RESUMEN
Microtubules are a vital component of the cell's cytoskeleton and their organization is crucial for healthy cell functioning. The use of label-free SH imaging of microtubules remains limited, as sensitive detection is required and the true molecular origin and main determinants required to generate SH from microtubules are not fully understood. Using advanced correlative imaging techniques, we identified the determinants of the microtubule-dependent SH signal. Microtubule polarity, number and organization determine SH signal intensity in biological samples. At the molecular level, we show that the GTP-bound tubulin dimer conformation is fundamental for microtubules to generate detectable SH signals. We show that SH imaging can be used to study the effects of microtubule-targeting drugs and proteins and to detect changes in tubulin conformations during neuronal maturation. Our data provide a means to interpret and use SH imaging to monitor changes in the microtubule network in a label-free manner.
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Microscopía Intravital/métodos , Microtúbulos/ultraestructura , Imagen Molecular/métodos , Microscopía de Generación del Segundo Armónico , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Células Cultivadas , Colchicina/farmacología , Estudios de Factibilidad , Guanosina Trifosfato/metabolismo , Ratones , Microscopía Electrónica , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Neurogénesis , Cultivo Primario de Células , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestructuraRESUMEN
Previously, we showed histamine-mediated sensitization of transient receptor potential (TRP) vanilloid 1 (TRPV1) in patients with irritable bowel syndrome (IBS). Sensitization of TRP ankyrin 1 (TRPA1) and TRP vanilloid 4 (TRPV4) are also involved in aberrant pain perception in preclinical models of somatic pain. Here, we hypothesize that in parallel with TRPV1, histamine sensitizes TRPA1 and TRPV4, contributing to increased visceral pain in patients with IBS. Rectal biopsies were collected from patients with IBS and healthy subjects (HS) to study neuronal sensitivity to TRPA1 and TRPV4 agonists (cinnamaldehyde and GSK1016790A) using intracellular Ca2+ imaging. In addition, the effect of supernatants of rectal biopsies on patients with IBS and HS was assessed on TRPA1 and TRPV4 responses in murine dorsal root ganglion (DRG) sensory neurons. Finally, we evaluated the role of histamine and histamine 1 receptor (H1R) in TRPA1 and TRPV4 sensitization. Application of TRPA1 and TRPV4 agonists evoked significantly higher peak amplitudes and percentage of responding submucosal neurons in biopsies of patients with IBS compared with HS. In HS, pretreatment with histamine significantly increased the Ca2+ responses to cinnamaldehyde and GSK1016790A, an effect prevented by H1R antagonism. IBS supernatants, but not of HS, sensitized TRPA1 and TRPV4 on DRG neurons. This effect was reproduced by histamine and prevented by H1R antagonism. We demonstrate that the mucosal microenvironment in IBS contains mediators, such as histamine, which sensitize TRPV4 and TRPA1 via H1R activation, most likely contributing to increased visceral pain perception in IBS. These data further underscore H1R antagonism as potential treatment for IBS. NEW & NOTEWORTHY We provide evidence for histamine-mediated transient receptor potential (TRP) ankyrin 1 and TRP vanilloid 4 sensitization in irritable bowel syndrome (IBS) via histamine 1 receptor (H1R) activation, most likely contributing to increased visceral pain perception. Our results reveal a general role of sensory TRP channels as histamine effectors in the pathophysiology of IBS and provide novel mechanistic insights into the therapeutic potential of H1R antagonism in IBS.
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Histamina/metabolismo , Canales Catiónicos TRPV/metabolismo , Adulto , Animales , Femenino , Humanos , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Células Receptoras Sensoriales/metabolismo , Transducción de Señal/fisiología , Canales Catiónicos TRPV/genética , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
Tiredness of life in older adults can lead to a request for the wish to die. This article provides a practical approach for physicians of this problem on the basis of a flow chart. The main causes of tiredness of life should be identified and evaluated for their reversibility and treatment options. The first group are the physical factors which, besides organ pathology, should also take frailty into account as a possible cause. A second important group are the psychological risk factors such as psychiatric disorders, loneliness, dignity, subjective well-being, coping and spiritual power. These factors also determine the complaint and needs of the patient. Here is a multidisciplinary assessment and approach desirable. This multidisciplinary approach also applies to the socioeconomic risk factors. In addition, the caregiver should examine if the weariness of life indeed gives rise to the suffering of the older person and to what extent this is hopeless and unbearable suffering. Hopelessness is a professional judgment about the remaining treatment and care perspective and is often objectified; unbearable is a matter of the patient and therefore always subjective and personal. The current legislation on euthanasia, the reversibility of the underlying causes and the unbearable suffering will determine whether the request of the patient with tiredness of life can be considered. Some questions will not fit within the proposed framework. For those a multidisciplinary advice of an ethics committee may be desirable.
