Effects of alosetron on spontaneous migrating motor complexes in murine small and large bowel in vitro

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, 89557-0046

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Alosetron (Lotronex) is a serotonin subtype 3 (5-HT₃) receptor antagonist that alleviates symptoms of irritable bowel syndrome (IBS) in female patients. Alosetron may act centrally, involve the alteration of ascending pain sensation, or modulate peristaltic, secretory, or sensory function. To investigate further the mechanisms underlying its action and gender selectivity we recorded the effect of increasing concentrations of alosetron or ondansetron on spontaneous migrating motor complexes (MMCs) from isolated terminal ileum or colon from C57BL/6 mice. Both antagonists inhibited MMC frequency before affects on duration or amplitude. The threshold of inhibition for alosetron was 100-fold less in small intestine from females (20 nM) than from males. The opposite effect of gender was observed with ondansetron in the colon. All MMCs were abolished by either drug at 10 µM. Our results demonstrate that alosetron selectively inhibits MMC frequency in isolated preparations of murine bowel. Because contractile events in the ileum correlate with symptoms of IBS in humans, the gender selectivity of alosetron may be caused by a direct action within the small intestine.

ondansetron; serotonin; enteric nervous system; motility

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

IRRITABLE BOWEL SYNDROME (IBS) is a functional gut disorder characterized by abdominal pain, discomfort, and altered bowel function (41). The syndrome affects ~15% of the population (9, 17) and is characterized by a female predominance of ~70% (35, 47). Although the pathophysiology remains unclear, it is most likely multifactorial. Dysmotility of both the large and small bowel of IBS patients has been described (12, 30, 31, 45). Inappropriate secretory activity is also a feature of IBS (2). Increasingly, however, visceral hypersensitivity has been implicated in IBS (16, 36, 49). Unfortunately, current therapies for IBS, including dietary fiber, opioids for diarrhea, smooth muscle relaxants, and psychotropic and psychological agents, are not selective and do not give consistent relief of symptoms (8).

Several lines of evidence support the use of serotonin subtype 3 (5-HT₃) receptor antagonists for the treatment of IBS. Originally developed for suppressing chemotherapy-induced nausea, 5-HT₃ receptor antagonists attenuate 5-HT-induced signaling in visceral afferents (23). Intestinal distension in animals leads to activation of reflex behavior that is used to model visceral pain. 5-HT₃ antagonists potently suppress these reflexes (34). These compounds also suppress gut motility in IBS patients, particularly in the large bowel (26, 38, 46). Additional therapeutic effects of 5-HT₃ receptor antagonists may be a consequence of their antianxiolytic properties (14, 28).

Numerous studies have investigated the effects of 5-HT₃ receptor antagonists on human bowel function in health and disease. Rectal desensitization by granisetron in IBS patients occurred in the absence of effect on rectal tone (38). In addition, postprandial motility was inhibited but distension-induced motility was unaffected. On the other hand, ondansetron significantly slowed whole gut transit in healthy men (22) but did not influence any index of colonic function in five patients with diarrhea-predominant IBS (25). A single intravenous dose of ondansetron failed to affect rectal or gastric sensitivity in IBS, although compliance of the colon was increased (51). A gender selectivity of action for ondansetron has not been reported.

Recent studies demonstrated that alosetron (Lotronex), a novel and highly potent 5-HT₃ antagonist, gives relief of symptoms selectively in female patients with IBS, although in both studies the number of male patients included was low (1, 10). A larger study, conducted entirely in women, has demonstrated 1 mg b.d. as the most effective dose for improving abdominal pain and discomfort, urgency, and stool frequency and consistency (11). Alosetron did not change the perception of colonic distension, yet it significantly increased the compliance of the colonic wall to distension in IBS patients (15). In carcinoid diarrhea, alosetron did not affect gastric emptying or small bowel transit but significantly slowed proximal colon emptying, the most abnormal physiological parameter of this disease (43, 48). In both healthy volunteers and IBS patients, alosetron delayed colonic transit in the absence of an effect on small bowel transit (26).

