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JNM J Neurogastroenterol Motil, Vol. 21 No. 3 July, 2015 pISSN: 2093-0879 eISSN: 2093-0887http://dx.doi.org/10.5056/jnm14157 Journal of Neurogastroenterology and Motility Distribution of 5-HT3, 5-HT4, and 5-HT7 Receptors Along the Human Colon Nor S Yaakob,1,2 Kenneth A Chinkwo,1,3 Navinisha Chetty,1 Ian M Coupar,1 and Helen R Irving1*
1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville campus), Parkville Victoria, Australia; 2Drug and Herbal Research Center, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia (Current address); and 3School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia (Current address) Several disorders of the gastrointestinal tract are associated with abnormal serotonin (5-HT) signaling or metabolism where the 5-HT3 and 5-HT4 receptors are clinically relevant. The aim was to examine the distribution of 5-HT3, 5-HT4, and 5-HT7 receptors in the normal human colon and how this is associated with receptor interacting chaperone 3, G protein coupled receptor kin- ases, and protein LIN-7 homologs to extend previous observations limited to the sigmoid colon or the upper intestine. Samples from ascending, transverse, descending, and sigmoid human colon were dissected into 3 separate layers (mucosa, lon- gitudinal, and circular muscles) and ileum samples were dissected into mucosa and muscle layers (n = 20). Complementary DNA was synthesized by reverse transcription from extracted RNA and expression was determined by quantitative or end point polymerase chain reaction.
The 5-HT3 receptor subunits were found in all tissues throughout the colon and ileum. The A subunit was detected in all sam - ples and the C subunit was expressed at similar levels while the B subunit was expressed at lower levels and less frequently. The 5-HT3 receptor E subunit was mainly found in the mucosa layers. All splice variants of the 5-HT4 and 5-HT7 receptors were expressed throughout the colon although the 5-HT4 receptor d, g, and i variants were expressed less often. The major differences in 5-HT receptor distribution within the human colon are in relation to the mucosa and muscular tissue layers where the 5-HT3 receptor E subunit is predominantly found in the mucosal layer which may be of therapeutic relevance.
(J Neurogastroenterol Motil 2015;21:361-369)
Key Words
Colon; G protein coupled receptor kinases; Ileum; Receptors, serotonin
Received: December 22, 2014 Revised: March 31, 2015 Accepted: April 5, 2015 CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
*Correspondence: Helen R Irving, PhD Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkvil e campus), 381 Royal Parade, Parkvil e, VIC 3052, Australia Tel: +61-3-9903-9565, Fax: +61-3-9903-9581, E-mail: [email protected] Financial support: None.
Conflicts of interest: None.
Author contributions: Ian M Coupar and Helen R Irving conceived and designed the study; Nor S Yaakob, Kenneth A Chinkwo, Navinisha Chetty, and Helen R Irving were involved in designing the experiments, acquiring, analysing, and interpreting data; Helen R Irving drafted the manuscript; and all authors were involved in critically revising the manuscript.
ORCID: Nor S Yaakob, http://orcid.org/0000-0003-4726-1972; Kenneth A Chinkwo, http://orcid.org/0000-0002-7369-1880; Navinisha Chetty, http://orcid.org/0000-0002-3524-4385; Helen R Irving, http://orcid.org/0000-0002-1514-0909.
