Expression and function of 5-hydroxytryptamine 4 receptors in smooth muscle preparations from the duodenum, ileum, and pelvic flexure of horses without gastrointestinal tract disease

Andrea S. Prause Divisions of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Berne, 3012 Berne, Switzerland

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Christophe T. Guionaud Divisions of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Berne, 3012 Berne, Switzerland

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Michael H. Stoffel Veterinary Anatomy, Vetsuisse Faculty, University of Berne, 3012 Berne, Switzerland

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Christopher J. Portier Environmental Systems Biology and Risk Assessment, National Institute of Environmental Health Sciences, 111 TW Alexander Dr, Research Triangle Park, NC 27709

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Meike Mevissen Divisions of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Berne, 3012 Berne, Switzerland

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Abstract

Objective—To evaluate the expression of the 5-hydroxytryptamine 4 (5-HT4) receptor subtype and investigate the modulating function of those receptors on contractility in intestinal tissues obtained from horses without gastrointestinal tract disease.

Sample Population—Smooth muscle preparations from the duodenum, ileum, and pelvic flexure collected immediately after slaughter of 24 horses with no history or signs of gastrointestinal tract disease.

Procedures—In isometric organ baths, the contractile activities of smooth muscle preparations in response to 5-hydroxytryptamine and electric field stimulation were assessed; the effect of tegaserod alone or in combination with 5-hydroxytryptamine on contractility of intestinal specimens was also investigated. Presence and distribution of 5-HT4 receptors in intestinal tissues and localization on interstitial cells of Cajal were examined by use of an immunofluorescence technique.

Results—Widespread 5-HT4 receptor immunoreactivity was observed in all intestinal smooth muscle layers; 5-HT4 receptors were absent from the myenteric plexus and interstitial cells of Cajal. In electrical field–stimulated tissue preparations of duodenum and pelvic flexure, tegaserod increased the amplitude of smooth muscle contractions in a concentration-dependent manner. Preincubation with tegaserod significantly decreased the basal tone of the 5-HT–evoked contractility in small intestine specimens, compared with the effect of 5-HT alone, thereby confirming that tegaserod was acting as a partial agonist.

Conclusions and Clinical Relevance—In horses, 5-HT4 receptors on smooth muscle cells appear to be involved in the contractile response of the intestinal tract to 5-hydroxytryptamine. Results suggest that tegaserod may be useful for treatment of reduced gastrointestinal tract motility in horses.

Abstract

Objective—To evaluate the expression of the 5-hydroxytryptamine 4 (5-HT4) receptor subtype and investigate the modulating function of those receptors on contractility in intestinal tissues obtained from horses without gastrointestinal tract disease.

Sample Population—Smooth muscle preparations from the duodenum, ileum, and pelvic flexure collected immediately after slaughter of 24 horses with no history or signs of gastrointestinal tract disease.

Procedures—In isometric organ baths, the contractile activities of smooth muscle preparations in response to 5-hydroxytryptamine and electric field stimulation were assessed; the effect of tegaserod alone or in combination with 5-hydroxytryptamine on contractility of intestinal specimens was also investigated. Presence and distribution of 5-HT4 receptors in intestinal tissues and localization on interstitial cells of Cajal were examined by use of an immunofluorescence technique.

Results—Widespread 5-HT4 receptor immunoreactivity was observed in all intestinal smooth muscle layers; 5-HT4 receptors were absent from the myenteric plexus and interstitial cells of Cajal. In electrical field–stimulated tissue preparations of duodenum and pelvic flexure, tegaserod increased the amplitude of smooth muscle contractions in a concentration-dependent manner. Preincubation with tegaserod significantly decreased the basal tone of the 5-HT–evoked contractility in small intestine specimens, compared with the effect of 5-HT alone, thereby confirming that tegaserod was acting as a partial agonist.

Conclusions and Clinical Relevance—In horses, 5-HT4 receptors on smooth muscle cells appear to be involved in the contractile response of the intestinal tract to 5-hydroxytryptamine. Results suggest that tegaserod may be useful for treatment of reduced gastrointestinal tract motility in horses.

Serotonin (ie, 5-HT), a ubiquitous neurotransmitter, exerts its action by interacting with 7 receptor subtypes.1 In the intestinal tract, 5-HT mediates a variety of physiologic effects on enterocytes, smooth muscle cells, and enteric neurons through its effects on several 5-HT receptor subtypes that regulate gastrointestinal tract motility, vascular tone, and secretion. Serotoninergic signaling abnormalities have also been putatively implicated in the pathogenesis of functional intestinal diseases.2,3 In humans, 5-HT receptor subtypes are established targets for drugs used in the treatment of CNS disorders (eg, mental disorders) as well as gastrointestinal disorders (eg, gastroesophageal reflux, functional dyspepsia, irritable bowel disease, and functional constipation) and for the prevention of nausea.4 Although activation of several different receptors is responsible for 5-HT–mediated responses within the gastrointestinal tract, the 5-HT4 receptor subtype is considered particularly important, both physiologically and pathophysiologically.5,6 Activation of 5-HT4 receptors enhances the peristaltic reflex, and 5-HT4 receptor agonists have gastrointestinal prokinetic activities.7–9 In equine veterinary medicine, the application of serotoninergic drugs is primarily limited to modulation of the peristaltic activity of the gastrointestinal tract. The evaluation of possible targets for the development of new prokinetic drugs for use in horses is emerging because gastrointestinal disorders, such as postoperative ileus, are common causes of death in horses and often involve considerable economic losses.10

