RESUMEN
The serotonin-gated ion channel (5-HT3R) mediates excitatory neuronal communication in the gut and the brain. It is the target for setrons, a class of competitive antagonists widely used as antiemetics, and is involved in several neurological diseases. Cryo-electron microscopy (cryo-EM) of the 5-HT3R in complex with serotonin or setrons revealed that the protein has access to a wide conformational landscape. However, assigning known high-resolution structures to actual states contributing to the physiological response remains a challenge. In the present study, we used voltage-clamp fluorometry (VCF) to measure simultaneously, for 5-HT3R expressed at a cell membrane, conformational changes by fluorescence and channel opening by electrophysiology. Four positions identified by mutational screening report motions around and outside the serotonin-binding site through incorporation of cysteine-tethered rhodamine dyes with or without a nearby quenching tryptophan. VCF recordings show that the 5-HT3R has access to four families of conformations endowed with distinct fluorescence signatures: 'resting-like' without ligand, 'inhibited-like' with setrons, 'pre-active-like' with partial agonists, and 'active-like' (open channel) with partial and strong agonists. Data are remarkably consistent with cryo-EM structures, the fluorescence partners matching respectively apo, setron-bound, 5-HT bound-closed, and 5-HT-bound-open conformations. Data show that strong agonists promote a concerted motion of all fluorescently labeled sensors during activation, while partial agonists, especially when loss-of-function mutations are engineered, stabilize both active and pre-active conformations. In conclusion, VCF, though the monitoring of electrophysiologically silent conformational changes, illuminates allosteric mechanisms contributing to signal transduction and their differential regulation by important classes of physiological and clinical effectors.
Asunto(s)
Fluorometría , Técnicas de Placa-Clamp , Conformación Proteica , Receptores de Serotonina 5-HT3 , Receptores de Serotonina 5-HT3/metabolismo , Receptores de Serotonina 5-HT3/química , Receptores de Serotonina 5-HT3/genética , Fluorometría/métodos , Humanos , Serotonina/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Sitios de Unión , Activación del Canal IónicoRESUMEN
Visceral and somatic hypersensitivity is a common cause of functional dyspepsia. Marine bioactive components have been revealed to possess numerous valuable abilities. However, as a kind of polysaccharide extracted from brown algae, the study focused on the biological properties of laminarin is still limited, especially in gastrointestinal disorders. In our study, indicators associated with visceral sensational function and gastrointestinal microecology were determined to investigate the modulatory effects of laminarin on functional dyspepsia induced by iodoacetamide. Mice with visceral hypersensitivity were orally administrated with laminarin (50 and 100 mg per kg bw) for fourteen days. The results indicated that laminarin partly alleviated the dysfunction by regulating corticosterone secretion, the expression of 5HT3 receptors at both protein and mRNA levels, and mechanical transduction through the PIEZO2-EPAC1 axis. Furthermore, laminarin administration moderated the imbalanced gut microbial profile, including modulating the abundance of Bacteroidetes and Firmicutes. Our findings revealed that laminarin may restore the overexpression of 5HT3 receptors, the abnormal mechanical transduction, and impaired gut microecology. In conclusion, we provide evidence to support the utilization of laminarin as the ingredient of complementary and alternative medicine of regulating visceral and somatic hypersensitivity.
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Dispepsia , Microbioma Gastrointestinal , Glucanos , Yodoacetamida , Receptores de Serotonina 5-HT3 , Animales , Receptores de Serotonina 5-HT3/metabolismo , Receptores de Serotonina 5-HT3/genética , Ratones , Microbioma Gastrointestinal/efectos de los fármacos , Dispepsia/tratamiento farmacológico , Dispepsia/metabolismo , Glucanos/farmacología , Masculino , Yodoacetamida/farmacología , Corticosterona/sangreRESUMEN
Anxiety disorders are the most common psychiatric condition, but the etiology of anxiety disorders remains largely unclear. Our previous studies have shown that neuroplastin 65 deficiency (NP65-/-) mice exhibit abnormal social and mental behaviors and decreased expression of tryptophan hydroxylase 2 (TPH2) protein. However, whether a causal relationship between TPH2 reduction and anxiety disorders exists needs to be determined. In present study, we found that replenishment of TPH2 in dorsal raphe nucleus (DRN) enhanced 5-HT level in the hippocampus and alleviated anxiety-like behaviors. In addition, injection of AAV-NP65 in DRN significantly increased TPH2 expression in DRN and hippocampus, and reduced anxiety-like behaviors. Acute administration of exogenous 5-HT or HTR3 agonist SR57227A in hippocampus mitigated anxiety-like behaviors in NP65-/- mice. Moreover, replenishment of TPH2 in DRN partly repaired the impairment of long-term potentiation (LTP) maintenance in hippocampus of NP65-/- mice. Finally, we found that loss of NP65 lowered transcription factors Lmx1b expression in postnatal stage and replenishment of NP65 in DRN reversed the decrease in Lmx1b expression of NP65-/- mice. Together, our findings reveal that NP65 deficiency induces anxiety phenotype by downregulating DRN-hippocampus serotonergic-HTR3 transmission. These studies provide a novel and insightful view about NP65 function, suggesting an attractive potential target for treatment of anxiety disorders.