ⓒ 2015 The Korean Society of Neurogastroenterology and Motility J Neurogastroenterol Motil, Vol. 21 No. 3 July, 2015 Nor S Yaakob, et al logous desensitization involving the G protein coupled receptor (GPCR) kinases (GRKs) down regulates surface expression of 5-HT4 receptors.31-33 Proteins such as protein LIN-7 (LIN7) Most of the body serotonin stores are found in the gastro- homolog A to C of Caenorhabditis elegans (also known as Veli1-3 intestinal tract, where abnormalities in serotonin release, trans- in mammals) are likely to be involved in processing 5-HT4 and port and metabolism are associated with dyspepsia, nausea and 5-HT7 receptors to the plasma membrane.34-37 Receptor interact- vomiting, coeliac disease, inflammatory bowel disease, and irrita- ing chaperone 3 (RIC3) assists cell surface assembly of 5-HT3 ble bowel syndrome (IBS).1-5 Inflammation contributes to these receptors.38,39 Therefore, transcripts of RIC3, LIN7 homologs, disorders due to underlying inflammatory states in coeliac disease and GRKs were measured as these proteins contribute to receptor and inflammatory bowel disease and in part because it modulates processing. Although subtle differences in distribution of the re- serotonin levels.1,4,6 ceptors and associated proteins occurred along the colon, only the The serotonin (5-HT) 4 receptor is expressed in several dif- 5-HT3 receptor subunits exhibited any major differences in ex- ferent cell types in the human colon7-16 where it stimulates in- pression between the tissue layers which may be of therapeutic testinal activity (ie, prokinetic action) and is a target for treating constipation predominant IBS (IBS-C) and chronic constipation. The 5-HT4 receptor partial agonist, tegaserod was used for treat-ment of IBS-C until withdrawn in 2007 as it was associated with Materials and Methods
rare adverse cardiovascular effects.1 Currently, the high affinity 5-HT 4 receptor agonist, prucalopride is used to treat chronic or idiopathic constipation refractory to at least two other classes of Full thickness specimens from different regions of the intes- laxatives in the European Union, Canada, and Australia.17 Intes- tine of 24 patients (11 male and 13 female ranging in age from 50 tinal 5-HT7 receptors are often located near 5-HT4 receptors to 88 years [median 73]) were collected immediately following where they augment 5-HT induced responses.11,18 surgical resection and transported to the laboratory in 4oC 5-HT3 receptor antagonists decrease colonic motility, secre- Krebs-Henseleit solution. The patients were undergoing surgical tion and nociception and are used to treat diarrhea predominant resection for colonic cancer and the pathologist indicated that the IBS (IBS-D).1,3 The 5-HT3 receptor is a ligand gated ion chan- specimens obtained as far from the tumor as possible appeared to nel composed of 5 subunits that form homomeric (al 3A sub- be disease free following gross visual examination. The mucosa units) or heteromeric (mixture of 3A and 3B, 3C, 3D, or 3E sub- and associated mesentery plus fat were removed before the longi- units) functionally active channels.19-21 All of the human 5-HT3 tudinal muscle bands (taeniae coli) were dissected and the re- receptor subunit genes are expressed in the gastrointestinal maining intertaenial tissue was cut into strips in the orientation of tract.22-26 Immunohistochemistry studies have pinpointed co-ex- the circular smooth muscle. All tissue was stored in RNAlaterⓇ pression of A, B, C, D, and E subunits in enterocytes and also the (Ambion, Austin Texas, USA) at −80oC. The project was ap- nerves of the myenteric plexus24,27 indicating that heteromeric proved by the Human Research Ethics Committee of the hospi- 5-HT3 