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Keywords:

  • colorectal hypersensitivity;
  • 5-HT4 receptors;
  • rat;
  • tegaserod

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References

Abstract  Tegaserod is a 5-HT4 receptor partial agonist approved for the treatment of irritable bowel syndrome in women with constipation and in both men and women with chronic constipation. The efficacy of tegaserod is based on the importance of 5-HT4 receptors regulating intestinal peristalsis and secretion, and possibly visceral sensory pathways. Our aim was to investigate the effect of tegaserod on colorectal sensitivity using models of normal and exaggerated responsiveness to colorectal distension (CRD). The visceromotor responses (VMR) to CRD at graded pressures (0–60 mmHg) were measured by the number of reflex abdominal contractions. Acute colorectal hypersensitivity was induced by intracolonic infusion of dilute acetic acid. Chronic hypersensitivity was observed in rats following spontaneous resolution of trinitrobenzenesulfonic acid-induced colitis. Rats with normosensitive colons served as controls. Tegaserod (0.1–10 mg kg−1) caused dose-dependent reduction of the VMR to CRD in control rats and in those with colonic hypersensitivity. 5-HT4 antagonists reversed the effects of tegaserod in rats with normosensitive colons, and partially inhibited effects in rats with colonic hypersensitivity. Central administration of tegaserod had no inhibitory effect. These results support the assumption that colonic hypersensitivity could be normalized by tegaserod acting, at least in part, through peripheral 5-HT4 receptors.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References

Irritable bowel syndrome (IBS) is a gastrointestinal (GI) disorder characterized by abdominal discomfort or pain and abnormal bowel habits. GI dysmotility and altered visceral sensitivity in the absence of organ pathology is the most common pathophysiological correlates identified in IBS patients. Studies investigating motility abnormalities in IBS patients indicated exaggerated motility in the large intestine following rectosigmoid distension,1 a greater increase in colonic phasic contractions in response to a meal2 and increased myoelectrical activity associated with diarrhoea.3 Recent evidence suggests that increased visceral sensitivity is a leading contributor to IBS symptoms. Balloon distension studies indicated that IBS patients become aware of distension and perceive pain at distending pressures and volumes that are lower compared with controls.4–6 The increased perception of intestinal distension or contraction has been attributed to lower perception thresholds.7,8 It is now considered that an abnormal interaction between normal or disordered motility and visceral hypersensitivity (low thresholds of visceral perception) may be a determining factor for the symptoms of IBS. It is also proposed that stress, anxiety or the memories of aversive events can further exacerbate visceral hypersensitivity and pain perception in IBS patients.9,10

5-Hydroxytryptamine (5-HT) released from enterochomaffin cells in the mucosa in response to intraluminal stimuli plays a key role in the normal functioning of the GI tract. Activation of 5-HT receptors, including the 5-HT4 subtype, is associated with the release of neurotransmitters, which initiate peristaltic reflex pathways, facilitate propulsive activity in the colon11–14 and induce Clsecretion, increasing the fluid in the lumen.15,16 The importance of 5-HT4 receptors as modulators of intestinal peristalsis and secretion, and their possible involvement in afferent neurotransmission provide a solid rationale for the potential benefit of selective 5-HT4 agonists in the treatment of IBS.

Tegaserod, an aminoguanidine indole acting as a partial agonist of 5-HT4 receptors has been approved for the treatment of women with constipation-predominant IBS (IBS-C),17,18 and both men and women with chronic constipation.19 Clinical studies indicated that tegaserod (6 mg b.i.d.) induced a sustained improvement of symptoms, including abdominal pain, in female patients with IBS-C and was well tolerated.20,21 However, the mechanisms of action of tegaserod on visceral hypersensitivity have not been thoroughly assessed. The aim of the present study was to investigate the effects of tegaserod on colorectal sensitivity using rat models of normal and exaggerated responsiveness to colorectal distension (CRD). In addition, experiments using selective 5-HT4 antagonist and different routes of administration of tegaserod were performed to identify the receptor mechanisms and the site of action of tegaserod.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References

Experimental animals

Male Sprague–Dawley rats (200–250 g) were housed under controlled conditions (21–22 °C, 50 ± 10% humidity, 12 h light/dark cycle) with rodent chow and water provided ad libitum. Following 1 week acclimation to the facility, the rats were brought into the laboratory daily between 8:00 am and 4:00 pm for 1 week to acclimate to the environment and experimenter. Prior to the experiments, the rats were fasted with free access to water for 14 to 18 h. The experimental protocol was approved by the Institutional Animal Care and Use Committees at the University of Oklahoma Health Science Center and the Oklahoma City VA Medical Center.

