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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2010 Mar 5;159(8):1623–1625. doi: 10.1111/j.1476-5381.2010.00653.x

Activation of submucosal 5-HT3 receptors elicits a somatostatin-dependent inhibition of ion secretion in rat colon

N Yang 1,*,†, SM Liu 2,*, LF Zheng 1, T Ji 1, Y Li 1, XL Mi 1, H Xue 1, W Ren 3, JD Xu 1, XH Zhang 1, LS Li 1, Y Zhang 1, JX Zhu 1
PMCID: PMC2925486  PMID: 20233224

Abstract

Background and purpose:

5-Hydroxytryptamine (5-HT) is a key regulator of the gastrointestinal system and we have shown that submucosal neuronal 5-HT3 receptors exerted a novel inhibitory effect on colonic ion transport. The aim of the present study was to investigate the precise mechanism(s) underlying this inhibitory effect.

Experimental approach:

Mucosa/submucosa or mucosa-only preparations from rat distal colon were mounted in Ussing chambers for measurement of short-circuit current (Isc) as an indicator of ion secretion. Somatostatin release was determined with radioimmunoassay. Intracellular cAMP content was measured with enzyme-linked immunoadsorbent assay (elisa). Immunohistochemical techniques were used to study the expression of 5-HT3 receptors, somatostatin and somatostatin receptors in colonic tissue.

Key results:

In rat distal colonic mucosa/submucosa preparations, pretreatment with 5-HT3 receptor antagonists enhanced 5-HT-induced increases in Isc. However, in mucosa-only preparations without retained neural elements, pretreatment with 5-HT3 receptor antagonists inhibited 5-HT-induced ΔIsc. Pretreatment with a somatostatin-2 (sst2) receptor antagonist in mucosa/submucosa preparations augmented 5-HT-induced ΔIsc. Combination of sst2 and 5-HT3 receptor antagonists did not cause further enhancement of 5-HT-induced ΔIsc. Moreover, both sst2 and 5-HT3 receptor antagonists enhanced 5-HT-induced increase in intracellular cAMP concentration in the mucosa/submucosa preparations. 5-HT released somatostatin from rat colonic mucosa/submucosa preparations, an effect prevented by pretreatment with 5-HT3 receptor antagonists. Immunohistochemical staining demonstrated the presence of 5-HT3 receptors on submucosal somatostatin neurons and of sst2 receptors on colonic mucosa.

Conclusion and implications:

Activation of neuronal 5-HT3 receptors in the submucosal plexus of rat colon suppressed 5-HT-induced ion secretion by releasing somatostatin from submucosal neurons.

Keywords: 5-HT3 receptor, somatostatin, colonic ion transport

Introduction

5-Hydroxytryptamine (5-HT) is an important biogenic regulator, widely distributed in the gastrointestinal (GI) tract. 5-HT mediates responses as diverse as nausea, vomiting, secretion, peristalsis, and has been implicated in many GI dysfunctions such as irritable bowel syndrome and inflammatory bowel disease (Hicks et al., 2002; Spiller, 2008). More than 95% of the GI 5-HT is present in the enterochromaffin cells, which are widely dispersed over the intestinal mucosa. In addition, 5-hydroxytryptaminergic neurons also exist in the enteric nervous system (ENS), but the amount of 5-HT in enteric neurons is very low in comparison with the amount in enterochromaffin cells (Racke et al., 1996). 5-HT secreted by enterochromaffin cells or enteric neurons acts on 5-HT receptors (5-HT1, 5-HT2, 5-HT3, 5-HT4 and 5-HT7; nomenclature follows Alexander et al., 2009) located on smooth muscles, enterocytes and nerves to regulate GI motility, secretion and sensation (O'hara et al., 2004; Moskwa and Boznanska, 2007).

In vitro studies suggested that 5-HT-evoked intestinal secretion can be described by two pathways: neural and non-neural (Budhoo et al., 1996; Hansen and Skadhauge, 1997; Kiso et al., 1997). The non-neural pathway involves activation of the 5-HT4 receptors residing at the level of the colonocyte, while the neural pathway is mainly mediated by 5-HT3 receptors (Ning et al., 2004; King et al., 2004a,b;). 5-HT4 receptors belong to G protein-coupled receptor superfamily that stimulate cAMP production in response to 5-HT (Ning et al., 2004). Unlike other 5-HT receptor subtypes, the 5-HT3 receptor is a ligand-gated cation channel and usually exerts excitatory influences within the GI tract (Camilleri et al., 1999; Crowell, 2001). Activation of 5-HT3 receptors promotes GI motility, secretion and sensation (Rahimi et al., 2008). As a possible therapeutic target in bowel disorders, 5-HT3 receptors have become a centre of attention (Budhoo et al., 1996; Kiso et al., 1997; Glatzle et al., 2002). 5-HT3 receptor antagonists have been shown to slow down colonic transit and increase fluid absorption, thus improving symptoms in some diarrhoea-predominant irritable bowel syndrome patients (Camilleri et al., 1999; 2001;). Understanding the 5-HT3 receptor regulatory pathways in GI secretion is likely to provide mechanistic insight into the aetiology and treatment of GI disorders or diseases.

Our recent study demonstrated an inhibitory effect on colonic ion secretion mediated by activation of 5-HT3 receptors in the submucosal plexus (Yang et al., 2008). This finding provides novel insights into the role of 5-HT3 receptors in GI secretion. However, the mechanism(s) underlying the inhibitory effect of 5-HT3 receptors is not fully understood.