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Ansiedad , Núcleo Dorsal del Rafe , Hipocampo , Ratones Noqueados , Receptores de Serotonina 5-HT3 , Serotonina , Triptófano Hidroxilasa , Animales , Núcleo Dorsal del Rafe/metabolismo , Hipocampo/metabolismo , Ansiedad/metabolismo , Serotonina/metabolismo , Ratones , Masculino , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismo , Triptófano Hidroxilasa/deficiencia , Receptores de Serotonina 5-HT3/metabolismo , Receptores de Serotonina 5-HT3/genética , Ratones Endogámicos C57BL , Fenotipo , Potenciación a Largo PlazoRESUMEN
The N6-methyladenosine (m6A) modification of RNA is an emerging epigenetic regulatory mechanism that has been shown to participate in various pathophysiological processes. However, its involvement in modulating neuropathic pain is still poorly understood. In this study, we elucidate a functional role of the m6A demethylase alkylation repair homolog 5 (ALKBH5) in modulating trigeminal-mediated neuropathic pain. Peripheral nerve injury selectively upregulated the expression level of ALKBH5 in the injured trigeminal ganglion (TG) of rats. Blocking this upregulation in injured TGs alleviated trigeminal neuropathic pain, while mimicking the upregulation of ALKBH5 in intact TG neurons sufficiently induced pain-related behaviors. Mechanistically, histone deacetylase 11 downregulation induced by nerve injury increases histone H3 lysine 27 acetylation (H3K27ac), facilitating the binding of the transcription factor forkhead box protein D3 (FOXD3) to the Alkbh5 promoter and promoting Alkbh5 transcription. The increased ALKBH5 erases m6A sites in Htr3a messenger RNA (mRNA), resulting in an inability of YT521-B homology domain 2 (YTHDF2) to bind to Htr3a mRNA, thus causing an increase in 5-HT3A protein expression and 5-HT3 channel currents. Conversely, blocking the increased expression of ALKBH5 in the injured TG destabilizes nerve injury-induced 5-HT3A upregulation and reverses mechanical allodynia, and the effect can be blocked by 5-HT3A knockdown. Together, FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain through m6A-dependent stabilization of Htr3a mRNA in TG neurons. This mechanistic understanding may advance the discovery of new therapeutic targets for neuropathic pain management.
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Neuralgia , Neuralgia del Trigémino , Animales , Ratas , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Neuralgia/genética , Neuralgia/metabolismo , ARN Mensajero/metabolismo , Células Receptoras Sensoriales/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Receptores de Serotonina 5-HT3/genéticaRESUMEN
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.
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Receptores de Serotonina 5-HT3 , Serotonina , Humanos , Serotonina/metabolismo , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/metabolismo , Células HEK293 , Ondansetrón/farmacología , Mitocondrias/metabolismoRESUMEN
Serotonin or 5-hydroxytryptamine type 3 (5-HT3) receptors belong to the family of pentameric ligand-gated ion channels (pLGICs) that are therapeutic targets for psychiatric disorders and neurological diseases. Due to structural conservation and significant sequence similarities of pLGICs' extracellular and transmembrane domains, clinical trials for drug candidates targeting these two domains have been hampered by off-subunit modulation. With the present study, we explore the interaction interface of the 5-HT3A subunit intracellular domain (ICD) with the resistance to inhibitors of choline esterase (RIC-3) protein. Previously, we have shown that RIC-3 interacts with the L1-MX segment of the ICD fused to maltose-binding protein. In the present study, synthetic L1-MX-based peptides and Ala-scanning identify positions W347, R349, and L353 as critical for binding to RIC-3. Complementary studies using full-length 5-HT3A subunits confirm that the identified Ala substitutions reduce the RIC-3-mediated modulation of functional surface expression. Additionally, we find and characterize a duplication of the binding motif, DWLR VLDR, present in both the MX-helix and the transition between the ICD MA-helix and transmembrane segment M4. Analogous Ala substitutions at W447, R449, and L454 disrupt MAM4-peptide RIC-3 interactions and reduce modulation of functional surface expression. In summary, we identify the binding motif for RIC-3 in 5-HT3A subunits at two locations in the ICD, one in the MX-helix and one at the MAM4-helix transition.
