application and that was sustained in its continued presence for up to
25 min. SRIF (300 nM, n = 6) had no effect on MMCs in
the presence of L-NAME (Fig.
5B).

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Fig. 5.
Effect of nitro-L-arginine methyl ester
(L-NAME) on the response to SRIF in the rat jejunum.
A: representative trace showing the absence of the
inhibitory effect of SRIF on contraction complexes in the presence of
the nitric oxide synthase inhibitor L-NAME (100 µM).
L-NAME augmented both the amplitude and frequency of MMCs
and blocked the effect of SRIF (300 nM) on MMC interval.
B: histograms representing group data from 6 experiments.
** P < 0.01 compared with predrug control.
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MMC in the mouse jejunum.
Contractile activity in isolated mouse jejunum followed a similar
pattern to that observed in the rat, although lower intraluminal pressures were required for their initiation (2.5-3.5
cmH20). Contractions in mouse tissue (based on a sample of
16) had a mean duration of 71.5 ± 4 s and an amplitude of
1.6 ± 0.1 cmH2O and were separated by a mean interval
of 131.2 ± 8 s.
Inhibition of contractions by SRIF in mouse tissue involves more
than one receptor subtype.
As observed in the rat, SRIF produced a concentration-dependent
increase in the MMC interval (0.01-100 nM, n = 4;
Fig. 6). Although we were unable to
determine maximum effective concentrations, and thus EC50 values, in
either mouse or rat tissue (due to the 600-s ceiling imposed by
experimental protocol) the effect of SRIF in mouse tissue appeared to
be more potent than that in rat, because equivalent concentrations
produced a greater increase in interval in the former tissue. Indeed,
with 100 nM SRIF, there was a complete absence of contractile activity
in the majority of mouse jejunal segments. In contrast to observations
in rat tissue, in which the selective agonists produced a greater
effect than SRIF at the equivalent concentration, a higher
concentration of BIM-23027 was required to inhibit MMCs in mouse tissue
(Fig. 7). Furthermore, although the
inhibitory action of BIM-23027 (30 nM, n = 7) in mouse
tissue was abolished by prior administration of Cyn (1 µM), the
antagonist only partially prevented the inhibition of MMCs produced by
the nonselective agonist SRIF (10 nM, n = 5; Fig. 7).
The sst5-receptor antagonist BIM-23056 did not prevent the
inhibition of contractions by SRIF (10 nM, n = 4; data
not shown).

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Fig. 6.
Concentration-dependent effect of SRIF in the mouse
jejunum. Histograms showing the MMC interval before ( ) and after
( ) administration
of SRIF (0.01-100 nM, n = 4). Note that equivalent
concentrations of SRIF had a greater magnitude of effect in the mouse
jejunum compared with the rat. * P < 0.05, *** P < 0.001 compared with predrug
control.
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Fig. 7.
The effect of the sst2-receptor antagonist
Cyn in the mouse. Histograms showing increase in MMC intervals in the
mouse by SRIF (10 nM, n = 4) and BIM-23027 (30 nM,
n = 5). Note that in this species, the magnitude of the
response to the selective sst2-receptor agonist is less
than that to SRIF itself, in contrast to findings in the rat (see Fig.
3A). The sst2-receptor antagonist Cyn abolished
the inhibitory action of the sst2-selective agonist
BIM-23027 (30 nM, n = 7) but only attenuated the
inhibition produced by SRIF (10 nM, n = 5).
* P < 0.05 compared with predrug control;
#P < 0.05 compared with Cyn untreated
tissues.
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Studies in sst2-receptor knockout mice.
A comparison of the actions of SRIF and BIM-23027 on MMCs was made in
tissue taken from mice lacking the sst2 receptor
(Sstr2
/
) and their wild type
littermates. There was no difference in the parameters of the
contractile activity between the tissues taken from
sst2-receptor knockout and wild-type animals
(P > 0.05 n = 5; Fig.
