Expression and role of 5-HT7 receptor in brain and intestine

Original articleExpression and role of 5-HT7 receptor in brain and intestine in rats with irritable bowel syndromeZOU Bai-cang, DONG Lei, WANG Yan, WANG Sheng-hao and CAO Ming-boKeywords: irritable bowel syndrome; 5-hydroxytryptamine; 5-hydroxytryptamine7 receptor; cyclicadenosine monophosphateBackground The 5-hydroxytryptamine7 receptor (5-HT7 receptor, 5-HT7R) plays an important role in the regulation of smooth muscle relaxation and visceral sensation and might be involved in the pathogenesis of the gastrointestinal dyskinesia, abdominal pain and visceral paresthesia in irritable bowel syndrome (IBS). The aim of this study was to investigate the role of the 5-HT7 receptor in the pathogenesis of IBS.Methods A rat model of irritable bowel syndrome with diarrhea (IBS-D) was established by colonic instillation of acetic acid and restraint stress. A rat model with irritable bowel syndrome with constipation (IBS-C) was established by stomach irrigated with 0–4°C cool water daily for 14 days. The content and distribution of 5-HT in the brain and gut were examined by immunohistochemistry and the mRNA expression of the 5-HT7 receptor was determined by fluorescent quantitative reverse transcription polymerase chain reaction. The accumulation of cyclic adenosine monophosphate (cAMP) in all the same tissues was measured by radioimmunity.Results The models of IBS were reliable by identification. The immunohistochemistry results showed that there were significantly more 5-HT positive cells in the IBS-D group than in the control group in the hippocampus, hypothalamus, jejunum, ileum, proximate colon and distal colon (P<0.05), as well as more than were found in the IBS-C group in jejunum and ileum (P<0.05). There were more 5-HT positive cells in the IBS-C group than in the control hippocampus, hypothalamus, ileum, proximate colon, and distal colon (P<0.05). Real time-PCR results showed that the expression level of the 5-HT7 receptor in both the IBS-C and IBS-D groups were enhanced compared with the control group in the hippocampus and hypothalamus (P<0.05). The expression level of 5-HT7 receptors in the IBS-C group was notably greater when compared with the controls in the ileum and colon (P<0.05). The cAMP accumulation in the hippocampus and hypothalamus in both the IBS-C and IBS-D groups was higher than that in the control group (P<0.01 and P<0.05).The cAMP accumulation in the IBS-C group was higher than that in the control group in the proximal and distal colon (P<0.05).Conclusions The increased 5-HT content in the brain and intestine is related to the IBS pathogenesis. The up-regulated expression of the 5-HT7 receptor in the brain and colon might play an important role in the pathogenesis of IBS-C.Chin Med J 2007;120(23):2069-2074rritable bowel syndrome (IBS) is present in 10% to 22% of the general population and is the most common cause for patient visits to gastroenterologists. According to the clinical symptoms it is mainly divided into two subgroups: irritable bowel syndrome with diarrhea (IBS-D) and irritable bowel syndrome with constipation (IBS-C). The pathogenesis of IBS is not completely clear.5-hydroxytryptamine (5-HT) is an important neurotrans- mitter regulating the function of the gut. 5-HT and its receptors are extensively distributed in the central nervous system, peripheral nervous system and gastrointestinal tract and participate in regulating the functions of psychology, nerves and gut. The biological effect of 5-HT is activated through a combination of different receptors located in all the effector tissues and cells. The functions of 5-HT are diverse in different organisms because of different receptors. It is known there are 7 types of 5-HT receptors (5-HT1-7R), one of the receptor, 5-HT7R, a G-protein-coupling receptor, has been recently shown to have at least 4 subtypes1 and plays a role in regulating smooth muscle relaxation in the gastrointestinal and peripheral nociceptive pathways.2-6 5-HT7R may be involved in the pathological mechanisms of gastrointestinal dyskinesia, abdominal pain and visceral paresthesia in IBS.7 However its role in the pathogenesis of IBS hasn’t been reported. Our study aimed to investigate the role of 5-HT7R in the pathogenesis of IBS by observing the difference in expression of 5-HT, 5-HT7R and cAMP in the brain and intestine in two rat models of IBS.IDepartment of Gastroenterology, Second Hospital of Xi'anJiaotong University, Xi’an 710004, China (Zou BC, Dong L, WangY, Wang SH and Cao MB)Correspondence to: Prof. DONG Lei, Department ofGastroenterology, second hospital of Xi'an Jiaotong University,Xi’an 710004 (Tel: 86-029-********. Email: donglei4488@sina.com)This study was supported