To investigate whether alosetron has a direct action on the gut and to determine whether this is a site of gender selectivity, we examined the effects of alosetron on spontaneous migrating motor complexes (MMCs) in the terminal ileum and colon of the C57BL/6 mouse in vitro. These events recorded from the murine bowel in vitro share pharmacological characteristics with phase III activity of the human MMC in vivo (20). Our earlier work (5) revealed no gender differences in frequency, amplitude, or duration of MMCs for either tissue. We therefore recorded the effects of alosetron and ondansetron on spontaneous MMCs in tissue from both males and females.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Measurement of contractile activity. Nonfasted C57BL/6N mice (Simonsen Labs, Gilroy, CA) of either gender, 7-12 wk of age, were killed by cervical dislocation. The phase of the ovarian cycle in female mice was not determined. The entire colon and, separately, the terminal ileum of a comparable length (6-7 cm), were removed into modified Krebs solution. The luminal contents were flushed gently, a stainless steel rod (1.0-mm diameter) was inserted into the lumen of the bowel, and the tissue was secured to mounting posts fixed securely to the bottom of the bath using rubber O rings. The bath contained prewarmed Krebs solution at 37.0 ± 0.5°C and was gassed (3% CO₂-97% O₂ vol/vol) throughout the experiment. The Krebs solution was replaced approximately every hour until activity became coordinated.

Two stainless steel clips (micro-serrefines; Fine Science Tools, Foster City, CA) were attached to the tissue within 1.5-2 cm of the oral and anal ends. Suture silk was used to connect each clip to a force transducer (model TST125C; Biopac Systems, Santa Barbara, CA). Initial tension was routinely set to <5 mN to minimize local reflex stimulation of the bowel. Tension was monitored continuously using an MP100 interface and recorded on a PC running Acqknowledge software 3.2.6 (Biopac Systems).

Solutions and drugs. The composition of the Krebs solution was (in mM) 120.35 NaCl, 5.9 KCl, 15.5 NaHCO_3, 1.2 NaH₂PO₄, 1.2 MgSO₄, 2.5 CaCl₂, and 11.5 glucose. The solution was gassed continuously with a mixture of 3% CO₂-97% O₂ (vol/vol) to give a final pH of 7.3-7.4.

Stock solutions of 1 mM alosetron and ondansetron were made up in distilled water, aliquoted, and stored frozen. Dilution series were prepared daily and added sequentially to the bath.

Analysis of data and statistical methods. Contractile activity was analyzed from computer traces. Frequency and duration of coordinated peaks were assessed from the anal trace using established experimental parameters (5, 19). The anal trace was selected because peaks were more uniform and were more stable with increasing time ex vivo compared with the oral trace (see Figs. 2-4). The duration of each contraction was determined as the time between the half-maximal amplitude points on the rising and falling phases. The interval between contractions was determined by the time between the half-maximal amplitude points on the rising phase of consecutive contractions (Fig. 1). Amplitude, duration, and frequency were expressed as percentage of control values for statistical analysis. Control values were obtained from a minimum of five peaks immediately preceding the application of antagonist to the bath. Data are presented as means ± SE. Log IC₅₀ and Hill coefficient values were obtained using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). Statistical significance was determined using Student's t-test (paired or unpaired as appropriate) or Mann-Whitney rank correlations.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Migrating motor complex (MMC) parameter analysis. Amplitude was defined as the difference between precomplex tension and maximum peak height. Duration was measured between the half-maximum amplitude points of the rising and falling phases of the complex. Interval was measured between the half-maximum amplitude points of the rising phase of consecutive complexes. Measurements were made only in propagated complexes in anal traces.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Stability of MMC activity in large and small bowel. To demonstrate that stability of MMC activity exceeded the duration of the antagonist experiments, preparations were monitored continuously in the absence of any manipulation (Figs. 2A and 3A). Although duration and frequency remained constant, there was a tendency for the amplitude to wane. This effect was significant in oral traces but did not reach significance in anal traces (data not shown). All measurements of effects of antagonists were measured using recording of activity in anal traces.

View larger version (54K): [in this window] [in a new window]   Fig. 2.   Effect of alosetron on MMC activity in male colon. Once established, MMC activity was sustained in colonic preparations for >3 h (A). Sequential addition of alosetron caused a pronounced slowing of MMC frequency (B).

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Effect of alosetron on MMC activity in male ileum. MMC activity in the ileum could be maintained for >4 h (A). Increasing concentrations of alosetron caused a slowing of MMC frequency (B).

