Rat gastroduodenal motility in vivo: involvement of NO and ATP
in spontaneous motor activity
Ian
Glasgow,
Kamal
Mattar, and
Anthony
Krantis
Digestive Diseases Research Group, University of Ottawa, Ottawa,
Ontario, Canada K1H 8M5
 |
ABSTRACT |
Our studies of
fasted anesthetized rats have shown that all spontaneous relaxations of
the antrum are nitric oxide (NO) dependent. Duodenal motility is
patterned into propagating "grouped" motor activity interposed
with "intergroup" periods of nonpropagating motor activity; in
the duodenum, only intergroup relaxations are NO dependent. We examined
the involvement of NO and ATP in spontaneous motor activities of the
gastroduodenum in vivo: contractions and relaxations were recorded and
analyzed simultaneously from the antrum
(S1) and proximal duodenum
(D1) of anesthetized
Sprague-Dawley rats (n = 10/group),
using extraluminal foil strain gauges. Treatment with the NO synthase
inhibitor
NG-nitro-L-arginine
methyl ester (L-NAME; 10 mg/kg
iv) attenuated (P < 0.05)
antral and intergroup relaxations, whereas grouped relaxations were
enhanced (P < 0.05). These effects
were reversed with L-arginine
(300 mg/kg iv). L-NAME also
increased (P < 0.05) the amplitude
of duodenal contractions. ATP (8 mg · kg
1 · min
1
iv) stimulated relaxations at S1
and D1 that were blocked by the
P2-purinoceptor antagonist suramin
(60 mg/kg iv). This treatment did not affect spontaneous antral
relaxations; however, duodenal grouped relaxations were attenuated.
Desensitization to the
P2x-purinoceptor agonist
,
-methylene ATP (300 µg/kg iv) gave results similar to suramin.
In contrast, the P2y-purinoceptor
agonist 2-methylthio-ATP (2-MeS-ATP; 360 µg/kg iv) evoked duodenal
relaxations that were attenuated by
L-NAME, and desensitization to
2-MeS-ATP attenuated intergroup relaxations. Spontaneous relaxations of
the rat antrum and duodenal intergroup relaxations are NO dependent.
Both gut regions relax in response to systemically administered ATP;
this response is sensitive to suramin. Grouped duodenal relaxations display functional sensitivity to suramin and
P2x- purinoceptor desensitization,
indicative of the involvement of ATP and
P2x purinoceptors.
P2y purinoceptors must also be
present; however, these occur on elements releasing NO. Although NO
does not mediate grouped relaxations or duodenal contractions, the
sensitivity of these responses to
L-NAME indicates that
the pathway(s) controlling these responses is modulated by NO.
nitric oxide;
,
-methylene-ATP; 2-methylthio-ATP; gastroduodenum; relaxations
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INTRODUCTION |
SINCE THE FIRST PROPOSAL that all relaxations of
mammalian gastrointestinal smooth muscle are mediated by extrinsic
adrenergic nerves, overwhelming pharmacological and physiological
evidence has accumulated to show that the mammalian gut wall contains a distinct class of intrinsic inhibitory motoneurons, the so-called nonadrenergic, noncholinergic (NANC) neurons (3, 8, 13). These neurons
mediate functional relaxations of the gut. However, the identity of the
neurotransmitter of this NANC inhibitory motor innervation is
controversial, with three main candidates:
1) a purinergic neurotransmitter,
involving ATP, 2) a nitrergic
neurotransmitter, acting via release of nitric oxide (NO), and
3) a peptidergic neurotransmitter,
such as vasoactive intestinal peptide (VIP).
Both neural stimulation and exogenous application of ATP have been
shown to produce relaxations in isolated preparations of enteric smooth
muscle from the rat duodenum and fundus and from the guinea pig fundus,
small intestine, and taenia coli (16-19, 25).