La fatigue de vie chez la personne âgée peut susciter le désir de mourir. Ce document, destiné aux médecins, est un manuel pratique sur cette problématique. Il faut rechercher les facteurs de risque physiques, psychiques et socioéconomiques de la lassitude pour évaluer dans quelle mesure ils sont réversibles et peuvent être traités. L'étiologie de la lassitude étant souvent multifactorielle, il est souhaitable que son évaluation soit multidisciplinaire, tout comme son approche. Le prestataire de soins doit chercher à savoir si la lassitude est responsable d'une souffrance sans issue et insupportable. Le fait que la souffrance soit sans issue doit être évalué de manière professionnelle pour répondre à la question de savoir si une perspective de traitement et de soins est encore présente, ce qui est souvent objectivable. Le fait que la souffrance soit insupportable est strictement personnel et toujours subjectif. L'éventuelle prise en compte d'une demande d'euthanasie en cas de fatigue de vivre est déterminée par les critères de la législation actuelle sur l'euthanasie, la réversibilité des causes sous-jacentes de la lassitude et le caractère insupportable de la souffrance. Si, comme cela arrive de temps à autre, la demande d'euthanasie se situe en dehors du cadre proposé, l'avis multidisciplinaire mûrement réfléchi d'un comité d'éthique est alors souhaitable.
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Envejecimiento/psicología , Fatiga/psicología , Estrés Psicológico/etiología , Anciano , Anciano de 80 o más Años , Eutanasia/psicología , Eutanasia/estadística & datos numéricos , Fatiga/epidemiología , Fatiga/etiología , Humanos , Trastornos Mentales/epidemiología , Estrés Psicológico/epidemiologíaRESUMEN
Post-infectious irritable bowel syndrome (PI-IBS) is a common gastrointestinal disorder characterized by persistent abdominal pain despite recovery from acute gastroenteritis. The underlying mechanisms are unclear, although long-term changes in neuronal function, and low grade inflammation of the bowel have been hypothesized. We investigated the presence and mechanism of neuronal sensitization in a unique cohort of individuals who developed PI-IBS following exposure to contaminated drinking water 7 years ago. We provide direct evidence of ongoing sensitization of neuronal signaling in the bowel of patients with PI-IBS. These changes occur in the absence of any detectable tissue inflammation, and instead appear to be driven by pro-nociceptive changes in the gut micro-environment. This is evidenced by the activation of murine colonic afferents, and sensitization responses to capsaicin in dorsal root ganglia (DRGs) following application of supernatants generated from tissue biopsy of patients with PI-IBS. We demonstrate that neuronal signaling within the bowel of PI-IBS patients is sensitized 2 years after the initial infection has resolved. This sensitization appears to be mediated by a persistent pro-nociceptive change in the gut micro-environment, that has the capacity to stimulate visceral afferents and facilitate neuronal TRPV1 signaling.
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Síndrome del Colon Irritable/diagnóstico , Adulto , Animales , Capsaicina/farmacología , Estudios de Casos y Controles , Colon/patología , Citocinas/metabolismo , Femenino , Ganglios Espinales/patología , Gastroenteritis/complicaciones , Gastroenteritis/patología , Humanos , Mucosa Intestinal/metabolismo , Síndrome del Colon Irritable/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Receptores Histamínicos H1/metabolismo , Transducción de Señal , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismoRESUMEN
BACKGROUND: Promoter methylation of N-myc Downstream-Regulated Gene 4 (NDRG4) in fecal DNA is an established early detection marker for colorectal cancer (CRC). Despite its connection to CRC, NDRG4 is predominantly studied in brain and heart, with little to no knowledge about its expression or role in other organs. In this study, we aimed to determine the whole-body expression of NDRG4, with a focus on the intestinal tract. METHODS: We investigated NDRG4 expression throughout the body by immunohistochemistry, Western Blotting and in situ mRNA hybridization using tissues from NDRG4 wild-type, heterozygous and knockout mice and humans. In addition, we explored cell-specific expression of NDRG4 in murine whole-mount gut preparations using immunofluorescence and confocal microscopy. KEY RESULTS: NDRG4 is specifically expressed within nervous system structures throughout the body. In the intestinal tract of both mouse and man, NDRG4 immunoreactivity was restricted to the enteric nervous system (ENS), where it labeled cell bodies of the myenteric and submucosal plexuses and interconnecting nerve fibers. More precisely, NDRG4 expression was limited to neurons, as NDRG4 always co-localized with HuC/D (pan-neuronal marker) but never with GFAP (an enteric glial cell marker). Furthermore, NDRG4 was expressed in various neuropeptide Y positive neurons, but was only found in a minority (~10%) of neurons expressing neuronal nitric oxide synthase. CONCLUSIONS AND INFERENCES: NDRG4 is exclusively expressed by central, peripheral and enteric neurons/nerves, suggesting a neuronal-specific role of this protein. Our findings raise the question whether NDRG4, via the ENS, an understudied component of the tumor microenvironment, supports CRC development and/or progression.