Despite the fact that a variety of prokinetic drugs is on the market, none of those drugs are considered sufficient in terms of efficacy and safety. Cisapride, a 5-HT4 receptor agonist that has 5-HT3 and dopamine receptor (D2) antagonistic properties, has proven effective to improve impaired gastrointestinal tract motility in horses.11 In 2000, cisapride was withdrawn from the market because of adverse prolongation of the QT interval in humans.12 In addition, the partial 5-HT4 agonist tegaserod—a drug that is highly effective in humans with constipation-predominant irritable bowel disease—was withdrawn in 2007 because of concerns regarding an increased incidence of myocardial infarction, angina, and stroke in humans, although the mechanism of adverse effects is still unclear and the adverse effects were not 5-HT4 receptor related. Despite the risk potential of the aforementioned compounds, 5-HT4 receptors remain promising targets in the treatment of gastrointestinal tract motility disorders. A therapeutic value of tegaserod in horses has been suggested by recently published reports.13–15 In vitro, tegaserod increases intestinal smooth muscle contraction in specimens of the pelvic flexure (a hairpin turn in the large colon that is predisposed to obstruction) of horses.13,14 Moreover, it was demonstrated in vivo that the 5-HT4 partial agonist tegaserod accelerates the gastrointestinal transit time and increases the frequency of defecation and scores of intestinal sounds in healthy horses.15 Therapeutic plasma concentrations of tegaserod were achieved following oral administration of a single dose of 0.27 mg/kg.14 However, controversial findings regarding the general impact of 5-HT4 receptors on equine gastrointestinal tract motility have been reported.16 Delesalle et al16 found a lack of evidence for the presence of 5-HT4 receptors in the small intestine of horses; their conclusion was based on the observation that selected 5-HT4 receptor antagonists could not alter serotonin-induced tonic contractions of equine jejunal tissue in vitro.

Besides tegaserod, other 5-HT4 receptor ligands have been investigated with respect to their potential for treatment of gastrointestinal tract motility disorders in humans and horses. One of the most relevant of them is prucalopride, which is anticipated to be released soon onto the European market for the treatment of chronic constipation in humans. In addition to proven efficacy across different patient groups, prucalopride provides a favorable cardiovascular and overall safety profile.17 Partial 5-HT4 receptor agonists such as PF-0135408218 or CJ-03346619 that are currently in early stages of pharmaceutical development are expected to exert a favorable effect on gastrointestinal motor disorders with reduced adverse effects mediated by other related receptors.

Efficient gastrointestinal tract motility requires the coordinated activity of several cell types including enteric nerves, smooth muscle cells, and ICCs.20 Interstitial cells of Cajal are oval or stellate cells of mesenchymal origin. They are widely distributed in the smooth muscle layers of the gastrointestinal tract. The ICCs are predominantly located between the circular and longitudinal muscle layers as well as in the deep muscular plexus. Their cytoplasm is scarce but extends to make contacts with autonomic nerve fibers of the myenteric plexus through varicose nerve terminals and to establish gap junctions with adjacent smooth muscle cells. The ICCs have a key role in the regulation of intestinal peristalsis by generating spontaneous, rhythmic electrical oscillations (called slow waves) and by mediating enteric motor neurotransmission and afferent signaling. The ICCs mediate gastrointestinal tract motility through cholinergic excitatory and nitrergic inhibitory motor neurotransmissions.20–22 Interstitial cells of Cajal have been detected in circular muscle layer and the myenteric plexus of horses.23 Their pacemaker function, in terms of generating slow waves, is essential for gastrointestinal tract motility.20

Given that differing distributions of 5-HT4 receptors along the gastrointestinal tract in different species might explain the different responses of either smooth muscle contraction or relaxation, the purpose of the study reported here was to evaluate the expression of the 5-HT4 receptor subtype and investigate the modulating function of those receptors on intestinal contractility in selected intestinal tissues obtained from horses. Furthermore, the effect of the 5-HT4 partial agonist tegaserod alone or in combination with serotonin on contractility of intestinal specimens was investigated. Our investigations were focused on tissues at 3 locations in the intestinal tract: the duodenum, ileum, and pelvic flexure. The duodenum and ileum were selected for investigation because the small intestine is frequently associated with gastrointestinal tract disorders in horses. In addition, pelvic flexure was chosen because of its established motility pacemaker activity, which regulates colonic aboral and retropropulsive transit of digesta.24 Also, we evaluated the expression and possible colocalization of 5-HT4 receptors and c-kit by use of a double-labeling immunofluorescence technique. The c-kit marker is a tyrosine kinase receptor that is a well-established marker of ICCs.

Materials and Methods

Tissue samples and preparation—Specimens of duodenum, ileum, and pelvic flexure were obtained from 24 horses with no history or signs of gastrointestinal tract disease after they had been slaughtered at local abattoirs. The horses (males and females aged 1 to 30 years) were of various breeds, and specimens were collected within 15 minutes after the horses were slaughtered by use of a captive bolt.

In each horse, 2 to 6 tissue specimens (including all muscle layers) were obtained from a location 20 cm aboral to the pylorus (duodenum), from the proximal insertion site of the plica ileocaecalis (ileum), and from the pelvic flexure at the position where tenia were no longer evident. Subsequently, the intestinal content was disposed of by rinsing of the specimens with cooled (5°C) KH solution (NaCl, 118.4mM; KCl, 4.7mM; KH2PO4, 1.2mM; MgSO4, 1.2mM; CaCl2, 2.5mM; NaHCO3, 25mM; and glucose, 11mM) or PBS solution (phosphate buffer, 10mM [pH, 7.4]; NaCl, 140mM; and KCl, 3mM). Intestinal specimens were kept in either cooled KH or PBS solution during transport (20 minutes' duration) to the laboratory. At the laboratory, the mucosa was removed, and rectangular tissue samples (approx 2 × 4 cm) were prepared and kept in cooled PBS solution until used in the immunofluorescence experiments or in cooled KH solution aerated with carbogen (95% O2 and 5% CO2) until used in organ bath experiments.

Immunofluorescence experiments—The most adequate antibody for detection of 5-HT4 receptors in horses was selected on the basis of protein sequence data deduced from coding regions extracted from the Horse Genome Project sequencesa and from the equine HTR4 partial cDNA sequence obtained by our group from an equine colon sample (GenBank accession No. AY263357). A commercial affinity-purified polyclonal antibodyb against the third cytoplasmic domain (aa 214–260) of the human 5-HT4 receptor was used. The third cytoplasmic domain of the human protein differs only by 4 residues along the same region of the horse 5-HT4 receptor (91% identity; data not shown). To investigate the possible colocalization of 5-HT4 receptors and c-kit on ICCs, the affinity-purifed polyclonal goat anti-mouse stem cell factor receptor (SCF R)/c-kit antibody was used.

