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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 280:154 –161, 1997
Vol. 280, No. 1
Printed in U.S.A.
Dual Excitatory and Inhibitory Effect of Nitric Oxide on
Peristalsis in the Guinea Pig Intestine1
P. HOLZER, I.TH. LIPPE, A. LOTFI TABRIZI, L. LÉNÁRD, JR. and L. BARTHÓ
Department of Experimental and Clinical Pharmacology, University of Graz, Universitätsplatz 4, A-8010 Graz, Austria (P.H., I.Th.L.) and
Department of Pharmacology, University Medical School Pécs P.O.B. 99, H-7643 Pécs, Hungary (A.L.T., L.L., L.B.)
Accepted for publication September 11, 1996
The enteric neural pathways subserving intestinal peristalsis involve sensory neurons and interneurons as well as
ascending excitatory and descending inhibitory motor neurons (Furness and Costa, 1987; Gershon et al., 1994; Waterman et al., 1994b). It has only recently been demonstrated
that inhibitory neural pathways causing relaxation of the
longitudinal and circular muscle layers play a crucial role in
the coordination and propagation of peristalsis (Waterman et
al., 1994a). In terms of their transmitters, enteric inhibitory
motor neurons are nonadrenergic noncholinergic neurons,
and in the guinea pig small intestine two mechanisms of
nonadrenergic noncholinergic inhibitory transmission to the
circular muscle have been distinguished (Niel et al., 1983;
Costa et al., 1986). One mechanism relies on fast inhibitory
junction potentials that are blocked by apamin and are most
probably mediated by adenosine triphosphate or a related
purine (Niel et al., 1983; Bywater and Taylor, 1986; Costa et
al., 1986; Crist et al., 1992). Apamin-insensitive inhibitory
Received for publication April 29, 1996.
1
This study was supported by the Austrian-Hungarian Foundation (Grant
16u3), the Austrian Science Foundation (Grant P9473-MED) and the Hungarian Grants ETT T-04739/93, OTKA T-013045 and OTKA T-016945.
ester (100 –300 mM) facilitated peristalsis, an effect that was
reduced by L-arginine (1 mM) but left unaltered by atropine (10
nM). Blockade of inhibitory neuromuscular transmission by
successive exposure of the ileum to apamin (0.5 mM) and
NG-nitro-L-arginine methylester (300 mM), in this or reverse
order, disrupted the coordinated pattern of peristalsis and
caused irregular nonpropulsive contractions of the circular
muscle. It is concluded that NO has a dual excitatory and
inhibitory effect on intestinal motility. The excitatory effect involves cholinergic motor neurons, whereas the inhibitory effect
reflects relaxation of intestinal muscle. Abolition of peristalsis
by combined exposure to NG-nitro-L-arginine methylester and
apamin attests to an essential role of enteric inhibitory motor
neurons in the coordination of propulsive motility in the intestine.
transmission involves slow inhibitory junction potentials
(Niel et al., 1983; Bywater and Taylor, 1986) that are brought
about by vasoactive intestinal polypeptide and nitric oxide
(NO) acting in series (He and Goyal, 1993) and that are
prevented by NO synthase inhibitors (Lyster et al., 1992).
NO synthase occurs in both enteric inhibitory motor neurons as well as in descending interneurons of the guinea pig
small intestine (Costa et al., 1992; Furness et al., 1994;
Young et al., 1995), from which NO is released after nerve
stimulation (Wiklund et al., 1993b). The most widely reported action of NO in the gut is relaxation of smooth muscle
(Sanders and Ward, 1992), which is consistent with the ability of NO to induce slow inhibitory junction potentials in the
muscle of the guinea pig small intestine (Lyster et al., 1992;
He and Goyal, 1993). However, authentic NO can also cause
acetylcholine-mediated contractions of the resting guinea pig
ileum (Barthó and Lefebvre, 1994) although the stimulusevoked release of acetylcholine and tachykinins from enteric
neurons is inhibited by NO (Knudsen and Tottrup, 1992;
Wiklund et al., 1993a; Kilbinger and Wolf, 1994). These multiple actions of NO suggest that manipulation of the NO
system influences enteric motor reflexes and peristalsis of
ABBREVIATIONS: D-NAME, NG-nitro-D-arginine methylester; L-NAME, NG-nitro-L-arginine methylester; L-NNA, NG-nitro-L-arginine; NO, nitric
oxide; Pa, Pascal; SNP, sodium nitroprusside.