Visceromotor responses (VMR) to CRD

On the day of experiment, rats were anaesthetized with isoflurane (2–3%) and a strain gauge force transducer (RB Product, Inc., Stillwater, MN, USA) was sutured to the external oblique muscle. A 5-cm latex balloon was inserted 11 cm into the distal colon and held in place by taping the balloon catheter to the base of the tail. The catheter was attached to a programmable rigid piston barostat with zero intrinsic compliance (Distender Series II; G & J Electronics, Toronto, ON, Canada). The strain gauge was connected to a DC preamplifier of a Grass Model-7 Polygraph (Grass Instrument Co., Quincy, MA, USA). The animals were allowed 30 min to recover completely from anaesthesia and the viscerosomatic behavioural response, i.e. the reflex contractions of the external oblique muscle during CRD was recorded in freely moving animals. The distension protocol involved 10-min distensions at constant pressures of 0, 15, 30, 45 and 60 mmHg separated by 10-min intervals without distension. The number of abdominal muscle contractions during each distension period was used to measure the VMR.

Induction of postinflammatory colonic hypersensitivity

Rats fasted overnight were anaesthetized with isoflurane to receive an enema of trinitrobenzenesulfonic acid (TNBS) (50 mg kg−1, 0.5 mL in 25% ethanol). The rats were then returned to their home cage. TNBS induces an acute inflammatory response in the colon, which heals spontaneously within 2 to 3 weeks. Postinflammatory visceral hypersensitivity was characterized in pilot experiments showing that by day 30 post-TNBS, the rats had recovered from colitis. The colon did not show morphological signs of mucosal inflammation and tissue myeloperoxidase (MPO) activity was normal. However, the rats treated with TNBS had exaggerated VMR to CRD compared to rats that received a saline enema and did not develop colonic inflammation. In the present study, on the day of experiment the rats pretreated with TNBS underwent three CRDs at 30 mmHg, applied at 10-min intervals and the VMRs were recorded. Those rats that showed more than 50% increase in the number of abdominal contractions compared to the average value from the saline-treated rats in the pilot experiments were considered hypersensitive and were included in the study. Between 5 and 10% of the TNBS-treated animals did not meet the criteria for colonic hypersensitivity and were excluded.

Acute acetic acid-induced colonic hypersensitivity

Acute colonic hypersensitivity based on the sensitizing effect of a mild irritant was induced as described by Langlois et al.22 and Plourde et al.23 The rats were fasted overnight and dilute acetic acid (0.6%, 1.5 mL) was infused into the colon via a silastic tube (inner diameter 0.86 mm) placed in parallel with the balloon distension catheter. The number of abdominal contractions in response to CRD measured 1 h after the administration of acetic acid was increased by more than 50% compared with the responses recorded prior to treatment with acetic acid or in naïve rats. In pilot experiments we found that the hypersensitive responses to CRD remained constant for 3 h following acetic acid infusion. Colonic tissue isolated within this time interval showed no morphological abnormalities or significant changes in MPO activity.

Experimental design and drug treatment

The experiments were designed to examine the effect of tegaserod on colonic sensitivity in naïve (untreated) rats with normosensitive colons, in rats with acute acetic acid-induced colonic hypersensitivity and in rats with postinflammatory hypersensitivity. The VMRs to an initial series of CRD was recorded and a 50-min period was allowed before the administration of tegaserod or the vehicle. The effect of tegaserod on VMRs induced by a second series of CRD was investigated 10 min after intraperitoneal (i.p.) administration of tegaserod at doses of 0.1, 1 or 10 mg kg−1, or vehicle (propylene glycol). In separate experiments designed to define a possible central mechanism of action, tegaserod or the vehicle were administered directly to the brain via intracerebroventricular (i.c.v.) infusion of 10 or 100 μg kg−1 tegaserod or 5 μL propylene glycol. Colonic sensitivity was investigated 10 min after i.c.v. infusion. The receptor subtype mediating the action of tegaserod in these models was tested in rats pretreated with selective 5-HT4 antagonists GR 113808 (1–10 mg kg−1) or SB203186 (1–10 mg kg−1) administered subcutaneously (s.c.) 10 min prior to the administration of tegaserod. Each animal received a single dose of tegaserod or vehicle and was killed at the end of the experiment.