receptors are likely to form in the human colon. tal and academic institute and all patients gave informed consent Although 5-HT4 and 5-HT7 receptor splice variants have prior to surgery. been identified in separate studies in the small intestine and co-lon,22,28-30 no study has looked at their tissue distribution along the colon. However, one earlier study showed that the 5-HT3 re- Approximately 18-25 mg excised tissue was homogenized ceptor subunit transcript distribution differed in the sigmoid colon22 using a PRO 200 homogenizer (PRO Scientific Inc, Oxford, providing evidence that specific subunits may be potential targets. CT, USA) and total RNA was extracted using RNeasy Fibrous Therefore the aim of this study was to investigate the distribution kit (Qiagen, Chadstone Center, Victoria, Australia) and Turbo of transcripts of 5-HT receptors and their associated proteins in DNA-freeTM Kit (Life Technologies, Mulgrave, Victoria, the ileum and along the length of the colon. Membrane bound re- Australia) was used to degrade any contaminating DNA. RNA ceptors are subject to strict processing to be positioned correctly was quantified pre and post-DNAse treatment with a Nanodrop and to regulate their responsiveness following activation. Homo- 1000 using ND-1000 software (Thermo Scientific, Wilmington, Journal of Neurogastroenterology and Motility Human Intestinal Serotonin Receptors DE, USA). The reverse transcription step was carried out using plate controls and negative reverse transcriptase controls consist- 1 g of DNAse-treated RNA, SuperScriptⓇIII Reverse ing of RNA samples where reverse transcriptase was not added Transcriptase (Life Technologies) and oligo dT15 primers in a (such that no cDNA was produced) to test for DNA contami- total reaction volume of 20 L. Separate negative reverse-tran- scriptase controls were included for every reaction. Statistical Methods Polymerase Chain Reaction Conditions Each quantitative PCR sample was analyzed in duplicate In end-point polymerase chain reactions (PCR), the primers and efficiency of reactions was determined using linear re- described in a prior study22 were used to amplify GRK, 5-HT4, gression of the Log (fluorescence) per cycle number data with and 5-HT7 receptor genes while the fol owing primers were used to the LinRegPCR program40 and ranged from 2.008 ± 0.006 amplify LIN7A-C and glyceraldehyde dehydrogenase (GAPDH) (-actin) to 1.877 ± 0.008 (5-HT3 receptor C subunit genes. GAPDH (NM_002046.5) forward 5'-ACCACAGTC- [HTR3C]). Expression data was calculated relative to -actin CATGCCATCAC-3' (714-734) and reverse 5'-TCCACCAC- and GAPDH and expressed as the following ratio: Ratio = CCTGTTGCTGTA-3' (1165-1146); LIN7A (NM_004664.2) (Efficiencyreference)Ct reference/(Efficiencysample)Ct sample, where Ct is forward 5'-CAGCTAGTGAAGGCCACTCC-3' (486-505) the crossing point threshold of the sample for the amplified and reverse 5'-GCAGCTGGTCTCCTCTTTTG-3' (659-639); LIN7B (NM_022165.2) forward 5'-CAGCTTTATGACA- Relative expression data was further analyzed after log trans- CGCTGGA-3' (210-229) and reverse 5'-GATGACCCGG- formation using one-way ANOVA followed by Tukey's multiple GAGATGT-3' (413-397); and LIN7C (NM_018362.3) for- comparisons test. One-way ANOVA and Tukey's multiple com- ward 5'-AACAGAAGAGGGCCTTGGAT-3' (516-496) and parisons were used to analyze the number of patients expressing reverse 5'-GCGGCTTTCAGCAGTTCTAC-3' (321-340) which genes detected using end point PCR. The number of ob- wil produce products of 451 (for al 4 splice variants), 174, 203, servations used to derive mean values is expressed by n and arith- and 195 bp respectively. It should be noted that the LIN7A pri- metic mean values are given as mean ± SEM.