154
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ABSTRACT
The implications of the enteric neurotransmitter nitric oxide
(NO) in intestinal peristalsis were investigated. Propulsive motility in isolated segments of the guinea pig ileum was triggered
by intraluminal fluid infusion to distend the intestinal wall, and
the pressure threshold for eliciting peristaltic waves was used
to quantify facilitation (decrease in threshold) or inhibition (increase in threshold) of peristalsis. The NO donor sodium nitroprusside (0.1–100 mM serosally) caused a prompt facilitation of
peristalsis, which in the presence of a threshold concentration
of atropine (10 nM) was followed by a concentration-related
blockade of peristalsis. Further analysis showed that sodium
nitroprusside (10 and 100 mM) first relaxed, then contracted,
and finally relaxed the longitudinal muscle of the guinea pig
isolated ileum, the contraction being blocked by atropine (1
mM). Inhibition of NO synthase by NG-nitro-L-arginine methyl-
1997
NO and Intestinal Peristalsis
Methods
Basic preparation common to all experiments. Adult guinea
pigs of either sex and 350 to 450 g body weight were stunned and
bled. The ileum was excised, flushed of luminal contents and placed,
for up to 4 hr, in Tyrode solution kept at room temperature and
oxygenated with a mixture of 95% O2 and 5% CO2. The composition
of the Tyrode solution was (mM): NaCl 136.9, KCl 2.7, CaCl2 1.8,
MgCl2 1.0, NaHCO3 11.9, NaH2PO4 0.4 and glucose 5.6. After dissection, ileal segments were mounted in organ baths that contained
oxygenated Tyrode solution maintained at 37°C.
Peristalsis. Peristalsis was studied with a constant intraluminal
perfusion system that has been described in detail previously (Costall et al., 1993; Holzer and Maggi, 1994). Briefly, ileal segments
(approximately 10 cm in length) were secured horizontally in a
silanized glass organ bath containing 30 ml of Tyrode solution.
Prewarmed Tyrode solution was continuously infused into the intestinal lumen; the infusion rate was 0.5 ml min21. The fluid passing
the gut lumen was directed into a vertical outlet tubing (Costall et
al., 1993) which ended 4 cm above the fluid level in the organ bath.
This arrangement required the peristaltic effector system to raise
the intraluminal pressure (recorded with a pressure transducer at
the aboral end of the segments and displayed on a pen recorder)
above 400 Pa to empty the intestinal segments.
The infusion of fluid caused gradual filling of the intestine as
shown by a slow rise of the intraluminal pressure (fig. 1, A and B).
When the intraluminal pressure reached a threshold an aborally
moving wave of circular muscle contraction, measured as a spike-like
increase in intraluminal pressure, propelled the intraluminal fluid to
leave the system and thus caused emptying of the segment (fig. 1, A
and B). The pressure threshold for eliciting peristaltic waves was
used to quantify effects of drugs on peristalsis (Costall et al., 1993;
Holzer and Maggi, 1994). Stimulation of peristalsis was reflected by
a decrease in the pressure threshold whereas inhibition was mirrored by an increase in the threshold and abolition of peristalsis
manifested itself in a lack of propulsive motility despite an intraluminal pressure of 400 Pa.
The preparations were allowed to equilibrate with the bathing
solution for a period of 20 min during which they were kept in a
quiescent state. Thereafter the outlet tubing was raised such that
peristalsis was initiated, after which the segments were equilibrated
for another 20 min before they were exposed to any drug. The drugs
to be tested were administered into the bath, i.e., to the serosal
surface of the intestinal segments, at volumes not exceeding 1% of
the bath volume. The corresponding vehicle solutions were devoid of
any effect.
Two parameters of peristalsis were evaluated: the frequency of
peristaltic waves (min21) and the pressure threshold (measured in
Pa relative to the zero base-line pressure) which is the intraluminal
pressure level at which a peristaltic wave is elicited (Holzer and
Maggi, 1994). The amplitude of the peristaltic waves was not assessed because this parameter was least sensitive to the manipulations under study. The term “peristalsis” (Waterman and Costa,
1994) is used to describe the fluid propulsion that resulted from the
regular occurrence of peristaltic waves (fig. 1A). The term “peristaltic
wave” is meant to denote the aborally moving wave of circular
muscle contraction that accomplished the propulsion of fluid.
Longitudinal muscle activity. Segments of 1.5 cm length were
suspended vertically in organ baths (capacity 5 ml). The preparations were kept under a resting load of 5 mN, and the mechanical
activity of the longitudinal muscle was recorded with isotonic lever
displacement measuring systems (HSE, March-Hugstetten, Germany) and displayed on a pen recorder. After a 30-min period of
equilibration, the preparations were primed by repeatedly testing
their responses to SNP (10 or 100 mM, contact time 6 min) at
intervals of 30 min. After reproducible responses had been obtained,
the ileal segments were repeatedly exposed to the same concentration of SNP (contact time 20 min) with washout periods of 30 min
between the exposures. Atropine (1 mM) was administered to the
bath 20 min before the second 20-min challenge of the preparations
with SNP, although its vehicle (physiological saline, 1 ml ml21 bath
fluid) was given 20 min before the first 20-min challenge with SNP.
At the end of the experiments the preparations were standardized
by recording their reactions to histamine and isoproterenol. The
changes in mechanical activity evoked by the drugs under study
were expressed as percentages of the maximal contraction evoked by
histamine (1 mM, contact time 1 min), 0% being the level of the
maximal relaxation caused by isoproterenol (1 mM) given as soon as
the segments had relaxed after challenge with histamine.
