1Department of Pharmacology, University of Bologna, 40126 Bologna; and 2Department of Internal Medicine and Therapeutics, University of Pavia, 27100 Pavia, Italy
Submitted 12 July 2002 ; accepted in final form 18 March 2003
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ABSTRACT |
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gastric motility; gastric compliance; barostat; serotonin receptors; dog
Animal models have allowed us to get more insight into the possible
mechanism mediating the gastric motor effects of sumatriptan. Coulie et al.
(13), using an in vivo cat
model, suggested that the sumatriptan-induced increase in gastric volume is
due to activation of a nitrergic inhibitory pathway, because the nitric oxide
synthase inhibitor N-nitro-L-arginine
methyl ester (L-NAME) antagonized the effect of sumatriptan.
However, they did not investigate the possible involvement of
5-HT1B/D receptors, for which sumatriptan has high affinity
(2). Thus we compared the
effects of sumatriptan alone and combined with
N-[4-methoxy-3-(4-methyl-1-piperazinyl)
phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-[1,1-biphenyl]-4-carboxamide
hydrocloride (GR-127935), N-[3-[3
(dimethylamino)-ethoxy]-4-methoxyphenyl]-2'-[methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)]-[1,1-biphenyl]-4-carboxamide
hydrocloride (SB-216641 hydrochloride), or
3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol hydrocloride
(BRL-15572 hydrochloride) (respectively, nonselective 5-HT1B/D,
selective 5-HT1B, and selective 5-HT1D receptor
antagonists) on canine gastric accommodation to isobaric distensions performed
with a barostat. Atropine and bethanechol were used as reference drugs because
of their well-known relaxing and contracting effect on the stomach,
respectively (14,
15). Indeed, canine gastric
tone is known to be maintained by a vagally mediated, cholinergic input
(6).
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MATERIALS AND METHODS |
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The barostat can operate in two modes. If the barostat mode is used, a constant pressure is maintained in the intragastric bag, so that when the stomach contracts, the system withdraws air and when the stomach relaxes, the system injects air (maximal rate of airflow 50 ml/s). Thus the barostat allows quantitative measurement of variations in gastric tone by recording changes in the volume of air within the intragastric bag. Alternatively, the barostat can be used in the distension mode to assess the ability of the stomach to adapt to a distending stimulus (i.e., its compliance). This mode allows measurement of volume changes induced by a pressure increase (isobaric distensions). The slope of the pressure-volume curve (dV/dP) provides an estimate of gastric compliance.
Experimental model. Experiments were carried out on four female Beagle dogs (1216 kg body wt) purchased from an authorized local breeder (Green Hill, Montichiari, Italy) and housed in single, air-conditioned boxes. All experiments were performed at the University of Pavia according to European Union Directive 86/609 on the care and use of experimental animals (authorizations of the Italian Ministry of Health 10/96C, 60/99C, 61/99B). The protocol was approved by the Ethics Committee of the Department of Internal Medicine and Therapeutics of the University of Pavia. The animals were fed with dog food in pellets (Stefano Morini, S. Polo d'Enza, Italy). Water was available ad libitum. The dogs were operated on under general thiopental anesthesia (2540 mg/kg iv) with aseptic technique and assisted respiration. Through a midline laparotomy, we inserted a modified Thomas cannula into the stomach at the greater curvature opposite to the incisura angularis and exteriorized in the anterior aspect of the abdomen. Antibiotic prophylaxis consisted of intramuscular amoxycillin (0.05 ml/kg Clamoxyl; Pfizer, Rome, Italy) for 3 days starting the day of surgery.
Experimental procedure. Dogs were trained to stand quietly in a sling without sedation. Experiments were performed on conscious dogs after allowing at least 15 days for recovery after surgery. Before each experimental session, the dogs were fasted for at least 18 h; water was available ad libitum. Between consecutive experimental sessions with the same animal, a washout period of at least 72 h was allowed. The dogs were observed throughout the experiment, and any sign of discomfort or anomalous behavior was noted. The gastric cannula was opened, and after verifying that the stomach did not contain any food residues, we introduced the bag of the barostat into the proximal stomach (position of the bag checked fluoroscopically). Before and at the end of the in vivo tests, the bag was checked for air leaks by increasing the pressure to 20 mmHg.
At the beginning of the experimental session, intrabag pressure was maintained at a baseline level of 2 mmHg, because in all dogs, it represented the minimal distending pressure, i.e., the pressure at which the mean intrabag volume was >25 ml and continuous respiratory fluctuations were first detected. The very small baseline pressure applied by the barostat system allowed accurate recording of gastric volume changes without distortion of the physiological pattern of gut motor activity (4, 5).