Drugs and chemicals

Tegaserod, supplied by Novartis Pharmaceuticals (East Hanover, NJ, USA), was dissolved in propylene glycol (100%) and used fresh for each experiment. GR113808 and SB203186 hydrochloride (soluble in water) were purchased from Tocris Cookson, Inc. (Ellisville, MO, USA). Isoflurane was obtained from Abbot Laboratories (North Chicago, IL, USA).

Data analysis and statistics

The VMR to CRD was evaluated as the number of abdominal muscle contractions during the 10-min distension period. Responses were measured for two consecutive series of CRD at 0, 15, 30, 45 and 60 mmHg performed 60 min apart. Data are reported as mean ± SEM for each group (n = 6 rats). Statistical significance between the VMR induced by the first and second series of CRD was assessed using two-way anova with repeated measures followed by Bonferroni post-test to compare the differences at each distension pressure. One-way anova was applied to assess the significance of differences between colorectal compliance in rats with postinflammatory colonic hypersensitivity treated with tegaserod (0.1, 1 or 10 mg kg−1 i.p.) or the vehicle. Differences were considered significant at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References

Effect of tegaserod in naïve rats

In naïve rats with normosensitive colons, the initial series of colonic distensions induced a graded VMR characterized by a pressure-dependent increase in the number of abdominal contractions. The rats were allowed to rest for 50 min and were then treated with tegaserod (0.1, 1 or 10 mg kg−1 i.p.) or the vehicle. The results indicated a dose-dependent reduction in the number of abdominal contractions (Fig. 1), showing a greater degree of inhibition with the highest dose of 10 mg kg−1 tegaserod. At a dose of 1 mg kg−1, tegaserod caused a small but significant (P = 0.008) inhibitory effect on colonic sensitivity while no effect of tegaserod was observed at a dose of 0.1 mg kg−1. Further experiments were performed to evaluate the role of 5-TH4 receptors in tegaserod-induced changes in colonic sensitivity in rats with normosensitive colon. The inhibition of the VMR induced by 1 mg kg−1 tegaserod was prevented completely in animals pretreated with the selective 5-HT4 receptor antagonist GR113808 at a dose of 3 mg kg−1 s.c. (Fig. 2).

image

Figure 1. Effect of tegaserod on visceromotor responses (VMR) to graded pressure of colorectal distension (CRD) in naïve rats. An initial series of CRD (open symbols) was applied in the absence of tegaserod followed by a second series of CRD (filled symbols) applied after intraperitoneal administration of the vehicle (A) or tegaserod at doses of 0.1 mg kg−1 (B), 1 mg kg−1 (C) or 10 mg kg−1 (D). Statistical significance of the differences between the first and the second distension series was assessed using two-way anova (P and F-values indicated for comparison). Bonferroni post-test was used to evaluate the difference between VMR to each distension pressure. Data are mean ± SEM of six experiments, **P < 0.01, ***P < 0.00, Bonferroni post-test.

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image

Figure 2. Inhibitory effect of the selective 5-HT4 receptor antagonist GR113808 on tegaserod-induced reduction of the visceromotor responses (VMR) to graded pressure of colorectal distension (CRD) in naïve rats. In all animals an initial series of CRD (open symbols) was applied prior to drug treatment, while the second series of CRD (filled symbols) was applied either after the administration of saline (vehicle for GR113808) followed by 1 mg kg−1 tegaserod (A) or after the administration of 3 mg kg−1 GR113808 followed by 1 mg kg−1 tegaserod (B). The significance of the effects of tegaserod in rats treated with the vehicle (A) or with GR113808 (B) was evaluated by two-way anova (P and F-values indicated for comparison). Bonferroni post-test was used to evaluate the difference between the VMR to each distension pressure. Data are mean ± SEM of six experiments, *P < 0.05, **P < 0.01, Bonferroni post-test.