Effects of alosetron on spontaneous MMC activity in large and small bowel. Alosetron dose-dependently decreased the frequency of MMC activity in both the large and small bowel (Figs. 2B and 3B). The effect on frequency occurred before effects on either amplitude or duration. Although there was no effect of gender on colonic frequency, the small intestine from females showed a significantly greater sensitivity than tissue from males (Fig. 4). There was a 100-fold difference in threshold concentrations required to slow MMC activity in females and males (20 nM and 2 µM, respectively). MMC activity in both tissues was abolished by 10 µM alosetron. In both intestinal regions, there was a tendency for reduced amplitude in the presence of 2 µM alosetron, but this was only significant in the small bowel (Fig. 4). With increasing doses of alosetron, the duration of each MMC appeared to decrease in the male colon. In contrast, there was a significant increase in the duration of MMCs in female tissue. No effect of gender on duration of MMC in the small intestine was observed.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of alosetron on MMC parameters. Alosetron showed a dose-dependent decrease in frequency of MMC activity in both the small and large bowel. No effect of gender was observed in the colon. However, the threshold for slowing of MMCs occurred at a 100-fold lower dose in female compared with male small intestine (20 nM and 2 µM, respectively). All MMC activity was abolished by 105 M alosetron. *P < 0.05, **P < 0.01 compared with control activity. SI, small intestine.

Effects of ondansetron on spontaneous MMC activity in large and small bowel. The effects of ondansetron on spontaneous MMC activity in both the small and large bowel of the mouse were qualitatively indistinguishable from the effects of alosetron (Fig. 5). As was observed for alosetron, the threshold for effect of ondansetron on MMC frequency occurred before an effect on either amplitude or duration. No effect of gender was observed on the decrease in MMC frequency in the small intestine. At low concentrations of ondansetron (<5 nM), we observed a small but significant increase in MMC frequency selectively in male small intestine preparations (Fig. 6). In the colon, however, we observed a large gender difference in the response to ondansetron. The threshold for inhibition of MMC frequency was 100-fold lower in the male colon compared with the female colon (Fig. 6; 20 nM and 2 µM, respectively).

View larger version (54K): [in this window] [in a new window]   Fig. 5.   Effect of ondansetron on MMC activity in male ileum and colon. In both ileal (A) and colonic (B) preparations ondansetron decreased MMC frequency in a manner qualitatively indistinguishable from the effect of alosetron.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of ondansetron on MMC parameters. Ondansetron showed a dose-dependent decrease in frequency of MMC activity in both the small and large bowel. No effect of gender was observed in the small intestine. In the colon, and the reverse of the selectivity of alosetron, the threshold at which MMC frequency was inhibited in the male occurred at a 100-fold lower dose then in the female (20 nM and 2 µM, respectively). All MMC activity was abolished by 105 M ondansetron. *P < 0.05, **P < 0.01 compared with control activity.

The effects of ondansetron on amplitude were not affected by gender in either tissue. In contrast, and similar to the trend observed with alosetron, ondansetron appeared to reduce the duration of MMC activity in the colon selectively in male tissue. This effect was not observed in the small intestine.

Gender selectivity of effect of 5-HT₃ antagonists on MMC activity in large and small bowel. To determine IC₅₀ values for each drug, the inhibition of MMC frequency was plotted against drug concentration (Fig. 7). The values ranged from 0.1 to 1.5 µM (Table 1). No gender differences were apparent between any drugs or tissues, with the exception that ondansetron was significantly more potent in the male compared with the female colon. Although no differences in IC₅₀ values for alosetron were observed (Table 1), the inhibitory effect in the female small intestine was significantly greater than that in male tissue (Fig. 7). This was reflected in the large difference between the Hill coefficients for these tissues (Table 2).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Inhibition of MMC frequency by serotonin subtype 3 receptor (5-HT3) antagonists. Although effects of alosetron were indistinguishable in colonic preparations from both genders, the threshold for effects of alosetron was lower in small intestine preparation from female mice compared with males. In contrast, gender had no effect on the efficacy of ondansetron in the small intestine preparations from both genders but was significantly move efficacious in the male colon. *P < 0.05, **P < 0.01 compared with equivalent gender group.