NO is a potent inhibitory neurotransmitter (9), and NO-synthesizing
neurons are distributed extensively in the myenteric and submucosal
nerve networks of guinea pigs, rats, and humans (1, 20-22).
Functional in vitro and in vivo studies provide convincing evidence in
support of the notion that NO is released by NANC inhibitory
motoneurons mediating relaxations of the mammalian gut (2, 5, 7, 23).
Taken together, this evidence suggests that no single neurotransmitter
is responsible for mediating all NANC relaxations in the mammalian gut.
These putative transmitters may represent distinct inhibitory nerve
types or may be colocalized and as such may represent a single
inhibitory nerve type with a variety of transmitters released under
differing stimulus conditions (6). In addition to their direct actions
on enteric smooth muscle, evidence exists that some of these putative
NANC neurotransmitters may be present in other types of intrinsic
neurons, including interneurons (5, 10). As transmitters of
interneurons, these agents could mediate stimulation of inhibitory or
excitatory motoneurons, as well as modulate motoneurons.
Because there is compelling evidence that more than one population of
intrinsic inhibitory innervation exists, it is important to identify
1) how these innervations and their
transmitters are involved in spontaneous gut motor activity and
2) whether these neurotransmitters
can be differentiated on the basis of their involvement within specific
patterns of motor activity. This study characterizes relaxant activity
in the rat gastroduodenum in vivo and investigates the involvement of
nitrergic and purinergic mechanisms in the pathways controlling this
motor activity. Follow-up studies of the involvement of VIP and the
putative transmitter of gut interneurons,
-aminobutyric acid (GABA),
are presented by Krantis et al. (9a) in a companion study.
 |
METHODS |
The methodology used was developed in our laboratory for recording
motor activity from the gastroduodenum of Sprague-Dawley rats (13). A
brief description of the recording and analysis techniques is presented
in Data recording and analysis.
Male Sprague-Dawley rats (250-350 g) were fasted for 24 h with
free access to water before surgery. Rats were then anesthetized with
halothane (3-4%) and transferred to a scavenging table maintained at 37°C. The level of anesthesia was reduced to 2% for the
surgical procedure. A midline incision was made in the neck region of
the animal to allow access to the right jugular vein and the right carotid artery. The right jugular vein was cannulated for the purpose
of intravenous drug infusion. Mean arterial pressure (MAP) was
monitored continuously by means of a pressure transducer (P23 ID; Gould
Statham) connected to a cannula inserted into the right carotid artery.
The blood pressure transducer was subsequently attached to an IBM PC
data acquisition system via an interface box.
After midline laparotomy, the stomach and ~6-8 cm of the
proximal duodenum were exteriorized onto saline-soaked gauze pads. Two
foil strain gauges (attached to 30-cm-long fine 32 silver wires) were
then glued to the serosal surface of the stomach and the proximal
duodenum, using Vetbond glue (tissue adhesive no. 1469; 3M). The first
strain gauge (S1) was glued onto
the gastric antrum, ~2 cm proximal to the pyloric sphincter, and the
second gauge (D1) was glued onto
the antimesenteric border of the duodenum, 2 cm distal to the
sphincter. Both strain gauges were oriented parallel to the
longitudinal muscle. In the antrum, this orientation preferentially
detects circular muscle activity. In the duodenum, the tubular
structure confines the gauge (irrespective of the axis of orientation)
to detect only circumferential muscle force, and alignment in the
longitudinal axis affords the greatest sensitivity (13). The fine wire
leads attached to the strain gauges were advanced caudally to the
distal end of the midline laparotomy, where they were exteriorized. The
abdominal incision was then closed, and the animal was carefully
rotated to a prone position to allow the exteriorized wires to be
attached to the IBM-based data acquisition system via an interface box
(channel 1: S1; channel 2:
D1). The animal was then covered
with a blanket to help maintain normal body temperature, and anesthesia
was reduced to 1% halothane and maintained at that level for the
remainder of the experiment. The animal was monitored carefully and a
blood pressure between 70 and 100 mmHg was deemed acceptable. After
surgery, the animal was allowed a 1-h period of stabilization before
the motility recordings began. Experiments consisted of a 60-min
control period of recording, at which point drug treatment was
administered, followed by motility recordings for up to 4 h. For all
drugs except suramin, the treatment period started at the time of drug
injection (time 0). Suramin is a
slowly equilibrating competitive antagonist at
P2x purinoceptors with maximum
effect in vitro evident within 90 min (14). On this basis, the
treatment period for animals injected with suramin in this study
started at 120 min after suramin.