mer set is within one exon. Amplifications were undertaken in a MyCycler thermal cycler (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and a hot start involving 15 minutes at 94oC was made. To keep within an exponential range during amplification, 20 to 25 cycles were made with denaturation at 94oC, annealing at Distribution of 5-HT3 Receptor Subunits in 55oC, and extension at 72oC all for 1 minute. Products were vi- the Human Intestine sualized following separation on agarose gels and staining with The distribution of 5-HT3 receptor subunits was examined GelRedTM nucleic acid stain (Biotium, Hayward, CA, USA). using quantitative reverse transcript PCR on samples obtained Quantitative PCR was undertaken using similar amplifica- from throughout the length of the colon and the ileum area of the tion conditions as described in a prior study22 except that the 30 small intestine. In all regions, expression was examined in both L reactions contained 1 L cDNA, 0.5 mol/L forward and the mucosal and muscular tissue layers and reported relative to reverse primers, 4 mmol/L MgCl2, and 2× SensiMix SYBR expression of -actin. GAPDH was used as a second house- Green PCR Master Mix (Bioline, Sydney, NSW, Australia). keeping gene for comparison with a previous study where the rel- The cDNA was amplified by 1 cycle at 95oC for 15 minutes fol- ative expression of the 5-HT3 receptors in the sigmoid colon was lowed by 36 cycles of 95oC for 15 seconds (denaturing), 55oC for reported.22 Control RNA samples incubated without reverse 20 seconds (annealing), and 72oC for 25 seconds (extension) us- transcriptase and then amplified with GAPDH primers were also ing a C1000TM Thermal Cycler and CFX96TM Real-Time undertaken to demonstrate that there was no DNA con- System (Bio-Rad Laboratories). Melting point analysis indicated tamination of the RNA extractions (data not shown). GAPDH that only a single product was produced in each reaction and con- levels of expression were consistently and significantly higher firmed by preliminary runs with gel electrophoresis which also es- than those of the 5-HT3 receptor subunits or RIC3 in both ileum tablished that only one product of the predicted size was pro- and colon (P < 0.05 one-way ANOVA; Fig. 1) which is con- duced. Control runs of all PCR experiments contained non-tem- sistent with the previous study.22 The 5-HT3 receptor D subunit Vol. 21, No. 3 July, 2015 (361-369) Nor S Yaakob, et al Figure 1. Distribution of serotonin type 3 (5-HT3) receptor subunits and receptor interacting chaperone 3 (RIC3) transcripts in human intestinal tissue layers. (A) Comparison of the relative expression levels of transcripts of RIC3 and 5-HT3 receptor subunits in the mucosal and muscle tissue layers in the human ileum (n = 4). (B) Comparison of the relative expression levels of transcripts of RIC3 and 5-HT3 receptor subunits in the mucosaland muscle layers (circular and longitudinal) in tissue samples obtained from throughout the human colon (n = 16). Data are expressed as a ratio relative to -actin as described in the Material and Methods section. Bars indicate the mean. 5-HT3 receptor D subunit (HTR3D) transcripts were not detected in any tissue tested. RIC3 and 5-HT3 receptor subunit transcripts are expressed at significantly lower levels than glyceraldehyde dehydrogenase (GAPDH) transcripts (P < 0.001 one-way ANOVA followed by Tukey's multiple comparisons test) in all tissues. The letters above the x-axis (v, w, x, y, and z representing the highest to lowest level respectively) indicate that the transcripts are found at significantly different levels (P < 0.05 one-way ANOVA followed by Tukey's multiple comparisons test) in the ileum or colon tissue layers (ie, transcripts in the different layers with an x underneath are expressed at the same level; x is the highest and z the lowest level in [A] while v is the highest and z the lowest level in [B]).