Somatostatin is an important regulatory peptide in the mammalian GI tract, where it inhibits a wide variety of processes, including cell proliferation, motility, release of digestive enzymes/regulatory peptides and electrogenic ion secretion (Warhurst et al., 1993; Samson et al., 2000). The effects of somatostatin are mediated by five G protein-coupled high affinity receptors (sst1 to sst5) (Hope et al., 2001). In vitro studies suggest that somatostatin-induced inhibition of basal or forskolin/carbachol-stimulated ion secretion in the rat isolated distal colonic mucosa is mediated by somatostatin receptor-2 (sst2) (Mckeen et al., 1995). Moreover, regulation of somatostatin release by 5-HT has been reported in the GI tract (Koop and Arnold, 1984; Racke et al., 1996). These results suggest that somatostatin might be involved in the inhibitory action of 5-HT3 receptor on rat colonic ion transport. Thus, the aim of the present study was to test the hypothesis that activation of the neuronal 5-HT3 receptors in the submucosal plexus inhibits colonic secretion through somatostatin release.

Methods

Animal care and tissue preparation

The animal care and experimental protocols were approved by the Animal Care and Use Committee of Capital Medical University and met NIH guidelines. Adult male Sprague-Dawley rats (Laboratory Animal Services Center, Capital Medical University) ranging 200–300 g had free access to standard rodent laboratory food and water until the day of experiments. The animals were killed by cervical dislocation. The distal colon was removed and defined as the ∼7 cm long segment proximal to the lymph node (typically situated 3 cm from the anus). The distal colon was then divided into four segments, termed DC1 (adjacent to the lymph node), DC2, DC3 and DC4 respectively. Preliminary results indicated that the responses to 5-HT were different in the four segments (Yang et al., 2006); however, the responses in DC3 and DC4 were more reliable and consistent. Thus, DC3 and DC4 segments were used in the present study. Each segment was cut along the mesenteric border into a flat sheet and flushed with ice-cold oxygenated Krebs-Henseleit solution (K-HS) containing (in mM): 117 NaCl, 4.7 KCl, 1.2 MgCl2·6H2O, 1.2 NaH2PO4, 25 NaHCO3 and 2.5 CaCl2·2H2O. The tissue was pinned flat with the mucosal side down in a Petri dish containing ice-cold oxygenated K-HS. The serosa and the muscle layers were carefully removed under a dissecting microscope to obtain the mucosa/submucosa preparation. For the mucosa preparation, the submucosal plexus was also removed.

Ussing chamber experiments

Flat sheets of colonic mucosa-only or mucosa/submucosa preparations were mounted in modified Ussing chambers with a cross-sectional area of 0.5 cm2. The mucosal and serosal surfaces of the tissue were bathed with 5 mL K-HS circulated from a reservoir maintained at 37°C and bubbled with 95% O2 and 5% CO2 to maintain the pH at 7.4. Drugs were added directly to the basolateral sides of the samples, and responses were recorded continuously. Transepithelial potential difference for every colonic sample was measured with Ag/AgCl reference electrodes (Physiologic Instruments, San Diego, CA, USA; P2020S) connected to a preamplifier that was in turn connected to a voltage-clamp amplifier (Physiologic Instruments, San Diego, CA, USA; VCC MC6). The change in short-circuit current (Isc) was calculated as the difference between before and after drug application. Isc was normalized as current per unit area of epithelium (µA·cm−2), which allowed the area under the curve for 15 min to be calculated (µA·min).

Immunofluorescence

Frozen sections

Segments of rat distal colon were fixed for 10 min at room temperature in acetone and then washed (3 × 10 min) in phosphate buffer solution (PBS). The segments were embedded in optimum cutting temperature medium (McCormick, St. Louis, MO, USA) and cut at 8 µm thickness with a Cryostat microtome (Leica CM1850, St. Gallen, Switzerland). The sections were air dried for 1 h at room temperature, permeabilized with 0.3% Triton X-100 overnight at 4°C, blocked with 10% normal goat serum (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature, followed by primary antibody incubation (4°C, overnight). Then, sections were washed in PBS (3 × 10 min) and incubated in the secondary antibody diluted in 0.3% Triton X-100 PBS for 1 h. After the final wash, the tissue sections were coverslipped and observed under a fluorescence microscope (Leica DM LB2, St. Gallen, Switzerland).

Whole-mount preparations

For preparing whole-mount submucosal plexus, the distal colon segments were opened along the mesenteric border, stretched tautly, pinned out flat with mucosa side up onto a Sylgard-coated Petri dish. Preparations were fixed in Zamboni's fixative (4% formaldehyde plus 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.0) for 3 h at room temperature. Whole mounts of the submucosal plexuses were dissected from these segments under a stereoscope. The mucosa was removed with fine-tip dissecting forceps, and the submucosal plexus was cut off using a pair of microscissors (Liu S et al., 2008; Xue et al., 2009). Whole-mount preparations were permeabilized with 0.3% Triton X-100 in PBS or 10 min and blocked with 10% normal donkey serum (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature. The preparations were then incubated with the primary antibody for 5-HT3 receptors for 48 h at 4°C. After washing in PBS, the tissues were incubated in FITC-labelled donkey anti-rabbit IgG at room temperature for 1 h. The tissue was washed in PBS and coverslipped with VECTASHIELD Mounting Medium (Vector lab, Burlingame, CA, USA). Fluorescence labelling was examined under the fluorescence microscope to ensure the quality of the labelling. The tissues were then washed in PBS and subsequently incubated with the appropriate primary and secondary antibodies for somatostatin or Hu C/D with intermittent washes. After a thorough rinse, the tissues were coverslipped with VECTASHIELD mounting medium and examined and photographed on a Nikon EclipseTE300 microscope using a Spot-RT digital camera (Diagnostic Instruments, Sterling Heights, MI, USA). Preabsorption of the 5-HT3 receptor antibodies with their corresponding immunizing peptides (Calbiochem, San Diego, CA, USA) was carried out to determine if this would abolish the immunostaining. Immunostaining was also performed by omitting either the primary or the secondary antibody, and the antibodies were replaced by the solution for antibody dilution. Information on primary and secondary antibodies used in this study is summarized in Tables 1 and 2.

