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Reverse transcriptase polymerase 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 stained with GelRed. A negative control is shown for the LIN7A sample as the primers are within one exon. Letters represent samples obtained from M (mucosa), C (circular muscle), and L (longitudinal muscle).
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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...
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... 5-HT 3 receptor E subunit tran- scripts were nearly always only detected in the mucosa samples at levels significantly greater than the B subunit (Fig. 1B). Only one longitudinal muscle sample showed any E subunit expression (Fig. 1B), so to determine if the 5-HT 3 receptor E subunit was expressed in the muscle layers but below the reliable detection levels of the quantitative PCR conditions, the amplified PCR samples were run in gels (Supplementary Fig. 2). Only very low levels of expression were observed in two additional colon mucosa samples and two longitudinal muscle samples as well as an addi- tional ileum muscle sample. ...
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... us- ing partial or nested PCR. GAPDH expression was used as a control for this analysis as it has previously been shown to be comparable between human colonic tissue layers 11 ( Fig. 1 and Supplementary Fig. 3). An example of the expression of different transcripts following end point RT-PCR analysis is shown for a descending colon sample (Fig. 2). The samples were obtained from ileum, ascending, transverse, descending and sigmoid colon and overall the distribution of transcripts appeared independent 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 ...
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... samples that contained readily detectable levels of GAPDH (and -actin for quantitative PCR) and were not contaminated by genomic DNA (no bands detected in the negative reverse transcript assays) were processed further to exclude the probability of undetectable expression due to storage-related RNA degradation ( Supplementary Fig. 3). The robustness of the quantitative PCR was ensured by adhering to the MIQE guidelines 47 and samples were run on gels to ensure that only the expected products were generated ( Supplementary Fig. 2). The end point PCR studies were designed to ensure that the products were still within the linear amplification range. ...
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... ensured by adhering to the MIQE guidelines 47 and samples were run on gels to ensure that only the expected products were generated ( Supplementary Fig. 2). The end point PCR studies were designed to ensure that the products were still within the linear amplification range. Where expression levels were low (eg, 5-HT 4 receptor splice var- iant d; Fig. 2), independent observers confirmed the presence of detectable bands in the gels. The distribution of 5-HT 3 receptor A subunits is in keeping with previous studies where this subunit was widespread and thought to reflect the distribution of functional 5-HT 3 receptor ...
Citations
... The expression of serotonin receptor 4 (5-HT4R) was evaluated by the expression of the "a" splice variant of this protein (5-HT4a). It was confirmed that this variant of the receptor was the most widely represented throughout the colon [17]. We amplified the housekeeping GAPDH gene in each sample to eliminate sample-to-sample differences during RNA extraction and conversion to cDNA. ...
... Moreover, mucosal 5-HT7 expression in patients with diverticulosis was comparable to healthy controls. Activation of 5-HT7 on dendric cells within the gut was previously suggested as a modulator of colitis [17,22]. Taken together, we may conclude that observed 5-HT and SERT changes in the sigmoid colon in patients with diverticulosis did not result from neuroinflammation. ...
Background and Objectives: Diverticulosis is frequently accompanied by altered bowel habits. The biogenic amines within colonic mucosa control bowel motility, and in particular, alterations in serotonin signaling may play a role in colon diverticulosis. The aim of the study was to assess the concentration of biogenic amines and serotonin receptor expression in the colonic mucosa in patients with diverticulosis and healthy controls. Materials and Methods: This prospective, comparative study included 59 individuals: 35 with sigmoid diverticulosis and 24 healthy controls. The study was held at the Department of Gastroenterology and Internal Medicine, Medical University of Warsaw, Poland. Mucosal samples were taken from the right and left colon during a colonoscopy in all patients. Concentrations of norepinephrine, 3-methoxy-4-hydroxyphenylglycol, dopamine, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid were measured with high-performance liquid chromatography. Expressions of human 5-hydroxytryptamine receptor 3A, 5-hydroxytryptamine receptor 4, 5-hydroxytryptamine receptor 7, solute carrier family 6 member 4 (SERT) for serotonin, as well as the neuroglia activation markers glial fibrillary acidic protein, S100 calcium-binding protein B, and proteolipid protein 1, were assessed with polymerase chain reaction. Results: The median age and sex distribution were comparable in both study groups (median 69 y vs. 52 y; p < 0.455 and males/females in cases 11/17 vs. 18/19 in controls; p < 0.309). In diverticulosis patients, there was a higher concentration of serotonin in the left affected colon compared to the right healthy part of the colon (median 8239 pg/mg vs. 6326 pg/mL; p < 0.01). The SERT expression was lower in the affected left segment compared to the right colon (median 0.88 vs. 1.36; p < 0.01). There was a higher colonic mucosa concentration of serotonin (median 8239 pg/mg vs. 6000 pg/mL; p < 0.02) and 5-hydroxyindoleacetic acid/serotonin ratio (median 0.27 vs. 0.47; p < 0.01) in diverticulosis patients compared to controls in the left side of the colon. Conclusions: The concentration of serotonin in the mucosa of the colon segment affected by diverticula is higher than in the healthy segment in the same individuals and higher than in healthy controls. These results underline serotonin signaling in colon diverticulosis pathophysiology.