by the key clinical project of ChineseMinistry of Health (2004-2006).METHODSAnimal model and experimental groupsForty-five adult Sprague-Dawley rats (220―250 g) were obtained from the Animal Supplied Centre of Xi’an Jiaotong University, Xi’an, China. All procedures were in accordance with the guidelines of Xi’an Jiaotong University Animal Care and Use Committee. The rats were randomly separated into 3 groups: control group (n=15), IBS-C group (n=15) and IBS-D group (n=15). Rats of the IBS-C group were pretreated with 0–4°C cool water (2 ml) to irrigate the stomach daily for 14 days.8 Rats of IBS-D group, after fasting for 12 hours, were anesthetized by inhaling ether and then a silica gel pipe was inserted into the colon about 8 cm from the anus. 4% acetic acid (1 ml) was instilled into the colon through the pipe and retained for 30 seconds then the colon was flushed with 2 ml PBS buffer. After 6 days of recovery, their forelegs were constrained to limit activities for 3 hours.9 The control group did not receive any intervention. All rats were maintained under controlled temperature (21–24°C) and light (lights on 8:00–20:00) conditions with free access to water and laboratory chow pellets. Tissue preparationEight rats from each group were deeply anesthetized with chloralhydrate (2 ml/kg, ip) and received intracardiac perfusion with 300 ml of cool normal saline followed by 500 ml 4% p-formaldehyde. The brain tissues, 2 cm segments of jejunum, ileum, proximal colon and distal colon were removed, post-fixed for 12 hours in 4% p-formaldehyde and immersed in 25% sucrose until the tissue sank to the bottom. All tissues were sectioned with a cryostat at –20°C and mounted on gel-coated slides. The other 7 rats from each group were deeply anesthetized with chloralhydrate and then the hippocampus, hypothalamus, jejunum, ileum, proximal colon and distal colon were carefully removed to determine mRNA by real time-PCR and to assay cAMP by radioimmunity. Immunohistochemical staining for 5-HT5-HT staining was performed by the method of immunohistochemical ABC. The primary antibody was rabbit anti-rat 5-HT antibody (Boster Bio-project Co, Wuhan, China) and the biotin-labeled secondary antibody was sheep anti-rabbit IgG (Boster Bio-project Co. Wuhan, China). Tissue sections were incubated in the primary antibodies (1:1500 dilution) at 4°C for 24 hours and then incubated in the secondary antibody at room temperature for 1 hour and finally with the ABC complex for 30 minutes. Tissue sections were stained by DAB (Zhongshan Bio-project Co, Beijing, China) and every slide was observed under a light microscope and positive cells were counted in 5 random visual fields. Phosphate buffered saline (PBS) was substituted for the first antibody as the negative control.Determination of 5-HT7 receptor mRNATotal mRNA was extracted from all tissues with the Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions and 1.5 μg of total RNA was used for reverse transcription with ExSccript TM RT reagent Kit (TaKaRa Biotechnology Co, Dalian, China). Real Time RT-PCR was performed with Master mix (TOYOBO Co, LTD, Japan) following the manufacturer’s protocol. The 5-HT7 receptor primers were designed as Genebank accession #NM 022938: forward primer: 5’- GCTCATCACGCTGCTGACGAT-3’; reverse primer: 5’-CGCCAGGGACACAATCAGG-3’, product length: 106 bp; the primer of GAPDH gene as control was designed as Genenbank Accession #NM 17701: forward primer: 5’-CAGTGCCAGCCTCGTCTCATA-3’; reverse primer: 5’-TGCCGTGGGTAGAGTCA TA-3’, product length: 184 bp. Samples were incubated at 95°C for 15 seconds, followed by 40 cycles; each cycle consisted of 58°C for 16 seconds, 72°C for 45 seconds, followed by a final extension at 72°C for 5 minutes with an ABI prism 7000 thermal cycler (ABI, USA). Each sample of GAPDH and 5-HT7R were amplified under the same conditions and every reaction were designed with a free control (reacting system without template) to eliminate contamination. We used the relative minimum cycle number to calculate the comparative gene copy of 5-HT7R: 2Ct(GAPDH) - Ct(5-HT7R).Radioimmunity assay of cAMP accumulationAbout 50 mg of tissue from each sample was homogenated in cool acetic acid, centrifuged and torrefied according to the Kit instructions (125I-cAMP RIA Kit, Shanghai University of Chinese Medicine, Shanghai, China), then assayed with a γ-immunity indicator (FM 2000, Xi’an, China).Statistical analysisAll data were expressed as mean ± standard deviation (SD). The significance of differences was analyzed by Student t test and One-way ANOV A using SPSS 10. 