Drugs. The following drugs were used. Apamin, histamine dihydrochloride (both from Serva, Heidelberg, Germany), atropine sulfate and sodium nitroprusside (both from Merck, Darmstadt, Germany) were dissolved in water and diluted in Tyrode solution.
Isoproterenol hydrochloride was used in the form of Isuprel injections (0.2 mg ml21 stabilized aqueous solution, Winthrop, New York,
NY). Tyrode solution was used to dissolve L-NNA (10 mM), L-NAME
(100 mM), its enantiomer D-NAME (100 mM) and L-arginine (100
mM; all from Bachem, Bubendorf, Switzerland). For completely dissolving L-NNA the solution was sonicated for 2 min followed by
vortex stirring.
Statistics. Quantitative data are presented as means 6 S.E.M.
Statistical evaluation of the results was made with the Mann-Whitney U test, the Wilcoxon test for pair differences or the Quade test
(Theodorsson-Norheim, 1987) as appropriate. Probability values P ,
.05 were regarded as significant.
Results
Effect of SNP on peristalsis. Administration of SNP
(0.1–100 mM) to the organ bath caused a prompt concentration-dependent stimulation of peristalsis as portrayed by a
decrease in the pressure threshold, a response that lasted 10
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the guinea pig small intestine in a complex manner. The
reported data attest to this complexity inasmuch as in one
study SNP and other NO donors were found to stimulate
peristalsis (Sugisawa et al., 1991) although in other laboratories SNP was shown to inhibit ascending and descending
enteric motor reflexes (Yuan et al., 1995) and peristaltic
activity (Waterman and Costa, 1994). Furthermore, inhibition of endogenous NO synthesis depresses neuromuscular
transmission in the descending inhibitory motor reflex of the
guinea pig ileum (Yuan et al., 1995) whereas the peristaltic
reflex is facilitated (Ciccocioppo et al., 1994; Suzuki et al.,
1994; Waterman and Costa, 1994).
Some of these discrepancies are likely to be the result of
differences in experimental conditions and recording protocol. Because in most studies intervals of 10 to 20 min were
allowed to elapse between addition of the drugs and recording of their effects (Ciccocioppo et al., 1994; Waterman and
Costa, 1994; Yuan et al., 1995), it was the aim of our study 1)
to continuously record the immediate and delayed effects of
the NO donor SNP and two inhibitors of NO synthase, LNNA and L-NAME, on peristalsis of the guinea pig isolated
ileum, 2) to analyze the dual excitatory/inhibitory action of
SNP on peristalsis and, for comparison, on the motor activity
of the longitudinal muscle, 3) to examine the time course
with which successive exposure of the ileum to L-NAME and
apamin, in this or reverse order, abolishes peristalsis and 4)
to analyze whether SNP or combined addition of L-NAME
and apamin inhibits peristalsis via a similar or a different
type of action.
155
156
Holzer et al.
Fig. 1. Recording of the effect of sodium nitroprusside (SNP) on peristalsis in the guinea pig isolated ileum. A, Effect of SNP recorded 25
min after addition of vehicle (Tyrode solution) to the bath. B, Effect of
SNP recorded 25 min after addition of atropine to the bath. The pressure threshold of peristalsis is marked by arrow heads. The initial
stimulation of peristalsis (decrease in pressure threshold) is followed by
inhibition of fluid propulsion (elevation of pressure threshold) when
atropine is present in the bath (B).
in the pressure threshold became maximal 20 to 30 min after
the administration of SNP (table 1) and was not accompanied
by any appreciable change in the frequency of peristaltic
waves (data not shown). When all data for the delayed effect
of SNP on the pressure threshold were averaged it turned out
that SNP failed to significantly enhance the pressure threshold (figs. 2, A and C and 3B) and to change the frequency of
peristaltic waves (data not shown). This was also true for the
delayed response to 1 mM SNP (n 5 6, data not shown). A
relationship between the initial excitatory and delayed inhibitory effect of SNP on the pressure threshold was therefore
not evident (fig. 3, A and B).