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Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/metabolismo , Sistema Nervioso Entérico/metabolismo , Proteínas Musculares/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/metabolismo , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Musculares/análisis , Proteínas del Tejido Nervioso/análisisRESUMEN
Co-ordinated gastrointestinal function is the result of integrated communication between the enteric nervous system (ENS) and "effector" cells in the gastrointestinal tract. Unlike smooth muscle cells, interstitial cells, and the vast majority of cell types residing in the mucosa, enteric neurons and glia are not generated within the gut. Instead, they arise from neural crest cells that migrate into and colonise the developing gastrointestinal tract. Although they are "later" arrivals into the developing gut, enteric neural crest-derived cells (ENCCs) respond to many of the same secreted signalling molecules as the "resident" epithelial and mesenchymal cells, and several factors that control the development of smooth muscle cells, interstitial cells and epithelial cells also regulate ENCCs. Much progress has been made towards understanding the migration of ENCCs along the gastrointestinal tract and their differentiation into neurons and glia. However, our understanding of how enteric neurons begin to communicate with each other and extend their neurites out of the developing plexus layers to innervate the various cell types lining the concentric layers of the gastrointestinal tract is only beginning. It is critical for postpartum survival that the gastrointestinal tract and its enteric circuitry are sufficiently mature to cope with the influx of nutrients and their absorption that occurs shortly after birth. Subsequently, colonisation of the gut by immune cells and microbiota during postnatal development has an important impact that determines the ultimate outline of the intrinsic neural networks of the gut. In this review, we describe the integrated development of the ENS and its target cells.
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Sistema Nervioso Entérico/embriología , Tracto Gastrointestinal/inervación , Mesodermo/embriología , Cresta Neural/embriología , Animales , Comunicación Celular/fisiología , Diferenciación Celular , Movimiento Celular/fisiología , Tracto Gastrointestinal/embriología , Humanos , Cresta Neural/citología , Neuronas/citología , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: With an increasing number of people dying in old age, collaboration between palliative care and geriatric medicine is increasingly being advocated in order to promote better health and health care for the increasing number of older people. The aim of this study is to identify barriers and facilitators and good practice examples of collaboration and integration between palliative care and geriatric medicine from a European perspective. METHODS: Four semi-structured group interviews were undertaken with 32 participants from 18 countries worldwide. Participants were both clinicians (geriatricians, GPs, palliative care specialists) and academic researchers. The interviews were transcribed and independent analyses performed by two researchers who then reached consensus. RESULTS: Limited knowledge and understanding of what the other discipline offers, a lack of common practice and a lack of communication between disciplines and settings were considered as barriers for collaboration between palliative care and geriatric medicine. Multidisciplinary team working, integration, strong leadership and recognition of both disciplines as specialties were considered as facilitators of collaborative working. Whilst there are instances of close clinical working between disciplines, examples of strategic collaboration in education and policy were more limited. CONCLUSIONS: Improving knowledge about its principles and acquainting basic palliative care skills appears mandatory for geriatricians and other health care professionals. In addition, establishing more academic chairs is seen as a priority in order to develop more education and development at the intersection of palliative care and geriatric medicine.
