ObjectiveWe assessed whether the bicarbonate-rich mineral water Staatl. Fachingen STILL is superior over conventional mineral water in relieving heartburn.DesignMulticentre, double-blind, randomised, ...placebo-controlled trial STOMACH STILL in adult patients with frequent heartburn episodes since ≥6 months and without moderate/severe reflux oesophagitis. Patients drank 1.5 L/day verum or placebo over the course of the day for 6 weeks. Primary endpoint was the percentage of patients with reduction of ≥5 points in the Reflux Disease Questionnaire (RDQ) score for ‘heartburn’. Secondary endpoints included symptom reduction (RDQ), health-related quality of life (HRQOL, Quality of Life in Reflux and Dyspepsia (QOLRAD)), intake of rescue medication and safety/tolerability.ResultsOf 148 randomised patients (verum: n=73, placebo: n=75), 143 completed the trial. Responder rates were 84.72% in the verum and 63.51% in the placebo group (p=0.0035, number needed to treat=5). Symptoms improved under verum compared with placebo for the dimension ‘heartburn’ (p=0.0003) and the RDQ total score (p=0.0050). HRQOL improvements under verum compared with placebo were reported for 3 of 5 QOLRAD domains, that is, ‘food/drink problems’ (p=0.0125), ‘emotional distress’ (p=0.0147) and ‘vitality’ (p=0.0393). Mean intake of rescue medication decreased from 0.73 tablets/day at baseline to 0.47 tablets/day in week 6 in the verum group, whereas in the placebo group it remained constant during the trial. Only three patients had treatment-related adverse events (verum: n=1, placebo: n=2).ConclusionSTOMACH STILL is the first controlled clinical trial demonstrating superiority of a mineral water over placebo in relieving heartburn, accompanied by an improved HRQOL.Trial registration numberEudraCT 2017-001100-30.
Within the family of serotonin receptors, the 5-hydroxytryptamine-3 (5-HT(3)) receptor is the only ligand-gated ion channel. It is composed of five subunits, of which the 5-HT(3A) and 5-HT(3B) ...subunits are best characterized. Several studies, however, have reported on the functional diversity of native 5-HT(3) receptors, which cannot solely be explained on the basis of the 5-HT(3A) and 5-HT(3B) subunits. After our discovery of further putative 5-HT(3) serotonin receptor-encoding genes, HTR3C, HTR3D, and HTR3E, we investigated whether these novel candidates and the isoform 5-HT(3Ea) are able to form functional 5-HT(3) receptor complexes. Using immunofluorescence and immunoprecipitation studies of heterologously expressed proteins, we found that each of the respective candidates coassembles with 5-HT(3A). To investigate whether the novel subunits modulate 5-HT(3) receptor function, we performed radioligand-binding assays and calcium-influx studies in human embryonic kidney 293 cells. Our experiments revealed that the 5-HT(3C),5-HT(3D), 5-HT(3E), and 5-HT(3Ea) subunits alone cannot form functional receptors. Coexpression with 5-HT(3A), however, results in the formation of functional heteromeric complexes with different serotonin efficacies. Potencies of two agonists and antagonists were nearly identical with respect to homomeric 5-HT(3A) and heteromeric complexes. However, 5-HT showed increased efficacy with respect to 5-HT(3A/D) and 5-HT(3A/E) receptors, which is consistent with the increased surface expression compared with 5-HT(3A) receptors. In contrast, 5-HT(3A/C) and 5-HT(3A/Ea) receptors exhibited decreased 5-HT efficacy. These data show for the first time that the novel 5-HT(3) subunits are able to form heteromeric 5-HT(3) receptors, which exhibit quantitatively different functional properties compared with homomeric 5-HT(3A) receptors.