After a 15-min equilibration period, we increased intrabag pressure with
2-mmHg increments every 3 min, starting from the baseline value of 2 mmHg up
to 12 mmHg (or until an intrabag volume of 1,000 ml was achieved), without
deflating the barostat bag at each pressure step. We did not exceed the
pressure of 12 mmHg for two reasons: 1) preliminary experiments
indicated that dogs started to show the first signs of discomfort (stretching
of the legs) at 1618 mmHg, and 2) a pressure of 12 mmHg
leads to a significant increase in transient lower esophageal sphincter
relaxations (TLESRs; at a rate of
7 every 30 min) in dogs of the same
body weight (9). Although
TLESRs are physiological events, their occurrence at this rate is considered a
sign of excessive distension, and we decided not to exceed this pressure.
Although distending pressures between 7 and 12 mmHg are above the normal
physiological range, the same pressure range of 212 mmHg above minimal
distending pressure was used in human volunteers
(28).
In each experiment, four pressure-volume curves were obtained, allowing a 40-min interval between the end of a distension cycle and the beginning of the subsequent cycle. During these intervals, pressure was maintained at 2 mmHg. The first distension cycle was used to unfold the intragastric bag, and the values recorded were discarded. Preliminary experiments showed that the next three cycles gave reproducible pressure-volume curves (coefficient of variation <10%). Therefore, when studying drug effects, we performed a first distension cycle to unfold the bag. The next two distension cycles were used as controls (Fig. 1). Test drugs (sumatriptan, atropine, or L-NAME) were administered 15 min before starting the fourth distension cycle. 5-HT1B/D receptor antagonists were injected intravenously 30 min before the fourth distension cycle. Bethanechol was administered by intravenous infusion (15-min infusion, starting 15 min before starting the fourth distension cycle).
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In preliminary experiments, it was found that, as in cats and humans,
sumatriptan could induce a rapid-onset relaxation (immediately after the end
of the intravenous injection). However, this response was short-lasting (the
baseline gastric volume recorded at the distending pressure of 2 mmHg
invariably returned to the preinjection value within 15 min) and was observed
in all dogs only at the doses of 400 and 800 nmol/kg. Because the effect of
sumatriptan on intragastric volume may depend on the distending pressure, we
devised an experimental procedure that covers the 2- to 12-mmHg pressure range
and takes into account both the duration of the acute relaxation (the 15-min
period elapsing between sumatriptan administration and the next distension
cycle allows recovery to the initial baseline gastric volume) and the
pharmacokinetics of sumatriptan [the half-life of sumatriptan in dogs is
2 h (21)].
In each dog, we carried out three sets of experiments, performing each
experiment in duplicate. In the first set of experiments, we tested the
effects of intravenous administration of sumatriptan, atropine, bethanechol,
and L-NAME. The following doses of sumatriptan were used: 100, 200,
400, 800, 1,600, 2,400 nmol/kg iv (i.e., 0.516 times the
subcutaneous dose used in migraine). Atropine was injected intravenously at
the doses of 100 and 300 nmol/kg on the basis of a previous dose-response
study showing that they provided partial and full blockade of muscarinic
transmission, respectively
(17). The dose of bethanechol
(508
nmol·kg1·h1
for 15 min) was chosen on the basis of a previous dose-response study showing
that this was the minimal dose required to significantly counteract the
gastric relaxation induced by vagal blockade in dogs
(15). The dose of
L-NAME (37.04 µmol/kg iv) was selected from a review of the
literature (1,
3).
In a second set of experiments, we tested the effects of intravenous administration of 5-HT1B/D receptor antagonists alone (GR-127935, SB-216641, or BRL-15572 at doses of 54, 559, and 676 nmol/kg, respectively).
In a third set of experiments, we combined a 5-HT1B/D receptor antagonist (GR-127935, SB-216641, or BRL-15572 at doses of 54, 559, and 676 nmol/kg, respectively) with sumatriptan (800 nmol/kg iv). The doses of antagonists were chosen on the basis of a review of the literature (18, 23, 43).
Drugs. Atropine sulfate, bethanechol chloride, and L-NAME hydrochloride were purchased from Sigma (St. Louis, MO). Sumatriptan succinate and GR-127935 were kindly donated by GlaxoSmithKline (Stevenage, Hertfordshire, UK). SB-216641 hydrochloride and BRL-15572 hydrochloride were purchased from Tocris Cookson (Bristol, UK). All drugs were dissolved in distilled water.
