Tegaserod Stimulates 5-HT4 Serotonin Receptors in the Isolated Human Atrium.

Tegaserod (1-{[(5-methoxy-1H-indol-3-yl)methyliden]amino}-3-pentylguanidine) is a potent agonist at human recombinant 5-HT4 serotonin receptors. Consequently, tegaserod is utilized in the treatment of bowel diseases. The objective of this study was to test the hypothesis that tegaserod stimulates human cardiac atrial 5-HT4-receptors via cyclic adenosine monophosphate (cAMP)-dependent pathways. Tegaserod exerted positive inotropic effects (PIEs) and positive chronotropic effects (PCEs) in isolated left and right atrial preparations, respectively, from mice with cardiac-specific overexpression of the human 5-HT4 serotonin receptor (5-HT4-TG) in a concentration- and time-dependent manner. However, no effect was observed in the hearts of littermates of wild-type mice (WT). Western blot analysis revealed that the expression of 5-HT4 receptors was significantly higher in 5-HT4-TG mice compared to WT mice. The specificity of the signal for the 5-HT4 receptor was confirmed by the absence of the signal in the hearts of 5-HT4 receptor knockout mice. Furthermore, tegaserod increased the force of contraction (at concentrations as low as 10 nM), reduced the time of tension relaxation, and increased the rate of tension development in isolated electrically stimulated (at a rate of 60 beats per minute) human right atrial preparations (HAPs, obtained during open-heart surgery) when administered alone. The potency and efficacy of tegaserod to raise the force of contraction were enhanced in the presence of cilostamide, a phosphodiesterase III inhibitor. The positive inotropic effect of tegaserod in HAPs was found to be attenuated by the 5-HT4 serotonin receptor antagonist GR 125487 (0.1 µM). The efficacy of tegaserod (10 µM) in raising the force of contraction in HAPs was less pronounced than that of serotonin (10 µM) or isoprenaline (1 µM). Tegaserod shifted the concentration-response curve of the force of contraction to serotonin to the right in HAPs, indicating that it is a partial agonist at 5-HT4 serotonin receptors in this model. We propose that the mechanism of action of tegaserod in HAPs involves cAMP-dependent phosphorylation of cardiac regulatory proteins.

Effects and mechanisms of sciadonic acid on colonic transit function through regulating 5-HT4/cAMP/PKA/AQP4 signaling pathway in STC model mice.

Torreya grandis (T. grandis) oil has been reported to alleviate symptoms of slow transit constipation (STC). However, the impact of sciadonic acid (SA), a distinctive fatty acid found in T. grandis oil, on the pathological progression of STC remains unclear. This study aimed to evaluate the effect of SA on STC and uncover the underlying mechanisms. The STC model was established by feeding Balb/c mice with loperamide. After 2 weeks of intervention, SA significantly improved weight loss and intestinal motility decline induced by STC, along with enhancing plasma indices and reducing colon pathological damage. SA effectively reversed the STC-induced decrease in the 5-HT4/cAMP/PKA/AQP4 signaling pathway genes and expression. Furthermore, 16S rRNA analysis demonstrated that SA mitigated the imbalance of the intestinal microbiota induced by STC, by reducing the ratio of Firmicutes to Bacteroidetes (F/B) and increasing the abundance of beneficial bacteria such as Akkermansia. In conclusion, SA intervention alleviated colonic dysfunction in STC mice. The activation of the SA-mediated 5-HT4/cAMP/PKA/AQP4 signaling pathway may serve as a potential target for STC treatment. These findings suggest that SA holds promise as a treatment option for STC and could potentially be extended to other related gut diseases for further investigation.

Effects of psilocin and psilocybin on human 5-HT4 serotonin receptors in atrial preparations of transgenic mice and humans.