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Servicios de Salud para Ancianos , Comunicación Interdisciplinaria , Cuidados Paliativos , Anciano , Anciano de 80 o más Años , Actitud del Personal de Salud , Conducta Cooperativa , Europa (Continente)/epidemiología , Femenino , Geriatría , Necesidades y Demandas de Servicios de Salud , Servicios de Salud para Ancianos/organización & administración , Servicios de Salud para Ancianos/normas , Humanos , Masculino , Persona de Mediana Edad , Cuidados Paliativos/organización & administración , Cuidados Paliativos/normas , Guías de Práctica Clínica como Asunto , Investigación CualitativaRESUMEN
The intake of free fructose has increased substantially since the development of high-fructose corn syrup. This has not only been associated with metabolic disorders but recent evidence also indicates that chronic fructose consumption can affect neuronal and cognitive function. In this study we investigated the effects of fructose consumption on serotonergic signaling and neuronal activity in the mouse submucous plexus. Male mice were put on a control or fructose (23% solution) diet for 6 weeks or were assigned to a recovery group that received normal water (2 weeks) after 4 weeks of fructose. At the end of the diet, gene expressions and enteric neuronal activity, after depolarization with high K(+) and 5-HT, were measured using Ca(2+) imaging and RT-qPCR, respectively. Even in the lack of gain weight and the absence of changes in duodenal permeability, the total number of 5-HT-responding neurons and the depolarization and 5-HT-evoked Ca(2+) amplitudes were significantly lower after fructose consumption. Expression of synaptobrevin CaV 2.1 and CaV 2.2 mRNA did not differ after fructose intake; however, CaV 2.1 mRNA levels were significantly higher in the recovery animals. SERT mRNA concentration, isolated from submucosal plexus containing mucosal epithelium, was significantly decreased after fructose consumption. Chronic fructose consumption impairs serotonergic signaling in the mouse submucous plexus, prior to weight gain and detectable intestinal permeability problems.
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Sistema Nervioso Entérico/efectos de los fármacos , Fructosa/administración & dosificación , Neuronas Serotoninérgicas/efectos de los fármacos , Serotonina/metabolismo , Transducción de Señal/efectos de los fármacos , Plexo Submucoso/efectos de los fármacos , Animales , Calcio/metabolismo , Dieta , Sistema Nervioso Entérico/metabolismo , Ratones , Neuronas Serotoninérgicas/metabolismo , Plexo Submucoso/metabolismoRESUMEN
BACKGROUND: Congenital enteric neuropathies of the distal intestine (CEN) are characterized by the partial or complete absence of enteric neurons. Over the last decade, zebrafish has emerged as a leading model organism in experimental research. Our aim was to demonstrate that the mutant zebrafish, lessen, expressing CEN characteristics, is an equally valuable animal model alongside mammalian models for CEN, by studying its enteric phenotype. METHODS: The effect of the lessen mutation on the development of the enteric nervous system (ENS), interstitial cells of Cajal (ICC), and intestinal motility in each intestinal region of mutant and wild-type (wt) zebrafish embryos at 3-6 dpf, was analyzed by immunofluorescent detection of neurochemical markers and motility assays. KEY RESULTS: Development of intestinal motility in the mutant was delayed and the majority of the observed contractions were disturbed. A significant disturbance in ENS development resulted in a distal intestine that was almost free of neuronal elements, in reduced neuronal density in the proximal and mid-intestine, and in a defect in the expression of neurochemical markers. Furthermore, markedly disturbed development of ICC gave rise to a less dense network of ICC. CONCLUSIONS & INFERENCES: The observed alterations in intestinal motility, intrinsic innervation and ICC network of the mutant in comparison with the wt zebrafish, are similar to those seen in the oligo- and aganglionic regions of the intestine of CEN patients. It is concluded that the zebrafish mutant lessen is an appropriate animal model to investigate CEN.
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Sistema Nervioso Entérico/fisiopatología , Motilidad Gastrointestinal/genética , Seudoobstrucción Intestinal/genética , Transactivadores/genética , Proteínas de Pez Cebra/genética , Animales , Modelos Animales de Enfermedad , Inmunohistoquímica , Células Intersticiales de Cajal/metabolismo , Células Intersticiales de Cajal/patología , Seudoobstrucción Intestinal/fisiopatología , Mutación , Pez CebraRESUMEN
BACKGROUND: Correct neuronal identification is essential to study neurons in health and disease. Although commonly used as pan-neuronal marker, HuC/D's expression pattern varies substantially between healthy and (patho)physiological conditions. This heterogenic labeling has received very little attention. We sought to investigate the subcellular HuC/D localization in enteric neurons in different conditions. METHODS: The localization of neuronal RNA-binding proteins HuC/D was investigated by immunohistochemistry in the mouse myenteric plexus using different toxins and caustic agents. Preparations were also stained with Sox10 and glial fibrillary acidic protein (GFAP) antibodies to assess enteric glial cell appearance. KEY RESULTS: Mechanically induced tissue damage, interference with the respiratory chain and oxygen (O2 ) deprivation increased nuclear HuC/D immunoreactivity. This effect was paralleled by a distortion of the GFAP-labeled glial network along with a loss of Sox10 expression and coincided with the activation of a non-apoptotic genetic program. Chemically induced damage and specific noxious stimuli did not induce a change in HuC/D immunoreactivity, supporting the specific nature of the nuclear HuC/D localization. CONCLUSIONS & INFERENCES: HuC/D is not merely a pan-neuronal marker but its subcellular localization also reflects the condition of a neuron at the time of fixation. The functional meaning of this change in HuC/D localization is not entirely clear, but disturbance in O2 supply in combination with the support of enteric glial cells seems to play a crucial role. The molecular consequence of changes in HuC/D expression needs to be further investigated.