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Receptores de Serotonina 5-HT3 , Serotonina , Humanos , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/química , Dominios ProteicosRESUMEN
BACKGROUND: Previous studies have shown that depression was associated with HTR3B gene. The aim of this study was to investigate the relationship between polymorphisms of the HTR3B gene and depression and its executive dysfunction in Chinese Han population. METHODS: A total of 229 patients with depressive disorder and 202 healthy controls were enrolled. Six Single nucleotide polymorphism sites (SNPs) including rs10789970, rs4938056, rs12421126, rs1176744, rs2276305 and rs12795805 were genotyped by Snapshot. Clinical features were collected using a general demographic questionnaire. The 24-item Hamilton Depression Scale (HAMD) was used to assess the symptoms' severity of the patients. The patients' executive function was assessed using a series of cognitive tests including Maze Test, Symbolic Coding Test, Spatial Span Inverse Order Test, Linking Test, and Emotional Management Test. RESULTS: The genotypic and allelic distributions of rs1176744 in HTR3B gene were significantly different (χ2 = 11.129, P = 0.004, χ2 = 9.288, P = 0.002, respectively) between patients and controls. The A allele was positively correlated with depression. The proportion of A carriers was significantly higher and that of C carriers was lower in patients than those in controls. Patients had significantly lower scores of Spatial Span Inverse Order Test in carriers of A allele at locus rs1176744 and higher scores in carriers of C alleles at locus rs1176744 and rs12795805. CONCLUSIONS: The polymorphisms of HTR3B gene may be associated with depression in Chinese Han population. The A allele of rs1176744 may increase the risk of developing depression and executive dysfunction while C alleles of rs1176744 and rs12795805 may be the protective factors for executive dysfunction in patients with depression.
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Disfunción Cognitiva , Depresión , Humanos , Estudios de Casos y Controles , Depresión/genética , Polimorfismo de Nucleótido Simple , Genotipo , Receptores de Serotonina 5-HT3/genéticaRESUMEN
5-HT3 receptors are members of the family of pentameric ligand-gated ion channels. Each subunit has an extracellular, transmembrane, and intracellular domain. Only part of the intracellular domain structure has been solved, revealing it contains two α-helical segments; one, the MA helix, is an extension of M4, while the other, the MX helix, is formed from residues located close to the end of M3. This MX helix is in distinct locations in open and closed receptor structures, suggesting it may play a role in function. Here, we explore this hypothesis using functional responses of Ala-substituted mutant receptors expressed in HEK293 cells. The data show altering many of the MX residues results in a small decrease in EC50 (up to 5-fold), although in one (H232A) this is increased. Radiolabeled ligand binding on selected mutants showed no change in binding affinity, indicating an effect on gating and not binding. In addition, five mutations (P316A, V317A, P318A, D319A, and H323A) initially resulted in nonfunctional receptors, but the function could be rescued by coexpression with a chaperone protein, suggesting a likely role in assembly or folding. Examination of previously obtained MD simulation data shows that the extent of MX encompassed by membrane lipids differs considerably in the open and closed structures, suggesting that lipid-protein interactions in this region could have a major effect on channel opening propensity. We conclude that the MX helix can modulate the function of the receptor and propose that its interactions with membrane lipids play a major role in this.
Asunto(s)
Receptores de Serotonina 5-HT3 , Serotonina , Secuencia de Aminoácidos , Células HEK293 , Humanos , Lípidos de la Membrana , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/metabolismoRESUMEN
BACKGROUND: Single-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). AIM: To assess the association of HTR3 polymorphisms with depressive, anxiety, and somatization symptoms in individuals with IBS. METHODS: In 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. RESULTS: Depressive 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. CONCLUSION: We 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.