Table 1.

Primary antibodies

Antibody Immunizing antigen Host species Dilution Source/catalogue no.
5-HT3 receptor A synthetic peptide (SLEKRDEMREVARD) corresponding to amino acids 444–457 of rat 5-HT3 receptor, conjugated to KLH Rabbit 1:1000 Calbiochem/PC347
Somatostatin Somatostatin conjugated to KLH using glutaraldehyde method Sheep 1:4000 American research Products/13-2366
sst2 receptor Epitope corresponding to amino acids 320–369 mapping at the C-terminus of sst2a human receptor Rabbit 1:500 Santa Cruz/sc-25676
HuC/D 12 amino acid synthetic peptide representing amino acids 240–251 from human HuD (monoclonal 16Al1) Mouse 1:50 Mol. Probes/A21271
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5-Hydroxytryptamine; Hu, antihuman neuronal protein; sst2, somatostatin receptor-2.

Table 2.

Secondary antibodies

Secondary antibody Conjugation Dilution Source/catalogue no.
Goat anti-rabbit IgG FITC 1:200 Sigma/F9887
Donkey anti-sheep IgG Cy3 1:500 Jackson/713-166-147
Donkey anti-rabbit IgG FITC 1:100 Jackson/713-095-147
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Cy3, indocarbocyanin; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G.

Antibody characterization

Information on primary antibodies used in this study is summarized in Table 1. Further details regarding the specificity of each of the primary antibodies are given below.

The anti-5-HT3 receptor antibody was raised against to a synthetic peptide corresponding to amino acids 444–457 of rat 5-HT3 receptor. No Western blotting data are available for this antibody. Nevertheless, as detailed in Results, the 5-HT3 receptor antibody displayed the same pattern of cellular morphology and distribution as previously published for the rat ENS (Glatzle et al., 2002). Specificity of the 5-HT3 receptor antibody was also tested by preadsorption of the 5-HT3 receptor antibody with the corresponding blocking peptide provided by the manufacturers and by omitting the secondary antibody. Both abolished immunostaining in the submucosal plexus (not shown).

The sheep somatostatin antibody was raised against a synthetic somatostatin peptide conjugated to KLH. No Western blotting data are available for this antibody. Nevertheless, the somatostatin antibody displayed the same pattern of cellular morphology and distribution as previously published for the ENS (Liu et al., 2005).

The anti-sst2 receptor antibody was raised against the C-terminus of the sst2a receptor of human origin (amino acids 320–369). This antibody identified a single band of 54 kDa by Western blotting in the rat cardiomyocytes (Bell et al., 2008). Ablation of sst2 receptor immunoreactivity (IR) by omitting the primary antibody of sst2 receptor or by preadsorption with an excess of the corresponding synthetic peptide confirmed the specificity of the antibody (data not shown).

Anti-human neuronal protein C/D (HuC/D) monoclonal (clone 16A11) antibody labelled neuronal cell nuclei and perikarya (Marusich et al., 1994). The staining pattern of cellular morphology and distribution in the present work was the same as previously described for the ENS (Lin et al., 2002).

cAMP measurement

Rat colonic mucosa/submucosa samples (about 150 mg) were incubated in K-HS solution for 30 min for equilibration. The samples were pretreated with vehicle (saline), 5-HT3 receptor antagonists or sst2 receptor antagonist for 5 min before adding 5-HT (10 µM, 5 min). All the samples were frozen in liquid nitrogen immediately, then homogenized on ice in saline (0.9% NaCl) and centrifuged (10 620×g, 5 min). Intracellular levels of cAMP were determined by enzyme-linked immunoadsorbent assay (elisa) according to the manufacturer's instructions (Sigma). The stimulation of cAMP formation was occurring in enterocytes and that none of the changes in cAMP were related to ganglion cells in the submucosal plexus. This is because activation of the neuronal 5-HT3 receptor, which is a type of ligand-gated ion channel, does not stimulate cAMP formation.

Somatostatin release

Rat colonic mucosa/submucosa tissues (about 150 mg) were incubated in tubes containing 500 µL K-HS solution for 30 min for equilibration. The tissues were pretreated with vehicle (0.9% NaCl), 5-HT3 receptor antagonists or sst2 receptor antagonist for 5 min before adding 5-HT (10 µM, 5 min). At the end of the incubation, all samples in the tubes were frozen in liquid nitrogen immediately, then homogenized on ice in saline (0.9% NaCl) and centrifuged (10 620×g, 5 min). Somatostatin levels in the supernatant were measured by a commercial radioimmunoassay (RIA) kit (Beijing Sinouk Institute of Biological Technology, Beijing, China).