View this table: [in this window] [in a new window]   Table 1.   Effect of 5-HT3 antagonists on MMC frequency

View this table: [in this window] [in a new window]   Table 2.   Hill coefficient values for inhibition of MMC frequency by 5-HT3 receptor antagonists

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Recent clinical trials have demonstrated that the 5-HT₃ receptor antagonist alosetron (Lotronex) relieves symptoms of IBS and that this occurs selectively in female patients (1, 10, 11). However, the sites of action and mechanisms that underlie both the action and gender selectivity of alosetron remain uncertain.

In this study, we found that alosetron and ondansetron had a direct, selective inhibitory effect on the frequency of spontaneous MMC in isolated small and large intestine from C57BL/6 mice. In addition, with the exception of human studies, this is the first study to demonstrate a gender difference in bowel response to 5-HT₃ antagonism.

Gender selectivity of action. Our previous work (5) demonstrated that gender had no effect on the parameters of MMC activity. We therefore made comparisons between drugs, genders, and bowel regions. A striking and reciprocal effect of gender was observed for the two drugs. The threshold for effects of alosetron in the female small intestine (20 nM) was 100-fold lower than for male small intestine. There was no effect of gender in the colon. Ondansetron, on the other hand, showed no gender effect in the small intestine, but the threshold in the male colon (20 nM) was 100-fold lower than in the female colon. The threshold for effect and IC₅₀ values, ranging from 0.1 to 1.5 µM, strongly suggest that 5-HT₃ receptor activation regulates the frequency of MMCs in the murine bowel.

The therapeutic effect of alosetron in female IBS patients can be observed at 1 (11) and 2 (1) mg b.d. Pharmacokinetic studies have demonstrated that the peak plasma concentration after oral administration of 2 mg is between 10 and 20 ng/ml (approximating 30-60 nM) (24). However, because the alosetron shows a large first-pass effect and the gastrointestinal tract may be the site of this effect, the local concentration within the gastrointestinal tract may be much higher. The pharmacokinetic profiles of ondansetron and alosetron are similar. Systemic exposure after oral or intravenous dosing with ondansetron was significantly higher in women than in men (39). A higher systemic exposure in women is also observed for alosetron (24). Three mechanisms may be responsible. First, women cleared the drug more slowly than men. Second, women showed higher bioavailability, presumably because of reduced first-pass uptake of drug. Third, the weight-adjusted volume of distribution was smaller in women. However, increasing the dose of alosetron in male IBS patients was ineffective in alleviating symptoms, suggesting that factors other than bioavailability are responsible for the gender selectivity of alosetron efficacy (10).

It is therefore currently unclear why alosetron shows efficacy selectively in female IBS patients. This is the first study to demonstrate a gender difference in the effects of 5-HT₃ antagonists in an animal model of gut physiology. The principal gender difference in response to alosetron was a 100-fold lower threshold for decreasing frequency of ileal MMC activity in female mice. Because the distal small intestine has been implicated as the origin of symptoms in some patients with IBS (30, 31), it is tempting to speculate that the gender selectivity of alosetron is based on a direct effect on the female small intestine. Alosetron did not affect ileal transit in clinical studies, suggesting that effects on ileal motility are not the mechanism of its therapeutic efficacy (26). Our data also show that there are gender differences between individual 5-HT₃ antagonists in this assay. One explanation for this effect is a differential distribution of 5-HT₃ receptor subtypes. Recently the pharmacological properties of a shorter splice variant of the murine 5-HT₃ receptor have been reported (4). Although most drugs exhibited almost identical properties at the native and variant receptor, ondansetron was more potent at the shorter splice variant. Whether gender differences in the expression patterns of the full-length 5-HT₃ receptor and the splice variant exist in either mice or humans is currently unknown.

Extensive preclinical pharmacology has demonstrated the potency and selectivity of alosetron. The pK_i values for the rat (9.8) and human (9.4) 5-HT₃ receptors are very similar, with no other receptor demonstrating binding with a pK_i >6 (13). The pK_B for alosetron on rat 5-HT₃ receptors was 9.8 (13), whereas in the same assay ondansetron had a pK_B of 8.6 (7). Unfortunately, interspecies comparisons must be interpreted with caution because large species difference in 5-HT₃ receptor pharmacology have been reported. In particular, binding of antagonists at the guinea pig 5-HT₃ receptor appears to require several orders of magnitude greater concentrations of drug compared with binding at the rat receptor (6). Although the binding affinity for the murine 5-HT₃ receptor has not been assessed directly, in vivo and tissue culture studies have shown that its pharmacological properties are similar to those of the rat receptor (4, 32, 50). In vivo studies have shown that ondansetron (3.2 mg/kg) does not disturb normal colonic transit in the rat (29). However, lower doses (0.1-1 mg/kg) dose dependently increased whole gut transit time in male mice (37). Similarly, the concentrations required to demonstrate the antianxiolytic effects of ondansetron are similar in rats and mice (28). Together, these results strongly suggest that the mouse 5-HT₃ receptor more closely resembles that of the rat than that of the guinea pig.