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Proteínas ELAV/metabolismo , Plexo Mientérico/metabolismo , Neuronas/metabolismo , Animales , Hipoxia de la Célula , Colon/inervación , Colon/metabolismo , Proteína 3 Similar a ELAV , Proteína 4 Similar a ELAV , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones , Ratones Endogámicos C57BL , Plexo Mientérico/patología , Neuroglía/metabolismo , Neuronas/patologíaRESUMEN
BACKGROUND: Ghrelin is the only known peripherally active orexigenic hormone produced by the stomach that activates vagal afferents to stimulate food intake and to accelerate gastric emptying. Vagal sensory neurons within the nodose ganglia are surrounded by glial cells, which are able to receive and transmit chemical signals. We aimed to investigate whether ghrelin activates or influences the interaction between both types of cells. The effect of ghrelin was compared with that of leptin and cholecystokinin (CCK). METHODS: Cultures of rat nodose ganglia were characterized by immunohistochemistry and the functional effects of peptides, neurotransmitters, and pharmacological blockers were measured by Ca(2+) imaging using Fluo-4-AM as an indicator. KEY RESULTS: Neurons responded to KCl and were immunoreactive for PGP-9.5 whereas glial cells responded to lysophosphatidic acid and had the typical SOX-10-positive nuclear staining. Neurons were only responsive to CCK (31 ± 5%) whereas glial cells responded equally to the applied stimuli: ghrelin (27 ± 2%), leptin (21 ± 2%), and CCK (30 ± 2%). In contrast, neurons stained more intensively for the ghrelin receptor than glial cells. ATP induced [Ca(2+) ]i rises in 90% of the neurons whereas ACh and the NO donor, SIN-1, mainly induced [Ca(2+) ]i changes in glial cells (41 and 51%, respectively). The percentage of ghrelin-responsive glial cells was not affected by pretreatment with suramin, atropine, hexamethonium or 1400 W, but was reduced by l-NAME and by tetrodotoxin. Neurons were shown to be immunoreactive for neuronal NO-synthase (nNOS). CONCLUSIONS & INFERENCES: Our data show that ghrelin induces Ca(2+) signaling in glial cells of the nodose ganglion via the release of NO originating from the neurons.
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Señalización del Calcio/fisiología , Ghrelina/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Comunicación Paracrina/fisiología , Animales , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Ghrelina/farmacología , Inmunohistoquímica , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Ganglio Nudoso , Comunicación Paracrina/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
BACKGROUND: Menthol reduces intestinal motility in animal studies, an effect that is probably mediated by transient receptor potential channels. Peppermint oil (PO), with menthol as a major constituent, is widely used as a spasmolytic agent in irritable bowel syndrome. In the current study, we investigated the effect of acute PO administration on intragastric pressure (IGP) profiles and gastric sensorimotor functions in health. METHODS: Healthy volunteers underwent IGP measurement before and during continuous intragastric infusion of a nutrient drink (n = 13), and gastric barostat studies (n = 13). A single capsule of PO (182 mg) or placebo was administered during the studies in a randomized controlled crossover design. Throughout the studies, healthy volunteers scored 11 epigastric symptoms on a visual analogue scale (VAS); satiation was scored on a 6-point Likert scale during intragastric infusion. KEY RESULTS: During fasting, IGP and motility index (MI) of the proximal stomach decreased significantly after PO administration compared with placebo (P < 0.0001 and <0.05, respectively). In contrast, during intragastric infusion of the nutrient drink, no significant differences were detected between PO and placebo in IGP profiles, MI, satiation scores, and epigastric symptoms. The maximum infused volume, gastric compliance or sensitivity to balloon distention did not differ between both treatment arms. However, reduced appetite scores were seen during fasting after PO treatment, as compared with placebo (P = 0.01). Postprandial VAS scores were similar between PO and placebo. CONCLUSIONS & INFERENCES: Peppermint oil reduces IGP, proximal phasic contractility, and appetite, with negligible effects on gastric sensitivity, tone, accommodation, and nutrient tolerance in health.