Ex vivo experiments.
Ex vivo experiments were conducted to allow comparison of the effects
of drugs previously tested intravenously. Sprague-Dawley rats were
surgically prepared as for the in vivo studies described above, except
the animals were stabilized for 60 min in a supine position. After the
stabilization period, the abdominal cavity was carefully reopened and
exposed using two hemostat clamps. With the use of saline-soaked
cotton-tipped applicators, the stomach and a segment of proximal
duodenum were very gently exteriorized onto saline-soaked gauze pads.
The gut maintained on the gauze was kept moist with saline (37°C).
Drugs were applied serosally close to the strain gauge of interest.
Fresh gauze pads and cotton-tip applicators were used to absorb excess
drug, so as to prevent the applied drug from entering the abdominal
cavity.
Data recording and analysis.
Measurement and analysis of long-term recordings was based on the
method of Krantis et al. (13). During the experiment, an IBM data
acquisition system (acquisition software provided by Dr. Frank Johnson,
Institute of Medical Engineering, University of Ottawa) acquired,
digitized, and stored the motility data for both channels 1 (S1) and 2 (D1). Data are recorded as
events and are related to a change in voltage due to the bending of the
foil gauge in either an upward deflection (contraction) or a downward deflection (relaxation). The total displacement range of the foil strain gauge is
5 to +5 V, which corresponds to a range of
2,048 to +2,048 digital units (A/D units). The actual
displacement (grams tension) was determined using the equation
y = 1.31 × 10
3x,
where y represents the grams and
x represents the A/D units. The
digitized files contained 60 min of control motor activity followed by
up to 4 h of motor activity. Files (motility recordings) for each
channel were simultaneously analyzed for contractions and relaxations
using a proprietary software program (GI-Analysis: CGIQ TRANS, provided
by Drs. F. Johnson and C. Wood, Institute of Medical Engineering,
University of Ottawa). This program allowed us to simultaneously
analyze the files for contractions (positive deflections above the
baseline) and relaxations (deflections below the baseline). The
baseline is defined by the tone of the smooth muscle at the point
before initiation of a response. Measurement of absolute tone is
problematic. There is a trade-off between obtaining a representation of
absolute tone and getting an accurate representation of motor events.
The recording system used here minimizes this problem by allowing us to
increase the scale for recording coupled with increased accuracy of
recording motor activity. Typical recordings presented in the results
always show the recording scale (grams tension) in the ordinate and
allow assessment of relative change in tone only. For each animal, the
ability of the gauges to detect relaxations and contractions was tested
using papaverine (10 mM) and carbachol (1 mM), respectively, applied directly ex vivo onto the gut serosa (13). Only animals in which relaxations and contractions were recorded were subsequently used for
our studies.
Statistical analysis.
Experimental data for each channel were first grouped according to the
event being analyzed (contractions or relaxations) and then further
grouped into control or treatment period and then according to the
parameter being analyzed. For this study, we examined
1) the amplitude (grams tension) of
the event and 2) the frequency of
the events. All tabulated results are expressed as means ± SE of
either amplitude or frequency. Each animal served as its own control,
and for pooled data the treatment period means are expressed as a
percentage of the control mean, where the control mean was represented
by 100%. An ANOVA with a Tukey multiple comparison test (Statsgraphics
Plus, version 5.2) was used to determine significance between the raw
data from the treatment period and the control period.