Several fungi belonging to the genus Psilocybe, also called "magic mushrooms", contain the hallucinogenic drugs psilocybin and psilocin. They are chemically related to serotonin (5-HT). In addition to being abused as drugs, they are now also being discussed or used as a treatment option for depression. Here, we hypothesized that psilocybin and psilocin may act also on cardiac serotonin receptors and studied them in vitro in atrial preparations of our transgenic mouse model with cardiac myocytes-specific overexpression of the human 5-HT4 receptor (5-HT4-TG) as well as in human atrial preparations. Both psilocybin and psilocin enhanced the force of contraction in isolated left atrial preparations from 5-HT4-TG, increased the beating rate in isolated spontaneously beating right atrial preparations from 5-HT4-TG and augmented the force of contraction in the human atrial preparations. The inotropic and chronotropic effects of psilocybin and psilocin at 10 µM were smaller than that of 1 µM 5-HT on the left and right atria from 5-HT4-TG, respectively. Psilocybin and psilocin were inactive in WT. In the human atrial preparations, inhibition of the phosphodiesterase III by cilostamide was necessary to unmask the positive inotropic effects of psilocybin or psilocin. The effects of 10 µM psilocybin and psilocin were abrogated by 10 µM tropisetron or by 1 µM GR125487, a more selective 5-HT4 receptor antagonist. In summary, we demonstrated that psilocin and psilocybin act as agonists on cardiac 5-HT4 receptors.

No association between peripheral serotonin-gene-related DNA methylation and brain serotonin neurotransmission in the healthy and depressed state.

BACKGROUND: Methylation of serotonin-related genes has been proposed as a plausible gene-by-environment link which may mediate environmental stress, depressive and anxiety symptoms. DNA methylation is often measured in blood cells, but little is known about the association between this peripheral epigenetic modification and brain serotonergic architecture. Here, we evaluated the association between whole-blood-derived methylation of four CpG sites in the serotonin transporter (SLC6A4) and six CpG sites of the tryptophan hydroxylase 2 (TPH2) gene and in-vivo brain levels of serotonin transporter (5-HTT) and serotonin 4 receptor (5-HT4) in a cohort of healthy individuals (N = 254) and, for 5-HT4, in a cohort of unmedicated patients with depression (N = 90). To do so, we quantified SLC6A4/TPH2 methylation using bisulfite pyrosequencing and estimated brain 5-HT4 and 5-HTT levels using positron emission tomography. In addition, we explored the association between SLC6A4 and TPH2 methylation and measures of early life and recent stress, depressive and anxiety symptoms on 297 healthy individuals. RESULTS: We found no statistically significant association between peripheral DNA methylation and brain markers of serotonergic neurotransmission in patients with depression or in healthy individuals. In addition, although SLC6A4 CpG2 (chr17:30,236,083) methylation was marginally associated with the parental bonding inventory overprotection score in the healthy cohort, statistical significance did not remain after accounting for blood cell heterogeneity. CONCLUSIONS: We suggest that findings on peripheral DNA methylation in the context of brain serotonin-related features should be interpreted with caution. More studies are needed to rule out a role of SLC6A4 and TPH2 methylation as biomarkers for environmental stress, depressive or anxiety symptoms.

Lysergic acid diethylamide stimulates cardiac human H2 histamine and cardiac human 5-HT4-serotonin receptors.

Lysergic acid diethylamide (LSD) is an artificial hallucinogenic drug. Thus, we hypothesized that LSD might act 5-HT4 serotonin receptors and/or H2 histamine receptors. We studied isolated electrically stimulated left atrial preparations, spontaneously beating right atrial preparations, and spontaneously beating Langendorff-perfused hearts from transgenic mice with cardiomyocyte-specific overexpression of the human 5-HT4 receptor (5-HT4-TG) or of the H2-histamine receptor (H2-TG). For comparison, we used wild type littermate mice (WT). Finally, we measured isometric force of contraction in isolated electrically stimulated muscle strips from the human right atrium obtained from patients during bypass surgery. LSD (up to 10 µM) concentration dependently increased force of contraction and beating rate in left or right atrial preparations from 5-HT4-TG (n = 6, p < 0.05) in 5-HT4-TG atrial preparations. The inotropic and chronotropic effects of LSD were antagonized by 10 µM tropisetron in 5-HT4-TG. In contrast, LSD (10 µM) increased force of contraction and beating rate in left or right atrial preparations, from H2-TG. After pre-stimulation with cilostamide (1 µM), LSD (10 µM) increased force of contraction in human atrial preparations (n = 6, p < 0.05). The contractile effects of LSD in human atrial preparations could be antagonized by 10 µM cimetidine and 1 µM GR 125487. LSD leads to H2-histamine receptor and 5-HT4-receptor mediated cardiac effects in humans.