Possible enteric sites and modes of drug action. One of the original criteria for diagnosing IBS is abdominal pain relieved by defecation (33). It has been shown that pain associates temporally with eating but not defecation in IBS (40). A striking correlation between prolonged propagating contractions (PPCs) and abdominal pain was observed in IBS patients (30). Interestingly, this study also reported that PPCs were also associated with postprandial pain, similar to that regularly experienced in 44% of IBS patients. Together, these studies suggest that in some patients the ileum may be the site of origin of symptoms of IBS.

Although 5-HT₃ receptors have central, peripheral, and enteric locations, our data suggest that alosetron and ondansetron have direct actions on both the small and large intestine. Recent studies also showed that 5-HT₃ receptor antagonists can act directly on the large intestine, because LY-278584 slows the propulsion of pellets in the isolated guinea pig distal colon (27) and ondansetron reduces MMC activity in the isolated colon of nonaffected littermates of the piebald lethal mouse (18).

5-HT₃ receptor antagonists could affect neuro-neuronal transmission down the bowel, because some descending interneurons in the mouse are serotonergic and ligand-gated 5-HT₃ receptors have been demonstrated on different functional classes of enteric neurons, at least in the guinea pig (42, 44). However, both alosetron and ondansetron reduced MMC frequency more effectively than complex amplitude, suggesting that 5-HT₃ receptors were more likely to be involved in the pacemaker responsible for the generation of the MMC than in the conduction of MMCs down the bowel. This latter phenomenon is critically dependent on cholinergic neurotransmission (5, 19). Alosetron and ondansetron could be blocking 5-HT₃ receptors on the mucosal processes of intrinsic sensory neurons, which are readily activated by mucosally applied 5-HT (3). The generation of the MMC may therefore be dependent on spontaneous release of serotonin from enterochromaffin (EC) cells in the mucosa, which contain over 90% of the body's serotonin, that excites intrinsic sensory neurons (44). EC cells themselves express 5-HT₃ receptors that appear to enhance 5-HT release (21). 5-HT₃ antagonism at this site would therefore reduce the frequency of 5-HT release and may in turn reduce MMC frequency.

In conclusion, our results show clear gender differences in the action of both alosetron and ondansetron on spontaneous migrating motor activity in the murine bowel. Compared with the male small intestine, alosetron had a significantly lower threshold for effects in the female small intestine. The ileum has been identified as a site of origin of symptoms in IBS in humans. Gender differences in the distribution of the 5-HT₃ receptor or its splice variants have the potential to contribute to the gender selectivity of action of alosetron in IBS.

	ACKNOWLEDGEMENTS

Support for this project was provided by the National Institute of Diabetes and Digestive and Kidney Diseases (DK-10793 to T. K. Smith, PO1-DK-41315 to K. M. Sanders and T. K. Smith) and by Glaxo Pharmaceuticals (K. M. Sanders and T. K. Smith).

	FOOTNOTES

Preliminary results of this study were presented at the annual meeting of the American Gastroenterological Society, San Diego, CA, 2000, and at the International Society for Autonomic Neuroscience, London, UK, 2000.

Address for reprint requests and other correspondence: T. K. Smith, Dept. of Physiology and Cell Biology, Univ. of Nevada, School of Medicine, Anderson Medical Bldg. MS 352, Reno NV 89557-0046 (E-mail: tks{at}physio.unr.edu).

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.

Received 8 February 2001; accepted in final form 21 May 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

1.   Bardhan, KD, Bodemar G, Geldof H, Schutz E, Heath A, Mills JG, and Jacques LA. A double-blind, randomized, placebo-controlled dose-ranging study to evaluate the efficacy of alosetron in the treatment of irritable bowel syndrome. Aliment Pharmacol Ther 14: 23-34, 2000[ISI][Medline].