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Síndrome del Colon Irritable , Alelos , Humanos , Síndrome del Colon Irritable/genética , Síndrome del Colon Irritable/metabolismo , Polimorfismo de Nucleótido Simple , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/metabolismo , Estudios Retrospectivos , Serotonina/genética , Serotonina/metabolismoRESUMEN
Several studies have examined the acquisition and extinction of fear in PTSD in the context of Pavlovian conditioning. However, research examining reconditioning of fear following extinction, a form of post-extinction re-emergence of conditioned behavior is limited. Although the 5-HT3A receptor gene polymorphism has been linked to trauma responses, its influence on the re-emergence of conditioned fear among those exposed to trauma remain unclear. In the present study, combat-exposed veterans (N = 114) completed a differential fear conditioning task in which one colored rectangle (CS+) predicted a loud scream (US), whereas a different colored rectangle (CS-) predicted no US. Acquisition, extinction, and post-extinction reconditioning effects indexed by conditioned anxiety, US expectancy, and skin conductance response were examined. Associations with allelic variation in the serotonin 5-HT3 gene, HTR3A (rs1062613) were also examined. Participants rated the CS+ as significantly more anxiety inducing and associated with greater US expectancy than the CS- during acquisition. The CS+ also elicited a stronger skin conductance response than the CS- during acquisition. A significant decrease in anxiety and US expectancy in response to the CS+ was observed after extinction and a re-emergence of conditioned responses to the CS+ was observed during reacquisition. Although a diagnosis of PTSD was characterized by greater anxiety to the CS + but not the CS- during acquisition and extinction, those with and without a PTSD diagnosis did not differ in the reacquisition of fear following extinction. Subsequent preliminary analysis did show that increased posttraumatic symptoms and cognitions were associated with increased US expectancy at reacquisition for the CS+ and CS- among CC carriers but not among T carriers of HTR3A (rs1062613). These findings suggest that posttraumatic symptoms among trauma exposed veterans with the CC polymorphism of the HTR3A gene may be associated with stronger reconditioning of fear following extinction.
Asunto(s)
Extinción Psicológica , Miedo , Receptores de Serotonina 5-HT3 , Trastornos por Estrés Postraumático , Ansiedad/genética , Condicionamiento Clásico/fisiología , Extinción Psicológica/fisiología , Miedo/fisiología , Respuesta Galvánica de la Piel , Humanos , Receptores de Serotonina 5-HT3/genética , Trastornos por Estrés Postraumático/genéticaRESUMEN
Monoamine serotonin is a major neurotransmitter that acts on a wide range of central nervous system and peripheral nervous system functions and is known to have a role in various processes. Recently, it has been found that 5-HT is involved in cognitive and memory functions through interaction with cholinergic pathways. The natural flavonoid kaempferol (KAE) extracted from Cudrania tricuspidata is a secondary metabolite of the plant. Recently studies have confirmed that KAE possesses a neuroprotective effect because of its strong antioxidant activity. It has been confirmed that KAE is involved in the serotonergic pathway through an in vivo test. However, these results need to be confirmed at the molecular level, because the exact mechanism that is involved in such effects of KAE has not yet been elucidated. Therefore, the objective of this study is to confirm the interaction of KAE with 5-HT3A through electrophysiological studies at the molecular level using KAE extracted from Cudrania tricuspidata. This study confirmed the interaction between 5-HT3A and KAE at the molecular level. KAE inhibited 5-HT3A receptors in a concentration-dependent and voltage-independent manner. Site-directed mutagenesis and molecular-docking studies confirmed that the binding sites D177 and F199 are the major binding sites of human 5-HT3A receptors of KAE.