The monophasic action of SNP to reduce the pressure
threshold was in all experiments converted to a distinctly
biphasic action when SNP was tested in the presence of a
threshold concentration of atropine (10 nM, added to the bath
25 min before SNP). Atropine itself caused a slight but significant elevation of the pressure threshold, which rose from
92 6 4 to 133 6 8 Pa (n 5 30, P , .01), and a reduction of the
frequency of peristaltic waves, which fell from 0.46 6 0.02 to
0.40 6 0.02 min21 (n 5 30, P , .01). In the presence of
atropine, the initial decrease in the pressure threshold
evoked by SNP (0.1–100 mM) was invariably followed by a
marked increase in the pressure threshold (figs. 1B, 2, B and
D and 3B, table 1). With 100 mM SNP the delayed rise of the
pressure threshold was so large that peristalsis was abolished in all six segments that were tested (figs. 1B, 2D and
3B), and the same effect was seen with 1 mM SNP (n 5 6,
data not shown). The initial SNP-evoked decrease in the
pressure threshold was not altered by atropine in any consistent manner (figs. 1B, 2, B and D and 3A), and the variability in the influence of atropine on the SNP-induced decrease in the pressure threshold (fig. 3A) needs to be seen in
the light of the atropine-induced elevation of the base-line
pressure threshold (fig. 1B). Atropine (10 nM) failed to alter
the initial effect of SNP (0.1 mM, 1 mM, 10 mM, 100 mM, 1
mM) to raise the frequency of peristaltic waves and the
residual pressure (data not shown).
Effect of SNP on longitudinal muscle activity. To
shed more light on the ability of atropine to unmask SNPinduced inhibition of peristalsis, the action of SNP on the
mechanical activity of the quiescent longitudinal muscle of
the guinea pig isolated ileum was examined. Administration
of SNP (10 and 100 mM) to the organ bath had a biphasic
effect on the activity of the longitudinal muscle (fig. 4A).
Although the base-line tone of the preparations was low, SNP
initially relaxed the muscle, a response that soon was followed by a longer-lasting but transient contraction of the
muscle (fig. 4A). Although the relaxation caused by 100 mM
SNP was not larger than that caused by 10 mM SNP, the
magnitude of the contraction was related to the concentration of SNP and amounted to 10 to 20% of the maximally
possible contraction (fig. 4B, table 2). Once the contractile
response to SNP had faded away, the tone of the preparations
was invariably lower than before exposure to SNP (fig. 4A).
The delayed SNP-evoked contraction was inhibited by an
effective concentration of atropine (1 mM, fig. 4B), and table
2 shows that in the presence of atropine SNP (10 and 100
mM) was no longer able to significantly increase the contractile state of the preparations. In contrast, the initial relaxant
response to SNP did not seem to be inhibited by atropine (fig.
4B, table 2), because SNP led to a significant relaxation of the
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to 15 min (figs. 1A, 2, A and C and 3A). This facilitatory effect
of SNP was accompanied by an increase in the frequency of
peristaltic waves, which with 100 mM SNP rose from 0.42 6
0.06 to 0.69 6 0.07 min21 (maximal change, n 5 6, P , .01),
and an elevation of the residual pressure (fig. 1A). The latter
parameter, which is the intraluminal pressure (relative to
the zero base-line pressure) measured immediately after the
completion of a peristaltic wave, increased from 5 6 2 to 11 6
2 Pa (maximal change, n 5 6, P , .05) after administration
of 100 mM SNP. In addition, SNP reduced the amplitude of
the peristaltic waves in all experiments (fig. 1) but this parameter was not quantified in our study. The administration
of a 10-fold higher dose of SNP (1 mM, n 5 6, data not shown)
failed to evoke effects that were larger than those induced by
100 mM SNP.
The SNP-induced facilitation of peristalsis, which took
place in all experiments in a concentration-dependent manner (fig. 3A), was sometimes followed by a small depression of
fluid propulsion as deduced from a moderate increase in the
pressure threshold (fig. 1A, table 1). Table 1 shows that a
slight rise of the pressure threshold by $20 Pa was occasionally observed when the intestinal segments were exposed to
10 mM or higher SNP concentrations. This delayed increase
Vol. 280
1997
NO and Intestinal Peristalsis
157
TABLE 1
Effect of SNP to cause a delayed increase in the pressure threshold of peristalsisa
Concentration of
SNP (mM)
0.1
1
10
100
1000
Vehicle-Treated Preparations
Responding to SNP with Increase in
Pressure Threshold by 20 Pa or
More
0 of 7
0 of 6
3 of 6
1 of 6
4 of 6
Latency of Maximal SNP
Effect in Presence of
Vehicle (min)
28.2 6 3.2 (6)
20.6 (1)
23.7 6 3.2 (4)
Atropine-Treated Preparations
Responding to SNP with Increase in
Pressure Threshold by 20 Pa or More
4 of 6
6 of 6
6 of 6
6 of 6
6 of 6
Latency of Maximal SNP
Effect in Presence of
Atropine (min)
20.1 6 5.7 (4)
25.2 6 3.2 (6)
27.8 6 2.8 (6)
22.0 6 4.3 (6)
24.2 6 4.0 (6)
a
The data summarize the effect of SNP to increase the pressure threshold of peristalsis after the initial decrease in the pressure threshold has waned. The effects
of SNP were quantified by giving the number of preparations that, in the presence of vehicle or atropine, responded to SNP with an increase in the pressure threshold
by 20 Pa or more. Vehicle (Tyrode solution) or atropine (10 nM) was added to the bath 25 min before the preparations were exposed to SNP. The latencies of the
maximal effect of SNP correspond to the time interval between administration of SNP and the maximal increase in the pressure threshold and are given as means 6
S.E.M., the number of experiments are indicated in parentheses.
muscle in both vehicle- and atropine-treated preparations
(table 2). The observation that the SNP-evoked relaxation
was numerically smaller in the atropine- than in the vehicletreated segments (table 2) is related to the numerically lowered base-line tone in the atropine-treated preparations (fig.