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Suplementos Dietéticos , Motilidad Gastrointestinal/efectos de los fármacos , Estado de Salud , Aceites de Plantas/administración & dosificación , Células Receptoras Sensoriales/efectos de los fármacos , Adulto , Estudios Cruzados , Método Doble Ciego , Femenino , Vaciamiento Gástrico/efectos de los fármacos , Vaciamiento Gástrico/fisiología , Motilidad Gastrointestinal/fisiología , Humanos , Masculino , Mentha piperita , Células Receptoras Sensoriales/fisiología , Resultado del TratamientoRESUMEN
BACKGROUND: The intimate association between glial cells and neurons within the enteric nervous system has confounded careful examination of the direct responsiveness of enteric glia to different neuroligands. Therefore, we aimed to investigate whether neurotransmitters known to elicit fast excitatory potentials in enteric nerves also activate enteric glia directly. METHODS: We studied the effect of acetylcholine (ACh), serotonin (5-HT), and adenosine triphosphate (ATP) on intracellular Ca(2+) signaling using aequorin-expressing and Fluo-4 AM-loaded CRL-2690 rat and human enteric glial cell cultures devoid of neurons. The influence of these neurotransmitters on the proliferation of glia was measured and their effect on the expression of c-Fos as well as glial fibrillary acidic protein (GFAP), Sox10, and S100 was examined by immunohistochemistry and quantitative RT-PCR. KEY RESULTS: Apart from ATP, also ACh and 5-HT induced a dose-dependent increase in intracellular Ca(2+) concentration in CRL-2690 cells. Similarly, these neurotransmitters also evoked Ca(2+) transients in human primary enteric glial cells obtained from mucosal biopsies. In contrast with ATP, stimulation with ACh and 5-HT induced early gene expression in CRL-2690 cells. The proliferation of enteric glia and their expression of GFAP, Sox10, and S100 were not affected following stimulation with these neurotransmitters. CONCLUSIONS & INFERENCES: We provide evidence that enteric glial cells respond to fast excitatory neurotransmitters by changes in intracellular Ca(2+). On the basis of our experimental in vitro setting, we show that enteric glia are not only directly responsive to purinergic but also to serotonergic and cholinergic signaling mechanisms.
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Sistema Nervioso Entérico/fisiología , Neuroglía/metabolismo , Neurotransmisores/metabolismo , Transmisión Sináptica/fisiología , Animales , Calcio/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Sistema Nervioso Entérico/efectos de los fármacos , Humanos , Inmunohistoquímica , Neuroglía/efectos de los fármacos , Neurotransmisores/farmacología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transmisión Sináptica/efectos de los fármacosRESUMEN
Slow changes in [Ca(2+)](i) reflect increased neuronal activity. Our study demonstrates that single-trial fast [Ca(2+)](i) imaging (≥200 Hz sampling rate) revealed peaks each of which are associated with single spike discharge recorded by consecutive voltage-sensitive dye (VSD) imaging in enteric neurones and nerve fibres. Fast [Ca(2+)](i) imaging also revealed subthreshold fast excitatory postsynaptic potentials. Nicotine-evoked [Ca(2+)](i) peaks were reduced by -conotoxin and blocked by ruthenium red or tetrodotoxin. Fast [Ca(2+)](i) imaging can be used to directly record single action potentials in enteric neurones. [Ca(2+)](i) peaks required opening of voltage-gated sodium and calcium channels as well as Ca(2+) release from intracellular stores.
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Potenciales de Acción/fisiología , Calcio/fisiología , Plexo Mientérico/fisiología , Neuronas/fisiología , Compuestos de Anilina/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Potenciales Postsinápticos Excitadores/fisiología , Colorantes Fluorescentes/farmacología , Cobayas , Humanos , Íleon/fisiología , Masculino , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Compuestos de Piridinio/farmacología , Rojo de Rutenio/farmacología , Tetrodotoxina/farmacología , Imagen de Colorante Sensible al Voltaje , Xantenos/farmacología , omega-Conotoxinas/farmacologíaRESUMEN
BACKGROUND: From a classical point of view, gastric motility acts to clear the stomach between meals, whereas postprandial motility acts to provide a reservoir for food, mixing and grinding the food and to assure a controlled flow of food to the intestines. AIM: To summarise findings that support the role of gastric motility as a central mediator of hunger, satiation and satiety. METHODS: A literature review using the search terms 'satiety', 'satiation' and 'food intake' was combined with specific terms corresponding to the sequence of events during and after food intake. RESULTS: During food intake, when gastric emptying of especially solids is limited, gastric distension and gastric accommodation play an important function in the regulation of satiation. After food intake, when the stomach gradually empties, the role of gastric distension in the determination of appetite decreases and the focus will shift to gastric emptying and intestinal exposure of the nutrients. Finally, we have discussed the role of the empty stomach and the migrating motor complex in the regulation of hunger signals. CONCLUSIONS: Our findings indicate that gastric motility is a key mediator of hunger, satiation and satiety. More specifically, gastric accommodation and gastric emptying play important roles in the regulation of gastric (dis)tension and intestinal exposure of nutrients and hence control satiation and satiety. Correlations between gastric accommodation, gastric emptying and body weight indicate that gastric motility can also play a role in the long-term regulation of body weight.