P < 0.05 was considered
statistically significant.
Chemicals.
ATP,
,
-methylene-ATP (
,
-Me-ATP),
L-arginine, and
NG-nitro-L-arginine
methyl ester (L-NAME) were
obtained from Sigma (Toronto, ON, Canada). Suramin was obtained from CB
Chemicals (Woodbury, CT). 2-Methylthio-ATP (2-MeS-ATP) was obtained
from Research Biochemicals International (Natick, MA).
 |
RESULTS |
Control motor activity.
Spontaneous patterns of gastroduodenal motility recorded under control
conditions in anesthetized rats (n = 6) included nonpropagating single and propagating "grouped" motor
events. In the gastric antrum
(S1), motor activity consisted
primarily of periodic relaxations (Fig. 1).
These relaxations were similar in amplitude (0.07 ± 0.02 g), with a
frequency of ~5 per minute, and were often oscillatory in appearance.
Contractions of 0.04 ± 0.01 g were also observed, but these were
typically less frequent (~3 per minute) and their occurrence was
random.

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Fig. 1.
Typical recording of rat antral
(S1) spontaneous motor activity
before and after injection of
NG-nitro-L-arginine
methyl ester (L-NAME; 10 mg/kg
iv). The 3 panels represent a recording from
t = 40 min to
t = 90 min.
L-NAME was injected as a single
bolus at t = 60 min. Control activity
consisted predominantly of relaxations and a few contractions. After
L-NAME injection, relaxations
were inhibited within 10 min. Tension scale shown on
y-axis can be used to compare
alteration in tone and amplitude of responses.
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In the proximal duodenum (D1),
motor activity was clearly different, consisting of two obvious
patterns that we have termed grouped and "intergroup" activity
(13), as shown in Fig. 2. Grouped activity
was characterized by distinct groups of large-amplitude, high-frequency
relaxations and/or contractions. Each grouped activity period
lasted 0.5-3.0 min and was periodic, occurring approximately every
6.6 ± 0.4 min (minimum 4.1 min, maximum 10.5 min). Within this
period of grouped activity, responses appeared to build in intensity
reminiscent of migrating myoelectric complexes (MMCs), and these
coincided with motor activity that could be seen to propagate caudally
down the duodenum, as can be seen in the recordings obtained from
multiple foil strain gauges in the proximal duodenum (Fig.
3). Recorded motor activity occurring
between the episodes of grouped activity, herein termed intergroup,
consisted of both contractions and relaxations. Compared with
relaxations within the grouped activity, intergroup relaxations
occurred with a lower frequency, and the majority of these relaxations
were smaller in amplitude, approximately one-third that of grouped
relaxation amplitude. Quantitative parameters of the duodenal motor
activities are presented in Table 1.
Injection of saline vehicle (0.9% iv) or direct application of saline
onto the serosa in the ex vivo protocol (not shown here) had no effect
on gastroduodenal motor activity.

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Fig. 2.
Effects of L-NAME (10 mg/kg iv)
on spontaneous motor activity in rat duodenum
(D1). In these typical
recordings obtained from different experiments, relaxations in the
intergroup period are reduced and contraction amplitude is increased.
Grouped motor activity (within dashed lines) is enhanced. Tension scale
shown on y-axis should be used to
compare responses.
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Fig. 3.
Motor activity recorded at multiple sites in proximal duodenum.
Position of the foil strain gauges (D) is shown alongside the
respective recording. Gauges are numbered according to distance (mm)
from the pylorus. Dashed lines connecting grouped motor activity
periods should be used to judge propagation velocity. Note change in
predominant activity within the grouped period at the most distal
recording site.
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Table 1.
Characteristics of the spontaneous interdigestive motor pattern of the
duodenum in anesthetized Sprague-Dawley rats
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Effects of treatment with the NO synthase inhibitor
L-NAME.