4B). Figure 4B demonstrates that the contractility level to
which the segments were relaxed by 100 mM SNP in the
presence of atropine was significantly lower than that seen in
the vehicle-treated segments, which confirms that the relaxant activity of SNP was not compromised by atropine.
Effect of NO synthase inhibitors on peristalsis. Administration of the NO synthase inhibitor L-NAME (100 mM)
to the organ bath stimulated peristalsis as shown by a de-
crease in the pressure threshold from 92 6 7 to 67 6 6 Pa
(n 5 7, P , .01) and an increase in the frequency of the
peristaltic waves from 0.48 6 0.04 to 0.60 6 0.05 min21 (n 5
7, P , .01). The effect of a 3-fold higher dose of L-NAME (300
mM) to decrease the pressure threshold of peristalsis (fig. 5A)
and to increase the frequency of peristaltic waves from
0.57 6 0.06 to 0.76 6 0.06 min21 (n 5 9, P , .01) was not
different from that caused by 100 mM L-NAME. The inactive
enantiomer, D-NAME (300 mM), failed to influence the pressure threshold (fig. 5A) and the frequency of peristaltic waves
(0.45 6 0.07 min21 before exposure to D-NAME, 0.44 6 0.06
min21 after exposure to D-NAME, n 5 7). As with L-NAME,
the NO synthase inhibitor L-NNA (30 mM) reduced the pressure threshold (fig. 5B) and increased the frequency of peristaltic waves from 0.53 6 0.08 to 0.71 6 0.08 min21 (n 5 7,
P , .05).
The facilitatory effects of L-NAME (300 mM) and L-NNA
(30 mM) on peristalsis, which were sustained for more than
20 min, were reduced by L-arginine (1 mM) (fig. 5, A and B).
L-Arginine (1 mM) alone caused some inhibition of peristalsis as shown by a rise of the pressure threshold (fig. 5B) and
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Fig. 2. Time course of the effect of sodium nitroprusside (SNP, 1 and
100 mM) on the pressure threshold of peristalsis in the guinea pig
isolated ileum recorded in the presence of vehicle (A and C) or atropine
(B and D). Vehicle (Tyrode solution) or atropine (10 nM) was added to
the bath 25 min before SNP. Abscissa: time relative to the addition of
SNP at time 0. Ordinate: pressure threshold of peristalsis relative to the
zero base-line pressure. SNP first decreased the pressure threshold, an
effect that in the presence of atropine was followed by a marked
increase in the pressure threshold. The figures recorded in the presence of SNP denote the minimal and maximal pressure thresholds
measured in the presence of SNP, and the time values given underneath the post-SNP bars refer to the time points when on average the
SNP-induced decrease and delayed increase in pressure threshold was
maximal. In D peristalsis was invariably abolished by SNP, i.e., even at
the intraluminal pressure of 400 Pa no peristalsis was elicited. The bars
shown are means 6 S.E.M. of six experiments. *P , .05, **P , .01 vs
time 25 min.
Fig. 3. Effect of atropine on the concentration-dependent effect of
SNP to first decrease (A) and then increase (B) the pressure threshold
of peristalsis in the guinea pig isolated ileum. Ordinate: maximal SNPinduced change (D) in pressure threshold of peristalsis relative to the
zero base-line pressure. Abscissa: concentration of SNP. Vehicle (Tyrode solution) or atropine (10 nM) was added to the bath 25 min before
SNP. The bars shown are means 6 S.E.M. of six to seven experiments.
*P , .05, **P , .01 vs the respective values recorded in the presence
of the vehicle.
158
Holzer et al.
Vol. 280
TABLE 2
Changes in the mechanical activity of the guinea pig ileum longitudinal muscle caused by SNP in the absence and presence of
atropinea
Effect and Condition
SNP (10 mM)
SNP (100 mM)
Initial relaxation in the presence of vehicle
Initial relaxation in the presence of atropine
Delayed contraction in the presence of vehicle
Delayed contraction in the presence of atropine
21.42 6 0.54% (n 5 8)b
21.19 6 0.31% (n 5 8)c
18.83 6 0.93% (n 5 8)c
11.93 6 2.29% (n 5 8) NS
21.78 6 0.63% (n 5 6)b
20.67 6 0.29% (n 5 6)b
115.80 6 4.83% (n 5 6)b
14.14 6 4.27% (n 5 6) NS
a
The data show the SNP-induced changes in the contractile state of the preparations, the changes are expressed as the differences between the maximally altered
contractile state and the base-line state measured before administration of SNP. The contractile state of the preparations was recorded as a percentage of the
contraction in response to 1 mM histamine (100%) relative to the relaxation caused by 1 mM isoproterenol (0%). Vehicle (Tyrode solution) or atropine (1 mM) was added
to the bath 20 min before the preparations were exposed to the test concentration of SNP. The figures shown are means 6 S.E.M., the number of experiments are
given in brackets.