Strips of intestinal tissue (approx 15 mm in length and 5 mm in width) obtained from each location in each horse were prepared, pinned on foam plates, and flash frozen in liquid nitrogen. Tissues were cryostat sectioned at a thickness of 5 μm. The immunofluorescence staining procedure was performed at room temperature (approx 20°C), and PBS solution was used to wash sections thoroughly between each step. Briefly, the slides were fixed in 2% paraformaldehyde in PBS solution for 30 minutes, treated with 0.2M glycine in PBS solution for 20 minutes, and made permeable in 0.3% Triton X-100 for 20 minutes. Sections then underwent immunostaining procedures for detection of 5-HT4 receptors and c-kit. The sections were washed for 5 minutes 3 times in PBS solution with 0.1% Tween 20 before being blocked with 5% donkey serum with streptavidinc for 1 hour at room temperature, according to the manufacturer's protocol. Primary antibodies (rabbit anti-human 5-HT4 polyclonal antibodyb diluted 1:200 [5 ng/μL] and goat anti-mouse SCF R/c-kit polyclonal antibodyb diluted 1:25 [4 ng/μL]) supplemented with biotin were applied overnight at room temperature. After washing for 5 minutes 3 times, sections were incubated with secondary antibodies (biotin-SP–coupled donkey anti-rabbit IgGd diluted 1:3,000) and Cy2-labeled donkey anti-goat IgGd diluted 1:100) for 1 hour at room temperature. Further incubation with (Cy3-labeled streptavidin diluted 1:3,000)d was performed for 1 hour at room temperature. Sections were treated with 4′,6-diamidino-2-phenylindolee to stain nuclei. All immunolabeling reagents were diluted in an antibody diluent.b

After immunostaining, the slides were washed in PBS solution and covered with mounting medium.f Negative control experiments included omission of the primary antibodies and use of an irrelevant primary antibody (rabbit anti-calcitonin receptor antibodyg diluted 1:100). Images were captured by use of a fluorescence microscopeh equipped with a digital camera.h All experimental and corresponding control images were obtained with identical camera settings. Differential interference contrast microscopy was used to observe structural details in the tissue sections. Computer softwarei was used to adjust the contrast equally for experimental and control images.

Experimental protocols—Intestinal specimens were obtained as described. The intestinal specimens were kept in cooled KH solution aerated with carbogen (95% O2 and 5% CO2) until used in an organ bath experiment.

Strips of intestinal tissue (1 cm in length and 3 mm in width) cut parallel to the longitudinal muscle layer were prepared. Each tissue strip was arbitrarily allocated to an individual Schuler tissue bath chamberj filled with aerated KH solution at 37°C. One end of the muscle preparation was fixed to a hook and the other attached to an isometric force transducer,k as described previously.13,25 Smooth muscle contractility data were acquired, transferred to a personal computer, and analyzed by use of computer software.l To avoid development of tachyphylaxis, each preparation was used once only.

Spontaneous contractions—After 10 minutes of equilibration in the organ bath, each tissue strip was challenged with 10–6M carbacholm to check for viability. Subsequently, the organ bath was flushed 3 times, and the initial muscle tension was routinely adjusted to 1 g. Each smooth muscle preparation was allowed to equilibrate for 60 minutes before the start of the experiment, during which time the KH solution in the organ bath chambers was replaced every 30 minutes and the tension was readjusted after 60 minutes. The specimen was examined for spontaneous activity. Only specimens that had constant frequency, amplitude, and basal tone of contraction for at least 10 minutes prior to the start of the experiment were used for analysis. Variables such as basal tone or amplitude at this time were designated as predrug values. Cumulative concentration-response curves following treatment with 5-HTn in the absence or presence of tegaserodo were generated. At 5-minute intervals, the concentration of 5-HT in the organ bath was increased logarithmically (from 10–10 to 10–5M). For experiments performed in the presence of tegaserod, preparations were preincubated for 20 minutes with tegaserod at a concentration of 1 × 10–6M. Subsequently, 5-HT was added as described. Solvent control experiments (by use of methylpyrrolidone for tegaserod and H2O for 5-HT) were conducted similarly.

Electrical field stimulation—The tissue preparations were routinely set to a tension of 1 g and allowed to equilibrate for 30 minutes. The bath solution was changed and the tension was readjusted every 15 minutes. After stabilization of the tissue, EFS was applied. Electrical impulses (rectangular wave pulses [duration, 5 milliseconds] each with an amplitude of 125 mA and current frequency of 50 Hz; train duration, 5 seconds; interval between trains, 20 seconds) were generated by a stimulator.p After EFS was initiated, the specimens were checked for activity (amplitude of contractions). To evoke submaximal contractions, the current intensity was gradually increased every 5 minutes until contractility was initiated. To generate a concentration-response curve for tegaserod and the corresponding solvent methylpyrrolidone, only specimens for which constant frequency, amplitude, and basal tone of contraction were maintained for at least 10 minutes prior to the start of the experiment were selected. At 5-minute intervals, tegaserod was added in half-logarithmic steps reaching concentrations ranging from 10–10 to 10–6M. Solvent controls were conducted similarly.

Data and statistical analyses—The contractility variables of basal tone (in g), Amax (in g), and frequency (in counts/min [for spontaneous contractions]) were recorded in 5-minute intervals. The data acquisition of contractility variables started 5 minutes prior to the first addition of 5-HT or tegaserod (designated as the predrug period) and ceased 5 minutes after the last addition. All data are reported relative to the values obtained during the predrug period.

The data collected over the period of cumulative addition of 5-HT or tegaserod followed by 5-HT were examined by use of a Friedman testq for each experimental protocol and orientation of the muscle layers separately. Observed effects for all variables and all experiments over time were compared with the effect of solvent by use of an ANOVA for repeated measures followed by the Bonferroni multiple comparison test.r Values are expressed as mean ± SEM; a value of P < 0.05 was considered significant.

Concentration-response curves for 5-HT and tegaserod plus 5-HT were calculated by use of the Hill function, and estimation by use of the least squares method was applied.s The underlying equation for the Hill function is:

article image

where C is the compound concentration, K represents the EC50 value, and the exponent α describes the shape of the function.