2.   Bearcroft, CP, Perrett D, and Farthing MJ. Postprandial plasma 5-hydroxytryptamine in diarrhoea predominant irritable bowel syndrome: a pilot study. Gut 42: 42-46, 1998[Abstract/Free Full Text].

3.   Bertrand, PP, Kunze WA, Bornstein JC, Furness JB, and Smith ML. Analysis of the responses of myenteric neurons in the small intestine to chemical stimulation of the mucosa. Am J Physiol Gastrointest Liver Physiol 273: G422-G435, 1997[Abstract/Free Full Text].

4.   Bruss, M, Molderings GJ, Bonisch H, and Gothert M. Pharmacological differences and similarities between the native mouse 5-HT3 receptor in N1E-115 cells and a cloned short splice variant of the mouse 5-HT3 receptor expressed in HEK 293 cells. Naunyn Schmiedebergs Arch Pharmacol 360: 225-233, 1999[ISI][Medline].

5.   Bush, TG, Spencer NJ, Watters N, Sanders KM, and Smith TK. Spontaneous migrating motor complexes occur in both the terminal ileum and colon of the C57BL/6 mouse in vitro. Auton Neurosci 84: 162-168, 2000[ISI][Medline].

6.   Butler, A, Elswood CJ, Burridge J, Ireland SJ, Bunce KT, Kilpatrick GJ, and Tyers MB. The pharmacological characterization of 5-HT3 receptors in three isolated preparations derived from guinea-pig tissues. Br J Pharmacol 101: 591-598, 1990[ISI][Medline].

7.   Butler, A, Hill JM, Ireland SJ, Jordan CC, and Tyers MB. Pharmacological properties of GR38032F, a novel antagonist at 5-HT3 receptors. Br J Pharmacol 94: 397-412, 1988[ISI][Medline].

8.   Camilleri, M. Clinical evidence to support current therapies of irritable bowel syndrome. Aliment Pharmacol Ther 13, Suppl2: 48-53, 1999.

9.   Camilleri, M, and Choi MG. Irritable bowel syndrome. Aliment Pharmacol Ther 11: 3-15, 1997[ISI][Medline].

10.   Camilleri, M, Mayer EA, Drossman DA, Heath A, Dukes GE, McSorley D, Kong S, Mangel AW, and Northcutt AR. Improvement in pain and bowel function in female irritable bowel patients with alosetron, a 5-HT3 receptor antagonist. Aliment Pharmacol Ther 13: 1149-1159, 1999[ISI][Medline].

11.   Camilleri, M, Northcutt AR, Kong S, Dukes GE, McSorley D, and Mangel AW. Efficacy and safety of alosetron in women with irritable bowel syndrome: a randomised, placebo-controlled trial. Lancet 355: 1035-1040, 2000[ISI][Medline].

12.   Chaudhary, NA, and Truelove SC. Human colonic motility: a comparative study of normal subjects, patients with ulcerative colitis, and patients with the irritable bowel syndrome. Gastroenterology 40: 1-17, 1961[ISI][Medline].

13.   Clayton, NM, Sargent R, Butler A, Gale J, Maxwell MP, Hunt AA, Barrett VJ, Cambridge D, Bountra C, and Humphrey PP. The pharmacological properties of the novel selective 5-HT3 receptor antagonist, alosetron, and its effects on normal and perturbed small intestinal transit in the fasted rat. Neurogastroenterol Motil 11: 207-217, 1999[ISI][Medline].

14.   Costall, B, and Naylor RJ. Anxiolytic potential of 5-HT3 receptor antagonists. Pharmacol Toxicol 70: 157-162, 1992[ISI][Medline].

15.   Delvaux, M, Louvel D, Mamet JP, Campos-Oriola R, and Frexinos J. Effect of alosetron on responses to colonic distension in patients with irritable bowel syndrome. Aliment Pharmacol Ther 12: 849-855, 1998[ISI][Medline].

16.   Drossman, DA. An integrated approach to the irritable bowel syndrome. Aliment Pharmacol Ther 13, Suppl2: 3-14, 1999.

17.   Everhart, JE, and Renault PF. Irritable bowel syndrome in office-based practice in the United States. Gastroenterology 100: 998-1005, 1991[ISI][Medline].

18.   Fida, R, Bywater RA, Lyster DJ, and Taylor GS. Chronotropic action of 5-hydroxytryptamine (5-HT) on colonic migrating motor complexes (CMMCs) in the isolated mouse colon. J Auton Nerv Syst 80: 52-63, 2000[ISI][Medline].