Activation of the 5-hydroxytryptamine 4 receptor ameliorates tight junction barrier dysfunction in the colon of type 1 diabetic mice.

Hyperglycemia drives dysfunction of the intestinal barrier. 5-Hydroxytryptaine 4 receptor (5-HT 4R) agonists have been considered therapeutics for constipation in clnic. However, the roles of 5-HT 4R activation in mucosa should be fully realized. Here, we investigate the effects of 5-HT 4R activation on diabetes-induced disruption of the tight junction (TJ) barrier in the colon. Not surprisingly, the TJ barrier in diabetic mice with or without 5-HT 4R is tremendously destroyed, as indicated by increased serum fluorescein isothiocyanate (FITC)-dextran and decreased transepithelial electrical resistance (TER). Simultaneously, decreased expressions of TJ proteins are shown in both wild-type (WT) and 5-HT 4R knockout (KO) mice with diabetes. Notably, chronic treatment with intraperitoneal injection of a 5-HT 4R agonist in WT mice with diabetes repairs the TJ barrier and promotes TJ protein expressions, including occludin, claudin-1 and ZO-1, in the colon, whereas a 5-HT 4R agonist does not improve TJ barrier function or TJ protein expressions in 5-HT 4R KO mice with diabetes. Furthermore, stimulation of 5-HT 4R inhibits diabetes-induced upregulation of myosin light chain kinase (MLCK), Rho-associated coiled coil protein kinase 1 (ROCK1), and phosphorylated myosin light chain (p-MLC), which are key molecules that regulate TJ integrity, in the colonic mucosa of WT mice. However, such action induced by a 5-HT 4R agonist is not observed in 5-HT 4R KO mice with diabetes. These findings indicate that 5-HT 4R activation may restore TJ integrity by inhibiting the expressions of MLCK, ROCK1 and p-MLC, improving epithelial barrier function in diabetes.

Cardiovascular effects of bufotenin on human 5-HT4 serotonin receptors in cardiac preparations of transgenic mice and in human atrial preparations.

It is unclear whether bufotenin (= N,N-dimethyl-serotonin = 5-hydroxy-N,N-dimethyl-tryptamine), a hallucinogenic drug, can act on human cardiac serotonin 5-HT4 receptors. Therefore, the aim of the study was to examine the cardiac effects of bufotenin and for comparison tryptamine in transgenic mice that only express the human 5-HT4 receptor in cardiomyocytes (5-HT4-TG), in their wild-type littermates (WT) and in isolated electrically driven (1 Hz) human atrial preparations. In 5-HT4-TG, we found that both bufotenin and tryptamine enhanced the force of contraction in left atrial preparations (pD2 = 6.77 or 5.5, respectively) and the beating rate in spontaneously beating right atrial preparations (pD2 = 7.04 or 5.86, respectively). Bufotenin (1 µM) increased left ventricular force of contraction and beating rate in Langendorff perfused hearts from 5-HT4-TG, whereas it was inactive in hearts from WT animals, as was tryptamine. The positive inotropic and chronotropic effects of bufotenin and tryptamine were potentiated by an inhibitor of monoamine oxidases (50 µM pargyline). Furthermore, bufotenin concentration- (0.1-10 µM) and time-dependently elevated force of contraction in isolated electrically stimulated musculi pectinati from the human atrium and these effects were likewise reversed by tropisetron (10 µM). We found that bufotenin (10 µM) increased the phosphorylation state of phospholamban in the isolated perfused hearts, left and right atrial muscle strips of 5-HT4-TG but not from WT and in isolated human right atrial preparations. In summary, we showed that bufotenin can increase the force of contraction via stimulation of human 5-HT4 receptors transgenic mouse cardiac preparations but notably also in human atrial preparations.