Significance of comparisons made on the basis of this model was determined by use of the likelihood ratio statistic, which yields a χ2 test. Standard deviations reported for variables in the Hill model are based on the Cramer-Rao statistic.26 The results were expressed as Vmax and EC50 of Amax.

Results

Immunofluorescence experiments—In all enteric locations examined, marked c-kit immunoreactivity was detected between circular and longitudinal muscle layers in the myenteric plexus (Figure 1). Strong 5-HT4 receptor immunoreactivity was observed in the walls of blood vessels of the myenteric region, but not in ICCs because no colocalization of 5-HT4 receptors with c-kit was identified. Furthermore, weak to moderate 5-HT4 immunoreactivity was detected in both intestinal muscle layers (Figure 2). Nevertheless, staining was more pronounced in the circular muscle layer than in the longitudinal muscle layer. Moderate 5-HT4 immuno-reactivity was observed in the mucosal region, where crypts are expected to be located, and strong staining was observed in round-shaped areas within the mucosal villi (Figure 3). Submucosal blood vessels were also highly stained for the 5-HT4 receptor, and the immunofluorescence signals were assigned to the tunica media and tunica intima. The negative controls were devoid of specific immunolabeling.

Figure 1—
Figure 1—

Representative photomicrographs illustrating the localization of 5-HT4 receptors and c-kit by use of immunofluorescence staining performed on cryostat sections of duodenum, ileum, and pelvic flexure obtained from 2 horses. For each tissue type, findings for 1 experimentally treated section (columns marked Exp) and 1 control section (columns marked Con) are provided. Experimentally treated sections were double labeled with anti–c-kit and anti–5-HT4 receptor antibodies, and Cy2- and Cy3-tagged streptavidin were used to detect c-kit (green fluorescence; top-row images) and 5-HT4 receptors (red fluorescence; middle-row images), respectively. In matching control sections, primary antibodies were omitted. For the experimentally treated and control sections for each tissue type, top- and middle-row images were merged to create the bottom-row images. Notice that merged images revealed no colocalization of 5-HT4 and c-kit immunoreactivity. CM = Circular muscle layer. LM = Longitudinal muscle layer. MP = Myenteric plexus. Bar = 100 μm (applies to all images).

Citation: American Journal of Veterinary Research 71, 12; 10.2460/ajvr.71.12.1432

Figure 2—
Figure 2—

Representative photomicrographs illustrating the localization of 5-HT4 receptors by use of immunofluorescence staining performed on 2 cryostat sections of ileum obtained from 2 horses. Findings for 1 experimentally treated section (column marked Exp) and 1 control section (column marked Con) are provided. The experimentally treated section was labeled with anti–5-HT4 receptor antibody and Cy3-labeled streptavidin; primary antibody was omitted in the control section. Differential interference contrast microscopy was used to observe structural details in the tissue sections (top-row images). In the middle-row images, receptor localization is indicated by red fluorescence. For each section, top- and middle-row images were merged to create the bottom-row images. In the merged image for the experimentally treated section, expression of 5-HT4 receptors is localized in longitudinal and circular muscle layers as well as in myenteric blood vessels. Bar = 200 μm (applies to all images). See Figure 1 for key.

Citation: American Journal of Veterinary Research 71, 12; 10.2460/ajvr.71.12.1432

Figure 3—
Figure 3—

Representative photomicrographs (obtained by use of a 10× [A and B] and 40× [C] objective lens) illustrating the localization of 5-HT4 receptors by use of immunofluorescence staining performed on cryostat sections of ileum obtained from 2 horses. Each section was labeled with anti–5-HT4 receptor antibody and Cy3-labeled streptavidin (receptor localization indicated by red fluorescence [top-row images]) and also with 4',6-diamidino-2-phenylindole (nuclei indicated by blue fluorescence [middle-row images]). Primary antibodies were omitted from control sections (not shown). For each section, top- and middle-row images were merged to create the bottom-row images. In the merged images, 5-HT4 receptor immunoreactivity is evident in mucosal villi (A) and blood vessels (B and C) in the mucosa (M) and tela submucosa (SM), respectively. Bar = 200 μm (applies to all images).

Citation: American Journal of Veterinary Research 71, 12; 10.2460/ajvr.71.12.1432

Organ bath experiments—Intestinal tissue preparations that had regular contractile activity 10 minutes before the start of the experiment were included in the study. In total, 38 duodenal preparations, 42 ileal preparations, and 52 pelvic flexure preparations were collected from 13 horses. Overall, 27 of 38 (71%) duodenal preparations, 29 of 42 (69%) ileal preparations, and 31 of 52 (60%) pelvic flexure preparations met the study inclusion criterion. With regard to the spontaneous contractions at the start of the experiment of duodenal, ileal, and pelvic flexure specimens, mean frequency was 11.48, 7.18, and 3.75 counts/min, respectively, and mean Amax was 1.46, 1.14, and 2.35 g, respectively; basal tone was 1.08, 1.13, and 1.23 g, respectively.

In the experiments involving spontaneous contractions, treatment with 5-HT caused concentration-dependent increases in basal tone in duodenal and ileal tissue strips, compared with the effect of solvent, whereas no similar effect on basal tone was evident in pelvic flexure preparations (Figure 4). Significant (P < 0.001) differences between 5-HT–treated duodenal specimens and solvent controls were evident at 5-HT concentrations of 10–9 to 10–6M. Significant (P < 0.05) differences between 5-HT–treated ileal specimens and solvent controls were evident at 5-HT concentrations of 10–7 to 10–5M. In preparations of duodenum, the shape of the concentration-response curve was biphasic with a first plateau at a concentration of 10–9M and a maximum effect at 10–6M. A biphasic concentration-response curve was also obtained for ileal preparations with a first plateau at concentrations of 10–10 to 10–8M followed by increases in basal tone at higher concentrations (10–7 to 10–5M).