L-NAME is widely used as an
inhibitor of NO synthase activity both in vitro and in vivo (5, 7). A
previous in vivo study of anesthetized rats (15) has determined that
L-NAME (10 mg · kg
1 · ml
1
iv) is effective in inhibiting gastric fundus relaxations, with only a
transient effect on MAP. On this basis, we chose to test L-NAME (10 mg · kg
1 · ml
1
iv) for its effects on motor activity of the rat gastroduodenum in vivo
under conditions of anesthesia.
L-NAME always caused a transient
(up to 10 min) 10 mmHg increase in MAP. This served as an indicator of
effective injection of the drug. Antral contractions showed no
significant (P > 0.05) change in
either amplitude or frequency after
L-NAME injection (Table
2). However, within 10-15 min of
L-NAME injection, spontaneous antral relaxations were significantly
(P < 0.05) reduced in both amplitude
and frequency (Fig.
4A). The
effects of L-NAME lasted for the
duration of the experiment (up to 3 h) with no recovery of motility
patterns to those observed in the control recording period.

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Fig. 4.
Effects of L-NAME and
L-arginine on amplitude and
frequency of spontaneous relaxations in rat antrum
(S1)
(A) and duodenum
(D1)
(B). Treatment groups
(n = 6-8/group) include control,
L-arginine (300 mg/kg iv) alone,
L-NAME (10 mg/kg iv) alone, and
L-NAME followed 60 min later by
L-arginine (300 mg/kg iv). Data
were collated for each minute and averaged over 10-min periods, and
test data are expressed as a percentage of control (means ± SE).
* P < 0.05 compared with
control.
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In the proximal duodenum, the two types of motor activity patterns,
grouped and intergroup, were affected differently by
L-NAME (Fig. 2). Grouped
relaxations were enhanced by
L-NAME, characterized by a
significant (P < 0.05) increase in
amplitude and frequency (Fig. 4B).
In contrast, intergroup relaxations were either reduced or abolished on
L-NAME treatment. Although
antral contractions were not affected by
L-NAME treatment, in the
duodenum, the amplitude of grouped contractions was significantly
increased (Table 2). Comparable results were obtained with
L-NAME (3.7 mM, 100 µl) administered onto the serosa of the antrum or duodenum (data not shown).
When injected 60 min after
L-NAME (10 mg/kg iv)
administration, L-arginine (300 mg/kg iv) transiently reversed (for up to 10 min) the effects of
L-NAME in both the antrum and
duodenum (Fig. 4). Injection of
L-arginine alone (300 mg/kg iv)
generally caused effects opposite to those of
L-NAME treatment. In the antrum, L-arginine significantly
increased the amplitude and frequency of spontaneous relaxations (Fig.
4A). In contrast to the antrum, duodenal grouped relaxation amplitudes were significantly reduced (Fig.
4B). The action of
L-arginine in the duodenum was
confined to the grouped relaxations, and all of the effects of
L-arginine were transient,
lasting 4-10 min.
L-Arginine did not affect
gastroduodenal spontaneous motor contractions.
Effects of ATP, suramin,
,
-Me-ATP,
and 2-MeS-ATP.
ATP (8 mg · kg
1 · min
1
iv, for 1 min) caused a transient (up to 10 min) decrease (~20 mmHg)
in MAP. In the gastric antrum, ATP infusion always evoked
large-amplitude (215% of control) relaxations (Fig.
5). These were easily distinguishable from
control spontaneous relaxations and lasted for ~10-15 min before
a return to control patterns. Similarly, in the duodenum, ATP always
evoked relaxations. However, in contrast to the antrum, the duodenum
usually only responded with a single, transient relaxation within 1 min
of ATP injection. In addition, this response occurred (or was visible) only if ATP was injected during the intergroup activity. This relaxation could easily be distinguished from the random spontaneous relaxations during intergroup activity by its long duration and large
amplitude. ATP did not affect the spontaneous activity of either the
grouped or intergroup periods.