Domesticated HERV-W env contributes to the activation of the small conductance Ca2+-activated K+ type 2 channels via decreased 5-HT4 receptor in recent-onset schizophrenia.

The human endogenous retroviruses type W family envelope (HERV-W env) gene is located on chromosome 7q21-22. Our previous studies show that HERV-W env is elevated in schizophrenia and HERV-W env can increase calcium influx. Additionally, the 5-HTergic system and particularly 5-hydroxytryptamine (5-HT) receptors play a prominent role in the pathogenesis and treatment of schizophrenia. 5-hydroxytryptamine receptor 4 (5-HT4R) agonist can block calcium channels. However, the underlying relationship between HERV-W env and 5-HT4R in the etiology of schizophrenia has not been revealed. Here, we used enzyme-linked immunosorbent assay to detect the concentration of HERV-W env and 5-HT4R in the plasma of patients with schizophrenia and we found that there were decreased levels of 5-HT4R and a negative correlation between 5-HT4R and HERV-W env in schizophrenia. Overexpression of HERV-W env decreased the transcription and protein levels of 5-HT4R but increased small conductance Ca2+-activated K+ type 2 channels (SK2) expression levels. Further studies revealed that HERV-W env could interact with 5-HT4R. Additionally, luciferase assay showed that an essential region (-364 to -176 from the transcription start site) in the SK2 promoter was required for HERV-W env-induced SK2 expression. Importantly, 5-HT4R participated in the regulation of SK2 expression and promoter activity. Electrophysiological recordings suggested that HERV-W env could increase SK2 channel currents and the increase of SK2 currents was inhibited by 5-HT4R. In conclusion, HERV-W env could activate SK2 channels via decreased 5-HT4R, which might exhibit a novel mechanism for HERV-W env to influence neuronal activity in schizophrenia.

Functional interaction of H2-receptors and 5HT4-receptors in atrial tissues isolated from double transgenic mice and from human patients.

In the past, we generated transgenic mice that overexpress the human histamine 2 (H2)-receptor (H2-TG) or that overexpress the human serotonin 4 (5-HT4)-receptor (5-HT4-TG) in the heart. Here, we crossbred these lines of mice to generate double transgenic mice that overexpress both receptors (DT). This was done to study a conceivable interaction between these receptors in the mouse heart as a model for the human heart. When in left atria, initially, force of contraction was elevated maximally with 1 µM serotonin, and subsequently, histamine was cumulatively applied; a biphasic effect of histamine was noted: the force of contraction initially decreased, maximally at 10 nM histamine, and thereafter, the force of contraction increased again at 1 µM histamine. Notably, functional interaction between 5-HT and histamine was also identified in isolated electrically stimulated trabeculae carneae from human right atrium (obtained during cardiac surgery). These functional and biochemical data together are consistent with a joint overexpression of inotropically active H2-receptors and 5-HT4-receptors in the same mouse heart. We also describe an antagonistic interaction on the force of contraction of both receptors in the mouse atrium (DT) and in the human atrial muscle strips. We speculate that via this interaction, histamine might act as a "brake" on the cardiac actions of 5-HT via inhibitory GTP-binding proteins acting on the activity of adenylyl cyclase.

The proarrhythmic effects of hypothermia in atria isolated from 5-HT4-receptor-overexpressing mice.