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Digestión/fisiología , Ingestión de Alimentos/fisiología , Vaciamiento Gástrico/fisiología , Hambre/fisiología , Saciedad/fisiología , Estómago/fisiología , HumanosRESUMEN
Serotonin is one of the most abundant molecules in the gastrointestinal tract and it plays a crucial role in the regulation of several physiological functions, such as motility, secretion and visceral sensitivity. Besides this well documented physiological role, increasing evidence supports the concept that 5-HT is directly involved in pathological mechanisms, as well as the modulation of immune/inflammatory responses within the gut. The wide range of pathophysiological actions exerted by 5-HT are mediated by several different serotonergic receptor types and subtypes. Depending on the receptor bound and its localization, 5-HT evokes different and, sometimes, opposite responses. Therapeutic interventions aiming at modulating 5-HT signaling are mainly focused on the development of receptor agonists/antagonists, characterized by high affinity and selectivity for serotonergic receptors in the gut, to avoid the presence of adverse effects in the brain, where 5-HT is important in control mood. This review summarizes the vast current knowledge on 5-HT as a physiological mediator and analyzes the increasing body of literature describing 5-HT signaling abnormalities in functional and inflammatory disorders both in animal models and in humans. Finally, an overview on the therapeutic agents used in clinical practice is provided.
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Enfermedades Gastrointestinales/fisiopatología , Tracto Gastrointestinal/fisiología , Serotonina/fisiología , Animales , Estreñimiento/fisiopatología , Dispepsia/fisiopatología , Sistema Nervioso Entérico/fisiología , Gastroenteritis/fisiopatología , Enfermedades Gastrointestinales/tratamiento farmacológico , Motilidad Gastrointestinal/efectos de los fármacos , Motilidad Gastrointestinal/fisiología , Humanos , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Síndrome del Colon Irritable/fisiopatología , Neuronas/fisiología , Percepción del Dolor/fisiología , Receptores de Serotonina/clasificación , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/fisiología , Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Antagonistas de la Serotonina/uso terapéutico , Proteínas de Transporte de Serotonina en la Membrana Plasmática/fisiología , Agonistas de Receptores de Serotonina/farmacología , Agonistas de Receptores de Serotonina/uso terapéutico , Nervio Vago/fisiología , Dolor Visceral/fisiopatologíaRESUMEN
BACKGROUND: Little is known about the physiological role of the endocannabinoid system in the regulation of the motility and the sensitivity of the stomach. Endocannabinoid system dysfunction has been hypothesized to contribute to the control of food intake and the pathogenesis of functional dyspepsia. AIM: To study the influence of rimonabant, the endocannabinoid 1 (CB1) receptor antagonist, on gastric sensorimotor function in healthy controls. METHODS: After 4 days of pre-treatment with rimonabant 20 mg/day or placebo, 12 healthy volunteers (mean age 34 +/- 12 years, six men) participated in a placebo-controlled, double-blind, randomized, crossover study with a gastric barostat assessment of gastric sensitivity to distension, gastric compliance, gastric accommodation and phasic motility on day 3 and a liquid nutrient challenge test on day 4. RESULTS: Rimonabant did not influence gastric compliance and sensitivity to distension. The meal-induced gastric accommodation reflex was significantly inhibited by rimonabant (154.3 +/- 30.9 vs. 64.3 +/- 32.4 mL, P = 0.02). Rimonabant did not affect maximal nutrient tolerance or meal-related symptoms during the satiety drinking test. CONCLUSION: Endocannabinoids acting on the CB1 receptor are involved in the control of gastric accommodation in man.