b
P , .05
c
P , .01 vs. base-line values measured before administration of SNP.
a decrease in the frequency of the peristaltic waves from
0.49 6 0.04 to 0.42 6 0.05 min21 (n 5 6, P , .05). In another
series of experiments it was found that the stimulant influence of L-NAME on peristalsis was not inhibited by a threshold concentration of atropine (10 nM). In the absence of
atropine, L-NAME (300 mM) reduced the pressure threshold
from 109 6 7 to 69 6 6 Pa (n 5 5, P , .05) whereas in the
presence of atropine (10 nM) the pressure threshold fell from
148 6 11 to 91 6 9 Pa (n 5 5, P , .05) in response to
L-NAME.
Effect of L-NAME in combination with apamin on
peristalsis. In one of two sets of experiments the ileal segments were exposed to L-NAME (300 mM) 10 min before
apamin (0.5 mM) was administered into the organ bath. As
described above, L-NAME stimulated peristalsis (fig. 6A) by
decreasing the pressure threshold from 109 6 7 to 70 6 4 Pa
(n 5 9, P , .01) and increasing the frequency of the peristaltic waves from 0.57 6 0.06 to 0.76 6 0.06 min21 (n 5 9, P ,
.01). Addition of apamin instantly disrupted the regular pattern of fluid propulsion and caused nonpropulsive contrac-
tions of the circular muscle. As can be seen from figure 6A,
periods of incoordinated nonpropulsive contractions alternated with brief periods of coordinated peristalsis.
In the other set of experiments the order with which the
ileal segments were exposed to L-NAME and apamin was
reversed, and apamin was added to the bath 10 min before
L-NAME was given. In this instance, apamin (0.5 mM) facilitated peristalsis (fig. 6B) as shown by a reduction of the
pressure threshold from 129 6 12 to 77 6 6 Pa (n 5 7, P ,
.01) and a rise of the frequency of the peristaltic waves from
0.59 6 0.04 to 0.67 6 0.04 min21 (n 5 7, P , .05). Addition of
L-NAME instantly disrupted the regular pattern of peristalsis and caused nonpropulsive spasms of the circular muscle,
which were interrupted by brief periods of apparently coordinated peristalsis (fig. 6B).
Discussion
The results of this study show that interference with inhibitory neuroeffector transmission in the guinea pig isolated
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Fig. 4. Effect of SNP on the contractile state of the
guinea pig ileum longitudinal muscle in the absence and
presence of atropine. A, Recording of the effect of SNP in
the presence of vehicle. B, Effect of SNP (10 and 100 mM)
in the presence of vehicle and atropine (1 mM, added to
the bath 20 min before exposure to SNP). The contractile
state of the preparations was recorded as a percentage of
the contraction in response to 1 mM histamine (100%)
relative to the relaxation caused by 1 mM isoproterenol
(0%). The first bar in each triplet denotes the contractile
state of the preparation before exposure to SNP, the
second bar denotes the maximal relaxation and the third
bar the peak contraction, in response to SNP. The bars
shown are means 6 S.E.M. of six to eight experiments.
*P , .05, **P , .01 vs respective bar recorded in the
presence of vehicle.
1997
ileum modifies fluid propulsion in a distinct manner. Peristalsis was facilitated by apamin, a blocker of certain calcium-dependent potassium channels, and by the NO synthase
inhibitor L-NAME, whereas combined administration of both
drugs stopped regular peristalsis. It was unexpected to see
that the NO donor SNP also stimulated peristalsis as shown
by a decrease in the pressure threshold of the peristaltic
waves. Only when peristalsis was compromised by a low
concentration of atropine did SNP cause a delayed inhibition
of peristalsis. The pattern of peristaltic shutdown caused by
SNP, however, was profoundly different from that caused by
combined administration of L-NAME plus apamin. Whereas
SNP abolished motor activity by locking the intestinal segment in a state of complete relaxation in which it was unable
to contract, exposure of the intestine to L-NAME plus
apamin prevented peristalsis by abolishing the coordination
of peristaltic waves although the segment was still able to
contract and actually appeared hyperactive but failed to produce regular peristaltic waves.
159
The analysis of drug effects on peristalsis is complicated by
the multiplicity of sites at which drugs can interfere with the
neural and muscular effector systems of propulsive motility.