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Fig. 5.
Representative recordings of motor activity in rat antrum
(S1)
(A) and duodenum
(D1)
(B), showing effect of intravenously
injected ATP. Large-amplitude relaxations were induced in the gastric
antrum, and control activity returned within 12 min. In the duodenum,
grouped and intergroup activity is evident, and ATP caused a
long-lasting relaxation together with a reduction in grouped relaxation
responses.
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To further elucidate the involvement of ATP in gastroduodenal motor
activity, parallel experiments were conducted using the P2-purinoceptor antagonist
suramin. To our knowledge, this antagonist, which has been used to
investigate ATP sites of action in the gut in vitro, has not been used
previously for in vivo motility studies. Preliminary in vivo
experiments in this laboratory, using a dose of 30 mg/kg [which
is slightly higher than the dosage effective against parasites in
humans (19)], found the effects of suramin on spontaneous motor
activity to be inconsistent. However, suramin at a dose of 60 mg/kg iv
was consistently effective without altering vital signs in the test
animals (n = 6). Suramin (60 mg/kg)
administered as a single intravenous bolus injection attenuated the
amplitude of the ATP-induced antral relaxation by 49 ± 6% compared
with control. In the duodenum, ATP-induced relaxations were reduced by
57 ± 4%. Spontaneous motor activity of the antrum and duodenal intergroup activity were not affected by suramin treatment. However, suramin caused changes in grouped motor activity evident from 90 min
and maximal at 120 min after drug injection. This long equilibration
time for suramin action is comparable to that observed in other studies
(14). Suramin actions were characterized by an increase in the average
duration of grouped activity periods (5-7 min) and inhibition
(P < 0.05) of grouped relaxation
amplitude and frequency (Fig. 6). Grouped
contractile activity was not affected.

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Fig. 6.
Effects of ATP agonist and antagonist drugs on spontaneous duodenal
relaxations. Treatment groups (n = 6-10/group) are shown on x-axis.
Collated data are expressed as percent change ± SE compared with
control. Amplitude and frequency data are presented separately.
2-MeS-ATP, 2-methylthio-ATP. * P < 0.05 compared with control.
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The P2x-purinoceptor agonist
,
-Me-ATP (300 µg/kg iv) caused a transient increase in the
amplitude of grouped relaxations (Fig. 7).
This effect lasted up to 2 min and was followed by a dramatic reduction
in both the amplitude and frequency of grouped relaxations (Figs. 6 and
7).
,
-Me-ATP-induced inhibition lasted until the end of the
experiment, indicative of a persistent desensitization. At a lower
dosage (150 µg/kg iv),
,
-Me-ATP exerted similar effects, but
these were not sustained, and in some cases there was recovery as early
as 30 min after injection. There were no significant changes to the
spontaneous antral activity or duodenal contractile activity.

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Fig. 7.
Effects of , -methylene-ATP on duodenal motor activity. Grouped
and intergroup periods are shown, before and after treatment with
, -methylene-ATP (300 µg · kg 1 · min 1
iv). In this example, grouped periods occurred relatively close
together just before drug injection. These can be used to compare the
effects of , -methylene-ATP, which transiently augmented and then
reduced grouped motor activity.