Figure 4—
Figure 4—

Effect (determined in organ bath experiments) of 5-HT on basal tone in longitudinal specimens of duodenum (n = 9; A), ileum (10; B), and pelvic flexure (10; C) obtained from 24 horses following preincubation of specimens for 20 minutes without (black circles) or with (inverted black triangles) tegaserod (1 × 10–6M). Following the preincubation period (60 minutes), the concentration of 5-HT in the organ bath was increased logarithmically (from 10–10 to 10–5M); control specimens were preincubated without tegaserod and exposed only to solvent (H2O [white circles]). Data are reported as mean basal tone (as a percentage of the predrug period value) and SEM. *Response to 5-HT alone (without tegaserod preincubation) is significantly (P < 0.05) different from the corresponding response to solvent. Response to 5-HT following tegaserod preincubation is significantly (P < 0.05) different from the corresponding response to 5-HT alone. Significant (P < 0.05) effect over time in this treatment group.

Citation: American Journal of Veterinary Research 71, 12; 10.2460/ajvr.71.12.1432

Preincubation of tissue preparations with tegaserod resulted in decreases in basal tone in all intestinal specimens, compared with the effect of 5-HT alone, although the change was significant only for specimens from the duodenum at 5-HT concentrations of 10–7 and 10–6M (Figure 4). In preparations from the ileum and pelvic flexure, tegaserod did not significantly change basal tone with respect to the effect of 5-HT alone.

Treatment with 5-HT at all concentrations except 10–7M resulted in significant (P < 0.05) increases (as a percentage of the predrug period value) in Amax in pelvic flexure specimens, compared with the effect of solvent, whereas no significant increases in Amax were detected in preparations from the duodenum and ileum (Figure 5). Exposure to tegaserod did not change Amax values, compared with the effect of 5-HT alone, in specimens from any intestinal location.

Figure 5—
Figure 5—

Effect (determined in organ bath experiments) of 5-HT on Amax of spontaneous contractions in longitudinal specimens of duodenum (n = 9; A), ileum (10; B), and pelvic flexure (10; C) obtained from 24 horses following preincubation of specimens for 20 minutes without (black circles) or with (inverted black triangles) tegaserod (1 × 10 –6M). Following the preincubation period (60 minutes), the concentration of 5-HT in the organ bath was increased logarithmically (from 10 10 to 10–5M); control specimens were preincubated without tegaserod and exposed only to solvent (H2O [white circles]). Data are given as mean Amax (as a percentage of the predrug period value) and SEM. See Figure 4 for remainder of key.

Citation: American Journal of Veterinary Research 71, 12; 10.2460/ajvr.71.12.1432

Analysis of the EC50 values for basal tone induced by treatment with 5-HT with or without preincubation with tegaserod revealed no differences among intestinal locations. Regardless of experimental conditions, values of Vmax for basal tone were significantly (P < 0.001) higher in specimens from the duodenum and ileum, compared with specimens from the pelvic flexure.

Compared with specimens exposed to 5-HT and tegaserod, the Vmax of basal tone induced by 5-HT alone was significantly higher in preparations of duodenum (all values of P < 0.01) and ileum (all values of P < 0.05), but not in preparations of pelvic flexure (Table 1). Analysis of EC50 and Vmax values for Amax did not reveal any significant differences among the intestinal preparations, regardless of experimental conditions (data not shown). Neither 5-HT nor tegaserod treatment affected the frequency of contractions in tissue preparations from any of the 3 intestinal locations.

Table 1—

Mean Vmax and EC50 values of the basal tone (determined in organ bath experiments) in specimens of duodenum, ileum, and pelvic flexure obtained from 24 horses following preincubation of specimens for 20 minutes without or with tegaserod (1 × 10–6M [Teg]).

TissueTreatmentNo. of experimentsVmax (g [95% confidence interval])EC50 (M)
Duodenum5-HT80.79a (0.36–1.72)4.1 × 10−9
5-HT and Teg90.47a (0.09–2.40)5.6 × 10−7
Ileum5-HT101.05b (0.01–165.5)9.2 × 10−6
5-HT and Teg100.15b (7.43 × 10−6 −2.86 × 103)1.00 × 10−5
Pelvic flexure5-HT10NANA
5-HT and Teg10NANA

NA = Not applicable (ie, 5-HT did not induce an effect on spontaneous contractions).

Within a column, values with the same superscript are significantly (P < 0.05) different from each other.

EFS data—Preincubation with tegaserod did not significantly change basal tone in preparations from any of the intestinal locations, compared with the effect of solvent (data not shown). In all preparations investigated, tegaserod induced a significant and concentration-dependent increase in Amax over time (duodenum and ileum, P < 0.001; pelvic flexure, P < 0.05; Figure 6). Compared with the effect of solvent, values of Amax were significantly (P < 0.05) increased in duodenal preparations at tegaserod concentrations of 10–7 through 10–6M and in pelvic flexure preparations at all but 1 tegaserod concentration in the range of 10–9 through 10–6.5M.

Figure 6—
Figure 6—

Effect (determined in organ bath experiments) of tegaserod (black circles) on Amax of EFS-induced contractile activity in longitudinal specimens of duodenum (n = 12; A), ileum (14; B), and pelvic flexure (6; C) obtained from 24 horses. Each smooth muscle preparation was allowed to equilibrate for 60 minutes before tegaserod was added to the organ bath. The concentration of tegaserod in the organ bath was increased logarithmically (from 10–10 to 10–6M); control specimens were incubated without tegaserod and exposed only to solvent (methylpyrrolidone [white circles]). Data are reported as mean and SEM. *Response to tegaserod is significantly (P < 0.05) different from the corresponding response to solvent. Significant (P < 0.05) effect over time in this treatment group.

Citation: American Journal of Veterinary Research 71, 12; 10.2460/ajvr.71.12.1432

In EFS tissue preparations, the values of EC50 and Vmax for basal tone did not differ among intestinal locations. With regard to Amax, the value of Vmax for the duodenal specimens was significantly (P < 0.05) lower than the value for the ileal or pelvic flexure preparations; the values for the ileal and pelvic flexure preparations did not differ significantly (Table 2).

Table 2—

Mean Vmax and EC50 values of the Amax of EFS-induced contractions (determined in organ bath experiments) in specimens of duodenum, ileum, and pelvic flexure obtained from 24 horses following treatment of specimens with tegaserod at concentrations ranging from 10−10 to 10−6M.