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Quempferoles/farmacología , Triterpenos Pentacíclicos/farmacología , Receptores de Serotonina 5-HT3/metabolismo , Antagonistas del Receptor de Serotonina 5-HT3/farmacología , Sitios de Unión , Relación Dosis-Respuesta a Droga , Humanos , Quempferoles/química , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Triterpenos Pentacíclicos/química , Unión Proteica , Receptores de Serotonina 5-HT3/química , Receptores de Serotonina 5-HT3/genética , Antagonistas del Receptor de Serotonina 5-HT3/química , Relación Estructura-ActividadRESUMEN
Mutations in serotonin pathway genes, especially the serotonergic receptor subunit gene HTR3A, are associated with autism. However, the association of HTR3A deficiency with autism and the underlying mechanisms remain unknown. Methods: The Htr3a knockout (KO) mice were generated using transcription activator-like effector nuclease technology. Various behavior tests, including social interaction, social approach task, olfactory habituation/dishabituation, self-grooming, novel object recognition, contextual fear conditioning, elevated plus maze, open field and seizure susceptibility, were performed to assess the phenotypes. Transcriptome sequencing was carried out to search for molecular network and pathways underlying the phenotypes. Electrophysiological recordings, immunoblotting, immunofluorescence staining, immunoprecipitation, and quantitative real-time PCR were performed to verify the potential mechanisms. The N-methyl-D-aspartate receptor (NMDAR) antagonist memantine was used to treat the KO mice for rescuing the phenotypes. Results: The Htr3a KO mouse model showed three phenotypic domains: autistic-like behaviors (including impaired social behavior, cognitive deficits, and increased repetitive self-grooming), impaired memory, and attenuated susceptibility to pentylenetetrazol-induced seizures. We observed enhanced action potential-driven γ-aminobutyric acid-ergic (GABAergic) transmission in pyramidal neurons and decreased excitatory/inhibitory (E/I) ratio using the patch-clamp recording. Transcriptome sequencing on the hippocampus revealed the converged pathways of the dysregulated molecular networks underlying three phenotypic domains with upregulation of NMDAR. We speculated that Htr3a KO promotes an increase in GABA release through NMDAR upregulation. The electrophysiological recordings on hippocampal parvalbumin-positive (PV+) interneuron revealed increased NMDAR current and NMDAR-dependent excitability. The NMDAR antagonist memantine could rescue GABAergic transmission in the hippocampus and ameliorate autistic-like behaviors of the KO mice. Conclusion: Our data indicated that upregulation of the NMDAR in PV+ interneurons may play a critical role in regulating GABAergic input to pyramidal neurons and maybe involve in the pathogenesis of autism associated with HTR3A deficiency. Therefore, we suggest that the NMDAR system could be considered potential therapeutic target for autism.
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Trastorno del Espectro Autista/genética , Neuronas GABAérgicas/metabolismo , Receptores de Serotonina 5-HT3/genética , Animales , Trastorno del Espectro Autista/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Expresión Génica/genética , Hipocampo/metabolismo , Masculino , Memoria/fisiología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Células Piramidales/metabolismo , Receptores de GABA/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de Serotonina 5-HT3/metabolismo , Convulsiones/fisiopatología , Serotonina/metabolismo , Conducta Social , Transcriptoma/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Since westernized diet-induced insulin resistance is a risk factor in Alzheimer's disease (AD) development, and lipopolysaccharide (LPS) coexists with amyloid ß (Aß)1-42 in these patients, our AD novel model was developed to resemble sporadic AD by injecting LPS into high fat/fructose diet (HFFD)-fed rats. The neuroprotective potential of palonosetron and/or methyllycaconitine, 5-HT3 receptor and α7 nAChR blockers, respectively, was evaluated after 8 days of daily administration in HFFD/LPS rats. All regimens improved histopathological findings and enhanced spatial memory (Morris Water Maze); however, palonosetron alone or with methyllycaconitine promoted animal performance during novel object recognition tests. In the hippocampus, all regimens reduced the expression of glial fibrillary acidic protein and skewed microglia M1 to M2 phenotype, indicated by the decreased M1 markers and the enhanced M2 related parameters. Additionally, palonosetron and its combination regimen downregulated the expression of ASC/TMS1, as well as levels of inflammasome downstream molecules and abated cleaved caspase-1, interleukin (IL)-1ß, IL-18 and caspase-11. Furthermore, ACh and 5-HT were augmented after being hampered by the insult. Our study speculates that blocking 5-HT3 receptor using palonosetron overrides methyllycaconitine to combat AD-induced neuroinflammation and inflammasome cascade, as well as to restore microglial function in a HFFD/LPS novel model for sporadic AD.