Statistical analysis

The data were expressed as means ± SEM (standard error of mean); n refers to the number of rats/tissues used. Comparisons between groups of data were made via Student's paired or unpaired t-test as appropriate. A P-value of less than 0.05 was considered statistically significant.

Materials

5-HT, indomethacin, MDL72222 and tetrodotoxin (TTX) were obtained from Sigma Chemical Company. CYN 154806, tropanyl-3, 5-dimethylbenzoate and 2-methyl-5-HT hydrochloride were purchased from Tocris Cookson Inc. (Ellisville, MO, USA). The agonists and antagonists concentrations were chosen based upon previous reports. Stock solutions of some chemicals (indomethacin, MDL72222, GR113808, tropanyl-3, 5-dimethylbenzoate) were dissolved in dimethyl sulphoxide (DMSO), with final DMSO concentrations less than 0.1% (vol/vol). Preliminary experiments indicated that the vehicle did not alter any baseline electrophysiological parameters.

Results

Effects of 5-HT3 receptor antagonists on 5-HT-induced ΔIsc in colonic mucosa/submucosa preparations

Previous studies have reported that 5-HT produced a concentration-dependent increase in Isc with an EC50 of 5.4 ± 0.8 µM in the rat colon (Budhoo and Kellum, 1994). Thus, 10 µM was chosen as the concentration for 5-HT in the present study. As in our previous findings, addition of 5-HT (10 µM) to the basolateral side of the colonic mucosa/submucosa preparations elicited an increase in Isc, while apical application of 5-HT had a negligible effect (Ning et al., 2004). Therefore, in the present study, 5-HT and 5-HT receptor agonists/antagonists were added only to the basolateral side of the tissue. After equilibration for 30 min, the baseline Isc and the transepithelial resistance (Rt) of the mucosa/submucosa preparations of rat distal colon were 45.2 ± 4.6 µA·cm−2 and 39.6 ± 4.0 Ω·cm2 (n= 17).

The cyclooxygenase (COX) pathway plays a major role in mediation of the secretory response to exogenous 5-HT in vitro (King et al., 2004a). Indomethacin (10 µM), a COX inhibitor, was routinely used to abolish endogenous prostaglandin production involved in this process (Gierse et al., 1995). Basolateral application of indomethacin resulted in a significant reduction of baseline Isc values by 26.3 ± 3.7 µA·cm−2 (n= 17).

To investigate the role of 5-HT3 receptors in the submucosal plexus in 5-HT-induced colonic secretion, 5-HT3 receptor antagonists or agonists were added to the basolateral side of the preparations. Pretreatment with 5-HT3 receptor antagonists MDL72222 (0.1 µM; Figure 1A) or tropanyl-3, 5-dimethylbenzoate (0.1 µM; Figure 1B) for 5 min did not inhibit, but increased 5-HT (10 µM)-induced ΔIsc (P < 0.01). Effects of different doses of MDL72222 (0.001, 0.01, 0.1, 1 and 10 µM) on the 5-HT (10 µM)-induced ΔIsc in rat mucosa/submucosa preparations were also tested. Pretreatment with MDL72222 at lower doses (0.01 and 0.1 µM) significantly enhanced 5-HT-evoked ΔIsc (Figure 1C). The enhancing effect of MDL72222 on 5-HT-evoked ΔIsc became weaker when its concentration increased to 1.0 and 10 µM. In more than half of the preparations, pretreatment with MDL72222 at 1.0 and 10 µM inhibited, instead of enhancing, 5-HT-induced Isc response (Figure 1C).

Figure 1.

Figure 1

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Effect of 5-HT3 receptor antagonists on 5-HT-induced Isc responses in rat distal colon mucosa/submucosa preparations. (A1) Representative Isc recording with arrow indicating the basolateral application of 5-HT (10 µM) to the mucosa/submucosa preparations pretreated with MDL72222 (0.1 µM). (A2) Summary of the effects of MDL72222 (0.1 µM) on 5-HT-induced ΔIsc. (B1) Representative Isc recording showing the effect of tropanyl-3, 5-dimethylbenzoate (0.1 µM) on 5-HT-induced Isc responses. (B2) Summary of the effects of Tropanyl-3, 5-dimethylbenzoate (0.1 µM) on 5-HT-induced ΔIsc. Values are means ± SEM; **P < 0.01; n= 12. (C1) Effects of different concentrations of MDL72222 on 5-HT-induced Isc responses in rat distal colonic mucosa/submucosa preparations. Numbers in the table indicate the number of tissues demonstrating the variable effect on Isc at different doses of MDL72222. (C2) Summary of the effects of different doses of MDL72222 on 5-HT-induced ΔIsc. Values are means ± SEM; *P < 0.05; **P < 0.01; n= 6 (0.001 µM), n= 12 (0.01 µM), n= 11 (0.1 µM), n= 14 (1.0 µM), n= 12 (10 µM). 5-HT, 5-Hydroxytryptamine; Isc, short-circuit current.

Effects of tetrodotoxin

5-HT3 receptors are found not only on neurons, but also on epithelial cells (Day et al., 2005). To determine the origin of the inhibitory effect on colonic ion secretion mediated by activating the 5-HT3 receptors, TTX (1 µM), a voltage-gated Na+ channel blocker, was applied to the basolateral side to block the neurally mediated colonic ion secretion in the mucosa/submucosa preparations (Pérez-navarro et al., 2005). TTX pretreatment did not significantly change the baseline Isc or the Rt. In the presence of TTX, 5-HT (10 µM)-induced ΔIsc was significantly inhibited by the 5-HT3 antagonist, MDL72222 (0.1 µM) (P < 0.01; Figure 2A). These results, combined with the results in the above section, suggested that the neuronal 5-HT3 receptor in the submucosal plexus mediated an indirectly inhibitory action on colonic secretion, while the non-neuronal 5-HT3 receptors on the epithelium mediated a directly stimulatory action on colonic secretion.