TissueNo. of experimentsVmax (g [95% confidence interval])EC50 (M)
Duodenum120.74a,b (0.06–9.61)1.5 × 10−10
Ileum140.93a (0.10–8.80)9.46 × 10−10
Pelvic flexure61.08b (0.13–8.74)2.16 × 10−9

See Table 1 for key.

Discussion

The results of the present study indicate that 5-HT4 receptors are present in the duodenum, ileum, and pelvic flexure of horses. The distribution of 5-HT4–immunoreactive cells was consistent throughout the 3 regions of the gastrointestinal tract examined. Although 5-HT4 receptor immunoreactivity was detected in the circular and the longitudinal muscle layers of the intestine, 5-HT4 receptors were not present in the myenteric plexus. These observations are in agreement with the results of Streutker et al,t who identified 5-HT4 receptors by use of immunohistochemical analysis and in situ hybridization in specimens of human nonneuronal cells in the gastrointestinal tract. In that study,t 5-HT4 receptors were localized in the circular and longitudinal layer of the tunica muscularis, whereas neurons did not stain specifically for 5-HT4 receptors. However, in specimens of small intestine from guinea pigs and colon from rats and mice, 5-HT4 receptor immunoreactivity was associated with intrinsic primary afferent neurons and motor neurons in the myenteric ganglia.27 Use of other detection methods, such as autoradiography, has revealed that neuronal 5-HT4 receptor expression is detectable not only in colonic tissue from guinea pigs but also in colonic tissue from humans, even though myenteric localization in human tissue was less distinct than that in guinea pig tissue.28 In agreement with our findings, Streutker et alt also detected 5-HT4 receptors in blood vessels. The strong immunoreactivity in the blood vessels could indicate an important role of 5-HT4 receptors in the regulation of mesenteric blood flow in horses. Among species, 5-HT4 receptor expression and function in blood vessels is variable. Cocks and Arnold29 reported that relaxation of isolated pulmonary veins obtained from sheep is mediated via activation of 5-HT4 receptors, but there was no evidence for 5-HT4–mediated relaxation in dog, pig, or human pulmonary veins.29 Furthermore, 5-HT4 receptor mRNA has been detected via reverse transcriptase PCR analysis in rat blood vessels but not in vascular tissues from pigs.30 In cultured human endothelial cells from pulmonary and coronary arteries, umbilical vein, and aorta, the same group reported30 weak expression of 5-HT4 receptor mRNA. In contrast, results of functional and radio-ligand-binding experiments involving isolated intra-pulmonary arteries and veins obtained from humans did not provide evidence for the involvement of 5-HT4 receptors in vasoconstriction,31 and in pulmonary arteries obtained from rabbits, 5-HT4 receptor mRNA could not be detected.32

In the present study, 5-HT4 receptor immunoreactivity was evident in the tunica intima and tunica media of enteric blood vessels. This finding is in agreement with findings of other studies30,33 in humans in which 5-HT4 receptors were detected on endothelial cells and vascular smooth muscle cells. Currently, the potential impact of the presence of 5-HT receptors in blood vessels on modulation of gastrointestinal tract motility has not been investigated, to our knowledge.

A recent study34 performed by our group revealed that ganglion cells of the myenteric plexus of horses react with antibodies against c-kit, thereby providing evidence that ICCs were present in all intestinal locations tested (the same locations as those investigated in the present study). The ICCs are considered important for gastrointestinal tract motility because of their role as electrical pacemakers, generating spontaneous electrical slow waves that propagate to smooth muscle cells to evoke phasic contractions. In contrast to the findings for 5-HT7 receptors in horses,34 results of the present study indicated that 5-HT4 receptors are not colocalized with the ICC marker c-kit. In other studies27,35 in mice and guinea-pigs, 5-HT4 receptors were expressed by myenteric ICCs.

The expression of 5-HT4 receptors in equine intestinal mucosa raises the question whether these receptors have a role in mucosal secretion. In the rat colon as well as in guinea pig and human ileum, the transmucosal short-circuit current response to 5-HT is mediated by a receptor of the 5-HT4 type.36–38 In rats and mice, it has been reported39,40 that 5-HT4 receptors are involved in secretion of bicarbonate from the duodenal mucosa, which is crucial to maintain mucosal integrity. Although Safsten et al39 postulated an exclusively myenteric location of 5-HT4 receptors on the basis of data obtained from the duodenum of rats, the findings of a study in mice by Tu o et al40 indicated that 5-HT4 receptor mRNA is present in duodenal mucosa, which supports our observations in equine intestinal tissue. The impact of mucosal 5-HT4 receptors in secretory processing in horses has to be investigated further. In humans, expression of 5-HT4 receptors in the mucosa of the duodenum was greater than expressions in mucosa of the stomach, which indicates a prominent role of serotoninergic signaling at the mucosal level in this part of the intestine.41 Possible pathways include the arachidonic acid pathway and activation of sensory dendrites in the lamina propria, which leads to a sensory reflex arc.42