8). MMCs in knockout mouse tissue
(n = 5) had a mean duration of 72.3 ± 11 s,
mean amplitude of 2.2 ± 0.5 cmH2O, and were separated
by a mean interval of 83.5 ± 18 s. Those in wild-type tissue
had a mean duration of 85.8 ± 18 s, mean amplitude of
1.8 ± 0.8 cmH2O, and were separated by a mean
interval of 113.8 ± 24 s. Due to restricted tissue supply,
single doses of SRIF (10 nM) and BIM-23027 (30 nM) were chosen that
would be expected to produce an inhibitory effect based on the data
above. In wild-type jejunum, both agonists SRIF (10 nM) and BIM-23027
(30 nM) produced a significant increase in the MMC interval (139.2 ± 30 vs. 526 ± 58 s, n = 5, P < 0.01 and 116 ± 24 vs. 256.2 ± 62 s, n = 5, P < 0.05, respectively), although the response to
BIM-23027 was significantly less than that to SRIF (P < 0.05). In contrast, BIM-23027 was without effect in the knockout
mouse jejunum, whereas SRIF produced a significant increase in the MMC
interval, the magnitude of which was significantly less than that in
the wild-type animal (526 ± 58 compared with 320 ± 84 s,
P < 0.05, n = 5; Fig. 9).

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Fig. 8.
MMC in the wild-type and sst2-receptor
knockout mouse. Representative traces showing MMCs evoked by distension
(intraluminal pressure 2.5-3.5 cmH2O) in the knockout
( / ) mice (A) and wild-type littermates +/+
(B). There is no difference in the properties of the MMCs
between the 2 groups.
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Fig. 9.
Effect of SRIF and BIM-23027 on MMC intervals in the mouse jejunum.
Individual data points for 5 wild-type (A) and 5 knockout
animals (B) showing MMC interval before and after addition
of SRIF (10 nM) and BIM-23027 (30 nM). Both BIM-23027 and SRIF
significantly increased the interval between MMCs in the wild-type
tissue, but only SRIF was effective in the knockout tissue.
* P < 0.05 compared with predrug control.
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Inhibition of contractions in mouse jejunum by SRIF-receptor
activation does not involve an NOS pathway.
L-NAME (100 µM) decreased the interval between MMCs from
136.6 ± 25 to 103.1 ± 13 s (n = 8, P < 0.05) and increased the mean amplitude from
2.6 ± 0.5 to 3.6 ± 0.6 cmH2O (n = 8, P < 0.05). The effect of SRIF (10 nM,
n = 4) and BIM-23027 (30 nM, n = 4) on
the MMC interval was not influenced by prior treatment with L-NAME, both agonists still producing a significant
increase in the MMC interval (Fig. 10,
A and B).

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Fig. 10.
Effect of L-NAME on the inhibition of MMCs
produced by SRIF and BIM-23027 in the mouse jejunum. A:
L-NAME (100 µM) augmented the amplitude of MMCs and
decreased the intervals (data in text). In contrast to observations in
rat tissue (see Fig. 4), the effect of BIM-23027 (30 nM) and SRIF (10 nM) on MMC interval was maintained in the presence of
L-NAME. B: histograms showing the group data
illustrating the magnitude of the effect of BIM-23027 (30 nM,
n = 4) and SRIF (10 nM, n = 4) on MMC
interval in jejunal segments pretreated with L-NAME
(n = 4). * P < 0.05 compared with
predrug control.
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DISCUSSION |
The ability of SRIF to inhibit intestinal peristalsis peripherally
is well documented. However, the site of action of SRIF and the
receptor subtype(s) responsible have not been fully characterized. In
this study, we have demonstrated that SRIF caused a
concentration-dependent inhibition of peristaltic activity evoked by
luminal distension in isolated segments of rat and mouse jejunum, with
a major role for sst2 receptors in this effect. However,
whereas in rat jejunum, SRIF mediated its inhibitory action via
NOS-dependent pathways, this was not the case in mouse jejunum.
Moreover, experiments with receptor-selective ligands and in the
sst2 knockout mouse revealed a non-sst2
receptor-mediated inhibition of intestinal peristalsis in mouse tissue
that was not apparent in the rat.
Mechanisms underlying the inhibitory effect of SRIF in the rat and
mouse.
Somatostatin is widely distributed in the gastrointestinal tract and
can be visualized immunocytochemically in mucosal D cells, in some
subpopulations of sympathetic nerve terminals, and in the cell bodies
and nerve fibers of submucosal and myenteric neurones (10, 13,
31, 38). Colocalization and fiber tracing studies have shown
that somatostatin is present in interneurones that form descending
chains of neurones that connect to muscle motor neurones (10, 31,
35). This topography is consistent with transmitter release
studies by Grider and co-workers (20-23) implicating somatostatin in a complex interaction with GABAergic and
enkephalinergic neuronal mechanisms that ultimately regulate NO
and VIP release during descending relaxation in the rat colon. A
similar neuronal interaction is suggested to regulate tachykinin
release from longitudinal muscle motoneurones (22). Thus
somatostatin is likely to act within the enteric circuits controling
peristalsis rather than at the level of the neuromuscular junction, and
this would explain the ability of SRIF to attenuate neurogenically
mediated contraction of the guinea pig ileum (14, 18, 24,
36, 45).