Figure 2.

Figure 2

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Effects of 5-HT3 receptor antagonist on 5-HT-induced Isc responses in rat colonic mucosa/submucosa and mucosa-only preparations in the presence of TTX. (A1) Representative Isc recording with arrow indicating the basolateral application of 5-HT (10 µM) to the mucosa/submucosa preparations pretreated with MDL72222 (0.1 µM) plus TTX (1 µM). (A2) Summary of the effects of MDL72222 (0.1 µM) plus TTX (1 µM), on 5-HT-induced ΔIsc. Values are means ± SEM; **P < 0.01; n= 9. (B1/C1) Representative Isc recording with arrows indicating the basolateral application of 5-HT (10 µM) to rat mucosa-only preparations pretreated with MDL72222 (0.1 µM, B1) or tropanyl-3, 5-dimethylbenzoate (0.1 µM, C1) plus TTX (1 µM) respectively. (B2/C2) Summary of the effects of MDL72222 (0.1 µM, B2) and tropanyl-3, 5-dimethylbenzoate (0.1 µM, C2) on 5-HT-induced ΔIsc. Values are means ± SEM; *P < 0.05; **P < 0.01; n= 5 respectively. 5-HT, 5-Hydroxytryptamine; Isc, short-circuit current; TTX, tetrodotoxin.

To further test our hypothesis, rat colonic mucosa-only preparations (submucosa was removed) were also used. After 30 min equilibration, the baseline Isc and Rt of the rat distal colonic mucosa were 54.2 ± 6.0 µA·cm−2 and 37.9 ± 4.3 Ω·cm2 (n= 10) respectively. To avoid the effects of any remaining enteric neurons and endogenous prostaglandins, both TTX (1 µM) and indomethacin (10 µM) were applied to the basolateral side of the mucosa-only preparations. Treatment with indomethacin resulted in a significant reduction of the baseline Isc to 30.6 ± 2.6 µA·cm−2 (n= 10) and TTX did not have distinct influence on Rt or baseline Isc. Basolateral pretreatment with MDL72222 (0.1 µM) or tropanyl-3, 5-dimethylbenzoate (0.1 µM) significantly inhibited 5-HT (10 µM)-induced increase in Isc (P < 0.05, Figure 2B) and (P < 0.01, Figure 2C) respectively. Similar to a previous reported by Day et al. (Day et al., 2005), basolateral addition of 5-HT3 receptor agonist, 2-methyl-5-HT (100 µM), to the mucosa-only preparations induced an increase in Isc (294.5 ± 84.5 µA·min; n= 7), which was blocked by pretreatment with the 5-HT3 receptor antagonist, MDL72222 (0.1 µM, n= 4). 2-methyl-5-HT, given at 10 µM, failed to induce an increase in Isc. (Ning et al., 2004).

Effects of the sst2 receptor antagonist, CYN 154806

Somatostatin is expressed in the submucosal plexus and has been known to decrease basal as well as forskolin/carbachol-induced Cl- secretion in rat colonic mucosa via activation of sst2 receptors (Mckeen et al., 1995). The sst2 receptor antagonist CYN 154806, when used at 0.1–1 µM, was able to inhibit somatostatin-induced decrease in Isc (Holliday et al., 2007). In the present study, basolateral application of CYN 154806 (0.5 µM) significantly increased 5-HT-induced ΔIsc in rat mucosa/submucosa preparations (P < 0.01, Figure 3A). Comparing with CYN 154806 (0.5 µM) or MDL72222 (0.1 µM) alone, the combination of CYN 154806 (0.5 µM) and MDL72222 (0.1 µM) did not cause any further enhancement of 5-HT-induced Isc response (Figure 3B). The similar enhancing effects on 5-HT-induced ΔIsc by 5-HT3 receptor antagonists and sst2 receptor antagonist suggest that somatostatin might be involved in the inhibitory effect on colonic secretion through activation of the neuronal 5-HT3 receptors.

Figure 3.

Figure 3

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Effect of the sst2 receptor antagonist CYN 154806 on 5-HT-induced Isc responses in rat distal colon mucosa/submucosa preparations. (A1) Representative Isc recording with arrow indicating the basolateral application of 5-HT (10 µM) to the mucosa/submucosa preparations pretreated with CYN 154806 (0.5 µM). (A2) Summary of the effect of CYN 154806 (0.5 µM) on 5-HT-induced ΔIsc. Values are means ± SEM; **P < 0.01; n= 7. (B1) Representative Isc recording with arrow indicating the basolateral application of 5-HT (10 µM) to the mucosa/submucosa preparations pretreated with CYN 154806 (0.5 µM) and MDL72222 (0.1 µM). (B2) Summary of the effect of CYN 154806 (0.5 µM) and MDL72222 (0.1 µM) on 5-HT-induced ΔIsc. Values are means ± SEM; **P < 0.01; n= 4. 5-HT, 5-Hydroxytryptamine; Isc, short-circuit current; sst2, somatostatin receptor-2.