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Effects of tegaserod in rats with acute colonic hypersensitivity

The development of acute colonic hypersensitivity in rats receiving intracolonic 0.6% acetic acid was confirmed by an exaggerated VMR to graded pressure of CRD compared with naïve control rats (Fig. 3). To investigate whether tegaserod has an inhibitory effect on the VMR in animals with heightened responsiveness to colonic stimulation, the rats were pretreated with tegaserod or the vehicle. Pretreatment with the vehicle had no significant effect on the VMR (Fig. 3A) demonstrating an identical level of colonic hypersensitivity during the first and second series of distensions. In contrast, when rats were treated with increasing doses of 0.1, 1 or 10 mg kg−1 tegaserod administered 10 min prior to the second distension series, the VMR was inhibited in a dose-dependent manner (Fig. 3B–D). At the lower doses of 0.1 or 1 mg kg−1 the VMR induced by distension pressures of 45 and 60 mmHg was reduced to a level similar to that recorded at matching pressures in naïve animals. The highest dose of 10 mg kg−1 caused a greater reduction in the number of abdominal contractions at all distension pressures, with responses being reduced below the level recorded in naïve animals. As observed in the naïve animals, there appeared to be a greater degree of inhibition of the VMR produced by higher distension pressures.

image

Figure 3. Effect of tegaserod on visceromotor responses (VMR) to graded pressure of colorectal distension (CRD) in rats with acute acetic acid-induced colonic hypersensitivity. In all animals 0.6% acetic acid was infused into the colon to induce hypersensitivity and a series of CRD was applied 60 min after acetic acid infusion. Rats in the separate treatment groups received the vehicle (A) or tegaserod at doses of 0.1 mg kg−1 (B), 1 mg kg−1 (C) or 10 mg kg−1 (D) 10-min prior to initiation of a second series of CRD. Statistical significance of the difference between the VMR induced prior (open symbols) and after (filled symbols) administration of tegaserod or vehicle was evaluated using two-way anova (P and F-values indicated for comparison. Bonferroni post-test was used to evaluate the difference between the VMR to each distension pressure. Data are mean ± SEM of six experiments, *P < 0.05, ***P < 0.001, Bonferroni post-test. The VMR in naïve rats with normosensitive colons are presented (dashed line) to serve as a reference to control.

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Experiments using the 5-HT4 receptor antagonist GR113808 were performed to investigate the role of 5-HT4 receptors in the inhibition of colonic hypersensitivity. Pretreatment with GR113808 at a dose of 3 mg kg−1, which completely inhibited the effect of tegaserod in naïve rats, did not cause a complete reversal of the effect induced by 1 mg kg−1 tegaserod (Table 1). These results were confirmed through the examination of a different 5-HT4 receptor antagonist SB203186 (1 or 10 mg kg−1 s.c.), which also induced only partial reversal of the effect of tegaserod (Fig. 4).

Table 1.  VMR to CRD in rats with acetic acid-induced colonic hypersensitivity. Effects of tegaserod (1 mg kg−1 i.p.) administered in rats pretreated with vehicle (saline, s.c.) or GR113808 (3 mg kg−1, s.c.). The VMR in untreated rats with acetic acid-induced colonic hypersensitivity is presented as a reference control
TreatmentVMR to CRD at increasing intraluminal pressure
0 mmHg15 mmHg30 mmHg45 mmHg60 mmHg
  1. VMR, visceromotor responses; CRD, colorectal distension. Data are mean ± SEM. Two-way anova comparing the effect of tegaserod in vehicle or GR113808 treated rats: P = 0.002, F = 11.2. Bonferroni post-test for each distension pressure: P > 0.05 (ns).