The pattern of contractile activity observed in the isolated segments
of rat and mouse jejunum was similar to the migrating motor complexes
described by others (5, 7, 26) in the mouse ileum and
colon. The MMCs described here consist of regularly recurring aborally
propagating waves of activity separated by longer periods of quiescence
and, similar to observations made by Bush and colleagues
(7), were dependent on cholinergic mechanisms because they
were blocked by hexamethonium and atropine. Bercik et al.
(5) described a similar pattern in the rat ileum that was
tetrodotoxin sensitive and superimposed on myogenic activity, which was
the main focus of their investigations. This myogenic activity, similar
to that describe earlier by Benard et al. (4), occurred at
a frequency similar to the waves of contraction observed during the
MMCs described in the present study. Clearly neural mechanisms are
necessary to organize the pattern of activity into the MMCs that can be
observed both in vitro and in vivo.
SRIF inhibited MMCs by increasing the interval between them rather than
by attenuating the magnitude or duration of the individual contractile
events. Thus it is unlikely that SRIF is acting in our model at the
level of the neuromuscular junction or on the muscle itself. However,
effects of SRIF have been observed on isolated gastric and colonic
smooth muscle, which were shown to be mediated predominantly via
sst1 and sst3 receptors (8). SRIF
sst2 receptors have also been localized
immunocytochemically on interstitial cells of Cajal (ICC) in the deep
muscular plexus (39), which are believed to play role in
nitrergic transmission (37, 41). An action of SRIF at
either smooth muscle or ICC sites would be expected to attenuate the
magnitude of MMCs. The fact that such an attenuation was not observed
is more consistent with Grider's hypothesis (21, 22) that
somatostatin leads to activation of mechanisms that augment descending
relaxation. However, how this is brought about is not clear. The
colocalization of somatostatin with acetylcholine in descending
interneurones in the guinea pig (35) would imply a
neuromodulatory role. In this respect, somatostatin acts
presynaptically to inhibit acetylcholine release (24, 45)
and postsynaptically to increase K+-channel activity, so
reducing neuronal excitability (34). All five sst
receptors appear to be preferentially coupled to pertussis toxin-sensitive G proteins of the Gi/Go type
(28). However, the effect of somatostatin on enteric
neuronal K+ conductance appears not to depend on inhibition
of adenylate cyclase but may involve a GTP binding protein
(34). In Grider's model (21) for the way
somatostatin augments descending relaxation, he proposes that
inhibitory motoneurones receive an inhibitory input that itself is
inhibited by somatostatin. The mechanism leading to descending
relaxation in a variety of species, including the rat, involves the
release of NO (9, 12, 19, 26). That SRIF was acting via
this pathway was confirmed by the observation in the present study that
inhibition of NO production with the L-arginine analog
L-NAME completely prevented the inhibitory effect of SRIF
in the rat jejunum. Interestingly, this was not the case in the mouse
jejunum, in which L-NAME had no effect on the SRIF-mediated inhibition of MMC generation. This is despite the wealth of evidence implicating NO in descending inhibition in the mouse intestine (7, 12, 37) and the observed augmentation of peristaltic activity brought about by treatment with L-NAME. Thus there
appears to be a fundamental difference between the mechanism of SRIF
inhibition in the mouse and rat jejunum.
Receptor characterization in the rat jejunum.
SRIF mediates its actions through a family of G protein-coupled
receptors that have been recently cloned (28). All five receptors (sst 1-5) have been shown to be expressed in the
gastrointestinal tract (32, 44), with high levels of
sst2 receptor in the rat jejunum. Our attempts to
pharmacologically characterize the receptor(s) involved in the
inhibition of jejunal peristalsis centered on the use of ligands that
have selectivity for the sst2 receptor. Both octreotide and
BIM-23027 are agonists that show selectivity for sst2
receptors but also have some affinity for the sst5-receptor subtype, and both mimicked the effect of SRIF in inhibiting contraction complex generation. Although EC50 values could not be
calculated, lower concentrations of octreotide and BIM-23027 than of
SRIF were necessary to inhibit MMCs, consistent with an action at
sst2 receptors. BIM-23027 was about three times more potent
than SRIF at inhibiting neurogenic contractions of the guinea pig ileum (15), and a similar potency order was observed for
sst2-mediated inhibition of rat parietal cell secretion
(43) and rat colon contractions (33).