Data analysis. Baseline gastric tone was defined as the intragastric bag volume when pressure was maintained at 2 mmHg and was calculated by averaging the barostat volume tracing over 30-s periods. A change in gastric tone was defined as a change in intragastric volume >30 ml lasting >10 s. Changes in intragastric volume occurring after administration of sumatriptan, atropine, bethanechol, or L-NAME were measured at the peak of the response. During stepwise distensions, the maximal volume achieved at each pressure level was calculated by averaging the intrabag volume for 30 s at the peak of the response. Data obtained in the same animal with duplicate experiments were averaged to calculate grand means (±SE) for the four dogs and perform the statistical analysis.
Pressure-volume curves were fitted by regression analysis (GraphPad Prism
version 3.00 for Windows, GraphPad Software, San Diego, CA) using a linear (V
= V0 + k2P) or an exponential (V =
V0ek1P) equation, where V is volume
(in ml), V0 is theoretical volume when P = 0 mmHg, P is pressure
(in mmHg), k1 is the rate constant in the exponential
equation, and k2 is the slope in the linear model. The
overall fit for each curve was summarized as an r2 value.
The first derivative of the pressure-volume curve (dV/dP) is a
measure of gastric compliance. If a linear model is used, the slope of the
regression line is a constant (k2), whereas if an
exponential model is used, the first derivative is y' =
V0k1ek1P and
increases with pressure. Therefore, to compare compliance values obtained with
the two models, we calculated the slope at Pmax (6 mmHg).
The parameters estimated for each dog according to the exponential model were
used to calculate the pressure corresponding to half-maximum volume
(Phalf), which is also a measure of gastric compliance
(8).
Volumes obtained at different pressures with different treatments were compared using repeated-measures twoway analysis of variance (GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego, CA): columns identified treatment and rows different pressure levels. Post-tests were performed applying the Bonferroni's correction for multiple comparisons. The other comparisons were performed using repeated-measures one-way analysis of variance with the Bonferroni's correction for multiple comparisons. P values <0.05 were considered significant.
To detect differences among doses of sumatriptan and to select the dose to be tested with 5-HT1B/D receptor antagonists, the mixed-effects, repeated-measures analysis of variance, in which dogs are random effects and dose and pressure fixed effects, was applied. The dependent variable was the delta between the values obtained before and after sumatriptan at each dose level, using the lowest dose of sumatriptan as baseline. The mixed procedure from the SAS package (SAS/STAT Software Release 6.11, SAS Institute, Cary, NC) was used.
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RESULTS |
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Effect of bethanechol, atropine, and L-NAME on the
pressure-volume relationship. The baseline gastric volume recorded at 2
mmHg was 45 ± 18 and 25 ± 5 ml before and after bethanechol
administration, respectively (P > 0.05). After bethanechol, the
pressure-volume curve was significantly (P < 0.01) shifted toward
significantly lower volumes and the curve that best fitted the experimental
data was still exponential (Fig.
2). Table 1 reports
the values obtained with the exponential and the linear model. With the use of
the exponential model, V0 and slope at Pmax
calculated after bethanechol were significantly lower with respect to
controls, whereas Phalf was significantly higher
(Table 1).
Both doses of atropine decreased baseline gastric tone (V at 2 mmHg
= 165 ± 43 and 145 ± 44 ml, respectively, for the lower and
higher dose; both P < 0.05) and significantly (P <
0.01) shifted the pressure-volume curve toward higher volumes
(Fig. 3). After atropine, the
linear model provided a better fit of the experimental data
(Fig. 3).
Table 1 reports the values
obtained with the exponential and the linear model. Atropine significantly
(P < 0.05) increased V0 and tended to decrease
k1 (rate constant), although the latter effect did not
achieve statistical significance and no significant change in the slope at
Pmax was observed. However, both doses of atropine
significantly decreased Phalf
(Table 1).
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Baseline gastric volume recorded at 2 mmHg was 61 ± 5 and 26 ± 5 ml before and after L-NAME administration, respectively (P > 0.05). After L-NAME, the pressure-volume curve was significantly (P < 0.01) shifted toward lower volumes and the curve that best fitted the experimental data was exponential (Fig. 4). Table 1 reports the values obtained with the exponential and the linear model: a significant decrease in V0 and an increase in Phalf was observed after L-NAME.
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Effect of sumatriptan on the pressure-volume relationship.