We investigated whether hypothermia would be arrhythmogenic in mice that overexpress the human 5-HT4 receptor only in their cardiac myocytes (5-HT4-TG). Contractile studies were performed in isolated, electrically driven (1 Hz) left and spontaneously beating right atrial preparations of 5-HT4-TG and littermate wild-type control mice (WT). Hypothermia (23 °C) decreased the force of contraction in the mouse right and left atrial preparations. Moreover, the concentration-dependent positive inotropic effects of 5-HT were blunted but still shifted to lower 5-HT concentrations in the left 5-HT4-TG atria in hypothermia compared to normothermia (37 °C). Furthermore, hypothermia increased the incidence of right atrial arrhythmias in 5-HT4-TG more than in WT mice. In contrast, at 37 °C, lowering the potassium concentration from 5.2 to 2.0 mM also induced arrhythmias in the right atrium, but with a similar incidence in WT and 5-HT4-TG mice. In contrast, 10 µM d,l-sotalol and 300 µM erythromycin did not induce arrhythmias. Hypothermia was accompanied by the increased expression of heat shock protein 70 (HSP70) in WT but not in 5-HT4-TG mice. We concluded that without the stimulation of 5-HT4-receptors by exogenous agonists, a simple temperature reduction can increase arrhythmias in 5-HT4-TG mice. It is tempting to speculate that in human patients, 5-HT4 receptors might contribute to potentially deadly hypothermia-induced arrhythmias.

Cardiovascular effects of metoclopramide and domperidone on human 5-HT4-serotonin-receptors in transgenic mice and in human atrial preparations.

It is unclear whether metoclopramide and domperidone act on human cardiac serotonin 5-HT4-receptors. Therefore, we studied transgenic mice that only express the human 5-HT4 receptor in cardiomyocytes in the atrium and in the ventricle (5-HT4-TG), their wild type-littermates (WT) and isolated human atrial preparations. We found that only metoclopramide but not domperidone enhanced the force of contraction in left atrial preparations (pEC50 = 6.0 ± 0.1; n = 7) from 5-HT4-TG, isolated spontaneously beating right atrial preparations (pEC50 = 6.1 ± 0.1; n = 7) from 5-HT4-TG, Langendorff perfused hearts from 5-HT4-TG, living 5-HT4-TG and human right atrial muscle preparations obtained during bypass surgery of patients suffering from coronary heart disease. The maximum inotropic effect of metoclopramide was smaller (81 ± 2%) than that of 5-HT on the left atria from 5-HT4-TG. The maximum increase in the beating rate due to metoclopramide was 93 ± 2% of effect of 5-HT on right atrial preparations from 5-HT4-TG. Metoclopramide and domperidone were inactive in WT. We found that metoclopramide but not domperidone increased the phosphorylation state of phospholamban in the isolated perfused hearts or muscle strips of 5-HT4-TG, but not in WT. Metoclopramide, but not domperidone, shifted the positive inotropic or chronotropic effects of 5-HT in isolated left atrial and right atrial preparations from 5-HT4-TG dextrally, resp., to higher concentrations: the pEC50 of 5-HT for increase in force was in the absence of metoclopramide 8.6 ± 0.1 (n = 5) versus 8.0 ± 0.3 in the presence of 1 µM metoclopramide (n = 5; P < 0.05); and the beating rate was 7.8 ± 0.2 (n = 7) in the absence of metoclopramide versus 7.2 ± 0.1 in the presence of 1 µM metoclopramide (n = 6; P < 0.05). These results suggested that metoclopramide had an antagonistic effect on human cardiac 5-HT4 receptors. In summary, we showed that metoclopramide, but not domperidone, was a partial agonist at human cardiac 5-HT4-receptors.

Serotonin receptor 4 in the hippocampus modulates mood and anxiety.