0. A P value < 0.05 was considered significantly different.RESULTSReliability of IBS modelThe methods to establish the IBS models have been used in many study and the models had the clinical characteristics of the IBS subgroups confirmed by experimental identification.8-14 In this experiment, the models were also identified from the feces conditions, the bowel transit time and visceral sensation. They were consistent with the pathological characteristic of IBD (described in another paper in press). We found the IBS models were reliable disease models for this study. Immunohistochemical staining of the 5-HTThe number of 5-HT positive cells in both the IBS-C and IBS-D groups were higher than in the control group in both the hippocampus and hypothalamus (P<0.01). 5-HT positive cells in IBS-D rats were also more numerous than in the control group in the jejunum, ileum, proximal colon, and distal colon (P<0.05) and more numerous than in the IBS-C group in the jejunum and ileum. In theFig. 1. Immstunohiochemical positive staining for the 5-HT (original magnification × 400). A: hippocampus; B: hypothalamus; C: jejunum; D: ileum; E: proximal colon; and F: distal colon.Table 1. Comparison of 5-HT positive cells (Mean ± SD, n =8)Groups Hippocampus HypothalamusJejunum Ileum Proximal colon Distal colon IBS-D 85.13±20.59 81.75±3.64 49.56±4.53 35.76±5.09 30.17±3.14 33.09±5.44 IBS-C 78.63±14.82∗∗ 95.63±27.5∗∗ 34.89±7.40 23.20±4.02 ∗∗ 30.43±3.81∗∗ 32.58±5.04 ∗∗ Control 53.00±10.56 52.5±11.6 33.19±2.2316.33±3.1620.45±3.37 11.98±3.76*P <0.05, **P <0.01 vs control group; #P <0.05, ##P <0.01 vs IBS-C group.IBS-C group 5-HT positive cells in the ileum, proximal colon and distal colon were increased compared with the same tissues in the control group (P <0.05, Fig. 1 and Table 1).Expression of the 5-HT 7 receptor in the two subgroups of the IBS modelBecause the quality of RNA greatly affects the results, RNA was evaluated with a spectrophotometer and a 260/280 value above 1.80 was required. By electropho- resis in 1.0% agarose gel, the electrophoresis strip of real time RT-PCR products were consistent with the predict length of the primers (Fig. 2). The expression level of 5-HT 7R in the hippocampus and hypothalamus in boththe IBS-C and IBS-D groups was enhanced comparedFig. 2. Electrophoresis picture of 5-HT 7R and GAPDH. M: marker; lanes 1-4: distal colon, proximal colon, ileum and jejunum of control group; lanes 5-8: distal colon, proximal colon, ileum and jejunum of IBS-C; and lanes 9-11: hippocampus of IBS-C, IBS-D and control group.with the control (P <0.05). The expression level of 5-HT 7R in the ileum, proximal colon and distal colonTable 2. Comparison of 5-HT7 receptor mRNA copies (Mean ± SD, n=7)Group Hippocampus(×10) Hypothalamus(×10)Jejunum (×10)Ileum (×10)Proximal colon (×10) Distal colon (×10)IBS-C 15.75±7.08 16.46±8.2329.42±12.5034.07±15.99 18.79±10.5717.85±10.5IBS-D 16.32±6.43* 15.93±3.54*22.83±11.2724.27±8.59 9.05±4.15 10.97±4.05 Control 8.77±3.59 9.49±3.5926.44±8.1918.01±8.819.89±5.48 9.11±5.01 *P<0.05 vs control; #P<0.05 vs IBS-D.Table 3. Comparison of cAMP content (pmol/mg)(Mean ± SD, n=7)Group Hippocampus Hypothalamus Jejunum Ileum Proximal colon Distal colonIBS-C0.36±0.66 0.35±0.050.27±0.09 0.23±0.07 0.33±0.03 0.40±012IBS-D0.25±0.03# 0.26±0.04# 0.19±0.020.25±0.040.25±0.070.20±0 05Control0.19±0.050.21±0.030.20±0.070.22±0.090.23±0.060.24±0.09P<0.05, P<0.01 vs control; P<0.05, P<0.01 vs IBS-D.of IBS-C was higher than that of the control (P<0.05), but there was no statistical difference between IBS-C and the control group in the gut (Table 2).Radioimmunity assay of cAMPThe cAMP accumulation in the hippocampus and hypothalamus in both the IBS-C and IBS-D groups was higher than the control (P<0.01 and P<0.05). The cAMP accumulation of IBS-C was higher than the control at the proximal colon and distal colon (P<0.05). The change of cAMP accumulation in each group was parallel with 5-HT7 receptor expression (Table 3).DISCUSSIONSerotonin (5-HT) is an important neurotransmitter and paracrine signaling molecule in the gastrointestinal tract. The enteric nervous system (ENS) comprises a semiautonomous effector system that is connected to the central autonomic network. Parasympathetic and sympathetic nerves modulate the ENS via afferent and efferent communications. Bidirectional brain-gut interactions involving the 5-HT pathways occur that significantly influence the effector systems. In the gut, 5-HT release from enterochromaffin cells (EC) initiates peristaltic, secretion, vascular tone and vagal and nociceptive reflexes. Recently researches found that intestinal mucosa caused gut peristalsis and evokes the release of 5-HT after receiving irritation. 