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On injection, 2-MeS-ATP (360 µg/kg iv) evoked a single large
relaxation (~30 s duration) easily distinguishable from control spontaneous activity. 2-MeS-ATP-induced relaxations were almost abolished by L-NAME treatment
(Table 3). Within 3 min of injection, 2-MeS-ATP significantly reduced intergroup relaxation amplitude and
frequency (Fig. 6); grouped relaxations were not affected. In addition,
there were no significant changes to the spontaneous motor activity of
the antrum or contractile duodenal activity.
 |
DISCUSSION |
The results obtained in this study show intravenous injection or direct
serosal application of the NO synthase inhibitor
L-NAME to differentially alter
spontaneous relaxant motor activity in the rat gastric antrum and
proximal duodenum in vivo. Spontaneous antral relaxations were either
significantly attenuated or abolished. However, in the duodenum, only
the relaxations of the intergroup period were attenuated. These
intergroup periods of motor activity comprise sparsely distributed
small-amplitude responses and are easily distinguished from the regular
periods of intense propagating motor activity, referred to herein as
grouped activity. The profile and duration of this grouped activity in
the duodenum are similar to those described previously by researchers
in this laboratory (13). Grouped activity consisted of both relaxations
and contractions, and often one type of response predominated. This may
be due to the intrinsic tissue tone, since it is well established that
localized regions of the gut are under a preset tone. Together, the
alternating periods of grouped vs. intergroup activity accounted for
all of the spontaneous duodenal motor activity in the anesthetized rat. Whereas intergroup relaxations were attenuated or abolished by L-NAME, grouped relaxations were
enhanced. All of the L-NAME
effects were reversed by the NO synthase substrate
L-arginine. These results indicate that NO is not the primary transmitter of the pathway controlling relaxations occurring within grouped activity. However, our
results clearly show that NO has a primary role in relaxations of the
antrum and within the duodenal intergroup activity.
The involvement of NO in relaxations of the stomach has been previously
reported (15) in studies in which
L-NAME either abolished or
greatly reduced vagally stimulated relaxations of the gastric fundus in
anesthetized rats. In addition,
L-NAME treatment enhanced
vagally stimulated gastric contractions. However, we found no
significant change in spontaneous antral contractions with
L-NAME treatment. It is likely
that the contractions evoked by vagal stimulation involve pathways
distinct from those mediating the spontaneous contractile activity
described in this study and that vagally stimulated contractions are in
some way modulated by NO.
The effect of L-NAME on grouped
relaxations was unexpected and contrary to the notion that NO functions
only as a transmitter of NANC inhibitory motoneurons or as a relaxant
factor within smooth muscle cells. In an in vivo study of the rat
jejunum, Calignano et al. (5) reported an increase in motility in vivo
after administration of a NO synthase inhibitor and proposed that NO
modulates the release of certain mediators from local neuronal or
intestinal cellular sources. Likewise, studies of anesthetized rats
show that inhibition of NO synthesis causes an increase in phasic
activity of the small intestine (11), indicative of a neuromodulatory role for NO in these regions. We believe that NO is not only a transmitter of NANC motoneurons in the gastroduodenum but is also released by inhibitory interneurons targeting neurons in the pathway(s) controlling propagating motor activity.
NO-synthesizing varicose fibers occur within almost all intestinal
ganglia of the rat, guinea pig, and human myenteric and submucosal
nerve layers (20, 21), which strongly supports the notion that NO may
also be a transmitter of enteric interneurons. Functional proof of this
has been provided by Young et al. (26), who showed that NO stimulates
cGMP production within both myenteric and submucosal ganglion cells.
This notion is further strengthened by the results of a recent anatomic
study in this laboratory in which a subpopulation of intestinal
GABAergic ganglion cells, and therefore by definition enteric
interneurons (22), was found to display the capacity to constitutively
synthesize NO.
Direct serosal application of
L-NAME onto the antrum in the
anesthetized rat caused effects identical to those of intravenous L-NAME. This strongly suggests
the involvement of local NO in the generation of these responses.