In the present study, increasing concentrations of 5-HT were applied to spontaneously contracting intestinal tissue specimens from horses, and concentration-dependent increases in basal tone in duodenal and ileal preparations were observed. Interestingly, this effect of 5-HT was not evident in pelvic flexure specimens. Because there is no information about the distribution of 5-HT receptor subtypes within the equine gastrointestinal tract, it is not possible to find plausible explanations for this observation. What is known, however, is that 5-HT immunoreactivity varies among intestinal locations in adult horses; the levels of 5-HT immunoreactivity in duodenum and ileum are twice as high as the level in the large intestine.43 In guinea pigs, 5-HT and tegaserod cause the same maximum stimulation in colon peristalsis.44 Data from an in vitro study45 involving rectal tissue from dogs indicated that tegaserod induces 55% of the maximum response induced by 5-HT, which is in agreement with the results of the present study. Most available data indicate that tegaserod is a potent partial 5-HT4 receptor agonist. Nevertheless, it has recently been reported46 that tegaserod blocks 5-HT2B receptors as well. Because of their mixed-drug–specific and tissue-dependent properties, 5-HT4 agonists are able to express tissue selectivity (ie, behave as partial agonists in some tissues and as full agonists in other tissues).47 In fact, agonist properties of tegaserod against recombinant receptors and receptors in isolated tissue preparations differ,48 although the extent to which these findings impact the clinical efficacy of tegaserod as a prokinetic agent remains to be determined. Results of a previous study16 of samples of small intestine obtained from horses are in concordance with findings of the present study; 5-HT increased the basal tone in a concentration-dependent fashion when administered as single doses of various concentrations. However, when serotonin was added to longitudinal jejunal preparations in a cumulative manner (10–10 to 10–6M) in that study,16 a bell-shaped concentration-response curve was generated, whereas we did not encounter a desensitization problem over time in our study. Similarly, in another previous study,49 desensitization of equine jejunal specimens did not occur as a result of cumulative administration of 5-HT. In the present study, preincubation of tissue preparations from the small intestine with tegaserod resulted in a significant decrease in basal tone, compared with the effect of 5-HT alone. In in vitro experiments in guinea-pig ileum, tegaserod revealed an intrinsic activity of 0.2, which corresponds to a fifth of that possessed by the full agonist 5-HT.50 On the basis of that finding, tegaserod can induce a submaximal tissue response (acting as a partial agonist) and may competitively block the effect of serotonin (acting as a full agonist), causing a decrease in contraction activity. In duodenal preparations, the effect of tegaserod on basal tone at different 5-HT concentrations was significant.

The results of the EFS experiments involving cumulative application of tegaserod also indicated that tegaserod has the functional characteristics of a partial agonist. The EFS protocol was set up to induce sub-maximal contractions, therefore, the amplitude of contractions could be augmented or decreased via application of tegaserod to the organ bath. Tegaserod caused an increase in Amax (compared with the effect of solvent) in equine intestinal specimens from all 3 locations. These observations are at least partly conflicting with results obtained in a previous study13 in which the ability of tegaserod to initiate contraction activity in preparations of ileum and pelvic flexure from horses was investigated. In that study,13 tegaserod increased the incidence of contractions in pelvic flexure specimens, but had no effect on contractility in ileal specimens. The discrepancy in findings between that investigation and the present study might be due to a difference in study design; in 1 study, the initiation of spontaneous contractility in noncontracting specimens was assessed, and in the other, modulation of the amplitude of existing spontaneous contractions was investigated. The prokinetic effect of tegaserod in equine pelvic flexure specimens is supported by data reported by Delco et al.14

On the basis of the results of the present study, it appears that the effect of tegaserod depends on the contractile status of the tissue. The partial 5-HT4 agonist caused both attenuation of 5-HT–induced contractions and stimulation of submaximal contractions elicited by EFS. The pharmacological properties of partial agonists might be exploited to develop effective promotility drugs.51 The features of tegaserod render it a promising candidate for treatment of gastrointestinal tract motility disorders in horses,52 particularly because acceleration in gastrointestinal transit in healthy horses following administration of tegaserod has been reported recently.15 Moreover, because of their drug-specific and tissue-related properties, 5-HT4 receptor agonists are tissue selective.47 Given the variable effects of selective compounds that target the different 5-HT receptor subtypes, an evaluation of the structure, function, and occurrence of 5-HT receptor subtypes within the gastrointestinal tract of horses could provide better understanding of the intestines' location-dependent differences in serotonin response.

Abbreviations

5-HT

5-hydroxytryptamine

Amax

Maximal amplitude of contractions

EC50

Concentration that causes 50% of the maximal effect

EFS

Electrical field stimulation

ICC

Interstitial cell of Cajal

KH

Krebs-Henseleit

Vmax

Maximal attainable response

a.

Horse Genome Project, University of Veterinary Medicine Hannover, Hannover, Germany. Available at: www.tiho-hannover.de/einricht/zucht/hgp/index.htm. Accessed May 2008.

b.

Novus Biologicals, Littleton, Colo.

c.

Streptavidin/Biotin Blocking Kit SP 2002, Vector Laboratories Inc, Burlinghame, Calif.

d.

Jackson Immuno Research Laboratories, West Grove, Pa.

e.

DAPI, Sigma Chemical Co, St Louis, Mo.

f.

Fluoprep, Biomérieux, Marcy l'Etoile, France.

g.

Abcam, Cambridge, Mass.

h.

Zeiss Axio Imager Z.1 equipped with an AxioCam MRm digital camera, Carl-Zeiss AG, Feldback, Switzerland.

i.

Image J, version 1.4.3, National Institutes of Health, Bethesda, Md. Available at: rsbweb.nih.gov/ij/index.html. Accessed July 15, 2009.

j.

Hugo Sachs Electronics, March-Hugstetten, Germany.

k.

Type 351, Hugo Sachs Electronics, March-Hugstetten, Germany.

l.

Power Lab and Chart, ADInstruments, Spechbach, Germany.

m.

Sigma-Aldrich, Buchs, Switzerland.

n.

Sigma, St Louis, Mo.

o.

Novartis AG, Basel, Switzerland.

p.

Grass S 88, Grass Instruments, Quincy, Mass.

q.

SYSTAT, 9.0.6.1, SPSS Inc, Chicago, Ill.

r.

NCSS 2007, Number Cruncher Statistical Systems, Kaysville, Utah.

s.

MATLAB Simulation Software, version R2009a, The Math-Works, Natick, Mass.

t.

Streutker CJ, Colley EC, Hillsley K, et al. 5HT4 receptor-immunoreactivity (5HT4-IR) is expressed by non-neuronal cells, including mast cells, in human, rat and mouse gastrointestinal tracts (abstr). Mod Pathol 2007;20:131A

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  • Figure 1—

    Representative photomicrographs illustrating the localization of 5-HT4 receptors and c-kit by use of immunofluorescence staining performed on cryostat sections of duodenum, ileum, and pelvic flexure obtained from 2 horses. For each tissue type, findings for 1 experimentally treated section (columns marked Exp) and 1 control section (columns marked Con) are provided. Experimentally treated sections were double labeled with anti–c-kit and anti–5-HT4 receptor antibodies, and Cy2- and Cy3-tagged streptavidin were used to detect c-kit (green fluorescence; top-row images) and 5-HT4 receptors (red fluorescence; middle-row images), respectively. In matching control sections, primary antibodies were omitted. For the experimentally treated and control sections for each tissue type, top- and middle-row images were merged to create the bottom-row images. Notice that merged images revealed no colocalization of 5-HT4 and c-kit immunoreactivity. CM = Circular muscle layer. LM = Longitudinal muscle layer. MP = Myenteric plexus. Bar = 100 μm (applies to all images).