5-HT then combines with its receptor to provoke peristalsis and regulate intestinal secretions.15 5-HT is known to markedly influence bowel function by activating at least 5 receptor types (5-HT(1, 2, 3, 4, 7)R). Among all 5-HT receptors those belonging to the 5-HT3, an ionotropic receptor and 5-HT4, a metabotropic receptor type, are the most extensively studied in gastroenterology; resulting in commercially available serotonergic agents for the treatment of IBS and functional dyspepsia. Recently, 5-HT7 receptors have been found to participate in the accommodation process of the circular muscles during the preparatory phase of ileal peristalsis.16 An exaggerated accommodation of the gut wall may contribute to abdominal distension, bloating and other symptoms about gastrointestinal dyskinesia.Irritable bowel syndrome (IBS) is a complex disorder thatis associated with altered gastrointestinal motility, secretion and sensation. The underlying mechanisms of IBS are unknown. As all of these aspects of gastrointestinal function involve serotonin signaling between enterochromaffin cells and sensory nerve fibers in the mucosal layer of the gut and potential alterations in mucosal serotonin signaling have been explored as a possible mechanism of altered motility function and sensation in irritable bowel syndrome. In a recent study IBS-C patients had raised platelet-depleted plasma 5-HT concentrations under fasting and fed conditions.17 Enterochromaffin cells in the colon of IBS patients are increased remarkably compared with health controls.185-HT dysfunction is involved in IBS.19 But research into the role of 5-HT and the 5-HT7 receptor in IBS pathogenesis hasn’t been reported.In this study, 5-HT positive staining was extensively seenin the neurons, astrocytes and in some nerve fibers at the cortex, hippocampus, thalamus and hypothalamus. In the gut staining was located mainly in the intestinal gland anda few places in the enteric neurons. We found that 5-HT positive cells in the hippocampus, hypothalamus and gut,in both the IBS-C and IBS-D group, were obviously increased. It implies that there is altered 5-hydroxytryptamine signaling in the brain and gut in all subgroups of irritable bowel syndrome. But there are different clinical manifestation in IBS-C and IBS-D because the different change of the gastrointestinal motility. How does the 5-HT signaling exert its effect in each subgroup of IBS? We hypothesized that the role of5-HT in the pathomechanism of IBS might depend on the difference of its receptor in the brain and gut.The 5-HT7 receptor is present in humans and other animals and is linked with depression, circadian rhythm, neuroendocrine function, affective behaviour, body temperature regulation and smooth muscle relaxation.20 It plays a role in mediating stress and glucocorticoid- induced effects on hippocampal neurogenesis which have been implicated in mood.21 Forebrain hyperactivity due to anxiety might be the psychology-biological mechanism of mental disorders in IBS patients which effect symptom severity and the course of disease.22 In the gut, 5-HT7R was found first in effector cells of the bowel. It takes partin regulating the smooth muscle relaxation of the gastrointestinal tract and restraining gut peristalsis.2-5 Blocking the 5-HT7 receptor may decrease the thresholdpressure volume of evoking enterokinesia.23 Our study showed the expression of 5-HT7R at the hippocampus and hypothalamus in the IBS-C and IBS-D group was enhanced compared with the control group. The expression level of the 5-HT7 receptor in the BS-C group was higher than that of the control group in the ileum and proximal and distal colon. It implies that the high expression of the 5-HT7 receptor might be related to the psychology-biological mechanism of mental disorder in IBS and the mechanism of gastrointestinal dyskinesis in IBS-C.The 5-HT7 receptor is one of the most studied members of the family of G-protein-coupled receptors. Its stimulation promotes the synthesis of cAMP as a second messenger to exert its biological effect. Our radioimmunoassay results showed that the change of cAMP content in each group was paralleled to the change of 5-HT7 receptor. 5-HT7 receptor stimulation caused the activation of adenylate cyclase 1 and adenylate cyclase 8. Activation of adenylate cyclase 1 and adenylate cyclase 8 are expressed in areas of the brain, such as the hippocampus and hypothalamus, where the 5-HT7 receptors are localized. This suggested that 5-HT might regulate cAMP in certain areas of the brain by mobilizing intracellular Ca2+ after activation of 5-HT7(a) receptors to regulate mood and affective behaviour.24 The 5-HT7 receptor can couple adenylate cyclase and regulate cAMP synthesis to cause muscle relaxation.23 Therefore the change in the expression of the 5-HT7 receptor paralleled with cAMP in brain and gut might play an important rolein the pathogenesis of IBS. 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