Serotonin receptor 4 (5-HT4R) plays an important role in regulating mood, anxiety, and cognition, and drugs that activate this receptor have fast-acting antidepressant (AD)-like effects in preclinical models. However, 5-HT4R is widely expressed throughout the central nervous system (CNS) and periphery, making it difficult to pinpoint the cell types and circuits underlying its effects. Therefore, we generated a Cre-dependent 5-HT4R knockout mouse line to dissect the function of 5-HT4R in specific brain regions and cell types. We show that the loss of functional 5-HT4R specifically from excitatory neurons of hippocampus led to robust AD-like behavioral responses and an elevation in baseline anxiety. 5-HT4R was necessary to maintain the proper excitability of dentate gyrus (DG) granule cells and cell type-specific molecular profiling revealed a dysregulation of genes necessary for normal neural function and plasticity in cells lacking 5-HT4R. These adaptations were accompanied by an increase in the number of immature neurons in ventral, but not dorsal, dentate gyrus, indicating a broad impact of 5-HT4R loss on the local cellular environment. This study is the first to use conditional genetic targeting to demonstrate a direct role for hippocampal 5-HT4R signaling in modulating mood and anxiety. Our findings also underscore the need for cell type-based approaches to elucidate the complex action of neuromodulatory systems on distinct neural circuits.

Duodenal mucosal secretory disturbances in functional dyspepsia.

BACKGROUND: There is increased recognition of duodenal disturbances (inflammation, altered mucosal protein expression, and chemosensitivity) in functional dyspepsia (FD). Besides sensorimotor functions, enteric submucosal neurons also regulate epithelial ion transport. We hypothesized that duodenal mucosal ion transport and expression of associated genes are altered in FD. METHODS: Duodenal mucosal ion transport (basal and acetylcholine- and glucose-evoked changes in short-circuit current [Isc]) and expression of associated genes and regulatory miRNAs were evaluated in 40 FD patients and 24 healthy controls. RESULTS: Basal Isc (FD: 88.2 [52.6] µA/cm2 vs healthy: 20.3 [50.2] µA/cm2 ; P ≤ .0001), acetylcholine-evoked Isc (FD: Emax 50.4 [35.8] µA/cm2 vs healthy: 16.6 [15] µA/cm2 ; P ≤ .001), and glucose-evoked Isc responses (FD: Emax 69.8 [42.1] µA/cm2 vs healthy: 40.3 [24.6] µA/cm2 ; P = .02) were greater in FD than in controls. The Emax for glucose was greater in FD patients on selective serotonin reuptake inhibitors. In FD, the mRNA expression of SLC4A7 and SLC4A4, which transport bicarbonate into cells at the basolateral surface, and the apical anion exchanger SLC26A3 were reduced (false discovery rate <0.05), the serotonin receptor HTR4 was increased, and the serotonin transporter SLC6A4 was decreased. Selected miRNAs (hsa-miR-590-3p, hsa-miR-32-5p) that target genes associated with ionic transport were upregulated in FD. CONCLUSIONS: Compared to controls, FD patients had greater baseline and agonist-evoked duodenal mucosal secretory responses. These findings may be explained by reduced gene expression, which would be anticipated to reduce luminal bicarbonate secretion. The upregulated miRNAs may partly explain the downregulation of these genes in FD.

Expression of serotonin receptor HTR4 in glucagon-like peptide-1-positive enteroendocrine cells of the murine intestine.

Serotonin (5-hydroxytryptamine [5-HT]) synthesized and released in enterochromaffin (EC) cells participates in various functions in the gastrointestinal tract by acting on a diverse range of 5-HT receptors (HTRs) expressed on smooth muscle, enteric neurons, and epithelial cells. We previously observed that genes encoding HTR2A, HTR2B, and HTR4 are expressed in murine intestinal organoids, suggesting the expression of these HTRs in intestinal epithelial cells. The present study investigated the localization of these HTRs in the murine intestine by immunofluorescence staining. HTR2A, HTR2B, and HTR4 localized in individual solitary cells in the epithelium, while HTR2C was observed in the lamina propria. In the epithelium, HTR2A, HTR2B, and HTR4 colocalized with 5-HT, and HTR4 colocalized with glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). Murine intestinal organoids show a colocalization pattern that is similar to in vivo HTR2A and HTR4 with 5-HT, GLP-1, and PYY. Intraperitoneal and intragastric administration of tegaserod, an HTR4 agonist, failed to alter plasma GLP-1 levels in fasted mice. However, intragastric but not intraperitoneal administration of tegaserod reduced dietary lipid-induced increases of plasma GLP-1 levels. This action of tegaserod was inhibited by co-administration of RS39604, an HTR4 antagonist. These results suggest that murine ileal GLP-1/PYY-producing enteroendocrine (EE) cells express HTR4, while 5-HT-producing EC cells express HTR2A, HTR2B, and HTR4. In addition, the observations regarding in vivo GLP-1 secretion suggest that HTR4 signaling in ileal EE cells suppresses dietary lipid-induced GLP-1 secretion. We thus propose that EC and EE cells may interact with each other through paracrine signaling mechanisms.