Theoretically, the facilitatory influence of L-NAME, apamin
and SNP may result from enforcement of excitatory enteric
pathways or partial blockade of inhibitory pathways. By taking account of their known actions it would appear that both
L-NAME and apamin facilitate peristalsis by interfering
with inhibitory neuroeffector transmission. This interpretation is based on the concept that, in the guinea pig small
intestine, inhibitory transmission to the circular muscle depends on fast inhibitory junction potentials that are blocked
by apamin and on slow inhibitory junction potentials that are
prevented by inhibitors of NO synthase (Bywater and Taylor,
1986; Lyster et al., 1992; He and Goyal, 1993). It would follow
that interruption of one of these inhibitory transmission
mechanisms shifts the balance between excitatory and inhibitory pathways such that stimulation of peristalsis prevails,
although this imbalance is not severe enough to distort the
basic coordination of regular peristaltic waves.
The stimulant effect of L-NAME and L-NNA on peristalsis,
as recorded here by a decrease in the pressure threshold of
peristaltic waves, was reduced by L-arginine, although Larginine alone enhanced the pressure threshold. These observations are in line with those of Waterman and Costa
(1994) who saw analogous effects of L-NAME and L-arginine
on the volume threshold of peristalsis. These characteristics
and the inability of the inactive enantiomer D-NAME to
influence peristaltic motility indicate that the actions of LNAME and L-NNA were due to inhibition of NO synthesis
(Kerwin and Heller, 1994). The action of L-NAME, which was
only partially counteracted by L-arginine, has previously
been found to be relatively resistant to inhibition by L-arginine (Rees et al., 1990).
It is very likely that there are multiple sites of action by
which blockade of NO synthase facilitates peristalsis in the
guinea pig small intestine as seen here and in other studies
(Ciccocioppo et al., 1994; Suzuki et al., 1994; Waterman and
Costa, 1994). The facilitatory influence of L-NAME and LNNA on peristalsis may be closely related to the action of NO
synthase inhibitors to enhance the descending inhibitory reflex by facilitating the transmission between sensory neurons
and interneurons (Yuan et al., 1995). However, NO synthase
inhibitors also block neuromuscular transmission within the
descending inhibitory reflex while the ascending excitatory
motor reflex remains unaltered (Yuan et al., 1995). How
these effects and the ability of NO synthase blockers to facilitate the release of acetylcholine and substance P from myenteric neurons (Knudsen and Tottrup, 1992; Wiklund et al.,
1993a; Kilbinger and Wolf, 1994) relates to their facilitatory
influence on peristalsis remains to be elucidated. This multiplicity of actions is consistent with the presence of NO
synthase in descending interneurons and motor neurons and
with the proposed role of NO as neuroneuronal and neuromuscular transmitter substance (Costa et al., 1992; Furness
et al., 1994; Waterman and Costa, 1994; Yuan et al., 1995;
Young et al., 1995).
When both mechanisms of inhibitory neuroeffector transmission in the guinea pig ileum are blocked by combined
administration of L-NAME and apamin, the balance between
excitation and inhibition is grossly distorted in favor of excitation, and the intestine becomes hyperactive and may even
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Fig. 5. Effect of NG-nitro-D-arginine methylester (D-NAME), NG-nitroL-arginine methylester (L-NAME), NG-nitro-L-arginine (L-NNA) and Larginine, alone or in combination, on the pressure threshold of peristalsis in the guinea pig isolated ileum. Abscissa: time relative to the
addition of drugs at time 0 and concentration of the drugs in the bath.
The recordings of the pressure threshold were taken at the indicated
time points. A, Left side: D-NAME was administered at time 0. A, Right
side: L-NAME was given at time 0, L-arginine at 20 min. B, Left side:
L-NNA was given at time 0, L-arginine at 10 min. B, Right side: Larginine was administered at time 0. Ordinate: pressure threshold of
peristalsis relative to the zero base-line pressure. The bars shown are
means 6 S.E.M. of six to seven experiments. **P , .01 vs time 25
(baseline value); †P , .05, ††P , .01 vs time 20 min (A) or time 10 min
(B).
NO and Intestinal Peristalsis
160
Holzer et al.
Vol. 280
show episodes of sustained spasm. Hyperexcitability and
maintained contraction of the muscle is one factor in the loss
of coordinated peristalsis (Waterman et al., 1994b). Another
factor is the blockade of inhibitory neurotransmission itself,
which has only recently been recognized as being crucial to
the alternating cycle of contraction and relaxation moving
anally in the peristaltically active gut (Ciccocioppo et al.,
1994; Waterman and Costa, 1994). Successive exposure of
the guinea pig ileum to L-NAME plus apamin, in this or
reverse order, was found to result in prompt disruption of
coordinated peristalsis and replacement of regular peristaltic
waves by multiple nonpropulsive contractions. This observation extends other studies in which gut distension failed to
elicit propulsive peristalsis in intestinal segments that had
been incubated with apamin and L-NAME or L-NNA for a
period of 10 to 20 min (Ciccocioppo et al., 1994; Waterman
and Costa, 1994) and emphasizes the importance of inhibitory motor neurons for the coordination of peristalsis.