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Aconitina/análogos & derivados , Enfermedad de Alzheimer/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Palonosetrón/farmacología , Aconitina/farmacología , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Cognición/efectos de los fármacos , Dieta Occidental/efectos adversos , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Hipocampo/patología , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Inflamación/genética , Inflamación/patología , Resistencia a la Insulina/genética , Interleucina-18/genética , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Microglía/patología , Fragmentos de Péptidos/genética , Piroptosis/efectos de los fármacos , Ratas , Receptores de Serotonina 5-HT3/genética , Factores de Riesgo , Memoria Espacial/efectos de los fármacosRESUMEN
The autonomic nervous system derives from the neural crest (NC) and supplies motor innervation to the smooth muscle of visceral organs, including the lower urinary tract (LUT). During fetal development, sacral NC cells colonize the urogenital sinus to form pelvic ganglia (PG) flanking the bladder neck. The coordinated activity of PG neurons is required for normal urination; however, little is known about the development of PG neuronal diversity. To discover candidate genes involved in PG neurogenesis, the transcriptome profiling of sacral NC and developing PG was performed, and we identified the enrichment of the type 3 serotonin receptor (5-HT3, encoded by Htr3a and Htr3b). We determined that Htr3a is one of the first serotonin receptor genes that is up-regulated in sacral NC progenitors and is maintained in differentiating PG neurons. In vitro cultures showed that the disruption of 5-HT3 signaling alters the differentiation outcomes of sacral NC cells, while the stimulation of 5-HT3 in explanted fetal pelvic ganglia severely diminished neurite arbor outgrowth. Overall, this study provides a valuable resource for the analysis of signaling pathways in PG development, identifies 5-HT3 as a novel regulator of NC lineage diversification and neuronal maturation in the peripheral nervous system, and indicates that the perturbation of 5-HT3 signaling in gestation has the potential to alter bladder function later in life.
Asunto(s)
Cresta Neural/metabolismo , Receptores de Serotonina 5-HT3/metabolismo , Transducción de Señal , Sistema Urinario/inervación , Sistema Urinario/metabolismo , Animales , Sistema Nervioso Autónomo , Diferenciación Celular , Biología Computacional/métodos , Perfilación de la Expresión Génica , Ratones , Cresta Neural/embriología , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuritas/metabolismo , Neurogénesis , Proyección Neuronal , Neuronas/metabolismo , Receptores de Serotonina/metabolismo , Receptores de Serotonina 5-HT3/genética , Transcriptoma , Sistema Urinario/embriologíaRESUMEN
Among mammals, serotonin is predominantly found in the gastrointestinal tract, where it has been shown to participate in pathway-regulating satiation. For the stomach, vascular serotonin release induced by gastric distension is thought to chiefly contribute to satiation after food intake. However, little information is available on the capability of gastric cells to synthesize, release and respond to serotonin by functional changes of mechanisms regulating gastric acid secretion. We investigated whether human gastric cells are capable of serotonin synthesis and release. First, HGT-1 cells, derived from a human adenocarcinoma of the stomach, and human stomach specimens were immunostained positive for serotonin. In HGT-1 cells, incubation with the tryptophan hydroxylase inhibitor p-chlorophenylalanine reduced the mean serotonin-induced fluorescence signal intensity by 27%. Serotonin release of 147 ± 18%, compared to control HGT-1 cells (set to 100%) was demonstrated after treatment with 30 mM of the satiating amino acid L-Arg. Granisetron, a 5-HT3 receptor antagonist, reduced this L-Arg-induced serotonin release, as well as L-Arg-induced proton secretion. Similarly to the in vitro experiment, human antrum samples released serotonin upon incubation with 10 mM L-Arg. Overall, our data suggest that human parietal cells in culture, as well as from the gastric antrum, synthesize serotonin and release it after treatment with L-Arg via an HTR3-related mechanism. Moreover, we suggest not only gastric distension but also gastric acid secretion to result in peripheral serotonin release.