was consistently not detected in either ileum or colon samples pressed at significantly lower levels than RIC3 or the A subunits which is in agreement with prior studies where transcripts of in all tissue layers. The mucosal levels of the C subunit were sig- HTR3D were only evident at very low levels.23,25 In the ileum, nificantly higher than the B subunit in any tissue and the C sub- RIC3 and 5-HT3 receptor A subunit transcripts were found in unit in the muscular layers. The 5-HT3 receptor E subunit tran- all samples with the other subunits being less prevalent (Fig. 1A). scripts were nearly always only detected in the mucosa samples at Transcript levels of RIC3 were significantly higher than 5-HT3 levels significantly greater than the B subunit (Fig. 1B). Only receptor B subunits in both mucosa and muscle layers and also one longitudinal muscle sample showed any E subunit expression the mucosa levels of the A subunit and the C subunit in the mus- (Fig. 1B), so to determine if the 5-HT3 receptor E subunit was cle layer. No significant differences were observed in the ex- expressed in the muscle layers but below the reliable detection pression levels of the 5-HT3 receptor subunits with the major ex- levels of the quantitative PCR conditions, the amplified PCR ception that the E subunit was only found in the mucosa layer samples were run in gels (Supplementary Fig. 2). Only very low levels of expression were observed in two additional colon mucosa The colonic tissue was dissected into mucosa, circular and samples and two longitudinal muscle samples as well as an addi- longitudinal muscular layers and a similar distribution of the spe- tional ileum muscle sample. cific transcripts occurred in the different areas (Supplementary Fig. 1). Therefore, the regional data was pooled to see if there Distribution of 5-HT4 and 5-HT7 Receptors in were any differences in gene expression at the tissue layer level the Human Intestine (Fig. 1B). Generally a similar distribution pattern of RIC3 and Adjacent tissue samples to those used in the analysis of the the 5-HT3 receptor subunits in the colonic tissue layers was ob- 5-HT3 receptor and RIC3 gene expression were used to examine served to that seen in the ileum. However some variations were the distribution of 5-HT4 and 5-HT7 receptors. Since several seen in 5-HT3 receptor subunit expression between the different splice variants with overlapping 3' coding regions exist for the tissue layers (Fig. 1B). The 5-HT3 receptor B subunit was ex- 5-HT4 and 5-HT7 receptors, their distribution was examined us- Journal of Neurogastroenterology and Motility Human Intestinal Serotonin Receptors Figure 2. Reverse transcriptase poly-merase chain reaction (RT-PCR) analysis of expression of protein LIN-7 homologs G protein coupled receptor kinase (GRK), serotonin type 4 (5-HT4) and 5-HT7 receptor gene products in the human descending colon of one patient. The products of predicted sizes are indicated by arrows and the size was correlated to 100 bp (molecular weight)markers run on 1.5% agarose gels stain-ed with GelRed. A negative control is shown for the LIN7A sample as the pri-mers are within one exon. Letters repre-sent samples obtained from M (mucosa),C (circular muscle), and L (longitudinal muscle). ing partial or nested PCR. GAPDH expression was used as a ing and transverse colon compared to the descending and sig- control for this analysis as it has previously been shown to be moid colon (Supplementary Table). Both GRK5 and GRK6 are comparable between human colonic tissue layers11 (Fig. 1 and expressed less often in all of the tissue layers compared to the Supplementary Fig. 3). An example of the expression of different 5-HT7 receptor a/b variants and in the mucosa and longitudinal transcripts following end point RT-PCR analysis is shown for a muscle than the 5-HT4 receptor a, b, c and n variants (P < 0.05, descending colon sample (Fig. 2). The samples were obtained one-way ANOVA; Fig. 3). However, no differences in the fre- from ileum, ascending, transverse, descending and sigmoid colon quency of expression were observed between the different GRK and overall the distribution of transcripts appeared independent or LIN7 genes in the different tissue layers (P > 0.05, one-way of the region of the colon and similar across the types of tissue lay- ers (Supplementary Table 1). Therefore the number of patients expressing any of the genes was compared with the colon tissue layers (Fig. 3). One-way ANOVA indicates that the only differ- ences are in the expression of the gene transcripts (P < 0.05) and Despite many similarities in its general functionality, the hu- not