Vehicle & tegaserod (n = 4)2.0 ± 0.42.5 ± 0.47.8 ± 0.812.0 ± 0.615.2 ± 0.3
GR113808 & tegaserod (n = 4)3.0 ± 0.34.3 ± 0.98.5 ± 0.614.3 ± 0.516.8 ± 1.4
No treatment (n = 6)2.8 ± 0.510.0 ± 1.415.7 ± 0.921.2 ± 1.625.7 ± 2.5
image

Figure 4. Effect of the selective 5-HT4 receptor antagonist SB203186 on the visceromotor responses (VMR) to graded pressure of colorectal distension (CRD) in rats with acute acetic acid-induced colonic hypersensitivity pretreated with 1 mg kg−1 tegaserod. All animals received intracolonic infusion of 0.6% acetic acid and following a 50-min period were treated with 1 mg kg−1 tegaserod. An initial series of CRD (open symbols) was applied 10 min after the administration of tegaserod and a second series of CRD (filled symbols) was applied either after the administration of saline (vehicle for SB203186) (A) or after the administration of SB203186 at doses of 1 mg kg−1 (B) or 10 mg kg−1 (C). The significance of the effects of SB203186 or vehicle on VMR in rats treated with tegaserod was evaluated by comparing the data from the first and the second distension series using two-way anova (P and F-values indicated for comparison). Bonferroni post-test was used to evaluate differences at each distension pressure. Data are mean ± SEM of six experiments, *P < 0.05, **P < 0.01, Bonferroni post-test. The VMR in rats with acetic acid-induced colonic hypersensitivity not treated with tegaserod are presented (dashed line) to serve as a reference to control.

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To define whether tegaserod may act via central mechanisms to reduce colonic hypersensitivity, experiments were performed with tegaserod applied directly to the brain of rats implanted with i.c.v. cannulae. Colonic hypersensitivity induced by acetic acid was not affected by i.c.v. administration of either the vehicle or tegaserod at doses of 10 or 100 μg kg−1 (Fig. 5).

image

Figure 5. Effect of tegaserod, administered via intracerebroventricular (i.c.v.) infusion, on visceromotor responses (VMR) to graded pressure of colorectal distension (CRD) in rats with acute acetic acid-induced colonic hypersensitivity. In all animals 0.6% acetic acid was infused into the colon to induce hypersensitivity and a series of CRD (open symbols) was performed 60 min after acetic acid infusion. Rats in the separate treatment groups received the vehicle (5 μL propylene glycol) (A) or tegaserod at doses 10 μg kg−1 (B) or 100 μg kg−1 (C) 10 min prior to initiation of a second series of CRD (filled symbols). Differences between the VMR induced prior and after administration of tegaserod or vehicle showed no statistical significance when evaluated using two-way anova (P and F-values indicated). Data are mean ± SEM of six experiments. The VMR in naïve rats with normosensitive colons are presented (dashed line) to serve as a reference to control.

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Effects of tegaserod in rats with postinflammatory colonic hypersensitivity

Postinflammatory colonic hypersensitivity was verified by the number of abdominal contractions induced by graded CRD in rats 30 days after the induction of TNBS colitis. Rats, which qualified as hypersensitive (see Material and methods) were pretreated with tegaserod or vehicle. While the vehicle had no significant effect (Fig. 6A), tegaserod (0.1, 1 or 10 mg kg−1) produced a dose-dependent inhibition of the VMR (Fig. 6B–D). Similar to our observation in rats with acute acetic acid-induced hypersensitivity, the lower doses of 0.1 or 1 mg kg−1 tegaserod reduced VMR to a level similar to that observed in naïve animals. The effect of the highest dose of 10 mg kg−1 was characterized by a more pronounced inhibition of the VMR at all distension pressures.

image

Figure 6. Effect of tegaserod on visceromotor responses (VMR) to graded pressure of colorectal distension (CRD) in rats with postinflammatory colonic hypersensitivity. Acute colitis was induced by a trinitrobenzenesulfonic acid (TNBS) enema and the rats were allowed to recover from the inflammatory insult. Colorectal hypersensitivity was tested on day 30 post-TNBS by measuring the VMR to a series of CRD. Following the first series of CRD (open symbols) the rats in the separate treatment groups received either the vehicle (A) or tegaserod at doses of 0.1 mg kg−1 (B), 1 mg kg−1 (C) or 10 mg kg−1 (D) 10 min prior to initiation of a second series of CRD (filled symbols). Statistical significance of the differences between the VMR induced prior and after administration of tegaserod or vehicle was evaluated using two-way anova (P and F-values indicated for comparison). Bonferroni post-test was used to evaluate the difference at each distension pressure. Data are mean ± SEM of six experiments, **P < 0.01, ***P < 0.001, Bonferroni post-test. The VMR in naïve rats with normosensitive colons are presented (dashed line) to serve as a reference to control.