  • Figure 2—

    Representative photomicrographs illustrating the localization of 5-HT4 receptors by use of immunofluorescence staining performed on 2 cryostat sections of ileum obtained from 2 horses. Findings for 1 experimentally treated section (column marked Exp) and 1 control section (column marked Con) are provided. The experimentally treated section was labeled with anti–5-HT4 receptor antibody and Cy3-labeled streptavidin; primary antibody was omitted in the control section. Differential interference contrast microscopy was used to observe structural details in the tissue sections (top-row images). In the middle-row images, receptor localization is indicated by red fluorescence. For each section, top- and middle-row images were merged to create the bottom-row images. In the merged image for the experimentally treated section, expression of 5-HT4 receptors is localized in longitudinal and circular muscle layers as well as in myenteric blood vessels. Bar = 200 μm (applies to all images). See Figure 1 for key.

  • Figure 3—

    Representative photomicrographs (obtained by use of a 10× [A and B] and 40× [C] objective lens) illustrating the localization of 5-HT4 receptors by use of immunofluorescence staining performed on cryostat sections of ileum obtained from 2 horses. Each section was labeled with anti–5-HT4 receptor antibody and Cy3-labeled streptavidin (receptor localization indicated by red fluorescence [top-row images]) and also with 4',6-diamidino-2-phenylindole (nuclei indicated by blue fluorescence [middle-row images]). Primary antibodies were omitted from control sections (not shown). For each section, top- and middle-row images were merged to create the bottom-row images. In the merged images, 5-HT4 receptor immunoreactivity is evident in mucosal villi (A) and blood vessels (B and C) in the mucosa (M) and tela submucosa (SM), respectively. Bar = 200 μm (applies to all images).

  • Figure 4—

    Effect (determined in organ bath experiments) of 5-HT on basal tone in longitudinal specimens of duodenum (n = 9; A), ileum (10; B), and pelvic flexure (10; C) obtained from 24 horses following preincubation of specimens for 20 minutes without (black circles) or with (inverted black triangles) tegaserod (1 × 10–6M). Following the preincubation period (60 minutes), the concentration of 5-HT in the organ bath was increased logarithmically (from 10–10 to 10–5M); control specimens were preincubated without tegaserod and exposed only to solvent (H2O [white circles]). Data are reported as mean basal tone (as a percentage of the predrug period value) and SEM. *Response to 5-HT alone (without tegaserod preincubation) is significantly (P < 0.05) different from the corresponding response to solvent. Response to 5-HT following tegaserod preincubation is significantly (P < 0.05) different from the corresponding response to 5-HT alone. Significant (P < 0.05) effect over time in this treatment group.

  • Figure 5—

    Effect (determined in organ bath experiments) of 5-HT on Amax of spontaneous contractions in longitudinal specimens of duodenum (n = 9; A), ileum (10; B), and pelvic flexure (10; C) obtained from 24 horses following preincubation of specimens for 20 minutes without (black circles) or with (inverted black triangles) tegaserod (1 × 10 –6M). Following the preincubation period (60 minutes), the concentration of 5-HT in the organ bath was increased logarithmically (from 10 10 to 10–5M); control specimens were preincubated without tegaserod and exposed only to solvent (H2O [white circles]). Data are given as mean Amax (as a percentage of the predrug period value) and SEM. See Figure 4 for remainder of key.

  • Figure 6—

    Effect (determined in organ bath experiments) of tegaserod (black circles) on Amax of EFS-induced contractile activity in longitudinal specimens of duodenum (n = 12; A), ileum (14; B), and pelvic flexure (6; C) obtained from 24 horses. Each smooth muscle preparation was allowed to equilibrate for 60 minutes before tegaserod was added to the organ bath. The concentration of tegaserod in the organ bath was increased logarithmically (from 10–10 to 10–6M); control specimens were incubated without tegaserod and exposed only to solvent (methylpyrrolidone [white circles]). Data are reported as mean and SEM. *Response to tegaserod is significantly (P < 0.05) different from the corresponding response to solvent. Significant (P < 0.05) effect over time in this treatment group.

  • 1

    Hoyer D, Hannon JP, Martin GR. Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol Biochem Behav 2002; 71:533554.

    • Search Google Scholar
    • Export Citation
  • 2

    Gershon MD & Tack J. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 2007; 132:397414.

    • Search Google Scholar
    • Export Citation
  • 3

    Gershon MD, Liu MT. Serotonin and neuroprotection in functional bowel disorders. Neurogastroenterol Motil 2007; 19(suppl 2):1924.

  • 4

    Spiller R. Serotonergic modulating drugs for functional gastrointestinal diseases. Br J Clin Pharmacol 2002; 54:1120.

  • 5

    Kim HS. 5-Hydroxytryptamine4 receptor agonists and colonic motility. J Smooth Muscle Res 2009; 45:2529.

  • 6

    Kim DY & Camilleri M. Serotonin: a mediator of the brain-gut connection. Am J Gastroenterol 2000; 95:26982709.

  • 7

    Ji S W, Park H, Chung J P, et al. Effects of tegaserod on ileal peristalsis of guinea pig in vitro. J Pharmacol Sci 2004; 94:144152.

  • 8

    Prather CM, Camilleri M, Zinsmeister AR, et al. Tegaserod accelerates orocecal transit in patients with constipation-predominant irritable bowel syndrome. Gastroenterology 2000; 118:463468.

    • Search Google Scholar
    • Export Citation
  • 9

    Tonini M & Pace F. Drugs acting on serotonin receptors for the treatment of functional GI disorders. Dig Dis 2006; 24:5969.

  • 10

    Kaneene JB, Ross WA & Miller R. The Michigan equine monitoring system. II. Frequencies and impact of selected health problems. Prev Vet Med 1997; 29:277292.

    • Search Google Scholar
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