Sumatriptan induced a rapid-onset relaxation, but this effect was observed in
all dogs only at the doses of 400 and 800 nmol/kg (V: 91 ± 52
and 112 ± 44 ml, P <0.05 vs. controls). This response was
short-lasting (usually <10 min), and baseline gastric volume invariably
returned to the baseline within 15 min, so that the baseline gastric volume
recorded at 2 mmHg just before starting the postdrug distension cycle was not
significantly different from controls with all doses of sumatriptan
(Fig. 5).
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After sumatriptan, the pressure-volume curve was shifted toward
significantly higher volumes (Fig.
5): at Pmax, the
V with 800 nmol/kg
sumatriptan vs. control was 163 ± 30 ml (P < 0.01). A
significant (P < 0.05) shift of the pressure-volume curve was
observed with doses ≥200 nmol/kg. The mixed-effects, repeated-measures
analysis of variance, used to detect differences among doses of sumatriptan,
revealed that only with the dose of 800 nmol/kg was the delta obtained before
and after sumatriptan significantly higher than that observed with the lowest
dose (100 nmol/kg) in at least four of six pressure levels. Therefore, the
dose of 800 nmol/kg was selected for the experiments with 5-HT1B/D
receptor antagonists.
Table 2 reports the values
obtained with the exponential model to fit the data obtained before and after
sumatriptan administration. Again, the exponential model provided a better fit
of the data points (data not shown). After sumatriptan, Phalf was
significantly decreased (Table
2). Although V0 and the slope at
Pmax tended to be higher after sumatriptan at doses ≥200 nmol/kg
(Table 2), this trend did not
achieve statistical significance.
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Effect of 5-HT1B/D receptor antagonists alone. GR-127935, SB-216641, and BRL-15572 per se had no effect on baseline gastric volume and on the volume-pressure curve (Fig. 6).
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Effect of combined 5-HT1B/D receptor
antagonists and sumatriptan. The gastric relaxation observed immediately
after injection of 800 nmol/kg sumatriptan was antagonized by GR-127935 and
SB-216641 (V: 28 ± 18 and 24 ± 17 ml, P <0.05
vs. sumatriptan alone) but not by BRL-15572 (
V: 121 ± 51
ml).
The shift of the pressure-volume curve observed with sumatriptan (800 nmol/kg) was fully reversed by GR-127935 and SB-216641 but not by BRL-15572 (Fig. 7).
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DISCUSSION |
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Although investigators often resort to a simpler, linear equation to fit pressure-volume data (2628, 3337), an exponential equation fits well with the concept of adaptive accommodation to a distending stimulus, at least within a narrow pressure range. Indeed, the proximal stomach can rapidly accept relatively large meals with only a minimal increase in pressure (adaptive relaxation). When, unlike in the present study, higher pressure levels are achieved, discomfort and pain are induced and the pressure-volume relationship does not fit an exponential model, probably because of the influence of the elastic properties of the gut wall (10). An advantage of the exponential with respect to the linear model is that the former does not provide negative values for V0, which have no biological meaning.
The exponential model proposed in this manuscript is simpler than the power exponential model, proposed by the Mayo group (7, 8, 39). The latter model has certainly the advantage of providing excellent fit for the sigmoid pressure-volume curves that tend to reach a plateau at the highest pressure levels (8). On the other hand, it is less straightforward than the simple exponential model, because it requires transformation of data (proportionate volume is expressed as a function of reciprocal pressure). In this study, we propose the simple exponential model for the following reasons: first, in our experiments, for both ethical and technical reasons (see MATERIALS AND METHODS), we investigated the pressure-volume curves in the 2- to 12-mmHg pressure range and, within this range, the curves were not sigmoid. Second, because the simple exponential model provided very good fit of the experimental data, it was unnecessary to resort to the more sophisticated power exponential model. However, we acknowledge that we investigated a limited pressure range that may not fully reflect the biomechanical properties of the stomach.
The shape of the exponential curve is consistent with a progressive activation of reflex mechanisms, which tend to prevent pressure increases in response to increasing gastric volumes and are mediated by neural pathways having nitric oxide as an important neuro-transmitter (20, 40). Several studies have documented the important role played by nitric oxide in mediating vagally induced gastric relaxations in different species, including guinea pigs (32) and dogs (30). Notably, the intravenous administration of the nitric oxide synthase inhibitor NG-nitro-L-arginine (L-NNA) shifted the pressure-volume curve toward lower volumes and decreased gastric compliance in dogs, suggesting the presence of a continuous nitrergic inhibitory tone (33). Our results with L-NAME confirm this hypothesis. The fact the L-NAME per se affected the pressure-volume curve prevented us from formally testing the effect of L-NAME against sumatriptan. In any case, the sumatriptan-induced shift of the pressure-volume curve toward higher volumes is consistent with the hypothesis put forward by Coulie et al. (13) that 5-HT1 receptors activate a nitrergic inhibitory pathway.