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Moduladores de Receptores de Cannabinoides/uso terapéutico , Endocannabinoides , Motilidad Gastrointestinal/efectos de los fármacos , Piperidinas/administración & dosificación , Pirazoles/administración & dosificación , Receptores de Cannabinoides/uso terapéutico , Adolescente , Adulto , Anciano , Estudios Cruzados , Método Doble Ciego , Ingestión de Alimentos/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Rimonabant , Saciedad/efectos de los fármacos , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND Infection and inflammatory diseases of the gut results in profound changes of intestinal motor function. Acute administration of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) was shown to have excitatory and neuromodulatory roles in the myenteric plexus. Here we aimed to study the effect of prolonged IL-1beta incubation on the response of myenteric neurones to different stimuli. METHODS Longitudinal muscle myenteric plexus preparations (LMMP's) of the guinea pig jejunum were incubated for 24 h in medium with or without IL-1beta. After loading with Fluo-4, calcium imaging was used to visualize activation of neurones. The response to application of serotonin (5-HT), substance P (SP) and ATP or to electrical fibre tract stimulation (eFTS) was tested. Expression of nNOS, HuD, calbindin and calretinin was compared by immunohistochemistry. KEY RESULTS IL-1beta concentration-dependently influenced the neuronal responsiveness and duration of the [Ca(2+)](i) rises to 5-HT and ATP, while it also affected the Ca(2+)-transient amplitudes induced by 5-HT, ATP and SP. Ca(2+)-transients in response to eFTS were observed in significantly more neurones per ganglion after IL-1beta (10(-10) and 10(-11) mol L(-1)). Peak [Ca(2+)](i) rise after eFTS was concentration-dependently decreased by IL-1beta. The duration of the [Ca(2+)](i) rise after eFTS was prolonged after IL-1beta 10(-12) mol L(-1). IL-1beta (10(-9) mol L(-1)) incubation did not affect the number of nNOS, calretinin and calbindin expressing neurones, nor did it induce neuronal loss (HuD). CONCLUSIONS & INFERENCES In this study, IL-1beta differentially modulates the neuronal response to eFTS and neurotransmitter application in the myenteric plexus of guinea pigs. This cytokine could be implicated in the motility disturbances observed during gastrointestinal inflammation.
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Calcio/metabolismo , Interleucina-1beta/farmacología , Yeyuno/efectos de los fármacos , Plexo Mientérico/efectos de los fármacos , Plexo Mientérico/fisiología , Neuronas/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Adenosina Trifosfato/farmacología , Análisis de Varianza , Animales , Calbindina 2 , Calbindinas , Relación Dosis-Respuesta a Droga , Proteínas ELAV/metabolismo , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Cobayas , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Yeyuno/fisiología , Masculino , Microscopía Confocal , Neuronas/fisiología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Serotonina/farmacología , Sustancia P/farmacología , Transmisión Sináptica/efectos de los fármacosRESUMEN
Cannabinoid (CB) receptors are expressed in the enteric nervous system (ENS) and CB(1) receptor activity slows down motility and delays gastric emptying. This receptor system has become an important target for GI-related drug development such as in obesity treatment. The aim of the study was to investigate how CB(1) ligands and antagonists affect ongoing activity in enteric neurone networks, modulate synaptic vesicle cycling and influence mitochondrial transport in nerve processes. Primary cultures of guinea-pig myenteric neurones were loaded with different fluorescent markers: Fluo-4 to measure network activity, FM1-43 to image synaptic vesicles and Mitotracker green to label mitochondria. Synaptic vesicle cluster density was assessed by immunohistochemistry and expression of CB(1) receptors was confirmed by RT-PCR. Spontaneous network activity, displayed by both excitatory and inhibitory neurones, was significantly increased by CB(1) receptor antagonists (AM-251 and SR141716), abolished by CB(1) activation (methanandamide, mAEA) and reduced by two different inhibitors (arachidonylamide serotonin, AA-5HT and URB597) of fatty acid amide hydrolase. Antagonists reduced the number of synaptic vesicles that were recycled during an electrical stimulus. CB(1) agonists (mAEA and WIN55,212) reduced and antagonists enhanced the fraction of transported mitochondria in enteric nerve fibres. We found immunohistochemical evidence for an enhancement of synaptophysin-positive release sites with SR141716, while WIN55,212 caused a reduction. The opposite effects of agonists and antagonists suggest that enteric nerve signalling is under the permanent control of CB(1) receptor activity. Using inhibitors of the endocannabinoid degrading enzyme, we were able to show there is endogenous production of a CB ligand in the ENS.
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Sistema Nervioso Entérico/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Receptor Cannabinoide CB1/metabolismo , Transducción de Señal/fisiología , Animales , Benzoxazinas/farmacología , Transporte Biológico/fisiología , Señalización del Calcio/fisiología , Moduladores de Receptores de Cannabinoides/metabolismo , Células Cultivadas , Sistema Nervioso Entérico/citología , Femenino , Colorantes Fluorescentes , Cobayas , Masculino , Morfolinas/farmacología , Naftalenos/farmacología , Neuronas/citología , Piperidinas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptores de Cannabinoides/metabolismo , Rimonabant , Vesículas Sinápticas/metabolismoRESUMEN
The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.