Effects of sacral nerve electrical stimulation on 5­HT and 5­HT3AR/5­HT4R levels in the colon and sacral cord of acute spinal cord injury rat models.

Spinal cord injury (SCI) often leads to defecation dysfunction. Sacral nerve electrical stimulation (SNS) therapy could improve defecation function. The present study aimed to assess SNS therapy, with regard to the levels of serotonin (5­HT) and its receptors (5­HT3AR and 5­HT4R) in the colon and sacral cord, a rat model of acute severe SCI was used. This rat model was made using the New York University Impactor device. Model rats were randomized to the SCI and SNS (electrical stimulation on the S3 nerve) groups. After 14 days of treatment, enteric transmission function was assessed. 5­HT and 5­HT3AR/5­HT4R were measured by ELISA, quantitative PCR, immunohistochemistry and western blotting. In SCI rats, SNS significantly increased the quantity of feces, shortened the time to the first fecal passage, and improved fecal texture and colon histology. SNS elevated 5­HT contents in the colon and spinal cord, and enhanced 5­HT3AR/5­HT4R protein expression and distribution in the colonic myenteric plexus and mucosa, sacral intermediolateral nucleus and dorsal horn. SNS upregulated the relative expression levels of 5­HT3AR/5­HT4R mRNA and protein in the colon and spinal cord. SNS can improve defecation and accelerate the recovery of colonic transmission functions in rat models of acute SCI. These effects involved upregulation of the 5­HT/5­HT3AR/5­HT4R axes.

Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin.

Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT4 (5-HT4R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT4R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT4R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections.

Association of human serotonin receptor 4 promoter methylation with autism spectrum disorder.

Human serotonin receptor 4 (HTR4) encodes a 5-HT4 receptor involved in learning, memory, depression, anxiety, and feeding behavior. The aim of this study was to investigate the association between the deoxyribonucleic acid (DNA) methylation of HTR4 promoter and autism spectrum disorder (ASD), a disease characterized by communication disorder and repetitive or restrictive behavior.Peripheral blood DNA was obtained from 61 ASD children and 66 healthy children, and the DNA methylation of HTR4 promoter was assessed by quantitative methylation-specific polymerase chain reaction. We used percentage of methylated reference (PMR) to represent DNA methylation level.Due to significant age differences between ASD cases and controls (3 [2, 5] years and 6 [5, 6] years, P = 3.34E-10), we used binary logistic regression analysis for adjustment. Our results showed that the DNA methylation levels of HTR4 promoter were significantly lower in children with ASD than in healthy children (median PMR: 66.23% vs 94.31%,P = .028, age-adjusted P = .034). In addition, the DNA methylation of HTR4 promoter was inversely associated with age in male ASD cases (total cases: r = -0.283, P = .027; male cases: r = -0.431, P = .002; female cases: r = -0.108, P = .752). Dual-luciferase reporter gene assay showed that the reporter gene expression in the strain with recombinant pGL3-promoter-HTR4 plasmid was significantly higher than that in the strain with pGL3-promoter plasmid (fold change = 2.01, P = .0065), indicating that the HTR4 promoter fragment may contain transcription factors to upregulate promoter activity.Our study suggested that hypomethylation of the HTR4 promoter is a potential biomarker for predicting the risk of male ASD.