In this study, sumatriptan facilitated gastric accommodation to a distending stimulus with a peak effect at 800 nmol/kg. Interestingly, the effect of sumatriptan was statistically significant already at the dose of 200 nmol/kg, which approximately corresponds to the single subcutaneous dose used in humans for the relief of a migraine attack (6 mg). Sumatriptan exerted its action on gastric accommodation through 5-HT1B receptors, because both GR-127935 and SB-216641 fully reversed the gastric motor effects of sumatriptan, whereas the lack of effect of BRL-15572 suggests that 5-HT1D receptors are not involved. Whether the location of the 5-HT1B receptors mediating the effect of sumatriptan is central or peripheral remains to be determined. The fact that sumatriptan penetrates poorly the blood-brain barrier (19) and can relax the guinea pig isolated stomach (31) would argue against a central site of action. We tentatively suggest a neuronal location of these 5-HT1B receptors, because the effect of sumatriptan in vitro was blocked by tetrodo-toxin (31). We should also consider 5-HT1P receptors, because their presence is reported in enteric neurons (29), and some authors have suggested that sumatriptan might act via this receptor subtype (36). However, 5-HT1P receptors are not included in the official International Union of Pharmacology classifi-cation of serotonin receptors [they are still considered "orphan" receptors (2, 25)], and to the best of our knowledge, none of the authors reporting the gastric motor effects of sumatriptan in vivo has ever tested the effect of 5-HT1P receptor antagonists because of the lack of selective agents suitable for in vivo use. The fact that, in our hands, the effect of sumatriptan was fully reversed by GR-127935 and SB-216641 supports the involvement of 5-HT1B receptors.
The fact that we studied drug effects in the 2- to 12-mmHg pressure range instead of working at a fixed pressure level [a fixed pressure level of 2 mmHg above the minimal distending pressure was used by Coulie et al. (13)], allowed us to appreciate some qualitative differences among the effects of sumatriptan, atropine, and bethanechol. Indeed, both the lower and higher dose of atropine induced a significant gastric relaxation (see also Ref. 16) at the baseline pressure of 2 mmHg and shifted the pressure-volume curve toward higher volumes with a change in the best-fit equation, which became linear. These observations confirm the important role played by vagal cholinergic pathways in maintaining gastric tone in the fasting dog (6, 16) and are in line with recent findings in humans (28). We interpret the change in the shape of the pressure-volume curve (i.e., from exponential to linear) after atropine as a phenomenon due to the significant gastric relaxation induced by muscarinic receptor blockade at baseline pressure, which masks the ability of the stomach to undergo the process of "adaptive accommodation." Notably, k1 values (rate constant) were lower after atropine, although this trend did not reach statistical significance.
By contrast, bethanechol did not significantly affect the shape of the curve (the exponential equation provided the best fit) but made the stomach less susceptible to undergo distension, as indicated by the shift of the pressure-volume curve to the right, the lower gastric compliance, and V0 values after bethanechol. The lack of a statistically significant difference in baseline gastric volume at 2 mmHg distending pressure after bethanechol is probably due to a type II error.
Finally, sumatriptan significantly shifted the pressure-volume curve toward higher volumes and enhanced gastric accommodation, an action that has potential application in the treatment of functional dyspepsia (36, 38). Indeed, contrary to the view that most dyspeptic patients have delayed gastric emptying and a relaxed stomach (hence, the use of prokinetic agents to relieve symptoms), a significant proportion of these subjects display impaired fundic relaxation to a meal or altered gastric sensitivity to distension (38). In these patients, prokinetics are contraindicated, whereas a gastric relaxing drug could decrease early satiety, a cardinal symptom of dysmotility-like dyspepsia.
In conclusion, sumatriptan facilitates gastric accommodation to a distending stimulus, and 5-HT1B receptors play an important role in mediating this effect. The finding that an exponential model fits gastric pressure-volume data better than a linear model well describes the concept of gastric adaptive relaxation.
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ACKNOWLEDGMENTS |
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This study was supported, in part, by a grant from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (Cofinanziamento 1999: Project No. 9905222532).
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FOOTNOTES |
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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.
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REFERENCES |
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