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.