From the facilitatory effect of NO synthase inhibition on
peristalsis it would seem predictable that exogenous NO,
administered by way of the NO donor SNP, inhibits peristaltic motility. However, the reverse was true, and SNP caused
a prompt facilitation of peristalsis, an effect that was also
noted by Sugisawa et al. (1991) but that is in contradiction
with the reported ability of SNP to inhibit peristalsis (Waterman and Costa, 1994) and to depress both ascending excitatory and descending inbitory motor reflexes (Yuan et al.,
1995). However, these discrepancies are very likely due to
the intervals of 15 to 20 min that were allowed to elapse
between drug addition and recording of its effect (Waterman
and Costa, 1994; Yuan et al., 1995) whereas in our study the
drug-induced changes of motility were continuously recorded. Closer analysis revealed that the action of SNP on
intestinal motility is composed of two distinct phases, an
initial period of excitation followed by inhibition of motility.
The contractile effect of SNP on the longitudinal muscle of
the guinea pig ileum is consistent with the ability of exogenous NO to contract the ileum of the guinea pig (Barthó and
Lefebvre, 1994) and other species (Barthó and Lefebvre,
1995). The SNP-evoked contraction involves cholinergic neurons, which is in keeping with the involvement of acetylcholine and tachykinins in the contractile response to exogenous
NO (Barthó and Lefebvre, 1994). In contrast, the delayed
relaxation caused by SNP is likely to mirror the direct inhibitory action of NO on intestinal muscle (Shuttleworth et al.,
1991; Lyster et al., 1992; He and Goyal, 1993; Wiklund et al.,
1993b; Barthó and Lefebvre, 1994) but may in addition be
related to the effect of SNP to depress slow synaptic excitation within the myenteric plexus (Tamura et al., 1993).
In view of the excitatory action of SNP on enteric neurons
it would appear that the SNP-induced facilitation of peristalsis results from stimulation of excitatory motor pathways or
from a permissive action of tonically released excitatory neurotransmitters. A delayed inhibitory effect of SNP on propulsive motility was seen, in a consistent manner, only when
peristaltic activity was compromised by a threshold concentration of atropine. This observation is at variance with the
finding of Waterman and Costa (1994) who noted a SNPevoked increase in the volume threshold of peristalsis in the
absence of atropine. It remains to be elucidated whether this
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Fig. 6. Recording of the effect of NG-nitro-L-arginine methylester (L-NAME) and apamin, in this or
reverse order, on peristalsis of the guinea pig isolated ileum. A, Effect of L-NAME given 10 min before apamin. B, Effect of apamin given 10 min before L-NAME. The pressure threshold is marked by
arrowheads. L-NAME or apamin given alone stimulate peristalsis (decrease in pressure threshold,
increase in the frequency of peristaltic waves) although combined presence of the drugs causes
nonpropulsive spasms of the circular muscle.
1997
Acknowledgments
The authors thank Wolfgang Schluet for his skillful help with the
experiments and Milana Jocič for her expert drawing of the graphs.
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Send reprint requests to: Dr. Peter Holzer, Department of Experimental
and Clinical Pharmacology, University of Graz, Universitätsplatz 4, A-8010
Graz, Austria.
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discrepancy arises from differences in animal strain, recording conditions or other factors. We assume that in our study
atropine caused a subtle shift in the balance between excitatory and inhibitory pathways of peristalsis such that the
depressant effect of SNP on peristaltic motility overrode the
drug’s stimulant effect. The pattern of peristaltic shutdown
caused by SNP in the presence of atropine indicates direct
relaxation of the muscle to an extent that the muscle is no
longer able to contract in response to the excitatory input
from enteric neurons, an action that would expectedly be
attributed to a transmitter of inhibitory enteric motor neurons. The observation that the effect of SNP on peristalsis
was changed by atropine although that of L-NAME remained
unaltered further attests to profound differences in the actions of SNP and L-NAME on peristalsis.
The multiple roles of NO in intestinal motor control highlight the difficulties that are encountered in the pharmacological analysis of drugs that may act at several sites, and in
an opposing manner, within the enteric pathways subserving
peristalsis. Our results illustrate that knowledge of specific
cellular effects of drugs such as SNP and L-NAME does in no
way allow the final outcome for the physiological process of
peristalsis to be readily predicted. As a consequence, complementary studies at the organ level are required to recognize
the full impact that a drug may have on propulsive motility.
Analysis of the complex motor effects of SNP and L-NAME
under this perspective has revealed that NO is an important
messenger molecule involved in the coordination of propulsive motility and that both overactivity of the NO system (as
reflected by the effects of SNP seen in the presence of atropine) as well as dysfunction of nitrergic inhibitory motor
neurons (as reflected by the effects of apamin plus L-NAME)
have deleterious effects on peristalsis. In the case of SNP
plus atropine it is loss of excitability, and in the case of
L-NAME plus apamin loss of the ability to relax, which
prevents the physiological process of peristalsis.
NO and Intestinal Peristalsis