the tissue layer. For instance, 5-HT4 receptor d and g splice man intestinal tract exhibits considerable differences to small ani- variants are significantly less likely to be detected in mucosa and mal models such as mice, rats, and guinea pigs.41 Serotonin re- longitudinal muscle compared to the 4a, b, c, and n splice variants ceptors for instance are widespread throughout the gastrointe- (P < 0.05 one-way ANOVA; Fig. 3). Like the 5-HT4 a, b, c, stinal tract in small animal models and humans but the proportion and n splice variants, the 5-HT7 receptor splice variants were and type of receptors at particular regions are different.41-43 In ad- widely expressed in all regions and tissues studied. Some minor dition, human 5-HT receptors are considerably different in their differences are evident with respect to GRK distribution. For ex- composition as evident by the diverse splice variants in 5-HT4 ample, GRK6 is less likely to be expressed in the ileum or ascend- and 5-HT7 receptors and the additional 5-HT3 receptor Vol. 21, No. 3 July, 2015 (361-369) Nor S Yaakob, et al Figure 3. Comparison of the number of patients expressing transcripts of G protein coupled receptor kinase (GRK), protein LIN-7 (LIN7) homologs , and 5-HT4 or 5-HT7 receptor splice variants in the mucosal (A), circular muscle (B), and longitudinal muscle (C) layers in tissue samples obtained from throughout the human colon (n = 16-18). Data are expressed as the number of patients where transcripts were detected. 5-HT4 receptor d and g splice variants in mucosa and longitudinal muscle, GRK5 and GRK6 (all tissue layers) were detected at signi-ficantly lower frequencies than 5-HT7 or 5-HT4 a, b, c, or n receptor splice variants (P < 0.05 one-way ANOVA followed by Tukey'smultiple comparisons test).
subunits.1,19,44 The function and pharmacological responses of to storage-related RNA degradation (Supplementary Fig. 3). 5-HT receptors in the intestine also varies between small animal The robustness of the quantitative PCR was ensured by adhering models and humans15,41,45,46 and therefore this study was under- to the MIQE guidelines47 and samples were run on gels to ensure taken to gain an insight to the distribution of 5-HT3, 5-HT4, and that only the expected products were generated (Supplementary 5-HT7 receptors expressed in the human ileum and throughout Fig. 2). The end point PCR studies were designed to ensure that the human colon. Although subtle differences in the regional dis- the products were still within the linear amplification range. tribution of the 5-HT receptors occurred, the main differences Where expression levels were low (eg, 5-HT4 receptor splice var- were between the mucosal and muscular tissue layers. iant d; Fig. 2), independent observers confirmed the presence of Quantitative and end point PCR were used to assess the dis- detectable bands in the gels. tribution of 5-HT3, 5-HT4, and 5-HT7 receptors using samples The distribution of 5-HT3 receptor A subunits is in keeping obtained from surgical resection that appeared to be disease free with previous studies where this subunit was widespread and following gross visual examination. Tissue samples were stored in thought to reflect the distribution of functional 5-HT3 receptor RNAlaterⓇ at −80oC until use. Only samples that contained subunits.11,22,23,25,48 The HTR3B gene which encodes for the readily detectable levels of GAPDH (and -actin for quantitative canonical 5-HT3 receptor B subunit was expressed at lower levels PCR) and were not contaminated by genomic DNA (no bands than HTR3A in all regions of colon but at similar levels in the detected in the negative reverse transcript assays) were processed ileum (Fig. 1). The actual occurrence of HTR3B in human co- further to exclude the probability of undetectable expression due lon and ileum (Supplementary Fig. 1) was almost 50% less than Journal of Neurogastroenterology and Motility Human Intestinal Serotonin Receptors the total occurrences of HTR3A and agrees with previous find- tory disorders.59,60 ings where lower levels but no distinct patterns of expression were Serotonin induces desensitization (tachyphylaxis) at different observed.23,25,27 Interestingly, the expression and occurrence of the rates and magnitudes in different tissues and this is the case with 5-HT3 receptor C subunit was similar to HTR3A while HTR3E 5-HT4 receptor agonists as highlighted by prucalopride desensi- was predominantly restricted to the mucosa confirming prior ob- tizing pig atrium but not pig stomach 5-HT4 receptors.61 servations in the colon, small intestine and stomach.22,23,25,26,38,49 Different GRKs are associated