Intracellular cAMP measurement

It has been reported that somatostatin inhibits colonic ion secretion through activation of sst2 receptors on epithelia cells and inhibition of intracellular cAMP accumulation (Warhurst et al., 1996). The 5-HT3 receptor is a ligand-gated cation channel that does not affect the intracellular cAMP concentration. In order to determine which neurotransmitter directly inhibits 5-HT-evoked colonic ion transport, we tested the effects of 5-HT3 antagonists and an sst2 receptor antagonist on 5-HT-stimulated intracellular cAMP accumulation in the colonic mucosa/ submucosa preparations. The basal level of cAMP was 6.9 ± 1.1 pM·mg−1 (n= 6). Pretreatment with the sst2 receptor antagonist, CYN 154806 (0.5 µM), augmented the 5-HT (10 µM)-induced increase in intracellular cAMP concentration (P < 0.01; Figure 4). Consistent with our hypothesis, after pretreatment with 5-HT3 antagonists, MDL72222 (0.1 µM) or tropanyl-3, 5-dimethylbenzoate (0.1 µM), 5-HT (10 µM)-stimulated intracellular cAMP production was similarly increased (P < 0.01; Figure 4). Pretreatment with the 5-HT4 receptor antagonist GR113803 (1 µM), which has been shown to totally block 5-HT-induced increase in Isc in rat colon mucosa preparations at this concentration (Ning et al., 2004), did not significantly inhibit 5-HT-induced accumulation of intracellular cAMP concentration.

Figure 4.

Figure 4

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Intracellular cAMP measurement. Summary of the effects of pretreatment with the sst2 receptor antagonist, CYN 154806 (0.5 µM), 5-HT3 receptor antagonists, MDL72222 (0.1 µM) and tropanyl-3, 5-dimethylbenzoate (0.1 µM), and 5-HT4 receptor antagonists, GR113808 (0.5 µM) on 5-HT (10 µM)-stimulated intracellular cAMP production in the rat colonic mucosa/submucosa preparations. **P < 0.01; n= 6 respectively. 5-HT, 5-Hydroxytryptamine; sst2, somatostatin receptor-2.

Expression of 5-HT3 receptor on colonic submucosal somatostatin neurons

In order to provide morphological evidence supporting the release of somatostatin from submucosal plexus as an inhibitory neurotransmitter following activation of the neuronal 5-HT3 receptors, rabbit anti-5-HT3 receptor and sheep anti-somatostatin were used to double-label the 5-HT3 receptor- and somatostatin-immunoreactive neurons in the submucosal plexus of the rat distal colon. In frozen sections of the rat distal colon, 5-HT3 receptor IR was observed in neurons in close proximity to mucosa and was weakly expressed in the crypts (Figure 5A). 5-HT3 receptor IR was observed mostly in somatostatin-positive neurons in the submucosal ganglia (Figure 5B,C). In whole-mount submucosal plexus of the rat distal colon, 5-HT3 receptor IR was detected in fibres as well as neuronal cell bodies (Figure 5D). The staining pattern, cellular morphology and distribution of 5-HT3 in the present study were the same as previously described for the rat ENS (Glatzle et al., 2002). Omission of the primary antibody or preadsorption of the primary antibody with the corresponding immunogen peptide abolished the immunostaining in the distal colon (data not shown). Double labelling with a pan-neuronal marker anti-human neuronal protein HuC/D (Figure 5E) revealed that 5-HT3 was localized to enteric neurons and did not appear in neighbouring glial cells (Figure 5F). Double labelling immunohistochemistry in the whole mounts demonstrated that almost all of the 5-HT3-immunopositive submucosal neurons contained somatostatin IR (Figure 5G–I).

Figure 5.

Figure 5

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Expression of 5-HT3 receptor in somatostatin neurons of the rat colonic submucosal plexus. (A–C) Immunoreactivity of 5-HT3 receptor (A) and somatostatin (B) in the frozen sections of the rat distal colon. (D–F) Immunoreactivity of 5-HT3 receptor in whole-mount preparations of the rat distal colon. 5-HT3 receptor (green) was expressed in two neurons of a submucosal ganglion and nerve fibres. Neuronal phenotype was confirmed using a pan-neuronal marker HuC/D (red). (G–I) 5-HT3 receptor (green) and somatostatin (red) was colocalized in neurons in whole-mount preparations of the submucosal plexus. Scale bars, 20 µm. 5-HT, 5-Hydroxytryptamine.

Expression of sst2 receptors in colonic mucosa

Experiments with antibody to sst2 receptors showed IR in the rat distal colonic mucosa and that this was widely located in the colonic crypts (Figure 6A and B). Ablation of sst2 receptor IR by omitting the primary antibody of the sst2 receptor and preadsorption with an excess of the corresponding synthetic peptide confirmed the specificity of the antibody (Figure 6C).

Figure 6.

Figure 6

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Expression of sst2 receptors in the epithelium of the rat distal colonic mucosa. sst2 receptor immunoreactivity was found to be abundantly expressed in the crypts of the rat distal colonic mucosa (A. lower magnification; B. higher magnification). (C) Omission of the primary antibody of sst2 receptors resulted in no immunostaining. sst2, somatostatin receptor-2.