... The 5HT3A and 5HT3B subunits are co-expressed in the human spleen, intestine, and some brain regions, such as the amygdala, telencephalon, and entorhinal cortex [16,17]. In humans, the A, B, C and E subunits are differentially expressed throughout the colon and ileum, where the E subunit in particular is expressed at high levels in the mucosal layers of the gut but not the muscular layers [18][19][20]. ...
The 5-hydroxytryptamine 3 (5-HT3) receptor belongs to the pentameric ligand-gated cation channel superfamily. Humans have five different 5-HT3 receptor subunits: A to E. The 5-HT3 receptors are located on the cell membrane, but a previous study suggested that mitochondria could also contain A subunits. In this article, we explored the distribution of 5-HT3 receptor subunits in intracellular and cell-free mitochondria. Organelle prediction software supported the localization of the A and E subunits on the inner membrane of the mitochondria. We transiently transfected HEK293T cells that do not natively express the 5-HT3 receptor with an epitope and fluorescent protein-tagged 5HT3A and 5HT3E subunits. Fluorescence microscopy and cell fractionation indicated that both subunits, A and E, localized to the mitochondria, while transmission electron microscopy revealed the location of the subunits on the mitochondrial inner membrane, where they could form heteromeric complexes. Cell-free mitochondria isolated from cell culture media colocalized with the fluorescent signal for A subunits. The presence of A and E subunits influenced changes in the membrane potential and mitochondrial oxygen consumption rates upon exposure to serotonin; this was inhibited by pre-treatment with ondansetron. Therefore, it is likely that the 5-HT3 receptors present on mitochondria directly impact mitochondrial function and that this may have therapeutic implications.
... 5-HT 3 receptors can form homomeric (all A subunits) or heteromeric (mixture of A and some combination of B, C, D, or E subunits) functionally active channels in humans with the orthosteric ligand binding site occurring in the extracellular N terminal region at the interface between two A subunits [71][72][73][74]. Subunit A and the other subunits (B, C, D and E) are differentially expressed in human brain regions and gastrointestinal layers [75][76][77][78][79], where their biophysical characteristics contribute to receptor function [80][81][82]. For example, 5-HT 3 AB receptor heteromers showed altered conductance and calcium ion channel permeability compared with homomeric 5-HT 3 A receptor during ligand binding [75,83,84]. ...
The 5-hydroxytrptamine 3 (5-HT 3 ) receptor is a member of the ’Cys-loop’ family and the only pentameric ligand gated ion channel among the serotonin receptors. 5-HT 3 receptors play an important role in controlling growth, development, and behaviour in animals. Several 5-HT 3 receptor antagonists are used to treat diseases (e.g., irritable bowel syndrome, nausea and emesis). Humans express five different subunits (A-E) enabling a variety of heteromeric receptors to form but all contain 5HT3A subunits. However, the information available about the 5-HT 3 receptor subunit occurrence among the metazoan lineages is minimal. In the present article we searched for 5-HT 3 receptor subunit homologs from different phyla in Metazoa. We identified more than 1000 5-HT 3 receptor subunits in Metazoa in different phyla and undertook simultaneous phylogenetic analysis of 526 5HT3A, 358 5HT3B, 239 5HT3C, 70 5HT3D, and 173 5HT3E sequences. 5-HT 3 receptor subunits were present in species belonging to 11 phyla: Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nematoda, Orthonectida, Platyhelminthes, Rotifera and Tardigrada. All subunits were most often identified in Chordata phylum which was strongly represented in searches. Using multiple sequence alignment, we investigated variations in the ligand binding region of the 5HT3A subunit protein sequences in the metazoan lineage. Several critical amino acid residues important for ligand binding (common structural features) are commonly present in species from Nematoda and Platyhelminth gut parasites through to Chordata. Collectively, this better understanding of the 5-HT 3 receptor evolutionary patterns raises possibilities of future pharmacological challenges facing Metazoa including effects on parasitic and other species in ecosystems that contain 5-HT 3 receptor ligands.