19.   Fida, R, Lyster DJ, Bywater RA, and Taylor GS. Colonic migrating motor complexes (CMMCs) in the isolated mouse colon. Neurogastroenterol Motil 9: 99-107, 1997[ISI][Medline].

20.   Galligan, JJ, Furness JB, and Costa M. Effects of cholinergic blockade, adrenergic blockade and sympathetic denervation on gastrointestinal myoelectric activity in guinea pig. J Pharmacol Exp Ther 238: 1114-1125, 1986[Abstract/Free Full Text].

21.   Gebauer, A, Merger M, and Kilbinger H. Modulation by 5-HT3 and 5-HT4 receptors of the release of 5-hydroxytryptamine from the guinea-pig small intestine. Naunyn Schmiedebergs Arch Pharmacol 347: 137-140, 1993[ISI][Medline].

22.   Gore, S, Gilmore IT, Haigh CG, Brownless SM, Stockdale H, and Morris AI. Colonic transit in man is slowed by ondansetron (GR38032F), a selective 5-hydroxytryptamine receptor (type 3) antagonist. Aliment Pharmacol Ther 4: 139-144, 1990[ISI][Medline].

23.   Gregory, RE, and Ettinger DS. 5-HT3 receptor antagonists for the prevention of chemotherapy-induced nausea and vomiting. A comparison of their pharmacology and clinical efficacy. Drugs 55: 173-189, 1998[ISI][Medline].

24.   Gunput, MD. Clinical pharmacology of alosetron. Aliment Pharmacol Ther 13, Suppl2: 70-76, 1999.

25.   Hammer, J, Phillips SF, Talley NJ, and Camilleri M. Effect of a 5HT3-antagonist (ondansetron) on rectal sensitivity and compliance in health and the irritable bowel syndrome. Aliment Pharmacol Ther 7: 543-551, 1993[ISI][Medline].

26.   Houghton, LA, Foster JM, and Whorwell PJ. Alosetron, a 5-HT3 receptor antagonist, delays colonic transit in patients with irritable bowel syndrome and healthy volunteers. Aliment Pharmacol Ther 14: 775-782, 2000[ISI][Medline].

27.   Jin, JG, Foxx-Orenstein AE, and Grider JR. Propulsion in guinea pig colon induced by 5-hydroxytryptamine (HT) via 5-HT4 and 5-HT3 receptors. J Pharmacol Exp Ther 288: 93-97, 1999[Abstract/Free Full Text].

28.   Jones, BJ, Costall B, Domeney AM, Kelly ME, Naylor RJ, Oakley NR, and Tyers MB. The potential anxiolytic activity of GR38032F, a 5-HT3-receptor antagonist. Br J Pharmacol 93: 985-993, 1988[ISI][Medline].

29.   Kadowaki, M, Nagakura Y, Tomoi M, Mori J, and Kohsaka M. Effect of FK1052, a potent 5-hydroxytryptamine3 and 5- hydroxytryptamine4 receptor dual antagonist, on colonic function in vivo. J Pharmacol Exp Ther 266: 74-80, 1993[Abstract/Free Full Text].

30.   Kellow, JE, and Phillips SF. Altered small bowel motility in irritable bowel syndrome is correlated with symptoms. Gastroenterology 92: 1885-1893, 1987[ISI][Medline].

31.   Kellow, JE, Phillips SF, Miller LJ, and Zinsmeister AR. Dysmotility of the small intestine in irritable bowel syndrome. Gut 29: 1236-1243, 1988[Abstract/Free Full Text].

32.   Lambert, JJ, Peters JA, Hales TG, and Dempster J. The properties of 5-HT3 receptors in clonal cell lines studied by patch-clamp techniques. Br J Pharmacol 97: 27-40, 1989[ISI][Medline].

33.   Manning, AP, Thompson WG, Heaton KW, and Morris AF. Towards positive diagnosis of the irritable bowel. Br Med J 2: 653-654, 1978.

34.   Mayer, EA, and Gebhart GF. Basic and clinical aspects of visceral hyperalgesia. Gastroenterology 107: 271-293, 1994[ISI][Medline].