5-HT-induced somatostatin release

To further test the hypothesis that somatostatin is involved in 5-HT3-mediated inhibition of colonic ion secretion, RIA was used to measure somatostatin release following 5-HT3 receptor activation in the rat mucosa/submucosa preparations (Figure 7). In samples without any stimulation, that is, basal conditions, there was a low level of somatostatin in the supernatant of rat colonic mucosa/submucosa preparations (Figure 7). Incubation of the rat colonic mucosa/submucosa preparations with 5-HT (10 µM) caused an increase in the concentration of somatostatin in the supernatant (P < 0.01). Pretreatment with the 5-HT3 antagonists MDL72222 (0.1 µM) or tropanyl-3, 5-dimethylbenzoate (0.1 µM) significantly inhibited the 5-HT-induced somatostatin release to about basal values (P < 0.05). The sst2 receptor antagonist, CYN 154806 (0.5 µM), did not affect 5-HT-induced somatostatin release.

Figure 7.

Figure 7

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Radioimmunoassay of somatostatin release in rat colonic mucosa/submucosa preparations. A summary of the effects of pretreatment with different concentrations of 5-HT3 antagonist, MDL72222 (0.1 µM, n= 6) and tropanyl-3, 5-dimethylbenzoate (0.1 µM, n= 5), as well as the sst2 receptor antagonist, CYN 154806 (0.5 µM, n= 5) on the 5-HT (10 µM, n= 6)-stimulated somatostatin release. n= 7. *P < 0.05 compared with 5-HT treatment alone. 5-HT, 5-Hydroxytryptamine; sst2, somatostatin receptor-2.

Discussion

The usual explanation of 5-HT action in the gut is that this amine modulates intestinal secretion predominantly by direct activation of the epithelia 5-HT4 receptors (Budhoo and Kellum, 1994; Ning et al., 2004; Fang et al., 2008), which are G protein-coupled receptors stimulating intracellular cAMP accumulation. An indirect pathway mediated by the 5-HT3 receptors, which are non-selective cationic channels on the intrinsic neural plexuses of the gut, has also been proposed (Cooke, 2000; Kozlowski et al., 2000). Recently, a non-neuronal 5-HT3 receptor-mediated chloride secretion was also reported in the rat distal colonic mucosa (Day et al., 2005). A combined blockade of 5-HT3 and 5-HT4 receptors completely inhibited 5-HT-induced diarrhoea in mouse and rat (Nagakura et al., 1997). Most recently, we found a novel dual role of 5-HT3 receptors in 5-HT-induced ion transport in rat distal colon (Yang et al., 2008). Besides the well-known stimulatory effect on intestinal secretion, activation of the submucosal neuronal 5-HT3 receptors also inhibits ion secretion. In the present study, we further investigated the mechanism(s) underlying the inhibitory effect mediated by the neuronal 5-HT3 receptors.

Submucosal 5-HT3 receptor-mediated inhibition of colonic ion secretion

Our results demonstrated that blocking 5-HT3 receptors increased, rather than decreased, 5-HT-induced ΔIsc in the rat distal colonic mucosa/submucosa preparations. This enhancing effect was abolished by the neuronal blocker TTX, suggesting that activation of neuronal 5-HT3 receptors in the submucosal plexus inhibits colonic ion transport in the mucosa/submucosa preparations. Similarly, when the submucosal plexus was removed, the enhancing effect of 5-HT3 antagonists on 5-HT-induced secretory response was diminished. These data confirm that 5-HT3 receptor-mediated inhibition of colonic ion secretion requires the presence of the submucosal plexus.

Submucosal plexus contains a variety of neurotransmitters involved in regulation of ion secretion. Activation of the neuronal 5-HT3 receptors in the submucosal plexus is expected to cause the release of one or more types of neurotransmitters, which in turn inhibit colonic secretion. Among these, somatostatin has been known to inhibit basal and stimulated chloride secretion in colonocytes, both by a direct action on the colonocytes and by inhibiting other enteric neurons (Mckeen et al., 1995; Warhurst et al., 1996; Cooke et al., 2003). Somatostatin is present in about 15–20% of secretomotor neurons in the submucosal plexus (Furness, 2000) and also in D cells of the gastric and duodenal epithelia (Booth et al., 2001). Once released, somatostatin is believed to function as an inhibitory neurotransmitter to inhibit colonic secretion by binding to the sst2 receptors on epithelial cells to reduce intracellular cAMP (Mckeen et al., 1995; Warhurst et al., 1996) and/or by hyperpolarizing the membrane potential and suppressing excitability of secretomotor neurons in the submucosal plexus (Shen and Surprenant, 1993;Liu et al., 2000) (Figure 8A). Several lines of evidence support our hypothesis that somatostatin is involved in neuronal 5-HT3-mediated inhibition of colonic ion secretion. First, as do 5-HT3 receptor antagonists, the sst2 receptor antagonist increased 5-HT-induced Isc responses in the colonic mucosa/submucosa preparations, suggesting that the neural 5-HT3 receptor and somatostatin in the submucosal plexus mediate inhibition of 5-HT-induced transepithelial ion transport. Second, pretreatment of rat submucosa/mucosa preparations with the sst2 receptor antagonist or 5-HT3 receptor antagonists further enhanced 5-HT-induced intracellular cAMP accumulation in the colonic mucosa/submucosa preparations. As 5-HT3 receptors are a ligand-gated cation channels and have no direct effect on intracellular cAMP concentration, other neurotransmitter(s)/neuromodulator(s) (e.g. somatostatin) must be involved. Moreover, immunohistochemical staining demonstrated the expression of 5-HT3 receptor on submucosal somatostatin neurons and sst2 receptors in colonic mucosa, providing histological evidence. Finally, 5-HT induced somatostatin release from the colonic mucosa/submucosa preparations, which was blocked by 5-HT3 receptor antagonists. All of these evidence suggest that activation of the 5-HT3 receptors at the level of the submucosal plexus inhibits colonic ion secretion by stimulating somatostatin release from submucosal neurons (Figure 8A). Once released, somatostatin activated sst2 receptors in intestinal epithelial cells to reduce intracellular cAMP concentration and inhibit ion secretion.