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The involvement of 5-HT4 receptors in the effect of tegaserod in rats with postinflammatory colonic hypersensitivity was studied in animals treated with increasing doses of 0.3, 1, 3 or 10 mg kg−1 of the selective 5-HT4 receptor antagonist GR113808. The results showed that even the highest dose of 10 mg kg−1 GR113808 was insufficient to completely reverse the inhibitory effect of 1 mg kg−1 tegaserod (Fig. 7).

image

Figure 7. Effect of the selective 5-HT4 receptor antagonist GR113808 on the visceromotor responses (VMR) to graded pressure of colorectal distension (CRD) in rats with postinflammatory colonic hypersensitivity treated with 1 mg kg−1 tegaserod. Postinflammatory colonic hypersensitivity was confirmed by measuring the VMR to an initial series of CRD (open symbols). The rats were pretreated with GR113808 at doses of 0.3 mg kg−1 (A), 1 mg kg−1 (B), 3 mg kg−1 (C) or 10 mg kg−1 (D) and 1 mg kg−1 tegaserod was administered 10 min after administration of the antagonist or the vehicle. A second series of CRD (filled symbols) was applied 10 min after the administration of tegaserod. The ability of GR113808 to restore the VMR to CRD in rats with postinflammatory colonic hypersensitivity treated with tegaserod was evaluated by comparing the data from the first and the second distension series using two-way anova (P and F values indicated for comparison). Bonferroni post-test was used to evaluate the difference at each distension pressure. Data are mean ± SEM of six experiments, *P < 0.05, **P < 0.01, Bonferroni post-test.

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Effects of tegaserod on colonic compliance

A final series of experiments was performed to elucidate whether the inhibition of colonic sensitivity by tegaserod was caused by changes in the compliance of the colon. The volume–pressure relationship during CRD at 15, 30, 45 and 60 mmHg was investigated in rats with postinflammatory hypersensitivity treated with increasing doses of 0.1, 1 or 10 mg kg−1 tegaserod. Changes in volume were measured for each CRD pressure and the results for each treatment group are summarized in Table 2. The data indicate that tegaserod did not induce any significant changes in colonic compliance.

Table 2.  Effects of tegaserod or the vehicle on colorectal compliance in rats with post-inflammatory colonic hypersensitivity. Colorectal compliance was assessed as the volume response to colorectal distension at 15 mmHg increments in balloon pressures ranging from 15 to 60 mmHg.
TreatmentVolume (mL) at increasing intraluminal pressure
15 mmHg30 mmHg45 mmHg60 mmHg
  1. Data are mean ± SEM. The differences between the treatment groups were not significant for each level of colorectal pressure (one-way anova, P > 0.05).

Tegaserod 0.1 mg kg−1 (n = 4)1.5 ± 0.32.9 ± 0.43.7 ± 0.34.3 ± 0.3
Tegaserod 1 mg kg−1 (n = 6)1.8 ± 0.43.7 ± 0.54.7 ± 0.75.2 ± 0.7
Tegaserod 10 mg kg−1 (n = 4)2.0 ± 0.42.2 ± 0.93.7 ± 0.64.1 ± 0.6
Vehicle (n = 4)1.6 ± 0.32.3 ± 0.33.9 ± 0.34.6 ± 0.3