However, NO-related elements may not be the final component in the
pathway mediating antral relaxations. NO could be stimulating the
release of ATP, since ATP injection also relaxed the gastric antrum and
ATP is known to have direct actions on gastrointestinal smooth muscle
(8, 16, 18). In our study, ATP caused relaxations in the antrum without
affecting spontaneous antral relaxations. In addition, treatment with
suramin at a dose that inhibited relaxations to systemically injected
ATP had no effect on spontaneous antral relaxations. Furthermore,
development of tachyphylaxis to
,
-Me-ATP or 2-MeS-ATP also had no
effect in the antrum. It would appear that NO and not ATP is the
transmitter of the inhibitory (presumably NANC) motor nerves mediating
spontaneous relaxation of the rodent stomach. This is further supported
by D'Amato et al. (7), who showed that the NO synthase inhibitor
NG-nitro-L-arginine
had no effect on ATP-evoked relaxations of rat gastric fundus muscle
strips.
For the rat small intestine, at least two different inhibitory motor
systems have been proposed to occur, with ATP being a major NANC
inhibitory transmitter in the duodenum (16). Our findings suggest that
the inhibitory transmitter responsible for grouped relaxations is not
NO. Rather, ATP mediates these relaxations, since treatment with
suramin attenuated grouped relaxations. We propose that grouped
relaxations are mediated (in part or in whole) by ATP and involve
P2 purinoceptors.
Although P2y purinoceptors are
considered to be the sites by which ATP mediates NANC relaxation of the
intestine (24), there is also evidence for ATP to induce relaxations
via P2x purinoceptors (17, 25).
Identification of the
P2-purinoceptor subtype involved in ATP-related responses of the gut in vivo is made difficult by the
lack of specific antagonists. Suramin may be suitable for antagonizing
P2 purinoceptors but cannot
distinguish between the subtypes. An alternative approach is to
differentiate these subtypes by their rank order of potency for ATP
analogs (4).
,
-Me-ATP exhibits greater affinity for the
P2x-receptor subtype, which readily desensitizes with continuous exposure to this ATP analog. Fortunately, 2-MeS-ATP is more potent at the
P2y purinoceptor. Our results show
that
,
-Me-ATP specifically blocked duodenal grouped relaxations
after an initial but transient augmentation of grouped relaxation
amplitude. Unexpectedly, 2-MeS-ATP induced a large relaxation in the
proximal duodenum that was found to be sensitive to
L-NAME. Like
,
-Me-ATP,
prolonged exposure to 2-MeS-ATP also caused a reduction in motor
activity; however, this was apparent only for the intergroup
relaxations.
In conclusion, the results presented here provide compelling evidence
that NO is the primary inhibitory neurotransmitter of the pathway(s)
mediating spontaneous relaxant motor activity in the rat gastric
antrum. There is more than one type of inhibitory motor innervation in
the proximal duodenum. These can be functionally separated with respect
to the distinct patterns of spontaneous motor activity within the
duodenum: intergroup relaxations, like antral relaxations, are mediated
by NO, whereas grouped relaxations are mediated by ATP. Since the
grouped motor activity is reminiscent of MMCs, we propose that ATP,
through P2x purinoreceptors, is the primary inhibitory transmitter involved in mediating propagatory motor activity in the duodenum of fasted rats. In addition, ATP, via
P2y purinoceptors, appears to be
targeting neurons in the pathway controlling NO-mediated intergroup
relaxations. The involvement of ATP within both types of duodenal motor
activity is also true of NO. NO appears to be more than just an
inhibitory motor neurotransmitter within the rat gastroduodenum, since
treatment with L-NAME enhanced grouped relaxations. We propose that NO exerts a tonic neuromodulatory control over ATP-mediated relaxations within grouped motor activity. Moreover, this tonic neuromodulatory action of NO extends to the excitatory motor innervation of the duodenum.
 |
ACKNOWLEDGEMENTS |
K. Mattar is a recipient of a National Science and Research Council
Industrial Postgraduate Scholarship.
 |
FOOTNOTES |
Address for reprint requests: A. Krantis, Dept. of Cellular and
Molecular Medicine, Univ. of Ottawa, 451 Smyth Rd., Ottawa, ON, Canada
K1H 8M5.
Received 17 March 1998; accepted in final form 24 June 1998.
 |
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