Asunto(s)
Arginina/farmacología , Ácido Gástrico/metabolismo , Células Parietales Gástricas/efectos de los fármacos , Protones , Serotonina/biosíntesis , Línea Celular Tumoral , Fenclonina/farmacología , Expresión Génica , Granisetrón/farmacología , Humanos , Concentración de Iones de Hidrógeno , Células Parietales Gástricas/citología , Células Parietales Gástricas/metabolismo , Inhibidores de Proteasas/farmacología , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/metabolismo , Antagonistas de la Serotonina/farmacología , Estómago/citología , Estómago/efectos de los fármacos , Técnicas de Cultivo de Tejidos , Triptófano Hidroxilasa/antagonistas & inhibidores , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismoRESUMEN
Schisandrin C (Sch C) is one of the main components of Schisandra chinensis (Schisandra). Since the olden times, Schisandra has been used as a traditional herbal medicine in Asia. Recent studies have shown that Schisandra is effective against irritable bowel syndrome (IBS) in an animal model and affects IBS through the 5-HT3A pathway in the IBS rat model. However, there lacks fundamental research on the interaction of specific components of Schisandra with the 5-HT3A receptor for the treatment of IBS. We hypothesized that a component of Schisandra binds to the 5-HT3A receptor and identified Sch C via a screening work using two electrode-voltage clamps (TEVC). Thus, we aimed to elucidate the neuropharmacological actions between Sch C and the 5-HT3A receptor at molecular and cellular levels. Co-treatment of Sch C with 5-HT inhibited I5-HT in a reversible, concentrate-dependent, like-competition, and voltage-independent manner, and IC50 values of Sch C. Besides, the main binding positions of Sch C were identified through 3D modeling and point mutation were V225A and V288Y on 5-HT3A receptor. Thus, we suggest the potential of Sch C in treating IBS in a manner that suppresses excessive neuronal serotonin signaling in the synapse of sensory neurons and enterochromaffin (EC) cells. In conclusion, the results demonstrate the mechanism of interaction between Sch C and 5-HT3A receptor and reveal Sch C as a novel antagonist.
Asunto(s)
Lignanos/farmacología , Compuestos Policíclicos/farmacología , Receptores de Serotonina 5-HT3/metabolismo , Antagonistas del Receptor de Serotonina 5-HT3/farmacología , Animales , Ciclooctanos/farmacología , Ciclooctanos/uso terapéutico , Células Enterocromafines/efectos de los fármacos , Células Enterocromafines/metabolismo , Humanos , Concentración 50 Inhibidora , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inervación , Mucosa Intestinal/patología , Síndrome del Colon Irritable/tratamiento farmacológico , Síndrome del Colon Irritable/patología , Lignanos/uso terapéutico , Simulación del Acoplamiento Molecular , Oocitos , Técnicas de Placa-Clamp , Compuestos Policíclicos/uso terapéutico , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Antagonistas del Receptor de Serotonina 5-HT3/uso terapéutico , Xenopus laevisRESUMEN
Evidence shows that long noncoding RNAs (lncRNAs) modulate mRNAs of multiple genes by post-transcriptional regulation. However, in esophageal squamous cell carcinoma, lncRNAs involvement in post-transcriptional regulation of mRNAs have been rarely reported. In this study, we investigated a novel mechanism of linc01305 promoting metastasis and proliferation of ESCC. The results for real-time quantitative reverse transcription PCR (qRT-PCR) and fluorescence in situ hybridization showed that linc01305 was highly expressed and predominantly located in cytoplasm of human esophageal cancer cells. Transwell and colony formation assays confirmed that linc01305 promoted migration and proliferation of esophageal cancer cells. RNA-seq, linc01305 pulldown, mass spectrometry, RNA immunoprecipitation and mRNA stability assays demonstrated that linc01305 stabilized mRNA of target gene HTR3A through interacting with IGF2BP2 and IGF2BP3. Taken together, our data unveils a novel mechanism in which cytoplasmic linc01305 stabilizes HTR3A mRNA through interacting with IGF2BP2 and IGF2BP3 and thereby promotes metastasis and proliferation of ESCC.