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References

5-Hydroxytryptamine, released by enterochromaffin cells in response to intraluminal stimulation, is the signalling molecule which initiates motor, secretory and sensory reflexes in the gut. In addition to direct effects on epithelial and smooth muscle cells, 5-HT regulates reflex activity via different 5-HT receptor subtypes expressed on afferent neurones. In the enteric nervous system, both sensory and motor neurones express a variety of 5-HT receptor subtypes including 5-HT1P, 5-HT3 and 5-HT411,24–27 while in the extrinsic afferents that signal to the CNS, the 5-HT3 receptor is well documented.28–30 Recent investigations suggest that changes in 5-HT signalling play a key role in IBS. An increased number of enterochromaffin cells might be one mechanism contributing to the symptoms in patients with postinfectious IBS.31 However, another investigation32 showed that colonic mucosal tryptophan hydroxylase 1 messenger RNA, serotonin transporter messenger RNA and serotonin transporter immunoreactivity were all significantly reduced in IBS patients, while the population of enterochromaffin cells was unchanged. Although the mechanisms remain unclear, IBS symptoms are related to deviations from normal GI motility and/or visceral sensitivity,33 implying that specific targeting of altered 5-HT signalling may prove effective in the treatment of IBS.

Based on the alteration in bowel habits, IBS is classified primarily as constipation, diarrhoea or alternating forms, with each subclass of patients demanding different therapeutic intervention to relieve the overall symptoms. Tegaserod (Zelnorm®, Novartis Pharmaceuticals, Basel, Switzerland) is currently approved by the FDA for the treatment of IBS in women whose primary bowel complaint is constipation17,18 and for the treatment of men and women with chronic constipation.19 Tegaserod mimics the physiological effects of 5-HT acting as a partial agonist of 5-HT4 receptors and promoting relief of the symptoms. The therapeutic effect is likely to be associated with stimulation of GI motor function and epithelial secretion induced by activation of 5-HT4 receptors on enterocytes, enterochromaffin cells, neurones and smooth muscle cells.17 Indeed, tegaserod was found to initiate the peristaltic reflex in isolated human, rat and guinea-pig intestine13,14 and stimulate GI motility and impaired gastric emptying in conscious dogs.34,35 More recent studies, however, suggest that 5-HT4 receptors are located on presynaptic terminals of cholinergic enteric neurones.26,36–38 and not on the mucosal terminals of intrinsic sensory neurones that first receive 5-HT released from enterochromaffin cells.39 These findings imply that 5-HT4 receptor activation causes facilitation rather than an initiation of enteric reflexes. Human studies showed that tegaserod accelerates GI transit in healthy adults40 and in patients with IBS-C.41 Surprisingly, although hyperalgesia to visceral stimuli is known as the hallmark of IBS, the relief of abdominal discomfort and pain in IBS patients is considered mainly a consequence of improvement in motility and secretion with little attention paid to the direct mechanisms through which tegaserod may reduce visceral sensitivity. An investigation42 of colorectal sensitivity and the mechanical parameters of the gut wall in IBS patients implied that visceral hypersensitivity is related to alterations in the nervous system rather than biomechanical parameters of the gut wall, suggesting that nociceptive pathways should be the main target for treatment of IBS pain. Studies in the cat have indicated that in the rectum, 5-HT4 receptors modulate the sensitivity of intramural mechanoreceptive afferents.43 With regard to these findings, few studies have found that tegaserod reduces rectal sensitivity in rats44 and humans.45 The present study provides further evidence showing that while 1 mg kg−1 tegaserod caused only a small inhibition of colorectal sensitivity to distension in naïve rats with normosensitive colons, it normalized the sensation to CRD in two models of visceral hypersensitivity. Supporting evidence for an antihyperalgesic effect of tegaserod in conscious rats was recently provided by Sun and Luo,46 who demonstrated that tegaserod caused a reduction in the number of Fos-labeled neurones in the lumbosacral spinal cord and an inhibition of substance P expression induced by colonic inflammation. Aiming to establish the mechanism of action of tegaserod at a dose resembling that used clinically, we found that in rats with a normosensitive colon, the small inhibitory effect of tegaserod at 1 mg kg−1 was completely reversed by the selective 5-HT4 receptor antagonist GR 113808. These findings suggest that 5-HT4 receptor-mediated mechanisms are responsible for the decrease in colonic sensitivity induced by the 1 mg kg−1 dose of tegaserod; however, these results do not exclude the possibility that other mechanisms may be involved in the inhibition of colonic sensitivity observed with the higher dose of tegaserod (10 mg kg−1) in rats with a normosensitive colon. In rats with acute acetic acid-induced or chronic postinflammatory colonic hypersensitivity, the inhibitory effect of tegaserod was more pronounced than that observed in naïve rats. There were two important differences between the results obtained in the hypersensitive and naïve animals. First, in the rats with hypersensitive colons, whether acute or chronic, there was an apparent leftward shift in the dose–response to tegaserod, with a significant inhibition of the VMR observed at 0.1 mg kg−1 tegaserod, a dose that was without effect in the naïve, non-sensitized animals. This observation suggests an increased role for tegaserod-sensitive 5-HT receptors in the visceral hypersensitivity displayed in these rat models. Considering the growing evidence for a disturbance in serotonin signalling in IBS, this correlation may provide some indirect support for the relevance of the models to the disease. Second, in the rats with colonic hypersensitivity, the selective 5-HT4 receptor antagonists GR113808 or SB203186 only partially reversed the effect of tegaserod. This suggests that a tegaserod-sensitive receptor, different from the 5-HT4 receptor subtype, may be involved in the inhibition of colorectal sensitivity in rats with hypersensitive colons. Indeed, recent findings indicating that tegaserod antagonizes 5-HT2B receptors with an affinity similar to that for its agonism of 5-HT4 receptors47 may provide some insight into the differences between colorectal sensory pathways in normal and sensitized rats. It is interesting to note that activation of 5-HT2B receptors has been shown to enhance NMDA receptor responses in frog spinal cord neurones,48 which are important for the development of peripheral sensitization and visceral hyperalgesia.49 Further experiments are warranted to assess whether 5-HT2B receptor antagonist activity plays a role in the antihyperalgesic effect of tegaserod demonstrated in the current study.