Figure 8.

Figure 8

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An illustration of the working model of 5-HT3 and 5-HT4 receptors in rat colonic submucosal plexus and mucosa. (A) Submucosal 5-HT3 receptor-mediated somatostatin-dependent inhibitory neural pathway. Activation of 5-HT3 receptors in the submucosal plexus leads to the release of somatostatin. Somatostatin binds with to the sst2 receptors on the colonic epithelium and reduces the intracellular cAMP concentration, which leads to a decrease of ion secretion in the rat colon. Somatostatin also inhibits secretomotor neuron activity, which leads to a decrease of ion secretion. (B) Epithelial non-neuronal pathway mediated by 5-HT3 and 5-HT4 receptors. In this pathway, 5-HT binds to 5-HT3 and 5-HT4 receptors in the rat colonic epithelium and stimulates ion secretion in rat colon. 5-HT, 5-Hydroxytryptamine; sst2, somatostatin receptor-2.

Contrary to our present finding in the rat distal colon, Cooke et al. (2003) have shown that activation of the neuronal 5-HT3 receptors in the submucosal plexus of the guinea pig distal colon stimulates colonic Cl- secretion, which is mainly mediated by the cholinergic neuronal pathway. Although this discrepancy is hard to resolve, species and regional differences may contribute to the divergent findings on the effect of 5-HT in vitro on electrolyte transport and Isc. In our previous study, we found that even within the same species, epithelial ion transport induced by stimulants demonstrated regional heterogeneity in the rat distal colon (Yang et al., 2006). The 5-HT uptake, metabolism and neuronal systems have need noted as potential sources of variation between species and between the regions of the same species (Chetty et al., 2006).

Epithelial 5-HT3 receptor-mediated stimulation of colonic ion secretion

5-HT3 receptors are not only expressed in enteric neurons, but also in non-neuronal cells, such as intestinal epithelium. In the rat distal colonic mucosa preparations without submucosal neural elements, the 5-HT3 receptor antagonists suppressed the 5-HT-induced secretory responses. Furthermore, the 5-HT3 receptor agonist, 2-methyl-5-HT, induced an increase in Isc in mucosa-only preparations, which was abolished by the 5-HT3 receptor antagonists. This observation is consistent with the report by Day et al. (2005) and suggests that the 5-HT3 receptors located at the colonic mucosa level stimulate intestinal secretion (Figure 8B). The exact cellular location and function of these non-neuronal 5-HT3 receptors are not known. Day et al. (2005) suggested that the epithelial 5-HT3 receptors may exist on the enterochromaffin cells as an autocrine receptor in releasing 5-HT.

Our pharmacological experiments with 5-HT3 antagonists did not demonstrate a concentration-dependent relationship on the enhancement of 5-HT-induced ion transport in the colonic mucosa/submucosa preparations (Figure 1C). A possible reason for this result could be related to the opposite effects by activating the 5-HT3 receptors at different locations within the GI tract. At lower concentrations, the 5-HT3 antagonists have easy access to the 5-HT3 receptors on submucosal neurons as the drugs were added to the basolateral side of the preparations. Blockade of the neuronal 5-HT3 receptors and enhancing 5-HT-induced ion transport would be the predominant effect. When the concentrations of the 5-HT3 antagonists were raised, the drugs would not only block the 5-HT3 receptors on the submucosal neurons, but also antagonize the 5-HT3 receptors on the colonic epithelial cells that would reduce 5-HT-induced colonic ion secretion. The weakening of the 5-HT-evoked responses by higher concentrations of MDL72222 might also reflect a non-selective local anaesthetic action on secretomotor neurons (Fozard, 1984). However, the exact mechanism still need to be clarified in future studies.

In summary, this study provides evidence that activation of the neuronal 5-HT3 receptor in the submucosal plexus suppressed colonic ion secretion by causing somatostatin release from submucosal neurons. Somatostatin activates sst2 receptors either on non-somatostatin submucosal neurons, which inhibit excitability of secretomotor neurons (Shen and Surprenant, 1993; Liu et al., 2000), or on colonocytes to decrease intracellular cAMP levels; both would lead to the inhibition of colonic ion secretion. Activation of the non-neuronal 5-HT3 receptor on the epithelium stimulates colonic ion secretion. These findings provide novel insights into the role of 5-HT signalling system in gut function and may also help to explain the complicated symptoms caused by the disturbance of the 5-hydroxytryptaminergic system in the gut.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (30770799; 30971076; 30911130139, JX Zhu), Beijing Natural Science Foundation Program and Scientific Research Key Program of Beijing Municipal Commission of Education (KZ200910025003, JX Zhu) and Beijing Municipal Project for Developing Advanced Human Resources for Higher Education (PHR201007110, JX Zhu).

Glossary

Abbreviations:

DMSO

dimethyl sulphoxide

elisa

enzyme-linked immunoadsorbent assay

ENS

enteric nervous system

GI

gastrointestinal

Isc

short-circuit current

K-HS

Krebs-Henseleit solution

PBS

phosphate buffer solution

RIA

radioimmunoassay

sst2 receptor

somatostatin receptor-2

TTX

tetrodotoxin

Conflict of interest

The authors have no financial, consultant, institutional and other relationships that might lead to bias or a conflict of interest.

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