... 28 Serotonin type-3 (5-HT 3 ) receptors have been found in colonic mucosal and muscularis tissue as well as autonomic and sensory nerves and brain. 29,30 Thus, alosetron was used to determine if evoked brain activation in the DSS-treated macaques was sensitive to a drug that is used to manage IBS symptoms. ...
... Thus, decreased Ins activation following alosetron treatment could have occurred due to inactivation of presynaptic structures enriched with 5-HT 3 receptors, including the gut, peripheral and CNS neurons or due to disinhibition of yet to be identified inhibitory interneurons. 29,60 At the same time, it is possible that peripheral tissues, including gut, peripheral visceral, sensory and autonomic nerves could have also been sensitized, which in turn led to initiating central sensitization or maintaining this state. Further examination of the role of peripheral tissues are needed to confirm their potential role. ...
Greater understanding of the mechanism that mediates visceral pain and hypersensitivity associated with irritable bowel syndrome (IBS) would facilitate the development of effective therapeutics to manage these symptoms. An objective marker associated with the underlying mechanisms of visceral pain and hypersensitivity could be used to guide therapeutic development. The current study examined brain activation evoked by rectal distention with functional magnetic resonance imaging (fMRI) in a cynomolgus macaque model of visceral hypersensitivity. Male, cynomolgus macaques underwent five four‐week treatments of dextran sodium sulfate (DSS)‐distilled water (DW), which induced mild‐moderate colitis with remission during each treatment cycle. Balloon rectal distention (RD) was performed under anesthesia 14 weeks after the final DSS‐DW treatment. Colonoscopy confirmed the absence of colitis prior to the start of RD. In naïve, untreated macaques, 10 ml, 20 ml and 30 ml RD did not evoke brain activation. However, insular cortex/somatosensory II cortex and cerebellum were significantly activated in DSS‐treated macaques at 20 ml and 30 ml rectal distention. Intra‐rectal pressure after DSS treatment was not significantly different from that of naïve, untreated macaques, indicating lack of alteration of rectal functioning following DSS‐treatment. Treatment with 5‐HT3 receptor antagonist alosetron (p.o.) reduced distension‐evoked brain activation and decreased intra‐rectal pressure. The current findings demonstrated activation of brain regions to RD following DSS treatments which was not present in naïve macaques, suggesting visceral hypersensitivity. Brain activation in turn was reduced by alosetron, which could underlie the analgesic effect alosetron in IBS patients.
... Drugs targeting 5-HT 3 and 5-HT 4 have been used for treatment of IBS symptoms but are associated with ischaemic side effects 22,23 . 5-HT 7 is a G-protein coupled receptor identified in enteric neurons, smooth muscles, and dendritic cells in the colon [24][25][26] . Stimulation of 5-HT 7 resulted in exaggerated relaxation of intestinal circular smooth muscle and activation of excitatory postsynaptic potential in colonic myenteric afferents 27,28 . ...
Irritable bowel syndrome (IBS) is characterized by visceral hypersensitivity (VH) associated with abnormal serotonin/5-hydroxytryptamine (5-HT) metabolism and neurotrophin-dependent mucosal neurite outgrowth. The underlying mechanisms of VH remain poorly understood. We investigated the role of 5-HT7 receptor in mucosal innervation and intestinal hyperalgesia. A high density of mucosal nerve fibres stained for 5-HT7 was observed in colonoscopic biopsy specimens from IBS patients compared with those from healthy controls. Staining of 5-HT3 and 5-HT4 receptors was observed mainly in colonic epithelia with comparable levels between IBS and controls. Visceromotor responses to colorectal distension were evaluated in two mouse models, one postinfectious with Giardia and subjected to water avoidance stress (GW) and the other postinflammatory with trinitrobenzene sulfonic acid-induced colitis (PT). Increased VH was associated with higher mucosal density of 5-HT7-expressing nerve fibres and elevated neurotrophin and neurotrophin receptor levels in the GW and PT mice. The increased VH was inhibited by intraperitoneal injection of SB-269970 (a selective 5-HT7 antagonist). Peroral multiple doses of CYY1005 (a novel 5-HT7 ligand) decreased VH and reduced mucosal density of 5-HT7-expressing nerve fibres in mouse colon. Human neuroblastoma SH-SY5Y cells incubated with bacteria-free mouse colonic supernatant, 5-HT, nerve growth factor, or brain-derived neurotrophic factor exhibited nerve fibre elongation, which was inhibited by 5-HT7 antagonists. Gene silencing of HTR7 also reduced the nerve fibre length. Activation of 5-HT7 upregulated NGF and BDNF gene expression, while stimulation with neurotrophins increased the levels of tryptophan hydroxylase 2 and 5-HT7 in neurons. A positive-feedback loop was observed between serotonin and neurotrophin pathways via 5-HT7 activation to aggravate fibre elongation, whereby 5-HT3 and 5-HT4 had no roles. In conclusion, 5-HT7-dependent mucosal neurite outgrowth contributed to VH. A novel 5-HT7 antagonist could be used as peroral analgesics for IBS-related pain. Current treatments, which mostly aim to restore bowel habits, are ineffective for intestinal pain in irritable bowel syndrome (IBS). Abnormal gut-derived serotonin metabolism and mucosal neurite outgrowth are linked to visceral hypersensitivity. Enhanced mucosal innervation dependent on 5-HT7 contributes to hyperalgesia in two IBS-like mouse models. A positive-feedback loop between serotonin and neurotrophin is involved in intensifying nerve fiber elongation. A novel 5-HT7 receptor antagonist may be administered orally as an intestinal analgesic for IBS-related pain.