Although 5-HT-receptors may modulate visceral sensation and pain perception at both peripheral and central sites, the prokinetic and antinociceptive effects of tegaserod have been considered restricted to its action on peripheral 5-HT4 receptors.17 The peripheral site of action of tegaserod was confirmed in our study by the lack of significant inhibition of the VMR to CRD following central administration of tegaserod. We also demonstrated that tegaserod, administered intraperitoneally at doses that reduced colonic sensitivity to distension, had no effect on colonic compliance. Together these results provide indirect evidence that tegaserod may act on primary afferents to reduce colonic sensitivity. Indeed, although no effect of tegaserod or another 5-HT4 receptor agonist, prucalopride, was observed upon rat jejunal afferents in vivo,50 evidence from in vitro studies of rat colonic afferent nerves has provided strong support to this hypothesis.51 It is unclear at this stage how activation of 5-HT4 receptors on spinal afferent nerves leads to an analgesic effect. The action of tegaserod as an agonist of 5-HT4 receptors appears to be in conflict with evidence from studies in isolated rat dorsal root ganglion showing a facilitation of tetrodotoxin-resistant sodium currents in a subpopulation of neurones.52,53 A possible explanation for this apparent conflict may be found in the non-specific nature of in vitro experiments, which do not focus on cells innervating the GI tract. Indeed the 5-HT4 receptor-mediated stimulation of TTX-resistant sodium currents may be specific to thermosensitive and not mechanosensitive sensory nerves.53 Furthermore, the role of TTX-resistant sodium currents in visceral pain processing is unknown and it is possible that an excitation of the afferent terminal may make the neurone refractory to further stimulation, resulting in reduced signalling to the spinal cord and analgesia. Clearly there is a need for further investigation of the precise mechanism by which the analgesic effect we observe occurs.

In conclusion, our findings are the first to demonstrate that the postinflammatory changes in a rat model of TNBS-induced colitis include a significant increase in colorectal sensitivity, similar to the acute hypersensitivity induced by a chemical irritant. Furthermore, colorectal hypersensitivity was inhibited by tegaserod via a peripheral site of action. Our data confirm the visceral analgesic property of 5-HT4 receptor agonists and demonstrate a key role for 5-HT4 receptors in colonic sensory signalling. The results support the assumption that hypersensitivity to colorectal distension, which is a major factor in IBS, could be normalized by tegaserod.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. References
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