The 5-hydroxytryptamine-3 (5-HT3) receptor, a ligand-gated ion channel, is known to be involved in gut motility and peristalsis, the mediation of pain and psychiatric diseases. 5-HT3 receptor ...antagonists are effectively used to treat chemotherapy-induced emesis and irritable bowel syndrome. We have characterized the impact of four naturally occurring variants in the HTR3B gene leading to amino acid exchanges within the respective subunit of heteromeric 5-HT3A/B receptors on a functional and expressional level.
For functional characterization, a Ca influx assay based on aequorin bioluminescence was used. Radioligand-binding studies with the 5-HT3 receptor antagonist HGR65630 were carried out to determine expression levels of heteromeric 5-HT3A/B receptors. Transiently transfected human embryonic kidney 293 cells using 5-HT3A and 5-HT3B complementary DNA constructs were shown to coexpress homopentameric 5-HT3A next to heteromeric 5-HT3A/B receptors. The variant p.V183I decreased surface expression, whereas p.Y129S and p.S156R led to pronounced increases of 5-HT maximum responses, despite nearly unaltered surface expression levels of heteromeric 5-HT3A/B receptors.
These results may help to explain earlier reported association findings of the frequent p.Y129S and p.V183I variants with psychiatric diseases. Replication studies with larger sample pools, especially regarding the rare p.S156R variant would be useful, to obtain an idea about the predisposing role of these single nucleotide polymorphisms as susceptibility variants.
The aim of this study was to analyze functional properties of the naturally occurring C23S variant of the human 5-HT2C receptor. In HEK293 cells transiently expressing the unedited forms of the ...variant receptor (VR) or the wild-type receptor (WTR), surface expression was determined by 3Hmesulergine binding to membrane fragments. Function was examined by an aequorin luminescence-based Ca2+ assay. Surface expression of the VR was 116% of that of the WTR. The 5-HT-induced increase in cytosolic Ca2+ (Ca2+i), and its inhibition by the inverse agonist SB 206553 did not differ between VR- or WTR-expressing cells. Preexposure of VR- or WTR-expressing cells to 0.5 μM 5-HT (3 min-4.5 h) led to a practically identical time course and extent in the reduction of the 5-HT-induced increase in Ca2+i. In contrast, prolonged preexposure to the inverse agonist SB 206553 (1 μM) elevated the 5-HT-induced increase in Ca2+i for both isoreceptors. A preexposure time of 4.5 h was necessary to significantly elevate the Ca2+ response of the WTR, but the VR produced this elevation within 1 h with virtually no further effect after 4.5 h of preexposure. In conclusion, prolonged preexposure to 5-HT caused equally rapid and strong desensitization of both isoreceptors. The different time course of SB 206553-induced resensitization of the two isoreceptors might be therapeutically relevant for drugs exhibiting inverse agonist properties at 5-HT2C receptors, such as atypical antipsychotics and certain antidepressants.
BACKGROUNDSingle-nucleotide polymorphisms (SNPs) of the serotonin type 3 receptor subunit (HTR3) genes have been associated with psychosomatic symptoms, but it is not clear whether these associations ...exist in irritable bowel syndrome (IBS). AIMTo assess the association of HTR3 polymorphisms with depressive, anxiety, and somatization symptoms in individuals with IBS. METHODSIn this retrospective study, 623 participants with IBS were recruited from five specialty centers in Germany, Sweden, the United States, the United Kingdom, and Ireland. Depressive, anxiety, and somatization symptoms and sociodemographic characteristics were collected. Four functional SNPs - HTR3A c.-42C>T, HTR3B c.386A>C, HTR3C c.489C>A, and HTR3E c.*76G>A - were genotyped and analyzed using the dominant and recessive models. We also performed separate analyses for sex and IBS subtypes. SNP scores were calculated as the number of minor alleles of the SNPs above. The impact of HTR3C c.489C>A was tested by radioligand-binding and calcium influx assays. RESULTSDepressive and anxiety symptoms significantly worsened with increasing numbers of minor HTR3C c.489C>A alleles in the dominant model (F depressive = 7.475, P depressive = 0.006; F anxiety = 6.535, P anxiety = 0.011). A higher SNP score (range 0-6) was linked to a worsened depressive symptoms score (F = 7.710, P-linear trend = 0.006) in IBS. The potential relevance of the HTR3C SNP was corroborated, showing changes in the expression level of 5-HT3AC variant receptors. CONCLUSIONWe have provided the first evidence that HTR3C c.489C>A is involved in depressive and anxiety symptoms in individuals with IBS. The SNP score indicated that an increasing number of minor alleles is linked to the worsening of depressive symptoms in IBS.