... The specificity of this neurotransmitter is presumably conserved in all vertebrate species . 5-HT is expressed in central nervous system and gastrointestinal tract Yaakob et al., 2015); here, 5-HT receptors are expressed by intestinal neurons, enterocytes, endocrine cells and MCs (Mandic & Volkoff, 2018). ...
The common goldfish is the most widespread teleosts in the world. Due to its peculiar characteristics, such as the high resistance, easy availability and stabulation, and for its evolutionary characteristics, this fish lends itself to be one of the most used experimental models. This study aimed to characterize the mast cells in the intestine of Carassius auratus using anti‐TLR‐2, anti‐S100, anti‐VIP, anti‐serotonin (5‐HT) and anti‐Piscidin antibodies. The intestine of goldfish, like that of all vertebrates, plays an important role in the immunology of the animal. The gut‐associated lymphoid tissue GALT is an immune component containing several specific cells such as lymphocytes, macrophages and mast cells. In addition, the presence of goblet cells in the intestinal epithelium strengthens the defence system, secreting many cytokines and chemokines and displaying antibacterial properties. Our results show mast cells labelled with antibodies that are highly conserved between fish and mammals, demonstrating an active role of these cells in the immune response.
... [96,104] 5-HT3 receptor subunit expression occurs in the enteric nervous system and other regions of the gut, and they are particularly known for their involvement in brain-gut circuitry. Gene transcripts encoding 5-HT3 receptor subunits have been detected in the human colon [13,14,[105][106][107][108] and the stomach [109]. Unexpectedly, there was a spatial differential in the tissue distribution of the E subunit being only present in the mucosal layers [108] and a single nucleotide polymorphism in the gene encoding E subunit is associated with diarrhea predominant irritable bowel syndrome [42]. ...
... Gene transcripts encoding 5-HT3 receptor subunits have been detected in the human colon [13,14,[105][106][107][108] and the stomach [109]. Unexpectedly, there was a spatial differential in the tissue distribution of the E subunit being only present in the mucosal layers [108] and a single nucleotide polymorphism in the gene encoding E subunit is associated with diarrhea predominant irritable bowel syndrome [42]. Other polymorphisms in the gene encoding C subunit are also useful in predicting treatment response [97] (Table 1). ...
5-hydroxytryptamine type 3 (5-HT3) receptors are ligand gated ion channels, which clearly distinguish their mode of action from the other G-protein coupled 5-HT or serotonin receptors. 5-HT3 receptors are well established targets for emesis and gastrointestinal mobility and are used as adjunct targets in treating schizophrenia. However, the distribution of these receptors is wider than the nervous system and there is potential that these additional sites can be targeted to modulate inflammatory and/or metabolic conditions. Recent progress in structural biology and pharmacology of 5-HT3 receptors have provided profound insights into mechanisms of their action. These advances, combined with insights into clinical relevance of mutations in genes encoding 5-HT3 subunits and increasing understanding of their implications in patient’s predisposition to diseases and response to the treatment, open new avenues for personalized precision medicine. In this review, we recap on the current status of 5-HT3 receptor-based therapies using a biochemical and physiological perspective. We assess the potential for targeting 5-HT3 receptors in conditions involving metabolic or inflammatory disorders based on recent findings, underscoring the challenges and limitations of this approach.
... DCs are located close to the intestinal mucosa and play a significant role in initiating and modulating the process of colitis. Although the 5HT7 receptor mRNA is expressed also on mucosa and muscle layer of the gut, its role in motility is rather equivocal [35,77]. ...