Cyn is a potent and selective sst2-receptor antagonist at
human, rat, as well as guinea pig receptors (2, 16, 17). This ligand prevented the inhibitory action of SRIF on rat jejunal MMC
generation, confirming a major role for sst2 receptors, but had no effect in its own right on baseline contractile activity. Our
data, therefore, strongly implicate the sst2-receptor
subtype in the observed action of SRIF in the rat jejunum; however, as discussed below, there may be additional receptor mechanisms that are
functional in the mouse jejunum.
Receptor characterization in the mouse jejunum.
Although SRIF exerted an inhibitory effect on MMC generation in the
mouse jejunum, there were some notable differences from the
observations in the rat. Firstly, whereas octreotide and BIM-23027 were
more effective at lower concentrations than SRIF in the rat, the
reverse was true in the mouse jejunum, in which SRIF was the most
effective of the agonists. One explanation for this is that the
different sst receptors are expressed in the rat and mouse. In this
report, although Cyn abolished the response to BIM-23027 in the mouse
jejunum, it only attenuated the response to SRIF. Thus, in the mouse,
there is an additional non-sst2 receptor that is
functionally linked to inhibition of intestinal peristalsis. The lack
of effect of BIM-23056, which has been shown to have antagonist
activity at human sst5 receptors, on either MMC intervals themselves or on the response to SRIF, would argue against the involvement of this receptor subtype.
Another difference between rat and mouse relates to the role of NO in
the inhibitory response to SRIF. In the rat, the
sst2-mediated response to SRIF is absent in tissue treated
with L-NAME, which blocks NO synthesis. In contrast, in the
mouse, the effect of both SRIF and BIM-23027 is unaffected by NOS
inhibition. This suggests that different mechanisms may contribute to
the inhibition of peristalsis in the mouse. Moreover, Cyn, although
reversing the effect of BIM-23027 in the mouse jejunum, does not
completely block the response to SRIF. It would appear that SRIF
receptors other than the sst2 receptor are functional in
the mouse jejunum, but neither sst2 receptor nor the
non-sst2 receptor-mediated inhibition is dependent on
nitrergic mechanisms.
Responses in the sst2 knockout mouse.
MMCs were not different in the sst2 receptor knockout mouse
compared with its wild-type littermate. Similarly, the sst2
antagonist had no effect on baseline MMC activity in the rat jejunum.
Thus, although exogenous SRIF can exert a profound inhibitory effect via sst2 receptors, there is little evidence that
endogenous SRIF is involved in the regulation of MMC generation under
the current experimental circumstances. This may reflect redundancy in
the sst-receptor mechanisms that influence intestinal contractile activity. Indeed, the observation that SRIF but not BIM-23027 was able
to evoke an inhibitory effect on MMC generation (despite the absence of
functional sst2 receptors) confirms that an additional receptor subtype is involved in the regulation of intestinal
peristalsis in the mouse.
In summary, from a pharmacological perspective, somatostatin is a
potent inhibitor of intestinal peristaltic activity, but because it
modulates the interval between contractions without any attenuation of
the magnitude of the pressure rise, it would appear that the site of
action is neither at the level of the muscle nor neuromuscular
transmission. Instead, it seems that the interneuronal enteric
circuitry that organizes the timing of motor activity is the site of
action. Although this is true for both mouse and rat, only for the
latter is the inhibition expressed through nitrergic pathways.
sst2 Receptors mediate this inhibitory action of SRIF in
both species, but our pharmacological data and the observations of
maintained responses to SRIF in the sst2 knockout mouse
would indicate that in this species, there is at least one other
mechanism involved. Thus from a physiological perspective, the role of
endogenous somatostatin in intestinal peristalsis remains enigmatic.
 |
ACKNOWLEDGEMENTS |
This work was supported by King Abdull-Aziz University, Saudi Arabia.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
D. Grundy, Dept. of Biomedical Science, Alfred Denny Bldg., Univ.
of Sheffield, Western Bank, Sheffield S10 2TN, UK.
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.
10.1152/ajpgi.00354.2001
Received 9 August 2001; accepted in final form 25 November 2001.
 |
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