Zelnorm, an agonist of 5-Hydroxytryptamine 4-receptor, acts as a potential antitumor drug by targeting JAK/STAT3 signaling.

The Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway plays central roles in cancer cell growth and survival. Drug repurposing strategies have provided a valuable approach for developing antitumor drugs. Zelnorm (tegaserod maleate) was originally designed as an agonist of 5-hydroxytryptamine 4 receptor (5-HT4R) and approved by the FDA for treating irritable bowel syndrome with constipation (IBS-C). Through the use of a high-throughput drug screening system, Zelnorm was identified as a JAK/STAT3 signaling inhibitor. Moreover, the inhibition of STAT3 phosphorylation by Zelnorm was independent of its original target 5-HT4R. Zelnorm could cause G1 cell cycle arrest, induce cell apoptosis and inhibit the growth of a variety of cancer cells. The present study identifies Zelnorm as a novel JAK/STAT3 signaling inhibitor and reveals a new clinical application of Zelnorm upon market reintroduction.

Inhibiting Acetylcholinesterase to Activate Pleiotropic Prodrugs with Therapeutic Interest in Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease which is still poorly understood. The drugs currently used against AD, mainly acetylcholinesterase inhibitors (AChEI), are considered clinically insufficient and are responsible for deleterious side effects. AChE is, however, currently receiving renewed interest through the discovery of a chaperone role played in the pathogenesis of AD. But AChE could also serve as an activating protein for pleiotropic prodrugs. Indeed, inhibiting central AChE with brain-penetrating designed carbamates which are able to covalently bind to the enzyme and to concomitantly liberate active metabolites in the brain could constitute a clinically more efficient approach which, additionally, is less likely to cause peripheral side effects. We aim in this article to pave the road of this new avenue with an in vitro and in vivo study of pleiotropic prodrugs targeting both the 5-HT4 receptor and AChE, in order to display a neuroprotective activity associated with a sustained restoration of the cholinergic neurotransmission and without the usual peripheral side effects associated with classic AChEI. This plural activity could bring to AD patients effective, relatively safe, symptomatic and disease-modifying therapeutic benefits.

Neural anti-inflammatory action mediated by two types of acetylcholine receptors in the small intestine.

Gastrointestinal prokinetic agents function as serotonin-4 receptor (5-HT4R) agonists to activate myenteric plexus neurons to release acetylcholine (ACh), which then induce anti-inflammatory action. Details of this pathway, however, remain unknown. The aim of this study is to clarify the anti-inflammatory mechanism underlying the 5-HT4R agonist, mosapride citrate (MOS)-induced anti-inflammatory action on postoperative ileus (POI). POI models were generated from wild-type C57BL6/J (WT), 5-HT4R knock-out (S4R KO), α7 nicotinic AChR KO (α7 R KO), and M2 muscarinic ACh receptor KO (M2R KO) mice. MOS attenuated leukocyte infiltration in WT. MOS-induced anti-inflammatory action was completely abolished in both S4R KO and S4R KO mice upon wild-type bone marrow transplantation. MOS-induced anti-inflammatory action against macrophage infiltration, but not neutrophil infiltration, was attenuated in α7 R KO mice. Selective α7nAChR agonists (PNU-282987 and AR-R17779) also inhibited only macrophage infiltration in POI. MOS-mediated inhibition of neutrophil infiltration was diminished by atropine, M2AChR antagonist, methoctramine, and in M2R KO mice. Stimulation with 5-HT4R inhibits leukocyte infiltration in POI, possibly through myenteric plexus activation. Released ACh inhibited macrophage and neutrophil infiltration likely by activation of α7nAChR on macrophages and M2AChR. Thus, macrophage and neutrophil recruitment into inflamed sites is regulated by different types of AChR in the small intestine.