The gastrointestinal (GI) tract contains the highest concentration of biogenic amines in the human body. Neurons located in the GI tract, modulated by biogenic amines and various peptide and non-peptide transmitters, are called Enteric Nervous System (ENS). That explains why many medications used in neurology and psychiatry present side effects from the gut. Serotonin (5-hyroxytrypatamine, 5-HT), 95% of which is synthesized in the gut, is the most important amine (beside epinephrine and norepinephrine) colon functionality but another substances such as histamine, dopamine and melatonin are also potent in modulating intestine’s actions. Over 30 receptors for 5-HT were described in the human body, and 5-HT3, 5-HT4 and 5-HT7 are known to have the highest influence on motility and are a potent target for the drugs for treatment GI disorders, such as Irritable Bowel Syndrome (IBS) and Inflammatory Bowel Diseases (IBD). Histamine is a key biogenic amine for pathogenesis of allergy also in the colon. Alteration in histaminergic system is found in patients with diarrhea and allergic enteropathy. Dopamine affects functions of the large intestine but its modulating actions are more presented in the upper part of GI tract. Melatonin is best known for regulating circadian circle, but may also be a potent anti-inflammatory agent within the gut. Despite many years of research, it seems that more studies are needed to fully understand human colon neurochemistry.
... The 5-HT 3 receptor A and C subunits are expressed in discrete regions of the human such as the dorsal root ganglion and gastrointestinal tract [5,6,8,32,[34][35][36]. Immunohistochemistry of human colonic samples revealed co-expression of A and C subunits in individual neurons and enterocytes [32]. ...
Five different subunits of the human serotonin 3 (5-hydroxytrptamine 3; 5-HT3) receptor exist and these are present in both central and peripheral systems. Different subunits alter the efficacy of 5-HT3 receptor antagonists used to treat diarrhoea predominant-irritable bowel syndrome, chemotherapy induced nausea and vomiting and depression. Cell surface arrangement of 5-HT3 receptor complexes and the contribution of C, D and E subunits to receptor function is poorly understood. Here, we examine interactions of A and C subunits using 5-HT3 receptor subunits containing fluorescent protein inserts between the 3rd and 4th transmembrane spanning region. HEK293T cells that do not normally express 5-HT3 receptor subunits, were transiently transfected with A or C or both subunits. Patch clamp experiments show that cells transfected with either fluorescent protein tagged A or A and C subunits generate whole cell currents in response to 5-HT. These findings correlate with the apparent distribution of fluorescent protein tagged A and C subunits at or near cell surfaces detected using TIRF microscopy. In co-transfected cells, the A and C subunits are associated forming AC heteromer complexes at or near the cell surface and a proportion can also form A or C homomers. In conclusion, it is likely that both A homomers and AC heteromers contribute to whole cell currents in response to 5-HT with minimal contribution from C homomers.
... Distribution of 5-HT 7 receptors 5-HT 7 receptors are expressed mainly in two compartments: the CNS (Hedlund and Sutcliffe 2004) and the gastrointestinal (GI) tract (Yaakob et al. 2015), although they are also expressed in other tissues including immune cells (see below). In the CNS, the receptor is broadly expressed in the spinal cord, suprachiasmatic nucleus of the hypothalamus, antedorsal thalamus, globus pallidus, prefrontal cortex, trigeminal nucleus caudalis, raphe nuclei area, amygdala and hippocampus, particularly in pyramidal cells of cornu ammonis (CA)1 and CA3, where they are expressed in both, neurons and glial cells, including microglia, the CNS-specific phagocytic cell (Chapin and Andrade 2001;Dogrul and Seyrek 2006;Gill et al. 2002;Horisawa et al. 2013;Thomas and Hagan 2004;Tokarski et al. 2003;Russo et al. 2005;Hedlund and Sutcliffe 2004;Lovenberg et al. 1993). ...
In mammalians, serotonin (5-HT) has critical roles in the central nervous system (CNS), including mood stability, pain tolerance, or sleep patterns. However, the vast majority of serotonin is produced by intestinal enterochromaffin cells of the gastrointestinal tract and circulating blood platelets, also acting outside of the CNS. Serotonin effects are mediated through its interaction with 5-HT receptors (5-HTRs), a superfamily with a repertoire of at least fourteen well-characterized members. 5-HT7 receptors are the last 5-HTR member to be identified, with well-defined functions in the nervous, gastrointestinal, and vascular systems. The effects of serotonin on the immune response are less well understood. Mast cells are known to produce serotonin, while T cells, dendritic cells, monocytes, macrophages and microglia express 5-HT7 receptor. Here, we review the known roles of 5-HT7 receptors in the immune system, as well as their potential therapeutic implication in inflammatory and immune-mediated disorders.