with 5-HT4 receptor desensitiza- The mucosa plays a role in fluid transport, while the muscular tion depending on the tissue type in the rat and significantly these layers are involved with motility so it is possible that physiological GRKs are not necessarily those predicted by studies using re- alterations in the different colon tissue layers may result in se- combinant proteins expressed in cell culture.32 GRK2 and GRK3 lective colon disease or dysfunction. Immunoreactants to both were relatively well distributed in all tissue layers while GRK5 5-HT3 receptor C and E subunits have been identified as being was less common and GRK6 was absent in the ileum but present co-expressed within the human colon (enterocytes, myenteric in the descending and sigmoid colon, so there may be regional plexus and muscular layer).24 Together these findings are sugges- and tissue layer variation in the GRKs involved in 5-HT4 re- tive that associations of 5-HT3 receptor A subunits occur with C ceptor desensitization in the human colon. The distribution of and/or E subunits in the gastrointestinal tissues. In fact, genetic LIN7 homologs in the human colon is of interest as LIN7C (Veli studies have revealed associations of HTR3C and HTR3E with 3) has been shown to interact with the 5-HT4a receptor splice several clinical conditions.50-53 Interestingly, a single nucleotide variant.35 In the human intestine, all 3 LIN7 homologues were polymorphism in the 5-HT3 receptor E subunit that is associated present in the different tissue regions except the region of the with female IBS-D where the 5-HT3 receptor subunit is transverse colon tissue layers. However, detection of the LIN7 up-regulated by microRNA (miR-510) co-expressed with the E homologues may be limited by their occurrence at pre-synaptic subunit in gut epithelium enterocytes.51 (axonal) and postsynaptic (dendritic) subcellular compartments.36 The 5-HT4 receptor splice variants have different prefer- In conclusion, this study demonstrates that the major differ- ences to ligands as shown by different potency and binding affin- ences in the localization of 5-HT receptors within the human co- ities for various 5-HT4 receptor agonists44,54-57 and their dis- lon are in relation to the mucosa and muscular tissue rather than tribution may influence drug responses. 5-HT4 receptor tran- the region of the intestine. The fact that the study proteins are scripts have been identified in human mucosa biopsies obtained distributed along the intestine, only serves to emphasize how from the duodenum, ileum and colon and this mucosal expression dominant the receptors and their associated proteins are, and so contributes to luminal responses in model animal systems.10 There any dysfunction of one is likely to manifest seriously in clinical are no significant patterns in the expression of 5-HT4 or 5-HT7 conditions. Only minor differences were observed in the distri- receptors although the number of patients expressing the 5-HT4 bution of 5-HT4 and 5-HT7 receptors and their splice variants. receptor d and g splice variants is consistently lower (Fig. 3). The Differences in the distribution of 5-HT3 receptor subunits were 5-HT4 receptor d splice variant in particular has been shown to evident with A, B, and C subunits occurring in all tissues whereas have low expression levels before in different parts of the body the E subunit was only significantly observed in the mucosal such as the central nervous system.30 It would be of interest to layer. The findings suggest that it may be possible to target learn if changes in distribution of 5-HT4 receptor splice variants 5-HT3 receptors in the mucosal or muscular layers if subunit spe- occur in gastrointestinal disorders as the 5-HT4 receptor a and b cific molecules can be generated to cater for different colon dis- splice variants are down regulated while the d variant is upregu- eases or dysfunction.
lated in adenomas.58 The 5-HT7 receptor splice variants in this study were expressed in all of the different tissue layers. The over-all expression patterns of 5-HT 4 and 5-HT7 receptors are in ac- cordance with their known functional properties in the human co- Note: To access the supplementary table and figures men- lon to regulate ascending contraction and descending relaxation tioned in this article, visit the online version of Journal of to generate peristaltic reflexes resulting in bowel movement.7-16,18 Neurogastroenterology and Motility at http://www.jnmjournal.org/, 5-HT7 receptors are also found on macrophages and have been and at http://dx.doi.org/10.5056/jnm14157.
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