35.   Mayer, EA, Naliboff B, Lee O, Munakata J, and Chang L. Review article: gender-related differences in functional gastrointestinal disorders. Aliment Pharmacol Ther 13, Suppl2: 65-69, 1999.

36.   Mayer, EA, and Raybould HE. Role of visceral afferent mechanisms in functional bowel disorders. Gastroenterology 99: 1688-1704, 1990[ISI][Medline].

37.   Nagakura, Y, Naitoh Y, Kamato T, Yamano M, and Miyata K. Compounds possessing 5-HT3 receptor antagonistic activity inhibit intestinal propulsion in mice. Eur J Pharmacol 311: 67-72, 1996[ISI][Medline].

38.   Prior, A, and Read NW. Reduction of rectal sensitivity and post-prandial motility by granisetron, a 5 HT3-receptor antagonist, in patients with irritable bowel syndrome. Aliment Pharmacol Ther 7: 175-180, 1993[ISI][Medline].

39.   Pritchard, JF, Bryson JC, Kernodle AE, Benedetti TL, and Powell JR. Age and gender effects on ondansetron pharmacokinetics: evaluation of healthy aged volunteers. Clin Pharmacol Ther 51: 51-55, 1992[ISI][Medline].

40.   Ragnarsson, G, and Bodemar G. Pain is temporally related to eating but not to defaecation in the irritable bowel syndrome (IBS). Patients' description of diarrhea, constipation and symptom variation during a prospective 6-week study. Eur J Gastroenterol Hepatol 10: 415-421, 1998[ISI][Medline].

41.   Sandler, RS. Epidemiology of irritable bowel syndrome in the United States. Gastroenterology 99: 409-415, 1990[ISI][Medline].

42.   Sang, Q, Williamson S, and Young HM. Projections of chemically identified myenteric neurons of the small and large intestine of the mouse. J Anat 190: 209-222, 1997.

43.   Saslow, SB, Scolapio JS, Camilleri M, Forstrom LA, Thomforde GM, Burton DD, Rubin J, Pitot HC, and Zinsmeister AR. Medium-term effects of a new 5HT3 antagonist, alosetron, in patients with carcinoid diarrhoea. Gut 42: 628-634, 1998[Abstract/Free Full Text].

44.   Smith, TK, and McCallum RW. Serotonin mediation of intestinal peristalsis. In: Regulatory Mechanisms in Gastrointestinal Function, edited by Gaginella TS.. New York: CRC, 1995, p. 219-240.

45.   Snape, WJ, Jr, Carlson GM, Matarazzo SA, and Cohen S. Evidence that abnormal myoelectrical activity produces colonic motor dysfunction in the irritable bowel syndrome. Gastroenterology 72: 383-387, 1977[ISI][Medline].

46.   Talley, NJ, Phillips SF, Haddad A, Miller LJ, Twomey C, Zinsmeister AR, MacCarty RL, and Ciociola A. GR 38032F (ondansetron), a selective 5HT3 receptor antagonist, slows colonic transit in healthy man. Dig Dis Sci 35: 477-480, 1990[ISI][Medline].

47.   Taub, E, Cuevas JL, Cook EW, Crowell M, 3rd, and Whitehead WE. Irritable bowel syndrome defined by factor analysis. Gender and race comparisons. Dig Dis Sci 40: 2647-2655, 1995[ISI][Medline].

48.   Von der Ohe, MR, Camilleri M, Kvols LK, and Thomforde GM. Motor dysfunction of the small bowel and colon in patients with the carcinoid syndrome and diarrhea. N Engl J Med 329: 1073-1078, 1993[Abstract/Free Full Text].

49.   Whitehead, WE, Holtkotter B, Enck P, Hoelzl R, Holmes KD, Anthony J, Shabsin HS, and Schuster MM. Tolerance for rectosigmoid distention in irritable bowel syndrome. Gastroenterology 98: 1187-1192, 1990[ISI][Medline].

50.   Yakel, JL, and Jackson MB. 5-HT3 receptors mediate rapid responses in cultured hippocampus and a clonal cell line. Neuron 1: 615-621, 1988[ISI][Medline].

51.   Zighelboim, J, Talley NJ, Phillips SF, Harmsen WS, and Zinsmeister AR. Visceral perception in irritable bowel syndrome. Rectal and gastric responses to distension and serotonin type 3 antagonism. Dig Dis Sci 40: 819-827, 1995[ISI][Medline].