Diarrhea predominant irritable bowel syndrome (IBS-D) is a complex disorder related to dysfunctions in the serotonergic system. As cis-regulatory variants can play a role in the etiology of complex ...conditions, we investigated the untranslated regions (UTRs) of the serotonin receptor type 3 subunit genes HTR3A and HTR3E. Mutation analysis was carried out in a pilot sample of 200 IBS patients and 100 healthy controls from the UK. The novel HTR3E 3′-UTR variant c.*76G>A (rs62625044) was associated with female IBS-D (P = 0.033, OR = 8.53). This association was confirmed in a replication study, including 119 IBS-D patients and 195 controls from Germany (P = 0.0046, OR = 4.92). Pooled analysis resulted in a highly significant association of c.*76G>A with female IBS-D (P = 0.0002, OR = 5.39). In a reporter assay, c.*76G>A affected binding of miR-510 to the HTR3E 3′-UTR and caused elevated luciferase expression. HTR3E and miR-510 co-localize in enterocytes of the gut epithelium as shown by in situ hybridization and RT–PCR. This is the first example indicating micro RNA-related expression regulation of a serotonin receptor gene with a cis-regulatory variant affecting this regulation and appearing to be associated with female IBS-D.
Oxymetazoline and xylometazoline are both used as nasal mucosa decongesting α‐adrenoceptor agonists during a common cold. However, it is largely unknown which of the six α‐adrenoceptor subtypes are ...actually present in human nasal mucosa, which are activated by the two alpha‐adrenoceptor agonists and to what extent. Therefore, mRNA expression in human nasal mucosa of the six α‐adrenoceptor subtypes was studied. Furthermore, the affinity and potency of the imidazolines oxymetazoline and xylometazoline at these α‐adrenoceptor subtypes were examined in transfected HEK293 cells. The rank order of mRNA levels of α‐adrenoceptor subtypes in human nasal mucosa was: α2A > α1A ≥ α2B > α1D ≥ α2C >> α1B. Oxymetazoline and xylometazoline exhibited in radioligand competition studies higher affinities than the catecholamines adrenaline and noradrenaline at most α‐adrenoceptor subtypes. Compared to xylometazoline, oxymetazoline exhibited a significantly higher affinity at α1A‐ but a lower affinity at α2B‐adrenoceptors. In functional studies in which adrenoceptor‐mediated Ca2+ signals were measured, both, oxymetazoline and xylometazoline behaved at α2B‐adrenoceptors as full agonists but oxymetazoline was significantly more potent than xylometazoline. Furthermore, oxymetazoline was also a partial agonist at α1A‐adrenoceptors; however, its potency was relatively low and it was much lower than its affinity. The higher potency at α2B‐adrenoceptors, i.e. at receptors highly expressed at the mRNA level in human nasal mucosa, could eventually explain why in nasal decongestants oxymetazoline can be used in lower concentrations than xylometazoline.
Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A–E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the ...pentameric ligand-gated nicotinic acetylcholine and 5-HT3 receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT3 receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca2+ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT3A receptors but leads to differential increases of 5-HT-induced maximum response (Emax) on cells expressing different subunits. Increases of Emax were due to analogously enhanced Bmax values for binding of the 5-HT3 receptor antagonist 3HGR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT3A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT3 receptor composition in vivo.
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