Asunto(s)
Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/secundario , Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/metabolismo , Receptores de Serotonina 5-HT3/química , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Humanos , Pronóstico , Proteínas de Unión al ARN/genética , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/metabolismo , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Rotavirus infection is highly prevalent in children, and the most severe effects are diarrhea and vomiting. It is well accepted that the enteric nervous system (ENS) is activated and plays an important role, but knowledge of how rotavirus activates nerves within ENS and to the vomiting center is lacking. Serotonin is released during rotavirus infection, and antagonists to the serotonin receptor subtype 3 (5-HT3 receptor) can attenuate rotavirus-induced diarrhea. In this study, we used a 5-HT3 receptor knockout (KO) mouse model to investigate the role of this receptor in rotavirus-induced diarrhea, motility, electrolyte secretion, inflammatory response, and vomiting reflex. The number of diarrhea days (P = 0.03) and the number of mice with diarrhea were lower in infected 5-HT3 receptor KO than wild-type pups. In vivo investigation of fluorescein isothiocyanate (FITC)-dextran transit time showed that intestinal motility was lower in the infected 5-HT3 receptor KO compared to wild-type mice (P = 0.0023). Ex vivo Ussing chamber measurements of potential difference across the intestinal epithelia showed no significant difference in electrolyte secretion between the two groups. Immediate early gene cFos expression level showed no difference in activation of the vomiting center in the brain. Cytokine analysis of the intestine indicated a low effect of inflammatory response in rotavirus-infected mice lacking the 5-HT3 receptor. Our findings indicate that the 5-HT3 receptor is involved in rotavirus-induced diarrhea via its effect on intestinal motility and that the vagus nerve signaling to the vomiting center occurs also in the absence of the 5-HT3 receptor. IMPORTANCE The mechanisms underlying rotavirus-induced diarrhea and vomiting are not yet fully understood. To better understand rotavirus pathophysiology, characterization of nerve signaling within the ENS and through vagal efferent nerves to the brain, which have been shown to be of great importance to the disease, is necessary. Serotonin (5-HT), a mediator of both diarrhea and vomiting, has been shown to be released from enterochromaffin cells in response to rotavirus infection and the rotavirus enterotoxin NSP4. Here, we investigated the role of the serotonin receptor 5-HT3, which is known to be involved in the nerve signals that regulate gut motility, intestinal secretion, and signal transduction through the vagus nerve to the brain. We show that the 5-HT3 receptor is involved in rotavirus-induced diarrhea by promoting intestinal motility. The findings shed light on new treatment possibilities for rotavirus diarrhea.
Asunto(s)
Diarrea/fisiopatología , Sistema Nervioso Entérico/fisiopatología , Motilidad Gastrointestinal/fisiología , Receptores de Serotonina 5-HT3/metabolismo , Infecciones por Rotavirus/patología , Vómitos/fisiopatología , Animales , Células Enterocromafines/metabolismo , Motilidad Gastrointestinal/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Serotonina 5-HT3/genética , Rotavirus/fisiología , Serotonina/metabolismo , Antagonistas del Receptor de Serotonina 5-HT3/farmacologíaRESUMEN
Cortical GABAergic inhibitory neurons are composed of three major classes, each expressing parvalbumin (PV), somatostatin (SOM) and 5-hydroxytryptamine receptor 3A (Htr3a), respectively. Htr3a+ inhibitory neurons are mainly derived from the caudal ganglionic eminence (CGE). This highly heterogeneous group of inhibitory neurons are comprised of many different subtypes with distinct molecular signatures, morphological and electrophysiological properties and connectivity patterns. In this review, we summarized recent research progress regarding cortical Htr3a+ inhibitory neurons, focusing on their molecular, morphological and electrophysiological diversity, and introduced some genetic mouse tools that were used to study Htr3a+ inhibitory neurons.
Asunto(s)
Interneuronas , Serotonina , Animales , Interneuronas/metabolismo , Ratones , Neuronas/metabolismo , Parvalbúminas/genética , Parvalbúminas/metabolismo , Receptores de Serotonina 5-HT3/genética , Somatostatina/metabolismoRESUMEN
Earlier, after our bioinformatic analysis of single-nucleotide polymorphisms of TATA-binding protein-binding sites within gene promoters on the human Y chromosome, we suggested that human reproductive potential diminishes during self-domestication. Here, we implemented bioinformatics models of human diseases using animal in vivo genome-wide RNA-Seq data to compare the effect of co-directed changes in the expression of orthologous genes on human reproductive potential and during the divergence of domestic and wild animals from their nearest common ancestor (NCA). For example, serotonin receptor 3A (HTR3A) deficiency contributes to sudden death in pregnancy, consistently with Htr3a underexpression in guinea pigs (Cavia porcellus) during their divergence from their NCA with cavy (C. aperea). Overall, 25 and three differentially expressed genes (hereinafter, DEGs) in domestic animals versus 11 and 17 DEGs in wild animals show the direction consistent with human orthologous gene-markers of reduced and increased reproductive potential. This indicates a reliable association between DEGs in domestic animals and human orthologous genes reducing reproductive potential (Pearson's χ2 test p < 0.001, Fisher's exact test p < 0.05, binomial distribution p < 0.0001), whereas DEGs in wild animals uniformly match human orthologous genes decreasing and increasing human reproductive potential (p > 0.1; binomial distribution), thus enforcing the norm (wild type).